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Background: Tumor necrosis factor (TNF)-α inhibitors represented by Etanercept (a fusion protein containing soluble TNF receptor II (sTNFRII) and the Fc segment of human IgG1) play a pivotal role in Rheumatoid arthritis (RA) treatment. However, long-term use increases the risk of infection and tumors for their systemic inhibition of TNF-α, which disrupts the regular physiological function of this molecular. Mesenchymal stem cells (MSCs)-based delivery system provides new options for RA treatment with their "homing" and immune-regulation capacities, whereas inflammatory environment (especially TNF-α) is not conducive to MSCs' therapeutic effects by inducing apoptosis/autophagy. Here, we constructed a strain of sTNFRII-Fc-expressing MSCs (sTNFRII-MSC), aiming to offset the deficiency of those two interventions., Methods: Constructed sTNFRII-Fc lentiviral vector was used to infect human umbilical cord-derived MSCs, and sTNFRII-MSC stable cell line was generated by monoclonal cultivation. In vitro and vivo characteristics of sTNFRII-MSC were assessed by coculture assay and an acute inflammatory model in NOD/SCID mice. The sTNFRII-MSC were transplanted into CIA model, pathological and immunological indicators were detected to evaluate the therapeutic effects of sTNFRII-MSC. The distribution of sTNFRII-MSC was determined by immunofluorescence assay. Apoptosis and autophagy were analyzed by flow cytometry, western blot and immunofluorescence., Results: sTNFRII-Fc secreted by sTNFRII-MSC present biological activity both in vitro and vivo. sTNFRII-MSC transplantation effectively alleviates mice collagen-induced arthritis (CIA) via migrating to affected area, protecting articular cartilage destruction, modulating immune balance and sTNFRII-MSC showed prolonged internal retention via resisting apoptosis/autophagy induced by TNF-α., Conclusion: sTNFRII-Fc modification protects MSCs against apoptosis/autophagy induced by TNF-α, in addition to releasing sTNFRII-Fc neutralizing TNF-α to block relevant immune-inflammation cascade, and thus exert better therapeutic effects in alleviating inflammatory arthritis., (© 2021. The Author(s).)

Tolerogenic dendritic cells (tolDCs) have received much attention because of their capacity to restore immune homeostasis. RNA interference techniques have been used in several studies to generate tolDCs by inactivating certain molecules that regulate DC maturation and immunologic function. BAFF is a key B cell survival factor that is not only essential for B cell function but also T cell costimulation, and DCs are the major source of BAFF. In this study, we determined whether BAFF gene silencing in mature DCs could lead to a tolerogenic phenotype as well as the potential therapeutic effect of BAFF-silenced DCs on collagen-induced arthritis (CIA) in mice. Meanwhile, CRISPR/Cas9-mediated BAFF -/- DC2.4 cells were generated to verify the role of BAFF in DC maturation and functionality. BAFF-silenced DCs and BAFF -/- DC2.4 cells exhibited an immature phenotype and functional state. Further, the transplantation of BAFF-silenced DCs significantly alleviated CIA severity in mice, which correlated with a reduction in Th17 populations and increased regulatory T cells. In vitro, BAFF-silenced DCs promoted Foxp3 mRNA and IL-10 expression but inhibited ROR-γt mRNA and IL-17A expression in CD4 + T cells. Together, BAFF-silenced DCs can alleviate CIA, partly by inducing Foxp3 + regulatory T cells and suppressing Th17 subsets. Collectively, BAFF plays an important role in interactions between DCs and T cells, which might be a promising genetic target to generate tolDCs for autoimmune arthritis treatment., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Rheumatoid arthritis (RA) is characterized by the massive infiltration of various chronic inflammatory cells in synovia. In synovial fluid of patients with RA, M1 macrophages are dominant among all subtypes of macrophages, the mechanisms of macrophages polarization imbalance in RA has not been fully illuminated. The prostaglandin E2 (PGE2) augments M2 polarization in part via the cyclic adenosine monophosphate (cAMP)-cyclic AMP responsive element binding (CREB) signaling. However, previous study found constant stimulus of PGE2 on fibroblast-like synovial cells of adjuvant arthritis rats induced the decrease of cAMP, which is primarily caused by G protein-coupled receptor kinase 2 (GRK2)-induced EP4 over- desensitization. Whether GRK2 mediated-EP4 over-desensitization reduces the level of cAMP and inhibits M2 polarization in RA is unclear. Here we observed M1 macrophages were dominant in peritoneal macrophages (PMs), bone-marrow-derived macrophages (BMMs) and synovial macrophages of collagen-induced arthritis (CIA) mice. PGE2 stimulated M2 polarization via the EP4-cAMP-CREB in normal mice, while failed to promote M2 polarization in the PMs of CIA mice. Further, we found the EP4 over-desensitization stimulated by PGE2 induced abnormal PGE2-cAMP-CREB signaling as well as the imbalance of macrophage polarization. Targeted disruption of GRK2 in Raw264.7 (RAW) through GRK2 siRNA or CRISPR/Cas9 downregulated the M1 macrophage markers, upregulated the M2 macrophage markers and the EP4 membrane localization. The reduced M1/M2 ratio and increased p-CREB expression were observed in BMMs and PMs of GRK2 +/- mice. This study highlighted a novel role of GRK2 in regulating macrophages function in RA and provided new idea for precision treatment of RA., Competing Interests: The authors have declared that no competing interests exist.

