international conference on myasthenia gravis paris, december 2-3, 2009 is there a link between...

International Conference on Myasthenia Gravis

Paris, December 2-3, 2009

Is there a link between innate and autoimmunity in MG?

Renato Mantegazza Dept. of Neuroimmunology and

Neuromuscular Diseases

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Innate Immunity & AutoimmunityInnate Immunity & Autoimmunity

Innate immunity is the first line of defense against infection.

The characteristics of the innate immune response include the following:

Responses are broad-spectrum (non-specific) There is no memory or lasting protective

immunity There is a limited repertoire of recognition

molecules The responses are phylogenetically ancient

Potential pathogens are encountered routinely, but only rarely cause disease.

The vast majority of microorganisms are destroyed within minutes or hours by innate defenses.

The acquired specific immune response comes into play only if these innate defenses are breached.

Toll Like Receptors:

Are central components of innate immune system

Recognize pathogen products and initiate signalling cascades leading to protective immune responses

May participate in alterated pathways leading from “self-protection” to “self-destruction” through the induction of a chronic pro-inflammatory state favourable to autoimmunity [Ehlers M. and Ravetch J.V., Trends Immunol 2007;28:74-79]

Toll-like Receptors (TLRs): from “self protection” to “self destruction”?Toll-like Receptors (TLRs): from “self protection” to “self destruction”?

► Analysis of TLR4 transcript levels in hyperplasia, involuted thymus, thymitis and thymoma by real-time PCR.

► Analysis of TLR4 transcript levels in hyperplasia, involuted thymus, thymitis and thymoma by real-time PCR.

TLR4 mRNA levels(mean ±SD)

HYPERPLASIA 1.58 ± 1.10

INVOLUTED YTHYMUS 8.85 ± 7.13

THYMITIS 9.79 ± 8.43

THYMOMA 3.66 ± 4.21

Bernasconi P et al Am J Pathol 2005;167:129-139

Overexpression of TLR4 mRNA in MG thymuses with thymitis and thymic involution

Overexpression of TLR4 mRNA in MG thymuses with thymitis and thymic involution

Thymitis Involuted thymus

Bernasconi P et al Am J Pathol 2005;167:129-139

TLR4 was detected on CK+ cells in close association with clusters of AChR+ myoid cells in thymic medulla and at the borders between cortical and medullary areas

B cells were never TLR4+

TLR4 was detected on CK+ cells in close association with clusters of AChR+ myoid cells in thymic medulla and at the borders between cortical and medullary areas

B cells were never TLR4+

Immunolocalization of TLR4 protein in MG thymusImmunolocalization of TLR4 protein in MG thymus

Increased expression of TLR7 and TLR9 in non-neoplastic MG thymusIncreased expression of TLR7 and TLR9 in non-neoplastic MG thymus

Thymic pathology TLR7 transcriptional levels(mean ±SD)

Normal 1.14 ± 0.67

Hyperplasia 9.54 ± 4.33

Thymitis 3.84 ± 1.67

Involuted 7.29 ± 4.74

Thymic pathology TLR9 transcriptional levels(mean ±SD)

Normal 1.08 ± 0.45

Hyperplasia 8.48 ± 6.28

Thymitis 2.11 ± 1.46

Involuted 6.71 ± 4.13

p=0.003

p=0.008

p=0.023TLR7

p=0.030

p=0.018TLR9

Immunohistochemistry: TLR7 and TLR9Immunohistochemistry: TLR7 and TLR9

Normal Hyperplasia Thymitis Involuted

TLR7

TLR9

Normal Hyperplasia Thymitis Involuted

HC

0

5

10

15

20

25

30

35

40

45

Thymitis Thymoma Involuted Hyperplasia

IL-2

0

100

200

300

400

500

600

700

800

900


IL-6

0

1

2

3

4

5

6

7

8

9


TNF-

0

2

4

6

8

10

12


IFN- TNF-

0

1

2

3

4

5

6

7

8


GITR

Up-regulation of pro-inflammatory cytokines in MG thymusUp-regulation of pro-inflammatory cytokines in MG thymus

Quantification of cytokine and chemokine expression by Low Density Array (TaqMan LDA microfluid card

technology, Applied Biosystems).

