the impact of siv infection on gut innate lymphocyte populations and gene expression

www.ias2013.org Kuala Lumpur, Malaysia , 30 June - 3 July 2013

The impact of SIV infection on gut innate lymphocyte populations and gene expression

Ronald S. Veazey, DVM, PhDDivision of Comparative Pathology

Tulane National Primate Research CenterCovington, Louisiana

Tulane University School of MedicineNew Orleans, Louisiana


Differences between innate and adaptive immunity?

• Adaptive immunity previously defined as having ”immunologic memory” but lines are increasingly blurred – innate lymphoid cells (ILC) do not have antigen specific receptors (CD3)

• Emerging evidence shows innate lymphoid cells develop independently in GALT, and develop immunologic memory

• Murine studies indicate GALT develops through immune responses to bacteria mediated through specialized ”Lymphoid tissue inducer” (Lti), possible stem cells, and other innate lymphoid cell (ILC) subsets


Innate lymphoid cellsNewly proposed nomenclature:

• Group 1: ILC1: ILCs that produce IFNγ. Prototypical member is the NK cell. NK cells display both cytotoxic activity, and produce IFNγ following activation.

• Group 2: ILC2: ILCs that produce type 2 cytokines (including IL-5 and IL-13) and are dependent on GATA-binding protein 3 (GATA3) and retinoic acid receptor- related orphan receptor-α (RORα) for development and function.

• Group 3: ILC3: ILCs that produce IL-17 and/or IL-22 and depend on the transcription factor RORγt for development and function. Lymphoid tissue inducer cells (LTi) are prototype.

Spits, H., D. Artis, et al. (2013). "Innate lymphoid cells--a proposal for uniform nomenclature." Nat Rev Immunol 13(2): 145-149.

“ILCs should be categorized into three groups based on the cytokines that they can produce and the transcription factors that regulate their development and function”


Innate lymphoid cells• Proinflammatory retinoic-acid-receptor-related orphan

receptor-gammat-positive (RORgammat+) innate lymphoid cells (ILCs) differentiate from distinct fetal liver RORgammat(+) precursors and are crucial for immune homeostasis (ILC17, 22, etc.). Sawa, Eberl et al., 2010. Lineage relationship analysis of RORgammat+ innate lymphoid cells. Science 330:665-669.

• Mucosal (RORgammat(+)innate lymphoid cells (ILCs) are an important innate lymphocyte population required for immunity to intestinal infections Klose, Diefenbach, et al., 2013. A T-bet gradient controls the fate and function of CCR6-RORgammat+ innate lymphoid cells. Nature 494:261-265.

• ILC regulate CD4+ T-cell responses to intestinal bacteria. Hepworth, Sonnenberg et al, 2013. Innate lymphoid cells regulate CD4+ T-cell responses to intestinal commensal bacteria. Nature 498:113-117.


PLoS Pathog. 2012 September; 8(9) Loss of Effector and Anti-Inflammatory Natural Killer T Lymphocyte Function in Pathogenic Simian Immunodeficiency Virus Infection. Namita Rout,…Amitinder Kaur

PLoS Pathog. 2012;8(8) ADCC develops over time during persistent infection with live-attenuated SIV and is associated with complete protection against SIV(mac)251 challenge. Alpert MD, ….Evans DT.

Blood. 2010 Jun 3;115(22):4439-46. Epub 2010 Mar 25.CD16- natural killer cells: enrichment in mucosal and secondary lymphoid tissues and altered function during chronic SIV infection. Reeves RK,……Johnson RP.

Blood. 2011 Sep 22;118(12):3321-30. Epub 2011 Jul 26.Gut inflammation and indoleamine deoxygenase inhibit IL-17 production and promote cytotoxic potential in NKp44+ mucosal NK cells during SIV infection. Reeves RK, …Johnson RP.

Recent studies of NK / ILC in SIV-infected macaques


Phenotyping innate lymphoid cells (ILC) in normal macaque blood(Xu, Veazey et al, Mucosal Immunol 2012)

Gating strategy: Lineage negative, CD3negCD8++

C3negCD8a neg(DC, B cells, etc.)

