Ningli Li

Role of IFN-γ in induction of Foxp3 and conversion of CD4+ CD25– T cells to CD4+ Tregs

IFN-gamma is an important Th1 proinflammatory cytokine and has a paradoxical effect on EAE in which disease susceptibility is unexpectedly heightened in IFN-gamma-deficient mice. In this study, we provide what we believe is new evidence indicating that IFN-gamma is critically required for the conversion of CD4(+)CD25(-) T cells to CD4(+) Tregs during EAE. In our study, the added severity of EAE in IFN-gamma knockout mice was directly associated with altered encephalitogenic T cell responses, which correlated with reduced frequency and function of CD4(+)CD25(+)Foxp3(+) Tregs when compared with those of WT mice. It was demonstrated in both human and mouse systems that in vitro IFN-gamma treatment of CD4(+)CD25(-) T cells led to conversion of CD4(+) Tregs as characterized by increased expression of Foxp3 and enhanced regulatory function. Mouse CD4(+)CD25(-) T cells, when treated in vitro with IFN-gamma, acquired marked regulatory properties as evidenced by suppression of EAE by adoptive transfer. These findings have important implications for the understanding of the complex role of IFN-gamma in both induction and self regulation of inflammatory processes.

A critical role of IFNgamma in priming MSC-mediated suppression of T cell proliferation through up-regulation of B7-H1.

Bone-marrow-derived mesenchymal stem cells (MSCs) have been shown to possess immunosuppressive properties, e.g., by inhibiting T cell proliferation. Activated T cells can also enhance the immunosuppression ability of MSCs. The precise mechanisms underlying MSC-mediated immunosuppression remain largely undefined, although both cell-cell contact and soluble factors have been implicated; nor is it clear how the immunosuppressive property of MSCs is modulated by T cells. Using MSCs isolated from mouse bone marrow, we show here that interferon gamma (IFNγ), a well-known proinflammatory cytokine produced by activated T cells, plays an important role in priming the immunosuppressive property of MSCs. Mechanistically, IFNγ acts directly on MSCs and leads to up-regulation of B7-H1, an inhibitory surface molecule in these stem cells. MSCs primed by activated T cells derived from IFNγ−/− mouse exhibited dramatically reduced ability to suppress T cell proliferation, a defect that can be rescued by supplying exogenous IFNγ. Moreover, siRNA-mediated knockdown of B7-H1 in MSCs abolished immunosuppression by these cells. Taken together, our results suggest that IFNγ plays a critical role in triggering the immunosuppresion by MSCs through up-regulating B7-H1 in these cells, and provide evidence supporting the cell-cell contact mechanism in MSC-mediated immunosuppression.

Role of osteopontin in amplification and perpetuation of rheumatoid synovitis

Osteopontin (OPN) is an extracellular matrix protein of pleiotropic properties and has been recently recognized as a potential inflammatory cytokine. In this study, we demonstrate, for the first time to our knowledge, that overexpression of OPN in synovial T cells is associated with local inflammatory milieu and that OPN acts as an important mediator in amplification and perpetuation of rheumatoid synovitis. The study revealed that mRNA expression of OPN was highly elevated in CD4(+) synovial T cells derived from patients with RA, which correlated with increased OPN concentrations in synovial fluid (SF). The pattern of OPN overexpression was confined to rheumatoid synovium and correlated with coexpression of selected OPN receptors in synovial T cells, including integrins alphav and beta1 and CD44. RA-derived SF stimulated the expression of OPN in T cells, which was attributable to IL-10 present in SF and abrogated by anti-IL-10 antibody. Among the more than 300 autoimmune and inflammatory response genes examined, OPN selectively induced the expression of proinflammatory cytokines and chemokines known to promote migration and recruitment of inflammatory cells. Furthermore, it was evident that OPN activated transcription factor NF-kappaB in mononuclear cells. The study has important implications for understanding the role of OPN in rheumatoid synovitis and other inflammatory conditions.

IL-21 regulates Th17 cells in rheumatoid arthritis.

IL-21 is a type I cytokine that like IL-2, IL-4, IL-7, IL-9, and IL-15 uses the common gamma chain of cytokine receptor. IL-21 has been shown to regulate the function of T cells, B cells, natural killer cells, and dendritic cells in immune responses. Although activated CD4(+) T cells produce IL-21, recent data suggest that novel subsets of effector T cells are the major producers in immune responses. In this study, we show that IL-21 expression correlates with the presence of Th17 cells in synovial fluid (SF) and peripheral blood in rheumatoid arthritis patients. Human CCR6+ CD4(+) T cells produce high levels of both IL-21 and IL-17. Similar to mouse T cells, IL-21 auto-regulates its own production in human CD4(+) T cells. IL-21 potently enhances Th17 proliferation and suppresses Foxp3 expression, leading to the expression of RORC. IL-21 is therefore an autocrine cytokine that regulates human Th17 cells in rheumatoid arthritis, and serves as a good target for treating this autoimmune disease.

