Mouse Model Of Arthritis Research Articles

Circular RNA CircCDKN2B−AS_006 Promotes the Tumor-like Growth and Metastasis of Rheumatoid Arthritis Synovial Fibroblasts by Targeting the miR−1258/RUNX1 Axis

Rheumatoid arthritis (RA) is an autoimmune polyarthritis in which synovial fibroblasts (SFs) play a major role in cartilage and bone destruction through tumor-like proliferation, migration, and invasion. Circular RNAs (circRNAs) have emerged as vital regulators for tumor progression. However, the regulatory role, clinical significance, and underlying mechanisms of circRNAs in RASF tumor-like growth and metastasis remain largely unknown. Differentially expressed circRNAs in synovium samples from patients with RA and patients with joint trauma were identified via RNA sequencing. Subsequently, in vitro and in vivo experiments were performed to investigate the functional roles of circCDKN2B-AS_006 in RASF proliferation, migration, and invasion. CircCDKN2B-AS_006 was upregulated in synovium samples from patients with RA and promoted the tumor-like proliferation, migration, and invasion of RASFs. Mechanistically, circCDKN2B-AS_006 was shown to regulate the expression of runt-related transcription factor 1 (RUNX1) by sponging miR-1258, influencing the Wnt/β-catenin signaling pathway, and promoting the epithelial-to-mesenchymal transition (EMT) in RASFs. Moreover, in the collagen-induced arthritis (CIA) mouse model, intra-articular injection of lentivirus-shcircCDKN2B-AS_006 was capable of alleviating the severity of arthritis and inhibiting the aggressive behaviors of SFs. Furthermore, the correlation analysis results revealed that the circCDKN2B-AS_006/miR-1258/RUNX1 axis in the synovium was correlated with the clinical indicators of RA patients. CircCDKN2B-AS_006 promoted the proliferation, migration, and invasion of RASFs by modulating the miR-1258/RUNX1 axis.

Therapeutic Potential of Targeting the NLRP3 Inflammasome in Rheumatoid Arthritis.

NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is a cytoplasmic multiprotein complex composed of the innate immune receptor protein NLRP3, the adapter protein apoptosis-associate speck-like protein containing a caspase recruitment domain (ASC), and the inflammatory protease cysteine-1. Pathogen-associated molecular patterns (PAMPs) or other endogenous danger-associated molecular patterns (DAMPs) activate the NLRP3 inflammasome. As part of the innate immune response, activated NLRP3 promotes GSDMD-dependent pyroptosis, and IL-1β and IL-18 are released during inflammation. Aberrantly activated NLRP3 is deeply involved in various inflammatory diseases. Due to its interaction with adaptive immunity. NLRP3 inflammation has increasingly received attention in autoimmune diseases. Rheumatoid arthritis (RA) is a classic autoimmune disease, which mainly causes bone and cartilage damage. Elevated levels of NLRP3 can be detected in the synovium of RA patients. NLRP3 overactivation is strongly associated with RA activity. Mouse models of spontaneous arthritis has shown that NLRP3/IL-1β axis is implicated in periarticular inflammation in RA. In this review, we describe the current understanding of NLRP3 activation in RA pathogenesis and dissect its impact on innate and adaptive immunity. We also discuss the potential application of specific inhibitors of NLRP3 to provide new therapeutic strategies for treating RA.

Immunomodulatory Microparticles Epigenetically Modulate T Cells and Systemically Ameliorate Autoimmune Arthritis.

Disease modifying antirheumatic drugs (DMARDs) have improved the prognosis of autoimmune inflammatory arthritides but a large fraction of patients display partial or nonresponsiveness to front-line DMARDs. Here, an immunoregulatory approach based on sustained joint-localized release of all-trans retinoic acid (ATRA), which modulates local immune activation and enhances disease-protective T cells and leads to systemic disease control is reported. ATRA imprints a unique chromatin landscape in T cells, which is associated with an enhancement in the differentiation of naïve T cells into anti-inflammatory regulatory T cells (Treg ) and suppression of Treg destabilization. Sustained release poly-(lactic-co-glycolic) acid (PLGA)-based biodegradable microparticles encapsulating ATRA (PLGA-ATRA MP) are retained in arthritic mouse joints after intra-articular (IA) injection. IA PLGA-ATRA MP enhance migratory Treg which in turn reduce inflammation and modify disease in injected and uninjected joints, a phenotype that is also reproduced by IA injection of Treg . PLGA-ATRA MP reduce proteoglycan loss and bone erosions in the SKG and collagen-induced arthritis mouse models of autoimmune arthritis. Strikingly, systemic disease modulation by PLGA-ATRA MP is not associated with generalized immune suppression. PLGA-ATRA MP have the potential to be developed as a disease modifying agent for autoimmune arthritis.

