thesis - unige

Thesis

Reference

Genetic mechanisms for the expression of endogenous retroviral

envelope glycoprotein gp70 implicated in murine systemic lupus

erythematosus

BAUDINO, Lucie Clementine

Abstract

The endogenous retroviral envelope glycoprotein, gp70, implicated in murine lupus nephritis is

secreted by hepatocytes as an acute phase protein. To better understand the genetic basis of

the expression of serum gp70, we analyzed the abundance of Xeno, PT or mPT gp70 RNAs

in livers in various strains of mice. Our results demonstrated that the expression of different

gp70 RNAs was remarkably heterogeneous among mouse strains and that serum gp70

production was regulated by multiple genes in physiological vs. inflammatory conditions. In

addition, we observed a contribution of PT and mPT gp70s, in addition of Xeno gp70, to

serum gp70. Furthermore, we observed an increased expression of intact mPT env RNA,

regulated by the Sgp3 locus, in all lupus-prone mice, as compared with non-autoimmune

strains of mice. Finally, we demonstrated that TLR7 played a critical role in the expression of

gp70 and in the production of anti-gp70 autoantibodies. These data suggest that lupus-prone

mice may possess a unique genetic mechanism responsible for the expression of mPT

retroviruses, which could act as a triggering factor through activating [...]

BAUDINO, Lucie Clementine. Genetic mechanisms for the expression of endogenous

retroviral envelope glycoprotein gp70 implicated in murine systemic lupus

erythematosus. Thèse de doctorat : Univ. Genève, 2010, no. Sc. 4234

URN : urn:nbn:ch:unige-107313

DOI : 10.13097/archive-ouverte/unige:10731

Available at:

http://archive-ouverte.unige.ch/unige:10731

Disclaimer: layout of this document may differ from the published version.

1 / 1

http://archive-ouverte.unige.ch/unige:10731

UNIVERSITÉ DE GENÈVE Département de Biologie Cellulaire Département de Pathologie et Immunologie

FACULTÉ DES SCIENCES Prof. Didier Picard FACULTÉ DE MEDECINE Prof. Shozo Izui

Genetic Mechanisms for the Expression of Endogenous Retroviral Envelope Glycoprotein gp70 Implicated

in Murine Systemic Lupus Erythematosus

THÈSE présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès Sciences, mention biologie

Par

Lucie BAUDINO

D’Annecy (France)

Thèse n° 4234

Genève Atelier d’impression ReproMail

2010

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Remerciements

Je tiens à exprimer ma profonde gratitude:

Au Professeur Shozo Izui qui m’a accueillie et soutenue depuis mon master. Je le

remercie d’avoir toujours été disponible afin de me transmettre sa passion pour la

recherche ainsi qu’une partie de son incroyable connaissance. Son dynamisme, ses idées

(une par seconde!) ainsi que son enthousiasme à toute épreuve, m’ont été indispensables

tout au long de ma thèse.

Aux Professeurs Daniel Pinschewer, Gilbert Fournié et Didier Picard d’avoir accepté de

faire partie de mon jury de thèse.

Aux Professeurs Daniel Kolakofsky et Walter Reith qui ont parrainé ma thèse.

A Giuseppe Celetta, Guy Brighouse, Montserrat Alvarez, Marie-Laure Santiago-Raber

dont la présence scientifique et amicale a été indispensable à la réussite de ma thèse. A

Mahdia Benkhoucha, dont nos rythmes de thèse étaient symbiotiques, pour avoir

partagé les moments de rires mais aussi de stress (merci aussi pour les macroutes!).

Ainsi qu’à tous mes autres collaborateurs du laboratoire Izui, passés ou présents,

Eduardo Martinez-Soria, Céline Manzin, Gregory Schneiter, Ngoc Lan Tran, Harris

Lemopoulos, Kumiko Yoshinobu, Naoki Morito, Gregg Sealy, Sandra Frayard, Evelyne

Homberg et Patrice Lalive, merci pour cette extraordinaire ambiance de travail.

A mes collaborateurs du Département de Pathologie et Immunologie pour le partage du

matériel et de leurs connaissances ainsi que pour les bons moments du café. Je remercie

tout particulièrement Isabelle Dunand-Sauthier pour son soutien moral et scientifique,

pour tous les fous rires (Ken Lee!) mais aussi pour son amitié. Un grand merci à

Isabelle Tchou pour l’aide et la compagnie au cours des longues journées à isoler les

hépatocytes primaires. Merci à Cécile Guichard pour tous ses conseils ainsi que pour les

soirées sushi-confocales lors de notre collaboration.

Au Professeur Leonard Evans, pour avoir partagé sa grande connaissance sur les

rétrovirus au cours de nombreuses discussions et aussi pour les corrections de

manuscripts.

A Léti, Co, Hélo, Virginie, Puce et tous les copains pour m’avoir encouragée.

Aux personnes qui me sont les plus chères, mes parents, mes sœurs Marion et Bertille et

surtout Quentin, qui m’ont apporté leur précieux soutien durant toutes ces années de

thèse.

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TABLE OF CONTENTS

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Table of contents Remerciements p1 Abbreviations p9 Résumé p13 Summary p23

I. Introduction p33

I.1. Systemic Lupus Erythematosus p33

I.1.1. Autoimmunity p33

I.1.2. Etiology of Human SLE p34

I.1.3. Murine Models of SLE p35

I.1.4. Multigenic Features of Murine SLE p36

I.1.4.1. Spontaneous Mutations Predisposing to SLE in Lupus Mice p37

I.1.4.1.1 The Fas and Fas ligand gene p37

I.1.4.1.2 The Yaa Mutation p37

I.1.4.2. MHC Association of Murine SLE p38

I.1.4.3. Non-MHC-linked Lupus Susceptibility Loci p41

I.1.4.3.1. Lupus Susceptibility Loci Mapped to Chromosome 1 p42




I.1.5. Development of Autoimmune Responses in Murine SLE p47

I.1.5.1. Hyperactive Phenotype of B cells in Murine SLE p47

I.1.5.2. Defective Clearance of Apoptotic Cells in Murine SLE p49

I.1.5.3. Critical Role of TLR7 in the Development of Autoimmune

Responses in Murine SLE p50

I.2. Endogenous retroviruses in SLE p55

I.2.1. Retroviruses p55

I.2.2. Murine ERVs p59

I.2.3. Role of ERVs in Murine SLE p61

II. Aims of the Study p67

II.1. Dissection of Genetic Mechanisms Governing the Expression of Serum

Retroviral gp70 Implicated in Murine Lupus Nephritis p67

II.2. Selective Up-Regulation of Intact, but Not Defective env RNAs of Endogenous

Modified Polytropic Retrovirus by Sgp3 Locus of Lupus-Prone Mice p68

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II.3. TLR-mediated Up-Regulation of Serum Retroviral gp70 is Controlled by

the Sgp Loci of Lupus-Prone Mice p68

II.4. The Sgp3 Locus Derived from the 129 Strain is Responsible for Enhanced p69

Endogenous Retroviral Expression in macroH2A1-deficient Mice

III. Results p73

III.1. Dissection of Genetic Mechanisms Governing the Expression of Serum

Retroviral gp70 Implicated in Murine Lupus Nephritis p73

III.2. Selective Up-regulation of Intact, but Not Defective env RNAs of Endogenous

Modified Polytropic Retrovirus by Sgp3 Locus of Lupus-Prone Mice p83

III.3. TLR-mediated Up-Regulation of Serum Retroviral gp70 is Controlled by

the Sgp Loci of Lupus-Prone Mice p94

III.4. The Sgp3 Locus Derived from the 129 Strain is Responsible for Enhanced

Endogenous Retroviral Expression in macroH2A1-deficient Mice p126

IV. General Discussion p153

IV.1. Genetic Origin of Serum Retroviral gp70 p153

IV.2. Polygenic Control of the Expression of Serum Retroviral gp70 p154

IV.3. Sgp3-mediated Control of Enhanced gp70 Production during

Acute Phase Responses p157

IV.4. Search for the Candidates Genes for Sgp3 p161

IV.5. Role of TLR7 and ERVs in Murine SLE p163

V. References p173

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ABBREVIATIONS

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Abbreviations

AIHA autoimmune hemolytic anemia

APP acute-phase protein

APR acute-phase response

B10 C57BL/10

BCR B-cell receptor

B6 C57BL/6 mice

CFS chronic fatigue syndrome

DC dendritic cell

EBV Epstein-Barr virus

Eco ecotropic (virus)

env envelope (gene)

ERV endogenous retrovirus

FcγγγγRs IgG Fc receptors

gag group specific antigen (gene)

gp70 glycoprotein (of) 70 (kDa molecular weight)

Gv1 Gross virus antigen 1 (locus)

IC immune complexes

IFNs interferons

IL interleukin

IL-6-RE interleukin-6-responsive element

KRAB-ZFP Krüppel-associated box-zinc-finger proteins

lpr lymphoproliferation

LPS lipopolysaccharides

LTR long terminal repeat sequence

MHC major histocompatibility complex

mPT modified polytropic (virus)

MS multiple sclerosis

MSRV multiple sclerosis-associated retroviral agent

NC nucleoproteins

NF-κκκκB nuclear factor-kappa B

NZB new zealand black (mice)

NZW new zealand white (mice)

pDC plasmacytoid dendritic cell

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PMN polymorphonuclear (cell)

PT polytropic (virus)

RNaseL ribonuclease L

Rsl regulator of sex limitation (gene)

Rsrc1 arginine/serine-rich coiled-coil 1 (gene)

Sgp serum gp70 production (gene)

SLAM signaling lymphocyte activation molecule

SLE systemic lupus erythematosus

SLP sex-limited protein

SU surface envelop glycoprotein

TLR toll-like receptor

TM transmembrane envelope glycoprotein

TNFαααα tumor necrosis factor α

Xeno xenotropic (virus)

XMRV xenotropic murine leukemia virus-related virus

XPR1 xenotropic and polytropic retrovirus receptor

Yaa Y-linked autoimmune acceleration (gene)

Zfp zinc-finger protein (gene)

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RESUME

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Résumé

Introduction

Les rétrovirus endogènes (ERVs) sont impliqués dans la pathogénèse du lupus

érythémateux disséminé (SLE). Cette relation a été suggérée lorsque des antigènes de

virus de la leucémie murine ont été trouvés dans les dépôts de complexes-immuns (IC)

des glomérules de souris lupiques NZB et (NZB x NZW)F1 hybrides. Par la suite, il a

été démontré qu’une grande quantité de l’enveloppe de la glycoprotéine 70 (gp70),

dérivées des ERVs, est présente dans le sérum des souris lupiques (NZB x NZW)F1,

MRL-Faslpr et BXSB et que seules les souris lupiques développent spontanément des

anticorps contre la gp70 rétrovirale sérique. En effet, des ICs gp70-anti-gp70 (gp70 ICs)

sont observés dans la circulation sanguine dès l’apparition de la maladie rénale et dans

les glomérules des souris lupiques en corrélation avec le développement de la néphrite

lupique sévère, confirmant un rôle pathogénique des gp70 ICs dans le lupus murin.

La concentration de la gp70 sérique varie grandement entre les différentes

souches de souris. Toutes les souches de souris lupiques ont une concentration sérique

de la gp70 relativement élevée (>15 µg/ml), alors que les souris C57BL/6 (B6),

C57BL/10 (B10) et BALB/c produisent une faible quantité de la gp70 sérique (<5

µg/ml). Des études génétiques ont révélé la présence d’au moins deux loci liés au

niveau basal de la gp70 sérique. Un locus majeur Sgp3 (serum gp70 production 3) est

localisé au milieu du chromosome 13. Notons que Sgp3 se superpose au gène Gv1

(Gross virus antigen 1) qui contrôle l’expression de l’antigène gp70 GIX du thymus. De

plus, un deuxième locus, Sgp4, est localisé sur la partie distale du chromosome 4. La

g70 sérique partage des propriétés immunologiques et biochimiques avec l’antigène

GIX impliqué dans la différenciation thymocytaire. Cependant, la glycoprotéine gp70

GIX n’est pas la source principale de la gp70 sérique. En effet, la gp70 rétrovirale

sérique est secrétée par les hépatocytes et se comporte comme une protéine de la phase

aigüe (APP). Contrairement aux APPs conventionnelles, seules les souris qui possèdent

un niveau basal élevé de la gp70 sérique présentent une augmentation de l’expression

celle-ci en réponse au LPS, suggérant que la gp70 sérique est sous contrôle génétique.

Les ERVs sont classés en trois groupes de virus, écotropiques (Eco),

xénotropiques (Xeno) ou polytropiques suivant leur capacité à infecter différents hôtes

en fonction de leurs protéines gp70 respectives. De plus, quatre sous-groupes de

provirus Xeno (Xeno-I, Xeno-II, Xeno-III et Xeno-IV), ainsi que deux sous-groupes de

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virus polytropiques, polytropiques (PT) et polytropiques modifiés (mPT), ont été

caractérisés en fonction de la différence de la séquence nucléotidique de leurs gp70. Des

analyses sérologiques ont exclu l’implication de la gp70 Eco comme étant une source de

la gp70 sérique. De plus, des analyses de cartes de peptides tryptiques ont montré que la

molécule de la gp70 sérique ressemble à celle de l’enveloppe du virus NZB-X1, l’un

des deux virus Xeno isolés de souris NZB. Cependant, les empreintes des gp70 du

sérum présentent aussi des marqueurs peptidiques détectables dans les gp70 d’autres

virus Xeno, comme le deuxième virus Xeno isolé de souris NZB, NZB-X2, et les gp70

exprimées sur les thymocytes et les lymphocytes de la rate.

Objectifs

Le but de cette étude est de définir l’origine de l’autoantigène gp70 rétroviral

sérique impliqué dans le lupus murin et les mécanismes génétiques régulant

l’expression de la gp70 sérique dans des conditions physiologiques ou inflammatoires.

1) La glycoprotéine de l’enveloppe de rétrovirus endogène, gp70, impliquée

dans la néphrite lupique murine, est sécrétée par les hépatocytes. Cette protéine gp70 a

été décrite comme étant le produit de virus endogènes Xeno, NZB-X1. Cependant,

comme les virus endogènes PT et mPT codent pour des gp70 qui sont fortement

apparentées à la gp70 de virus Xeno, ces virus peuvent être considérés comme des

sources additionnelles de gp70 sérique. Afin d’identifier l’origine génétique de la gp70

dans le sérum, nous avons déterminé la composition des provirus Xeno, PT et mPT et

l’abondance de leurs ARNs codant pour la gp70 dans le foie de différentes souches de

souris, dont les souris congéniques Sgp3 et Sgp4 dans des conditions physiologiques et

inflammatoires.

2) En vue d’examiner la possibilité qu’une classe particulière de ERVs soit

associée à la pathogénèse du SLE, nous avons comparé l’expression des virus Eco,

Xeno, PT et des trois variants de mPT récemment découverts dans des souris lupiques,

Sgp3 et Sgp4 congéniques ou non-autoimmunes. De plus, étant donné le rôle émergent

de TLR7 dans la pathogénèse du SLE, nous avons défini le rôle potentiel de TLR7 dans

le développement des réponses autoimmunes anti-gp70.

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3) Afin de mieux définir les mécanismes moléculaires, cellulaires et génétiques

responsables de la production de la gp70 sérique dans des conditions inflammatoires,

nous avons comparé l’effet du LPS sur l’expression de la gp70 sérique lors de la phase

aigüe avec l’effet de cytokines inflammatoires, connues comme inducteurs de APP,

dans différentes souches de souris dont les souris Sgp congéniques. Etant donné que

TLR7 et TLR9 jouent un rôle important dans la pathogénèse du SLE, nous avons aussi

exploré l’implication de TLR7 et TRL9 dans l’expression de la gp70 sérique lors de la

phase aigüe.

4) Une récente découverte a démontré que l’expression des ERVs est réprimée

par les variants de macroH2A1, dont le gène est localisé dans l’intervalle Sgp3. Nous

avons donc exploré la possibilité que le gène macroH2A1 soit un gène candidat de Sgp3.

Nous avons déterminé le niveau de la gp70 sérique et l’abondance des ARNs des gp70

des ERVs dans le foie de deux souris sauvages ou déficientes pour le gène macroH2A1

dans un fond génétique B6 ou 129 en relation avec le locus Sgp3 dérivé de souris 129.

Résultats

1) Dissection des Mécanismes Génétiques Gouvernant l’Expression de la gp70

Rétrovirale Sérique Impliqués dans la Néphrite Lupique Murine

Afin de mieux comprendre les bases génétiques de l’expression de la gp70

sérique, nous avons analysé l’abondance des ARNs messagers de la gp70 de Xeno, PT

et mPT dans le foie et la composition génomique des provirus correspondants dans

différentes souches de souris, dont les deux différentes souris Sgp congéniques. Nos

résultats ont démontré que l’expression des ARNs des gp70 sériques est très hétérogène

entre les différentes souches de souris et que le niveau de production de la gp70 sérique

est régulé par de nombreux gènes structuraux et régulateurs. De plus, une contribution

significative de la gp70 de PT et mPT à la gp70 sérique a été révélée. En effet, les

transcrits de gp70 de PT et mPT, mais pas ceux de Xeno, ont été détectés dans les souris

129. De plus, le niveau de gp70 sériques montre une plus grande corrélation avec

l’abondance des ARNs codant pour les gp70 de PT et mPT qu’avec celle de Xeno dans

les souris Sgp3 congéniques. Par ailleurs, l’injection de LPS augmente sélectivement

l’expression des ARNs codants pour les gp70 de Xeno et mPT, mais pas celle de PT.

Nos résultats indiquent que l’origine de la gp70 sérique est plus hétérogène que nous

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pensions, et que les différentes gp70 rétrovirales sont régulées différemment dans des

conditions physiologiques et inflammatoires.

2) Augmentation Sélective de l’ARN de l’Enveloppe Intacte de Rétrovirus

Endogènes Polytropiques Modifiés par le Locus Sgp3 dans les Souris Lupiques

Comme les quatre classes de rétrovirus endogènes, Eco, Xeno, PT ou mPT, sont

exprimées chez la souris, nous avons examiné la possibilité qu’une classe de rétrovirus

endogène particulière soit associée avec le développement du SLE murin. Nous avons

observé une augmentation de plus de 15 fois de l’expression de l’ARN de l’enveloppe

de mPT dans le foie des quatre souches de souris lupiques, en comparaison avec les

neuf souches de souris non-autoimmunes. Ceci n’était pas le cas pour les trois autres

classes de rétrovirus. De plus, nous avons observé que, en plus de transcrits intacts de

mPT, de nombreuses souches de souris expriment deux transcrits défectueux de

l’enveloppe mPT, D1 et D2, qui présentent une délétion dans la partie 3’ de la séquence

de la protéine de surface de l’enveloppe gp70 et dans la partie 5’ de la protéine

transmembranaire p15E, respectivement. Remarquablement, à la différence des souches

de souris non-autoimmunes, les quatre souches de souris lupiques expriment des

niveaux abondants du transcrit de l’enveloppe intacte de mPT, mais des niveaux faibles

non détectables des transcrits des mutants de l’enveloppe mPT. Le locus Sgp3 dérivé de

souris lupiques est responsable de l’augmentation sélective de l’ARN de la gp70 de

mPT intact. Finalement, nous avons observé que TLR7, spécifique de la reconnaissance

des ARN simples brins, joue un rôle critique dans la production des autoanticorps anti-

gp70. Ces résultats suggèrent que les souris lupiques possèdent un mécanisme génétique

unique contrôlant l’expression des rétrovirus mPT, qui peuvent agir comme un facteur

déclenchant, à travers l’activation de TLR7, du développement de réponses

autoimmunes chez les souris prédisposées au SLE.

3) L’Augmentation de la gp70 Rétrovirale Sérique Induite par TLR est Contrôlée

par les Loci Sgp des Souris Lupiques

La gp70 rétrovirale sérique est secrétée par les hépatocytes comme une APP et

cette réponse est sous contrôle génétique. Etant donné le rôle de TLR7 et TLR9 dans la

pathogénèse du SLE, nous avons évalué leur contribution dans l’expression de la gp70

sérique lors de la phase aigüe, et exploré le rôle central des loci Sgp3 et Sgp4 dans cette

réponse. Nos résultats démontrent que le niveau de la gp70 sérique est augmenté dans

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les souris lupiques NZB injectées avec les agonistes de TLR7 et TLR9 et atteint des

niveaux comparables à ceux induits par l’injection d’IL-1, IL-6 ou TNF. De plus,

l’étude des souris B6 Sgp3 ou Sgp4 congéniques à permis de définir un rôle majeur de

ces deux loci dans l’augmentation de la production de la gp70 sérique au cours des

réponses de la phase aigüe. Finalement, l’analyse de souris Sgp3 congéniques suggère

fortement la présence d’au moins deux facteurs génétiques distincts dans l’intervalle

Sgp3, dont un contrôle le niveau basal de l’expression de la gp70 de Xeno, PT et mPT et

l’autre contrôle l’augmentation de la production des gp70 de Xeno et mPT durant les

réponses de la phase aigüe. Nos résultats ont démontré un nouveau rôle pathogénique de

TLR7 et TLR9 en favorisant l’expression de l’autoantigène néphritogénique de la gp70

impliqué dans la néphrite lupique. De plus, ils révèlent la participation de multiples

gènes régulateurs dans l’expression des autoantigènes de la gp70 dans des conditions

basales ou inflammatoires.

4) Le Locus Sgp3 Dérivé de Souris 129 est Responsable de l’Augmentation de

l’Expression Rétrovirale Endogène dans les Souris Déficientes pour le Gène

macroH2A1

L’expression de la gp70 sérique est régulée par le locus Sgp3 sur le chromosome

13. Etant donné que le gène macroH2A1, codant pour un variant de l’histone macroH2A,

est localisé dans l’intervalle Sgp3 et que la transcription des séquences rétrovirales

endogènes est augmentée dans les souris B6 déficientes pour le gène macroH2A1, nous

avons examiné la possibilité que le gène macroH2A1 soit un gène candidat du locus

Sgp3. Les souris B6 déficientes pour le gène macroH2A1, portant le locus Sgp3 dérivé

de souris 129 qui a été co-transféré avec le gène mutant macroH2A1 de souris 129,

présentent des niveaux élevés de la gp70 sérique et d’ARNs rétroviraux hépatiques de

gp70 comparables à ceux des souris B6.NZB-Sgp3 congéniques. Au contraire,

l’abondance des ARNs rétroviraux de la gp70 dans les souris 129 déficientes pour le

gène macroH2A1 n’est pas affectée par rapport à celle des souris 129 sauvages. De plus,

les souris Sgp3 subcongéniques, dépourvues du gène macroH2A1 dérivé de souris NZB,

présentent un phénotype Sgp3 identique à celui des souris B6.NZB-Sgp3 congéniques

portant le gène macroH2A1 dérivé de souris NZB. Ceci exclut donc le gène macroH2A1

comme gène candidat de Sgp3. Par ailleurs, des niveaux comparables observés d’ARN

messager de macroH2A1 entre les souris subcongéniques B6.NZB-Sgp3 et les souris B6

sauvages démontrent que Sgp3 ne contribue pas à la diminution de la répression des

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rétrovirus endogènes via la diminution de l’expression du gène macroH2A1. Nos

résultats indiquent que l’augmentation de la transcription de séquences rétrovirales

endogènes observée dans les souris B6 déficientes pour le gène macroH2A1 n’est pas

due à la mutation macroH2A1, mais due à la présence du locus Sgp3 dérivé des souris

129.

Discussion

L’analyse de l’abondance des ARNs spécifiques de groupes et sous-groupes de

gp70 rétrovirales et la présence des provirus correspondants révèlent que l’origine

génétique de la gp70 sérique est plus hétérogène que nous pensions. En effet, nous

observons une contribution non négligeable des gp70 de PT et mPT, en plus de celle de

Xeno, à la gp70 sérique. Cette hétérogénéité observée dans différentes souches de souris

est en partie due à l’absence de certains provirus dans leurs génomes respectifs, et

d’autre part, due probablement à leurs sites d’intégration ou à la régulation de la

transcription. En effet, l’expression de la gp70 sérique est fortement dépendante de la

présence de gènes régulateurs. Nos études des souris Sgp congéniques ont révélé que

Sgp3 n’est pas un gène structural mais agit plutôt comme un gène régulateur majeur du

contrôle du niveau basal de la gp70 sérique via la régulation transcriptionelle des

provirus Xeno, PT and mPT. De plus, nous avons confirmé une contribution modeste

mais significative du locus Sgp4 dans la production de la gp70 sérique en régulant le

niveau d’expression basale d’un sous-groupe de provirus Xeno (Xeno-I). L’analyse de

souris B6 double congéniques portant les loci Sgp3 et Sgp4 a révélé un effet synergique

de ces deux loci Sgp sur la production de la gp70 sérique. Cependant, leur niveau basal

de la gp70 sérique n’a pas atteint celui des souris lupiques NZB et NZW, suggérant

qu’un autre locus pourrait contribuer à la gp70 sérique. En effet, nos études actuelles

des souris B6 portant l’intervalle de la partie proximale du chromosome 12 provenant

des souris NZB ont montré une hausse modeste de la gp70 sérique via une augmentation

sélective de l’ARN de la gp70 de Xeno. De plus, nos résultats ont démontré que le locus

Sgp3 est responsable de l’expression prédominante et abondante des provirus mPT

portant le gène de l’enveloppe intact, et d’une absence presque totale de l’expression

des provirus mPT portant les gènes de l’enveloppe défectueux (D1 or D2) dans les

quatre souches de souris lupiques. Ceci indique que les souris lupiques possèdent

probablement un mécanisme génétique unique responsable de l’expression des

rétrovirus mPT. Nos résultats montrent que les différents niveaux de gp70 sérique dans

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les différentes souches de souris peuvent être expliqués par la présence de différents

gènes régulateurs et structuraux impliqués dans la production de la gp70 sérique dans le

foie.

L’expression de la gp70 sérique est augmentée par les inducteurs de APP,

indiquant que la gp70 sérique se comporte comme une APP. Cette notion a été

confirmée par notre étude qui a montré que les cytokines proinflammatoires IL-1, IL-6

et TNF induisent de façon similaire une augmentation du niveau des gp70 dans le sérum

de souris NZB. Cependant, contrairement aux APPs conventionnelles, la réponse de la

gp70 sérique est dépendante des souches de souris, puisque seules les souris ayant un

niveau basal élevé de la gp70 sérique montrent une augmentation de la production de la

gp70 sérique en réponse au LPS. Nos études des souris Sgp3, Sgp4 et Sgp3/4

congéniques ont révélé que les deux loci Sgp agissent de manière synergique et jouent

un rôle majeur dans l’expression de la gp70 sérique lors de la phase aigüe.

Remarquablement, seuls les ARNs des gp70 de Xeno et mPT ont été induits durant les

réponses de la phase aigüe. L’effet sélectif du LPS sur les ARNs des gp70 de Xeno et

mPT est probablement lié à l’importante hétérogénéité des régions régulatrices U3 des

séquences terminales longues répétées (LTR) entre les différentes classes de ERVs.

Contrairement à l’effet sélectif de Sgp3 sur l’expression des provirus mPT portant

le gène de l’enveloppe intacte dans les conditions basales, Sgp3 est aussi impliqué dans

l’augmentation de l’expression d’un des provirus mPT défectueux portant l’enveloppe

mutante D1 en réponse au LPS. De plus, l’injection de LPS dans les souris B6.Sgp4 a

induit une augmentation de l’ARN de la gp70 de Xeno-I mais aussi de Xeno-II et Xeno-

III, alors que le locus Sgp4 n’a induit qu’une augmentation de l’ARN de la gp70 de

Xeno-I dans des conditions non-inflammatoires. Ces résultats suggèrent que les loci

Sgp3 et Sgp4 portent probablement au moins deux éléments régulateurs distincts, qui

contrôlent indépendemment l’expression des ERVs dans des conditions physiologiques

versus inflammatoires.

Il a été démontré récemment que la transcription des séquences rétrovirales

endogènes dans le foie de souris B6 déficientes pour le gène de macroH2A1 est

augmentée. Puisque le gène macroH2A1 est présent dans l’intervalle Sgp3, nous avons

testé l’hypothèse que macroH2A1 soit un gène candidat du locus Sgp3. Cependant, nos

analyses ont exclu l’implication du gène macroH2A1 dans l’expression des ERVs. En

effet, l’augmentation de la transcription des séquences rétrovirales endogènes observée

dans les souris B6 déficientes pour le gène macroH2A1 n’était pas due à la mutation de

macroH2A1, mais due à la présence du locus Sgp3 dérivé de souris 129, qui a été co-

- 20 -

transféré avec le gène mutant de macroH2A1 de souris 129 par rétrocroisement. Nos

études actuelles ont permis de réduire le locus Sgp3 dérivé de souris NZB à un

intervalle de 5.42 Mb. Notablement, cette région contient un groupe de 21 gènes Zfp,

qui codent pour des protéines à doigt de zinc, mais les gènes cibles régulés par la

plupart de ces gènes Zfp restent inconnus. Il est possible que plusieurs de ces gènes Zfp

présents dans le locus Sgp3 participent à la régulation de l’expression des ERVs dans

des conditions physiologiques ou inflammatoires.

Finalement, nos études ont révélé que TLR7, un récepteur pour l’ARN simple brin,

joue un rôle critique dans le développement des réponses autoimmunes contre les gp70

sériques, confirmant l’idée que les ERVs jouent un rôle actif dans le SLE murin.

Notablement, nous avons observé que le locus Sgp3 promeut l’expression abondante et

préférentielle des provirus mPT possédant un gène de l’enveloppe intacte dans les souris

lupiques. Ainsi, une augmentation de la production des ERVs pourrait induire une

activation des cellules B autoréactives anti-gp70 et des cellules dendritiques via la

stimulation de TLR7, impliqués dans le SLE murin. De plus, notre démonstration de

l’augmentation de la production de la gp70 sérique après l’injection d’agoniste de TLR7

a révélé un rôle nouveau de ce récepteur dans la pathogénèse de la néphrite lupique

murine. TLR7 est impliqué dans l’augmentation de la gp70 sérique au cours de la

maladie, probablement via l’activation de macrophages en réponse à des IC IgG

contenant de l’ARN, fournissant ainsi une source supplémentaire pour des stimulations

antigéniques et la formation de IC néphritogéniques. Des recherches supplémentaires

sur les bases moléculaires responsables de l’expression des ERVs dans le SLE murin

vont permettre de déterminer la pertinence chez l’humain, procurant ainsi un indice

pour un rôle potentiel des ERVs dans le SLE humain.

