Toll-like receptor gene polymorphisms are associated with susceptibility to graves' ophthalmopathy in Taiwan males

Graves' disease (GD) is an organ-specific autoimmune thyroid disease, one of the manifestations of which is ophthalmopathy [1]. Graves' ophthalmopathy (GO) is characterized by inflammation and fat deposition in the eye muscles and the connective tissue surrounding the eye. It is known that multiple factors contribute to the etiology and severity of GD, including the host's genetic factors as well as environmental factors [2, 3]. Female sex, old age, and smoking history are common risk factors for GD [4‐8]. With regard to genetic factors, the classical major histocompatibility complex class II genes and cytotoxic T cell antigen-4 genes (CTLA-4) [9‐11] have been consistently reported to be associated with GD. Also, there were published studies on association of GO and genes such as CD103 [12], CTLA-4 and IL-13 [13]. However, the findings of most genes effect in GD or GO were inconsistent. Recently, toll-like receptors (TLRs) which play important roles in eliciting human innate/adaptive immune responses and developing chronic inflammation [14] are a new area of basic immunological investigation and could be associated with autoimmune thyroiditis [15].

TLRs are a family of pattern-recognition receptors and can be expressed in several types of cells and tissues such as macrophages, dendritic cells (DCs), B cells, T cells and monocytes. A total of 10 human TLRs have been identified and the functions of human TLR-1 to TLR-9 have been characterized [16, 17, 14, 15]. TLRs are well known to recognize a variety of microbial molecules such as TLR-4 can recognize lipopolysaccharides (LPSs) in gram-negative bacteria and TLR-9 activation can be triggered by unmethylated CpG DNA of bacteria. Once TLRs are activated by microbial molecules, downstream signalling pathway via myeloid differentiation factor 88 (MyD88) and interleukin-1R (IL-1R)-associated kinase (IRAK) activates nuclear factor κB (NF-κB), leading to cytokine production, and even to apoptosis. TLRs could response to not only exogenous, microbe derived pathogen associated molecular patterns (PAMPs) but also endogenous or self ligands. Recently, the ability of TLRs to recognize host-derived danger signals, which are produced on cell damage and necrosis, was also recognized [18, 19]. Moreover, studies have found that the immune complexes containing self-RNA and/or self-DNA can act as endogenous triggers for the activation of TLRs [20‐22].

Recently, release of endogenous TLR ligands during inflammation and the consequent activation of TLR signaling pathways have been indicated and polymorphism of the TLR4 and TLR9 gene have been reported to be associated with many autoimmune diseases, such as systemic lupus erythematosus (SLE), atherosclerosis, asthma, type 1 diabetes, multiple sclerosis, and rheumatoid arthritis (RA) [14, 23‐31]. However, to the best of our knowledge, there were no reports about TLR4 and TLR9 in GO. Therefore, our aim in the present study was to investigate the potential association between GO and single-nucleotide polymorphisms (SNPs) of TLR genes--TLR-4 and TLR-9 genes--in a Chinese population in Taiwan.

A total of 471 GD patients comprising 200 patients with GO and 271 patients without GO were enrolled in this study. The female to male ratio was 3.75 and the mean age was 39.41 ± 12.42 years. There were statistically significant differences in the GO phenotype and smoking history between the male and female patients. The percentage of male and female GD patients with GO was 52% and 40%, respectively (p = 0.04). None of the frequency distribution for other anatomic sites (goiter, nodular hyperplasia, myxedema and vitiligo) was significant different between two genders. A higher percentage of male patients had smoking history than female patients (66.67% vs. 12.10%, p < 0.0001) (Table 1).

In this study, we investigated the effect of TLR-4 and TLR-9 gene polymorphisms on the organ-specific autoimmune disease GD in a Taiwanese population. TLR-9 polymorphisms and haplotype were significant associated with susceptibility to GO in males. However, none of the TLR-4 and TLR-9 polymorphism or haplotypes was associated with overall GO risk. The TLR4 is known to activate the NF-κB and subsequent gene expression such as cytokines and adhesion molecules. A TLR-4 polymorphism that prevents ligands binding and subsequent cellular signaling would result in lower NF-κB activation and subsequent NF-κB-dependent proinflammatory gene expression. However, we did not find any association between TLR4 polymorphism and GO in present study. Low statistical power may be the reason for this non significant results in TLR4 association in GO. For TLR9, our results were consistent with those of the previous studies with regard to the association between TLR9 gene polymorphisms and systemic autoimmune diseases, such as SLE in Japan [30, 28] and China [38, 36]. From SLE model, Tao et al study [30, 28] found diminished expression of TLR9 could increase anti-dsDNA antibody in SLE mice model which may increase the risk of developing autoimmune disease. Also, it has been hypothesized that nucleic acid from dying cells may act as ligands for TLR9 to trigger IFN-1 production in SLE [39, 37]. In this study, 2 TLR-9 SNPs, one (-1486TàC, rs187084) is in the promoter region and the other (1635AàG, rs352140) is in exon 2. The variation of TLR-9 gene in those region may down regulate TLR9 expression and involved in production of autoantibodies or IFN-1 which may increase the risk of GO. Therefore, future studies are required to investigate if variation of TLR9 gene, especially in exon region may affect TLR9 protein expression or function which increase the risk of GO.

TLR-9 polymorphisms were associated with increase the risk of GO in males in present study. In general, GD is more common among women; however, male sex also faces the risk of developing progressive and severe thyroid-associated orbitopathy [4, 7, 4, 7] or responds poorly to treatment [40, 38]. A previous study has reported a female to male ratio of 9.3 for patients with mild GO and of 3.2 for those with moderate GO, with the ratio being 1.4 for patients with severe GO [4, 4]. Our data suggest that the role of TLR-9, which plays an important role in conferring innate immunity, would be more critical in men with GO. The susceptibility factor seems to play a different role depending on sex, or the effect could be more apparent in men and not in women because of the difference in the basic immune responses between the 2 sexes [41, 39]. Further studies are required to investigate that if the TLR-9 activity and its potential relevance cytokines being more prone to male with GO.

As the smoking is potential confounding factor for susceptibility of GO, we adjusted the variable of smoking history in the multivariate model to eliminate the smoking effect on susceptibility of GO. However, the significant association between TLR-9 gene polymorphism and susceptibility to GO in the male patients was no changed. Furthermore, the strength association between TLR-9 polymorphism and GO in male was modest in present study. Therefore, a larger sample sized, especially in male population will be needed in the future study to confirm the importance of these polymorphisms as genetic markers of GD in Taiwanese population. Furthermore, the interpretation of our study results is limited because the patients were recruited from Taiwan only. Studies in ethnically disparate populations are needed before firm conclusions can be drawn.

In conclusion, our findings suggested that TLR-9 gene polymorphisms were significantly associated with susceptibility to GO in the male GD patients in Taiwan.