Cytokines

Expression values for MG pathological thymus are normalized with respect to normal thymus (calibrator).

0

1

2

3

4

5

6


CXCR3

0

1

2

3

4

5

6


CCR4

0

0,5

1

1,5

2

2,5

3


MCP1

0

1

2

3

4

5

6

7

8


MIP3c

0

10

20

30

40

50

60

70

80

90


CCR7

Up-regulation of pro-inflammatory chemokines in MG thymusUp-regulation of pro-inflammatory chemokines in MG thymus

Expression values for MG pathological thymus are normalized with respect to normal thymus (calibrator).

In vitro study: response to TLR4 stimulation by LPS in MG TECs In vitro study: response to TLR4 stimulation by LPS in MG TECs

0

2

4

6

8

10

0 3 6 24 48

IL6 m

RN

A r

elat

ive

valu

e HYPERPLASIA

INVOLUTED

THYMOMA

time (hours)

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

0 3 6 24 48

TLR

4 m

RN

A re

lati

ve v

alue HYPERPLASIA

INVOLUTED

THYMOMA

time (hours)

0

1

2

3

4

5

6

7

0 3 6 24 48

AChR

m

RN

A r

elat

ive

valu

e

HYPERPLASIA

INVOLUTED

THYMOMA

time (hours)

0

2

4

6

8

10

12

0 3 6 24 48

CCL2

2 m

RN

A re

lati

ve v

alue

time (hours)

TECs from hyperplasia

TLR4 stimulation: a pro-inflammatory response by TECsTLR4 stimulation: a pro-inflammatory response by TECs

*: p<0.05

Quantification of cytokines, growth factors and chemokines in supernatants of TECs from

hyperplastic MG thymus by Bio-Plex system (Bio-Rad)

Cytokines and growth factors

0

5

10

15

20

25

30

PD

GF

pg

/ML

0

50

100

150

200

250

300

IL-1

b p

g/M

L

oh

24h LPS

48h LPS

0

200

400

600

800

1000

1200

IL-1

5 p

g/M

L

0

20

40

60

80

100

120

IFN

-g p

g/M

L

0

20

40

60

80

100

120

140

160

G-C

SF

pg

/ML

0

5

10

15

20

25

30

35

40

45

50

IL-2

pg

/ML

21000

21500

22000

22500

23000

23500

24000

24500

25000

IL-6

pg

/ML

0

20

40

60

80

100

120

140

160

TNF-

a pg

/ML

* * *

*

*

* * **

Chemokines

*: p<0.05

**: p<0.005

*

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

MC

P-1

0

5

10

15

20

25

30

MIP

-1a

pg

/ML

0

10

20

30

40

50

60

70

80

90

MIP

-1b

0

5000

10000

15000

20000

25000

30000

IL-8

pg

/ML

0

20

40

60

80

100

120

140

160

IP-1

0 p

g/M

L

0

100

200

300

400

500

600

700

800

RA

NT

ES

pg

/ML

* **

*

* ***

**

*

TLR4 stimulation: a pro-inflammatory response by TECsTLR4 stimulation: a pro-inflammatory response by TECs

The TLRs differential/over expression within thymus and

the TLR4 ability to promote pro-inflammatory molecules may suggest a link between innate immunity and autoimmunity in

Myasthenia Gravis

The TLRs differential/over expression within thymus and

the TLR4 ability to promote pro-inflammatory molecules may suggest a link between innate immunity and autoimmunity in

Myasthenia Gravis





We then searched for the presence of viruses within thymus

We then searched for the presence of viruses within thymus

A dysregulated EBV infection might be a common feature of autoimmune diseases

characterized by B cell activation and lymphoid neogenesis in pathological

tissues

A dysregulated EBV infection might be a common feature of autoimmune diseases

characterized by B cell activation and lymphoid neogenesis in pathological

tissues

Therefore it was straightforward for us to search for EBV in the pathological thymus associated with MG

Therefore it was straightforward for us to search for EBV in the pathological thymus associated with MG

