hIgD-Fc-Ig fusion protein regulates T cell functions by inhibiting TCR signaling pathway in adjuvant arthritis rats.

This study aimed to investigate the effect of hIgD-Fc-Ig on TCR-Lck-Erk activated by IgD in adjuvant arthritis (AA) rats. Wistar rats were divided into the normal, AA model, hIgD-Fc-Ig (1 mg/kg, 3 mg/kg and 9 mg/kg) and Etanercept (3 mg/kg) groups. The overall index of AA rats was measured every 3 days. The pathologic examination of knee joints and the proliferation of the spleen and thymus of AA rats were detected by H&E staining and CCK-8. The blood flow signal of knee joints of experimental rats was examined by US. The articular bone injury was detected by X-ray. The changes in PBMCs and spleen T cell subsets were detected by flow cytometry. The expression of CD3ε, p-Lck, p-Zap70, Ras, and p-Erk in rat spleens was detected by immunofluorescence and WB. Rat spleen T cells or Jurkat cells treated by IgD to observe the effect of hIgD-Fc-Ig on TCR and its downstream protein expression. The results showed that hIgD-Fc-Ig had a therapeutic effect on AA rats by reducing the secondary inflammation, improving pathological changes. hIgD-Fc-Ig can reduce the ratio of Th cells of PBMCs of AA rats, the ratio of Th, Th1, Th17 cells and increase the ratio of Th2, Treg cells of AA rat spleens. hIgD-Fc-Ig could down-regulate the expression of CD3ε, p-Lck, p-Zap70, Ras, p-Erk in vivo or in vitro. In conclusion, hIgD-Fc-Ig could alleviate the symptoms of AA rats and regulate T cells through TCR-Lck-Erk signaling pathway and maybe a new promising biological agent for RA.

BAFF Promotes FLS Activation Through BAFFR-Mediated Non-canonical NF-κB Pathway and the Effects of CP-25.

B cell activating factor (BAFF) has been shown to play a key role in regulating B cell function, but little is known about whether BAFF affects the function of fibroblast-like synoviocyte (FLS), an effector cell of rheumatoid arthritis (RA). CP-25, a new ester derivative of paeoniflorin, could alleviate the arthritis symptoms of collagen-induced arthritis (CIA) mice by inhibiting BAFF-mediated abnormal activation of B cells. In this study, we aimed to understand the mechanism by which BAFF activates FLS and the effect of CP-25 on FLS function. Therefore, the proliferation and migration abilities of FLS and key proteins on the non-canonical NF-κB pathway were examined. The results showed that compared with the FLS of normal rats/OA patients, the expression of BAFF-R, TRAF2, NIK, p-IKKα, P100, and P52 was higher in the FLS of AA rats/RA patients, while the expression of TRAF3 was lower. And, BAFF promotes FLS activation by activating the non-canonical NF-κB signaling pathway. Meanwhile, BAFFR-siRNA inhibited the proliferation of FLS and the activation of non-canonical NF-κB signaling in FLS induced by BAFF. Additionally, CP-25 could inhibit abnormal proliferation and migration of FLS by regulating non-canonical NF-κB signaling. We concluded that BAFF may act as an important role in facilitating the function of FLS through the BAFFR-mediated non-canonical NF-κB pathway, which would be useful for revealing the pathological mechanism of RA. And CP-25 may become a potential new drug for the treatment of RA, providing a scientific basis for the development of new drugs to treat RA.