- 21 -

SUMMARY

- 22 -

- 23 -

Summary Introduction

Endogenous retroviruses (ERVs) are implicated in the pathogenesis of murine

systemic lupus erythematosus (SLE). This relationship was first suggested when murine

leukemia viral antigens were found in immune deposits of diseased glomeruli from

lupus-prone mice NZB and (NZB x NZW)F1 hybrid mice. Subsequently, it was

demonstrated that relatively large amounts of the envelope glycoprotein gp70 (encoded

by the env gene), derived from ERVs, are present in the sera from lupus-prone (NZB x

NZW)F1, MRL-Faslpr and BXSB mice and that only lupus-prone mice spontaneously

develop autoantibodies against serum retroviral gp70. Indeed, gp70-anti-gp70 immune

complexes (gp70 IC) are found in the circulating blood close to the onset of renal

disease and within diseased glomeruli of lupus mice in correlation with the development

of severe lupus nephritis, further supporting the pathogenic role of gp70 IC in murine

SLE.

Serum concentrations of gp70 vary greatly among different inbred strains of

mice. Significantly, all SLE-prone strains have relatively high concentrations of gp70 in

their sera (>15 µg/ml), whereas C57BL/6 (B6), C57BL/10 (B10) and BALB/c mice

produce low serum levels of gp70 (<5 µg/ml). Genetic studies revealed the presence of

at least two loci linked with steady-state level of serum retroviral gp70. A major locus

Sgp3 (serum gp70 production 3) is located on mid chromosome 13, which overlaps with

the Gv1 (Gross virus antigen 1) gene controlling the expression of thymic GIX gp70

antigen, and a second locus, Sgp4, on distal chromosome 4. Serum gp70 shares

immunological and biochemical properties with the thymocyte differentiation antigen

GIX. However, the GIX gp70 is not a major source for serum retroviral gp70. Instead,

serum retroviral gp70 is secreted by hepatocytes and behaves like an acute-phase

protein (APP). However, unlike conventional APP, only mice having high basal levels

of serum gp70 displayed an up-regulated expression of serum gp70 in response to LPS,

indicating that the acute phase expression of serum gp70 is apparently under a genetic

control.

ERVs are classified as ecotropic (Eco), xenotropic (Xeno) or polytropic viruses

according to their host range dictated by their respective gp70 proteins. In addition,

based on differences in gp70 nucleotide sequences, four subgroups of Xeno proviruses

(Xeno-I, Xeno-II, Xeno-III and Xeno-IV), as well as two subgroups of polytropic

proviruses, termed PT (polytropic) and mPT (modified polytropic), are present in the

- 24 -

mouse genome. Serological analysis excluded the involvement of Eco gp70 as a source

of serum gp70. Tryptic peptide mapping analysis showed that serum gp70 molecule

resembles the envelope protein of NZB-X1 virus, one of the two distinct Xeno viruses

isolated from NZB mice. However, the fingerprint of serum gp70 also displayed

additional marker peptides detectable in gp70 of other Xeno viruses, including the

second NZB Xeno virus, NZB-X2, and gp70 expressed on thymocytes and splenic

lymphocytes.

Aims of the Study

The present study aims to define the genetic origin of serum retroviral gp70

autoantigen implicated in murine SLE and the genetic mechanisms governing the

expression of serum gp70 under steady-state or inflammatory conditions.

1) The endogenous retroviral envelope glycoprotein, gp70, implicated in murine

lupus nephritis is secreted by hepatocytes, and has been believed to be a product of an

endogenous Xeno virus, NZB-X1. However, since endogenous PT and mPT viruses

encode gp70s that are closely related to Xeno gp70, these viruses can be additional

sources of serum gp70. To identify the genetic origin of serum gp70, we determined the

genomic composition of Xeno, PT and mPT proviruses and the abundance of their gp70

RNAs in livers from various strains of mice, including Sgp3 and Sgp4 congenic mice,

under physiological and inflammatory conditions.

2) We explored whether a particular class of ERVs is associated with the

development of SLE. To address this question, we compared the expression of Eco,

Xeno, PT and mPT retroviruses in lupus-prone mice, Sgp3 and Sgp4 congenic mice and

non-autoimmune mice. Furthermore, in view of an emerging role of TLR7 in the

pathogenesis of SLE, we defined the role of TLR7 in the development of anti-gp70

autoimmune responses.

3) In order to better understand the molecular, cellular and genetic mechanisms

responsible for the production of serum gp70 under inflammatory condition, we

compared the effect of LPS on the acute phase expression of serum gp70 with that of

inflammatory cytokines, known as inducers of APP, in different stains of mice,

including Sgp congenic mice. Furthermore, since TLR7 and TLR9 play important roles

- 25 -

in the pathogenesis of SLE, we explored the implication of TLR7 and TLR9 in the acute

phase expression of serum gp70.

4) Based on a recent finding which claimed that the expression of ERVs is

repressed by macroH2A1 histone variants, the gene of which is located within the Sgp3

interval, we explored the possibility of macroH2A1 gene as a candidate for Sgp3. We

determined serum levels of gp70 and the abundance of endogenous retroviral gp70

RNAs in livers from two different macroH2A1-suficient and -deficient mice bred into

the B6 or 129 background in relation to the 129-derived Sgp3 locus.

Results

1) Dissection of Genetic Mechanisms Governing the Expression of Serum

Retroviral gp70 Implicated in Murine Lupus Nephritis

To better understand the genetic basis of the expression of serum gp70, we

analyzed the abundance of Xeno, PT or mPT gp70 RNAs in livers and the genomic

composition of corresponding proviruses in various strains of mice, including two

different Sgp congenic mice. Our results demonstrated that the expression of different

viral gp70 RNAs was remarkably heterogeneous among various mouse strains and that

the level of serum gp70 production was regulated by multiple structural and regulatory

genes. In addition, a significant contribution of PT and mPT gp70s to serum gp70 was

revealed by the detection of PT and mPT, but not Xeno transcripts in 129 mice and by a

closer correlation of serum levels of gp70 with the abundance of PT and mPT gp70

RNAs than with that of Xeno gp70 RNA in Sgp3 congenic mice. Furthermore, the

injection of LPS selectively up-regulated the expression of Xeno and mPT gp70 RNAs,

but not PT gp70 RNA. Our data indicate that the genetic origin of serum gp70 is more

heterogeneous than previously believed, and that distinct retroviral gp70s are

differentially regulated in physiological vs. inflammatory conditions.

2) Selective Up-Regulation of Intact, but Not Defective env RNAs of Endogenous

Modified Polytropic Retrovirus by the Sgp3 Locus in Lupus-Prone Mice

Since four different classes of ERVs, i.e. Eco, Xeno, PT or mPT, are expressed

in mice, we investigated the possibility that a particular class of ERVs is associated with

the development of murine SLE. We observed more than 15-fold increased expression

of mPT env RNA in livers of all four lupus-prone mice, as compared with those of nine

non-autoimmune strains of mice. This was not the case for the three other classes of

- 26 -

retroviruses. Furthermore, we found that in addition to intact mPT transcripts, many

strains of mice expressed two defective mPT env transcripts, D1 and D2, which carry a

deletion in the env sequence of the 3’ portion of the gp70 surface protein and the 5’

portion of the p15E transmembrane protein, respectively. Remarkably, in contrast to

non-autoimmune strains of mice, all four lupus-prone mice expressed abundant levels of

intact mPT env transcripts, but only low or non-detectable levels of the mutant env

transcripts. The Sgp3 locus derived from lupus-prone mice was responsible for the

selective up-regulation of the intact mPT env RNA. Finally, we observed that single-

stranded RNA-specific TLR7 played a critical role in the production of anti-gp70

autoantibodies. These data suggest that lupus-prone mice may possess a unique genetic

mechanism responsible for the expression of mPT retroviruses, which could act as a

triggering factor through activating TLR7 for the development of autoimmune

responses in mice predisposed to SLE.

3) TLR-mediated Up-Regulation of Serum Retroviral gp70 is Controlled by the

Sgp Loci of Lupus-Prone Mice

Serum retroviral gp70 is secreted by hepatocytes like an APP, the response of

which is under a genetic control. Given critical roles of TLR7 and TLR9 in the

pathogenesis of SLE, we assessed their contribution to the acute phase expression of

serum gp70, and defined a pivotal role of the Sgp3 and Sgp4 loci in this response. Our

results demonstrated that serum levels of gp70 were up-regulated in lupus-prone NZB

mice injected with TLR7 or TLR9 agonist at levels comparable to those induced by

injection of IL-1, IL-6 or TNF. In addition, studies of B6 Sgp3 and/or Sgp4 congenic

mice defined the major roles of these two loci in up-regulated production of serum gp70

during acute phase responses. Finally, the analysis of Sgp3 congenic mice strongly

suggests the presence of at least two distinct genetic factors in the Sgp3 interval, one of

which controlled the basal-level expression of Xeno, PT and mPT gp70 and the other

which controlled the up-regulated production of Xeno and mPT gp70 during acute

phase responses. Our results uncovered an additional pathogenic role of TLR7 and

TLR9 by promoting the expression of nephritogenic gp70 autoantigen implicated in

murine nephritis. Furthermore, they revealed the involvement of multiple regulatory

genes for the expression of gp70 autoantigen under steady-state and inflammatory

conditions.

- 27 -

4) The Sgp3 Locus Derived from the 129 Strain is Responsible for Enhanced


The expression of serum retroviral gp70 is largely regulated by the Sgp3 locus on

chromosome 13. Because of the localization of the macroH2A1 gene encoding

macroH2A histone variants within the Sgp3 interval and of an up-regulated transcription

of endogenous retroviral sequences in macroH2A1-deficient B6 mice, we investigated

the possibility of the macroH2A1 gene as a candidate for Sgp3. macroH2A1-deficient

B6 mice carrying the 129-derived Sgp3 locus, which was co-transferred with the 129

macroH2A1 mutant gene, displayed increased levels of serum gp70 and hepatic

retroviral gp70 RNAs comparable to those of B6.NZB-Sgp3 congenic mice. In contrast,

the abundance of retroviral gp70 RNAs in macroH2A1-deficient 129 mice was not

elevated at all as compared with wild-type 129 mice. Furthermore, Sgp3 subcongenic

mice devoid of the NZB-derived macroH2A1 gene displayed the Sgp3 phenotype

identical to that of B6.NZB-Sgp3 congenic mice carrying the NZB-derived macroH2A1

gene, excluding macroH2A1 as the candidate Sgp3 gene. Moreover, comparable levels

of macroH2A1 mRNAs between B6.NZB-Sgp3 subcongenic and wild-type B6 mice

ruled out the contribution of Sgp3 to the derepression of ERVs through the down-

regulated expression of macroH2A1. Collectively, our data indicate that enhanced

transcription of endogenous retroviral sequences observed in macroH2A1-deficient B6

mice was not a result of the macroH2A1 mutation, but due to the presence of 129-

derived Sgp3 locus, and rule out the implication of macroH2A1 in the expression of

ERVs.

Discussion

The analysis of the abundance of group- and subgroup-specific retroviral gp70

RNAs and the presence of corresponding proviruses disclosed that the genetic origin of

serum gp70 is more heterogeneous than previously believed, as we observed a

substantial contribution of PT and mPT gp70s, in addition of Xeno gp70, to serum gp70.

This heterogeneity observed in different strains of mice is in part due to the absence of

some of the proviruses in the respective genomes or possibly due to the site of

integration or transcriptional regulation. Indeed, the expression of serum gp70 is highly

dependent on the presence of regulatory genes. Our studies on Sgp congenic mice

revealed that Sgp3 is not the structural gene but acts as a major regulatory gene to

control basal serum levels of gp70 through the transcriptional regulation of Xeno, PT

- 28 -

and mPT proviruses. Furthermore, we confirmed a significant, but modest contribution

of Sgp4 locus to the production of serum gp70 by regulating the basal-level expression

of a subgroup of Xeno provirus (Xeno-I). The analysis of double congenic B6 mice

bearing both Sgp3 and Sgp4 revealed their synergic effect on the production of serum

gp70. However, their basal levels of serum gp70 were still lower than those in lupus-

prone NZB and NZW mice, suggesting that an additional locus can contribute to serum

gp70. Indeed, our on-going studies showed that B6 mice bearing the proximal

chromosome 12 interval from NZB mice displayed modest increases of serum gp70

through a selective up-regulation of Xeno gp70 RNA. Furthermore, our analysis

demonstrated that the Sgp3 locus is responsible for predominant and abundant

expression of mPT proviruses carrying the intact env gene at the near exclusion of the

expression of mPT proviruses bearing defective (D1 or D2) env gene in all four lupus-

prone strains of mice, but not in non-autoimmune strains of mice. This indicates that

lupus-prone mice may possess a unique genetic mechanism responsible for the

expression of mPT retroviruses. Collectively, our results indicated that diverse levels of

serum gp70 in various murine strains can be explained by the presence of a different

assortment of regulatory and structural genes implicated in the production of serum

gp70 in liver.

The expression of serum retroviral gp70 is enhanced by inducers of APP,

indicating that serum gp70 behaves like an APP. This notion was confirmed by present

studies showing that proinflammatory cytokines IL-1, IL-6 and TNF similarly induced

increased levels of serum gp70 in NZB mice. However, unlike conventional APPs, the

serum gp70 response is strain-dependent, as only mice having high basal levels of

serum gp70 displayed an up-regulated production of serum gp70 in response to LPS.

Our studies on Sgp3, Sgp4 and Sgp3/4 congenic mice revealed that both Sgp loci act

synergistically and play a major role in the acute phase expression of serum gp70.

Strikingly, only Xeno and mPT gp70 RNAs were induced during acute phase responses.

The selective effect of LPS on Xeno and mPT gp70 RNAs is likely to be related to the

remarkable heterogeneity of the U3 regulatory regions of the long terminal repeat (LTR)

among different classes of ERVs.

In contrast to the selective effect by Sgp3 on the expression of mPT proviruses

carrying the intact env gene under steady-state condition, Sgp3 was also involved in an

up-regulated expression of one of the defective mPT proviruses carrying the D1 env

mutant in response to LPS. Moreover, injection of LPS in B6.Sgp4 mice resulted in

increases of not only Xeno-I but also Xeno-II and Xeno-III gp70 RNAs, while the Sgp4

- 29 -

locus only up-regulated the level of Xeno-I gp70 RNA under non-inflammatory

condition. These results suggest that the Sgp3 and Sgp4 loci likely carry at least two

distinct regulatory elements, which independently control the expression of ERVs in

physiological versus inflammatory conditions.

Because of the recent demonstration of an up-regulated transcription of

endogenous retroviral sequences in livers of B6 mice deficient in macroH2A1, and

because of the presence of the macroH2A1 gene within the Sgp3 interval, we tested the

hypothesis that macroH2A1 is a candidate gene for Sgp3. However, our analysis ruled

out the implication of macroH2A1 in the expression of ERVs, since enhanced

transcription of endogenous retroviral sequences observed in macroH2A1-deficient B6

mice was not a result of the macroH2A1 mutation, but due to the presence of 129-

derived Sgp3 locus, which was co-transferred with the 129 macroH2A1 mutant gene

during backcross procedures. Our on-going studies have narrowed down the NZB-

derived Sgp3 locus within a 5.42 Mb NZB interval. Notably, this region contains a

cluster of 21 Zfp genes, which encode KRAB (Krüppel-associated box) zinc-finger

proteins, but the target genes regulated by most of these Zfp genes are still unknown. It

may be possible that several of the Zfp genes present in the Sgp3 locus are involved in

the regulation of the expression of ERVs under physiological and inflammatory

conditions.

Finally, our studies revealed that TLR7, an innate immune receptor for single-

stranded RNA, plays a critical role in the development of autoimmune responses against

serum gp70, supporting the idea that ERVs play an active role in murine SLE. Notably,

we observed that the Sgp3 locus promotes abundant and preferential expression of mPT

proviruses possessing an intact env gene in lupus-prone mice. Thus, an enhanced

production of ERVs could lead to an activation of anti-gp70-specific autoreactive B

cells and dendritic cells through the stimulation of TLR7, thereby implicating in murine

SLE. In addition, our demonstration of an enhanced production of serum gp70 after

injection of TLR7 agonist revealed a novel role of this innate receptor in the

pathogenesis of murine lupus nephritis. TLR7 is involved in an enhanced production of

serum gp70 during the course of disease, possibly through the activation of

macrophages in response to RNA-containing IgG IC, thereby providing an additional

source for antigenic stimulation and for nephritogenic IC formation. Further research on

molecular basis responsible for the expression of ERVs implicated in murine SLE will

enable us to address the relevance of their human counterparts, thus providing a clue for

a potential role of ERVs in human SLE.

- 30 -

- 31 -

I. INTRODUCTION

- 32 -

- 33 -

I. Introduction

I.1. Systemic Lupus Erythematosus

I.1.1. Autoimmunity

The detection and the clearing of pathogens are essential for the survival of

vertebrates. The first line of host defense against pathogens is the innate immune system.

The innate response, mediated by diverse effectors such as macrophages, monocytes,

neutrophils, dendritic cells (DCs), and complements, rapidly detect and remove

pathogens. Receptors of the innate immune response recognize structures common to

different pathogens. Initial innate immune response also activates the adaptive immune

response which is slower but more specific against one pathogen. The effectors of the

adaptive immune response are lymphocytes (T and B cells). Antigens receptors of

lymphocytes are encoded by genes that are cut, spliced and modified to produce

numerous variants, which enable any potential pathogens to be recognized.

Consequently, the adaptive immune response produces almost unlimited numbers of T

and B lymphocytes and each lymphocyte recognizes a specific antigen, which leads to

the proliferation and differentiation into effectors lymphocytes. This mechanism of

receptor gene rearrangement can generate T and B cells that could also recognize self

antigens. However, in normal individuals, those clones are negatively selected by a

mechanism of central tolerance (in the thymus and in the bone marrow) and of

peripheral tolerance by different mechanisms including clonal anergy, functional

ignorance and editing of antigen receptors (1-5).

The failure of regulatory mechanisms responsible for self tolerance leads to the

persistence and activation of potentially self-reactive lymphocytes and the development

of autoimmune diseases. Autoimmune diseases are classified according to predominant

effectors involved: T-cells or B-cells mediated autoimmune diseases. The first kind of

autoimmune disease is caused by effectors T-cells, which damage the pancreas in

insulin-dependent diabetis mellitus (IDDM), the joints in rheumatoid arthritis (RA) and

the central nervous system in multiple sclerosis (MS). Second effectors are

autoantibodies directed against components of cell surfaces or the extracellular matrix.

In autoimmune hemolytic anemia (AIHA), autoantibodies bind component of

erythrocyte surface, leading to the destruction of erythrocytes as a result of IgG Fc

receptors (FcγR)- or complement receptor-mediated erythrophagocytosis. In

Goodpasture’s syndrome, autoantibodies binds extracellular matrix (type IV collagen)

on renal glomerular basement membranes, resulting in the development of very severe

- 34 -

glomerulonephritis. The third kind of autoimmune diseases is associated with the

formation of soluble immune complexes (IC) that are deposited in tissues, such as

systemic lupus erythematosus (SLE), in which the deposition of IC provokes

glomerulonephritis, i.e. lupus nephritis.

I.1.2. Etiology of Human SLE

SLE was first named lupus erythematosus because of the characteristic facial

rash (erythema), which give the appearance of a wolf (lupus). SLE is considered to be

the prototypic systemic autoimmune disease as it involves various organs: skin, joints,

brains, kidney, lungs and heart. SLE is characterized by the formation of a variety of

autoantibodies following the activation of autoreactive B-cells. Association of some of

these autoantibodies with the corresponding antigens results in the formation of IC,

which are responsible of the development of glomerulonephritis leading to proteinuria,

chronic renal failure and end-stage renal disease which could induce death (6). Among a

number of autoantibodies produced in SLE, principal targets are nucleic acid-protein

complexes, such as chromatin and ribonucleoproteins (RNP).

The prevalence of lupus is approximately 40 cases per 100 000 in Northern

Europeans but the risk is higher in black population and female gender. Indeed, black

population has a prevalence of 200 per 100 000 persons and 90% of patients with lupus

are female (7). Genetics studies on twins showed that the concordance rate for lupus is

25% among monozygotic twins and approximately 2% among dizygotic twins (8).

These findings indicate the implication of environmental factors in addition to genetic

contribution to the development of SLE. Indeed, environmental factors are also

involved in the pathogenesis of SLE. The ratio of female to male developing SLE is due

to the effect of endogenous sex hormones (9). Studied in lupus-prone mice showed that

administration of exogenous estrogens exacerbates SLE, while androgens protect (10-

12). Furthermore, SLE can be induced by drugs such as procainamide, hydralazine and

quinidine. Clinical manifestations of drug-induced lupus are identical to SLE except that

they recover when the treatment with these drugs stopped (13). Additionally, the role of

infectious agents as an environmental factor that triggers SLE has been studied. Epstein-

Barr virus (EBV) could be associated with the development of SLE, since a case-control

study showed that anti-EBV antibodies were found in 99% of SLE patients (14).

Another environmental factor associated with SLE is ultraviolet radiation. Individuals

- 35 -

with particular sun-reactive skin type had an increased risk of developing SLE (15, 16).

In summary, both genetic and environmental factors play role in human SLE.

I.1.3. Murine Models of SLE

Several murine strains, which spontaneously develop symptoms common to

human SLE, NZB (New Zealand black), NZW (New Zealand white), MRL-Faslpr and

BXSB, have offered the opportunity to study the immunological and genetic basis

underlying the pathogenesis of SLE (17). They are characterized by a wide spectrum of

autoimmune manifestations culminating in the development of IC-mediated lupus

nephritis. The severity of kidney lesions is closely associated with the increase in serum

titers of IgG autoantibodies directed against various nuclear antigens. In addition, lupus-

prone mice spontaneously develop autoantibodies against serum glycoprotein gp70

derived from endogenous retroviruses. gp70-anti-gp70 IC (gp70 IC) are detected in

diseased glomeruli of lupus mice (17-19).

The NZB (H2d) and NZW (H2z) strains were developed in New Zealand from a

murine stock of undefined background by selection on black and white color,

respectively. NZB mice develop AIHA, but neither NZB nor NZW mice develop a

typical lupus-like syndrome. In contrast, (NZB x NZW)F1 hybrid mice develop a severe

autoimmune disease resembling human SLE, which affects the females earlier than the

males, and sex hormones have been shown to be responsible for the early development

of disease in the females. The MRL strain (H2k) is derived from a series of crosses

involving four strains (LG/J, AKR/J, C3H/Di and C57BL/6). The spontaneous and

recessive lpr (lymphoproliferation) mutation in the MRL strain results in a generalized

lymphadenopathy due to massive accumulation of a unique subset of T cells

(TCRαβ+,CD4–,CD8–,B220+) (20). The lpr mutation consists of an insertion of an

endogenous retrovirus in the Fas gene, which codes for a receptor implicated in

apoptosis of lymphocytes (21). The presence of the Faslpr mutation markedly

accelerates the progression of SLE-like autoimmune syndrome in MRL mice. The

BXSB mouse (H2b) is a recombinant inbred strain derived from a cross between a

C57BL/6 (B6) female and a SB/Le male. These mice spontaneously develop an SLE-

like disease that affects male animals much earlier than females. The male-determined

accelerated disease is independent of sex hormones, but due to the presence of the

genetic abnormality, Yaa (Y-linked autoimmune acceleration), presents in the Y

chromosome of BXSB mice, which is originally inherited from the SB/Le strain (22,

- 36 -

23). The Yaa mutation was recently identified to be a translocation from the telomeric

end of the X chromosome, containing the gene encoding Toll-like receptor 7 (TLR7),

onto the Y chromosome (24, 25). Accordingly, the Tlr7 gene duplication has been

proposed to be the disease-accelerating mechanism conferred by the Yaa mutation.

I.1.4. Multigenic Features of Murine SLE

The pathogenesis of SLE is a complex process in which major

histocompatibility complex (MHC)-linked and multiple non-MHC-linked genetic

factors contribute to the overall susceptibility and progression of the disease, along with

contributions of hormonal and environmental factors. The availability of several murine

strains with distinct genetic backgrounds, such as (NZB x NZW)F1, MRL and BXSB,

which all spontaneously develop an autoimmune syndrome resembling human SLE, has

offered an invaluable opportunity for elucidating the genetic basis underlying the

etiopathogenesis of SLE.

Since the development of a SLE-like syndrome was first reported in the F1

progeny of the NZB and NZW strains, the genetic basis for SLE in (NZB x NZW)F1

hybrids has been investigated in a number of laboratories. Classic progeny studies have

provided only limited information on the number, identity and chromosomal location of

the lupus susceptibility genes. However, the availability of polymorphic microsatellite

markers covering the entire mouse genome has permitted to map more precisely the

genetic loci linked with a wide spectrum of autoimmune traits, i.e. production of

autoantibodies, development of lupus nephritis and production of nephritogenic gp70

autoantigen. Genome-wide linkage analyses in mice obtained through by intercrosses or

backcrosses of different lupus-prone and non-autoimmune strains led to the

identification of multiple autoimmune susceptibility regions distributed all over the

murine genome (26-28). These analyses have shown that 1) lupus-like disease is

controlled by sets of susceptibility loci that independently or additively contribute to the

overall susceptibility and progression of the disease; 2) heterogeneous combinations of

multiple disease-promoting genes operate in a threshold-dependent manner to achieve

full expression of the disease; and 3) contributions are unlikely to be linked to “true”

genetic mutations, but are rather due to polymorphic alleles with subtle functional

differences, except for the Fas and Yaa mutations observed in MRL and BXSB mice,

respectively.

- 37 -

I.1.4.1. Spontaneous Mutations Predisposing to SLE in Lupus Mice

I.1.4.1.1. The Fas and Fas Ligand Gene

The identification of defects in Fas, mapped to chromosome 19, which is

involved in apoptosis, in lupus-prone MRL mice with the lpr phenotype, represented an

important contribution to our understanding of the genetic basis of SLE (21). Notably,

the gld (generalized lymphoproliferative disease) mutation, discovered in a colony of

the C3H/HeJ strain, induces marked lymphadenopathy phenotypically indistinguishable

from that induced by the lpr mutation (29). In fact, gld was identified as a mutation of

the gene encoding the Fas ligand (FasL), present in chromosome 1 (30). These Faslpr

and Faslgld mutations not only accelerate the progression of autoimmune disease in

lupus-prone MRL mice, but also induce the production of a broad spectrum of

autoantibodies in various strains of mice, including those not predisposed to SLE (17,

31, 32). Fas is highly expressed in activated B and T cells, while the expression of FasL

is limited to activated T cells (20). However, the Fas apoptosis pathway does not appear

to be essential for negative selection during T and B cell development in thymus and

bone marrow, respectively (33, 34). Therefore, it has been speculated that the abnormal

regulation of the Fas apoptotic pathway could result in a prevention of antigen-induced

apoptotic death of autoreactive lymphocytes in the periphery, thereby promoting the

development of lupus-like autoimmune responses. However, it should be stressed that

the mutation of Fas or FasL alone is not sufficient to induce severe autoimmune disease

in mice which are not predisposed to SLE (29, 31, 32), underlining the importance of

other still undefined lupus susceptibility background genes in the development of full-

blown SLE.

I.1.4.1.2. The Yaa Mutation

In contrast to the accelerated development of SLE in (NZB x NZW)F1 female

mice, male BXSB mice develop disease much more rapidly than their female

counterparts (17). This striking sexual dimorphism is not hormonally mediated, but

results from a mutant gene, Yaa, present in the Y chromosome of the BXSB strain (35-

37). The contribution of the Yaa mutation to lupus susceptibility remains limited

without other background genes, since non-autoimmune strains, such as CBA/J and B6,

were largely unaffected by the Yaa mutation (35, 36). Notably, when B6.Yaa consomic

males are mated with NZW females, which are phenotypically normal but have a

genetic potential to develop SLE, their F1 hybrid males bearing the Yaa mutation

- 38 -

develop typical SLE (36). In addition, studies in B6 or C57BL/10 (B10) mice carrying

different lupus susceptibility loci derived from either NZB, NZW or BXSB mice have

shown that the combination of a single lupus susceptibility locus with Yaa can be

sufficient to induce the development of lupus-like autoimmune syndrome, although the

severity of the disease was variable, depending on the individual lupus susceptibility

locus studied (38-40). These results indicate that the Yaa mutation by itself is unable to

promote SLE in mice which are not predisposed to autoimmune diseases, but in

combination with autosomal susceptibility alleles present in different lupus-prone

strains, it can induce or accelerate the development of SLE.

Studies of Yaa and non-Yaa double bone marrow chimeric mice have

demonstrated that anti-DNA autoantibodies are selectively produced by B cells bearing

the Yaa mutation, and that T cells from both Yaa and non-Yaa origin efficiently promote

anti-DNA autoantibody responses (41, 42). These data indicate that the Yaa defect is

functionally expressed in B cells, but not in T cells. Based on this finding, it has been

hypothesized that the action of Yaa may be to decrease the threshold for B-cell receptor

(BCR)-dependent stimulation, thereby promoting the activation of autoreactive B cells

(43). Recently, the Yaa mutation was shown to be a consequence of a translocation from

the telomeric end of the X chromosome onto the Y chromosome (24, 25). Based on the

presence of the gene encoding toll-like receptor 7 (TLR7) in this translocated segment

of the X chromosome, and the possible role of TLR7 in the activation of autoreactive B

cells (44, 45) and in the development of SLE (46, 47), the Tlr7 gene duplication has

been proposed to be the etiologic basis for the Yaa-mediated enhancement of disease

(24, 25, 48). Indeed, introduction of the Tlr7 null mutation on the X chromosome

significantly reduced serum levels of IgG autoantibodies against DNA and

ribonucleoproteins, and also incidence of lupus nephritis in lupus-prone mice bearing

the Yaa mutation (49). However, the protection was not complete, since these mice still

developed high titers of anti-chromatin and retroviral gp70 IC and lupus nephritis.

