Article

Increased Toll-Like Receptor 4 Expression in Thymus of Myasthenic Patients with Thymitis and Thymic Involution

Department of Neurology IV, Istituto Nazionale Neurologico Carlo Besta, Via Celoria 11, 20133 Milan, Italy.
American Journal Of Pathology (Impact Factor: 4.59). 08/2005; 167(1):129-39. DOI: 10.1016/S0002-9440(10)62960-4
Source: PubMed

ABSTRACT

Thymic abnormalities are present in approximately 80% of myasthenia gravis (MG) patients, and the thymus seems to be the main site of autosensitization to the acetylcholine receptor. In view of findings that the innate immune system can generate an autoimmune response, we studied the expression of Toll-like receptors (TLRs) 2 to 5, key components of innate immunity signaling pathways, in 37 thymuses from patients with autoimmune MG. TLR4 mRNA levels were significantly greater in thymitis (hyperplasia with diffuse B-cell infiltration) and involuted thymus than in germinal center hyperplasia and thymoma. By immunohistochemistry and confocal microscopy, cells positive for TLR4 protein were rarely detected in thymoma. However, in thymitis TLR4 protein was mostly found on epitheliomorphic (cytokeratin-positive) cells located in close association with clusters of acetylcholine receptor-positive myoid cells in thymic medulla and also at the borders between cortical and medullary areas. B cells were never TLR4-positive. TLR4 protein was also present in remnant tissue of involuted thymus. This is the first finding of a possible link between innate immunity and MG. We speculate that in a subgroup of MG patients, an exogenous or endogenous danger signal may activate the innate immune system and give rise to TLR4-mediated mechanisms contributing to autoimmunity.

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    • "Besides confirming up-regulated levels of TLR4 mRNA [4], qPCR analysis showed that CCL17 and CCL22 mRNA levels were significantly increased in both follicular and diffuse hyperplastic MG thymuses compared with controls, whereas CCR4 mRNA was increased in follicular hyperplastic MG thymuses only (Fig. 4A). Immunosuppressive treatment does not seem to affect thymic TLR4, CCL22 and CCR4 expression since the mRNA levels did not differ between untreated and corticosteroid-treated patients (Fig. 4B), whereas CCL17 transcriptional levels were significantly reduced in thymuses from corticosteroid-treated MG patients, thus suggesting that immunosuppressive treatment could normalize CCL17 levels in MG thymuses (Fig. 4B). "
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    ABSTRACT: The thymus is the main site of immune sensitization to AChR in myasthenia gravis (MG). In our previous studies we demonstrated that Toll-like receptor (TLR) 4 is over-expressed in MG thymuses, suggesting its involvement in altering the thymic microenvironment and favoring autosensitization and autoimmunity maintenance processes, via an effect on local chemokine/cytokine network. Here, we investigated whether TLR4 signaling may favor abnormal cell recruitment in MG thymus via CCL17 and CCL22, two chemokines known to dictate immune cell trafficking in inflamed organs by binding CCR4. We also investigated whether TLR4 activation may contribute to immunodysregulation, via the production of Th17-related cytokines, known to alter effector T cell (Teff)/regulatory T cell (Treg) balance. We found that CCL17, CCL22 and CCR4 were expressed at higher levels in MG compared to normal thymuses. The two chemokines were mainly detected around medullary Hassall's corpuscles (HCs), co-localizing with TLR4(+) thymic epithelial cells (TECs) and CCR4(+) dendritic cells (DCs), that were present in higher number in MG thymuses compared to controls. TLR4 stimulation in MG TECs increased CCL17 and CCL22 expression and induced the production of Th17-related cytokines. Then, to study the effect of TLR4-stimulated TECs on immune cell interactions and Teff activation, we generated an in-vitro imaging model by co-culturing CD4(+) Th1/Th17 AChR-specific T cells, naïve CD4(+)CD25(+) Tregs, DCs and TECs from Lewis rats. We observed that TLR4 stimulation led to a more pronounced Teff activatory status, suggesting that TLR4 signaling in MG thymic milieu may affect cell-to-cell interactions, favoring autoreactive T-cell activation. Altogether our findings suggest a role for TLR4 signaling in driving DC recruitment in MG thymus via CCL17 and CCL22, and in generating an inflammatory response that might compromise Treg function, favoring autoreactive T-cell pathogenic responses.
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    • "TLR 4 expression is increased in epithelioid cells in inflamed thymus from MG patients[66] and a recent study found that TLR9 levels in peripheral PBMCs from MG patients correlate with clinical severity[67]. TLR3 is a nucleotide-sensing receptor that recognizes double-stranded RNA (dsRNA) and is therefore important for viral detection. "
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    • "Some of them are potent inflammatory molecules mainly involved in acute inflammation (i.e., IL-6, IL-1β, TNF-α, and CSF); others are mainly involved in establishing chronic inflammation and promoting humoral and cellular immune response (i.e., IL-7, IL-10, IL-12, and IFN-γ) [25]. Upregulation of IL-6 and the chemokine RANTES in MG compared to normal thymus was in line with previous studies showing that these genes were abnormally overexpressed in MG TECs either at basal condition [37] or (IL-6) when stimulated by lipopolysaccharide (LPS) [38], a major activator of Toll-like receptor (TLR) 4 known to be upregulated in MG thymus [39]. IL-6 is a well-known proinflammatory agent with pathological regulatory function on growth and differentiation of T- and B-cells [40]; RANTES has been observed to regulate the transepithelial migration of T cells [41]. "
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    ABSTRACT: The thymus plays a major role in myasthenia gravis (MG). Our recent finding of a persistent Epstein-Barr (EBV) virus infection in some MG thymuses, combined with data showing that the thymus is in a proinflammatory state in most patients, supports a viral contribution to the pathogenesis of MG. Aim of this study was to gain further evidence for intrathymic chronic inflammation and EBV infection in MG patients. Transcriptional profiling by low density array and real-time PCR showed overexpression of genes involved in inflammatory and immune response in MG thymuses. Real-time PCR for EBV genome, latent (EBER1, EBNA1, LMP1) and lytic (BZLF1) transcripts, and immunohistochemistry for LMP1 and BZLF1 proteins confirmed an active intrathymic EBV infection, further supporting the hypothesis that EBV might contribute to onset or perpetuation of the autoimmune response in MG. Altogether, our results support a role of inflammation and EBV infection as pathogenic features of MG thymus.
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