EPO promotes the progression of rheumatoid arthritis by inducing desialylation via increasing the expression of neuraminidase 3.

OBJECTIVE: Erythropoietin (EPO) known as an erythrocyte-stimulating factor is increased in patients with rheumatoid arthritis (RA). Nevertheless, the function of EPO in the process of RA and relative mechanism needs to be further clarified. METHODS: The level of EPO in serum and synovial fluid from patients with RA and healthy controls was determined by . Collagen-induced arthritis (CIA) mice were constructed to confirm the role of EPO on RA pathogenesis. Differentially expressed genes (DEGs) of EPO-treated fibroblast-like synoviocyte (FLS) were screened by transcriptome sequencing. The transcription factor of neuraminidase 3 (NEU3) of DEGs was verified by double luciferase reporting experiment, DNA pulldown, electrophoretic mobility shift assay and chromatin immunoprecipitation-quantitative PCR (qPCR) assay. RESULTS: The overexpression of EPO was confirmed in patients with RA, which was positively associated with Disease Activity Score 28-joint count. Additionally, EPO intervention could significantly aggravate the joint destruction in CIA models. The upregulation of NEU3 was screened and verified by transcriptome sequencing and qPCR in EPO-treated FLS, and signal transducer and activator of transcription 5 was screened and verified to be the specific transcription factor of NEU3. EPO upregulates NEU3 expression via activating the Janus kinase 2 (JAK2)-STAT5 signalling pathway through its receptor EPOR, thereby to promote the desialylation through enhancing the migration and invasion ability of FLS, which is verified by JAK2 inhibitor and NEU3 inhibitor. CONCLUSION: EPO, as a proinflammatory factor, accelerates the process of RA through transcriptional upregulation of the expression of NEU3 by JAK2/STAT5 pathway.

Erythropoietin-mediated IL-17 F attenuates sepsis-induced gut microbiota dysbiosis and barrier dysfunction.

Septic gut damage is critical in the progression of sepsis and multiple organ failure, characterized by gut microbiota dysbiosis and epithelium deficiency in the gut barrier. Recent studies highlight the protective effects of Erythropoietin (EPO) on multiple organs. The present study found that EPO treatment significantly alleviated the survival rate, suppressed inflammatory responses, and ameliorated intestine damage in mice with sepsis. EPO treatment also reversed sepsis-induced gut microbiota dysbiosis. The protective role of EPO in the gut barrier and microbiota was impaired after EPOR knockout. Notably, we innovatively demonstrated that IL-17 F screened by transcriptome sequencing could ameliorate sepsis and septic gut damage including gut microbiota dysbiosis and barrier dysfunction, which was verified by IL-17 F-treated fecal microbiota transplantation (FMT) as well. Our findings highlight the protection effects of EPO-mediated IL-17 F in sepsis-induced gut damage by alleviating gut barrier dysfunction and restoring gut microbiota dysbiosis. EPO and IL-17 F may be potential therapeutic targets in septic patients.

Glycine increased ferroptosis via SAM-mediated GPX4 promoter methylation in rheumatoid arthritis.

OBJECTIVE: Over-proliferation of synovium is a key event of invasive pannus formation and cartilage damage in the progression of RA disease. At the same time, ferroptosis may play a pivotal role in maintaining the balance of proliferation and death of synovium. In this study, we firstly evaluated the ferroptosis level in RA fibroblast-like synoviocytes (FLS) and then explored the role of glycine in ferroptosis. METHODS: Ferroptosis was evaluated in RA synovium and FLS. The therapeutic effect of glycine on RA was evaluated by clinical and histopathological score and cytokine level in a CIA mouse model. The influence of glycine on ferroptosis was evaluated by mitochondrial morphology observation and membrane potential assay in RA FLS. Methylase expression was detected to explore the mechanism behind the effect of glycine on glutathione peroxidase 4 (GPX4) methylation. RESULTS: Compared with healthy controls, ferroptosis decreased in the RA synovium and FLS, with a decrease in Acyl Coenzyme A Synthetase Long Chain 4 (ACSL4) and an increase in Ferritin heavy chain 1 (FTH1), GPX4 and cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11). Although both oxidation and antioxidation levels of lipids were higher in RA FLS than in healthy controls, the increase in antioxidation was slightly higher than oxidation. RNA-seq and verification showed that glycine regulated the ferroptosis pathway through increase S-adenosylmethionine (SAM) concentration and decrease the expression of GPX4 and FTH1 by promoting SAM-mediated GPX4 promoter methylation and reducing FTH1 expression in RA FLS. CONCLUSIONS: In summary, we confirmed a decline in ferroptosis in RA and explored that glycine enhanced ferroptosis via SAM-mediated GPX4 promoter methylation and ferritin decrease.

