Type 1 diabetes-associated IL2RA variation lowers IL-2 signaling and contributes to diminished CD4+CD25+ regulatory T cell function

Numerous reports have demonstrated that CD4(+)CD25(+) regulatory T cells (Tregs) from individuals with a range of human autoimmune diseases, including type 1 diabetes, are deficient in their ability to control autologous proinflammatory responses when compared with nondiseased, control individuals. Treg dysfunction could be a primary, causal event or may result from perturbations in the immune system during disease development. Polymorphisms in genes associated with Treg function, such as IL2RA, confer a higher risk of autoimmune disease. Although this suggests a primary role for defective Tregs in autoimmunity, a link between IL2RA gene polymorphisms and Treg function has not been examined. We addressed this by examining the impact of an IL2RA haplotype associated with type 1 diabetes on Treg fitness and suppressive function. Studies were conducted using healthy human subjects to avoid any confounding effects of disease. We demonstrated that the presence of an autoimmune disease-associated IL2RA haplotype correlates with diminished IL-2 responsiveness in Ag-experienced CD4(+) T cells, as measured by phosphorylation of STAT5a, and is associated with lower levels of FOXP3 expression by Tregs and a reduction in their ability to suppress proliferation of autologous effector T cells. These data offer a rationale that contributes to the molecular and cellular mechanisms through which polymorphisms in the IL-2RA gene affect immune regulation, and consequently upon susceptibility to autoimmune and inflammatory diseases.     

Molecular screening and association study of IL15 gene polymorphisms in rheumatoid arthritis

Interleukin 15 (IL-15) is a pleiotropic pro-inflammatory cytokine known to play a relevant role in rheumatoid arthritis (RA) pathogenesis. In this study we aimed to investigate for the first time the contribution of IL15 gene to RA susceptibility. We screened 13 single nucleotide polymorphisms (SNPs) localised within IL15 regulatory regions (promoter, 5' UTR region and 3' UTR region) in a total of 420 individuals, who were genotyped by direct sequencing of PCR products. In addition, an association study of these IL15 SNPs was conducted in three independent case-control cohorts of Spanish Caucasian origin, including a total of 645 RA patients and 656 healthy controls. The presence of the 13 selected IL15 SNPs in our population was confirmed and no new genetic variants were found. The distribution of the IL15 selected SNPs in RA patients and controls showed no statistically significant deviation in any of the populations studied. Additionally, we performed a haplotype analysis that revealed three IL15 haplotype blocks. None of the haplotype blocks was associated with RA susceptibility or severity in the three cohorts analysed. Our results suggest that the IL15 gene polymorphisms do not appear to play a major role in RA genetic predisposition in our population.     

表达芯片筛选类风湿关节炎特异表达基因

为了寻找类风湿关节炎（rheumatoid arthritis,RA)新的特异性表达标记物,应用基因芯片对RA、骨关节炎（osteoarthritis,OA）和强直性脊柱炎（ankylosing spondylitis,AS）患者关节滑膜组织的基因表达谱进行比较,并采用实时定量PCR方法对芯片结果进行验证. 全基因组表达芯片的结果显示,与OA滑膜组织及AS滑膜组织相比,在RA患者的滑膜组织中,CD38,ANKRD38,E2F2,CFDP1, CD7, ISG20 和IL2RG基因表达异常|实时定量PCR结果证实,RA患者滑膜组织中,CD38、E2F2和IL2RG基因表达明显增高.     