Intra-articular delivery of AAV vectors encoding PD-L1 attenuates joint inflammation and tissue damage in a mouse model of rheumatoid arthritis.

Rheumatoid arthritis (RA) is the most common form of autoimmune inflammatory arthritis. Intra-articular gene delivery to block proinflammatory cytokines has been studied in pre-clinical models and human clinical trials. It has been demonstrated that the level of programmed death-ligand 1 (PD-L1) is associated with rheumatoid arthritis (RA). This study examined the therapeutic role of PD-L1 by intra-articular delivery via adeno-associated virus (AAV) vectors in the mouse collagen-induced arthritis (CIA) model. Mice were intra-articularly injected with AAV5 vectors encoding human PD-L1 on day 0 and immunized with bovine type II collagen to induce CIA simultaneously. On day 49 post AAV administration, joints were collected for histo-pathological and cytokine analysis. Additionally, the systemic impacts of intra-articular injection of AAV5/PD-L1 vectors were also studied. To study the therapeutic effect of PD-L1, AAV5/PD-L1 vectors were administered into the joints of RA mice on day 21. After administration of AAV5/PD-L1 vectors, strong PD-L1 expression was detected in AAV transduced joints. Joints treated with PD-L1 at the time of arthritis induction exhibited significantly less swelling and improved histopathological scores when compared to untreated joints. Additionally, the infiltration of T cells and macrophages was decreased in joints of CIA mice that received AAV5/PD-L1 vectors (P<0.05). The levels of pro-inflammatory cytokines, including IL-1, IL-6, IL-17 and TNFα, were lower in AAV5/PD-L1 treated than untreated joints (P<0.05). Furthermore, the administration of AAV5/PD-L1 vectors into the joints of CIA mice did not impact serum cytokine levels and the antibody titers to type II collagen. Biodistribution of AAV vectors after intra-articular injection showed undetectable AAV genomes in other tissues except for a low level in the liver. Similar to the results of AAV5/PD-L1 vector administration on day 0, decreased joint swelling and lower histopathological damage were observed in joints treated with AAV5/PD-L1 vectors on day 21. The results from this study demonstrate that local AAV mediated PD-L1 gene delivery into the joints is able to prevent the development and block the progression of arthritis in CIA mice without impacting systemic immune responses. This study provides a novel strategy to effectively treat inflammatory joint diseases using local AAV gene therapy by interference with immune checkpoint pathways.

A novel IRAK4/PIM1 inhibitor ameliorates rheumatoid arthritis and lymphoid malignancy by blocking the TLR/MYD88-mediated NF-κB pathway.

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a pivotal enzyme in the Toll-like receptor (TLR)/MYD88 dependent signaling pathway, which is highly activated in rheumatoid arthritis tissues and activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL). Inflammatory responses followed by IRAK4 activation promote B-cell proliferation and aggressiveness of lymphoma. Moreover, proviral integration site for Moloney murine leukemia virus 1 (PIM1) functions as an anti-apoptotic kinase in propagation of ABC-DLBCL with ibrutinib resistance. We developed a dual IRAK4/PIM1 inhibitor KIC-0101 that potently suppresses the NF-κB pathway and proinflammatory cytokine induction invitro and invivo. In rheumatoid arthritis mouse models, treatment with KIC-0101 significantly ameliorated cartilage damage and inflammation. KIC-0101 inhibited the nuclear translocation of NF-κB and activation of JAK/STAT pathway in ABC-DLBCLs. In addition, KIC-0101 exhibited an anti-tumor effect on ibrutinib-resistant cells by synergistic dual suppression of TLR/MYD88-mediated NF-κB pathway and PIM1 kinase. Our results suggest that KIC-0101 is a promising drug candidate for autoimmune diseases and ibrutinib-resistant B-cell lymphomas.

Telotristat Etiprate alleviates rheumatoid arthritis by targeting LGALS3 and affecting MAPK signaling.

