Frequency of Th17 CD4+ T Cells in Early Rheumatoid Arthritis: A Marker of Anti-CCP Seropositivity

Objective

To examine the frequency and phenotype of Th17 cells in the peripheral blood of early RA (eRA) patients.

Methods

CD4+ T cells were isolated from the peripheral blood of 33 eRA patients, 20 established RA patients and 53 healthy controls (HC), and from the synovial fluid of 20 established RA patients (RASF), by ficoll-hypaque gradient and magnetical negative selection. After polyclonal stimulation, the frequency of Th17 and Th1 cells was determined by flow cytometry and concentrations of IL-17, IFN-γ, TNF-α and IL-10 were measured by ELISA in cell-free supernatants.

Results

When all of our eRA patients were analyzed together, a significantly lower percentage of circulating Th17 cells and a lower CD4-derived IL-17 secretion were observed in comparison with HC. However, after stratifying by anti-CCP antibody status, circulating Th17 cells were decreased in anti-CCP(+) but not in anti-CCP(-)-eRA. All Th17 cells were CD45RO+CD45RA- and CCR6+. Dual Th17/Th1 cells were also exclusively decreased in anti-CCP(+)-eRA. Circulating Th17 and Th17/Th1 cells were negatively correlated with anti-CCP titres. When anti-CCP(+)-eRA patients were retested one year after initiating treatment with oral methotrexate, their circulating Th17 frequency was no longer different from HC. Of note, the percentage of circulating Th1 cells and the secretion of CD4-derived IFN-γ, TNF-α and IL-10 were not different between eRA patients and HC. In established RA patients, circulating Th17 and T17/Th1 cell frequencies were comparable to HC. In RASF, both Th17 and Th1 cells were increased when compared with blood of eRA patients, established RA patients and HC.

Conclusion

Decreased circulating Th17 levels in eRA seem to be a marker of anti-CCP seropositivity, and return to levels observed in healthy controls after treatment with methotrexate.     

白细胞介素17在类风湿性关节炎中意义的探讨

白细胞介素17（interleukin-17,IL-17）是近年来发现的一种重要的炎症性细胞因子。主要由CD4＋效应T细胞的亚群-辅助性T细胞17（T help cell 17,Th17）、CD8＋T细胞和γδT细胞产生,能调节并促进多种炎性介质的产生,有强大的招募中性粒细胞的作用,在白细胞的迁移、破骨细胞的活化和骨质的吸收等方面发挥重要作用。近年来研究发现,IL-17与多种炎性疾病、感染、肿瘤、过敏、及自身免疫性疾病均有密切关系,尤其在类风湿性关节炎（rheumatoid arthritis,RA）发病的各个环节中起重要作用。本文对IL-17在类风湿性关节炎的发生、发展、诊断及治疗等方面的作用进行综述。     

Gender-Specific Effects of Genetic Variants within Th1 and Th17 Cell-Mediated Immune Response Genes on the Risk of Developing Rheumatoid Arthritis

