骨蝕作用、骨形成、以及免疫耐受性對於台灣人僵直性脊椎炎發生之效應

Ankylosing spondylitis (AS) is a chronic inflammatiory disorder. Inflammation has been reported to promote the differentiation and maturation of the osteoclast, and bone resorption also affects the overexpression of the osteoblast resulting in the bone formation and syndesmophyte. In addition, imbalance of autoimmune tolerance is associated with the occurrence of autoimmune diseases. The higher expression of CD4+ and CD8+ T cells has also been found in AS patients than in healthy controls, and such expression may relate to the imbalance in the negative signal of activated T cells. Receptor activator of nuclear factor kappa B ligand (RANKL) binds to receptor activator of nuclear factor kappa B (RANK) could cause the activation of bone resorption. Osteoprotegerin (OPG) also competes with RANK by binding to RANKL and inhibiting bone absorptions. Therefore, we designed a case-control study to evaluate the association between occurance and clinical features of AS and RANK, RANKL and OPG genetic polymorphisms. A total of 330 AS patients and 330 age- and gender-matched healthy controls were recruited. Results revealed OPG GG genotype carriers had an elevated risk of AS compared with those with the GC and CC genotypes (odds ratio [OR] 1.74, 95% confidence interval [CI] 1.26-2.40). In addition, age of symptom onset and frequency of peripheral arthritis significantly differed among AS patients by different OPG G1181C genotypes. HLA-B27+ patients with the OPG C allele had the earliest age of symptom onset. Therefore, OPG G1181C polymorphism may be associated with AS development and clinical manifestations. MicroRNA (miR)-146a targets TNF-receptor-associated-factor-6 (TRAF-6) and interleukin-1 receptor-associated kinase 1 (IRAK1) to suppress nuclear factor kB (NF-kB) activity. We investigated whether miR-146a rs2910164 G/C, IRAK1 rs3027898 A/C and rs1059703 T/C genetic polymorphisms associated with AS development and clinical characteristics. A total of 450 Taiwanese AS patients and 438 healthy controls were recruited in our study. Pairwise analysis of the miR-146a rs2910164/IRAK1 rs3027898 alleles showed G/A (OR 2.84, 95% CI 1.34-6.03), G/C (OR 1.71, 95% CI 1.27-2.30), and C/A (OR 1.53, 95% CI 1.09-2.16) had a significantly greater risk of AS than the C/C alleles. Such results were found in males, but not in females. In addition, AS patients with miR-146a rs2910164/IRAK1 rs3027898 G/A pairwise alleles had a significantly higher risk of uveitis development than patients with miR-146a rs2910164/IRAK1 rs3027898 C/C pairwise alleles. Therefore, miR-146a rs2910164 and IRAK1 rs3027898 polymorphisms might be associated with the development of AS, as well as its clinical manifestations. Tumor necrosis factor (TNF)-α might induce the inflammation and osteoclastogenesis. In addition, miR-125b expression inhibits the TNF-α expression, and higher miR-155 expression inversely induces TNF-α expression by targeting suppressor of cytokine signaling-1 (SOCS-1). We compared the differences of TNF-α mRNA, miR-125b, miR-155, and SOCS-1 mRNA expressions between AS patients and healthy controls. The influence of rheumatologic drugs on the expressions of these molecules in AS patients was also assessed. Expressions of whole blood TNF-α mRNA, miR-125b, miR-155, and SOCS-1 mRNA were assessed by RT quantitative PCR (RT-qPCR) among 119 AS patients and 120 healthy controls. TNF-α mRNA expression was negatively correlated with miR-125b expression (r = -0.63, p = 0.03); positively correlated with Bath Ankylosing Spondylitis Functional Index (r = 0.82, p < 0.01), erythrocyte sedimentation rate (r = 0.70, p = 0.02), and C-reactive protein (r = 0.79, p < 0.01) in AS patients without taking nonsteroidal antiinflammatory drugs (NSAIDs) or disease-modifying antirheumatic drugs (DMARDs), respectively. But TNF-α mRNA expression was not correlated with bone metabolism