Rheumatoid arthritis (RA) is one of the most widespread chronic immune-mediated inflammatory diseases characterized by continuous erosion of bone and cartilage by synovial hyperplasia. Telotristat Etiprate is an inhibitor of tryptophan hydroxylase, a rate-limiting enzyme in the biosynthesis of serotonin. Telotristat Etiprate can be used in the treatment of carcinoid syndrome. The purpose of this study was to explore the effect of Telotristat Etiprate on RA and its mechanism. We investigated Telotristat Etiprate in collagen-induced arthritis (CIA) model mice and in rheumatoid arthritis synovial fibroblasts (RASFs). Results showed that Telotristat Etiprate had anti-inflammatory effects both in vitro and in vivo, can inhibit the invasion and migration of cells, inhibit the formation of pannus, and induce cell apoptosis. Transcriptome sequencing (RNA-seq) and mass spectrometry analysis showed that Galectins-3 (LGALS3) could be a newly identified target of Telotristat Etiprate, affecting the phosphorylation of the MAPK signaling pathway through UBE2L6, thereby improving RA.

SMSCs-derived sEV overexpressing miR-433-3p inhibits angiogenesis induced by sEV released from synoviocytes under triggering of ferroptosis

BackgroundFerroptosis is characterized by accumulation of lipid peroxides that leads to oxidative stress. In progressive rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) suffered from oxidative stress induced by generation of excess reactive oxygen species (ROS) and survived from elevated lipid oxidation. However the phenomenon of abnormal synovial fibroblasts proliferation under ferroptotic stress remain to be explained and the effects of this event on disease progression of RA need to be investigated. MethodsFLS from RA patients (RA-FLS) were stimulated with LPS as an inflammatory model in vitro, and simultaneously treated with ferroptosis inducer Erastin/RSL3 or inhibitor ferrostatin-1. Besides, small extracellular vesicles (sEV) from the supernatant of RA-FLS culture under Erastin/RSL3 management were isolated. The degree of ferroptosis in cells were evaluated by Lipid-ROS detection via flowcytometry and ferroptosis marker protein expression determined by western bloting. The expression of core component of ESCRT-III CHMP4A and CHMP5 was determined by western bloting, and knockdown of CHMP4A was further performed to detect the influence of ESCRT-III complex on ferroptosis as well as LPS/Erastin induced sEV (LPS/Erastin-sEV) releasing. Moreover, miR-433-3p level in the isolated sEV was evaluated by RT-qPCR and interaction of miR-433-3p with FOXO1/VEGF axis were evaluated. MiR-433-3p was overexpressed in synovial mesenchymal stem cells (SMSCs) via miR-433-3p mimics transfection. RA-FLS was co-cultured with human dermal microvascular endothelial cells (HDMECs). LPS/Erastin-sEV or sEV derived from miR-433-3p-overexpressing SMSCs (miR-433-3p-SMSCs-sEV) were added to the co-culture system, and supernatants from co-culture without sEV were given to HDMECs. Angiogenic activity of HDMECs were identified by transwell test and endothelial tube formation analysis. Erastin-sEV and miR-433-3p-SMSCs-sEV were also administrated in collagen-induced arthritis (CIA) mouse model respectively, and progression of arthritis were evaluated. ResultsFerroptosis of RA-FLS was triggered by LPS/Erastin and accompanied with increased expression of ESCRT-III core components as well as elevated release of sEV from RA-FLS. HDMECs’ migration and tube formation in vitro was significantly induced/suppressed by supernatants from co-culture under management of Erastin-sEV/miR-433-3p-SMSCs-sEV due to varied VEGF expression regulated by miR-433-3p targeting FOXO1. MiR-433-3p-SMSCs-sEV could inhibit the Erastin-sEV promoted VEGF expression and mitigated arthritis severity. ConclusionErastin-sEV could aggravate synovial angiogenesis and promote arthritis progression. Administration of miR-433-3p-SMSCs-sEV may be a potential novel therapeutic method as significant antagonism to Erastin-sEV for RA treatment.

Delivery of neutrophil membrane encapsulated non-steroidal anti-inflammatory drugs by degradable biopolymer microneedle patch for rheumatoid arthritis therapy