The present study was conducted to explore whether single nucleotide polymorphisms (SNPs) in Th1 and Th17 cell-mediated immune response genes differentially influence the risk of rheumatoid arthritis (RA) in women and men. In phase one, 27 functional/tagging polymorphisms in C-type lectins and MCP-1/CCR2 axis were genotyped in 458 RA patients and 512 controls. Carriers of Dectin-2 _rs4264222T allele had an increased risk of RA (OR = 1.47, 95%CI 1.10–1.96) whereas patients harboring the DC-SIGN _rs4804803G, MCP-1 _rs1024611G, MCP-1 _rs13900T and MCP-1 _rs4586C alleles had a decreased risk of developing the disease (OR = 0.66, 95%CI 0.49–0.88; OR = 0.66, 95%CI 0.50–0.89; OR = 0.73, 95%CI 0.55–0.97 and OR = 0.68, 95%CI 0.51–0.91). Interestingly, significant gender-specific differences were observed for Dectin-2 _rs4264222 and Dectin-2 _rs7134303: women carrying the Dectin-2 _rs4264222T and Dectin-2 _rs7134303G alleles had an increased risk of RA (OR = 1.93, 95%CI 1.34–2.79 and OR = 1.90, 95%CI 1.29–2.80). Also five other SNPs showed significant associations only with one gender: women carrying the MCP-1 _rs1024611G, MCP-1 _rs13900T and MCP-1 _rs4586C alleles had a decreased risk of RA (OR = 0.61, 95%CI 0.43–0.87; OR = 0.67, 95%CI 0.47–0.95 and OR = 0.60, 95%CI 0.42–0.86). In men, carriers of the DC-SIGN _rs2287886A allele had an increased risk of RA (OR = 1.70, 95%CI 1.03–2.78), whereas carriers of the DC-SIGN _rs4804803G had a decreased risk of developing the disease (OR = 0.53, 95%CI 0.32–0.89). In phase 2, we genotyped these SNPs in 754 RA patients and 519 controls, leading to consistent gender-specific associations for Dectin-2 _rs4264222, MCP-1 _rs1024611, MCP-1 _rs13900 and DC-SIGN _rs4804803 polymorphisms in the pooled sample (OR = 1.38, 95%CI 1.08–1.77; OR = 0.74, 95%CI 0.58–0.94; OR = 0.76, 95%CI 0.59–0.97 and OR = 0.56, 95%CI 0.34–0.93). SNP-SNP interaction analysis of significant SNPs also showed a significant two-locus interaction model in women that was not seen in men. This model consisted of Dectin-2 _rs4264222 and Dectin-2 _rs7134303 SNPs and suggested a synergistic effect between the variants. These findings suggest that Dectin-2, MCP-1 and DC-SIGN polymorphisms may, at least in part, account for gender-associated differences in susceptibility to RA.     

IL-26 Is Overexpressed in Rheumatoid Arthritis and Induces Proinflammatory Cytokine Production and Th17 Cell Generation

Interleukin-26 (IL-26), a member of the IL-10 cytokine family, induces the production of proinflammatory cytokines by epithelial cells. IL-26 has been also reported overexpressed in Crohn''s disease, suggesting that it may be involved in the physiopathology of chronic inflammatory disorders. Here, we have analyzed the expression and role of IL-26 in rheumatoid arthritis (RA), a chronic inflammatory disorder characterized by joint synovial inflammation. We report that the concentrations of IL-26 are higher in the serums of RA patients than of healthy subjects and dramatically elevated in RA synovial fluids compared to RA serums. Immunohistochemistry reveals that synoviolin⁺ fibroblast-like synoviocytes and CD68⁺ macrophage-like synoviocytes are the main IL-26-producing cells in RA joints. Fibroblast-like synoviocytes from RA patients constitutively produce IL-26 and this production is upregulated by IL-1-beta and IL-17A. We have therefore investigated the role of IL-26 in the inflammatory process. Results show that IL-26 induces the production of the proinflammatory cytokines IL-1-beta, IL-6, and tumor necrosis factor (TNF)-alpha by human monocytes and also upregulates the expression of numerous chemokines (mainly CCL20). Interestingly, IL-26-stimulated monocytes selectively promote the generation of RORgamma t⁺ Th17 cells, through IL-1-beta secretion by monocytes. More precisely, IL-26-stimulated monocytes switch non-Th17 committed (IL-23R⁻ or CCR6⁻ CD161⁻) CD4⁺ memory T cells into Th17 cells. Finally, synovial fluids from RA patients also induce Th17 cell generation and this effect is reduced after IL-26 depletion. These findings show that IL-26 is constitutively produced by RA synoviocytes, induces proinflammatory cytokine secretion by myeloid cells, and favors Th17 cell generation. IL-26 thereby appears as a novel proinflammatory cytokine, located upstream of the proinflammatory cascade, that may constitute a promising target to treat RA and chronic inflammatory disorders.     

辅助性T细胞17的研究进展

CD4 T细胞根据释放细胞因子不同可分为不同的亚型:辅助性T细胞(T helper cells, Th)1、Th2和调节性T细胞(regulatory T cells, Treg).最近发现了一种新的CD4 T细胞亚型:以分泌白细胞介素-17为主要特征的Th17细胞.Th17细胞的发现对CD4 T细胞分化、Th细胞漂移等传统理论提出了挑战,同样也为感染、免疫性疾病提供了新的研究思路,尤其是Treg、Th1、Th2和Th17细胞在体内的平衡对于维持正常免疫应答反应有重要意义.     