These results indicate that the Yaa-mediated acceleration of SLE cannot be explained by

the Tlr7 gene duplication alone, and suggest additional contributions from other

duplicated genes in the translocated X chromosome.

I.1.4.2. MHC Association of Murine SLE

The MHC region, called H2, is located on chromosome 17. Two isoforms of the

MHC class II molecule (I-A and I-E) are expressed in mice. MHC class II molecules are

- 39 -

heterodimers composed of an α-chain and a β-chain. I-A is encoded by the Aβ and Aα

genes, and I-E is encoded by Eβ and Eα genes (50). Since the development of SLE is

blocked by treatment with anti-I-A antibodies (51) and dependent on CD4+ T cells (52),

the implication of the MHC class II genes in murine SLE has been underlined.

Indeed, genetic studies in New Zealand mice have demonstrated a strong

association of H2d/z heterozygosity (vs. H2d/d or H2z/z) with the development of SLE,

indicating a co-dominant contribution from each strain, i.e. H2d from NZB and H2z

from NZW (53, 54). However, it is still unknown how this H2 heterozygosity

mechanistically contributes to murine SLE. It has been proposed that mixed haplotype

class II molecules produced by heterozygous pairing of an α-chain from one haplotype

with a β-chain from the other haplotype might play a critical role in the development of

SLE. However, the lack of disease enhancement by an Abz transgene introduced into

H2d homozygous (NZB x NZW.H2d)F1 mice (55) and by Ez or Az transgene in (B6 x

NZB)F1 x NZB backcross mice (56, 57) argue against this possibility. Significantly, a

comparative serological analysis of two different nephritogenic anti-DNA and anti-gp70

autoantibody productions in (NZB x NZW)F1 x NZW and (NZB x NZW)F1 x NZB

backcross mice revealed that in the F1 x NZW backcross, H2d/z (compared with H2z/z)

was associated preferentially with the production of anti-gp70 rather than anti-DNA

autoantibodies, whereas the opposite influence was noted for H2d/z (compared with

H2d/d) in the F1 x NZB backcross (57). These results suggest that enhancement of

disease by H2d/z heterozygosity is related to separate contributions from H2d and H2z,

thus providing one explanation as to why H2d/z heterozygosity is required for full

expression of disease in (NZB x NZW)F1 mice.

Another contribution of the MHC class II gene to the regulation of murine SLE

has been documented in BXSB and (NZB x BXSB)F1 mice, in which lupus

susceptibility was more closely linked with the H2b haplotype than with the H2d and

H2k haplotypes (58-60). However, this MHC effect was limited, as it was markedly

influenced by other factors in the genetic background of individual lupus-prone mice. In

the context of the BXSB background, in which the development of SLE is dependent on

the Yaa mutation, the H2d or H2k haplotype almost completely prevented the

development of autoimmune responses occurring in H2b-bearing conventional BXSB

mice. In contrast, (NZB x BXSB)F1 female hybrids homozygous for H2d still

developed typical SLE, although its development was markedly delayed as compared

with mice homozygous for H2b. This indicates that the genetic complementation of

NZB and BXSB genomes allows the development of spontaneous autoimmune

- 40 -

responses in the context of H2d, even without the Yaa mutation. More strikingly, the

Yaa mutation dramatically accelerated the progression of SLE in (NZB x BXSB)F1 H2d

mice to an extent comparable with that observed in F1 H2b mice. Thus, no more MHC

association was evident in these F1 hybrid males in the presence of the Yaa mutation.

Notably, similar results were observed in mice bearing the Faslpr mutation; the

production of autoantibodies in B6 mice bearing the Faslpr mutation was highly

dependent on H2b (61), while lupus-like disease was developed equally well in both H2k

and H2b lupus-prone MRL-Faslpr mice (62). All these experiments indicate that the

MHC class II genes likely provide at least some of the genetic requirements for the

predisposition to SLE, and that conventional MHC class II molecules are sufficient in

mice with an appropriate autoimmune genetic background. Most significantly, the

MHC-linked autoimmune promoting effect is no longer apparent in mice which are

highly predisposed to SLE, for example by powerful autoimmune accelerating genes,

such as Yaa or Faslpr.

The autoimmune inhibitory effect of the H2d and H2k haplotypes, as compared

with H2b, can be related at least in part to the expression of I-E molecules, since mice

bearing the H2b haplotype do not express I-E because of the deletion of the promoter

region of the Ea gene. The development of SLE was almost completely prevented in

BXSB (H2b) mice expressing two copies of an Ea transgene encoding I-E α-chains, as

is the case of H2d and H2k BXSB mice (38). In addition, the expression of two

functional Ea (one transgenic and the other endogenous) genes in either H2d/b (NZB x

BXSB)F1 or H2k/b (MRL x BXSB)F1 mice provided protection from SLE at levels

comparable to those conferred by the H2d/d or H2k/k haplotype (60). These results

suggest that the reduced susceptibility associated with the I-E+ H2d and H2k haplotypes

(vs. the I-E– H2b haplotype) is largely, if not exclusively, contributed by the Ea gene.

This idea is further supported by the demonstration that (NZB x NZW)F1 mice

expressing I-Ad but lacking I-E molecules developed a SLE as severe as that of wild-

type H2d/z heterozygous (NZB x NZW)F1 mice (63). However, it should be stressed

that since H2d/z (NZB x NZW)F1 mice express I-E, the unique autoimmune-promoting

effect conferred by the H2d/z heterozygosity apparently overcomes the protective effect

of I-E in this genetic background, as in the case of (NZB x BXSB)F1 mice expressing

the Yaa mutation and MRL mice bearing the Faslpr mutation.

The precise mechanism(s) responsible for the Ea gene-mediated protection from

SLE remains to be elucidated. Studies on Ea transgenic and non-transgenic mixed bone

marrow chimeras revealed that these chimeric mice developed a typical lupus-like

- 41 -

autoimmune syndrome, in which anti-DNA autoantibody production was preferentially

induced by non-transgenic B cells (64, 65). These results suggested that B cells are the

major target of Ea-mediated suppression of autoimmune responses, and that Ea gene

expression may interfere with an efficient interaction between autoreactive T and B

cells, possibly by modulating the presentation of pathogenic self-peptides by MHC class

II molecules. This could occur as a result of induction or increased expression of I-E

heterodimers following combination of transgenic I-E α-chains with endogenous I-E β-

chains or of increased formation of peptides derived from the I-E α-chains, since one of

the peptides, Eα52-68, has been identified as one of the major self-peptides presented

by I-A molecules (66, 67). However, recent studies in BXSB mice bearing the H2q

haplotype (i.e. unable to express I-E heterodimers because of a deficiency in I-E β-

chains) have demonstrated that the Ea transgene expression resulted in a marked

suppression of the development of SLE in H2q BXSB mice despite the absence of I-E

expression, and that the observed protection was not associated with any detectable

levels of T-cell depletion and regulatory T-cell expansion (68). Furthermore, in vitro

analysis using different model antigen-MHC class II combinations revealed that a high

transgene expression in B cells markedly inhibited the activation of T cells in an

epitope-dependent manner, and that this inhibition was related to the relative affinity of

Eα52-68 peptides vs. antigenic peptides to individual MHC class II molecules (69).

Taken together, these results support a model of autoimmunity prevention based on

competition for antigen presentation, in which generation of I-E α-chain-derived

peptides prevents, because of their high affinity to the I-A molecules, activation of

autoreactive T and B cells.

I.1.4.3. Non-MHC-linked Lupus Susceptibility Loci

More than 20 genome-wide linkage analyses led to the identification of a

number of non-MHC-linked lupus susceptibility loci in all 19 autosomal chromosomes,

associated with autoantibody production and/or lupus nephritis (70). However, it is

important to note that several major loci identified in independent studies are co-

localized in essentially identical chromosomal regions in different lupus-prone mice.

Among them, four non-MHC regions on chromosome 1, 4, 7 and 13 have been more

extensively studied: Nba2 (New Zealand black autoimmunity 2), Sle1 (Systemic lupus

erythematosus 1), Lbw7 (Lupus-NZB x NZW 7) and Bxs3 (BXSB 3) on chromosome 1,

Nba1, Sle2, Lbw2, Imh1 (IgM hyper 1), Adnz1 (Anti-dsDNA antibody in NZM2328

- 42 -

locus 1) and Sgp4 (Serum gp70 production 4) on chromosome 4, Sle3, Nba5, Lbw5 and

Lmb3 (Lupus in (MRL-Faslpr x B6-Faslpr)F2 cross 3) on chromosome 7, and Sgp3 and

Bxs6 on chromosome 13 (Figure 1). Although they have not yet been well characterized,

additional susceptibility loci have been mapped on other chromosomes, and some of

them are apparently strain-specific. This implies that different clusters of genes confer

lupus susceptibility in different strains of mice, though some loci are likely to be

common to several murine models of SLE. In addition, the existence of SLE

suppressive alleles within the genome of lupus-prone mice reveals an additional level of

complexity of the genetic analysis (71, 72).

Figure 1: Genetic map of major loci and genes implicated in SLE. Loci (■) are shown

in their approximate positions: Nba2 (New Zealand black autoimmunity 2), Sle1

(Systemic lupus erythematosus 1), Lbw7 (Lupus-NZB x NZW 7) and Bxs3 (BXSB 3) on

chromosome 1, Nba1, Sle2, Lbw2, Imh1 (IgM hyper 1), Adnz1 (Anti-dsDNA antibody in

NZM2328 locus 1) and Sgp4 (Serum gp70 production 4) on chromosome 4, Sle3, Nba5,

Lbw5 and Lmb3 (Lupus in (MRL-Faslpr x B6-Faslpr)F2 cross 3) on chromosome 7, and

Sgp3 and Bxs6 on chromosome 13. Genes candidate (in blue) corresponding to these

loci have been identified.

I.1.4.3.1. Lupus Susceptibility Loci Mapped to Chromosome 1

An NZB locus, Nba2, was initially mapped to the distal region of chromosome 1

by an analysis of (NZB x SM/J)F1 x NZB backcross mice (28), and is likely to be

identical to Lbw7 of NZB origin (27) (Figure 1). Since this locus was found to be

linked with the production of various autoantibodies, including anti-DNA, anti-

Fcgr2b, SALM/CD2

1

Nba2, Sle1, Lbw7, Bxs3

Cr2

4

Sle2

Nba1, Lbw2, Sgp4

C1qa

Adnz1

7

Sle3

Nba5, Lbw5,Lmb3

Cd22

13

Sgp3, Bxs6

17

H2

Fcgr2b, SALM/CD2

1


Cr2

Fcgr2b, SALM/CD2

1


Cr2

4

Sle2

Nba1, Lbw2, Sgp4

C1qa

Adnz1

4

Sle2

Nba1, Lbw2, Sgp4

C1qa

Adnz1

7

Sle3

Nba5, Lbw5,Lmb3

Cd22

7

Sle3

Nba5, Lbw5,Lmb3

Cd22

13

Sgp3, Bxs6

17

H2

17

H2

- 43 -

chromatin and anti-gp70, it apparently controls overall autoantibody production in SLE,

and thereby the development of lupus nephritis (40, 73). The Sle1 locus, derived from

the NZW strain, overlaps with the same region on chromosome 1, and was also linked

to autoantibody production and lupus nephritis (26, 74). In addition, the genetic analysis

involving the BXSB and B10 strains has identified a lupus susceptibility interval

situated in chromosome 1, designated Bxs3, which overlaps directly with Nba2, Lbw7

and Sle1 (72). B6 or B10 mice congenic for the Nba2, Sle1 or Bxs3 interval developed

elevated titers of anti-DNA and anti-chromatin autoantibodies, but failed to develop

lupus nephritis, while these congenic mice are able to develop severe lupus nephritis in

the presence of the Yaa mutation (38, 40). Significantly, the analysis of subinterval

congenic mice carrying different portions of the Sle1 locus has revealed that three non-

overlapping loci within Sle1, termed Sle1a, Sle1b and Sle1c, can independently cause

loss of self tolerance (75). This indicates that Sle1 is represented by a cluster of

functionally-related lupus susceptibility genes.

One important candidate gene, which is shared by NZB, BXSB and MRL strains,

is the NZB-type defective allele of the Fcgr2b gene encoding the inhibitory type II

FcγR (FcγRIIB) (Figure 1). The presence of promoter region polymorphism has been

shown to result in defective expression of FcγRIIB on activated B cells in germinal

centers of NZB mice (76, 77). Since its co-ligation to the BCR through IgG-containing

IC prevents the activation of BCR signaling (78), the defect of FcγRIIB expression in

activated B cells in lupus-prone mice is implicated in enhanced autoantibody production

due to excessive activation of autoreactive B cells. Notably, the development of SLE

was markedly prevented as a result of partial restoration of FcγRIIB levels (79) or of

congenic expression of the wild-type Fcgr2b allele in BXSB mice (80). Furthermore, it

has been shown that monocytes and macrophages bearing the NZB-type Fcgr2b allele

expressed much lower levels of FcγRIIB than those bearing the wild-type allele (70, 81).

Since activating FcγR apparently plays a critical role in the development of lupus

nephritis (70, 81), the defective FcγRIIB expression on monocytes/macrophages in

lupus-prone mice could additionally contribute to the effector phase of IC-mediated

lupus nephritis due to excessive activation of FcγR-bearing effector cells. In addition,

lupus susceptibility has been shown to be associated with extensive polymorphisms of

the signaling lymphocyte activation molecule SLAM/CD2 gene family (Cd244, Cd229,

Cs1, Cd48, Cd150, Ly108 and Cd84), as all lupus-prone mice share the same SLAM

haplotype, which is different from that of B6 mice (82). Since these genes encode cell

surface molecules that play a role in the modulation of cellular activation and signaling

- 44 -

in the immune system, they are also good candidates for promoting lupus-like

autoimmune responses. Among the SLAM family of receptors, the strongest candidate

appears to be Ly108, since the Ly108.1 allelic form expressed in lupus-prone mice was

found to be less efficient to sensitize immature autoreactive B cells to deletion than the

normal Ly108.2 allele (83). The contribution of Fcgr2b and SLAM/CD2

polymorphisms to the development of lupus-like autoimmune syndrome was further

supported by a recent analysis of B6.Nba2 subcongenic mice, which revealed that

Fcgr2b and SLAM intervals independently controlled the severity of autoantibody

production and renal disease, yet are both required for lupus susceptibility (84).


The Nba1 and Lbw2 loci were mapped to the mid-distal region of NZB

chromosome 4 and identified as loci contributing to the development of lupus nephritis,

but not to the production of IgG anti-nuclear autoantibodies (27, 85-87) (Figure 1). We

have recently identified a locus, designated Sgp4, in the distal region of the NZB

chromosome 4 overlapping with the Nba1 locus, which was linked to the production of

nephritogenic retroviral gp70 antigens (88, 89). Therefore, the association of lupus

nephritis with Nba1/Lbw2 could in part be a consequence of increased production of

nephritogenic gp70 autoantigens, as in the case of the contribution of Sgp3 locus in

chromosome 13 to lupus nephritis (see below). Additionally, the Nba1/Lbw2 genetic

contribution may be operating distal to autoantibody production by affecting IC

localization or inflammatory responses to deposited IC. In this regard, it is worth

mentioning that the Nba1/Lbw2 interval contains the C1qa gene encoding the first

component of complement C1q. Significantly, it has been shown that an insertion

polymorphism in the NZB sequence upstream of C1qa appears to downregulate the

serum levels of C1q (90). This could result in an impairment of IC clearance, thereby

promoting the deposition of IC and hence the development of lupus nephritis.

One additional study using NZM2328 mice, one of the recombinant inbred

NZM (New Zealand mixed) strains derived from a cross of NZB female and NZW male

(91) defined an NZB-derived locus, Adnz1, on the mid chromosome 4 (Figure 1), which

contributed to the production of anti-DNA autoantibodies, but not to lupus nephritis

(92). Strikingly, NZM2328 mice bearing the B6-type Adnz1 interval failed to produce

anti-nuclear autoantibodies, but still developed severe lupus nephritis in kinetics

comparable to those seen in wild-type NZM2328 mice (93). This raises a question

- 45 -

concerning the pathogenic role of anti-DNA autoantibodies in lupus nephritis. In fact, it

has repeatedly been shown that the development of murine lupus nephritis was

associated with an increased production of retroviral gp70 IC much more than anti-

DNA autoantibodies (23, 37, 86, 88, 94, 95). In addition, one might also consider the

involvement of other IC systems. For example, it has been claimed that anti-C1q

autoantibodies were associated with the presence of human lupus nephritis, amplifying

glomerular injury in SLE (96). The striking difference in autoimmune phenotypes

conferred by Sle2 vs. Adnz1, both of which are derived from the same chromosomal

region of NZW mice, further illustrates the complexity of lupus susceptibility.


The centromeric region of chromosome 7 contains lupus susceptibility genes

regulating autoantibodies and lupus nephritis, which are the Sle3 and Lbw5 loci derived

from the NZW strain (26, 27), the Nba5 locus from the NZB strain (40) and the Lmb3

locus from the MRL strain (97) (Figure 1). The contribution of these loci to murine

SLE has been confirmed by the analysis of Sle3 or Nba5 congenic B6 mice (40, 74) and

of Lmb3-congenic MRL mice (98).

One possible candidate gene present in this region is Cd22 (Figure 1), which

codes for a B cell-restricted adhesion molecule that recongnizes α2,6-linked sialic acid-

bearing glycans and functions as a negative regulator of BCR signaling (99). The

analysis of B6 x (NZW x B6.Yaa)F1 backcross males has provided evidence that an

NZW locus peaking at Cd22a was strongly linked with autoantibody production and

lupus nephritis (100). A link between dysregulated CD22 expression and lupus-like

autoimmune disease has also been suggested by the findings that mice with a disrupted

Cd22 gene developed increased serum titers of IgG anti-DNA autoantibodies (101) and

that partial CD22 deficiency, i.e. heterozygous level of CD22 expression, in B6 mice

can result in an induction of IgG anti-DNA autoantibody production in the presence of

the Yaa mutation (102). Significantly, NZW and NZB mice carry the defective Cd22a

allele. CD22 expression on Cd22a B cells is lower at steady state and less upregulated

following B cell activation than that of Cd22b B cells (102, 103). It is also worth

mentioning that B cells derived from BXSB mice bearing the Cd22c allele (104)

displayed the same defect as Cd22a B cells (102). Since CD22 functions primarily as a

negative regulator of BCR-mediated signal transduction, a limited upregulation of

CD22 in activated B cells may have significant functional consequences in B-cell

- 46 -

responses. In addition, Cd22a and Cd22c B cells appear to express aberrant forms of

CD22, differing in the N-terminal sequences constituting the ligand-binding site, due to

the synthesis of abnormally processed Cd22 mRNA as a result of the insertion of a short

interspersed nucleotide element in the second intron (102). Indeed, CD22a molecules

were less efficient in the binding to CD22 ligand (CD22L) than their CD22b

counterparts and that Cd22a B cells displayed a phenotype reminiscent of constitutively

activated B cells (103). In view of the importance of the CD22-CD22L interaction in the

regulation of B-cell activation (105), these data support the idea that the expression of

defective CD22a and CD22c could contribute to enhanced B-cell activation, and thus

favor the development of autoimmune responses in combination with other

susceptibility alleles present in lupus-prone mice.

In addition, a more recent study identified the Coronin-1A gene, which regulates

cytoskeletal structure, as a lupus-susceptibility gene present in the Lmb3 locus of the

MRL strain (106). The mutation of this gene was associated with multiple abnormalities

in T-cell function including activation, survival and migration. This indicates that

abnormalities in T-cell function or regulation can additionally contribute to the

development of SLE.

It should be stressed that unlike Sle3 and Lmb3, Nba5 was unable to promote the

production of anti-DNA and anti-chromatin autoantibodies, and that its autoimmune-

promoting effect was selective for nephritogenic gp70 autoantigens (40). Notably, the

Nba5 locus is located more than 10 cM distal to Cd22. Thus, the candidate gene for

Nba5 is most likely to be different from those that promote general hyperresponsiveness

of B and/or T cells to enhance overall autoimmune responses. Since B6.Nba5 congenic

mice did not have higher levels of serum gp70, it is improbable that this locus is

implicated in overall production of serum gp70. However, it is possible that the Nba5

locus could regulate the expression of a subpopulation(s) of gp70 that is critically

involved in anti-gp70 autoantibody responses and gp70 IC formation, given the possible

heterogeneity of serum gp70 proteins.


Additional genes which are involved in the pathogenesis of SLE are those

encoding nephritogenic autoantigens or regulating their expression. One of these

autoantigens, which plays an important role in the development of murine lupus

nephritis, is the endogenous retroviral envelope glycoprotein gp70 (18). This is

- 47 -

illustrated by the fact that the gp70 antigen is found in circulating IC and glomerular

immune deposits within diseased kidneys of lupus mice (19). gp70 IC become apparent

in the circulation close to the onset of disease, and their concentrations rise with the

progression of lupus nephritis (23, 37, 86, 88, 94, 95), thereby providing good evidence

that gp70 IC are implicated in renal injury of lupus-prone mice.

Interval mapping of backcross progeny between lupus-prone mice (NZB, NZW

and BXSB) and B6 or B10 mice identified a major locus controlling gp70 production on

the middle of chromosome 13, designated Sgp3 or Bxs6 (40, 107) (Figure 1). Notably,

these loci were also linked with anti-gp70 production and lupus nephritis, but not with

anti-DNA production (40, 100). Analysis in B6 or B10 mice congenic for the Sgp3 or

Bxs6 locus derived from either the NZB, NZW or BXSB strain revealed that all three

congenic mice had approximately 10-fold higher levels of gp70, as compared with B6

or B10 mice (40, 107), suggesting that the underlying allele of Sgp3 is shared among

these three different lupus-prone mice. However, serum concentrations of gp70 in Sgp3

congenic mice bearing the NZB- or NZW-derived Sgp3 locus were still lower than

those seen in NZB and NZW mice, indicating the presence of other loci controlling the

production of serum gp70. Notably, the presence of additional loci regulating the

production of gp70 has been identified on distal chromosome 4, designated Sgp4, and

proximal chromosome 12 of both NZB and NZW mice (88, 89).

I.1.5. Development of Autoimmune Responses in Murine SLE

I.1.5.1. Hyperactive Phenotype of B Cells in Murine SLE

Numerous studies have provided important insights into identifying crucial

checkpoints and the molecular pathways that mediate the loss of tolerance during

differentiation of B and T cells. Studies on several Ig transgenic mice have

demonstrated that the development of autoreactive B cells can be regulated by several

different mechanisms including clonal deletion, clonal anergy and receptor editing in

the bone marrow or peripheral lymphoid organs, depending on the nature of the

autoantigen, its concentration in different sites and the affinity of the autoantibodies.

Autoreactive B cells can be generated in germinal centers as a result of somatic

hypermutations of BCR during T-cell dependent immune responses against exogenous

antigens. However, such autoreactive B cells are likely to be eliminated by apoptosis in

immunologically normal individuals (108). Thus, it can be speculated that one of the

defects in lupus-prone mice may be the failure to efficiently eliminate autoreactive B

- 48 -

cells upon interaction with autoantigens. The resistance of mature B cells to BCR-

mediated apoptosis has been reported in lupus-prone (NZB x NZW)F1 mice (109).

Notably, mice deficient in B-cell apoptosis such as MRL-Faslpr and C3H/HeJ-Faslgld

mice develop spontaneous lupus-like autoimmune manifestations (20). In addition, the

genetic mutation of the Ly108 gene that modifies the strength of BCR in immature B

cells could be involved in the loss of central tolerance in lupus-prone mice (83).

Moreover, loss of peripheral tolerance due to the presence of the NZB-type defective

FcγRIIB allelic form apparently has an important role in the final differentiation and

activation of autoreactive B cells (79, 110). This concept is consistent with the findings

that transgenic overexpression of the anti-apoptotic proto-oncogene bcl-2 in B cells led

to spontaneous development of a SLE-like autoimmune syndrome in certain strains of

mice (111), and that the constitutive expression of the bcl-2 gene is able to counteract

the apoptotic death of autoreactive B cells upon interaction with autoantigens in the

periphery (112).

The BCR-mediated activation of B cells is positively and negatively regulated

by different co-receptors (Figure 2). Studies of single gene mutations in mice have

shown that defects in negative regulators of BCR signaling, such as FcγRIIB (113),

CD22 (114), leading low thresholds for B cell activation, are consistently associated

with the development of lupus-like autoimmune syndrome. Conversely, overexpression

of positive BCR regulators, such as CD19, promoted the production of lupus

autoantibodies (115). As stated above, the expression of defective alleles of Fcgr2b and

Cd22 in lupus-prone mice contributes to the hyperactive phenotype of B cells, and the

pathogenic role of the NZB-type defective Fcgr2b allele in murine SLE has been well

documented (79, 80).

- 49 -

Figure 2: Negative and positive regulators of BCR. BCR signals are modulated

negatively by CD22 and FcγRIIB which contains immunoreceptor tyrosine-based

inhibitory motifs (ITIMs) recruiting phosphatase such as SHIP and SHP-1. On the

contrary, CD21, a receptor of C3 cleavage fragments, forms a complex with CD19

leading to the recruitment of Vav, PI3-kinase and Fyn which positively regulate BCR

signaling.

I.1.5.2. Defective Clearance of Apoptotic Cells in Murine SLE

Cell death can be caused by apoptosis or necrosis. Apoptosis, which is a

physiological process, lead to the activation of intracellular proteases and DNases,

which degrade intracellular component, followed by cell shrinkage and membrane

blebbing (116). Released apoptotic fragments are usually rapidly phagocytosed by

macrophages, polymorphonuclear cells (PMN) and immature DC. However, when

apoptotic cells are not cleared quickly, they undergo secondary necrosis. Necrosis,

which is induced by external factors such as infectious agent or ischaemia, result in the

release of intracellular component without cleavage, favoring inflammation (117).

Several studies observed that PMN and macrophages of lupus patients show abnormal

removal of apoptotic debris (118-121). Therefore, intracellular antigens, such as

nucleosomes, Ro, La and anionic phospholipids, are exposed on the surface of apoptotic

cells in lupus patients (122). Different molecules have been implicated in the process of

clearance. DNase1 is the enzyme that degrades DNA in nuclear antigens. DNase1

knock-out mice developed a lupus-like disease with antinuclear antibodies and nephritis

(123). It has been reported that DNase activity was reduced in serum of SLE patients

Suppression Amplification

BCR

C3d-Ag

CD21CD22 CD19

SHP-1SHIP

FcγγγγRIIBBCR

IgG-Ag IC Constitutiveassociation

BCR BCR

+−−

Suppression Amplification

BCR

C3d-Ag

CD21CD22 CD19

SHP-1SHIP

FcγγγγRIIBBCR

IgG-Ag IC Constitutiveassociation

BCR BCR

++−−

- 50 -

(123, 124). These results indicate that lack or reduction of DNase1 could promote the

development of anti-nuclear antibody responses. In addition, it has been demonstrated

that C1q played a substantial role for the clearance of apoptotic bodies (125). Notably,

deficiency in C1q strongly predisposes to SLE (126), and C1q-deficient mice are able to

develop lupus-like autoimmune syndrome in association with an accumulation of

apoptotic bodies in tissues (127). Thus, it has become clear that apoptotic cells are a

major source of nuclear autoantigens in SLE, and that the failure of efficient elimination

of apoptotic bodies could favor the development of autoimmune responses against

nuclear antigens characteristic for SLE. This notion was further supported by the

findings that other mice deficient in molecules implicated in clearance of apoptotic

bodies developed anti-nuclear autoantibodies and that such a mutation also enhanced

autoantibody production in lupus-prone mice (128). It should be stressed that the

impaired clearance of apoptotic cells in MRL-Faslpr mice was associated with increased

levels of free nucleosomal antigens present in the circulation (121). In addition, down-

regulation of serum levels of C1q due to the C1qa polymorphism in NZB mice could

result in an impairement of clearance of apoptotic cells, thereby promoting the

development of autoimmune immune responses against nuclear autoantigens (90).

I.1.5.3. Critical Role of TLR7 in the Development of Autoimmune Responses in

Murine SLE

The hallmark of SLE is elevated serum levels of antibodies to nuclear

constituents consisting primarily of nucleic acid-protein complexes. One important

question which has not yet been answered in the past is why nucleic acid-containing

antigens become the major autoantibody target in SLE. It was initially speculated that

the autoimmune repertoire of B cells may be restricted to receptors that recognize these

autoantigens. However, this possibility was excluded since the specificity of

autoantibodies is not determined by a particular set of Ig variable region genes, as

documented by the nucleotide sequence analysis of a panel of anti-DNA monoclonal

autoantibodies. Instead, numerous studies have provided new insights into the role of

the innate immune system, specifically TLR7 and TLR9, in the recognition of nuclear

autoantigens implicated in murine SLE.

TLR are a family of germ-line encoded receptors which recognize a diverse

range of conserved molecular motifs commonly found in microbial pathogens, and

recognition of microbial components by TLR is critical in host responses against

- 51 -

pathogens (129). At least, three TLR act as receptors for nucleic acids in mice: TLR3

for double-stranded RNA, TLR7 for single-stranded RNA and TLR9 for unmethylated

CpG (cytosine-phosphate-guanine) DNA (the specificity of TLR8 has not yet been well

defined in mice). Notably, unlike other TLR expressed on the cell surface, the three

nucleic acid-specific TLR are localized in the endosome, thereby limiting the access to

foreign nucleic acids only and hence contributing to the discrimination between self and

non-self nucleic acids. TLR-mediated signaling induces maturation and activation of

DC. Upon stimulation through TLR7 and TLR9, a subset of DC, called plasmacytoid

DC (pDC), produces unusually large amounts of IFN-α (interferon alpha) (130). IFN-α

promotes the differentiation of monocytes into myeloid DC (131), which are highly

efficient at antigen presentation, express high levels of costimulatory molecules, and

produce B-cell survival factors. Thus, TLR clearly play a crucial role in the activation of

innate immune responses and the subsequent stimulation of the adaptive immune

response.

The DNA sequences containing unmethylated CpG motifs that can trigger TLR9

are a prominent feature of bacterial and viral DNA, but uncommon in mammalian DNA.