Rheumatoid arthritis (RA) is a common chronic autoimmune disease and eventually ends in severe disability and death. RA has no cure, but there are some treatments for decreasing the severity of various symptoms. For example, pain management of RA by non-steroidal anti-inflammatory drugs (NSAIDs), neutralization of the inflammatory cytokines (or block their functions) of RA by specific biologics. However, the clinical benefit is limited due to their low permeability and poor bioavailability. Meanwhile, the system toxicity for long-term use of NSAIDs is another focal issue. Herein, degradable biopolymer microneedle patch was designed and synthesized. In this dual drug delivery patch, NSAIDs were encapsulated by polymeric nanoparticles, which were coated by the neutrophil membrane, followed by loaded into the microneedle patch for local transdermal delivery. When applied to the murine models, the microneedle patch gradually dissolves and releases the contained nanoparticles. Coating of neutrophil membrane provides the nanosized NSAIDs with the capability of cytokine binding and inflammatory joint tropism. Based on inflammatory microenvironment regulation by cytokine absorption and cyclooxygenase-2 inhibition, the microneedle shows a significant local anti-inflammatory therapeutic effect on mouse model of collagen-induced arthritis and rat model of adjuvant-induced arthritis. Further development based on this advanced strategy may provide additional therapeutic options for RA treatment.

Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue.

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central roles in the pathogenesis of rheumatoid arthritis. It is important to understand the functions of both cell populations to reveal the mechanisms underlying pathological progression and remission in inflammatory arthritis. In general, in vitro experimental conditions should mimic the in vivo environment as much as possible. Primary tissue-derived cells have been used in experiments characterizing synovial fibroblasts in arthritis. In contrast, in experiments investigating the biological functions of macrophages in inflammatory arthritis, cell lines, bone marrow-derived macrophages, and blood monocyte-derived macrophages have been used. However, it is unclear whether such macrophages actually reflect the functions of tissue-resident macrophages. To obtain resident macrophages, previous protocols were modified to isolate and expand both primary macrophages and fibroblasts from synovial tissue in an inflammatory arthritis mouse model. These primary synovial cells may be useful for in vitro analysis of inflammatory arthritis.

Alleviation of arthritis through prevention of neutrophil extracellular traps by an orally available inhibitor of protein arginine deiminase 4

Protein arginine deiminases (PAD) 4 is an enzyme that catalyzes citrullination of protein and its role in autoimmune diseases has been established through clinical genetics and gene knock out studies in mice. Further, studies with PAD4 – deficient mice have shown that PAD4 deficiency does not lead to increased infection or immune suppression, which makes PAD4 an attractive therapeutic target for auto-immune and inflammatory diseases. PAD4 has critical enzymatic role of promoting chromatin decondensation and neutrophil extracellular traps (NETs) formation that is associated with a number of immune-mediated pathological conditions. Here, we present a non-covalent PAD4 inhibitor JBI-589 with high PAD4 isoform selectivity and delineated its binding mode at 2.88 Å resolution by X-ray crystallography. We confirmed its effectiveness in inhibiting NET formation in vitro. Additionally, by using two mouse arthritis models for human rheumatoid arthritis (RA), the well-known disease associated with PAD4 clinically, we established its efficacy in vivo. These results suggest that JBI-589 would be beneficial for both PAD4 and NET-associated pathological conditions.

Identification of anti-citrullinated osteopontin antibodies and increased inflammatory response by enhancement of osteopontin binding to fibroblast-like synoviocytes in rheumatoid arthritis

BackgroundAnti-citrullinated protein/peptide antibodies (ACPAs) are present in patients at onset and have important pathogenic roles during the course of rheumatoid arthritis (RA). The characteristics of several molecules recognized by ACPA have been studied in RA, but the positivity rate of autoantibodies against each antigen is not high, and the pathogenic mechanism of each antibody is not fully understood. We investigated the role of anti-citrullinated osteopontin (anti-cit-OPN) antibodies in RA pathogenesis.MethodsEnzyme-linked immunosorbent assays on RA patients’ sera were used to detect autoantibodies against OPN. Fibroblast-like synoviocytes (FLS) isolated from RA patients were used to test the binding activity and inflammatory response of OPN mediated by anti-cit-OPN antibodies, and their effect was tested using an inflammatory arthritis mouse model immunized with cit-OPN. Anti-cit-OPN antibody positivity and clinical characteristics were investigated in the patients as well.ResultsUsing sera from 224 RA patients, anti-cit-OPN antibodies were positive in approximately 44% of RA patients, while approximately 78% of patients were positive for the cyclic citrullinated peptide (CCP2) assay. IgG from patients with anti-cit-OPN antibody increased the binding activity of OPN to FLSs, which further increased matrix metalloproteinase and interleukin-6 production in TNF-stimulated FLSs. Mice immunized with cit-OPN antibodies experienced severe arthritis. Anti-cit-OPN antibodies in RA patients decreased the drug survival rate of tumor necrosis factor (TNF) inhibitors, while it did not decrease that of CTLA4-Ig.ConclusionsAnti-cit-OPN antibodies were detected in patients with RA. IgG from patients with anti-cit-OPN antibodies aggravated RA, and anti-cit-OPN antibody was a marker of reduced the survival rate of TNF inhibitors in RA patients.