A critical role of Cyr61 in interleukin-17–dependent proliferation of fibroblast-like synoviocytes in rheumatoid arthritis

OBJECTIVE Fibroblast-like synoviocytes (FLS) are a major component of the hyperplastic synovial pannus that aggressively invades cartilage and bone during the course of rheumatoid arthritis (RA). Cyr61 (CCN1) is a product of a growth factor-inducible immediate early gene and is involved in cell adhesion, proliferation, and differentiation. However, the role that Cyr61 plays in FLS proliferation has remained undetermined. The aim of this study was to identify the role of Cyr61 in regulating the proliferation of FLS derived from patients with RA. METHODS Expression of Cyr61 in synovial tissue (ST) and in FLS was determined simultaneously using immunohistochemistry, real-time polymerase chain reaction, and Western blotting. Cyr61 levels in synovial fluid (SF) were determined by enzyme-linked immunosorbent assay. FLS proliferation stimulated by SF, Cyr61, and interleukin-17 (IL-17) was measured by thymidine incorporation. Activation of signal transduction pathways was determined by Western blotting and confocal microscopy. RESULTS Cyr61 was overexpressed in ST, FLS, and SF samples from RA patients as compared with samples from normal controls. Elevated levels of Cyr61 in RA SF promoted the proliferation of FLS, an effect that was abrogated by a neutralizing monoclonal antibody against human Cyr61. Furthermore, in samples from RA patients, Cyr61 was found to protect FLS from apoptosis and to sustain the expression of Bcl-2 in FLS. Most importantly, the expression of Cyr61 in FLS was regulated by IL-17 mainly via the p38 MAPK and NF-kappaB signaling pathways. Knockdown of expression of the Cyr61 gene inhibited IL-17-stimulated FLS proliferation. CONCLUSION Our findings indicate that Cyr61 plays a critical role in IL-17-mediated proliferation of FLS in RA and likely contributes to hyperplasia of synovial lining cells and eventually to joint destruction in patients with RA.

A Novel p53/microRNA-22/Cyr61 Axis in Synovial Cells Regulates Inflammation in Rheumatoid Arthritis

We previously showed that Cyr61 acts to promote fibroblast‐like synoviocyte (FLS) proliferation and Th17 cell differentiation, suggesting that Cyr61 plays an important role in mediating the joint inflammation and damage in rheumatoid arthritis (RA). The aim of this study was to investigate whether Cyr61 expression is regulated at the posttranscription level, and if so, how this regulation connects to other etiologic factors in RA.

Cyr61 Induces IL-6 Production by Fibroblast-like Synoviocytes Promoting Th17 Differentiation in Rheumatoid Arthritis

Cysteine-rich protein 61 (Cyr61)/CCN1 is a product of an immediate early gene and functions in mediating cell adhesion and inducing cell migration. We previously showed that increased production of Cyr61 by fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) promotes FLS proliferation and participates in RA pathogenesis with the IL-17–dependent pathway. However, whether Cyr61 in turn regulates Th17 cell differentiation and further enhances inflammation of RA remained unknown. In the current study, we explored the potential role of Cyr61 as a proinflammatory factor in RA pathogenesis. We found that Cyr61 treatment dramatically induced IL-6 production in FLS isolated from RA patients. Moreover, IL-6 production was attenuated by Cyr61 knockdown in FLS. Mechanistically, we showed that Cyr61 activated IL-6 production via the αvβ5/Akt/NF-κB signaling pathway. Further, using a coculture system consisting of purified CD4+ T cells and RA FLS, we found that RA FLS stimulated Th17 differentiation, and the pro-Th17 differentiation effect of RA FLS can be attenuated or stimulated by Cyr61 RNA interference or addition of exogenous Cyr61, respectively. Finally, using the collagen-induced arthritis animal model, we showed that treatment with the anti-Cyr61 mAb led to reduction of IL-6 levels, decrease of Th17 response, and attenuation of inflammation and disease progression in vivo. Taken together, our results reveal a novel role of Cyr61 in promoting Th17 development in RA via upregulation of IL-6 production by FLS, thus adding a new layer into the functional interplay between FLS and Th17 in RA pathogenesis. Our study also suggests that targeting of Cyr61 may represent a novel strategy in RA treatment.