Protectin DX restores Treg/Th17 cell balance in rheumatoid arthritis by inhibiting NLRP3 inflammasome via miR-20a.

Regulatory T-cell (Treg)/T-helper 17 (Th17) cell balance plays an important role in the progression of rheumatoid arthritis (RA). Our study explored the protective effect of protectin DX (PDX), which restored Treg/Th17 cell balance in RA, and the role of the nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome pathway in this process. Using mass spectrometry, we discovered that level of PDX decreased in active-RA patients and increased in inactive-RA patients compared with HCs, and serum PDX was a potential biomarker in RA activity detection (area under the curve [AUC] = 0.86). In addition, a collagen-induced arthritis (CIA) mice model was constructed and PDX obviously delayed RA progression in the CIA model, upregulating Tregs and anti-inflammatory cytokines while downregulating Th17 cells and pro-inflammatory cytokines. Moreover, NLRP3 knockout and rescue experiments demonstrated that NLRP3 participated in PDX-mediated Treg/Th17 cell balance restoration, joint injury amelioration and inflammatory-response attenuation using Nlrp3-/- mice. Furthermore, microarray and verified experiments confirmed that PDX reduced NLRP3 expression via miRNA-20a (miR-20a). In summary, we confirmed for the first time that PDX could effectively ameliorate CIA progression by restoring Treg/Th17 cell balance, which was mediated by inhibition of the NLRP3 inflammasome pathway via miR-20a.

TSP1 is the essential domain of SEMA5A involved in pannus formation in rheumatoid arthritis.

OBJECTIVE: In this study, we explored the effect of semaphorin5A (SEMA5A) on RA pathogenesis and its specific TSP1 domain on pannus formation. METHODS: The expression of SEMA5A was detected in the synovium, the fibroblast-like synoviocytes (FLSs) and the SF of RA patients and healthy controls (HCs) by real-time quantitative PCR (q-PCR), immunohistochemistry staining, western blot and ELISA. SEMA5A-mAb intervention was performed to appraise the severity of joints in the CIA model. Transcriptome sequencing and bioinformatics analysis in SEMA5A-transfected FLSs from HCs were performed to screen differentially expressed genes after SEMA5A overexpression. An MTT assay in RA-FLSs, a chicken embryo allantoic membrane experiment and a tube formation experiment were used to clarify the influence of SEMA5A on cell proliferation and angiogenesis. Furthermore, a rescue experiment verified the function of the TSP1 domain of SEMA5A in the progress of RA with Sema5a-/- CIA mice. RESULTS: The expression of SEMA5A increased in RA compared with that in HCs. Simultaneously, SEMA5A-mAbs significantly attenuated joint injury and the inflammatory response in CIA models. In addition, transcriptome sequencing and angiogenesis-related experiments verified the ability of SEMA5A to promote FLS proliferation and angiogenesis. Moreover, TSP1 was proved to be an essential domain in SEMA5A-induced angiogenesis in vitro. Additionally, rescue of TSP1-deleted SEMA5A failed to reduce the severity of arthritis in a CIA model constructed with Sema5a -/- mice. CONCLUSION: In summary, upregulation of SEMA5A was first confirmed in pathological lesions of RA patients. Furthermore, treatment with SEMA5A-mAbs attenuated the progress of RA in the CIA model. Moreover, TSP1 was indicated as the key domain of SEMA5A in the promotion of pannus formation in RA.

Secreted KIAA1199 promotes the progression of rheumatoid arthritis by mediating hyaluronic acid degradation in an ANXA1-dependent manner.