However, it has been reported that mammalian DNA in the form of chromatin-

containing IC can stimulate DC and B cells through TLR9 in vitro (132-135). This

suggests that mammalian DNA segments enriched for hypomethylated CpG motifs

(such as CpG islands or mitochondrial DNA) can be preferentially released by nucleases

that are active in apoptotic cells. Notably, DNA isolated from circulating IC of lupus

patients displayed a higher CG content (136). It has also been shown that endogenous

RNA in the form of RNA-protein complexes, such as RNP, can trigger pDC through

TLR7 to produce IFN-α (137, 138). All these data suggest that endogenous nucleic

acid-protein complexes released from apoptotic cells could act as ligands for TLR7 and

TLR9, and thus activate pDC and nuclear antigen-specific autoreactive B cells. This

idea is consistent with the notion that apoptotic cells are likely to be a major source of

nuclear autoantigens.

As mentioned above, the expression of TLR7 and TLR9 in the endosome is a

mechanism to restrict TLR responses to nucleic acids from microbial pathogens and to

prevent their activation by host nucleic acids. However, this protective mechanism may

be overcome when autoantigens in the form of IgG IC can be internalized through FcγR

and subsequently interact with endosomal TLR7 and TLR9 in DC (Figure 3). However,

it is unlikely that this is a mechanism to trigger DC and initiate autoimmune responses

against nuclear antigens in SLE, since the production of IgG autoantibodies (to form

- 52 -

stimulating IC) is prerequisite for this process. Instead, this mechanism can sustain the

production of IFN-α through IC-mediated activation of pDC, thereby establishing a

vicious cycle not only aggravating the autoimmune process but also promoting the

development of autoimmune responses against a wide array of autoantigens that do not

engage TLR. In contrast to FcγR, specific recognition of nuclear acid-containing

autoantigens by BCR on autoreactive B cells can lead to their internalization and to

activation of TLR7 or TLR9 (Figure 3). Indeed, it has been shown that the synergistic

engagement of TLR7/9 and the BCR in response to DNA- or RNA-containing antigens

could induce the activation of autoreactive B cells (44-46, 134, 135). A recent study

provided evidence that after antigen binding and internalization, the BCR signals,

through a phospholipase-D-dependent pathway, recruit TLR9 from multiple small

endosomes to large autophagosome-like compartments in a microtubule-dependent

process (139). This unique mechanism for BCR-induced TLR7 and TLR9 recruitment

to the MHC class II antigen-processing compartments could explain how

hyperresponsiveness to RNA- or DNA-containing antigens is achieved.

The possible activation of autoreactive B cells through recognition of

endogenous nucleic acids by TLR7 and TLR9 prompted a number of investigators to

define more precisely the roles of both receptors in autoimmune responses against

distinct nuclear antigens and in the subsequent development of lupus nephritis. It has

been shown that the development of SLE was markedly suppressed in (NZB x NZW)F1

mice treated with a dual inhibitor of TLR7 and TLR9 (140) as well as in B6-Faslpr mice

bearing the Unc93b1 mutation which impairs signaling via TLR7 and TLR9 (141). The

duplication of the Tlr7 gene in mice bearing the Yaa mutation and transgenic

overexpression of TLR7 in B6 FcγRIIB-/- mice resulted in an enhanced production of

RNA-specific autoantibodies (24, 25, 48, 142). Moreover, the production of RNA-

specific autoantibodies was markedly suppressed in TLR7-deficient MRL-Faslpr and

B6.Nba2 mice (47, 49). Thus, it is now clear that TLR7 is indeed involved in the

activation of autoreactive B cells specific for RNA-associated autoantigens in murine

SLE. On the other hand, the analysis of TLR9-deficient MRL-Faslpr mice demonstrated

the contribution of TLR9 to the production of anti-DNA and anti-chromatin

autoantibodies (47, 143), in agreement with the finding that TLR9-deficient anti-DNA

B cells fail to undergo class switching to the pathogenic IgG2a and IgG2b isotypes

(144). Although the production of anti-chromatin autoantibodies was consistently

downregulated in different models of TLR9-deficient lupus-prone mice, the effect of

TLR9 deficiency on anti-DNA autoimmune responses was somehow controversial.

- 53 -

Indeed, it has been reported that serum levels of anti-DNA autoantibodies were rather

increased in other studies with TLR9-/- MRL- and B6-Faslpr mice as well as B6.Nba2

mice (145-147). These data suggest the presence of TLR9-independent pathway for

anti-DNA autoimmune responses. Indeed, the absence of anti-DNA autoantibodies in

TLR7-/- TLR9-/- double-deficient B6.Nba2 mice revealed a critical role of TLR7 in this

autoimmune response (147).

Studies in different models of murine SLE either lacking TLR7 or expressing

increased levels of TLR7 have demonstrated that TLR7 is critically involved in the

development of autoantibodies against RNA-related nuclear antigens and of lupus

nephritis (24, 25, 47-49, 142). This clearly contrasts with the increased production of

various autoantibodies and accelerated development of lupus nephritis in TLR9-

deficient lupus-prone mice (47, 145, 146, 148). Although the underlying mechanism

behind the opposing effects of TLR7 and TLR9 on the development of murine SLE has

remained elusive, it has been shown that TLR9 may exert an inhibitory effect on TLR7.

Indeed, in vitro studies in HEK293 cells transfected with human TLR cDNAs revealed

that the expression of TLR9 inhibited the activation of TLR7, but not vice versa (149).

Although the precise mechanism for this phenomenon has not been totally elucidated, it

was suggested that TLR7 and TLR9 may physically interact (directly or indirectly) and

that this interaction results in the inhibition of TLR7 but not TLR9. Thus, a possible

functionally upregulated expression of TLR7 in TLR9-deficient mice could promote the

production of nephritogenic anti-nuclear autoantibodies, thereby accelerating the

progression of lupus nephritis. This idea is consistent with recent findings that enhanced

disease was associated with functionally upregulated expression of TLR7, as

documented by an increased TLR7-dependent activation of B cells and plasmacytoid

DCs (147). Most significantly, disease exacerbation in TLR9-deficient mice was

completely suppressed by the deletion of TLR7, indicating TLR7 has a pivotal role in a

wide variety of autoimmune responses against nuclear antigens implicated in murine

SLE.

- 54 -

Figure 3: Possible activation of pDC and autoreactive B cells through TLR7 and TLR9.

A. DNA- or RNA-containing IgG IC can be internalized through FcγR and subsequently

interact with endosomal TLR7 and TLR9 in pDC. B. Specific recognition of nuclear

acid-containing autoantigens (DNA, chromatin or RNP) by BCR on autoreactive B cells

can lead to their internalization and to activation of TLR7 or TLR9.

pDC

FcγγγγR

TLR7 TLR9

TLR Signaling

Nucleus

αααα-DNA

Endosome

EndosomeTLR Signaling

Nucleus

TLR7 TLR9

Autoreactive B-cells

A

B

αααα-chromatin

αααα-RNP

DNA

chromatin

RNP

αααα-DNA

αααα-chromatin

αααα-RNP

DNA

chromatin

RNP

pDC

FcγγγγR

TLR7 TLR9

TLR Signaling

Nucleus

αααα-DNA

Endosome


Nucleus

TLR7 TLR9

Autoreactive B-cells

A

B

αααα-chromatin

αααα-RNP

DNA

chromatin

RNP

αααα-DNA

αααα-chromatin

αααα-RNP

DNA

chromatin

RNP

- 55 -

I.2. Endogenous Retroviruses in SLE

I.2.1. Retroviruses

Retroviruses are RNA viruses that employ the virus enzyme reverse

transcriptase to transcribe the RNA genome into DNA, which is integrated into the host

cell genome (150, 151). Two identical long terminal repeat sequences (LTRs), flanking

the coding sequences of retroviruses, contain sequences termed U3 (3’ unique region),

R (repeat) and U5 (5’ unique region) (Figure 4). Transcription of the provirus initiates

at the U3-R boundary in the upstream LTR and RNA cleavage/polyadenylation occurs

at the R-U5 boundary in the downstream LTR. The U3 sequence located on the 5’-LTR

serves as a viral promoter. The retroviral env (envelope) gene encodes a precursor

polyprotein, which is cleaved to produce two subunits; a surface (SU) glycoprotein and

a transmembrane (TM) protein. Both subunits remain associated with each other on the

intact virions through disulfide linkage. The TM subunit contains an amino-terminal

hydrophobic peptide that is thought to mediate membrane fusion (152, 153), whereas

the SU subunit bears the receptor binding function (154, 155). In murine leukemia virus,

SU and TM are designated gp70 and p15E, respectively, corresponding to their

molecular weights. Coating the inner surface of the membrane, the viral matrix (MA),

the capsid (CA), and nucleoproteins (NC) are encoded by the gag (group specific

antigen) gene. The RNA genome and associated proteins: reverse transcriptase (RT),

integrase (IN) and protease (PR) encoded by the pol gene, which are inside the capsid,

comprise the virus core.

- 56 -

Figure 4: Structure of a retrovirus (adapted from (150, 151)). The retrovirus is

enveloped and contains positive-strand RNA bearing three genes: gag, pol and env

which encode respectively: capsid proteins [matrix (MA), capsid (CA) and

nucleoproteins (NC)]; viral enzymes [protease (PR), reverse transcriptase (RT) and

integrase (IN)]; and envelope proteins [surface (SU) and transmembrane (TM)].

The retrovirus life cycle is composed of early and late phases (150). The early

phase starts when a free particle of retrovirus infects new cells by binding to a cell

surface receptor (Figure 5). The envelope of the retrovirus determines the specificity of

the interaction with the cell. The viral envelope either fuses with the plasma membrane

or is endocytosed, releasing the virion core into the cytoplasm. Then, the single-

stranded virion RNA is copied into double-stranded DNA by the reverse transcriptase

and the viral integrase acts to randomly insert the double-stranded DNA into the host

genome. The proviral DNA becomes part of the host genome and therefore is replicated

with host DNA. The infection is spread by infection of new cells or by multiplication of

cells which already contain the provirus. Moreover, infection of germ cells lead to the

transmission of the provirus to the progeny and are therefore called endogenous

retroviruses (ERVs). The late phase of the retrovirus life cycle is composed of

expression of viral RNA, synthesis of viral proteins, and assembly of virions. The

- 57 -

expression of the proviral DNA in the host depends on their integration sites and the

transcription factors of this cell. Some full-length transcripts of the retroviral genome

are exported directly from the nucleus and serve as the genome to be packaged into the

progeny virion. Other transcripts are used for translation. All retroviruses make at least

two mRNAs: the Gag and Gag-Pol proteins are synthesized from unspliced RNA,

whereas the Env proteins are synthesized from a spliced RNA, removing gag and pol

reading frames. When the Env proteins are synthesized, they are translocated into the

endoplasmic reticulum and then transported to the Golgi, where glycosylation and

cleavage of the proteins occur. The precursor Env protein is cleaved into SU and TM by

a cellular enzyme during transport from the Golgi to the plasma membrane (156-158).

Gag proteins are synthetized as a Gag polyprotein precursor and Pol proteins are made

as a large Gag-Pol polyprotein precursor. Upon processing by the viral proteinase the

Gag and Pol proteins interact with each other. The reorganized proteins subsequently

bud through the plasma membrane where the envelope proteins (SU and TM) have

accumulated. The NC domain of Gag binds to psi (ψ) packaging sequence, which is

near the 5’ end of viral genome RNA but downstream the 5’ splice site allowing

encapsidation of only full-length viral RNA (159, 160). Specific sequences in the 5’ end

of the RNA (161), termed dimmer linkage sequences, allow RNA dimerization.

Incorporation of a host tRNA serves as the initiating primer for DNA synthesis (162).

At the end of the budding process a roughly spherical particle is finally pinched off and

release from the cells. The Gag and Gag-Pol precursors proteins are cleaved by protease

to release the smaller proteins present in the infectious virions (163). The virion

contains two copies of the RNA genome copackaged into one particle, and, upon

infection, reverse transcription occurs by a copy choice mechanism on both RNAs (164,

165). In this mechanism, if the polymerase encounters a break in the RNA or a region

that is difficult to transcribe, it will continue synthesis on the second strand. Generally,

the two copies of RNA are identical, so that strand switching has no consequence.

However, when the two RNAs are from two strains of virus, recombinant virus can be

created.

- 58 -

Figure 5: Retrovirus life cycle (adapted from (150, 151)). Retrovirus life cycle is

divided in an early phase and a late phase. The early phase starts when retrovirus binds

to cell surface receptor, penetrates in the cell and is uncoated. The single-stranded virion

RNA is copied into double-stranded DNA and integrates into the host genome. The late

phase of the retrovirus life cycle is composed of expression of viral RNA, synthesis of

viral proteins, and assembly of virions.

Retroviruses are classified following their properties (150, 151). The family of

Retroviridae can be divided in simple viruses, which encode only gag, pol, and env

genes, and complex viruses, which encode small regulatory proteins in addition to the

same genes of simple viruses. Simple retroviruses include alpharetroviruses,

betaretroviruses and gammaretroviruses, whereas complex retroviruses are comprised of

deltaretroviruses, epsilonretroviruses, lentiviruses and spumaviruses. The

alpharetroviruses are characterized by a C-type morphology consisting of retroviruses

that assemble at the plasma membrane and contain central, symmetrically placed

- 59 -

spherical inner cores. They are typified by the avian leukosis sarcoma virus. The

betaretroviruses has either a B-type morphology with a round eccentrically positioned

inner core or a D-type morphology which exhibit a distinctive cylindrical core and

assemble in the cytoplasm. Mouse mammary tumor virus and the Mason-Pfizer monkey

virus belong to this group. The gammaretroviruses, including the murine leukemia

viruses and the feline leukemia viruses, are characterized by a C-type morphology. The

deltaretroviruses has also a C-type morphology and contain, in addition, regulatory

genes, called rex and tax, which control the synthesis and processing of the viral RNA.

The best known examples are the human T-lymphocyte viruses and the bovine leukemia

virus. The epsilonretroviruses, like the walleye dermal sarcoma virus, has a C-type

morphology and contain additional genes ORF (open reading frame) A, B and C, which

regulate the cell cycle. The lentiviruses such as human immunodeficiency virus are

characterized by cylindrical or conical cores. They also express additional genes

controlling synthesis and processing of the viral RNA, virion assembly, host gene

expression, etc. Finally, the spumaviruses, which contain prominent spikes on the

surface and a central uncondensed core, assemble in the cytoplasm and bud into the ER

and plasma membrane. They are typified by the human foamy virus. They expressed

two additional genes, tas and bet. The Tas (Transactivator of spumaviruses) protein

transactivates virus transcription. Bet (Between-env-and-LTR-1-and-2) protein

functions as a inhibitor of the APOBEC3 family of innate antiretroviral defense factors

(166).

I.2.2. Murine ERVs

ERVs and other LTR retroelements constitute around 10% of the human and

mouse genome (167, 168). Most of these retrovirus elements are defective or consist

only of solo LTRs generated by recombination between the two LTRs during the

integration process. Others, which contain coding sequences, are either silenced or

expressed. Notably, studies have shown that the expression of retroviral sequences is

strongly affected by the state of DNA methylation (169-171). Even though the

expression of these viruses could have deleterious consequences, they have been

utilized for different physiological processes such as transcriptional control of several

genes (172-175). Notably, endogenous retroviral sequences control tissue-specific

expression of human salivary amylase gene (176) and ERVs provide the

polyadenylation signal for certain human genes (177, 178). Furthermore, viral RNAs

- 60 -

were shown to interact with proteins in steroidogenesis. In fact, PSF (polypyrimidine

tract-binding protein associated-splicing factor), a protein of spliceosomes, represses

transcription of the first gene in the steroidogenic pathway, P450scc, by binding to the

promoter region. The VL30 retroelement RNA forms a complex with PSF that

dissociates from the gene, activating transcription (179). Finally, ERV have been

utilized at the protein level such as the role of endogenous retrovirus envelope proteins

in the fusion of placental trophoblasts which might prevent an immune response against

the development of the embryo (180).

ERVs are found in wild mouse species, which are the progenitor of common

inbred laboratory strains (181-185). ERVs related to murine leukemia virus have been

extensively studied and are the most thoroughly characterized. They are classified as

ecotropic (Eco), xenotropic (Xeno) or polytropic according to the host range dictated by

their respective gp70 proteins (186). Eco viruses can replicate in mouse but not non-

mouse cells. Xeno viruses can replicate in non-mouse cells but not in cells derived from

laboratory mouse strains, and polytropic viruses can infect both murine and non-murine

cells (186). Moreover, the analysis of an AKR SL12.3 cell line identified the presence

of four subgroups of Xeno proviruses (Xeno-I, Xeno-II, Xeno-III and Xeno-IV), which

differ within a variable region of the 5’ portion of the env gene (187). Furthermore,

polytropic viruses are divided into two subgroups, termed polytropic (PT) and modified

PT (mPT), based on differences in their gp70 nucleotide sequences (188).

Eco viruses utilize the cationic amino acid transporter CAT-1 as an entry

receptor (189). PT viruses are comprised of a large group of ERVs that encode gp70s

closely related to Xeno gp70 (186), as both retroviruses share a common entry receptor,

XPR1 (Xeno and polytropic retrovirus receptor) (190, 191). However, the host range

difference observed between Xeno and PT retroviruses is explained by sequence

polymorphisms in Xpr1. There are three functionally distinct variants of this receptor in

mouse species: Xpr1n receptor variant found in laboratory mouse strains permits

infection with PT but not Xeno viruses, the Sxv receptor variant (Xpr1sxv) is found in

most wild mouse species and permits infection with both PT and Xeno retroviruses

(192), and the Xpr1Cas variant, which lacks receptor function (193), is present in the

Asian mouse species Mus castaneus, which is resistant to infection by both PT and

Xeno retroviruses (194).

The XprICas variant can be consider a receptor-mediated resistance gene. A

second group of receptor-mediated resistance genes function through an interference

mechanism as a result of the expression of unique Env proteins. Resistance to Eco

- 61 -

viruses in Mus castaneus, Mus museulus molossinus and Lake Casitas mice of

California is due to the presence of a resistance gene, Fv4, which encodes an Eco Env

glycoprotein, and its expression is thought to interfere with receptor binding of

exogenous Eco viruses (195, 196). An analogous interference mechanism may be

responsible for Rmcf (resistance to MCF virus)-mediated resistance to the infection by

exogenous PT viruses due to the expression of a unique cell surface PT provirus-derived

Env glycoprotein (197). Similarly, Xeno provirus-derived resistance gene, Rmcf2,

prevents entry of exogenous PT retroviruses in Mus castaneus (198, 199). Notably, this

type of provirus Env-mediated resistance represents an important defense mechanism in

the mouse.

I.2.3. Role of ERVs in Murine SLE

The relationship between ERVs and SLE was first suggested when murine

leukemia viral antigens were found in immune deposits of diseased glomeruli from

lupus-prone mice NZB and (NZB x NZW)F1 hybrid mice (200, 201). Subsequently, it

was demonstrated that relatively large amounts of the envelope glycoprotein gp70,

derived from ERVs, are present in the sera from lupus-prone (NZB x NZW)F1, MRL-

Faslpr and BXSB mice (17, 18). Strikingly, only lupus-prone mice spontaneously

develop autoantibodies against serum retroviral gp70, which are detected in sera as a

form of IC because of the excess of free gp70 antigens (19). gp70 IC are found in the

circulating blood close to the onset of renal disease and within diseased glomeruli of

lupus mice (17-19). Several genetic studies have revealed a remarkable correlation of

serum levels of gp70 IC with the development of severe lupus nephritis (86, 94, 95,

202), further supporting the pathogenic role of gp70 IC in murine SLE.

All inbred strains of mice carry numerous ERVs as chromosomal genes. No

copie to five copies of Eco proviruses are present in laboratory mouse strains (183, 184,

203, 204). Xeno, PT and mPT are present in about 20 copies each and are polymorphic

in their insertion sites (184, 205, 206). gp70 (encoded by the retroviral env gene) is

expressed, depending on its site of integration into the mouse genome and on the

differentiation state of the cells (207). gp70 is a constituent of the surface of various

epithelia, thymocytes and peripheral lymphocytes, and shares immunological and

biochemical properties with the thymocyte differentiation antigen GIX (207-210).

Lymphoid cells are, however, not a major source for serum retroviral gp70 because

neither thymectomy nor splenectomy affected serum levels of gp70 (211). Instead,

- 62 -

serum retroviral gp70 is secreted by hepatocytes and behaves like an acute-phase

protein (APP), since its expression is enhanced by different inducers of APP such as

LPS (lipopolysaccharides), turpentine oil or polyriboinosinic-polyribocytidylic acid

(212, 213). It could be possible that with the onset of SLE and the corresponding

systemic inflammation, the production of gp70 is boosted, thereby further accelerating

lupus nephritis. Significantly, this acute-phase response (APR) is also under genetic

control, in which only mice having high basal levels of serum gp70 displayed an

upregulated expression of serum gp70 in response to LPS (212, 214). It was reported

that LPS-induced upregulated production of serum gp70 was linked to a locus,

designated Sgp2, present in chromosome 7 (215). However, these results should be

interpreted with caution, considering that the number of mice analyzed in this genetic

study was relatively limited. More extensive studies with GIX congenic 129 mice and

with B6 mice bearing the NZW-Sgp3 locus suggested the possible involvement of Sgp3

locus in enhanced serum gp70 production after LPS stimulation (107, 212).

The genetic origin of serum retroviral gp70 is still unclear. Serological analysis

clearly excluded the involvement of Eco gp70 as a source of serum gp70 (216). Earlier

studies of tryptic peptide mapping analysis showed that serum gp70 molecule resembles

the Env protein of NZB-X1 virus, one of the two distinct Xeno viruses isolated from

NZB mice (217, 218). However, the fingerprint of serum gp70 also displayed additional

marker peptides detectable in gp70 of other Xeno viruses, including the second NZB

Xeno virus, NZB-X2, and gp70 expressed on thymocytes and splenic lymphocytes.

Since PT proviruses are comprised of a large group of ERVs that encode gp70s closely

related to Xeno gp70 (186), these ERVs are potentially additional sources of serum

gp70.

Serum concentrations of gp70 vary greatly among different inbred strains of

mice (17-19, 219). Significantly, all SLE-prone strains have relatively high

concentrations of gp70 in their sera (>15 µg/ml), whereas B6, B10 and BALB/c mice

produce low serum levels of gp70 (<5 µg/ml). By studying the progeny of crosses of

lupus-prone NZB, NZW and BXSB strains with non-autoimmune B6 or B10 strains, a

major quantitative trait locus, Sgp3 (or Bxs6), on mid chromosome 13 was found to be

strongly linked with basal levels of serum retroviral gp70 (40, 88, 95, 100, 107). It is

worth noting that the Gv1 (Gross virus antigen 1) gene, which overlaps with the Sgp3

locus, controls the expression of thymic GIX gp70 antigen (220), which is closely

correlated to serum levels of gp70 (212). As Gv1 likely regulates in trans the expression

of multiple endogenous retroviral transcripts in different tissues including the liver (220,

- 63 -

221), it is reasonable to assume that Gv1 and Sgp3 are identical or related genes

regulating the expression of endogenous retrovirus.

A recent study has claimed that macroH2A1 histone variants are important for

repressing the expression of ERVs in mouse liver by promoting the methylation of

DNA (222), in agreement with the findings that the expression of retroviral sequences is

strongly affected by the state of DNA methylation (169-171). macroH2A histone

variants have an N-terminal H2A domain and a C-terminal nonhistone domain, known

as the macrodomain (223). macroH2As are preferentially associated with

transcriptionally repressed or silent chromatin domains, including the inactivated X

chromosome (224, 225), centromeric heterochromatin (226) and senescence-associated

heterochromatic foci (227). The distribution of macroH2As in chromatin suggests its

role in repressing gene expression. Thus, the macroH2A1 gene could be a Sgp3

candidate gene, as it is located in the Sgp3 interval. In addition, two Rsl (Regulator of

sex limitation) genes, Rsl1 and Rsl2, which encode Krüppel-associated box zinc-finger

proteins (KRAB-ZFP) and control male-dependent upregulated gene expression in liver,

have been identified in this region (228). In addition to these two Rsl genes, there exist

in this region more than 20 Zfp genes, the function of which has not yet been identified.

Since the KRAB transcription repressor domain has been shown to suppress lentivirus

proviral transcription by inducing heterochromatization in the lentiviral integration sites

(229), it is possible that the expression of retroviral serum gp70 is regulated by one of

the Zfp genes. A second NZB and NZW locus, Sgp4, on distal chromosome 4 was found

to be linked to serum gp70 levels in crosses with B6 and BALB/c backgrounds (88, 89).

Moreover, the presence of an additional minor locus controlling the expression of serum

gp70 was revealed on the proximal region of chromosome 12 of NZB and NZW mice

(89). All these data indicate that serum levels of gp70 are under the control of multiple

regulatory genes.

NZB mice spontaneously produce a very high titer of replication-competent

Xeno viruses from birth (230), while they fail to express Eco viruses because of the lack

of Eco sequences in their genome (204). The coexistence of the predisposition to

autoimmune disease and a persistent and enhanced expression of the endogenous Xeno

viruses in NZB mice throughout their life has suggested a potential role of Xeno viruses

for the development of SLE (200). However, the analysis of genetic crosses between

NZB mice and virus-negative SWR mice showed that the development of

autoantibodies and glomerulonephitis was independent of production levels of Xeno

viruses (231). In addition, attempts to induce autoimmune disease by injection of Xeno

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viruses were unsuccessful. Since the polymorphic form of XPR1 in laboratory mouse

strains renders them resistant to infection by Xeno viruses, it is not surprising to see the

lack of correlation with the abundance of Xeno viruses with the development of murine

SLE. However, an infectious murine leukemia virus, which was not characterized

further, has been isolated from NZB mice, and neonatal infection with this virus

induced a lupus-like autoimmune syndrome in (BALB/c x NZB)F1 mice (232). In

addition, it has been reported that 8.4-Kb full-length RNA transcripts of endogenous

mPT virus are expressed in lupus-prone mice, but not in a number of non-autoimmune

strains of mice (233, 234). Although endogenous PT and mPT viruses are likely to be

replication defective, replication-competent and infectious recombinant viruses

containing the PT or mPT gp70 sequences can be generated and may possess the

pathogenic potential to induce disease in genetically susceptible hosts. Notably, the

Sgp3 locus contributes to the production of autoantibodies against nuclear antigens as

well as retroviral gp70 antigen in the presence of the Yaa mutation, in which the Tlr7

gene duplication is considered to be the etiologic basis for the Yaa-mediated

enhancement of autoantibody production (40, 107, 235). Considering the critical role of

TLR7 in autoimmune responses against RNA-containing nuclear antigens, it is of

importance to reassess the possible role of ERVs in the context of TLR7 as a triggering

factor for the development of autoimmune responses against retroviral gp70 and hence

the pathogenesis of murine SLE.

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II. AIMS OF THE STUDY

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- 67 -

II. Aims of the Study

All inbred strains of mice contain numerous ERVs as chromosomal genes. The

envelope glycoprotein gp70, encoded by the retroviral env gene, is secreted by

hepatocytes and behaves like an APP (212, 213). Lupus-prone mice spontaneously

develop autoantibodies against serum retroviral gp70 and form gp70 IC (19). The

appearance of circulating gp70 IC close to the onset of renal disease and their deposition

in diseased glomeruli support the pathogenic role of gp70 IC in murine SLE. However,

the precise genetic origin of serum retroviral gp70 and the genetic mechanisms

responsible for its expression has not yet been well defined. Moreover, it has been

shown that an enhanced expression of serum gp70 during APRs is also under a genetic

control, but the genetic mechanism underlying this response has not yet been

understood. The present study aims to define the genetic origin of serum retroviral gp70

autoantigen implicated in murine SLE and the genetic mechanisms governing the

expression of serum gp70 under physiological or inflammatory conditions. This has

been achieved by using various lupus-prone and non-autoimmune strains of mice

expressing high or low serum levels of gp70 and also by using B6 or B10 congenic

mice carrying two different genetic loci derived from lupus-prone mice, Sgp3 (40, 88,

95, 100, 107) and Sgp4 (88, 89), which are linked with increased serum levels of

retroviral gp70.

II.1. Dissection of Genetic Mechanisms Governing the Expression of Serum


The endogenous retroviral envelope glycoprotein, gp70, has been thought to be a

product of endogenous Xeno virus, NZB-X1, which is spontaneously produced at a high

titer in NZB mice (217, 218). Mice carry four different subgroups of Xeno proviruses

(Xeno-I, Xeno-II, Xeno-III and Xeno-IV), which exhibit distinct nucleotide sequences

of their env genes (187), but it has not yet been determined which of the four Xeno gp70

are expressed in hepatocytes and might contribute to serum gp70 in relation to the NZB-

X1 virus. Furthermore, because of the presence of multiple copies of PT and mPT

proviruses, which encode gp70s that are closely related to Xeno gp70, it is possible that

either of these two proviruses are additional sources of serum gp70. To address these

questions, we investigated the genomic composition of Xeno (Xeno-I, Xeno-II, Xeno-

III and Xeno-IV), PT and mPT proviruses in relation with the abundance of respective

gp70 RNAs in livers and the concentration of serum gp70 in various strains of mice. In

- 68 -

addition, we determined the abundance of different retroviral gp70 RNAs in B6 and

B10 mice congenic for Sgp3 or Sgp4, in order to define the genetic mechanism by

which the Sgp3 and Sgp4 loci contribute to the production of serum gp70. Furthermore,

we determined the effect of LPS on the abundance of three different gp70 RNAs to

define whether the expression of ERVs is differentially regulated under physiological

and inflammatory conditions.

II.2. Selective Up-Regulation of Intact, but Not Defective env RNAs of Endogenous

Modified Polytropic Retrovirus by Sgp3 Locus of Lupus-Prone Mice

Four different ERVs, i.e., Eco, Xeno, PT or mPT, are expressed in mice. In view

of the implication of ERVs in murine SLE, we investigated whether a particular class of

ERVs is associated with development of murine SLE. This question was addressed by a

comparison of the expression levels of gp70 RNAs of these four different ERVs in

livers of four lupus-prone mice (NZB, NZW, BXSB and MRL-Faslpr) with those in nine

non-autoimmune mice (NFS, 129, AKR, DBA/2, B6, B10, BALB/c, CBA and C3H/He).

