Rongfen Huo

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.

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.

A novel anti-Cyr61 antibody inhibits breast cancer growth and metastasis in vivo

Cysteine-rich protein 61(CCN1/Cyr61) has been implicated as an important mediator in proliferation and metastasis of breast cancer, which indicated that blockage of Cyr61 might be a potent target for breast cancer treatment. However, the antitumor effect of anti-Cyr61 antibodies on breast cancer in vivo has not been reported so far. In this study, we reported the effect and likely mechanism of generated anti-human Cyr61 monoclonal antibodies (mAb) on Cyr61 high expression line MDA-MB-231, known as a highly malignant and invasive human breast cancer cell line, at aspects of proliferation and migration in vitro and in vivo. We found the mAb, denoted as 093G9, revealed inhibitory effects on MDA-MB-231 cell proliferation, migration, and invasion through downregulation of both AKT and ERK phosphorylation in vitro compared with its isotype control. 093G9 also showed significant efficacy on suppressing primary tumor growth and spontaneous lymph node metastasis in in vivo mouse model. The specific epitope recognized by 093G9 was identified to be 140LPNLGCP146, adjacent to the VWC domain of Cyr61 by Ph.D.-C7C phage library display system. Our study provides direct evidence that Cyr61 can be a potent therapeutic target for patients who bear high Cyr61 expression breast cancer. Furthermore, the mAb, 093G9 developed in our laboratory, has shown a promising therapeutic characteristic in breast cancer.

Total glucosides of paeony attenuated functional maturation of dendritic cells via blocking TLR4/5 signaling in vivo.

It is well known that dendritic cells (DCs) play a critical role in the initiation and development of an immune response. Inhibitory effect on DC maturation alters immune-mediated inflammatory reaction in vivo. Total glucosides of paeony (TGP) are active compounds extracted from the roots of Paeonia lactiflora and have been widely used to ameliorate inflammation in therapy for autoimmune diseases. However, whether TGP act on DC maturation remains unknown. In this study, we investigated the effect of TGP on DC maturation in ovalbumin (OVA) immunized mice. Ear inflammation was inhibited by TGP (150 mgkg(-1), i.p.×11 days) obviously. The antigen presenting capacity of DC derived from TGP-treated mice was arrested. Meanwhile, OVA specific T cell proliferation was inhibited. In addition, we found that maturation of DCs was decreased by TGP treatment. Furthermore, OVA specific T cell proliferation was rescued by the adoptive transfer of mature DCs (mDCs) into TGP treated OVA-challenged mice. The research on the mechanism showed that TGP significantly inhibited activation of TLR4/5 singling. All these results demonstrated that TGP inhibited DC maturation and function by selectively blocking TLR4/5 activation in vivo, which in turn leads to reduce immune-mediated inflammation in vivo, adding a novel mechanism and therapeutic target of TGP for inflammatory and autoimmune disease treatment.

Paeoniflorin inhibits skin lesions in imiquimod-induced psoriasis-like mice by downregulating inflammation

Psoriasis is a common chronic immune-mediated inflammatory disease. It is well known that macrophages, neutrophils and T-helper 1 (Th1)/T-helper 17 (Th17) cells play important roles in skin lesions by provoking inflammation. Paeoniflorin (PF) is the major effective component extracted from the root of Paeonia lactiflora, which has been widely used in China to treat inflammatory and autoimmune diseases, including psoriasis. Although PF shows a clinical therapeutic effect on psoriasis patients, how PF affects infiltrated immune cells in psoriasis skin lesions is still unknown. In this study, using a generated imiquimod (IMQ)-induced psoriasis-like mouse model, we found that PF ameliorates inflammation and skin lesions. Subsequent analyses showed that PF decreases the number of F4/80(+)CD68(+) macrophages and their related cytokine production (TNF-α, IL-1β, IL-6, IL-12 and inducible nitric oxide synthase (iNOS)) in the skin of IMQ-challenged mice. Moreover, PF suppresses the number of CD11b(+)Gr-1(+) neutrophils and the expression of macrophage inflammatory protein-2 (MIP-2; a counterpart of human IL-8, which is responsible for the recruitment of neutrophils in mice). Finally, PF also down-regulates Th1- and Th17-related cytokine expression. Therefore, our new findings reveal that PF alleviates psoriatic skin lesions by inhibiting inflammation, which provides new insights into the immunomodulatory effect of PF in psoriasis treatment.