GC

GC

GC

GCGC

Cavalcante P et al Ann Neurol, in press

B cell and plasma cell localization in normal and MG non-neoplastic thymus

B cell and plasma cell localization in normal and MG non-neoplastic thymus

A lot of B lymphocytes and plasma cells in MG thymus

EBNA1, 2, 3a,

3b, 3c,

LMP1, 2a, 2b

EBNA1,

LMP1, 2aEBNA1,

LMP 2a

BZLF1,

BFRF1

p1

60

gp

35

0/2

20

EBV infection: persistence and reactivationEBV infection: persistence and reactivation

17 thymuses from MG patients:

6 with follicular hyperplasia

6 with diffuse B cell hyperplasia

5 with thymic involution

17 thymuses from MG patients:

6 with follicular hyperplasia

6 with diffuse B cell hyperplasia

5 with thymic involution

EBERs: small non coding RNAs expressed in nearly all EBV-infected cells and in all forms of latency

+

LYTIC PHASE

LATENT PHASE


Control thymus

MG thymus with follicular hyperplasiaMG thymus with follicular hyperplasia


Expression of EBV RNA and latency and lytic phase proteinsExpression of EBV RNA and latency and lytic phase proteins

* LMP2A expressed in a large proportion of intrathymic B cells

Hyperplasia Thymitis Involuted

with GC (diffuse hyperplasia) Thymus

EBER + + +

(in situ hyb.) [in follicles] scattered in med. inf. scattered or [in GC]

LMP1/2A* + + +

(Latency program) GC & perifollicular scattered in med. inf. scattered or [in GC]

BMRF1/BFRF1 + + ++

(Early lytic phase) around GC scattered n. in B cell inf.

& CD138 - occasionally +/-

p160/gp350-220 rare ++ +(Late lytic phase)


CD8+ T cells and NK cells in MG thymusCD8+ T cells and NK cells in MG thymus


Blood dendritic cell antigen-2 (BDCA-2) marker for pDC

Control Hyper. with GC

Diffuse Hyper.

Involuted thymus

Plasmacytoid dendritic cells (pDCs)Plasmacytoid dendritic cells (pDCs)


Abnormal accumulation of EBV (B cells/plasma cells) in MG but not in control thymus.

Both EBV latent (LMP1, 2a) and lytic (BFRF1, BMFR1) proteins are expressed in MG thymus

GC are the main sites of viral persistence, although a high frequency of EBV+ B cells are seen throughout the thymic medulla.

GC of the MG thymus are devoid of CD8+ T cells, NK cells and pDCs; this might indicate that GC represent immunoprivileged niches of EBV persistence.

Conclusions on EBV studiesConclusions on EBV studies

Dysregulated EBV infection in the pathological thymus is a common feature in MG and could contribute to the immunological alterations initiating and/or perpetuating the disease

Dysregulated EBV infection in the pathological thymus is a common feature in MG and could contribute to the immunological alterations initiating and/or perpetuating the disease

EBV gene expression is aberrantly regulated in MG thymus

An abnormal viral load in the thymus might contribute to the chronic B cell activation observed in this organ in MG.

Similarity between the MG thymus and the MS brain, with respect to the high proportion of tissue-infiltrating EBV-infected B cells and the formation of EBV-enriched ectopic lymphoid tissue, supports the idea that EBV might be pathologically relevant for autoimmune diseases characterized by B cell abnormalities.

Comments on EBV in MG thymusComments on EBV in MG thymus

We also searched for TLR-related viruses within MG thymus

We also searched for TLR-related viruses within MG thymus





Analysis of MG thymi (n = 29) for the presence of cytomegalovirus, varicella-zoster virus, herpes simplex virus types 1 and 2, eubacterial 16S rDNA,

respiratory syncytial virus and enteroviruses

Analysis of MG thymi (n = 29) for the presence of cytomegalovirus, varicella-zoster virus, herpes simplex virus types 1 and 2, eubacterial 16S rDNA,

respiratory syncytial virus and enteroviruses

Detection of poliovirus in four (13.8%) MG thymi (two with thymitis and two with thymoma)

a. Detection of enterovirus RNA in MG thymus specimens by nested reverse transcription PCR

a. Detection of enterovirus RNA in MG thymus specimens by nested reverse transcription PCR

Lanes 1-4: EV-positive MG thymi

Lane 5: EV-negative MG thymus

Lane 6: Normal thymus

Lane 7: Positive control

-actin (lanes 1’-6’): internal control for cDNA integrity

b. Minus- and plus-strand PV RNAs were detected in MG thymus specimens suggesting a persistent thymic infection.

b. Minus- and plus-strand PV RNAs were detected in MG thymus specimens suggesting a persistent thymic infection.