Cytokines genes polymorphisms in chronic hepatitis C: Impact on susceptibility to infection and response to therapy

BackgroundCytokines play a key role in the regulation of immune responses. In hepatitis C virus infection, the production of abnormal cytokine levels appears to contribute in the progression of the disease, viral persistence, and affects response to therapy. Cytokine genes polymorphisms located within the coding/regulatory regions have been shown to affect the overall expression and secretion of cytokines. The aim of the study was to evaluate the association of of IL28B rs12979860, TGF-β1-509, TNF-α 308, and IL-10-1082 polymorphisms with the susceptibility to hepatitis C virus genotype 4 infection and response to pegylated interferon-α and ribavirin therapy.MethodsIL28B, TGF-β1 and TNF-α genes polymorphisms were genotyped using polymerase chain reaction (PCR)-based restriction fragment length polymorphism assay while IL-10 gene polymorphism was detected by sequence specific primer-PCR in 220 healthy individuals and 440 hepatitis C infected patients (220 sustained virological response and 220 non-responder to combination therapy).ResultsIL28 B CT and TT, TGF-β1 CT and TT and TNF-α AG and AA genotypes were significantly associated with susceptibility to hepatitis C infection and response to therapy. While no association was found between IL-10 gene polymorphism and susceptibility to HCV infection and response to treatment.ConclusionsThese results suggested that inheritance of IL28B CT and TT, TGF-β1 CT and TT and TNF-α AG and AA genotypes which appear to affect the cytokine production may be associated with susceptibility to HCV infection and resistance to combined antiviral therapy.     

The immunogenetics of multiple sclerosis

The discoveries in the 1970s of strong associations between various diseases and certain human leukocyte antigen (HLA) factors were a revolution within genetic epidemiology in the last century by demonstrating for the first time how genetic markers can help unravel the genetics of disorders with complex genetic backgrounds. HLA controls immune response genes and HLA associations indicate the involvement of autoimmunity. Multiple sclerosis (MS) was one of the first conditions proven to be HLA associated involving primarily HLA class II factors. We review how HLA studies give fundamental information on the genetics of the susceptibility to MS, on the importance of linkage disequilibrium in association studies, and on the pathogenesis of MS. The HLA-DRB1*1501 molecule may explain about 50% of MS cases and its role in the pathogenesis is supported by studies of transgenic mice. Studies of polymorphic non-HLA genetic markers are discussed based on linkage studies and candidate gene approaches including complete genome scans. No other markers have so far rivaled the importance of HLA in the genetic susceptibility to MS. Recently, large international collaborations provided strong evidence for the involvement of polymorphism of two cytokine receptor genes in the pathogenesis of MS: the interleukin 7 receptor alpha chain gene (IL7RA) on chromosome 5p13 and the interleukin 2 receptor alpha chain gene (IL2RA (=CD25)) on chromosome 10p15. It is estimated that the C allele of a single nucleotide polymorphism, rs6897932, within the alternative spliced exon 6 of IL7RA is involved in about 30% of MS cases.     

Regulation of natural killer cell activity by macrophages in the rheumatoid joint and peripheral blood

Recently, in another study, we observed that indomethacin, a prostaglandin synthetase inhibitor, significantly increased NK activity in both normal and rheumatoid arthritis (RA) peripheral blood (PB) but not in RA synovial fluid (SF). Because macrophages are a major source of prostaglandins, we examined the effect of macrophage-enriched adherent cells (AC) on NK activity as measured by a 3-hr Cr-release assay with K 562 cells. The removal of AC resulted in increased (p less than 0.01) NK activity in both normal and RA PB. In contrast, the removal of AC from RA SF resulted in a significant decrease (p less than 0.001) of NK activity. By using only nonadherent cells (NAC), NK activity in RA SF and synovial tissue (ST) was significantly reduced when compared to autologous RA PB (p less than 0.001). Enhancement of NK activity of SF NAC by both poly I:C and IL 2 was not dependent on AC. Mixing experiments demonstrated that the addition of synovial AC for 16 hr increased NK activity of synovial NAC to a level similar to that of unseparated mononuclear cells, whereas autologous PB AC suppressed NK activity of PB NAC. PB AC, when added to SF NAC, also increased NK activity. Supernatants from synovial mononuclear cells were stimulatory of synovial NAC NK activity, whereas normal PB mononuclear supernatants were suppressive. These observations document 1) a significant reduction of NAC-mediated NK activity in the rheumatoid joint as compared to PB from the same patient, and 2) that AC modulate NK activity differently in the rheumatoid joint as compared to RA or normal PB.     