In addition, because we observed the expression of two defective env RNAs of mPT

provirus, in addition to mPT provirus carrying the intact env sequence, we determined

the correlation between the abundance of the intact mPT env transcripts with the

development of murine SLE in relation to two different Sgp loci. Furthermore, in view

of an emerging role of TLR7 in the pathogenesis of SLE, we defined the role of TLR7

in the development of anti-gp70 autoimmune responses in TLR7-deficient and -

sufficient lupus-prone B6 mice congenic for the Nba2 locus, the major lupus

susceptibility locus derived from the NZB strain.

II.3. TLR-mediated Up-Regulation of Serum Retroviral gp70 is Controlled by the


Serum retroviral gp70 expression is enhanced by different inducers of APP such

as LPS, turpentine oil or polyriboinosinic-polyribocytidylic acid, indicating that serum

gp70 behaves like an APP (212, 213). However, unlike conventional APPs, the serum

gp70 response is strain-dependent, as only mice having high basal levels of serum gp70

displayed an upregulated production of serum gp70 in response to LPS. To confirm

whether serum gp70 is indeed an APP, we determined whether injection of interleukin-

1β (IL-1β), IL-6 or TNFα (tumor necrosis factor α), well known inducers of APP, is

able to induce high serum levels of gp70. In addition, to elucidate the genetic

- 69 -

mechanism involved in LPS-induced gp70 production, we determined the contribution

of the Sgp loci to this response in non-responder B6 mice bearing either Sgp3 and/or

Sgp4 locus. Furthermore, given the critical roles of TLR7 and TLR9 in the SLE

pathogenesis, we explored whether the activation of TLR7 and TLR9 is able to promote

the production of serum gp70 in comparison with the stimulation with LPS.

II.4. The Sgp3 Locus Derived from the 129 Strain is Responsible for Enhanced


The macroH2A1 histone variant is preferentially associated with

transcriptionally repressed or silent chromatin domains, and its distribution in chromatin

suggests its role in repressing gene expression (224-227). More recently, an up-

regulated transcription of endogenous retroviral sequences was reported in livers of

macroH2A1-deficient B6 mice (222). Since macroH2A1 gene is located within the Sgp3

interval, we hypothesize that macroH2A1 is a candidate gene for Sgp3. To address this

question, we analyzed the expression of endogenous retroviral gp70 RNAs in livers

from two different macroH2A1-sufficient or -deficient mice bred into the B6 or 129

backgrounds in relation to the 129-derived Sgp3 locus. In addition, we have generated a

B6.NZB-Sgp3 subcongenic line devoid of the NZB-derived macroH2A1 gene, and

determined whether these mice displayed the Sgp3 phenotype identical to that of

B6.NZB-Sgp3 congenic mice carrying the NZB-derived macroH2A1 gene.

- 70 -

- 71 -

III. RESULTS

- 72 -

- 73 -

III.1. Dissection of Genetic Mechanisms Governing the Expression of Serum


Lucie Baudino, Kumiko Yoshinobu, Naoki Morito, Shuichi Kikuchi, Liliane Fossati-

Jimack, Benard J. Morley, Timothy J. Vyse, Sachiko Hirose, Trine N. Jorgensen,

Rebecca M. Tucker, Christina L. Roark, Brian L. Kotzin, Leonard H. Evans and Shozo

Izui

Published in: Journal of Immunology (2008), 181: 2846-2854

- 74 -

- 75 -

- 76 -

- 77 -

- 78 -

- 79 -

- 80 -

- 81 -

- 82 -

- 83 -

III.2. Selective Up-Regulation of Intact, but Not Defective env RNAs of

Endogenous Modified Polytropic Retrovirus by Sgp3 Locus of Lupus-Prone Mice

Kumiko Yoshinobu, Lucie Baudino, Marie-Laure Santiago-Raber, Naoki Morito,

Isabelle Dunand-Sauthier, Bernard J. Morley, Leonard H. Evans, and Shozo Izui

Published in: Journal of Immunology (2009), 182: 8094-8103

- 84 -

- 85 -

gp70

RN

Agp

70 R

NA

0.01

0.1

1

10

Xeno

0.001

0.01

0.1

1

10

Eco

gp70

RN

Agp

70 R

NA

0.01

0.1

1

10

PT

NZB NZW BXSB MRL NFS 129 AKR DBA B6 B10 BALB CBA C3H0.01

0.1

1

10

mPT

gp70

RN

Agp

70 R

NA

0.01

0.1

1

10

Xeno

0.001

0.01

0.1

1

10

Eco

gp70

RN

Agp

70 R

NA

0.01

0.1

1

10

PT


0.1

1

10

mPT

0.01

0.1

1

10

PT


0.1

1

10

mPT

- 86 -

- 87 -

- 88 -

- 89 -

- 90 -

- 91 -

- 92 -

- 93 -

- 94 -

III.3. TLR-mediated Up-Regulation of Serum Retroviral gp70 is Controlled by the


Lucie Baudino, Kumiko Yoshinobu, Isabelle Dunand-Sauthier, Leonard H.Evans and

Shozo Izui

Submitted for publication (to the Journal of Autoimmunity)

- 95 -

TLR-mediated Up-Regulation of Serum Retroviral gp70 is Controlled by the


Lucie Baudino a

, Kumiko Yoshinobu a

, Isabelle Dunand-Sauthier a

,

Leonard H. Evans b

, and Shozo Izui a,*

a Department of Pathology and Immunology, University of Geneva, Geneva,

Switzerland. b Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories,

National Institute of Allergy and Infectious Diseases,

Hamilton, Montana 59840

* Corresponding author. Tel.: +41-22-379-5741; Fax: +41-22-379-5746.

E-mail address: [email protected] (S. Izui)

- 96 -

Abstract

The endogenous retroviral envelope glycoprotein, gp70, implicated in murine

systemic lupus erythematosus (SLE), has been considered to be a product of xenotropic,

polytropic (PT) and modified PT (mPT) endogenous retroviruses. It is secreted by

hepatocytes like an acute phase protein, but its response is under a genetic control.

Given critical roles of TLR7 and TLR9 in the pathogenesis of SLE, we assessed their

contribution to the acute phase expression of serum gp70, and defined a pivotal role of

the Sgp3 (serum gp70 production 3) and Sgp4 loci in this response. Our results

demonstrated that serum levels of gp70 were up-regulated in lupus-prone NZB mice

injected with TLR7 or TLR9 agonist at levels comparable to those induced by injection

of IL-1, IL-6 or TNF. In addition, studies of C57BL/6 Sgp3 and/or Sgp4 congenic mice

defined the major roles of these two loci in up-regulated production of serum gp70

during acute phase responses. Finally, the analysis of Sgp3 congenic mice strongly

suggests the presence of at least two distinct genetic factors in the Sgp3 interval, one of

which controlled the basal-level expression of xenotropic, PT and mPT gp70 and the

other which controlled the up-regulated production of xenotropic and mPT gp70 during

acute phase responses. Our results uncovered an additional pathogenic role of TLR7 and

TLR9 in murine lupus nephritis by promoting the expression of nephritogenic gp70

autoantigen. Furthermore, they revealed the involvement of multiple regulatory genes

for the expression of gp70 autoantigen under steady-state and inflammatory conditions

in lupus-prone mice.

Key words: Toll-like receptor • Systemic lupus erythematosus • Endogenous retrovirus •

Acute phase protein

- 97 -

1. Introduction

Relatively large amounts of the envelope glycoprotein, gp70, of endogenous

retroviruses circulate free from any association with viral particles in the blood of

virtually all strains of mice [1-3]. The demonstration of retroviral gp70 in immune

deposits within diseased glomeruli of mice with systemic lupus erythematosus (SLE)

indicates the pathogenic role of gp70-anti-gp70 immune complexes (gp70 IC) in the

development of lupus nephritis [4, 5]. Notably, only lupus-prone (NZB x NZW)F1,

MRL and BXSB mice spontaneously develop autoantibodies against serum gp70,

detected as gp70 IC, and appearance and amounts of gp70 IC closely parallel the course

of disease in each lupus-prone mouse [6-8].

Endogenous retroviruses are classified as ecotropic, xenotropic or polytropic

according to the host range dictated by their respective envelope gp70 proteins [9].

Furthermore, based on differences in their gp70 nucleotide sequences [9, 10], the

polytropic proviruses have been divided into two subgroups, termed polytropic (PT) and

modified PT (mPT). Serological and tryptic peptide mapping analysis showed that the

serum gp70 molecule most closely resembles, but is not identical to the gp70 protein of

xenotropic viruses isolated from NZB mice [3, 11]. Recent analysis of the abundance of

retroviral gp70 RNAs in different strains indicated that PT and mPT proviruses that

encode gp70s closely related to xenotropic gp70 are additional important sources of

serum gp70 [12].

Serum retroviral gp70 is secreted by hepatocytes in the blood circulation [13], and

its expression is controlled by multiple structural and regulatory genes [12, 14]. Genetic

studies identified at least two loci, Sgp3 (serum gp70 production 3) on mid chromosome

13 and Sgp4 on distal chromosome 4, which control basal serum levels of gp70 [8, 15-

20] through the transcriptional regulation of multiple endogenous retroviral proviruses

[12]. Significantly, the expression of serum gp70 corresponds to that of acute phase

proteins, in which it is enhanced by agents, such as LPS, polyriboinosinic-

polyribocytidylic acid or turpentine oil, with kinetics identical to those of acute phase

proteins [13]. However, this response is strain-dependent, in which only mice having

high basal levels of serum gp70 displayed an up-regulated expression of serum gp70 in

response to LPS [13, 14, 21]. Increases in serum levels of gp70 in Sgp3 congenic mice

injected with LPS suggest that the Sgp3 locus contributed to LPS-induced enhanced

production of serum gp70 [12, 18]. However, it has not yet been determined if the basal-

level expression of serum gp70 and the LPS-mediated enhanced production of serum

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gp70 are co-regulated by the same genetic element present in the Sgp3 locus or by

different genes in the interval.

Toll-like receptors (TLRs) are a family of germ-line encoded receptors that

recognize a diverse range of conserved molecular motifs commonly found in microbial

pathogens. Recognition of microbial components by TLR is critical in host responses

against pathogens [22]. Three TLRs act as receptors for nucleic acids in mice: TLR3 for

double-stranded RNA, TLR7 for single-stranded RNA and TLR9 for unmethylated CpG

DNA. Notably, nucleic acids can act as endogenous ligands for TLR7 and TLR9 [23],

which both contribute to the development of autoimmune responses against nuclear

autoantigens as well as serum retroviral gp70 in murine SLE [24-27]. Injection of TLR

agonists, LPS for TLR4 and polyriboinosinic-polyribocytidylic acid for TLR3, induced

high serum levels of gp70 in lupus-prone mice [13]. Similarly, the activation of TLR7

and TLR9 by apoptotic cells accumulated in lupus-prone mice or IgG IC containing

nucleic acids might boost the production of serum gp70 during the course of the disease,

thereby further accelerating the progression of lupus nephritis.

In the present study, we have implicated TLR7 and TLR9 in the production of

serum gp70, and defined the contribution of the Sgp3 and Sgp4 loci to the acute phase

response of serum gp70. Our results demonstrated that the stimulation of TLR7 and

TLR9 enhanced the production of serum gp70, and that the Sgp3 and Sgp4 loci

synergistically contributed to the LPS-induced serum gp70 response. Furthermore, the

analysis of mPT transcripts in Sgp3 congenic mice provided evidence that the gene

involved in the LPS-induced up-regulated transcription of mPT proviruses was distinct

from that controlling the basal-level expression of xenotropic, PT and mPT proviruses.

This strongly suggested the presence of multiple genes in the Sgp3 locus regulating the

expression of different classes of endogenous retroviruses under physiological or

inflammatory condition.

2. Materials and methods

2.1. Mice

NZB and BXSB mice were purchased from the Jackson Laboratory (Bar Harbor,

ME). B6.NZB-Sgp3 (B6.Sgp3) and B6.NZB-Sgp4 (B6.Sgp4) congenic mice were

generated by backcross procedures, as described previously [12, 16]. B6 mice double

congenic for both Sgp3 and Sgp4 loci (B6.Sgp3/4) were generated by intercrossing F1

progeny from B6.Sgp3 and B6.Sgp4 mice, using marker-assisted selection for the NZB-

- 99 -

derived Sgp3 and Sgp4 intervals, as described previously [12]. All studies presented

were carried out in female mice. Animal studies described in the present study have

been approved by the Ethics Committee for Animal Experimentation of the Faculty of

Medicine, University of Geneva.

2.2. Injection of TLR agonists or cytokines

TLR4 agonist, LPS from Escherchia coli 0111:B4 (Sigma-Aldrich, Saint Louis,

MO), TLR7 agonist, 1V136 [28] (TLR7 Ligand II, Calbiochem, EMD Chemicals Inc.,

Darmstadt, Germany), TLR9 agonist, CpG-containing oligonucleotides (Type B CpG

1018; a kind gift of Dr. Eyal Raz, UCSD, San Diego, CA) or different cytokines were

diluted to the desired concentrations with sterile PBS and i.p injected into 2-3 mo-old

NZB, BXSB and B6 female mice. Recombinant human IL-1β, IL-6, TNFα and IFNβ

were kindly provided by Dr Jean-Michel Dayer, University of Geneva, Switzerland.

Livers and sera were collected 9 h after LPS injection.

2.3. Serological assays

Serum levels of retroviral gp70 from 2-3 mo-old female mice were determined by

ELISA, as described previously [29]. Results are expressed as µg/ml of gp70 by

referring to a standard curve obtained with a serum pool from NZB mice containing a

known concentration of gp70.

2.4. Quantitative real-time RT-PCR

RNA from livers was purified with TRIzol reagent (Invitrogen AG, Basel,

Switzerland) and treated with DNase I (Amersham Biosciences Corp., Piscataway, NJ).

The abundance of xenotropic, PT and mPT env RNAs (genomic RNA and mRNA) was

quantified by real-time RT-PCR, as described previously [30]. For the amplification of

xenotropic gp70 cDNA, Xeno1098F forward and Xeno1298R reverse primers were

used. For PT and mPT gp70 cDNA, a common PT/mPT730F forward primer, and

PT892R and mPT880R reverse primers specific for PT and mPT viruses, respectively,

were used. The two different deletion mutants (D1 and D2) env cDNAs were amplified

using the following primers: mPT1115F forward and D1-R reverse primers for the D1

mutant; mPT1317F forward and D2-R reverse primers for the D2 mutant, as described

[30]. Haptoglobin and arginine/serine-rich coiled-coil 1 (Rsrc1) mRNA levels were

- 100 -

quantified using the following primers: haptoglobin; forward primer (5’-

TGAACACAGTCGCTGGAGAG-3’) and reverse primer (5’-

GCTGCCTTTGGCATCCATAG-3’), and Rsrc1; forward primer (5’-

GCCACCCTGGTAGAACAAGTC-3’) and reverse primer (5’-

GCACTTCACTTGGTTCTACTGC-3’). PCR was performed using the iCycler iQ Real-

Time PCR Detection System (Bio-Rad, Philadelphia, PA) and iQ SYBR green

Supermix (Bio-Rad). Results were quantified using a standard curve generated with

serial dilutions of a reference cDNA preparation from NZB or BXSB liver and

normalized using TATA-binding protein (TBP) mRNA.

2.5. RT-PCR

The gp70-p15E junction region of mPT env cDNAs was amplified with mPT-

specific mPT858F gp70 forward primers and a common p15E-R reverse primer, as

described [30]. For the amplification of wild-type (WT) and two deletion mutants (D1

and D2) of mPT env RNAs, an mPT858F forward primer and reverse primers specific

for WT (mPT1447R) or deletion mutants (D1-R and D2-R) were used. Using these sets

of primers, the abundance of three different species of mPT env RNAs was semi-

quantified with 4-fold serially diluted cDNA templates. As a control, the abundance of

GAPDH cDNA was semi-quantified. PCR products were visualized by staining with

ethidium bromide after electrophoresis on 3.5% polyacrylamide or 2% agarose gels.

2.6. Statistical analysis

Unpaired comparison for hepatic levels of different RNAs, and paired comparison

for serum levels of gp70 before and after injection of LPS were analyzed by Student's t

test. Probability values <5% were considered significant.

3. Results

3.1. Increases in levels of serum gp70 and hepatic gp70 RNAs in NZB mice after

injection of 1V136, CpG or LPS

Injection of the TLR4 ligand, LPS, into lupus-prone NZB, NZW, BXSB and MRL

mice promotes the production of serum gp70 [12-14]. In view of the critical

involvement of TLR7 and TLR9 in the pathogenesis of SLE [24-27], we investigated

whether the stimulation of TLR7 and TLR9 could enhance the production of serum

gp70 in NZB mice. 9 h after injection of 50 µg of TLR7 agonist, 1V136, or TLR9

agonist, CpG, NZB mice displayed significant 3.2- and 4.6-fold increases of serum

- 101 -

concentrations of gp70, respectively (P < 0.001), as compared with preinjection levels

(Table 1). Time course studies confirmed that kinetics of the serum gp70 response

elicited by 1V136 or CpG were comparable to that induced with LPS (data not shown).

We quantified the changes in abundance of xenotropic, PT and mPT retroviral gp70

RNAs in livers after injection of 1V136 or CpG, in comparison with those after

injection of LPS. Injection of 1V136 or CpG in NZB mice led to substantial up-

regulation of xenotropic gp70 RNA levels (P < 0.0001), which were similar to those

observed in LPS-injected NZB mice (Table 2). As noted previously [12], NZB mice

treated with LPS also displayed modest increases in mPT gp70 RNA (P < 0.0005),

while no statistically significant elevation of mPT gp70 RNA was observed in mice

administered with 1V136 or CpG. In contrast, levels of PT gp70 RNA was not up-

regulated in NZB mice injected with 1V136, CpG or LPS. Notably, the injection of

either TLR agonist induced 5-6-fold increases in hepatic haptoglobin mRNA levels (P <

0.0001). It should be also stressed that the injection of 1V136 or CpG failed to enhance

serum levels of gp70 in B6 mice having low serum levels of gp70 (data not shown), as

in the case of B6 mice injected with LPS [13, 14].

3.2. Increases in levels of serum gp70 and hepatic gp70 RNAs in NZB mice after

injection of IL-1β, IL-6 or TNFα

The effect of IL-1, IL-6 and TNF on the production of serum gp70 in NZB mice

was next assessed, as these cytokines are well known inducers for acute phase proteins

in the liver. Consistent with the idea that serum gp70 behaves as an acute phase protein,

NZB mice displayed significant 2-3-fold increases in serum concentrations of gp70 9h

after injection of recombinant human IL-1β (1 µg), IL-6 (5 µg) or TNFα (5 µg) (IL-1β,

P < 0.005; IL-6, P < 0.005; TNFα, P < 0.001; Table 3). These increases paralleled 3.3-

to 4.5-fold up-regulation of xenotropic gp70 RNA levels (P < 0.0005 for all three

cytokines) in livers of NZB mice (Table 4). In contrast, no statistically significant

increases in the abundance of PT and mPT gp70 RNAs were observed after injection of

the cytokines. As expected, the injection of these three inflammatory cytokines induced

substantial increases in hepatic haptoglobin mRNA levels (IL-1β, P < 0.05; IL-6, P <

0.05; TNFα, P < 0.01). Notably, the extent of elevation in haptoglobin mRNA induced

by each cytokine was essentially identical to that observed for xenotropic gp70 RNA.

The stimulation of plasmacytoid dendritic cells by TLR7 and TLR9 is known to

trigger the secretion of relatively large amounts of type I interferon [31]. Therefore, we

assessed the possible effect of IFNβ on the stimulation of serum gp70 production.

- 102 -

Injection of recombinant human IFNβ (5 µg) failed to increase the synthesis of serum

gp70 and the abundance of hepatic retroviral gp70 RNAs and haptoglobin mRNA in

NZB mice (Tables 3 and 4). This finding indicated that the increase in the synthesis of

serum gp70 corresponds to an acute phase response rather than a secondary response to

IFNβ.

3.3. Synergistic effect of the Sgp3 and Sgp4 loci to LPS-induced up-regulated

expression of serum gp70 in B6 mice

Earlier studies indicated that LPS-induced serum gp70 responses were strain-

dependent [14]. Studies with B6.Sgp3 and B6.Sgp4 congenic mice have shown only

modest (~2-fold) increases of serum gp70 in response to LPS [12, 18]. As basal serum

levels of gp70 are regulated by multiple genes, it may be possible that stronger serum

gp70 responses upon the injection of LPS might be elicited through the combination of

Sgp3 and Sgp4 acting synergistically. To address this question, we generated B6.Sgp3/4

double congenic mice and assessed their ability to produce serum gp70 in response to

LPS. When compared to B6 and B6 mice congenic for only the Sgp3 or Sgp4 locus,

untreated B6.Sgp3/4 mice had substantially higher basal levels of serum gp70. Basal

levels observed for the B6.Sgp3/4 mice were 14.9-, 3.6- and 2.0-fold higher than those

observed with B6 (P < 0.0001), B6.Sgp4 (P < 0.0005) and B6.Sgp3 (P < 0.01) mice,

respectively (Table 5). Injection of LPS led to a greater increase in serum concentrations

of gp70 in B6.Sgp3/4 congenic mice than increases observed with B6 mice congenic for

either Sgp3 or Sgp4 alone (B6.Sgp3, P < 0.005: B6.Sgp4, P < 0.0005; Table 5). Notably,

mean increases in serum gp70 concentrations after injection of LPS in double congenic

mice (96.1 µg/ml) were 3.2 times more than added values obtained by these two single

congenic mice (29.7 µg/ml). These data indicated that Sgp3 and Sgp4 synergistically

acted to promote the production of serum gp70 in response to LPS.

3.4. Differential effect of TLR agonists or inflammatory cytokines on the abundance of

three different species of mPT env RNAs in NZB, BXSB and B6.Sgp3 mice

We have recently shown the presence in B6 mice of not only intact WT mPT env

transcripts but also two defective (D1 and D2) mPT env transcripts which carry a

deletion in the env sequence [30]. Furthermore, all four lupus-prone mice (NZB, NZW,

BXSB and MRL) predominantly expressed WT mPT env transcripts rather than the

defective env transcripts. Since the Sgp3, but not Sgp4 locus derived from lupus-prone

mice was responsible for the selective expression of the WT mPT env RNA, we

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examined LPS-induced increases in mPT env RNA levels to determine if the up-

regulation was restricted only to proviruses carrying WT mPT env genes. The

abundance of the two defective (D1 and D2) and the WT mPT env RNAs were semi-

quantified by RT-PCR specific for the three different mPT env sequences in BXSB as

well as NZB mice, as only BXSB mice carry the D2 mutant provirus and express D2

mPT env transcripts among four different lupus-prone mice [30]. The use of

semi-quantified RT-PCR was necessary since we are unable to design real-time RT-

PCR primers specific for WT mPT env cDNA due to the remarkable homology in the

gp70-p15E junction region between mPT and PT env genes [30]. The analysis with

serially diluted cDNA samples from NZB and BXSB mice injected with LPS showed

moderate and robust increases in WT and D1 mPT env RNAs, respectively, as

compared to PBS-injected mice (Fig. 1). In contrast, no appreciable increases in D2

mPT env RNA were observed in BXSB mice.

Semi-quantitative RT-PCR analysis suggested that injection of LPS in NZB and

BXSB mice more efficiently induced transcription of the D1 mPT env gene than those

of WT mPT env genes. Indeed, real-time RT-PCR analysis confirmed 12.0- and 15.4-

fold increases in D1 mPT env RNA levels in NZB and BXSB mice, respectively (Table

6). These results contrasted to only 3-fold increases in the abundance of total mPT env

RNAs, which include WT, D1 and D2 transcripts, and no changes in the levels of D2

mPT env RNA. Notably, similar results were obtained in LPS-injected B6.Sgp3

congenic mice, while levels of the three mPT env transcripts were not elevated in B6

mice in response to LPS (Table 6). These results indicated that LPS induction

influenced the expression of each of these transcripts in distinct manners with strong,

modest and no up-regulated expression of D1, WT and D2 mPT proviruses, respectively,

in mice bearing the Sgp3 locus derived from lupus-prone mice. This contrasted to the

selectively increased expression of WT mPT env gene by Sgp3 observed under steady-

state condition [30]. It was further confirmed that injection of 1V136, CpG and

inflammatory cytokines (IL-1β, IL-6 and TNFα) resulted in 3.5- to 10.0-fold elevation

of D1 env RNA levels in livers of NZB mice (Fig. 2).

The markedly enhanced levels of D1 mPT env RNA in NZB, BXSB and B6.Sgp3

mice during acute phase responses can be due to a unique integration site of this

particular provirus. BLAST search analysis revealed that the D1 mPT mutant provirus is

integrated in the right transcription direction within the 4th intron of the Rsrc1

(arginine/serine-rich coiled 1) gene in B6 chromosome 3. However, the levels of Rsrc1

mRNA were comparable in livers of untreated B6 and B6.Sgp3 mice (means of 3 mice

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± SD: B6, 0.77 ± 0.13; B6.Sgp3, 0.85 ± 0.23), and the injection of LPS failed to

enhance, but rather down-regulated levels of Rsrc1 mRNA in these mice (means of 3

mice ± SD: B6, 0.27 ± 0.06; B6.Sgp3, 0.32 ± 0.04). These results thus argued against

the possibility that the enhanced expression of the D1 mPT env gene in LPS-injected

B6.Sgp3 mice was a result of co-regulated transcription of the LPS-responsive host gene,

in which the D1 provirus is integrated.

4. Discussion

Endogenous retroviral gp70 has been shown as one of the major nephritogenic

autoantigens in murine SLE. Its expression is under a polygenic control and regulated

by inflammatory stimuli, as it behaves as an acute phase protein. In the present study,

we have shown that TLR7 and TLR9 were also involved in the enhanced production of

serum gp70 during acute phase responses. In addition, our results demonstrated that the

Sgp3 and Sgp4 loci play critical roles in up-regulated expression of serum gp70 under

inflammatory conditions as well as in its steady-state expression. Moreover, analysis of

B6.Sgp3 congenic mice revealed that the Sgp3 locus contains at least two distinct

genetic factors; one of which controls the basal-level expression of serum gp70 and the

other the up-regulated production of serum gp70 during systemic inflammation.

Induction of high serum levels of gp70 in NZB mice injected with TLR7 and

TLR9 agonists (1V136 for TLR7 and CpG for TLR9) is likely to be mediated by

cellular and molecular mechanisms responsible for the induction of acute phase

responses in livers, based on the following findings. First, levels of serum gp70 and of

hepatic haptoglobin mRNA in NZB mice injected with 1V136 or CpG were similarly

up-regulated, as in the case of NZB mice injected with IL-1β, IL-6 and TNFα, known to

be a potent inducer of acute phase responses. Second, kinetics of serum gp70 responses

induced by 1V136 or CpG was essentially identical to that induced by LPS or IL-1 β.

Notably, activation of TLR7 and TLR9 in monocytes/macrophages induced the

secretion of IL-6 and TNFα [32, 33], while no serum gp70 or haptoglobin responses

were induced by the type I interferon, which is a unique cytokine abundantly secreted

by plasmacytoid dendritic cells upon stimulation of TLR7 or TLR9. Furthermore, the

pattern of up-regulated expression of three different classes of endogenous retroviral

gp70 RNAs in NZB mice injected with 1V136 or CpG was essentially identical to that

observed after injection of inflammatory cytokines. As discussed previously [12], the

lack of up-regulated expression of PT proviruses can be in part related to the absence of

an IL-6-responsive element (IL6-RE), common to genes encoding acute phase protein

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[34], in the U3 regulatory region of the long terminal repeat (LTR). In addition, higher

responses of xenotropic gp70 RNA can be explained by the presence of NF-kB-binding

motif in the U3 region of xenotropic viruses [35], as NF-kB is involved in one of the

several distinct signaling pathways leading to the synthesis of acute phase proteins [36].

It should be stressed that as in the case of LPS-induced gp70 responses, 1V136-

and CpG-induced gp70 responses were also strain-dependent, as B6 mice having low

serum levels of gp70 failed to display any increases in serum gp70 after injection of

either 1V136 or CpG. Because of only modest gp70 responses in B6.Sgp3 and B6.Sgp4

single congenic mice injected with LPS [12], it was considered that the contribution of

the Sgp loci to LPS-induced serum gp70 responses was relatively limited. However,

substantial, synergistic increases in serum levels of gp70 in B6.Sgp3/4 double congenic

mice injected with LPS indicated that the Sgp loci do play a major role in the up-

regulated expression of serum gp70 during acute phase responses. Notably, serum levels

of gp70 in LPS-injected B6.Sgp3/4 mice were comparable to those observed with

LPS-injected BXSB mice and even higher than those of LPS-injected MRL mice [12,

14]. However, increases in serum gp70 were still less than those in LPS-injected NZB

and NZW mice.

Basal levels of serum gp70 in B6.Sgp3/4 congenic mice were comparable to those

of BXSB and MRL mice, but still lower than those in NZB and NZW mice. This could

be accounted for if NZB and NZW mice carry an additional Sgp locus. Indeed, the

genetic analysis involving BALB/c mice revealed a strong linkage of serum gp70 levels

with a locus on proximal chromosome 12 of both NZB and NZW mice [20].

Preliminary studies in B6 mice bearing the proximal chromosome 12 interval derived

from NZB mice showed modest, but significant increases in serum gp70, similar to

those observed in BALB/c mice congenic for this putative Sgp locus derived from NZW

mice [20]. The involvement of multiple loci in the up-regulated expression of serum

gp70 during acute phase responses is consistent with the finding that the extent of serum

gp70 responses after injection of LPS was highly variable among different strains of

mice [14, 21].