C3negCD8a HIGH(ILC’s)


SIV/IgG immune complexes in SIV-infected macaques block detection of CD16 but not cytolytic activity of natural killer cells. Wei and Fultz, et al, Clin and Vacc Immunol 2006

Comparison of anti-human CD16 mAb clones on SIV-infected macaque cells before and after “washing”

CD16 detection is artificially masked by some anti-SIV immune complexes

CD16 clone DJ130 (Dako) best results


Lymphocytes IL-17+

IL-17 CD3

CD3+CD4+(Th17)

CD3+CD8+(Tc17)

CD3-CD8high

(ILC)

IL-17

PBMC Jej LPL Colon LPL Spleen Tonsil Duodenum

ILC that secrete IL-17 are restricted to mucosal tissues in macaques


IL-17 secreting cells in jejunum of normal rhesus macaques

Blood. 2008 Oct 1;112(7):2826-35. Differential Th17 CD4 T-cell depletion in pathogenic and nonpathogenic lentiviral infections. Brenchley…Douek, et al.

*


Intestinal ILC17 cells are depleted in SIV infection


Summary:

Some ILC17 (ILC3) cells secrete IL-22, TNF-a, but not IFN-g nor granzyme B (not cytolytic)

SIV infection results in significant loss of ILC17 cells, especially in the jejunum, which persists throughout SIV infection.

Loss of ILC17 cells (and IL-17 in general) may contribute to loss of intestinal mucosal integrity and disease progression in human immunodeficiency virus (HIV)/SIV infection.


Small Intestine (Jejunum)

Epithelial cells (Enterocytes)

Intraepithelial Lymphocytes

Lamina propria Cells (LPC’s)

Fibrovascular stroma

Approach to reduce tissue complexitySeparation by Percoll gradients:

SIV infectionpre infection (6 weeks)Resection biopsy 6-8 cm

21 d post infectionResection biopsy 6-8 cm

90d post infectionResection biopsy 6-8 cm

N=5

Changes in intestinal gene expression in SIV: Experimental Design


Genome wide changes in the jejunum lamina propria in SIV infection

(measured by Affymetrix rhesus arrays - 54,675 capture probes)

1) Intestinal lamina propria


Upregulation of multiple genes associated with immune activation


Downregulated genes associated with oxidative phosphorylation, IFNg, IL-17, B cell “help”


Gene changes in lamina propria in chronic (90 days) SIV infection


LBP (lipopolysaccharide binding protein) Expressed by macrophages and Paneth cells in response to LPS LBP-LPS complex together with CD14 activates TLR4 pathway

CD70 Cytokine that belongs to the TNF family of ligands Expressed only on activated T cells (CD4 & CD8) and binds to CD27 (receptor) Induces proliferation of co-stimulated T cells May bind CD27 on memory B cells and induce plasma cell differentiation

(hypergammaglobulinemia) CD38

Ectoenzyme and an activation marker for CD4, CD8 and B cells JNK3 (Jun-N-terminal kinase 3)

Activated by LPS and proinflammatory cytokines

Important Up-regulated genes at 90 d PI


CXCL18 Chemotactic for naïve T cells and non activated lymphocytes May be a protective response (moderation of inflammation)

TLR8 Binds ssRNA (HIV/SIV) Signaling induces IFNa production leading to immune activation Downregulation may be a protective response or cellular dysfunction

(DC, mac)IL-8

Produced mainly by macrophages, dendritic cells Can Inhibit HIV replication in PBMCs and ectocervical tissue explants

AICD Required for somatic hypermutation and class switch recombination

Important Down-regulated genes at 90 d PI


Intestinal Epithelium

PLoS One. 2013; Intestinal Epithelium Reveals Transcriptional Signatures Consistent with Disturbances in Enterocyte Maturation and Differentiation during the Course of SIV Infection. Mohan, Veazey, Lackner et al.