Correlation of CD44v6 expression with ovarian cancer progression and recurrence

BackgroundPreviously some groups demonstrated that CD44 variant 6 (CD44v6) is correlated with progression and metastasis of ovarian cancer. However, a number of other groups failed to find such an association. Moreover, epithelial ovarian cancer is known to easily metastasize to distinct sites such as the pelvic and abdominal cavities, but the potential association of CD44v6 expression with site-specific metastasis of ovarian cancer has not been explored. This study sought to evaluate the expression of CD44 standard (CD44s) and CD44v6 in primary, metastatic and recurrent epithelial ovarian cancer to explore the potential association of CD44s and CD44v6 with tumor progression and recurrence.MethodsTumor specimens were procured from patients with advanced (FIGO III, G3) and recurrent ovarian serous adenocarcinoma. CD44s and CD44v6 expression in the tumor tissues was evaluated by real-time RT-PCR and Western blot. Moreover, serum soluble CD44s or CD44v6 concentrations of early stage (FIGO I, G1), advanced (FIGO III, G3) and recurrent ovarian serous adenocarcinoma patients were determined by enzyme-linked immunosorbent assays (ELISA). CD44v6 expression in a different set of tumor samples on an ovarian cancer tissue chip was evaluated by immunohistochemistry (IHC) and the correlation of CD44v6 expression with clinicopathologic features was analyzed. Finally, the effects of knockdown of CD44v6 in SKOV3 cells on cell adhesion, invasion and migration were assessed.ResultsThe expression of CD44v6, but not CD44s, is up-regulated in recurrent ovarian serous cancer compared to advanced primary tumor. CD44v6 expression is also preferentially increased in the tumor at the abdominal cavity metastasis site of advanced diseases. Consistently, serum soluble CD44v6 levels of recurrent ovarian cancer were higher than those of early stage and advanced primary diseases. The IHC data demonstrate that CD44v6 expression is correlated with clinicopathologic features and tumor progression. Lastly, knockdown of CD44v6 decreases the adhesion and migration but not invasion capacities of SKOV3 cells.ConclusionsCD44v6 expression levels are associated with epithelial ovarian cancer progression, metastasis and relapse. Moreover, serum soluble CD44v6 may be used as a potential marker for identifying tumor relapse. Finally, CD44v6 may play a role in ovarian cancer metastasis by mediating tumor cell adhesion and migration.

Total glucosides of paeony inhibits Th1/Th17 cells via decreasing dendritic cells activation in rheumatoid arthritis

Total glucoside of paeony (TGP), an active compound extracted from paeony root, has been used in therapy for rheumatoid arthritis (RA). Th1 and Th17 cells are now believed to play crucial roles in the lesions of RA. However, the molecular mechanism of TGP in inhibition of Th1 and Th17 cells remains unclear. In this study, we found that TGP treatment significantly decreased percentage and number of Th1 and Th17 cells in collagen induced arthritis (CIA) mice. Consistently, treatment with TGP decreased expression of T-bet and RORγt as well as phosphorylation of STAT1 and STAT3. In particular, TGP treatment inhibited dendritic cells (DCs) maturation and reduced production of IL-12 and IL-6. Moreover, TGP-treatment RA patients showed shank population of matured DCs and IFN-γ-, IL-17-producing cells. Taken together, our results demonstrated that TGP inhibited maturation and activation of DCs, which led to impaired Th1 and Th17 differentiation in vivo.

Cyr61 is involved in neutrophil infiltration in joints by inducing IL-8 production by fibroblast-like synoviocytes in rheumatoid arthritis

IntroductionIt is well known that neutrophils play very important roles in the development of rheumatoid arthritis (RA) and interleukin (IL)-8 is a critical chemokine in promoting neutrophil migration. We previously showed that increased production of Cyr61 by fibroblast-like synoviocytes (FLS) in RA promotes FLS proliferation and Th17 cell differentiation, thus Cyr61 is a pro-inflammatory factor in RA pathogenesis. In this study, we explored the role of Cyr61 in neutrophil migration to the joints of RA patients.MethodsRA FLS were treated with Cyr61 and IL-8 expression was analyzed by real-time PCR and ELISA. The migration of neutrophils recruited by the culture supernatants was determined by the use of a chemotaxis assay. Mice with collagen-induced arthritis (CIA) were treated with anti-Cyr61 monoclonal antibodies (mAb), or IgG1 as a control. Arthritis severity was determined by visual examination of the paws and joint destruction was determined by hematoxylin-eosin (H&E) staining. Signal transduction pathways in Cyr61-induced IL-8 production were investigated by real-time PCR, western blotting, confocal microscopy, luciferase reporter assay or chromatin immunoprecipitation (ChIP) assay.ResultsWe found that Cyr61 induced IL-8 production by RA FLS in an IL-1β and TNF-α independent pathway. Moreover, we identified that Cyr61-induced IL-8-mediated neutrophil migration in vitro. Using a CIA animal model, we found that treatment with anti-Cyr61 mAb led to a reduction in MIP-2 (a counterpart of human IL-8) expression and decrease in neutrophil infiltration, which is consistent with an attenuation of inflammation in vivo. Mechanistically, we showed that Cyr61 induced IL-8 production in FLS via AKT, JNK and ERK1/2-dependent AP-1, C/EBPβ and NF-κB signaling pathways.ConclusionsOur results here reveal a novel role of Cyr61 in the pathogenesis of RA. It promotes neutrophil infiltration via up-regulation of IL-8 production in FLS. Taken together with our previous work, this study provides further evidence that Cyr61 plays a key role in the vicious cycle formed by the interaction between infiltrating neutrophils, proliferated FLS and activated Th17 cells in the development of RA.