The Microbial Capsular Polysaccharide Galactoxylomannan Inhibits IL-17A Production in Circulating T Cells from Rheumatoid Arthritis Patients

The persistence of activated T cells in rheumatoid arthritis (RA) synovium may be attributable to increased homing, increased retention or a possible imbalance between cell proliferation and programmed cell death. Induction of apoptosis may represent a potential therapeutic approach. Galactoxylomannan (GalXM) from the opportunistic fungus Cryptococcus neoformans can interact with T cells and induce T-cell apoptosis through the inhibition of CD45 phosphatase activity. The aim of this study was to determine the effect of GalXM on circulating T cells from patients with RA and the underlying mechanisms. GalXM immunomodulating effect on apoptosis and signal transduction pathway involved in IL-17A production was evaluated on T cells. RA T-cell apoptosis, higher than that of control T cells, was further increased by GalXM through induction of caspase-3 activation. Activated T cells expressing the CD45RO molecule and producing IL-17A were the main target of GalXM-induced apoptosis. GalXM induced consistent impairment of IL-17A production and inhibition of STAT3, which was hyperactivated in RA. In conclusion, GalXM triggered apoptosis of activated memory T cells and interfered with IL-17A production in RA. These data suggest therapeutic targeting of deleterious Th17 cells in RA and other autoimmune diseases.     

Treg细胞和Th17细胞在多发性骨髓瘤中的研究进展

多发性骨髓瘤(MM)系血液系统的恶性肿瘤,以老年人多见.目前治疗以化疗和自身干细胞移植为主,仍难以治愈.多发性骨髓瘤的进展涉及到一系列基因和骨髓微环境的改变,这些改变恰好促进了肿瘤细胞的生长并瓦解了局部的免疫反应.CD4+和CD8+T细胞在多发性骨髓瘤患者体内数量和功能的改变都已经阐明.Treg细胞和Th17细胞的平衡在维持多发性骨髓瘤患者的抗肿瘤免疫中起着至关重要的作用.Treg细胞负责维持机体对外来抗原和自身抗原的免疫耐受.Th17细胞主要参与机体抵抗真菌和寄生虫感染、炎症反应和自身免疫.多发性骨髓瘤患者体内TGF-β和IL-6高水平表达,并可直接或通过其他促炎因子影响Th17细胞的分化,进而调节机体的抗肿瘤免疫应答.因此我们针对Treg细胞和Th17细胞在多发性骨髓瘤中的研究进展进行阐述.     

Reciprocity between Regulatory T Cells and Th17 Cells: Relevance to Polarized Immunity in Leprosy

T cell defect is a common feature in lepromatous or borderline lepromatous leprosy (LL/BL) patients in contrast to tuberculoid or borderline tuberculoid type (TT/BT) patients. Tuberculoid leprosy is characterized by strong Th1-type cell response with localized lesions whereas lepromatous leprosy is hallmarked by its selective Mycobacterium leprae specific T cell anergy leading to disseminated and progressive disease. FoxP3+ Regulatory T cells (Treg) which are essential for maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases also dampen proinflammatory T cells that include T helper 17 (Th17) cells. This study is aimed at evaluating the role of Treg cells in influencing other effector T cells and its relationship with the cytokine polarized state in leprosy patients. Peripheral blood mononuclear cells from of BT/TT (n = 15) and BL/LL (n = 15) patients were stimulated with M. leprae antigen (WCL) in presence of golgi transport inhibitor monensin for FACS based intracellular cytokine estimation. The frequency of Treg cells showed >5-fold increase in BL/LL in comparison to BT/TT and healthy contacts. These cells produced suppressive cytokine, IL-10 in BL/LL as opposed to BT/TT (p = 0.0200) indicating their suppressive function. The frequency of Th17 cells (CD4, CD45RO, IL-17) was, however, higher in BT/TT. Significant negative correlation (r = -0.68, P = 0.03) was also found between IL-10 of Treg cells and IL-17+ T cells in BL/LL. Blocking IL-10/TGF-β restored the IL-17+ T cells in BL/LL patients. Simultaneously, presence of Th17 related cytokines (TGF-β, IL-6, IL-17 and IL-23) decreased the number of FoxP3+ Treg cells concomitantly increasing IL-17 producing CD4+ cells in lepromatous leprosy. Higher frequency of Programmed Death-1/PD-1+ Treg cells and its ligand, PDL-1 in antigen presenting cells (APCs) was found in BL/LL patients. Inhibition of this pathway led to rescue of IFN-γ and IL-17 producing T cells. Results indicate that Treg cells are largely responsible for the kind of immunosuppression observed in BL/LL patients. This study also proves that Treg cells are profoundly affected by the cytokine milieu and this property may be utilized for benefit of the host.     