Hypermethylation of Smad7 in CD4+ T cells is associated with the disease activity of rheumatoid arthritis.

Smad7 is protective in a mouse model of rheumatoid arthritis. Here we investigated whether Smad7-expressing CD4+ T cells and the methylation of Smad7 gene in CD4+ T cells contribute to the disease activity of RA in patients. Peripheral CD4+ T cells were collected from 35 healthy controls and 57 RA patients. Smad7 expression by CD4+ T cells were determined and correlated with the clinical parameters of RA including RA score and serum levels of IL-6, CRP, ESR, DAS28-CRP, DAS28-ESR, Swollen joints and Tender joints. Bisulfite sequencing (BSP-seq) was used to determine the DNA methylation in Smad7 promoter (-1000 to +2000) region in CD4+ T cells. In addition, a DNA methylation inhibitor, 5-Azacytidine (5-AzaC), was added to CD4+ T cells to examine the possible role of Smad7 methylation in CD4+ T cell differentiation and functional activity. Compared to the heath controls, Smad7 expression was significantly decreased in CD4+ T cells from RA patients and inversely correlated with the RA activity score and serum levels of IL-6 and CRP. Importantly, loss of Smad7 in CD4+ T cell was associated with the alteration of Th17/Treg balance by increasing Th17 over the Treg population. BSP-seq detected that DNA hypermethylation occurred in the Smad7 promoter region of CD4+ T cells obtained from RA patients. Mechanistically, we found that the DNA hypermethylation in the Smad7 promoter of CD4+ T cells was associated with decreased Smad7 expression in RA patients. This was associated with overreactive DNA methyltransferase (DMNT1) and downregulation of the methyl-CpG binding domain proteins (MBD4). Inhibition of DNA methylation by treating CD4+ T cells from RA patients with 5-AzaC significantly increased Smad7 mRNA expression along with the increased MBD4 but reduced DNMT1 expression, which was associated with the rebalance in the Th17/Treg response. DNA hypermethylation at the Smad7 promoter regions may cause a loss of Smad7 in CD4+ T cells of RA patients, which may contribute to the RA activity by disrupting the Th17/Treg balance.

Gallic acid regulates immune response in a mouse model of rheumatoid arthritis.

Previous studies revealed that gallic acid (GA) exerts anti-inflammation and immuno-regulatory properties. This study aims to explore the pharmacological activities of GA in collagen-induced arthritis (CIA) mouse model. Male DBA/1J mice were used to construct the CIA model. The mice were administrated with GA for 3 weeks. Clinical arthritis scores and hind paw volume were evaluated over the experimental period. qPCR and Western blot analysis were used to determine the levels of matrix metallopeptidases (MMPs) and cytokines. In addition, flow cytometry was used to measure the populations of Th17 and Treg cells. ELISAs were used to determine the cytokines in the serum and ankle joint tissues. Treatment of GA (40 and 80 mg/kg/d) reduced clinical arthritis scores and hind paw volume in the CIA mouse model. Besides, treatment of GA reduced the overexpression of MMPs and modulated the dysregulation of inflammation-related cytokines. Flow cytometry showed that treatment of GA decreased the population of Th17 cells, and increased the population of Treg cells, as supported by treatment of GA regulated the Th17/Treg-related cytokines. GA attenuates symptoms in the CIA mouse model by anti-inflammation and regulating Th17/Treg cell imbalance.

Additive efficacy of a bispecific anti-TNF/IL-6 nanobody compound in translational models of rheumatoid arthritis.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases affecting primarily the joints. Despite successful therapies including antibodies against tumor necrosis factor (TNF) and interleukin-6 (IL-6) receptor, only 20 to 30% of patients experience remission. We studied whether inhibiting both TNF and IL-6 would result in improved efficacy. Using backtranslation from single-cell RNA sequencing (scRNA-seq) data from individuals with RA, we hypothesized that TNF and IL-6 act synergistically on fibroblast-like synoviocytes (FLS) and T cells. Coculture of FLS from individuals with RA and T cells supported this hypothesis, revealing effects on both disease-driving pathways and biomarkers. Combining anti-TNF and anti-IL-6 antibodies in collagen-induced arthritis (CIA) mouse models resulted in sustained long-term remission, improved histology, and effects on bone remodeling pathways. These promising data initiated the development of an anti-TNF/IL-6 bispecific nanobody compound 1, with similar potencies against TNF and IL-6. We observed additive efficacy of compound 1 in a FLS/T cell coculture affecting arthritis and T helper 17 (TH17) pathways. This nanobody compound transcript signature inversely overlapped with described RA endotypes, indicating a potential efficacy in a broader patient population. In summary, we showed superiority of a bispecific anti-TNF/IL-6 nanobody compound or combination treatment over monospecific treatments in both in vitro and in vivo models. We anticipate improved efficacy in upcoming clinical studies.