Strikingly, the analysis of three different species of mPT env RNAs in NZB,

BXSB and B6.Sgp3 mice revealed that inducers of acute phase responses

up-regulated the abundance of the D1 mPT env RNA more strongly than that of WT

mPT env RNA, and that levels of D2 mPT env RNA were not modulated. This

suggested that the expression of only a fraction of mPT proviruses was selectively

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enhanced during the acute phase response. BLAST search analysis revealed the

presence in the B6 genome of 11 mPT proviruses carrying the WT env gene, in addition

to the D1 and D2 mPT proviruses on chromosome 3 and 5, respectively. Notably, the

presence of the IL6-RE is not a determinant of the differential expression we have

observed, since this motif is conserved in the U3 sequence of all mPT proviruses,

including the D2 mPT provirus, present in B6 mice. However, we noted considerable

microheterogeneity in their U3 sequences. Notably, the D1 mutant carries two unique

mutations in the U3 regulatory region [30]: a substitution of G (guanine) with A

(adenine) in an SV40 core-like motif (GTGATCA instead of GTGGTCA) and an

insertion of T (thymine) in the UCR (upstream conserved region), which negatively

regulates the expression of endogenous retroviruses [37]. It remains to be determined

whether these two mutations contribute to the up-regulated transcription of the D1 mPT

provirus by the presence of inflammatory stimuli. Alternatively, the site of integration

of mPT proviruses may play a critical role in these responses. This possibility seems

unlikely for the D1 mPT provirus, since we observed that LPS failed to enhance the

expression of the Rsrc1 gene which contains the D1 mPT provirus in the correct

orientation. Another plausible explanation is that the enhancer element(s) of the U3

region, implicated in the increased expression in response to inflammatory stimuli, may

be selectively methylated in certain proviruses, as the expression of retroviral sequences

is strongly affected by the state of DNA methylation [38-40].

In contrast to the enhanced expression of the D1 mPT env RNA in

LPS-injected B6.Sgp3 congenic mice, the basal-level expression of this transcript was

the same as in WT B6 mice [30]. Since D1 mPT env RNA levels were enhanced in

B6.Sgp3, but not in WT B6 mice following injection of LPS, the Sgp3 locus by itself is

responsible for LPS-induced increases in this transcript. Thus, the simplest explanation

would be that the Sgp3 locus harbors at least two distinct genetic elements, which

control respectively the basal-level transcription of xenotropic, PT and mPT retroviral

sequences and the up-regulated expression of xenotropic and mPT retroviral sequences

during acute phase responses. In view of the remarkable differences in the U3 region of

LTR among xenotropic, PT and mPT retroviruses [35], the presence of several genetic

factors which differentially control the expression of individual classes of retroviruses

under steady-state or inflammatory condition might not be surprising. In this regard, it is

noteworthy that the Gv1 locus derived from the 129 strain was reported to regulate the

transcription of PT, but not mPT proviruses [41], and that Gv1 controls the expression

of gp70 in a semi-dominant fashion [42]. Consistent with these findings, our on-going

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studies on Sgp3 homozygous and heterozygous mice revealed that the basal-level

expression of xenotropic, PT and mPT viral sequences was regulated in a dominant,

semi-dominant and recessive manner, respectively. All these data underline the

complexity for the genetic control of the expression of different classes of endogenous

retroviruses in mice.

Our on-going studies have narrowed down the Sgp3 locus within a ~9.0 Mb NZB

interval flanked by markers D13Mit283 (63.4 Mb from the centromere) and D13Mit26

(72.4 Mb). Notably, this region contains approximately 20 Zfp genes, which encode

KRAB (Krüppel-associated box) zinc-finger proteins, but the target genes regulated by

most of these Zfp genes are still unknown [43]. The KRAB transcription repressor

domain has been shown to suppress lentivirus proviral transcription by inducing

heterochromatization in the lentiviral integration sites [44]. More recently, it has also

been shown that ZFP809 recognizes integrated retroviral DNAs and silences them

through the recruitment of TRIM28 in embryonic stem cells [45]. Thus, it may be

possible that several of the Zfp genes present in the Sgp3 locus are involved in the

regulation of the expression of endogenous retroviruses under physiological and

inflammatory conditions in mice.

The role of TLR7 and TLR9 for the development of autoimmune responses

against nuclear autoantigens and retroviral gp70, both of which are implicated in murine

lupus nephritis, has been established [24-27]. In addition, our present results

demonstrated that they are also involved in the enhanced production of nephritogenic

gp70 antigens during the course of SLE, possibly through the activation of

monocytes/macrophages in response to DNA- or RNA-containing IgG IC. Thus, TLR7

and TLR9 display dual effects on the development of SLE. On one hand, they promote

autoimmune responses against nuclear and retroviral antigens through the activation of

autoreactive B cells as well as dendritic cells, and on the other hand, they enhance the

production of serum gp70 in the presence of the Sgp loci, thereby providing an

additional source for antigenic stimulation and for nephritogenic IC formation.

Increased levels of serum gp70 during the course of SLE, in association with increases

in serum levels of gp70 IC and accelerated development of lupus nephritis, have

previously been demonstrated in lupus-prone BXSB mice [46]. The contribution of

TLR7 to the production of anti-gp70 antibodies also suggests the implication of

endogenous retroviruses in murine SLE. The eventual identification of the Sgp genes

will help elucidate a molecular basis responsible for the expression of endogenous

retroviruses implicated in murine SLE, and will enable us to address the relevance of

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their human counterparts, thus providing a clue for the potential role of endogenous

retroviruses in human SLE.

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Acknowledgments

We thank Mr Guy Brighouse and Ms Montserrat Alvarez for their excellent technical

assistance. This work was supported by a grant from the Swiss National Foundation for

Scientific Research. L.H.E. was supported by the intramural research program of the

National Institute of Allergy and Infectious Diseases, National Institutes of Health.

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Table 1. Serum levels of gp70 in NZB mice injected with 1V13, CpG or LPS

Serum gp70

Stimuli Before After

1V136 63.2 ± 3.7 199.4 ± 21.4 (3.2)

CpG 61.1 ± 11.4 270.0 ± 20.2 (4.6)

LPS 62.4 ± 11.7 449.2 ± 122.4 (7.3)

PBS 62.2 ± 9.0 63.5 ± 11.1 (1.0)

_________________________________________________________________

Serum levels of gp70 (µg/ml; mean ± SD of 4 mice) in 2-3 mo-old NZB female mice

before and 9 h after an i.p. injection of 1V136 (50 µg), CpG (50 µg), LPS (25 µg) or

PBS. Fold increases in serum levels of gp70 after injection of CpG, 1V136, LPS or PBS

are indicated in parentheses.

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Table 2. Fold increases of gp70 RNAs and haptoglobin mRNA in livers of NZB mice

injected with 1V13, CpG or LPS relative to those in PBS-injected NZB mice

Stimuli Xeno gp70 PT gp70 mPT gp70 Haptoglobin

1V136 3.53 ± 0.49 0.99 ± 0.20 1.31 ± 0.46 5.24 ± 0.79

CpG 4.66 ± 0.36 1.25 ± 0.63 1.47 ± 0.43 6.49 ± 0.55

LPS 5.49 ± 0.67 1.23 ± 0.12 2.95 ± 0.34 6.09 ± 0.71

PBS 1.00 ± 0.04 1.00 ± 0.14 1.00 ± 0.14 1.00 ± 0.19

______________________________________________________________________

Levels of gp70 RNAs and haptoglobin mRNA (mean ± SD of 4 mice) in livers of 2-3

mo-old NZB female mice 9 h after an i.p. injection of 1V136 (50 µg), CpG (50 µg),

LPS (25 µg) or PBS were quantified relative to a standard curve generated with serial

dilutions of a reference cDNA preparation and normalized using TBP mRNA. Results

are expressed as fold increases of each transcript relative to PBS-injected NZB mice.

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Table 3. Serum levels of gp70 in NZB mice injected with IL-1β, IL-6, TNFα or IFNβ

Serum gp70


IL-1β 55.6 ± 7.4 182.7 ± 33.2 (3.3)

IL-6 64.2 ± 10.6 111.5 ± 19.9 (1.7)

TNFα 62.1 ± 8.7 211.8 ± 28.9 (3.4)

IFNβ 58.3 ± 6.9 60.1 ± 9.9 (1.0)

PBS 60.9 ± 6.3 63.3 ± 3.5 (1.1)

_________________________________________________________________

Serum levels of gp70 (µg/ml; mean ± SD of 4 mice) in 2-3 mo-old NZB female mice

before and 9 h after an i.p. injection of IL-1β (1 µg), IL-6 (5 µg), TNFα (5 µg), IFNβ

(5 µg) or PBS. Fold increases in serum levels of gp70 after injection of cytokines or

PBS are indicated in parentheses.

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Table 4. Fold increases of gp70 RNAs and haptoglobin mRNA in livers of NZB mice

injected with IL-1β, IL-6, TNFα or IFNβ relative to those in PBS-injected NZB mice

Stimuli Xeno gp70 PT gp70 mPT gp70 Haptoglobin

IL-1β 3.93 ± 0.58 1.12 ± 0.54 1.47 ± 0.59 4.45 ± 1.29

IL-6 3.28 ± 0.56 1.05 ± 0.26 1.26 ± 0.30 3.38 ± 1.14

TNFα 4.50 ± 1.22 1.39 ± 0.26 1.94 ± 0.70 4.51 ± 1.33

IFNβ 1.03 ± 0.31 0.85 ± 0.22 0.81 ± 0.18 0.98 ± 0.28

PBS 1.00 ± 0.19 1.00 ± 0.11 1.00 ± 0.18 1.00 ± 0.34

______________________________________________________________________

Levels of gp70 RNAs and haptoglobin mRNA (mean ± SD of 4 mice) in livers of 2-3

mo-old NZB female mice 9 h after an i.p. injection of IL-1β (1 µg), IL-6 (5 µg), TNFα

(5 µg), IFNβ (5 µg) or PBS were quantified relative to a standard curve generated with

serial dilutions of a reference cDNA preparation and normalized using TBP mRNA.

Results are expressed as fold increases of each transcript relative to PBS-injected NZB

mice.

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Table 5. Serum levels of gp70 in B6 Sgp congenic mice injected with LPS

Serum gp70


B6 2.4 ± 0.5 2.8 ± 0.8 (1.2)

B6.Sgp4 10.0 ± 1.0 17.8 ± 2.5 (1.8)

B6.Sgp3 18.2 ± 5.7 40.1 ± 10.7 (2.2)

B6.Sgp3/4 35.8 ± 8.7 131.9 ± 19.0 (3.8)

_________________________________________________________________

Serum levels of gp70 (µg/ml; mean ± SD of 5 mice) in 2-3 mo-old B6 or B6 Sgp

congenic female mice before and 9 h after an i.p. injection of 25 µg of LPS.

Fold increases in serum levels of gp70 after injection of LPS are indicated in

parentheses.

- 120 -

Table 6. Fold increases of D1 and D2 mPT env RNAs in livers of NZB, BXSB,

B6.Sgp3 and B6 mice injected with LPS relative to those in PBS-injected mice

Mice Stimuli Totala P

b D1

a P

b D2

a

NZB LPS 2.95 ± 0.34 *** 12.03 ± 2.29 *** NDb

NZB PBS 1.00 ± 0.14 1.00 ± 0.14 ND

BXSB LPS 3.34 ± 0.04 **** 15.35 ± 3.96 ** 1.08 ± 0.35

BXSB PBS 1.00 ± 0.15 1.00 ± 0.15 1.00 ± 0.08

B6.Sgp3 LPS 2.36 ± 0.33 * 4.79 ± 1.00 ** 1.10 ± 0.15

B6.Sgp3 PBS 1.00 ± 0.27 1.00 ± 0.21 1.00 ± 0.12

B6 LPS 1.18 ± 0.16 1.70 ± 0.38 0.94 ± 0.22

B6 PBS 1.00 ± 0.23 1.00 ± 0.17 1.00 ± 0.10

__________________________________________________________________________ a Levels of total (WT, D1 and D2), D1 and D2 mPT env RNAs (mean ± SD of 4 mice)

in livers of 2-3 mo-old NZB, BXSB, B6.Sgp3 or B6 female mice 9 h after an i.p.

injection of different stimulators or PBS were quantified relative to a standard curve

generated with serial dilutions of a reference cDNA preparation and normalized using

TBP mRNA. Results are expressed as fold increases of each transcript relative to PBS-

injected NZB, BXSB, B6.Sgp3 or B6 mice. b P value of comparison between LPS- and PBS-injected mice. * P < 0.01;

** P < 0.005; *** P < 0.0005; **** P < 0.0001 c Not detectable.

- 121 -

Figure legends

Figure 1. Semi-quantitative RT-PCR analysis for WT, D1 and D2 mPT env RNAs in

NZB and BXSB mice.

Semi-quantitative RT-PCR analysis for WT, D1 and D2 mPT env RNAs with reverse

primers specific for the three different mPT env genes (mPT1447R, D1-R and D2-R)

and a common forward mPT-specific primer (mPT858F) was carried out with 4-fold

serially diluted cDNAs from 2-3 mo-old female mice. As a control, the abundance of

GAPDH mRNA was assessed in parallel. For the analysis of WT mPT env transcripts in

NZB and BXSB mice, the first dilution of cDNA was 1:40, and four serial dilutions of

cDNAs were examined, while for D1/D2 mPT env RNAs and GAPDH mRNA the first

dilution was 1:10. Representative results of three individual mice analyzed are shown.

Figure 2. Quantitative real-time RT-PCR analysis of D1 mPT env RNA in livers of

NZB mice injected with 1V136, CpG, IL-1β, IL-6, TNFα, IFNβ or PBS.

Levels of D1 mPT env RNA (mean ± SEM of 4 mice) in livers of 2-3 mo-old NZB

female mice 9 h after an i.p. injection of different stimulators or PBS were quantified

relative to a standard curve generated with serial dilutions of a reference cDNA

preparation and normalized using TBP mRNA. Results are expressed as fold increases

of D1 mPT env RNA relative to PBS-injected NZB mice. P value of comparison with

PBS-injected mice. * P < 0.05; ** P < 0.01; *** P < 0.0001

- 122 -

WT

D1

D2

GAPDH

BXSB BXSB + LPS

WT

D1

GAPDH

NZB NZB + LPS

Figure 1

WT

D1

D2

GAPDH

BXSB BXSB + LPS

WT

D1

GAPDH

NZB NZB + LPS

WT

D1

D2

GAPDH

BXSB BXSB + LPS

WT

D1

GAPDH

NZB NZB + LPS

Figure 1

- 123 -

Figure 2

0

5

10

15

LPS CpG1V136 IL-1ββββ IL-6 TNFαααα IFNββββ PBS

Fol

d ch

ange

*** ***

***

**

***

Figure 2

0

5

10

15


Fol

d ch

ange

*** ***

***

**

***

0

5

10

15


Fol

d ch

ange

*** ***

***

**

***

- 124 -

Supporting Information

Figure S1. Levels of serum gp70 and hepatic retroviral gp70 RNAs in B6.Sgp3

homozygous and heterozygous mice.

(A) Serum levels of gp70 in 2-3 mo-old Sgp3 homozygous (NN), Sgp3 heterozygous

(NB) and WT B6 (BB) mice (µg/ml; mean ± SEM of 7-10 mice). Note that

heterozygous mice had intermediate levels of serum gp70 between homozygous and

WT mice. P values of comparison between homozygous and heterozygous mice and

between heterozygous and WT mice: P< 0.0001.

(B) Levels of each gp70 RNA in livers of 2-3 mo-old Sgp3 homozygous (NN), Sgp3

heterozygous (NB) and WT B6 (BB) mice (mean ± SEM of 4 female mice) were

quantified relative to a standard curve generated with serial dilutions of a reference

cDNA preparation and normalized using TBP mRNA. Results are expressed as fold

increases of each transcript relative to B6 mice. P values of comparison for xenotropic

gp70 RNA between homozygous and WT mice and between heterozygous and WT

mice: P <0.001 and P < 0.005, respectively. P values of comparison for PT gp70 RNA

between homozygous and heterozygous mice and between heterozygous and WT mice:

P <0.0001. P values of comparison for mPT gp70 RNA between homozygous and

heterozygous mice and between homozygous and WT mice: P <0.005.

(C) The presence of three different species of mPT env RNAs in livers of 2-3

Sgp3 homozygous (NN), Sgp3 heterozygous (NB) and WT B6 (BB) mice was

determined by RT-PCR with mPT specific gp70 forward and p15E-R reverse primers.

Representative results of three individual animals are shown. As a control (Ctl ), a

mixture of three different plasmids containing WT, D1 and D2 clones obtained from B6

mice was included. Note the predominant expression of WT env transcripts in Sgp3

homozygous (NN) mice, as compared with heterozygous (NB) and WT B6 (BB) mice.

- 125 -

Figure S1

Serum gp70

NN NB BB0

10

20

30

gp70

(µµ µµg

/ml)

A

C

WT

D2D1

Ctl NN NB BB

Xeno RNA

NN NB BB0.0

2.5

5.0

7.5

PT RNA

NN NB BB0.0

2.5

5.0

7.5

mPT RNA

NN NB BB0

10

20

B

Fol

d ch

ange

Figure S1

Serum gp70

NN NB BB0

10

20

30

gp70

(µµ µµg

/ml)

A

C

WT

D2D1

Ctl NN NB BBC

WT

D2D1

Ctl NN NB BB

Xeno RNA

NN NB BB0.0

2.5

5.0

7.5

PT RNA

NN NB BB0.0

2.5

5.0

7.5

mPT RNA

NN NB BB0

10

20

B

Fol

d ch

ange

- 126 -

III.4. The Sgp3 Locus Derived from the 129 Strain is Responsible for Enhanced


Lucie Baudino, John R. Pehrson and Shozo Izui

Submitted for publication (to the Journal of Virology)

- 127 -

The Sgp3 Locus Derived from the 129 Strain is Responsible for Enhanced


Lucie Baudino,1 John R. Pehrson,

2 and Shozo Izui

1*

Department of Pathology and Immunology, University of Geneva, Geneva,

Switzerland,1 and Department of Animal Biology, School of Veterinary Medicine,

University of Pennsylvania, Philadelphia, Pennsylvania 191042

Running title: Regulation of Endogenous Retroviral Expression

* Corresponding author. Mailing address: Department of Pathology and Immunology,

Centre Médical Universitaire, 1211 Geneva 4, Switzerland.

Phone: (22) 379-5741. Fax: (22) 379-5746. E-mail: [email protected]

- 128 -

Abstract

The endogenous retroviral envelope glycoprotein, gp70, implicated in murine

lupus nephritis, is secreted by hepatocytes, and its expression is largely regulated by the

Sgp3 (serum gp70 production 3) locus on chromosome 13. Because of the localization

of the macroH2A1 gene encoding macroH2A histone variants within the Sgp3 interval

and of an up-regulated transcription of endogenous retroviral sequences in macroH2A1-

deficient C57BL/6 (B6) mice, we investigated the possibility of the macroH2A1 gene as

a candidate for Sgp3. macroH2A1-deficient B6 mice carrying the 129-derived Sgp3

locus, which was co-transferred with the 129 macroH2A1 mutant gene, displayed

increased levels of serum gp70 and hepatic retroviral gp70 RNAs comparable to those

of B6.NZB-Sgp3 congenic mice. In contrast, the abundance of retroviral gp70 RNAs in

macroH2A1-deficient 129 mice was not elevated at all as compared with wild-type 129

mice. Furthermore, Sgp3 subcongenic mice devoid of the NZB-derived macroH2A1

gene displayed the Sgp3 phenotype identical to that of B6.NZB-Sgp3 congenic mice

carrying the NZB-derived macroH2A1 gene, excluding macroH2A1 as the candidate

Sgp3 gene. Moreover, comparable levels of macroH2A1 mRNAs between B6.NZB-

Sgp3 subcongenic and wild-type B6 mice ruled out the contribution of Sgp3 to the

derepression of endogenous retroviruses through the down-regulated expression of

macroH2A1. Collectively, our data indicate that enhanced transcription of endogenous

retroviral sequences observed in macroH2A1-deficient B6 mice was not a result of the

macroH2A1 mutation, but due to the presence of 129-derived Sgp3 locus, and rule out

the implication of macroH2A1 in the expression of endogenous retroviruses.

- 129 -

Endogenous retroviruses are classified as ecotropic, xenotropic or polytropic

according to the host range dictated by their respective envelope gp70 proteins (35).

Furthermore, based on differences in their gp70 nucleotide sequences (35), the

polytropic proviruses have been divided into two subgroups, termed polytropic (PT) and

modified PT (mPT). The retroviral env (envelope) gene encodes a precursor polyprotein,

which is cleaved to produce two subunits; a surface gp70 protein and a membrane-

anchored p15E protein. Retroviral gp70 is expressed, depending on its site of integration

into the mouse genome and on the differentiation state of the cells (21). Indeed, gp70 is

a constituent of the surface of various epithelia, thymocytes and peripheral lymphocytes,

and shares immunological and biochemical properties with the thymocyte

differentiation antigen GIX (8, 21, 25, 34, 39). In addition, gp70 is secreted by

hepatocytes in the blood circulation and behaves as an acute phase protein (13).

Significantly, lupus-prone (NZB x NZW)F1, MRL and BXSB mice spontaneously

develop autoimmune responses against gp70. gp70-anti-gp70 immune complexes are

detected close to the onset of renal disease in the circulation and found as immune

deposits within glomerular lesions of lupus mice (17, 40), underlining the pathogenic

role of gp70-anti-gp70 immune complexes in murine systemic lupus erythematosus

(SLE).

The expression of serum gp70 is controlled by multiple structural and regulatory

genes (3), as its concentrations are highly variable among different strains of mice (1, 17,

40). Genetic studies identified at least two loci, Sgp3 (serum gp70 production 3) on mid

chromosome 13 and Sgp4 on distal chromosome 4, which controlled basal serum levels

of gp70 (15, 18, 20, 29-31, 38) through the regulation of the abundance of multiple

endogenous retroviral gp70 transcripts in trans (3). Serological and tryptic peptide

mapping analysis showed that the serum gp70 molecule resembles the gp70 protein of

xenotropic viruses isolated from NZB mice (9, 16). However, recent analysis of the

abundance of retroviral gp70 RNA in livers from different strains, including Sgp

congenic mice, indicated that PT and mPT proviruses that encode gp70s closely related

to xenotropic gp70 are additional important sources of serum gp70 (3).

It has previously been shown that the Gv1 (Gross virus antigen 1) locus controls

the levels of endogenous retroviral sequences in different tissues, including the liver

(22), and regulates the abundance of thymocyte differentiation GIX gp70 antigen (34),

the expression of which is closely correlated to serum levels of gp70 (14, 26). Since the

Gv1 locus, identified in the 129 strain (33), directly overlaps with the Sgp3 locus (18,

- 130 -

20, 27, 29), Gv1 and Sgp3 are likely to be identical or related genes regulating the

transcription of retroviral sequences, and the GIX+ 129 strain may share the Sgp3 allele

with lupus-prone mice. However, our recent studies revealed that many strains of mice,

including the 129 strain, expressed not only intact mPT env transcripts but also one or

two defective mPT env transcripts, while lupus-prone mice predominantly expressed

abundant levels of the intact mPT env RNA at the near exclusion of the defective

transcripts (41). This specific pattern of expression was regulated by the Sgp3 locus

derived from lupus-prone mice. These results suggest that the 129 strain might carry an

Sgp3 allele different from that in lupus-prone mice, or alternatively, the Sgp3 locus

regulates only a fraction of mPT proviruses, which are absent in the 129 strain.

macroH2A core histone variants have an N-terminal H2A domain and a C-

terminal nonhistone domain, known as the macrodomain (28). macroH2A have three

variants: macroH2A1.1 and macroH2A1.2 are formed by alternative splicing of

macroH2A1, and macroH2A2 is encoded by a separate gene (36). macroH2As are

preferentially associated with transcriptionally repressed or silent chromatin domains,

including the inactivated X chromosome (4, 7), centromeric heterochromatin (12) and

senescence-associated heterochromatic foci (42). The distribution of macroH2As in

chromatin suggests its role in repressing gene expression. More significantly, recent

studies have shown that macroH2A1 nucleosomes were enriched on endogenous

retroviruses, the expression of which was markedly up-regulated in livers from

C57BL/6 (B6) mice bearing the macroH2A1 null mutation (5). Since the macroH2A1

gene is located within the Sgp3 interval, one attractive hypothesis is that macroH2A1 is

the Sgp3 gene. However, we cannot exclude the possibility that the observed effect of

the macroH2A1 null mutation in B6 mice could be due to the presence of the 129-

derived Sgp3 locus co-transferred with the macroH2A1 mutant gene, since macroH2A1-

deficient B6 mice were established by backcrossing the mutated 129 interval to B6 mice.

To define the implication of the macroH2A1 gene in the Sgp3-mediated regulation

of endogenous retroviral expression, we determined the abundance of endogenous

retroviral gp70 RNAs in livers from two different macroH2A1-suficient and -deficient

mice bred into the B6 or 129 background in relation to the 129-derived Sgp3 locus. Our

results demonstrated that enhanced expression of endogenous retroviruses observed in

macroH2A1-deficient B6 mice is modulated by the presence of the 129-derived Sgp3

locus, but not due to the macroH2A1 mutation. Furthermore, the analysis of a B6.NZB-

Sgp3 subcongenic line lacking the NZB-derived macroH2A1 gene excluded

- 131 -

macroH2A1 as the Sgp3 gene.

MATERIALS AND METHODS

Mice

macroH2A1-deficient 129 mice were generated by gene targeting in

129-derived ES cells, and macroH2A1-deficient mice with a B6 background were

established by backcrossing with B6 mice for 10 generations, as described previously

(5). The production of B6.NZB-Sgp3 congenic mice was previously described (3). A

B6.NZB-Sgp3 subcongenic line was generated by backcrossing the NZB chromosome

13 interval onto B6 mice using microsatellite markers polymorphic between NZB and

B6 mice. All studies presented were carried out in female mice. Animal studies

described in the present study have been approved by the Ethical Committee for Animal

Experimentation of the Faculty of Medicine, University of Geneva.

Genotyping analysis

Genotypes were determined by PCR using selected microsatellite markers either

purchased from Research Genetics (Huntsville, AL) or Invitrogen (Carlsbad, CA).

DNAs were extracted from tail biopsies kept at -70 oC before use. PCR amplification

was conducted with RED Taq DNA polymerase (Sigma-Aldrich, Saint Louis, MO)

using a GeneAmp PCR system 9700 thermal cycler (Applied Biosystems, Foster City,

CA), as described (20). The positions of the microsatellite markers with respect to the

centromere were obtained from the Ensembl Genome Browser database

(www.ensembl.org/Mus_musculus/index.html).

Quantitative real-time RT-PCR

RNA from livers was purified with TRIzol reagent (Invitrogen AG, Basel,

Switzerland) and treated with DNase I (Amersham Biosciences Corp., Piscataway, NJ).

The abundance of xenotropic, PT and mPT env RNAs (genomic RNA and mRNA) was

quantified by real-time RT-PCR, as described previously (41). For the amplification of

xenotropic gp70 cDNA, Xeno1098F forward and Xeno1298R reverse primers were

used. For PT and mPT viral gp70 cDNA, a common PT/mPT730F forward primer, and

PT892R and mPT880R reverse primers specific for PT and mPT viruses, respectively,

were used. For the amplification of macroH2A1.1 and macroH2A1.2 cDNAs, a

- 132 -

common forward primer (5’-TCTCCACCAAGAGCCTCTTCC-3’), and macroH2A1.1-

specific (5’-ATGGCCTCCACCTCAAAGC-3’) and macroH2A1.2-specific (5’-

CAGTGTTTGTCGGGTGAACG-3’) reverse primers. PCR was performed using the

iCycler iQ Real-Time PCR Detection System (Bio-Rad, Philadelphia, PA) and iQ

SYBR green Supermix (Bio-Rad). Results were quantified using a standard curve

generated with serial dilutions of a reference cDNA preparation from NZB liver and

normalized using TATA-binding protein (TBP) mRNA.

RT-PCR and genomic PCR

The gp70-p15E junction region of mPT env cDNAs was amplified with mPT-

specific mPT858F gp70 forward primers and a common p15E-R reverse primer, as

described (41). For the amplification of wild-type (WT) and two deletion mutants (D1

and D2) of mPT env RNAs, an mPT858F forward primer and reverse primers specific

for WT (mPT1447R) or deletion mutants (D1-R and D2-R) were used. Using these sets

of primers, the abundance of three different species of mPT env RNAs was semi-

quantified with 5-fold serially diluted cDNA templates. As a control, the abundance of

GAPDH cDNA was semi-quantified in a parallel assay. The presence of ecotropic gp70

RNA was detected by RT-PCR, using a forward primer (5’-

AGGCTGTTCCAGAGATTGTG-3’) and a reverse primer (5’-

TTCTGGACCACCACATGAC-3’). The presence of mPT proviruses carrying the WT

and mutant env genes in the genome was determined by PCR on genomic DNA

prepared from livers, using mPT858F forward primer and mPT1447R, D1-R or D2-R

reverse primers. PCR products were visualized by staining with ethidium bromide after

electrophoresis on 3.5% polyacrlyamide or 2% agarose gels.

Serological assays

Serum levels of gp70 were quantified by ELISA, as described (24). Results are

expressed as µg/ml of gp70 by referring to a standard curve obtained from a serum pool

of NZB mice.

Statistical analysis

Unpaired comparison for levels of retroviral RNAs and macroH2A1 mRNAs was

analyzed by Student's t test. Analysis for serum levels of gp70 was performed with the

Mann-Whitney U-test. Probability values <5% were considered significant.

- 133 -

RESULTS

Presence of the 129-derived Sgp3 locus in macroH2A1-deficient B6 mice

Using selected simple sequence length polymorphism markers of the chromosome

13, we first defined the 129 interval present in B6 mice carrying the 129 macroH2A1

mutant gene, which is located at 56.18-56.24 Mb from the centromere. A ~24 Mb 129-

derived segment flanked by markers D13Mit248 (53.04 Mb) and D13Mit99 (76.94 Mb)

was co-transferred with the macroH2A1 mutant gene into B6 mice (Fig. 1). This 129

segment directly overlaps with the NZB-derived Sgp3 interval encompassing markers

D13Mit139 (51.86 Mb) and D13Mit254 (76.12 Mb) of B6.NZB-Sgp3 congenic mice.