Endurance Exercise Diverts the Balance between Th17 Cells and Regulatory T Cells

Endurance, marathon-type exertion is known to induce adverse changes in the immune system. Increased airway hyper-responsiveness and airway inflammation are well documented in endurance athletes and endurance exercise is considered a major risk factor for asthma in elite athletes. Yet, the mechanisms underlying this phenomenon are still to be deduced. We studied the effect of strenuous endurance exercise (marathon and half-ironman triathlon) on CD4+ lymphocyte sub-populations and on the balance between effector and regulatory CD4+ lymphocytes in the peripheral blood of trained athletes, Endurance exercise induced a significant increase in Th17 cells and a sustained decrease in peripheral blood regulatory T cells (Tregs). While interleukin (IL)-2 levels remained undetectable, post-race serum IL-6 and transforming growth factor (TGF) β levels were significantly elevated. Treg levels in sedentary controls'' decreased in vitro after incubation with athletes'' post-exercise serum, an effect that was attenuated by supplements of IL-2 or anti IL-6 neutralizing antibodies. Our data suggest that exercise-induced changes in serum cytokine levels promote alterations in Tregs and Th17 cell populations, which may divert the subtle balance in the immune system towards inflammation. This may explain allergic and autoimmune phenomena previously reported in endurance athletes and contribute to our understanding of exercise-related asthma.     

Benign Rheumatoid Arthritis of the Aged

A severe type of rheumatoid arthritis which is seen in 25% of cases that present with the disease after the age of 60 is a self-limiting illness. It can be well controlled during the acute severe phase and has a good prognosis. This paper describes 29 patients with this pattern of disease who were separated from a larger group of 110 elderly onset cases.     

Th22 Cells as Well as Th17 Cells Expand Differentially in Patients with Early-Stage and Late-Stage Myelodysplastic Syndrome

Background

Immunological mechanisms are increasingly recognized in the progression of myelodysplastic syndrome (MDS). Early-stage MDS (E-MDS) is characterized by autoimmune-mediated myelosuppression whereas late-stage MDS (L-MDS) involves immune evasion, giving dysplastic cells growth potential to progress into acute myeloid leukemia. T-helper (Th) 22 is involved in the pathogenesis of inflammatory autoimmunity and tumorigenesis. The roles of Th22 cells in the pathophysiology of E-MDS and L-MDS remain unsettled.

Design and Methods

We studied 37 MDS patients (E-MDS, n = 17; L-MDS, n = 20) and 20 healthy controls to characterize their peripheral blood (PB), as well as 25 MDS patients and 10 healthy controls to characterize their bone marrow(BM). The expression of Interleukin-22 (IL-22), IL-17 or interferon gamma (IFN-γ) was examined in E-MDS, L-MDS patients and controls by flow cytometry. The mRNA expression levels of RAR-related orphan receptor C (RORC), IL-6, tumor necrosis factor alpha (TNF-α) and IL-23 in peripheral blood mononuclear cells (PBMCs) were determined by real-time quantitative polymerase chain reaction. The levels of IL-22 and IL-17 both in PB and BM plasma were examined by enzyme-linked immunosorbent assay.

Results

In E-MDS, peripheral Th17 cells were significantly elevated and correlated with peripheral Th22 cells compared with healthy controls and L-MDS. Significantly higher levels of peripheral Th22 expansion, mRNA expression of IL-6, TNF-α and lower level of RORC mRNA expression were observed in L-MDS compared with E-MDS. No statistical difference was found in IL-23 mRNA expression or plasma IL-22, IL-17 levels among E-MDS, L-MDS and controls.

Conclusions

Our data demonstrated that L-MDS cohort had increased frequencies of peripheral Th22 cells and higher mRNA expression levels of IL-6 and TNF-α, indicating that Th22 cells along with Th17 cells or not are involved in the dynamic immune responses of MDS.