in AS patients. The binding of miR-21 and programmed cell death 4 (PDCD4) might inhibit the expression of PDCD4, and further induce the activation of osteoclasts. We compared the difference of miR-21 expression between AS patients and healthy controls, and evaluated the relationships of miR-21, PDCD4 mRNA, and bone erosion in AS patients. The influence of rheumatologic drugs on the expressions of miR-21 and PDCD4 mRNA in AS patients was also assessed. The expressions of serum miR-21 and PDCD4 mRNA were assessed by RT-qPCR among 122 AS patients and 122 healthy controls, and serum CTX was detected using enzyme-linked immunosorbent assay (ELISA). Compared to healthy controls, AS patients had significantly higher levels of miR-21, PDCD4 mRNA, and CTX. In AS patients who were taking neither NSAIDs nor DMARDs, miR-21 expression was negatively correlated with PDCD4 mRNA expression. In addition, positive correlation of miR-21 and CTX level was observed in AS patients (r = 0.35, p < 0.001), especially in those with disease duration < 10 years (r = 0.49, p < 0.001) and active disease (r = 0.51, p < 0.001). Bone morphogenic proteins (BMPs), is secreted by osteoblasts, might influence the bone formation in AS. MiR-29 suppresses Dickkopf 1 (DKK1) and secreted frizzled related protein 2 (sFRP2) of the Wingless (Wnt) inhibitors, and miR-27 also suppresses sFRP1, DKK2, and adenomatous polyposis coli (APC) of the Wnt inhibitors to regulate the Wnt signal pathway. When Wnt signal pathway was activated, it might further induced BMPs expression. We compared the differences of miR-29, miR-27, and their target genes expressions between AS patients and healthy controls. The influence of rheumatologic drugs on the expressions of these molecules in AS patients was also assessed. Results observed that 73 patients with spinal fusion had higher serum levels of BMP-4 and BMP-7 than either 62 healthy controls or 79 patients without spinal fusion (ps < 0.01), respectively. AS patients with spinal fusion and those without spinal fusion also had the significantly increased miR-27b, miR-29a, miR-29b, and lower sFRP1 mRNA and sFRP2 mRNA expressions than healthy controls; respectively. Compared to AS patients without receiving drug treatment, those with receiving drug treatment had a lower miR-27b expression Therefore, BMPs, miR-27, and Wnt inhibitor proteins might relate to the bone formation in AS. Cytotoxic T lymphocyte antigen-4 (CTLA-4) and protein tyrosine phosphatase, nonreceptor 22 (PTPN22) might play the roles in the inhibition of activated T cells in the initial period of autoimmune tolerance. Programmed cell death-1(PD-1) binds programmed cell death-1 ligand 1(PD-L1) and ligand 2 (PD-L2) to induce negative signals that might maintain the balance of immune tolerance during late period. Therefore, we evaluated the effects of CTLA-4, PTPN22, PD-1, PD-L1 and PD-L2 genptypes in AS occurrences. Results revealed subjects with both of PTPN22 CC/CTLA-4 AA or PTPN22 GC/CTLA-4 AA genotypes had a 2.10 fold (95% CI 1.07-4.13) greater risk of AS development than those with other combinations of PTPN22 and CTLA-4 genotypes. In addition, subjects carrying combinations of PD-1 GG + GA/PD-L1 CC/PD-L2 CC genotypes, those carrying combinations of PD-1 AA/PD-L1 CC/PD-L2 CC genotypes, and those carrying combinations of PD-1 GG + GA/PD-L1 AC + AA/PD-L2 CC genotypes had 6.63 (95% CI 1.28-34.46), 4.33 (95% CI 0.36-52.64), and 3.05 (95% CI 2.16-4.32) greater risks of AS development than those with other combinations of PD-1/PD-L1/PD-L2 genotypes; respectively. Our results suggest that PTPN22 G-1123C, CTLA-4 A49G, PD-1 G-536A, PD-L1 A8923C, and PD-L2 C47103T genotypes have the combined effects on AS development. In conclusion, bone resorption, bone formation, and imbalance of immune tolerance-related gene and miRNA, and their target genes might associate with the development of AS and clinical syndromes.