Objective: CP-25 attenuates arthritis progression in animal models by inhibiting G protein-coupled receptor kinase 2 (GRK2) membrane expression. This study compared groups treated with high-dose methotrexate (MTX)/leflunomide (LEF) and CP-25 combined with low-dose MTX/LEF in an adjuvant-induced arthritis (AA) rat model and investigated possible mechanisms., Methods: AA was induced in rats via complete Freund's adjuvant. Experimental groups were divided into a normal group; vehicle group; monotherapy groups, including CP-25 (50 mg/kg), MTX (0.25, 0.5 mg/kg), and LEF (5, 10 mg/kg); and CP-25 (50 mg/kg)-combined MTX (0.25 mg/kg)/LEF (5 mg/kg) groups. We measured cytokine levels, phosphorylation and protein expression, and interactions between proteins. The role of GRK2 on phosphorylated extracellular signal-regulated kinase (p-ERK) was determined via GRK2-siRNA using a high content imaging system., Results: Therapeutic effects, including pathology and cytokine balance, were equivalent between the CP-25-combination groups and the high-dose MTX/LEF groups. P38, ERK, and c-Jun N-terminal kinase (JNK) activation in AA fibroblast-like synoviocytes (FLS) was reduced in the treatment groups; GRK2 expression was only inhibited in the CP-25 group. Interactions between GRK2 and p-ERK decreased in the vehicle group and were restored in the CP-25 group. GRK2 membrane expression and p-ERK nuclear expression increased in FLS pre-treated with tumour necrosis factor alpha and stimulated with prostaglandin E2. Nuclear expression of p-ERK increased in GRK2-siRNA FLS., Conclusion: Equivalent therapeutic effects were observed between CP-25-combination groups and high-dose MTX/LEF groups. CP-25 inhibited p-ERK by reducing the membrane expression of GRK2 in FLS from AA rats., (Copyright © 2018 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Ethnopharmacological Relevance: Total glucosides of paeony (TGP) is the first anti-inflammatory immune regulatory drug approved for the treatment of rheumatoid arthritis in China. A novel compound, paeoniflorin-6'-O-benzene sulfonate (code CP-25), comes from the structural modification of paeoniflorin (Pae), which is the effective active ingredient of TGP. The aim of the present study is to investigate the effect of CP-25 on adjuvant arthritis (AA) fibroblast-like synoviocytes (FLS) co-cultured with BAFF-activated CD4(+) T cells and the expression of BAFF-R in CD4(+) T cells., Methods: The mRNA expression of BAFF and its receptors was assessed by qPCR. The expression of BAFF receptors in CD4(+) T cells was analyzed by flow cytometry. The effect of CP-25 on AA rats was evaluated by their joint histopathology. The cell culture growth of thymocytes and FLS was detected by cell counting kit (CCK-8). The concentrations of IL-1β, TNF-α, and IL-6 were measured by Enzyme-linked immunosorbent assay (ELISA)., Results: The mRNA expression levels of BAFF and BAFF-R were enhanced in the mesenteric lymph nodes of AA rats, TACI expression was reduced, and BCMA had no change. The expression of BAFF-R in CD4(+) T cells was also enhanced. CP-25 alleviated the joint histopathology and decreased the expression of BAFF-R in CD4(+) T cells from AA rats in vivo. In vitro, CP-25 inhibited the abnormal cell culture growth of BAFF-stimulated thymocytes and FLS. In the co-culture system, IL-1β, IL-6 and TNF-α production was enhanced by FLS co-cultured with BAFF-activated CD4(+) T cells. Moreover, BAFF-stimulated CD4(+) T cells promoted the cell culture growth of FLS. The addition of CP-25 decreased the expression of BAFF-R in CD4(+) T cells and inhibited the cell culture growth and cytokine secretion ability of FLS co-cultured with BAFF-activated CD4(+) T cells., Conclusion: The present study indicates that CP-25 may repress the cell culture growth and cytokine secretion ability of FLS, and its inhibitory effects might be associated with its ability to inhibit the expression of BAFF-R in CD4(+) T cells in a co-culture. These observations might provide a scientific basis for the development of new drugs for the treatment of autoimmune diseases by