In inflamed joints, enhanced hyaluronic acid (HA) degradation is closely related to the pathogenesis of rheumatoid arthritis (RA). KIAA1199 has been identified as a hyaladherin that mediates the intracellular degradation of HA, but its extracellular function remains unclear. In this study, we found that the serum and synovial levels of secreted KIAA1199 (sKIAA1199) and low-molecular-weight HA (LMW-HA, MW < 100 kDa) in RA patients were significantly increased, and the positive correlation between them was shown for the first time. Of note, treatment with anti-KIAA1199 mAb effectively alleviated the severity of arthritis and reduced serum LMW-HA levels and cytokine secretion in collagen-induced arthritis (CIA) mice. In vitro, sKIAA1199 was shown to mediate exogenous HA degradation by attaching to the cell membrane of RA fibroblast-like synoviosytes (RA FLS). Furthermore, the HA-degrading activity of sKIAA1199 depended largely on its adhesion to the membrane, which was achieved by its G8 domain binding to ANXA1. In vivo, kiaa1199-KO mice exhibited greater resistance to collagen-induced arthritis. Interestingly, this resistance could be partially reversed by intra-articular injection of vectors encoding full-length KIAA1199 instead of G8-deleted KIAA119 mutant, which further confirmed the indispensable role of G8 domain in KIAA1199 involvement in RA pathological processes. Mechanically, the activation of NF-κB by interleukin-6 (IL-6) through PI3K/Akt signaling is suggested to be the main pathway to induce KIAA1199 expression in RA FLS. In conclusion, our study supported the contribution of sKIAA1199 to RA pathogenesis, providing a new therapeutic target for RA by blocking sKIAA1199-mediated HA degradation.

Tofacitinib restores the balance of γδTreg/γδT17 cells in rheumatoid arthritis by inhibiting the NLRP3 inflammasome.

Objective: Tofacitinib (TOF) is a Janus kinase (JAK) inhibitor used in the treatment of rheumatoid arthritis (RA), but the mechanism of its action remains unclear. In this study, we investigated the influence of TOF on gamma delta regulatory T-cell (γδTreg)/γδT17 cell balance in RA and the role of the nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome in this process. Methods: We detected levels of inflammatory factors in the serum of RA patients before and after administration of TOF using an enzyme-linked immunosorbent assay (ELISA). A collagen-induced arthritis (CIA) model was constructed to investigate the effect of TOF on arthritis symptoms, γδTreg/γδT17 cell balance and the NLRP3 inflammasome. We used bone marrow-derived macrophages (BMDMs) to study the effect of TOF on NLRP3 inflammasome activation. Nlrp3-/- mice were introduced to assess the influence of NLRP3 on γδT17 cell activation in RA. Results: TOF treatment decreased levels of γδT17 cell-related cytokine interleukin-17 (IL-17) in RA patients. In addition, TOF intervention in the CIA model reduced joint inflammation and damage, rebalanced the γδTreg/γδT17 cell ratio and inhibited excessive NLRP3 inflammasome activation in draining lymph nodes and arthritic joints. BMDM intervention experiments demonstrated that TOF decreased the level of secreted IL-1ß via downregulation of NLRP3. Furthermore, experiments using Nlrp3-/- mice verified that the NLRP3 inflammasome mediated the effect of TOF on γδT17 cell activation. Conclusions: Recovery of γδTreg/γδT17 cell balance was a novel mechanism by which TOF alleviated RA. Meanwhile, NLRP3 played a pivotal role in the process of TOF-mediated γδT17 cell activation.

