Transcript R. Mantegazza

Is there a link between innate
and autoimmunity in MG?
Renato Mantegazza
Dept. of Neuroimmunology and
Neuromuscular Diseases
International Conference on Myasthenia Gravis
Paris, December 2-3, 2009
Innate 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.
QuickTime™ and a
decompressor
are needed to see this picture.
Toll-like Receptors (TLRs): from “self protection” to “self destruction”?
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]
Overexpression of TLR4 mRNA in MG thymuses with thymitis
and thymic involution
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
►
Analysis of TLR4 transcript levels in
hyperplasia, involuted thymus, thymitis and
thymoma by real-time PCR.
Bernasconi P et al Am J Pathol 2005;167:129-139
Immunolocalization of TLR4 protein in MG thymus
Thymitis
Involuted thymus
 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
Bernasconi P et al Am J Pathol 2005;167:129-139
 B cells were never TLR4+
Increased expression of TLR7 and TLR9 in non-neoplastic MG thymus
TLR7
p=0.023
p=0.008
p=0.003
Thymic pathology
TLR9
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
p=0.018
p=0.030
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
Immunohistochemistry: TLR7 and TLR9
TLR7
Normal
Hyperplasia
Thymitis
Involuted
HC
TLR9
Normal
Hyperplasia
Thymitis
Involuted
Up-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).
Expression values for MG pathological thymus are normalized with respect to normal thymus (calibrator).
Cytokines
IL-2
IL-6
TNF-a
IFN-g
TNF-b
GITR
Up-regulation of pro-inflammatory chemokines in MG thymus
CCR4
CXCR3
CCR7
6
5
4
3
2
1
0
Thymitis
MCP1
Thymoma
Involute d
MIP3c
90
8
80
7
70
6
60
5
50
4
40
30
3
20
2
10
1
0
Thymitis
Thymoma
Involute d
Hype rpla sia
0
T h ymitis
T h ymo ma
In vo lu te d
Hyp e rp la sia
Expression values for MG pathological thymus are normalized with respect to normal thymus (calibrator).
Hype rpla sia
In vitro study: response to TLR4 stimulation by LPS in MG TECs
TLR4 mRNA relative value
3,5
HYPERPLASIA
3,0
2,5
INVOLUTED
2,0
THYMOMA
1,5
1,0
0,5
0,0
0
3
6
time (hours)
24
12
CCL22 mRNA relative value
IL6 mRNA relative value
10
HYPERPLASIA
HYPERPLASIA
10
8
INVOLUTED
INVOLUTED
8
6
THYMOMA
6
THYMOMA
4
2
0
0
3
6
24
time (hours)
4
2
0
48
0
7
3
6
time (hours)
24
48
20
6
LPS
18
HYPERPLASIA
IL6 mRNA relative value
AChR a mRNA relative value
TECs from hyperplasia
48
5
LPS+CYP
16
14
INVOLUTED
4
12
THYMOMA
10
3
2
1
8
6
4
2
0
0
0
3
6
time (hours)
24
48
0h
3h
6h
time (hours)
24h
48h
TLR4 stimulation: a pro-inflammatory response by TECs
Quantification of cytokines, growth factors and chemokines in supernatants of TECs
hyperplastic MG thymus by Bio-Plex system (Bio-Rad)
Cytokines and growth factors
oh
24h LPS
300
50
* *
48h LPS
45
*
250
25000
160
24500
140
24000
120
100
30
25
20
15
23500
23000
22500
*
5
21500
20
0
21000
0
120
160
30
100
1000
* *
140
*
25
80
60
40
G-CSF pg/ML
120
IFN-g pg/ML
IL-15 pg/ML
*
400
60
40
100
80
60
PDGF pg/ML
1200
80
22000
0
600
100
10
50
800
TNF-a pg/ML
150
IL-6 pg/ML
35
200
IL-2 pg/ML
IL-1b pg/ML
40
20
15
10
40
*: p<0.05
200
20
0
0
20
0
5
0
*
from
TLR4 stimulation: a pro-inflammatory response by TECs
Chemokines
20000
80
MIP-1a pg/ML
70
14000
MCP-1
*
25
16000
12000
10000
8000
6000
*
60
20
MIP-1b
18000
90
30
* *
15
50
40
30
10
20
4000
5
10
2000
0
* * **
140
20000
15000
10000
IP-10 pg/ML
IL-8 pg/ML
120
100
80
60
40
5000
0
*: p<0.05
**: p<0.005
*
800
*
700
RANTES pg/ML
160
30000
25000
0
0
600
500
400
300
200
20
100
0
0
*
*
The TLRs differential/over
expression within thymus and
the TLR4 ability to promote proinflammatory molecules may
suggest a link between innate
immunity and autoimmunity in
Myasthenia Gravis
We then searched for the presence of
viruses within thymus
QuickTime™ and a
decompressor
are needed to see this picture.