CP-25., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Paeoniflorin-6'-O-benzene sulfonate (code: CP-25), a novel ester derivative of paeoniflorin (Pae), was evaluated in rats with adjuvant-induced arthritis (AA) to study its potential anti-arthritic activity. AA rats were treated with CP-25 (25, 50, or 100 mg/kg) from days 17 to 29 after immunization. CP-25 effectively reduced clinical and histopathological scores compared with the AA groups. CP-25-treated rats exhibited decreases in pro-inflammatory cytokines (IL-1β, IL-6, IL-17 and TNF-α) coupled with an increase in the anti-inflammatory cytokine TGF-β1 in the serum. CP-25 treatment inhibited M1 macrophage activation and enhanced M2 macrophage activation by influencing cytokine production. Decreases in Th17-IL-17 and the Th17-associated transcription factor RAR-related orphan receptor gamma (ROR-γt) dramatically demonstrated the immunomodulatory effects of CP-25 on abnormal immune dysfunction. In addition, CP-25 suppressed the production of receptor activator of nuclear factor kappa B ligand (RANKL) and matrix metalloproteinase (MMP) 9, which supported its anti-osteoclastic effects. The data presented here demonstrated that CP-25 significantly inhibited the progression of rat AA by reducing inflammation, immunity and bone damage. The protective effects of CP-25 in AA highlight its potential as an ideal new anti-arthritic agent for human RA.

This study is in order to clear the roles of BAFF and its receptors in the inflammation course of autoimmune arthritis. We used a T cell-mediated experimental autoimmune model adjuvant-induced arthritis (AA) rat to study the profiles of BAFF and its receptors in spleen during the inflammation arthritis induction and the effects of BAFF on DCs functions. In vivo, the levels of BAFF and the expression of BAFF-R, TACI were increased in spleen from very early stage of AA. The lesions of spleen were definite correlated with increased levels of BAFF in homogenization. The mature of DCs and increased number in spleen were mainly at early stage of arthritis. In addition, the levels of Interleukin (IL)-12 were found highest and IL-10 were found lowest at this time too. In vitro recombinant BAFF promoted maturation of DCs and inhibited the phagocytosis of DCs. Under stimulation of BAFF on DCs, the levels of tumor necrosis factor (TNF)-α, IL-6, IL-12 were increased, IL-10 and transforming growth factor (TGF)-β1 were decreased. Moreover, BAFF-treated DCs induced proliferation of CD4(+) T cell. These findings support the crucial pathogenic role of DCs, BAFF, and its receptors in the development of experimental arthritis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The objective of this study was to determine the expression and effects of B-lymphocyte stimulator (BLyS) in T cell-mediated autoimmune arthritis, a rat model of human rheumatoid arthritis (RA). Rat adjuvant-induced arthritis (AA) was induced by intradermal injection of 0.1ml complete Freund's adjuvant. Arthritis was evaluated by the histopathological examination of joint ankle. The BLyS expression was detected by immunohistochemical analysis. The level of BLyS and interleukin (IL)-17 were assayed by enzyme-linked immunosorbent assay. The gene expression of BLyS and its receptors (TACI, BCMA and BAFF-R) were assessed by quantitative reverse-transcription polymerase chain reaction. The effect of BLyS on the function of T cell was investigated by transwell assay. Using an AA rat model, we detected dysregulated expression and level of BLyS and its receptors in the local joint synovium tissue, peripheral lymphoid organs (spleen) and immune cells (macrophage, dendritic cells (DC) and T cell) at the peak of inflammation. In vitro, BLyS-treated DC induced IL-17 producing T cells. Neutralization of BLyS by the TACI-Ig fusion protein attenuated these stimulating effects of BLyS. These data suggest that the overproduction of BLyS may contribute to T cell responses and may be an attractive target for control of autoimmune diseases, such as RA, that involves both T and B cells., (Copyright © 2015 Elsevier B.V. All rights reserved.)