IL2RA Genetic Heterogeneity in Multiple Sclerosis and Type 1 Diabetes Susceptibility and Soluble Interleukin-2 Receptor Production

Multiple sclerosis (MS) and type 1 diabetes (T1D) are organ-specific autoimmune disorders with significant heritability, part of which is conferred by shared alleles. For decades, the Human Leukocyte Antigen (HLA) complex was the only known susceptibility locus for both T1D and MS, but loci outside the HLA complex harboring risk alleles have been discovered and fully replicated. A genome-wide association scan for MS risk genes and candidate gene association studies have previously described the IL2RA gene region as a shared autoimmune locus. In order to investigate whether autoimmunity risk at IL2RA was due to distinct or shared alleles, we performed a genetic association study of three IL2RA variants in a DNA collection of up to 9,407 healthy controls, 2,420 MS, and 6,425 T1D subjects as well as 1,303 MS parent/child trios. Here, we report “allelic heterogeneity” at the IL2RA region between MS and T1D. We observe an allele associated with susceptibility to one disease and risk to the other, an allele that confers susceptibility to both diseases, and an allele that may only confer susceptibility to T1D. In addition, we tested the levels of soluble interleukin-2 receptor (sIL-2RA) in the serum from up to 69 healthy control subjects, 285 MS, and 1,317 T1D subjects. We demonstrate that multiple variants independently correlate with sIL-2RA levels.     

Are genetic variations in IL-21–IL-23R–IL-17A cytokine axis involved in a pathogenic pathway of rheumatoid arthritis? Bayesian hierarchical meta-analysis

Inflammatory cytokines have been established to be involved in the pathogenesis of rheumatoid arthritis (RA). The genetic polymorphisms in the interleukin (IL) 23 receptor (IL23R), IL21, and IL17 have been associated with RA risk. However, there is no conclusive understanding of the genes encoding the immunoinflammatory IL-21–IL-23R–IL-17A pathway in RA aetiopathogenesis. This meta-analysis was conducted to attain this goal. A comprehensive literature search was conducted in Scopus and PubMed to look for the relevant case–control studies up until 2018. A Bayesian hierarchical meta-analysis was carried out to assess the association between the polymorphisms and the risk of RA. The association was estimated by calculating the logarithm of odds ratio (Log OR) and 95% credible interval (95% CI). In this meta-analysis, 37 case–control studies comprising 23,506 RA patients and 25,984 healthy individuals were found for analyzing the IL23R, IL21, and IL1A gene polymorphism and risk of RA. In the IL23R gene rs1343151 SNP, the minor A allele significantly increased the risk of RA (Log OR = 0.085, 95% CI = 0.008, 0.156). Moreover, the minor AA genotype was significantly associated with increased RA risk (Log OR = 0.176, 95% CI = 0.028, 0.321). In addition, the C allele of the IL23R gene rs2201841 SNP significantly decreased the disease risk (Log OR = −0.544, 95% CI = −1.0, −0.065). Since Bayesian meta-analysis is a powerful strategy to pool the data, it can be mentioned that genetic polymorphisms of IL23R, but not IL21 and IL17A, are involved in susceptibility to RA.     

Role of C-reactive protein in osteoclastogenesis in rheumatoid arthritis

IntroductionC-reactive protein (CRP) is one of the biomarkers for the diagnosis and assessment of disease activity in rheumatoid arthritis (RA). CRP is not only the by-product of inflammatory response, but also plays proinflammatory and prothrombotic roles. The aim of this study was to determine the role of CRP on bone destruction in RA.MethodsCRP levels in RA synovial fluid (SF) and serum were measured using the immunoturbidimetric method. The expression of CRP in RA synovium was assessed using immunohistochemical staining. CD14+ monocytes from peripheral blood were cultured with CRP, and receptor activator of nuclear factor-κB ligand (RANKL) expression and osteoclast differentiation were evaluated using real-time PCR, counting tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells and assessing bone resorbing function. CRP-induced osteoclast differentiation was also examined after inhibition of Fcγ receptors.ResultsThere was a significant correlation between CRP levels in serum and SF in RA patients. The SF CRP level was correlated with interleukin (IL)-6 levels, but not with RANKL levels. Immunohistochemical staining revealed that compared with the osteoarthritis synovium, CRP was more abundantly expressed in the lining and sublining areas of the RA synovium. CRP stimulated RANKL production in monocytes and it induced osteoclast differentiation from monocytes and bone resorption in the absence of RANKL.ConclusionsCRP could play an important role in the bony destructive process in RA through the induction of RANKL expression and direct differentiation of osteoclast precursors into mature osteoclasts. In the treatment of RA, lowering CRP levels is a significant parameter not only for improving disease activity but also for preventing bone destruction.     

