IntroductionInterleukin (IL)-21 is a member of type I cytokine family. Recent studies indicate that IL-21 can promote T follicular helper (Tfh) cell differentiation and survival, a specialized T cell subset which provides help for B cell. It can also regulate the activation, proliferation and differentiation of human B cell and immunoglobulin (Ig) production as well as isotype switching of plasma cell. Rheumatoid arthritis (RA) is characterized by auto-antibodies overproduction such as rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibody, suggesting a pivotal role of Tfh cell and B cell in the pathogenesis of RA. This study aimed to investigate whether IL-21 had a regulatory effect on Tfh cell and B cell in RA.MethodsSerum IL-21 concentrations were measured by ELISA. The correlations between serum IL-21 levels and clinical features of RA patients were analyzed by Spearman's rank test. The percentages of Tfh-like cells, IL-21 receptor (R) expression on Tfh-like cells and B cells in peripheral blood (PB) were analyzed by flow cytometry. Peripheral blood mononuclear cells (PBMC) were stimulated by rIL-21 (100 ng/ml) in the presence or absence of anti-CD40 and/or anti-IgM, and changes of IL-21R, activation-associated surface markers (CD25, CD69 and CD40), the proliferation, apoptosis and differentiation of B cells were analyzed by flow cytometry. Production of IgG and IgM in the culture supernatants was determined by ELISA.ResultsThe results showed that the serum IL-21 levels in RA patients were significantly higher than that of healthy controls (HC). IL-21 concentrations were positively correlated with 28-joint count disease activity score (DAS28) and anti-CCP antibody in RA patients with high IL-21 levels. Furthermore, the frequencies of peripheral CXCR5+PD-1+CD4+ Tfh-like cells markedly increased in RA patients and the percentages of Tfh-like cells were positively correlated with DAS28 and anti-CCP antibody levels. Moreover, elevated IL-21 levels were also correlated with the frequencies of Tfh-like cells. IL-21R expression on both Tfh-like cells and B cells were significantly enhanced in RA patients. In cultures vitro, exogenous IL-21 upregulated IL-21R expression and activation-associated surface markers on B cells and promoted more B cell proliferation in RA than in HC. This IL-21-mediated effect could be reversed by IL-21R-specific neutralizing antibody. Importantly, IL-21 promoted more differentiation of B cell into plasmablast and higher levels of IgG and IgM production in RA than in HC.ConclusionsIncreased serum IL-21 levels in RA patients correlate with DAS28, anti-CCP antibody and frequencies of Tfh-like cells. IL-21 supports B cell activation, proliferation and antibody secretion via IL-21R pathway. Thus, IL-21 may be involved in the pathogenesis of RA and antagonizing IL-21 could be a novel strategy for the therapy of RA.

The complement C5b-9 complexes can result in cell apoptosis, but the mechanism of sublytic C5b-9-mediated glomerular mesangial cell (GMC) apoptosis in Thy-1 nephritis (Thy-1N) remains largely unclear. The Gadd45 gene is involved in the cellular response to DNA damage and can promote cell apoptosis. In this study, both Gadd45c expression patterns and pathologic changes of renal tissue were examined in rat Thy-1N. Both Gadd45c expression and GMC apoptosis were significantly decreased in Thy-1N rats upon the depletion of complement with cobra venom factor. Our in vitro studies showed that Gadd45c over-expression increased sublytic C5b-9-induced GMC apoptosis, while Gadd45c gene knockdown by siRNA greatly reduced GMC apoptosis. Moreover, Gadd45c gene silencing in vivo markedly inhibited the pathologic changes in the renal tissue of Thy-1N rats. These data suggest that Gadd45c gene expression is involved in regulating GMC apoptosis mediated by sublytic C5b-9 in Thy-1N.Key words: Apoptosis . Gadd45c . Glomerular mesangial cells . Sublytic C5b-9 .Thy-1 nephritis Supporting Information available online IntroductionMesangioproliferative glomerulonephritis (MsPGN) is a disease with high incidence in humans. Proliferation of glomerular mesangial cells (GMC) in MsPGN appears to be crucial for a subsequent increase in mesangial matrix and development of glomerulosclerosis [1,2]. Rat Thy-1 nephritis (Thy-1N), namely anti-thymocyte serum (ATS) induced nephritis, is a widely used animal model for studying human MsPGN [3,4]. ATS administered in rats binds to Thy-1 antigen on the membrane of GMC and then activates the complement system resulting in immunopathologic injury [5][6][7][8]. Complement activation results in generation of chemotactic factors, such as C5a, and formation of the C5b-9 complex [6,9]. Because GMC damage in Thy-1N is complement-dependent and neutrophil-independent, complement is generally regarded as the principal mediator of GMC lesions in the rats with Thy-1N. Assembly of C5b-9 can result in formation of transmembrane channels or rearrangement of membrane lipid with loss of membrane integrity. However, injury to nucleated cells by C5b-9 is almost non-lytic (sublytic) because the cell surface has many homologous restriction factors such as CD59 and MCP. Sublytic C5b-9 complexes can trigger [4,6,9,10]. Complement deposits are observed in the glomeruli of patients with MsPGN [1,2]. Our previous studies have also revealed the deposition of sublytic C5b-9 complexes on GMC surfaces in Thy-1N rats [9], but the role of sublytic C5b-9 complexes in Thy-1N has not been fully elucidated. During the progression of Thy-1N, GMC undergo two phases of change: early apoptosis/necrosis and secondary proliferation [4,9,11]. The mechanisms governing GMC apoptosis during the early stage of Thy-1N, including the genes involved in GMC apoptosis, remain unclear [12,13]. The growtharrest-and DNA-damage-inducible protein 45 (Gadd45) gene family, which encodes proteins that play important roles in controlling cell growth, has been im...

Metabolomics has been applied to explore altered metabolite profiles in disease and identify unique metabolic signatures in recent years. We aim to characterize the metabolic profile of rheumatoid arthritis patients and explore its underlying pathological processes using metabolomics approach. Serum samples from 30 rheumatoid arthritis (RA) patients, 30 primary Sjogren's syndrome (pSS) patients, and 32 healthy controls (HC) were collected. The sample was analyzed by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS). Potential biomarkers were screened from orthogonal projection to latent structure discriminate analysis (OPLS-DA) and further evaluated by receiver operating characteristic analysis (ROC). Compared with HC and pSS patients, the RA patients had increased serum levels of 4-methoxyphenylacetic acid, glutamic acid, L-leucine, L-phenylalanine, L-tryptophan, L-proline, glyceraldehyde, fumaric acid, and cholesterol as well as decreased capric acid, argininosuccinic acid, and billirubin. A total of eight potential biomarkers were screened and tentatively identified for RA. A panel of three metabolites (4-methoxyphenylacetic acid, L-phenylalanine, and L-leucine) was identified as specific biomarkers of RA. ROC analysis showed that the panel had a sensitivity of 93.30% with a specificity of 95.20% in discrimination RA from other groups. UPLC-HRMS-based quantification of circulating metabolites was a useful tool for identifying RA patients from pSS patients and healthy controls. The potential biomarkers indicated that the RA metabolic disturbance might be associated with inflammation injury, amino acid metabolism, oxidative stress, and phospholipid metabolism.