IgD promotes pannus formation by activating Wnt5A-Fzd5-CTHRC1-NF-κB signaling pathway in FLS of CIA rats and the regulation of IgD-Fc-Ig fusion protein.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by joint inflammation, synovial hyperplasia, cartilage degeneration, bone erosion, and pannus. Immunoglobulin D (IgD) plays an important role in autoimmune diseases although the content of it in vivo is low. Increased concentrations of anti-IgD autoantibodies have been detected in many RA patients. IgD-Fc-Ig fusion protein is constructed by connecting human IgD Fc domain and IgG1 Fc domain, which specifically block the IgD/ IgDR pathway and regulate the function of cells expressing IgDR to treat RA. The expression levels of Wnt5A and Frizzled 5 are higher in RA synovial tissue specimens. The complex of Wnt5A-Fzd5-LRP5/6-CTHRC1 promotes the expression of hypoxia inducible factor-1α by activating nuclear factor kappa-B (NF-κB), leading to high expression of VEGF and participating in angiogenesis. VEGF is the strongest angiogenic factor found so far. Here, we aimed to explore whether IgD participates in synovitis by binding to IgDR and regulating the activation of Wnt5A-Fzd5-CTHRC1-NF-κB signaling pathway in fibroblast synovial cells (FLSs), whether IgD-Fc-Ig fusion protein inhibits VEGF production in FLS of CIA and explore mechanism. We found that IgDR is expressed on MH7A and FLS. IgD promotes VEGF expression by activating Wnt5A-Fzd5-CTHRC1-NF-κB signaling pathway in MH7A and FLS. After activation of Fzd5 with Wnt5A, IgD-Fc-Ig reduced VEGF-A level in the culture supernatant of MH7A stimulation by IgD. The expressions of CTHRC1, Fzd5, p-P65 and VEGF in MH7A and FLSs were down-regulated after IgD-Fc-Ig treatment. IgD-Fc-Ig suppressed the combination of CTHRC1 and Fzd5 as well. By using the animal model, we demonstrated that IgD-Fc-Ig suppress ankle CTHRC1 and Fzd5 production resulted in inhibition of index of joint inflammation of CIA rats, which were consistent with vitro results. Conclusively, IgD-Fc-Ig inhibits IgD and Wnt5A-induced angiogenesis and joint inflammation by suppressing the combination of CTHRC1 and Fzd5. Our results show that IgD-Fc-Ig exerts its suppressive effect on IgD and Wnt5A by Wnt5A-Fzd5-CTHRC1-NF-κB signaling pathway.

The monomer derivative of paeoniflorin inhibits macrophage pyroptosis via regulating TLR4/ NLRP3/ GSDMD signaling pathway in adjuvant arthritis rats.

OBJECTIVE: This study was aimed to investigate the effect of monomer derivative of paeoniflorin (MDP) on macrophage pyroptosis mediated by TLR4/NLRP3/GSDMD signaling pathway in adjuvant arthritis (AA) rats. METHOD: Wistar rats were divided into normal group, AA model group, MDP (50 mg/kg) group and MTX (0.5 mg/kg) group. The expression of TLR4, NLRP3 and GSDMD in macrophage were detected by immunofluorescence assay. The expression of TLR4 and the ratio of macrophage pyroptosis were analyzed by flow cytometry. Cell morphology was observed by scanning electron microscopy. The cytokine levels of IL-18 and IL-1ß were detected by ELISA. The expressions of proteins related to macrophage pyroptosis were detected by western blot. RESULTS: MDP has a therapeutic effect on rats AA by reducing the secondary inflammation and improving pathological changes. The results of X-ray imaging and ultrasound images showed that MDP could inhibit bone erosion, soft tissue swelling, and joint space narrowing. Macrophage pyroptosis was found in secondary inflammation of AA rats. The expressions of TLR4, MyD88, NLRP3, Caspase-1, ASC, and GSDMD-N in macrophage were increased, the levels of IL-18 and IL-1ß were enhanced, and the morphology of pyroptosis could be observed. MDP could inhibit macrophage polarization and macrophage pyroptosis, and down-regulated the cytokine levels of IL-18 and IL-1ß in AA rats. MDP could regulate the M1/M2 ratio, decreased the ratio of macrophage pyroptosis and down-regulated the expressions of TLR4, MyD88, NLRP3, Caspase-1, ASC, and GSDMD-N in vivo and in vitro. CONCLUSION: Abnormal activation of TLR4/NLRP3/GSDMD signaling pathway may be involved in macrophage pyroptosis in AA rats. The therapeutic effect of MDP on AA rats is related to the inhibition of macrophage pyroptosis by regulating the TLR4/NLRP3/GSDMD signaling pathway.

hIgDFc-Ig inhibits B cell function by regulating the BCR-Syk-Btk-NF-κB signalling pathway in mice with collagen-induced arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease targeting the synovium. Previous studies have found that IgD may be a potential target for the treatment of RA. We designed a new type of fusion protein, hIgDFc-Ig (DG), to block the binding of IgD to IgD receptor (IgDR). In this study, we found that DG has a significant therapeutic effect in mice with collagen-induced arthritis (CIA). DG improved the claw of irritation symptoms in these mice, inhibited the pathological changes in spleen and joint tissues, and had a moderating effect on B cell subsets at different inflammatory stages. Moreover, DG could also decrease the levels of IgA, IgD, IgM and IgG subtypes of immunoglobulin in the serum of mice with CIA. In vitro, B cell antigen receptor (BCR) knockout Ramos cells were established using the CRISPR/Cas9 technology to further study the activation of BCR signalling by IgD and the effect of DG. We found that the therapeutic effect of DG in mice with CIA may be achieved by inhibiting the activation of BCR signalling by IgD, which may be related to the activation of Igß. In summary, DG may be a potential biological agent for the treatment of RA and it has broad application prospects in the future.