Notably, this region also correspond to the Gv1 locus, which was previously identified

on the 129 chromosome 13 within a ~24 Mb segment between markers D13Mit248 and

D13Mit231 (77.19 Mb) and peaked close to the D13Mit39 marker (62.97 Mb) (27). This

indicates that macroH2A1-deficient B6 mice likely carry the Sgp3 and Gv1 loci derived

from the 129 strain.

Enhanced hepatic expression of xenotropic, PT and mPT gp70 RNAs in

macroH2A1-deficient B6 mice at levels comparable to B6.NZB-Sgp3 congenic mice

Serum levels of gp70 in macroH2A1-deficient B6 mice were comparable to those

in B6.NZB-Sgp3 congenic mice, and approximately 5-fold higher than those in B6 mice

(p<0.001; Table I). We measured the abundance of different retroviral gp70 RNA

transcripts in livers of macroH2A1-deficient B6 mice in comparison with B6.NZB-Sgp3

and WT B6 mice. Quantification of gp70 RNAs in macroH2A1-deficient mice revealed

marked (4- to 30-fold) increases in xenotropic (p<0.005), PT (p<0.0001), and mPT

(p<0.0001) gp70 RNAs, as compared with B6 mice (Table I). Notably, levels of these

three different gp70 RNAs in macroH2A1-deficient B6 mice were comparable to that of

B6.NZB-Sgp3 congenic mice. In contrast, ecotropic gp70 transcripts were hardly

detectable in B6 mice, independently of the presence of the macroH2A1 null mutation

and the NZB-Sgp3 locus (data not shown).

Enhanced hepatic expression of xenotropic, PT and mPT gp70 RNAs in B6.NZB-

Sgp3 subcongenic mice lacking the NZB-derived macroH2A1 gene

To determine whether the macroH2A1 gene is a candidate gene for Sgp3, we

generated a B6.NZB-Sgp3 subcongenic line, designated B6.NZB-Sgp3a, carrying a ~13

Mb NZB interval flanked by markers D13Mit283 (63.40 Mb) and D13Mit254, in which

- 134 -

the NZB-derived macroH2A1 gene is excluded (Fig. 1). This subcongenic line had high

serum levels of gp70 similar to those of B6.NZB-Sgp3 mice (Table I). In addition, the

abundance of retroviral gp70 RNA transcripts in livers of B6.NZB-Sgp3a subcongenic

mice was essentially identical to that of B6.NZB-Sgp3 mice (Table I). This indicated

that macroH2A1 is not the Sgp3 gene by itself. Notably, quantification of macroH2A1.1

and macroH2A1.2 mRNAs in livers of B6.NZB-Sgp3a subcongenic mice showed no

significant modulation as compared with WT B6 mice (Table I). This ruled out the

possibility that the Sgp3 gene down-regulates the expression of the macroH2A1 gene,

thereby up-regulating the transcription of endogenous retroviral sequences.

Predominant expression of WT mPT env RNA in macroH2A1-deficient B6 and

B6.NZB-Sgp3a subcongenic mice

Our recent analysis of RNA from livers of B6 mice revealed the presence of not

only intact WT mPT env transcript but also two defective (D1 and D2) mPT env

transcripts which carry a deletion in the env sequence of the 3’ portion of the gp70

surface protein and the 5’ portion of the p15E transmembrane protein, respectively (41).

In contrast, lupus-prone mice expressed predominantly the WT mPT env RNA at the

near exclusion of the defective transcripts. Since the Sgp3 locus derived from lupus-

prone mice was responsible for the selective up-regulation of the WT mPT env RNA,

we assessed the relative expression of the three different species of mPT env transcripts

in macroH2A1-deficient B6 mice. As shown in Fig. 2A, macroH2A1-deficient B6 mice

as well as B6.NZB-Sgp3a subcongenic mice displayed a predominant expression of the

WT mPT env RNA, as in the case of B6.NZB-Sgp3 congenic mice (41). The levels of

three different mPT env RNAs in livers were semi-quantified by RT-PCR specific for

the three different mPT env sequences, because the remarkable homology in the gp70-

p15E junction region between mPT and PT env genes precluded the design of a WT-

specific mPT primer suitable for real-time RT-PCR (41). The analysis with serially

diluted cDNA samples from macroH2A1-deficient B6 mice showed marked and

selective ~100-fold increases in WT mPT env transcripts, as compared to WT B6 mice,

while no appreciable increases in D1 and D2 mPT env RNAs were observed (Fig. 2B).

Notably, the results obtained with macroH2A1-deficient B6 mice were essentially

identical to those with B6.NZB-Sgp3a subcongenic mice.

- 135 -

Lack of enhanced hepatic expression of xenotropic, PT and mPT gp70 RNAs in

macroH2A1-deficient 129 mice

macroH2A1-deficient B6 mice likely carry the 129-derived Sgp3 locus, and the

129 strain might share the Sgp3 allele with lupus-prone mice. Thus, the observed up-

regulated expression of endogenous retroviral gp70 RNAs in macroH2A1-deficient

livers cannot unequivocally attribute to the consequence of the macroH2A1 null

mutation alone, if the latter is involved in the regulation of the transcription of

endogenous retroviral sequences. To address this question, we determined whether the

presence of the macroH2A1 null mutation can indeed promote the expression of

endogenous retroviral gp70 RNAs in 129 mice. The analysis of the abundance of PT

and mPT gp70 RNA transcripts in the liver of macroH2A1-deficient 129 mice showed

no significant up-regulation for these retroviral gp70 RNAs, as compared with WT 129

mice (Fig. 3A). In addition, the relative expression pattern of the three different species

of mPT env transcripts was unchanged in macroH2A1-deficient 129 mice, which failed

to display the predominant expression pattern of WT mPT env RNAs (Fig. 3B). Semi-

quantitative RT-PCR analysis confirmed that levels of WT and D1 mPT env RNAs

were not different between macroH2A1-sufficient and -deficient 129 mice (data not

shown). The lack of D2 mPT env transcripts in 129 mice was due to the absence of the

D2 mutant provirus in this strain, as documented by genomic PCR analysis (Fig. 3C).

Notably, as described previously (3, 41), 129 mice failed to express xenotropic and

ecotropic gp70 RNAs because of the absence of both proviruses in their genome.

DISCUSSION

Sgp3 present on mid chromosome 13 has been identified as the major genetic

locus to control the expression of serum retroviral gp70 and endogenous retroviral gp70

RNAs in livers. Recent findings that the transcription of endogenous retroviral

sequences was substantially enhanced in livers from macroH2A1-deficient B6 mice

prompted us to investigate the possibility of macroH2A1 as a candidate for Sgp3, since

macroH2A1 is localized within the Sgp3 interval. Results obtained through comparative

analysis of macroH2A1-deficient B6 and 129 mice demonstrate that macroH2A1-

deficient B6 mice exhibited markedly enhanced levels of retroviral gp70 RNAs, as

compared with WT B6 mice, while this was not the case in macroH2A1-deficient 129

mice. Notably, the analysis of the genomic composition of the chromosome 13 revealed

that macroH2A1-deficient B6 mice still carry the 129-derived Sgp3 locus, which was

co-transferred with the macroH2A1 mutant gene during the backcross procedure. Our

- 136 -

data thus indicate that elevated levels of endogenous retroviral gp70 RNAs in

macroH2A1-deficient B6 mice was not the result of the macroH2A1 null mutation, but

due to the presence of the 129-derived Sgp3 locus. This conclusion is consistent with

the finding that B6.NZB-Sgp3 subcongenic mice lacking the NZB-derived macroH2A1

gene displayed the typical Sgp3 phenotype indistinguishable with B6.NZB-Sgp3

congenic mice carrying the NZB-derived macroH2A1 gene.

We have previously observed that the expression pattern of three different species

(WT, D1 and D2) of mPT env transcripts in GIX+ 129 mice was clearly different from

B6 and B10 mice bearing the Sgp3 locus derived from lupus-prone NZB and BXSB

mice, respectively (41). Since the expression of thymocyte differentiation GIX gp70

antigen is regulated by the Gv1 locus (34), as a consequence of the transcription of

endogenous retroviral sequences in different tissues, including the liver (22), we

speculated that lupus-prone mice carry different regulatory elements in the Sgp3 interval,

which might independently control the levels of mPT, PT and xenotropic proviral

sequences, and that the presence of the regulatory element controlling the mPT proviral

expression may be unique in lupus-prone mice. However, the analysis of macroH2A1-

deficient B6 and 129 mice bearing the 129-Sgp3 allele revealed that the 129-Sgp3 allele

is able to promote the predominant and abundant expression of WT mPT env transcripts

in B6 mice. This indicates that the 129 strain likely shares the same Sgp3 allele with

lupus-prone mice. Thus, the lack of predominant expression of WT mPT env transcripts

in 129 mice is probably due to the absence of mPT proviruses carrying the intact env

sequence, the expression of which is strongly promoted by Sgp3. This interpretation is

consistent with our previous findings in SB/Le mice, which also failed to display the

predominant expression of WT mPT env RNAs (41), despite the fact that the Sgp3 allele

of BXSB mice is inherited from the SB/Le strain, as BXSB is a recombinant strain

derived from a cross of B6 and SB/Le mice. Indeed, the region covering the Sgp3 locus

in BXSB mice originates from SB/Le (29). Notably, these results also exclude the

possibility that the selectively up-regulated expression of WT mPT env RNA in lupus-

prone mice and in Sgp3 congenic mice is the result of the presence in the Sgp3 region of

a unique mPT provirus, which may be especially highly expressed because of its

particular integration site.

Our present and previous studies strongly suggest that only a particular fraction of

mPT proviruses encoding the intact env gene is selectively regulated by Sgp3. Notably,

other GIX+ strains of mice, such as AKR, DBA/2 and C3H/He (26), also displayed the

expression pattern of the three species of mPT env RNAs similar to that of 129 and

- 137 -

SB/Le mice. If we assume that all the GIX+ strains of mice carry the same Sgp3 allele,

the copy number of the unique mPT provirus responsive to Sgp3 and its strain

distribution must be very limited. As the estimated copy numbers of mPT proviruses in

the genome of NZB, B6, AKR and C3H/He mice are 7-11 (10, 11, 37), it is possible

that Sgp3 regulates the expression of only one or two mPT proviruses. BLAST search

analysis confirmed the presence in the B6 mouse genome of 11 mPT proviruses

carrying the intact env gene, in which 9 different microheterogeneities of the U3

regulatory region in the long terminal repeat are identified. An extensive analysis of the

U3 sequences of expressed mPT proviruses in B6.NZB-Sgp3 and macroH2A1-deficient

B6 mice, in comparison with WT B6 mice, might help identify the genetic origin of the

mPT provirus selectively up-regulated by Sgp3. This could define whether the selective

function of Sgp3 as a trans-activating factor is related to a unique U3 sequence of

endogenous retroviruses or to their integration sites. Notably, the identification of the

genetic locus of the highly expressed mPT provirus by the presence of Sgp3 would help

determine its pathogenic role in the development of murine SLE, if this locus would be

associated with the known lupus susceptibility loci.

A possible contribution of endogenous retroviruses to the development of SLE

has long been suspected. This possibility was further supported by recent findings that

single-stranded RNA-specific TLR7 played a critical role for the development of

autoimmune responses against retroviral gp70 as well as RNA-related nuclear

autoantigens in murine SLE (32, 41). In addition, endogenous retroviruses can

contribute to the formation of pathological retroviruses (2, 6, 19). A wide variety of

mechanisms are used to protect the genome from retroviral elements, and one of those is

the control for the transcription of endogenous retroviral sequences (23). Although our

present studies show that macroH2A1 is not involved in silencing of endogenous

retroviruses, it is important to pursue further search and eventual identification of the

Sgp3 gene. Clearly, this would help elucidate a molecular base for transcriptional

suppression of endogenous retroviruses.

- 138 -

ACKNOWLEDGMENTS

This work was supported by a grant from the Swiss National Foundation for Scientific

Research. We thank Mr Guy Brighouse for his excellent technical assistance.

- 139 -

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TABLE I. Levels of gp70 in sera, and gp70 RNAs and macroH2A1 mRNA in livers of B6 mice deficient in macroH2A1 or congenic for the

Sgp3 locus

Mice Serum gp70a Xeno gp70

b PT gp70

b mPT gp70

b macroH2A1.1

b macroH2A1.2

b

macroH2A1-/- 12.7 ± 0.7 13.89 ± 2.72 3.91 ± 0.02 25.04 ± 3.17 NTc NT

NZB-Sgp3 11.1 ± 0.7 12.92 ± 2.93 4.51 ± 0.81 19.15 ± 4.14 NT NT

NZB-Sgp3a 10.3 ± 0.6 14.12 ± 1.32 4.28 ± 0.40 14.25 ± 2.12 0.77 ± 0.10 0.98 ± 0.07

WT 2.3 ± 0.2 1.02 ± 0.16 1.02 ± 0.13 1.01 ± 0.10 1.00 ± 0.07 1.00 ± 0.06

_____________________________________________________________________________________________________________________ a Serum levels of gp70 (µg/ml; mean ± SEM of 7 female mice at 2-3 months of age).

b Levels of each gp70 RNA and macroH2A1 mRNA (mean ± SEM of 4 female mice at 2-3 months of age) were quantified relative to a

standard curve generated with serial dilutions of a reference cDNA preparation and normalized using TBP mRNA. Results are expressed as

fold increases of each transcript relative to B6 WT mice. c Not tested.

- 145 -

Figure legends

FIG. 1. Genetic map of the chromosome 13 in B6 macroH2A1-/-, B6.NZB-Sgp3 and

B6.NZB.Sgp3a mice. Diagrams indicate the segment of the chromosome 13 derived from

macroH2A1-/- 129 mice in macroH2A1-/- B6 mice (left panel), and from NZB mice in

B6.NZB-Sgp3 (middle panel) and B6.NZB-Sgp3a congenic (right panel) mice. Black sections

indicate the region that is definitely 129 (left panel) or NZB (middle and right panels), and

grey sections the region which cannot be defined as B6 or 129 (left panel) and as B6 or NZB

(middle and right panels). In each panel, the position of selected microsatellite markers from

the centromere is indicated as Mb.

FIG. 2. RT-PCR analysis for WT, D1 and D2 mPT env genes in B6 mice deficient in

macroH2A1 and B6.Sgp3a subcongenic mice.

(A) The presence of three different species of mPT env RNAs in livers of 2-3

mo-old B6 female mice was determined by RT-PCR with mPT specific gp70 forward and

p15E-R reverse primers. Representative results of three individual animals are shown. Note

the predominant expression of WT env transcripts in macroH2A1-/- B6 (KO ) and B6.Sgp3a

subcongenic mice, as compared with WT B6 mice. As a control (Ctl ), a mixture of three

different plasmids containing WT, D1 and D2 clones obtained from B6 mice was included.

(B) Semi-quantitative RT-PCR analysis for WT, D1 and D2 mPT env RNAs with reverse

primers specific for the three different mPT env genes (mPT1447R, D1-R and D2-R) and a

common forward mPT-specific primer (mPT858F) was carried out with 5-fold serially diluted

cDNAs from different B6 mice. As a control, the abundance of GAPDH mRNA was assessed

in parallel. Four 5-fold dilutions of cDNAs were examined for WT mPT env RNA, while

three 5-fold dilutions of cDNAs were examined for D1/D2 mPT env RNAs and GAPDH

mRNA. Representative results of three individual mice analyzed are shown.

FIG. 3. Analysis for PT and mPT RNAs in macroH2A1-/- and WT 129 mice.

(A) Levels of PT and mPT gp70 RNAs from livers of 2-3 mo-old 129 mice (means ± SEM of

5 mice) were quantified relative to a standard curve generated with serial dilutions of a

reference cDNA preparation and normalized using TBP mRNA. Results are expressed as fold

changes of each transcript in macroH2A1-/- mice (KO ) relative to WT mice.

(B) The presence of two different species of mPT env RNAs in livers of macroH2A1-/- and

WT 129 mice was determined by RT-PCR with mPT specific gp70 forward and p15E-R

- 146 -

reverse primers. Representative results of three individual animals are shown. Lane 1,

macrH2A1-/- 129; lane 2, WT 129; lane 3, macroH2A1-/- B6; lane 4, WT B6.

(C) The presence of WT, D1 and D2-specific mPT env proviral sequences in 129 and B6 mice

was analyzed by genomic PCR with reverse primers specific for the three different mPT env

genes and a common forward mPT-specific primer.

- 147 -

Figure 1

D13Mit254

D13Mit13

D13Mit139

macroH2A1

NZB-Sgp3

D13Mit26

Mb

80

70

60

50

D13Mit248

macroH2A1

macroH2A1 KO

D13Mit99

NZB-Sgp3a

macroH2A1

D13Mit26

D13Mit123

D13Mit254

D13Mit283

D13Mit313

80

70

60

50

80

70

60

50

Figure 1

D13Mit254

D13Mit13

D13Mit139

macroH2A1

NZB-Sgp3

D13Mit26

Mb

80

70

60

50

D13Mit248

macroH2A1

macroH2A1 KO

D13Mit99

NZB-Sgp3a

macroH2A1

D13Mit26

D13Mit123

D13Mit254

D13Mit283

D13Mit313

80

70

60

50

80

70

60

50

D13Mit254

D13Mit13

D13Mit139

macroH2A1

NZB-Sgp3

D13Mit26

Mb

80

70

60

50

80

70

60

50

D13Mit248

macroH2A1

macroH2A1 KO

D13Mit99

NZB-Sgp3a

macroH2A1

D13Mit26

D13Mit123

D13Mit254

D13Mit283

D13Mit313

80

70

60

50

80

70

60

50

80

70

60

50

80

70

60

50

- 148 -

WT

D2D1

Ctl B6 KO Sgp3a NZBA

Figure 2

WT

D1

D2

GAPDH

KO Sgp3 B6B

WT

D2D1


WT

D2D1


Figure 2

WT

D1

D2

GAPDH

KO Sgp3 B6B

WT

D1

D2

GAPDH

KO Sgp3 B6B

- 149 -

129 129 B6B6 129 B6

WT D1 D2C

Figure 3

B1 2 3 4

WTD2D1

KO WT KO WT0.0

0.5

1.0

1.5

Fol

d ch

ange

APT mPT

129 129 B6B6 129 B6

WT D1 D2C

129 129 B6B6 129 B6

WT D1 D2129 129 B6B6 129 B6

WT D1 D2C

Figure 3

B1 2 3 4

WTD2D1

B1 2 3 41 2 3 4

WTD2D1

KO WT KO WT0.0

0.5

1.0

1.5

Fol

d ch

ange

APT mPT

KO WT KO WT0.0

0.5

1.0

1.5

Fol

d ch

ange

APT mPT

- 150 -

- 151 -

IV. GENERAL DISCUSSION

- 152 -

- 153 -

IV. General Discussion

IV.1. Genetic Origin of Serum Retroviral gp70

ERVs are classified as Eco, Xeno or polytropic viruses according their host range

dictated by their respective gp70 proteins. Based on differences in their gp70 nucleotide

sequences, four subgroups of Xeno proviruses (Xeno-I, Xeno-II, Xeno-III and Xeno-IV) as

well as two subgroups of polytropic proviruses termed PT and mPT, are identified in the

mouse genome (187, 188). Tryptic peptide mapping analysis showed that serum gp70

molecule resembles the Env protein of NZB-X1 virus, one of the two distinct Xeno viruses

isolated from NZB mice (217, 218). However, the fingerprint of serum gp70 also displayed

additional marker peptides detectable in gp70 of other Xeno viruses, including the second

NZB Xeno virus, NZB-X2, and gp70 expressed on thymocytes and splenic lymphocytes. Our

cDNA nucleotide sequence analysis revealed that both NZB-X1 and NZB-X2 viruses belong

to the Xeno-I subgroup. Selective up-regulation of Xeno-I gp70 in association with a

moderate increase of serum gp70 in B6.NZB-Sgp4 congenic mice supports the contribution of

Xeno-I gp70 to serum gp70. However, the lack of expression of Xeno-I gp70 RNA in BXSB,

NFS and 129 mice, which have relatively high serum levels of gp70, clearly indicates that

retroviral gp70s other than Xeno-I gp70 contribute to basal levels of serum gp70. Notably, we

found considerable heterogeneity in the expression of the four subgroups of Xeno gp70 RNAs

among various strains of mice. Accordingly, we are able to classify five different groups of

mice: 1) NZB type expressing all four Xeno gp70 RNAs (NZB and NZW); 2) MRL type

expressing Xeno-I, Xeno-III and Xeno-IV; 3) BXSB type expressing Xeno-II, Xeno-III and

XenoIV; 4) NFS type expressing only Xeno-III; and 5) 129 type expressing no Xeno gp70

RNAs. In addition to these qualitative differences in the expression patterns, real-time RT-

PCR analysis revealed quantitative differences among the four Xeno viral gp70 RNAs and

between different strains of mice tested.

Serological analysis clearly excluded the involvement of Eco gp70 as a source of

serum serum gp70 (216). Instead, our studies revealed a substantial contribution of PT and

mPT proviruses, that encode gp70s closely related to Xeno gp70 (186), to serum gp70. Indeed,

129 mice having relatively high serum levels of gp70 express only PT and mPT gp70 RNAs,

but not Xeno and Eco gp70 RNAs, clearly indicating that PT and mPT viral gp70 are

additional important sources of serum gp70. This idea is consistent with the findings in Sgp3

congenic mice, which most prominently increased levels of PT and mPT gp70 RNAs

compared to Xeno gp70 RNA which remained very low. Moreover, serum gp70 in

B10.BXSB-Sgp3 mice reached levels close to that observed in BXSB mice, in correlation

- 154 -

with a prominent increase of PT gp70 RNA in B10.BXSB-Sgp3 mice to the same level as in

BXSB mice.

Taken together, our results indicate a heterogeneous origin of serum gp70 with a

contribution of PT and mPT gp70s, in addition of Xeno gp70.

IV.2. Polygenic Control of the Expression of Serum Retroviral gp70

Remarkable heterogeneity in the expression in different Xeno retroviral gp70 observed

in different strains of mice is in part due to the absence of some of the Xeno proviruses in the

respective genomes. For example, in contrast to NZB, NZW and BXSB mice, MRL and NFS

mice contain in their genome and express only three or one of the four subgroups of Xeno

retrovirus, respectively. However, despite the presence of the provirus in some strains of mice,

there is no expression of the gp70, possibly due to the site of integration or transcriptional

regulation. This is the case in BXSB mice which do not express Xeno-I, despite the presence

of the provirus, and in 129 male mice, which carry Xeno-I and Xeno-IV proviruses but do not

express their transcripts. In addition, the copy numbers of PT and mPT proviruses present in

the mouse genome is highly variable among different strains of mice (185). Accordingly,

variable levels of serum gp70 among different inbred strains of mice are in part attributed by

the heterogeneity of genetic composition of ERVs in the respective genomes.

In addition to the structural genes, the expression of retrovirus is highly dependent on

the presence of regulatory genes. Previous studies of the progeny of crosses of lupus-prone

NZB, NZW and BXSB strains with non-autoimmune B6 or B10 strains identified a major

quantitative trait locus, Sgp3 (or Bxs6), on mid chromosome 13, which was strongly linked

with basal levels of serum retroviral gp70 (40, 88, 95, 100, 107). In addition, a second NZB

and NZW locus, Sgp4, on distal chromosome 4 was found to be linked to serum gp70 levels

in crosses with B6 and BALB/c backgrounds (88, 89). Moreover, the presence of an

additional minor locus controlling the expression of serum gp70 was revealed on the proximal

region of chromosome 12 of NZB and NZW mice when crossed with BALB/c (89). All these

data indicate that serum levels of gp70 are under the control of multiple regulatory genes.

B6 and B10 mice bearing the NZB-Sgp3 or BXSB-Sgp3 allele, respectively, display

increased levels of serum gp70 (40, 235). Both congenic mice revealed an increase of Xeno-I,

Xeno-II, Xeno-III, PT and mPT gp70 RNAs but not Xeno-IV. However, NZB-Sgp3 and

BXSB-Sgp3 predominantly enhanced the expression mPT and PT gp70 RNAs, respectively.

The different effect of NZB-Sgp3 and BXSB-Sgp3 could be due to allelic variation of Sgp3

between NZB and BXSB, as it appears that the expression of PT and mPT gp70 RNAs is

- 155 -

regulated by separate genes present in the Sgp3 locus, as discussed below. It should be also

noted that B10 congenic mice bearing the BXSB-Sgp3 allele displayed increase in Xeno-I

gp70 RNA, despite the fact that BXSB do not express Xeno-I, supporting the idea that Sgp3 is

not the structural gene but rather acts as a regulatory gene to control the expression of

multiple endogenous retroviral transcripts in trans.

The envelope gp70 protein is associated with a membrane-anchored p15E protein

through intersubunit disulfide linkage on the intact virions (236). Therefore, we searched for

the expression of aberrant p15E proteins which are unable to form disulfide-linked envelope

complexes with gp70, thereby promoting the release of free gp70 in the circulating blood. We

observed the presence in B6 mice of two defective (D1 and D2) mPT env transcripts which

carry a deletion in the env sequence of the 3’ portion of gp70 and the 5’ portion of p15E.

However, the lack of up-regulated expression of these two deletion mutants in Sgp3-congenic

mice as well as in lupus-prone mice excluded these mutant proteins as a source of serum gp70.

Unexpectedly, this analysis revealed that in contrast to non-autoimmune strains of

mice, all four lupus-prone strains predominantly and abundantly expressed intact mPT env

transcripts at the near exclusion of the defective env transcripts. We demonstrated that the

Sgp3 locus derived from lupus-prone NZB and BXSB mice was responsible for the selective

up-regulation of the intact mPT env RNA. However, to our surprise, SB/Le mice failed to

display the predominant expression of the intact mPT env transcripts, since the Sgp3 allele of

BXSB mice is inherited from the SB/Le strain, as BXSB is a recombinant strain derived from

a cross of B6 and SB/Le mice. This was also the case for the GIX+ 129 strain of mice, which

is expected to share the Sgp3 allele with lupus-prone mice, because the 129 strain carries the

Gv1 locus controlling the expression of the GIX gp70 antigen (220, 221) and because Gv1

and Sgp3 are likely to be identical or related genes regulating the transcription of endogenous

retroviral sequences. However, our studies on macroH2A1-deficient B6 and 129 mice bearing

the 129-Sgp3 allele revealed that the 129-Sgp3 allele is indeed able to promote the

predominant and abundant expression of intact mPT env transcripts in B6 mice, confirming

that 129 mice shares the same Sgp3 allele with lupus-prone mice. Thus, we interpret that the

lack of predominant expression of intact mPT env transcripts in 129 as well as SB/Le mice is

probably due to the absence of mPT proviruses carrying the intact env gene, the expression of

which is highly regulated by Sgp3. In addition, our data indicate that Sgp3 apparently controls

the expression of only a fraction of mPT proviruses bearing the intact env gene.

It is intriguing that the Sgp3 locus derived from lupus-prone mice is responsible for

selective up-regulation of the WT mPT, but not the two defective (D1 and D2) mPT env

transcripts. We hypothesized that the regulatory elements modulated by Sgp3 may be less

- 156 -

efficient in the D1 and D2 mutants. Accordingly, we attempted to identify the molecular basis

responsible for the selective effect of Sgp3 through the analysis of the U3 regulatory

sequences of LTR in the D1 and D2 mutants. However, this effort is not fruitful at the

moment, since it has not yet been identified which mPT proviruses carrying the intact env

gene are indeed up-regulated by Sgp3, as Sgp3 apparently controls the expression of only a

fraction of mPT proviruses. Because of the presence of microheterogeneity of the U3

regulatory region, an extensive analysis of the U3 sequences of expressed mPT proviruses in

B6.NZB-Sgp3 mice, in comparison with WT B6 mice, might help identify the genetic basis

for the selective effect of Sgp3 on the expression of WT mPT env genes.

It has been claimed that the Gv1 locus derived from the 129 strain was reported to

regulate the transcription of PT, but not mPT proviruses (220), and that Gv1 controls the

expression of gp70 in a semi-dominant fashion (208). In view of the remarkable differences in

the U3 region of LTR among Xeno, PT and mPT retroviruses (Figure 6), the presence of

several genetic factors which differentially control the expression of individual classes of

retroviruses might not be surprising. Indeed, our on-going studies on Sgp3 homozygous and

heterozygous mice revealed that the basal-level expression of Xeno, PT and mPT viral

sequences was regulated in a dominant, semi-dominant and recessive manner, respectively.

Figure 6: Enhancer elements in the LTR U3 region of murine endogenous retrovirus: CArG

(CC,AT-rich,GG consensus motif), LVb (leukemia virus factor b), Core (SV40 core-like

motif), NF1 (nuclear factor 1), GRE (glucocorticoid response element), GATA

((A/T),GATA(A/G) consensus motif), MLPal (MCF-13LTR-palindrome), NF-κB (nuclear

factor-kappa B) and SEF1 (suppressor of the essential function1).

The second genetic locus linked to serum gp70 in the NZB strain is the Sgp4 locus on

chromosome 4. We confirmed the contribution of Sgp4 to the production of serum gp70,

although its effect was more modest than Sgp3. Sgp4 contributes to an up-regulation of only

Virus

Xeno

PT

mPT

CArG

+

–

+

LVb

+

–

+

Core

+

+

+

NF1

+

+

+

GRE

+

+

+

GATA

+

–

–

NF-kB

+

–

–

MLPal

+

–

–

SEF1

+

–

–

Virus

Xeno

PT

mPT

CArG

+

–

+

LVb

+

–

+

Core

+

+

+

NF1

+

+

+

GRE

+

+

+

GATA

+

–

–

NF-kB

+

–

–

MLPal

+

–

–

Virus

Xeno

PT

mPT

CArG

+

–

+

LVb

+

–

+

Core

+

+

+

NF1

+

+

+

GRE

+

+

+

GATA

+

–

–

NF-kB

+

–

–

MLPal

+

–

–

SEF1

+

–

–

- 157 -

Xeno-I gp70 RNA, and a suppression of Xeno-II and Xeno-III gp70 RNAs. Double congenic

mice bearing both NZB derived Sgp3 and Sgp4 revealed a synergic effect on the production of

serum gp70. Although basal level of serum gp70 in B6.NZB-Sgp3/4 congenic mice are

comparable to those of lupus-prone BXSB and MRL mice, they are still lower than in NZB

and NZW mice, suggesting that an additional locus can contribute to serum gp70. Indeed, the

presence of an additional Sgp locus on proximal chromosome 12 from NZB and NZW mice

has been identified (89). Preliminary studies showed that B6 mice bearing the proximal

chromosome 12 interval from NZB mice displayed modest, but significant increases of serum

gp70, at levels comparable to those observed in BALB/c mice congenic for this putative Sgp

locus derived from NZW mice. Furthermore, our on-going analysis revealed that this locus

selectively regulates the expression of Xeno gp70 RNAs, as in the case of the Sgp4 locus.