Interleukin-18 gene polymorphisms and rheumatoid arthritis: A meta-analysis

Interleukin-18 (IL-18) is a member of the IL-1 superfamily that enhances both innate and acquired immune responses. IL-18 is highly expressed in sera, synovial fluids and synovial tissues of patients with RA, and these IL-18 levels are correlated with RA disease activity, indicating an important role of IL-18 in the pathogenesis of RA. Several studies have examined the association of IL-18 gene polymorphisms with RA, but these studies have shown inconclusive and controversial results. To verify the association between IL-18 gene polymorphism and susceptibility to RA, we conducted a meta-analysis of all relevant reports cited in MEDLINE/PubMed before October 2012. A meta-analysis on the association between the IL-18 rs1946518 SNP and RA was performed for 2944 patients with RA and 2377 controls from 7 published studies and a meta-analysis on the association between the IL-18 rs187238 SNP and RA was performed for 1319 patients with RA and 1211 controls from 5 published studies. In addition, 2 studies involving 1873 RA patients and 1092 controls were considered in the meta-analysis of the association between the IL-18 rs360722 SNP and RA. No significant association was found between two IL-18 SNPs (rs1946518 and rs187238) and RA susceptibility in all subjects. In subgroup analysis stratified by ethnicity, there was still no significant association between these two IL-18 SNPs and RA susceptibility. However, the frequency of the T allele at rs360722 was found to be significantly lower in patients with RA compared with controls, although this finding was based on only 2 studies. The results of our meta-analysis suggest that IL-18 rs360722 SNP is only associated with RA susceptibility. However, due to only two studies included in our meta-analysis, large-scale well designed studies should be considered in future studies to confirm the exact role of IL-18 rs360722 SNP in RA susceptibility.     

ICOS and B7 costimulatory molecule expression identifies activated cellular subsets in rheumatoid arthritis.

To better define important cell subsets expressing activation markers in rheumatoid arthritis (RA), we compared selective lymphocyte and monocyte B7H1, B7H2, B7RP.1, B7RP.2, and inducible costimulatory molecule (ICOS) expression from normal peripheral blood (NL PB), RA PB, and RA synovial fluid (SF) by multicolor flow cytometry and immunohistochemistry. RA SF memory lymphocytes expressed B7RP.1 and B7RP.2, suggesting that T-cells may function as antigen presenting cells (APCs) in RA joints. We found similar results for ICOS expression. RA SF CD14+ monocytes also expressed B7RP.1 (an ICOS ligand) and the homologous ligand B7RP.2, identifying monocytes as potential mediators of antigen processing and lymphocyte activation in RA. Furthermore, we found an increased population of RA SF CD14+ monocytes expressing B7H1 and B7H2. [The FACS analysis was supported by immunohistochemistry, showing intense lymphocyte and APC (macrophages with dendritic morphology) ICOS staining in RA synovial tissue (ST). Overall, these results define elevated populations of memoryT-lymphocytes expressing proinflammatory B7 molecules in RA SF that either stimulate T cells through ICOS (via ICOS ligands B7RP.1 and B7RP.2), or down-regulate RA ST T-lymphocytes through B7H1 and B7H2.] Therefore, in the same joint, there may exist positive and negative influences on the inflammatory response, and perhaps, the negative signals dominate as joint inflammation resolves.     