Gene changes in intestinal epithelium in acute (21 days) SIV infection


Transcription factors and signaling pathways known to regulate intestinal epithelial gene expression

1. Wnt-TCF7L2 /TCF4 signaling: Crypt cell proliferation Paneth cell differentiation Directs epithelial cell migration along the Villi

2. NOTCH signaling: Crypt cell proliferation and cell fate decisions

3. Sonic and Indian Hedgehog signaling: Crypt formation, spacing and villus development

4. EPH/Ephrins: Regulated by Wnt signaling: Progenitor cell migration up the crypt

5. PTEN/PI3K: Crypt stem cell renewal

6. BMP signaling: Crypt stem cell proliferation independent of Wnt signaling

7. LKB1 signaling: Regulates epithelial cell polarity


WNTDkk

CK1

GSK3

Dsh/Dvl

CK1

GBP

P

CK1LR

P

Axin

-catenin

TCF4/LEFCBP

-cateninBCL9Pygo

GSK3

Dsh/Dvl

-catenin APCUb

-TrCP

Target GenesMyc, Cyclin D1TCF1, PPAR-MMP-7, Axin-2CD44

Axin

Frizzled

Wnt-catenin-TCF7L2 signaling pathway

Groucho

Downregulated d21:Wnt 10AFrizzledTCF7/TCF4


Gene changes in intestinal epithelium in chronic (90 days) SIV infection


Upregulated genes in the intestinal epithelium at 90 d PI(most involved with epithelial cell proliferation)

NOTCH and NOTCH target genes (HES4, HES7) and EZH2 (histone methyltransferase)

ETS homologous factor: Regulate epithelial cell proliferation and differentiation

FGF4 and 12: Fibroblast growth factor 4 and 12 Stimulates intestinal epithelial cell proliferation

Kruppel like factor 12: Induces cell proliferation (up in invasive gastric cancers)

PI3K regulatory subunit 2 (beta), Inositol polyphosphate-4-phosphatase, type 1 Increases cell survival, growth and proliferation (altered in 40% of colorectal cancers)

Defensin Beta 119 Antimicrobial peptide

Mucin 5B, BMPR1A Upregulated in H. pylori induced gastric disease (anti-microbial ?) Lubricates intestinal contents


Downregulated genes in the intestinal epithelium at 90 d PI(most involved in epithelial differentiation and defense)

Paraxonase: Epithelial Defense response to pathogens

PDX1: Pancreatic and duodenal homeobox 1 Regulates gene expression in Enteroendocrine cells

Focal Adhesion Kinase: Important for epithelial cell motility, survival and healing

Kruppel like factor 6 and 10 A ubiquitous transcription factor that induces cell differentiation

FOXP2 and P4: Epithelial cell specification and differentiation

TGF 3: Induces cell differentiation and possess anti-inflammatory properties

Mucin 13: Cell signaling in epithelial cells

Solute carrier family 5 and 15: Uptake and digestion of tripeptides and monocarboxylates


Tight Junctions Claudin 22 Angiomotin like-1

Adherens Junctions Cadherin 5, 11, 23 Catenin (Cadherin associated protein) alpha 1 (Anchor protein links Cadherins to actin filament)

Desmosomes Desmoglein 2: Component of Desomosomes Desmocollin: Component of Desomosomes Junctional Plakoglobin: Component of Desomosomes

Hemidesmosomes Laminin beta 3, 4, gamma 1, gamma 2

Required for hemidesmosome assembly Binding, attaching and migration of cells (epithelial healing)

Integrin alpha 1, 3 and 6 Receptor for laminins Plays a critical structural role in hemidesmosomes

Cell adhesion Molecules downregulated 90 d PI:


Summary:

Early SIV infection results in upregulation of genes associated with intestinal epithelial apoptosis, proliferation, and repair

Upregulation of numerous genes associated with lymphocyte activation, LPS pathways

Downregulation of most epithelial adhesion / tight junction molecules indicating early barrier dysfunction

Downregulation of anti-microbial genes


Acknowledgements

Tulane National Primate Research CenterHuanbin XuMahesh Mohan Terri RasmussenAndrew Lackner

Northwestern University, ChicagoTom Hope

Queens University BelfastKarl Malcolm

Washington University, MOHerbert “Skip” VirginScott Handley

Case Western Reserve UniversityMichael LedermanEric Arts

Cornell UniversityJohn Moore Pj Klasse

St George’s Hospital, LondonRobin Shattock

NCI-Frederick Jeff LifsonMike Piatak

NIH / NIAIDNancy MillerSusan Plaeger Opendra SharmaJim Turpin

the impact of siv infection on gut innate lymphocyte populations and gene expression

Documents

lymphoid cells ilcs

innate lymphoid cellsnewly

nk cells

possible stem cells

cd4 tcell responses

positive rorgammat

intestinal bacteria

mucosal rorgammat