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
B cell and plasma cell localization in normal and MG
non-neoplastic thymus
GC
GC
GC
GC
GC
A lot of B lymphocytes and plasma cells in MG thymus
Cavalcante P et al Ann Neurol, in press
EBV infection: persistence and reactivation
LATENT PHASE
EBNA1, 2, 3a,
3b, 3c,
LMP1, 2a, 2b
EBNA1,
LMP1, 2a
EBNA1,
+
EBERs: small
non coding RNAs
expressed in
nearly all EBVinfected cells and
in all forms of
latency
LMP 2a
17 thymuses from MG patients:
BZLF1, BFRF1
p160
gp350/220
LYTIC PHASE
6 with follicular hyperplasia
6 with diffuse B cell hyperplasia
5 with thymic involution
Cavalcante P et al Ann Neurol, in press
MG thymus with follicular hyperplasia
Control
thymus
Cavalcante P et al Ann Neurol, in press
Expression of EBV RNA and latency and lytic phase proteins
Hyperplasia
with GC
Thymitis
(diffuse hyperplasia)
Involuted
Thymus
+
[in follicles]
+
scattered in med. inf.
+
scattered or [in GC]
+
GC & perifollicular
+
scattered in med. inf.
+
scattered or [in GC]
+
around GC
+
scattered
++
 n. in B cell inf.
-
occasionally
+/-
rare
++
+
EBER
(in situ hyb.)
LMP1/2A*
(Latency program)
BMRF1/BFRF1
(Early lytic phase)
& CD138
p160/gp350-220
(Late lytic phase)
* LMP2A expressed in a large proportion of intrathymic B cells
Cavalcante P et al Ann Neurol, in press
CD8+ T cells and NK cells in MG thymus
Cavalcante P et al Ann Neurol, in press
Plasmacytoid dendritic cells (pDCs)
Blood dendritic cell antigen-2 (BDCA-2) marker for pDC
Control
Hyper.
with GC
Diffuse
Hyper.
Involuted
thymus
Cavalcante P et al Ann Neurol, in press
Conclusions on EBV studies
 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.
Comments on EBV in MG thymus

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 EBVinfected 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.
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
We also searched for TLR-related
viruses within MG thymus
QuickTime™ and a
decompressor
are needed to see this picture.
Detection of Poliovirus (PV) in MG Thymus
Analysis of MG thymi (n = 29) for the presence of cytomegalovirus, varicellazoster 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
Lanes 1-4: EV-positive MG thymi
Lane 5: EV-negative MG thymus
Lane 6: Normal thymus
Lane 7: Positive control
b-actin (lanes 1’-6’): internal control
for cDNA integrity
Cavalcante P et al Neurology, submitted
b. Minus- and plus-strand PV RNAs were detected in
MG thymus specimens suggesting a persistent thymic
infection.
TLR4 expression in PV-positive thymoma and
thymitis
PV+ Thymoma
PV+ Thymitis
 Co-localization of TLR4
on MØs (CD68+) both in
thymitis and thymoma
 TLR4 expression more
prominent in thymitis
 No TLR4/CD68 (MØs) costaining in PV negative
thymitis, thymoma and
normal thymus
Cavalcante P et al Neurology, submitted
VP1/TLR4 double positive cells detected in PV-positive MG
thymuses
Thymitis
PV+ thymoma
VP1/CD68
VP1/CD68
PV+ thymitis
(panel D toH)
VP1/CD68
VP1/CD68
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
Cavalcante P et al Neurology, submitted
COMMENTS
 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.
COMMENTS
 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.
Genetics
Immune dysregulation
Environmental factors
Thymus dedicated
MG Group
Collaborations:
Francesca Andreetta, PhD
Ospedali Riuniti di Bergamo, Bergamo
Carlo Antozzi, MD
Lorenzo Novellino, MD
Fulvio Baggi, PhD
Massimo Barberis, MD
Pia Bernasconi, PhD
Paola Cavalcante, PhD
Istituto Superiore di Sanità
Patrizia Carnevale, Nurse
Francesca Aloisi, PhD
Lorenzo Maggi, MD
Barbara Serafini, PhD
Ornella Simoncini, Technician
CNRS UMR 8162 Université Paris-Sud, Paris
Sonia Berrih-Aknin, PhD