Collectively, our results demonstrated that serum levels of gp70 are under the control

of multiple structural and regulatory genes. Thus, diverse levels of serum gp70 in various

murine strains can be explained by the presence of a different assortment of multiple

structural and regulatory genes implicated in the production of serum gp70 in liver.

IV.3. Sgp-mediated Control of Enhanced gp70 Production during Acute Phase

Responses

The expression of serum retroviral gp70 is enhanced by different inducers of APP such

as LPS, turpentine oil or polyriboinosinic-polyribocytidylic acid in lupus-prone mice,

indicating that serum gp70 behaves like an APP (212, 213). This notion has been confirmed

by present studies showing that cytokines IL-1, IL-6 and TNF which are well known inducers

for APP, induce similarly increased levels of serum gp70 in NZB mice. However, unlike

conventional APPs, the serum gp70 response is strain-dependent, as only mice having high

basal levels of serum gp70 displayed an upregulated production of serum gp70 in response to

LPS. Although it has not been very clear about the genetic basis for the serum gp70

production in response to LPS, studies on Sgp3, Sgp4 and Sgp3/4 congenic mice revealed that

the Sgp loci act synergistically and play a major role in the acute phase expression of serum

gp70. This indicates that the Sgp3 and Sgp4 loci control the expression of serum gp70 under

not only steady-state condition but also inflammatory condition.

Anaylsis of the abundance of gp70 RNAs in livers during acute phase responses has

shown that inflammatory stimuli seletively up-regulated the levels of Xeno and mPT gp70

RNAs, but not that of PT gp70 RNA, in NZB, BXSB and B6.Sgp3 mice, while the abundance

of only Xeno gp70 RNA was increased in B6.Sgp4 congenic mice. The lack of mPT

- 158 -

responses in B6.Sgp4 mice is consistent with the fact that this locus regulates the expression

of only Xeno gp70 RNA. The selective effect of LPS on Xeno and mPT gp70 RNA is likely

to be related to the remarkable heterogeneity of the U3 regulatory regions of LTR among

different classes of ERVs. The lack of up-regulated expression of PT proviruses can be in part

related to the absence of an IL-6-responsive element (IL6-RE), common to genes encoding

acute phase protein (237). However, the presence of the IL6-RE may not be a determining

element for the acute phase response for retroviral gp70, since this motif is conserved in the

U3 sequence of all mPT proviruses, including the D2 mPT provirus which fails to be up-

regulated in response to LPS. Higher responses of Xeno gp70 RNA can be explained by the

presence of nuclear factor-kappa B (NF-kB)-binding motif in the U3 region of Xeno viruses

(Figure 6), as NF-kB is involved in one of the several distinct signaling pathways leading to

the synthesis of acute phase proteins (181).

In contrast to the selective effect by Sgp3 on the steady-state level expression of WT

mPT env gene, it was unexpected that NZB, BXSB and B6.Sgp3 mice injected with LPS

displayed marked increases in D1 mPT env RNA, as compared with WT mPT env RNA,

while levels of D2 mPT env RNA were not modulated. Since increases in D1 mPT env RNA

was not observed in WT B6 mice, the LPS-induced up-regulated expression of D1 mPT env

RNA is regulated by Sgp3. These results indicate that the genetic factor involved in up-

regulated expression of mPT gp70 RNA in response to LPS is distinct from those controlling

the steady-state level of Xeno, PT or mPT RNAs. In this regard, it should be mentioned that

injection of LPS in B6.Sgp4 mice resulted in increases of not only Xeno-I but also Xeno-II

and Xeno-III gp70 RNAs, while the Sgp4 locus only up-regulated the level of Xeno-I gp70

RNA under non-inflammatory condition. These results also indicate that the Sgp4 locus likely

carries regulatory elements, which independently control the transcription of Xeno env RNAs

in physiological versus inflammatory conditions.

At present, we cannot offer straightforward explanation for a strong up-regulated

expression of the D1 mPT env RNA in response to LPS, as compared with other mPT env

RNAs. Since the D2 mPT provirus also carries the IL6-RE, its presence is not a determining

element. However, the D1 mutant has two unique mutations in the U3 region: a substitution

of G (guanine) with A (adenine) in a SV40 core-like motif (GTGATCA instead of

GTGGTCA) and an insertion of T (thymine) in the UCR (upstream conserved region), which

negatively regulates the expression of ERVs (238). It remains to be determined whether these

two mutations contribute to the up-regulated transcription of the D1 mPT provirus by the

presence of inflammatory stimuli. Alternatively, marked increases in D1 mPT env RNA in

response to LPS could be related to its integration of this particular provirus, as it is integrated

- 159 -

in the right transcription direction within the 4th intron of the Rsrc1 (arginine/serine-rich

coiled-coil 1) gene in B6 chromosome 3. However, this possibility is unlikely, since we

observed that the expression of the Rsrc1 gene, which contains the D1 mPT provirus in the

correct orientation, was up-regulated neither by the presence of Sgp3 nor by the injection of

LPS. Another plausible explanation is that the enhancer element(s) of the U3 region,

implicated in the increased expression in response to inflammatory stimuli, may be selectively

methylated in certain proviruses, as the expression of retroviral sequences is strongly affected

by the state of DNA methylation (169-171).

The demonstration that the expression of serum gp70 under steady-state and

inflammatory conditions is regulated by distinct genes further underlines the complexity for

the genetic control of the expression of different classes of ERVs in mice.

IV.4. Search for the Candidates Genes for Sgp3

We identified Sgp3 as the major genetic locus to control the expression of serum

retroviral gp70 and endogenous retroviral gp70 RNAs in livers under steady-state and

inflammatory conditions. Because of the recent demonstration of an up-regulated transcription

of endogenous retroviral sequences in livers of B6 mice deficient in macroH2A1 (222), which

likely plays a role in repressing gene expression (224-227), and because of the presence of

macroH2A1 gene in the Sgp3 interval, we tested the hypothesis that macroH2A1 is a

candidate gene for Sgp3. However, our analysis of the expression of endogenous retroviral

gp70 RNAs in livers from two different macroH2A1-sufficient or -deficient mice bred into

the B6 or 129 backgrounds in relation to the 129-derived Sgp3 locus revealed that enhanced

transcription of endogenous retroviral sequences observed in macroH2A1-deficient B6 mice

was not a result of the macroH2A1 mutation, but due to the presence of 129-derived Sgp3

locus, which was co-transferred with the 129 macroH2A1 mutant gene during backcross

procedures.

Based on the results obtained by our on-going analysis of Sgp3 subcongenic lines

using the SNP map (www.well.ox.ac.uk/mouse/INBREDS), the Sgp3 region has been

narrowed down to the 5.42 Mb between 64.54 and 69.96 Mb of chromosome 13. There are 30

genes mapped to this region in the NCBI database, and significantly, a cluster of 21 KRAB-

ZFP has been identified in this region, although the precise function of most of these Zfp

genes has not yet been identified (228, 239) (Figure 7A). KRAB-ZFP are composed of a

KRAB domain, which represses transcription by recruiting KAP-1 corepressor acting as a

scaffold for chromatin-condensing protein, and of a zinc-finger, which selectively recognize

- 160 -

target gene through recognition of specific regulatory sites within the DNA (240, 241). It has

recently been shown that KRAB transcription repressor domain suppressed lentivirus proviral

transcription by inducing heterochromatization in the lentiviral integration sites (229) and that

ZFP809 silences integrated retroviral DNAs through the recruitment of TRIM28 in embryonic

stem cells (242). Since Sgp3 is likely to be a regulatory locus that acts in trans to control

expression of multiple ERVs across the mouse genome, the Zfp genes are the primary

candidate for Sgp3.

In collaboration with the group of Dr Diane Robbins, University of Michigan Medical

School, Ann Arbor, who characterized the function of two Zfp genes, Rsl1 (Regulator of sex-

limitation) and Rsl2, present in the Sgp3 region, we have recently determined serum levels of

gp70 in B10.D2.PL mice bearing B6-derived BAC 45-N-22 transgene. Although the number

of sera tested was still limited, our preliminary results showed a significant reduction of

serum levels of gp70 in BAC 45-N-22 transgenic mice, as compared to non-transgenic mice

(Figure 7B). This observation rasied a possibility that a candidate gene for Sgp3 could be

localized within the BAC clone 45-N-22, which carries 6 Zfp genes: Zfp712, Zfp708, Zfp759,

Rsl1, Zfp455 and Zfp458 (Figure 7A).

- 161 -

Figure 7: A. Physical map of the Rsl locus at 37cM on mouse chromosome 13 in B6.NZB-

Sgp3a mice. Sgp3a locus (■) is delimited by markers D13Mit283 and D13Mit254. Numbered

black boxes represent Rslcan (Rsl candidate) genes (most of them are now called as Zfp) and

arrows below indicate direction of transcription. Zfp genes present in BAC 45-N-22

transgenic mice are represented in a blue quadrant (��). Adapted from (239). B. Serum levels

of gp70 in BAC 45-N-22 non-transgenic (Non-Tg) or transgenic (Tg) mice. Each symbol

represents an individual animal. Results are expressed as micrograms per milliliter for gp70.

Horizontal line, mean values.

The Rsl1 gene has been shown to regulate male-specific expression of Sex-limited

protein (Slp), which is a murine homologue of C4A, in liver (228, 239). Some strains of mice

carry the natural mutation of the Rsl1 gene, by which they cannot express a functional Rsl1

protein because of an aberrant splicing of Rsl1 mRNA. Consequently, female mice

inappropriately express the male-specific Slp. We observed that all mice tested (NZB, NZW,

MRL, BXSB, NFS, 129, AKR, DBA/2, C3H, CBA and BALB/c), except B6 and B10, carry

the Rsl1 mutation (Figure 8). Thus, we tentatively exclude the Rsl1 gene as a candidate for

Sgp3, although the possibility does remain that different repressors control levels of gp70 in

non-autoimmune strains other than B6 and B10. In addition, the Zfp759 gene exhibits strain-

Zfp

459

Rsl

1

Rsl

2

Zfp

457

Zfp

708

Zfp

759

Zfp712 16 5Z

fp45

5Z

fp45

8

Zfp

595

Zfp

456

Zfp

817

Zfp

87

Zfp

748

Zfp

85

Zfp

273

Rsl1 BAC Tg Mice

Zfp

712

Sgp3a

D13

Mit

254

D13

Mit

283A

B

0

10

20

30

gp70

(µµ µµg

/ml)

0

10

20

30

Rsl1BAC TgNon-Tg

p<0.01

Zfp

459

Rsl

1

Rsl

2

Zfp

457

Zfp

708

Zfp

759

Zfp712 16 5Z

fp45

5Z

fp45

8

Zfp

595

Zfp

456

Zfp

817

Zfp

87

Zfp

748

Zfp

85

Zfp

273

Rsl1 BAC Tg Mice

Zfp

712

Sgp3a

D13

Mit

254

D13

Mit

283

Zfp

459

Rsl

1

Rsl

2

Zfp

457

Zfp

708

Zfp

759

Zfp712 16 5Z

fp45

5Z

fp45

8

Zfp

595

Zfp

456

Zfp

817

Zfp

87

Zfp

748

Zfp

85

Zfp

273

Rsl1 BAC Tg Mice

Zfp

712

Sgp3a

D13

Mit

254

D13

Mit

283A

B

0

10

20

30

gp70

(µµ µµg

/ml)

0

10

20

30

Rsl1BAC TgNon-Tg

p<0.01

- 162 -

specific addition or subtraction of zinc finger unit (239). This difference may lead to

differential target gene regulation, quantitatively or qualitatively. NZB, BXSB, NFS, DBA/2,

C3H, CBA and BALB/c lacks the zinc finger unit of the Zfp759, unlike NZW, MRL, 129,

AKR, B6 and B10. But this polymorphism is not associated with high or low serum levels of

gp70 (Figure 8). On-going RT-PCR analysis revealed an increased expression of Zfp458 and

Zfp455 mRNAs in B6.Sgp3 congenic mice, as compared with WT B6 mice. However, the

observed expression difference is not consistent with the hypothesis of Sgp3 being the loss of

a repressor, as the expression would thus to be expected to be higher in B6 than in B6.Sgp3

mice. Thus, Zfp458 and Zfp455 are unlikely to be a candidate gene for Sgp3. In contrast, we

have observed approximately 5-fold and 2-fold lower levels of Zfp708 and Zfp712 mRNAs,

respectively, in B6.NZB-Sgp3 and B10.BXSB-Sgp3 congenic mice than in WT B6 and B10

mice.

As the Sgp3 locus contains at least four distinc genetic elements which regulate the

expression of ERVs under steady-state and inflammatory conditions, we will not limit our

search for the Sgp candidate genes to those located within the BAC 45-N-22 clone. The

Zfp457 gene presents polymorphism similar to that found in the Zfp759 gene (239). However,

we observed that the strain distribution of the polymorphic allele of the Zfp457 gene is

identical to that of the Rsl1 gene among various strains of mice, arguing against Zfp457 as a

candidate for Sgp3 (Figure 8). Rsl2 and Zfp456 sequences are intermingled in some strains of

mice, resulting in a hybrid gene (239). However, comparison between high-gp70 and low-

gp70 mice showed no association of this mutation with high-gp70 strains of mice, as this

mutation was not present in BXSB, NFS and DBA/2 mice (Figure 8). On-going analysis of

mRNA levels for different Zfp genes revealed that lack of the expression of Zfp595 mRNA in

B6.Sgp3 mice, while it is abundantly present in B6 mice, indicating that Zfp595 is an

additional candidate gene for Sgp3. Clearly, more extensive analysis of other Zfp genes

present in the Sgp3 locus should be able to help identify the additional candidate genes for

Sgp3.

Since Zfp708, Zfp712 and Zfp595 could be so far potential candidates for Sgp3, we

will explore this possibility in vitro and in vivo. Using primary hepatocytes isolated from

B6.Sgp3 and WT B6 mice, we will determine whether the transduction of the WT Zfp708,

Zfp712 or Zfp595 gene could reduce the secretion of gp70 in vitro and the abundance of Xeno,

PT or mPT gp70 RNA in Sgp3-bearing hepatocytes. Finally, the results obtained in vitro can

be confirmed by the generation and analysis of B6.NZB-Sgp3 mice overexpressing the wild-

type allele of the Sgp3 candidate gene in liver.

- 163 -

Figure 8: Polymorphism of Rsl1, Rsl2, Zfp759 and Zfp457 in several strains of mice in

relation with serum levels gp70. a: Mice homozygous for allele identical to NZB; b: Mice

homozygous for allele identical to C57BL/6. ND: not determined.

IV.5. Role of TLR7 and ERVs in Murine SLE

TLR7 is an innate immune receptor specific for single-stranded RNA and plays a

critical role in the development of autoimmune responses against nuclear autoantigens in

murine SLE (243). Our studies demonstrated that the formation of gp70 IC was completely

suppressed in TLR7-deficient B6 mice congenic for the Nba2 locus, an NZB-derived major

lupus susceptibility locus, which contributes to overall production of various lupus antibodies

(73, 100). This indicates the implication of TLR7 in the formation of gp70 IC. Our results

suggest an active role of ERVs through interaction with TLR7 for the development of

autoimmune responses against serum retroviral gp70. This idea is consistent with the finding

that B10.Yaa mice had increased levels of gp70 IC in sera by the presence of the Sgp3 locus,

which promotes the expression of ERVs (235).

NZB mice spontaneously produce a very high titer of replication-competent Xeno

viruses from birth (230), while they fail to express Eco viruses because of the lack of Eco

sequences in their genome (244). In addition, we observed a more than 100-fold increased

levels of mPT env RNA derived from mPT proviruses bearing the intact env gene, but not

Strain

NZB

NZW

MRL

BXSB

NFS

129

AKR

DBA/2

C57BL/6

C57BL/10

C3H/HeJ

CBA

BALB/c

gp70

High

High

High

High

High

High

High

High

Low

Low

Low

Low

Low

Sgp3

a

a

a

a

ND

a

ND

ND

b

b

b

b

b

Rsl1

a

a

a

a

a

a

a

a

b

b

a

a

a

Rsl2

a

a

a

b

b

a

a

b

b

b

b

b

b

Zfp759

a

b

b

a

a

b

b

a

b

b

a

a

a

Zfp457

a

a

a

a

a

a

a

a

b

b

a

a

a

Strain

NZB

NZW

MRL

BXSB

NFS

129

AKR

DBA/2

C57BL/6

C57BL/10

C3H/HeJ

CBA

BALB/c

gp70

High

High

High

High

High

High

High

High

Low

Low

Low

Low

Low

Sgp3

a

a

a

a

ND

a

ND

ND

b

b

b

b

b

Rsl1

a

a

a

a

a

a

a

a

b

b

a

a

a

Rsl2

a

a

a

b

b

a

a

b

b

b

b

b

b

Zfp759

a

b

b

a

a

b

b

a

b

b

a

a

a

Zfp457

a

a

a

a

a

a

a

a

b

b

a

a

a

- 164 -

those bearing the defective env gene, in NZB, BXSB and Sgp3-congenic B6 and B10 mice as

compared with B6 and B10 mice. Such an increase was also observed in two other lupus-

prone NZW and MRL mice, but not in non-autoimmune strains of mice we tested. This

indicates that lupus-prone mice possess a unique genetic mechanism responsible for a very

high-level expression of mPT retroviruses. Consistent with our findings, it has been reported

that an 8.4-kb transcript corresponding to the full-length size mPT retroviruses was expressed

uniquely in thymi of NZB, BXSB and MRL mice, while the expression of full-length

transcripts of Xeno and PT viruses was not limited to lupus-prone mice (234). Although

endogenous mPT viruses are likely to be replication defective, replication-competent and

infectious recombinant viruses containing the mPT gp70 sequence can be generated. These

recombinant viruses utilize the XPR1 cell-surface receptor for infection of mice (XPR1

expressed in the laboratory strains of mice confers susceptibility to PT and mPT retroviruses,

but not to Xeno virus due to the Xpr1 polymorphism). Therefore, one could speculate that

abundant and preferential expression of mPT proviruses possessing an intact env gene in

lupus-prone mice could facilitate the generation of replication-competent mPT-derived

infectious viruses through recombination with Xeno viruses, and these infectious viruses may

act as a triggering factor for the development of murine SLE. In fact, we have attempted to

isolate replication-competent infectious retroviruses containing mPT gp70 sequence from

lupus-prone mice through co-culturing spleen cells from NZB mice with two different target

cell lines, Mus dunni and Fischer rat embryo cells, both of which are devoid of endogenous

Xeno, PT and mPT retroviruses. Significantly, PCR analysis has shown the presence of mPT-

derived proviral sequences in both target cells co-cultivated with spleen cells from 5 mo-old

NZB mice, in addition to Xeno sequences. These data suggest that NZB mice might

spontaneously generate mPT-derived replication-competent infectious retroviruses or

recombinant virus carrying mPT env gene. Obviously, future studies will be to identify the

genetic origin of this infectious virus and determine whether such virus can promote the

development of SLE in mice predisposed to autoimmune diseases.

It has well been established that DC play a pivotal role in the induction and regulation

of the immune response, because immature, non-activated DC that capture autoantigens

induce self tolerance, while the activation of antigen-loaded DC triggers their maturation and

enables them to induce antigen-specific immunity. A particular role of pDC, a subset of DC

which highly express TLR7, in SLE has been proposed, since this DC subset has been

identified as the major source of IFNα (245), a cytokine that plays a substantial role in the

development of SLE (131, 246). Thus, one attractive hypothesis would be that ERVs could

enter in the pDC through endocytosis and gain access to TLR7, leading to the activation of

- 165 -

pDC. Activated pDC rapidly secrete copious amounts of IFNα, and to a lesser extent

proinflammatory cytokines such as IL-6 or TNFα (247). Notably, TNFα is responsible, in

part, for driving the differentiation of immature DC into mature antigen-presenting cells, and

IL-6 and IFNα promote differentiation of plasma cells. Thus, excessive activation of pDC by

ERVs could play an important role in the accelerated development of SLE.

Since TLR7 is not a cell surface receptor but expressed in endosome, the activation of

pDC by ERVs might be dependent on XPR1, which allows their internalization and

subsequent interaction with TLR7 in endosome. Notably, the expression of Xpr1 mRNA in

pDC has been confirmed by RT-PCR. In addition, one cannot exclude the possibility that non-

infectious Xeno viruses internalized through other receptors can activate pDC through

stimulating TLR7 in endosome. For example, Xeno viruses in the form of IC with IgG anti-

gp70 autoantibodies can be internalized through FcγR and subsequently interact with

endosomal TLR7 in pDC (Figure 9A). However, it is unlikely that this is a mechanism to

trigger pDC and initiate autoimmune responses against nuclear and gp70 antigens in SLE,

since the production of IgG anti-gp70 autoantibodies (to form stimulating gp70 IC) is

prerequisite for this process. Instead, this mechanism can sustain the production of IFNα

through IC-mediated activation of pDC, thereby establishing a vicious cycle not only

aggravating the autoimmune process but also promoting the development of autoimmune

responses against a wide array of autoantigens that do not engage TLR. This idea is

supporting by the observation that TLR7 is responsible for enhanced autoimmune responses

against not only DNA- and RNA-related antigens but also several glomerular matrix antigens

(147).

Furthermore, retroviruses can directly activate anti-gp70 autoreactive B cells. ERVs

could be recognized by anti-gp70 BCR, then endocytosed where RNAs of ERVs gain access

to TLR7, inducing TLR signaling cascade and the activation of anti-gp70 autoreactive B cells

(Figure 9B). In this regard, it should be stressed that the activation of anti-gp70 autoreactive

B cells does not necessarily require infectious retroviruses, since ERVs can be internalized

through BCR but not through XPR1. This is also the case for the activation of pDC following

the interaction of ERV-anti-gp70 IC with FcγR. Thus, if high titers of ERVs are produced by

the presence of the Sgp loci, this might be sufficient to trigger anti-gp70 autoimmune

responses in mice predisposed to SLE. These autoimmune responses can be further

accentuated during the course of SLE as a result of activation of pDC and macrophages in

response to IgG IC containing nuclear antigens and ERVs, thereby accelerating the

development of lupus nephritis.

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Figure 9: Possible activation by ERVs of pDC and anti-gp70 autoreactive B cells through

TLR7. A. IgG anti-gp70-ERV IC can be internalized through FcγR and subsequently interacts

with endosomal TLR7 in pDC. ERVs bearing PT or mPT gp70 can be also internalized

through the XPR1 entry receptor, leading to the activation of endosomal TLR7. B. Specific

recognition of ERVs by anti-gp70 BCR on autoreactive B cells can lead to their

internalization and to activation of TLR7.

pDC

FcγγγγR

TLR7

TLR7 Signaling

Nucleus

αααα-gp70

Endosome


Nucleus

TLR7

Anti-gp70 B cell

A

B

.

.

.

.

.

.

.

.

Retrovirus

αααα-gp70BCR

Retrovirus

pDC

FcγγγγR

TLR7

TLR7 Signaling

Nucleus

αααα-gp70

Endosome


Nucleus

TLR7

Anti-gp70 B cell

A

B

..

..

..

..

..

..

...

...

Retrovirus

αααα-gp70BCR

Retrovirus

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It has been shown that neonatal infection with a murine leukemia virus isolated from

NZB mice induced a lupus-like autoimmune syndrome in (BALB/c x NZB)F1 mice, although

the genetic origin of this virus was not studied (232). In addition, the possible importance of

ERVs as a triggering factor for autoimmune responses in SLE has also been suggested by the

production of anti-nuclear autoantibodies in Sgp3 and GIX congenic mice (248-250).

Furthermore, a more recent study has shown that Raltegravir, a drug which inhibits retroviral

integrase, induced accumulation of pre-integration cDNA of ERVs, which may increase type I

IFN responses, thereby accelerating the development of kidney disease in lupus-prone (NZB x

NZW)F1 mice (251). This finding is consistent with the demonstration that the absence of 3’

repair exonuclease 1 (Trex1) contributes to the development of lupus-like autoimmune

syndrome (252). Collectively, all theses results further support the implication of ERVs in

SLE.

In addition to the contribution of TLR7 and TLR9 to the development of autoimmune

responses against nuclear antigens as well as retroviral gp70, we observed that the stimulation

of TLR7 and TLR9 induced high levels of serum gp70 in NZB mice in kinetics identical to

those induced by LPS or inflammatory cytokines. Notably, activation of TLR7 and TLR9 in

monocytes/macrophages induced the secretion of IL-6 and TNFα (253, 254), both of which

are a good inducer of APP. These data indicate that TLR7 and TLR9 are implicated in the

acute phase expression of serum gp70. Thus, we can speculate that DNA- and RNA-

containing IgG IC activate macrophages through interaction with FcγR and then TLR7 and

TLR9, which induce secretion of cytokines such as IL-6 and TNFα, acting as a positive

feedback on the production of serum gp70 and ERVs. Thus, TLR7 and TLR9 display dual

effects on the development of SLE. On one hand, they promote autoimmune responses

against nuclear and retroviral antigens through the activation of autoreactive B cells as well as

pDC, and on the other hand, they enhance the production of serum gp70 in the presence of the

Sgp loci, thereby providing an additional source for antigenic stimulation and for

nephritogenic IC formation.

All these results allow us to propose the following mechanism (Figure 10). The

expression of ERVs depends on their presence in the genome and the site of integration or

transcriptional regulation. Xeno, PT and mPT viruses contribute to steady-state levels of

serum gp70. Sgp3 and Sgp4 are the major genetic loci controlling the expression of serum

gp70 and ERVs. ERVs internalized through XPR1 receptor and/or FcγR stimulate TLR7

signaling cascade in pDC. Activated pDC aggravate autoimmune process, leading to increases

of various autoantigen-autoantibody IC, such as DNA-anti-DNA IC, implicated in lupus

nephritis. Furthermore, retroviral gp70 can be recognized by anti-gp70 BCR resulting in the

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activation of gp70-specific autoreactive B cell through TLR7 signaling. The production of

IgG anti-gp70 autoantibodies and subsequent formation of gp70 IC further contributes to the

development and progression of lupus nephritis. In addition, IgG IC containing nucleic acids

activate macrophages via TLR signaling, resulting in the production of inflammatory

cytokines, which further enhance the production of serum gp70 and ERVs.

Figure 10: Model of the implication of ERVs in murine SLE.

A possible contribution of ERVs to the development of human SLE has long been

suspected. With the use of polyclonal antibodies raised against murine and feline leukemia

viruses, the presence of an antigen related to mammalian retroviral core protein, p30, was

reported in immune deposits of glomerular lesions from human SLE patients (255). In

addition, the presence of free anti-gp70 antibodies of the simian sarcoma virus-simian

sarcoma-associated virus (256) or woolly monkey type C virus has been described in humans

(257). However, the search for the presence of serum retroviral gp70 (or its counterpart) and

for gp70 IC has, until now, not been successful in human SLE patients. One possible

explanation for this failure may be a lack of appropriate antibodies to specifically detect

retroviral gp70 antigens implicated in human SLE.

Nevertheless, a member of human ERVs, called multiple sclerosis (MS)-associated

retroviral agent (MSRV), was isolated in leptomeninges, choroid plexus and monocyte

cultures of MS patients (258-261). The MSRV Env protein was shown to stimulate activation

Retrovirus

Provirus

gp70

Anti-gp70

gp70-αααα-gp70 IC

Lupus Nephritis

Sgp3/4 Sgp3

TLR7

Nba2

Anti-DNA

DNA-αααα-DNA IC

TLR7

Nba2

TLR7TLR7

MacrophagesTLR7/9Retrovirus

Provirus

gp70

Anti-gp70

gp70-αααα-gp70 IC

Lupus Nephritis

Sgp3/4 Sgp3

TLR7

Nba2

Anti-DNA

DNA-αααα-DNA IC

TLR7

Nba2

TLR7TLR7

MacrophagesTLR7/9

- 169 -

of T lymphocytes (262) and the production of inflammatory cytokines (263-265), suggesting

that MSRV are involved in the pathogenesis of MS. Moreover, the possible role of Xeno

murine leukemia virus-related virus (XMRV) has recently been claimed in the pathogenesis

of human prostate cancer and chronic fatigue syndrome (CFS) (266, 267). XMRVs were

linked to prostate cancer in patients deficient for ribonuclease L (RNase L), which is an

effector of innate anti-viral responses (266). R462Q RNase L variant, showing a decreased

activity compared to wild-type enzyme, was found in 13% of prostate cancer cases, and 40%

of patients homozygous for the R462Q allele harbored the genome of XMRV. Furthermore,

XMRV sequences essentially identical to those isolated from patients with prostate cancer

were found in 67% of CFS patients (267). Infectious XMRVs were detected in activated B

and T cells of CFS patients. CFS patient-derived XMRVs were infectious either by cell-

associated (through co-culture) or cell free (through the plasma) transmission. In contrast to

the prostate cancer study, CFS study did not reveal any link between XMRV infection and

RNase L polymorphism. It is worth noting that the XMRV sequence is not found in human

genome, suggesting that XMRVs must have been acquired exogenously from rodents.

However, transfer of XMRV from rodent to human would require unlikely high levels of

rodent exposure for our society. Therefore, it has been suggested that XMRV may have been

resident in the human population for some time (266). Clearly, further studies are awaited to

define whether XMRVs are indeed a contributing factor in the pathogenesis of prostate cancer,

CFS and possibly other human diseases. Nevertheless, the possible role of MSRV or XMRVs

in the pathogenesis of different human diseases argues in favor of a possible contribution of

either human or murine retroviruses to human SLE. Further research on molecular basis

responsible for the expression of ERVs implicated in murine SLE will enable us to address

the relevance of their human counterparts, thus providing a clue for a potential role of ERVs

in human SLE.

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