Paeoniflorin-6'-o-benzene sulfonate (CP-25) improves vasculitis through inhibiting IL-17A/JAK/STAT3 signaling pathway in endothelial cells of HFD CIA rats.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that affects not only joints but also multiple organ systems including cardiovascular system. Endothelial dysfunction plays an important role in cardiovascular diseases (CVD). In RA, endothelial dysfunction exists at both the macrovascular and the microvascular levels, which is a precursor to vasculitis. This study aimed to investigate the pathogenesis of vasculitis and the therapeutic effect of CP-25 on vasculitis in high-fat diet (HFD) collagen-induced arthritis (CIA) rats. Experimental groups were divided into normal group, HFD group, CIA group, HFD CIA group, CP-25 group and MTX group. In vitro, IL-17A was used to stimulate human umbilical vein endothelial cells (HUVECs), and then CP-25 was used to intervene. Results showed that CP-25 reduced global scoring (GS), arthritis index (AI), and swollen joint count (SJC) scores, improved histopathological score, reduced T cells percentage, and decreased IL-17A and ICAM-1 levels. Besides, CP-25 reduced the expression of p-STAT3 to normal levels in vascular of HFD CIA rats. In vitro, IL-17A promoted the expression of p-JAK1, p-JAK2, p-JAK3, pSTAT3, and ICAM-1, and CP-25 inhibited the expression of p-JAK1, p-JAK2, p-JAK3, p-STAT3, and ICAM-1. In conclusion, CP-25 might inhibit endothelial cell activation through inhibiting IL-17A/JAK/STAT3 signaling pathway, which improves vasculitis in HFD CIA rats.

Etanercept Inhibits B Cell Differentiation by Regulating TNFRII/TRAF2/NF-κB Signaling Pathway in Rheumatoid Arthritis.

OBJECTIVE: To explore the role of B cells in rheumatoid arthritis (RA) and the potential effects and mechanisms of etanercept on B cells. METHODS: In RA patients, the levels of tumor necrosis factor-α (TNF-α) and B cell activating factor (BAFF) were detected by ELISA. The percentage of B cell subsets was measured by flow cytometry. Laboratory indicators (rheumatoid factor, C-reactive protein, erythrocyte sedimentation rate) and clinical indicators (disease activity score in 28 joints, health assessment questionnaire score, swollen joint counts, tender joint counts) were measured. The correlation between B cell subsets and laboratory indicators or clinical indicators was analyzed. In mice, B cells proliferation was detected by CCK-8 kit. The expression of TNFRII and the percentage of B cell subsets in spleen were detected by flow cytometry. The expressions of TRAF2, p38, P-p38, p65, P-p65 in B cells were detected by WB. RESULTS: The percentage of CD19-CD27+CD138+ plasma B cells was positively correlated with ESR or RF. Etanercept could decrease the percentage of CD19+ total B cells, CD19+CD27+ memory B cells and CD19-CD27+CD138+ plasma B cells, reduce the levels of TNF-α, BAFF, relieve clinical and laboratory indicators in RA patients. In addition, etanercept could inhibit the proliferation of B cells, bate the differentiation of transitional B cells to mature B cells, down-regulate the expression of TNFRII, TRAF2, P-p38, P-p65 in B cells. CONCLUSION: B cells act a key role in the pathogenesis of RA. Etanercept inhibits B cells differentiation by down-regulating TNFRII/TRAF2/NF-κB signaling pathway.

The advances in pyroptosis initiated by inflammasome in inflammatory and immune diseases.

Pyroptosis is a programmed and inflammatory cell death initiated by inflammasome. During pyroptosis, cytosolic pattern recognition receptors, apoptosis-associated speck-like protein and pro-Caspase-1 form activated inflammasome together. Caspase-1 activated by inflammasome results in generating an N-terminal cleavage product of gasdermin D (GSDMD), which is a major executor of pyroptosis. As a consequence of pyroptosis, a large number of pro-inflammatory cytokines are released including IL-1ß and IL-18. Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and absent in melanoma 2 (AIM2)-like receptors (ALRs) belong to cytosolic pattern recognition receptors and assemble inflammasomes by detecting host cell damage signals. Pyroptosis pathways are divided into canonical and non-canonical pathways according to the identification of damage signals by cytoplasmic protein sensors. Pyroptosis not only plays an important role in infection, but also plays a vital role in inflammatory immune diseases. This article reviews the advances research of pyroptosis initiated by inflammasome in inflammatory and immune diseases.