Resolvin D1 suppresses pannus formation via decreasing connective tissue growth factor caused by upregulation of miRNA-146a-5p in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation and joint stiffness, finally leading to tissue destruction. Connective tissue growth factor (CTGF) is a critical factor in RA progression, which promotes fibroblast-like synoviocyte (FLS) proliferation, pannus formation, and the damage of cartilage as well as bone. Resolvin D1 (RvD1) can promote inflammation resolution in acute inflammatory diseases, and recently, effects of RvD1 on chronic inflammatory diseases also attracted attention. This study aimed to examine the effect of RvD1 on pannus formation in RA and the underlying mechanism. METHODS: Serum levels of RvD1 and CTGF were determined in RA patients and healthy persons by UPLC-MS/MS and ELISA respectively. The levels of CTGF and inflammatory factors were assessed by qRT-PCR and ELISA. MicroRNA expression profile was determined by miRNA microarray. The effects of CTGF, RvD1, and miR-146a-5p on angiogenesis were evaluated with tube formation and chick chorioallantoic membrane (CAM) assays. Collagen-induced arthritis (CIA) mice were constructed to detect the effects of RvD1 and miR146a-5p on RA. STAT3 activation was determined by Western blotting. RESULTS: RvD1 levels decreased while CTGF levels increased in RA patients' serum, and an inverse correlation of the concentrations of RvD1 and CTGF in the serum of RA patients was synchronously observed. In CIA mice, RvD1 suppressed angiopoiesis and decreased the expression of CTGF. Simultaneously, RvD1 significantly decreased CTGF and pro-inflammation cytokines levels in RA FLS. Furthermore, CTGF suppressed angiopoiesis and RvD1 inhibited the proliferation and migration of RA FLS and angiopoiesis. MiRNA microarray and qRT-PCR results showed that RvD1 upregulated miRNA-146a-5p. The transfection experiments demonstrated that miRNA-146a-5p could decrease inflammatory factors and CTGF levels. Moreover, miRNA-146a-5p decreased the proliferation of FLS and angiogenesis in vivo. MiRNA-146a-5p also suppressed angiogenesis and downregulated the expression of CTGF in CIA mice. Finally, Western blot results revealed that miRNA-146a-5p inhibited the activation of STAT3. CONCLUSION: RvD1 is prone to alleviate RA progression through the upregulation of miRNA-146a-5p to suppress the expression of CTGF and inflammatory mediators, thereby decreasing pannus formation and cartilage damage.

Poly(ADP-Ribose) Polymerase Enhances Infiltration of Mononuclear Cells in Primary Sjögren's Syndrome Through Interferon-Induced Protein With Tetratricopeptide Repeats 1-Mediated Up-Regulation of CXCL10.

OBJECTIVE: Mononuclear cell infiltration and type I interferon (IFN) system activation play an important role in primary Sjögren's syndrome (SS). We undertook this study to investigate the mechanism of poly(ADP-ribose) polymerase family member 9 (PARP-9) on mononuclear cell infiltration triggered by type I IFN. METHODS: A proteomic study was conducted in peripheral blood mononuclear cells from patients with primary SS (n = 30) and healthy controls (n = 30) to determine differentially expressed proteins (DEPs) (P < 0.05; fold change >1.20). Labial salivary glands (LSGs) were isolated for hematoxylin and eosin staining and immunohistochemical analysis. CD19+ B cells were purified by magnetic cell sorting for immunofluorescence staining, lentivirus-PARP-9 transfection, and IFNα treatment experiments. PARP-9 small interfering RNA (siRNA) and DTX3L siRNA were delivered into female NOD/LtJ female mice to determine their effect. RESULTS: The overexpression of PARP-9 and CXCL10 as well as their colocalization was confirmed in primary SS. PARP-9 levels in LSGs rose with increased Chisholm scores in patients with primary SS. PARP-9 and DTX3L were present in the infiltrating mononuclear cells from salivary glands in female NOD/LtJ mouse models. Additionally, Ingenuity Pathway Analysis networks of DEPs demonstrated that PARP-9, STAT1, and IFN-induced protein with tetratricopeptide repeats 1 (IFIT-1) participated in the IFN-related pathway. Furthermore, PARP-9 could up-regulate the expression of IFIT1 and CXCL10 in B cells. Moreover, PARP-9 and CXCL10 could be induced by IFNα in B cells. CONCLUSION: This study is the first to implicate PARP-9 as a regulator of infiltration of mononuclear cells in primary SS progression and to reveal that PARP-9 increases CXCL10 expression through up-regulating IFIT-1, which is mediated by the phosphorylation of STAT1. PARP-9 might therefore be a novel therapeutic target for primary SS.