Cavalcante P et al Neurology, submitted

Detection of Poliovirus (PV) in MG ThymusDetection of Poliovirus (PV) in MG Thymus

PV+ Thymoma

PV+ Thymitis

PV- Thymoma

PV- Thymitis

PV- norm

al

thymus

Co-localization of TLR4 on MØs (CD68+) both in thymitis and thymoma

TLR4 expression more prominent in thymitis

No TLR4/CD68 (MØs) co-staining in PV negative thymitis, thymoma and normal thymus

Co-localization of TLR4 on MØs (CD68+) both in thymitis and thymoma

TLR4 expression more prominent in thymitis

No TLR4/CD68 (MØs) co-staining in PV negative thymitis, thymoma and normal thymus

TLR4 expression in PV-positive thymoma and thymitis

TLR4 expression in PV-positive thymoma and thymitis


PV+ thymoma

PV+ thymitis (panel D toH)

PV- th

ymom

a

PV- th

ymiti

s

Co-localization of VP1 on CD68+ cells (MØs) both in thymitis and thymoma; preferential localization in medulla and around Hassal’s corpuscles

Co-localization of VP1 and TLR4 both in thymitis and thymoma

Co-localization of VP1 on CD68+ cells (MØs) both in thymitis and thymoma; preferential localization in medulla and around Hassal’s corpuscles

Co-localization of VP1 and TLR4 both in thymitis and thymoma

Thymitis

Thymoma

VP1/CD68 VP1/CD68

VP1/CD68VP1/CD68

VP1/TLR4 double positive cells detected in PV-positive MG thymuses

VP1/TLR4 double positive cells detected in PV-positive MG thymuses


We provided a demonstration of persistent Poliovirus infection in the thymus of some MG patients.

Our findings are consistent with a viral infection in thymus and a persistent activation of TLR4 signalling could create an intra-thymic inflammatory status favourable to the initiation or perpetuation of the autoimmune response in a susceptible background.

We provided a demonstration of persistent Poliovirus infection in the thymus of some MG patients.

Our findings are consistent with a viral infection in thymus and a persistent activation of TLR4 signalling could create an intra-thymic inflammatory status favourable to the initiation or perpetuation of the autoimmune response in a susceptible background.

COMMENTSCOMMENTS

Results from TLR and viruses (EBV, Polio) suggest a link between innate immunity and autoimmunity in MG.

These findings retrieve the viral hypothesis as a crucial step in MG pathogenesis.

Although, the complete fitting of the elements of the puzzle is still missing.

Results from TLR and viruses (EBV, Polio) suggest a link between innate immunity and autoimmunity in MG.

These findings retrieve the viral hypothesis as a crucial step in MG pathogenesis.

Although, the complete fitting of the elements of the puzzle is still missing.

COMMENTSCOMMENTS

Environmental factorsImmune dysregulation

Genetics

MG Group

Francesca Andreetta, PhD

Carlo Antozzi, MD

Fulvio Baggi, PhD

Pia Bernasconi, PhD

Paola Cavalcante, PhD

Patrizia Carnevale, Nurse

Lorenzo Maggi, MD

Ornella Simoncini, Technician

Thymus dedicated

Collaborations:

Ospedali Riuniti di Bergamo, Bergamo

Lorenzo Novellino, MD

Massimo Barberis, MD

Istituto Superiore di Sanità

Francesca Aloisi, PhD

Barbara Serafini, PhD

CNRS UMR 8162 Université Paris-Sud, Paris

Sonia Berrih-Aknin, PhD

international conference on myasthenia gravis paris, december 2-3, 2009 is there a link between...

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