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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