TH-17 cells in rheumatoid arthritis

Introduction  

The aim of this study was to quantify the number of T-helper (TH)-17 cells present in rheumatoid arthritis (RA) synovial fluid (SF) and to determine the level of interleukin (IL)-17 cytokine in RA, osteoarthritis (OA) and normal synovial tissue, as well as to examine SF macrophages for the presence of IL-23, IL-27 and interferon (IFN)-γ.     

Reduction of inflammatory cytokines and prostaglandin E2 by IL-13 gene therapy in rheumatoid arthritis synovium

The rheumatoid arthritis (RA) joint is characterized by an inflammatory synovial pannus which mediates tissue destruction. IL-13 is a cytokine that inhibits activated monocytes/macrophages from secreting a variety of proinflammatory molecules. The aim of this study was to examine whether gene therapy-delivered IL-13 could reduce the production of key proinflammatory mediators in RA synovial tissue (ST) explants. Adenoviral vectors encoding the genes for human IL-13 (AxCAIL-13) and bacterial beta-galactosidase were generated and examined for protein production. Vectors were used to infect RA ST explants and RA synovial fibroblasts, and conditioned medium (CM) was collected at various times for analysis by ELISA and competitive immunoassay. AxCAIL-13 decreased the production of RA ST explant proinflammatory IL-1beta by 85% after 24 h. Likewise, TNF-alpha levels were decreased by 82 and 75% whereas IL-8 levels were reduced 54 and 82% after 24 and 48 h, respectively, in RA ST explant CM. Monocyte chemotactic protein-1 concentrations were decreased by 88% after 72 h in RA ST explant CM. RA ST explant epithelial neutrophil-activating peptide-78 concentrations were decreased 85 and 94% whereas growth-related gene product-alpha levels were decreased by 77 and 85% at 24 and 48 h, respectively, by AxCAIL-13. Further, IL-13 significantly decreased PGE2 and macrophage inflammatory protein-1alpha production. These results demonstrate that increased expression of IL-13 via gene therapy may decrease RA-associated inflammation by reducing secretion of proinflammatory cytokines and PGE2.     

Study of chemotactic activity developed by neutrophils from rheumatoid arthritis patients

Neutrophils are the predominant cells accumulated in the synovial fluid (SF) of rheumatoid arthritis (RA) patients. Accumulation of neutrophils may be regarded as a possible way by which neutrophils exert cytotoxic functions. The aim of the present study was to analyze the chemotactic response of neutrophils (PMNs) isolated from the peripheral blood or SF of patients with RA by performing the chemotaxis assay, in which N-formyl-methionyl-leucyl-phenylalanine (FMLP) was used as chemotactic agent. Our results showed that FMLP induced response of peripheral blood neutrophils from 12 patients with RA was similar with the response of 15 healthy controls. A decreased chemotactic response to FMLP was, however, observed in PMNs isolated from the SF of RA patients as comlipared with peripheral blood cells. Therefore, this defective chemotactic ability of neutrophil, was inversely correlated with the number of infiltrating cells in SF. These results indicate that chemotactic ability of neutrophils may be reduced after migration to the SF. Because PMNs chemotaxis in vivo has likely occurred in the presence of serum or SF, we tried to simulate the same conditions in vitro. Therefore, we analyzed the effect of serum or SF on the RA-PMNs chemotaxis. Heat-inactivated serum produced a marked reduction of chemotactic activity developed by PMNs isolated from patients with RA. Notably, a significant increase of chemotactic activity was observed when FMLP and serum stimuli were used together, as compared with the same stimuli used alone. The results suggested that complement activation might interfere with neutrophils chemotaxis. SF amplifies the chemotactic activity of PMNs isolated from peripheral blood of RA patients, but does not affect the chemotaxis developed by PMNs isolated from SF. The data might suggest that several components of SF (IL-8, leukotrien B4, thrombin, platelet-activating factor, etc.) could serve as a potent stimulus for recruitment of neutrophils from periphery into the RA joint. In conclusion, serum or SF components seem to contribute to chemotaxis of neutrophils and play a role in differential killing of PMNs and incidence of infection.