Cyr61 participates in the pathogenesis of rheumatoid arthritis by promoting proIL-1β production by fibroblast-like synoviocytes through an AKT-dependent NF-κB signaling pathway.

IL-1β plays a major role in the development of rheumatoid arthritis (RA). We previously showed that Cyr61 participates in RA pathogenesis as a proinflammatory factor. Here, we found that the levels of IL-1β and Cyr61 were higher in RA SF than in osteoarthritis (OA) SF. IL-1β mRNA and proIL-1β protein levels were remarkably increased in Cyr61-stimulated FLS; however, IL-1β was hardly detectable in the supernatant. We also found that the level of adenosine triphosphate (ATP) in SF and ST was significantly increased in RA patients and that the level of IL-1β in supernatants from Cyr61-activated FLS increased significantly when we added exogenous ATP to the culture. Mechanistically, Cyr61 induced proIL-1β production in FLS via the AKT-dependent NF-κB signaling pathway, and ATP caused Cyr61-induced proIL-1β to generate IL-1β in a caspase-1-dependent manner. Our results reveal a novel role of Cyr61 in RA that involves the promotion of proIL-1β production in FLS.

CCN1, a Pro-Inflammatory Factor, Aggravates Psoriasis Skin Lesions by Promoting Keratinocyte Activation

Psoriasis is a common chronic skin disease characterized by epidermal hyperplasia and inflammation. The pathogenesis of psoriasis is multifactorial and is not fully understood. Here we demonstrate that CCN1 (also called Cyr61, which is short for cysteine-rich 61), an extracellular matrix protein that is also considered a pro-inflammatory factor, is highly expressed in the lesional skin of psoriasis patients, as well as in that of imiquimod (IMQ)- and IL-23-treated psoriasis-like mice. Then we show that blocking CCN1 function in vivo attenuates epidermal hyperplasia and inflammation in psoriasis-like mice. Further, in primary cultured normal human keratinocytes and HaCaT (human keratinocyte cell line) cells, CCN1 promotes keratinocyte activation, including the proliferation and expression of immune-related molecules. Finally, we observe that integrin α6β1 is the receptor of CCN1 in keratinocytes, and CCN1 stimulation activates the downstream phosphoinositide-3 kinase/Akt/NF-κB signaling pathway. Taken together, our findings reveal that CCN1 has a critical role in psoriasis pathogenesis. Moreover, as CCN1 is a secreted extracellular matrix (ECM) protein, our study also provides evidence that ECM, which is involved in psoriatic pathogenesis, could be a potent target for psoriasis treatment.

Up-regulation of Th17 cells may underlie inhibition of Treg development caused by immunization with activated syngeneic T cells.

Background Our previous work showed that mice immunized with attenuated activated syngeneic T cells (aTCV) led to damping Treg function which resulted in enhancing anti-tumor immunity. It is well known that DC plays a very important role in controlling Th cell differentiation; whether DC involves Treg attenuation in immunized mice remained unknown. In this study, we provided evidence that increased mature DC (mDC) after immunization with aTCV skewed Th17 differentiation, which resulted in inhibition of Treg differentiation through IL-6 signaling pathway. Principal Findings In the present study, we found that the frequency of mDCs increased dramatically in the immunized mice accompanied by lower Treg cells compared to the controls. Moreover, both DCs and serum derived from the immunized mice suppressed Treg differentiation in vitro, respectively. mDCs generated from bone marrow precursor cells in vitro strongly inhibited Treg development and simultaneously drove Th17 differentiation with elevated IL-6 production. However, PD-L1, a potent Treg inducer did not show effect on Treg down-regulation. Assay with transwell systems showed that cell-cell contact was necessary for IL-6 production to a threshold to activate Th17 transcriptional factor RORγt and to inhibit Treg counterpart Foxp3. Conclusions Our results implicate up-regulated Th17 development might be one of mechanisms of enhancing anti-tumor immunity induced by immunization with aTCV, which provide a novel insight in numerous mechanisms responsible for anti-tumor immunity.