Sesquiterpene lactones-enriched fractions from Xanthium mongolicum Kitag alleviate RA by regulating M1 macrophage polarization via NF-κB and MAPK signaling pathway.

Introduction: Rheumatoid arthritis (RA) is a chronic autoimmune disease, characterized by activated M1-like macrophage in the joint. Xanthium mongolicum Kitag (X. mongolicum) is a traditional medicinal plant that has long been used to treat RA and other immune diseases in China. Methods: Fractions of X. mongolicum were separated based on polarity. Anti-RA activity of the fractions were screened by LPS-stimulated RAW264.7 macrophage in vitro. The major active compounds were identified by UPLC-MS and quantified by HPLC. The anti-RA effects of the active fraction was evaluated in complete freund's adjuvant (CFA)-induced arthritis and collagen-induced arthritis (CIA) mouse models in vivo and LPS-stimulated macrophage in vitro. Results: Sesquiterpene lactones-enriched fraction from X. mongolicum (SL-XM) exhibited the strongest anti-RA activity among all components in vitro. Five major constituents i.e., Xanthinosin (1), Xanthatin (2), Mogolide D (3), Mogolide E (4), and Mogolide A (5) were identified as major compounds of SL-XM. SL-XM ameliorated symptoms of CFA and CIA induced arthritis mice model. Furthermore, SL-XM treatment inhibited LPS-induced M1 macrophages polarization. In addition, SL-XM inhibited the phosphorylation of NF-κB and MAPK signaling pathways in LPS-induced macrophage and CIA-challenged mice. Discussion: The main anti-RA active fraction of X. mongolicum may be the Sesquiterpene lactones, which includes five key compounds. SL-XM may exert its anti-RA effect by suppressing M1 macrophage polarization via the NF-κB and MAPK signaling pathway.

Modulation of Neutrophil Function by Recombinant Human IgG1 Fc Hexamer in the Endogenous K/BxN Mouse Model of Rheumatoid Arthritis

Introduction: Neutrophils are a pivotal cell type in the K/BxN mouse model of rheumatoid arthritis and play an essential role in the progression of the arthritis. They are readily activated by immune complexes (ICs) via their FcγRs to release IL-1β in addition to other cytokines, which are inducing cartilage destruction. Neutrophils also release neutrophil-active chemokines to recruit themselves in an autocrine manner to perpetuate tissue destruction. FcγR-expression on neutrophils is of crucial importance for the recognition of ICs. Methods: In this study, due to its high avidity for binding to FcγRs, we investigated the potential anti-inflammatory effect of a recombinant IgG1 Fc hexamer (rFc-µTP-L309C) on neutrophils in the K/BxN mouse model of endogenously generated chronic arthritis. 200 mg/kg rFc-µTP-L309C and human serum albumin (HSA), used as controls, were administered subcutaneously every other day. Mouse ankle joints were monitored daily to generate a clinical score. Immunohistology was used to evaluate neutrophil infiltration and TUNEL to assess apoptosis. ELISA was used to measure IL-1β. Results: Treatment with rFc-µTP-L309C, but not HSA, was able to significantly ameliorate the arthritis in the K/BxN mice. Significant neutrophil infiltration into the ankle joint was found, but treatment with rFc-µTP-L309C resulted in significantly less neutrophil infiltration. There was no significant influence of rFc-µTP-L309C on neutrophil death or apoptosis. Less neutrophil infiltration could not be correlated to chemokine-mediated migration. Significantly less IL-1β was measured in mice treated with rFc-µTP-L309C. Conclusion: In the endogenous K/BxN mouse model of rheumatoid arthritis, amelioration can be explained in part by inhibition of neutrophil infiltration into the joints as well as inhibition of IL-1β production. Given the observed inhibitory properties on neutrophils, rFc-µTP-L309C may be a potential therapeutic candidate to treat autoimmune and inflammatory conditions in which neutrophils are the predominant cell type involved in pathogenesis.

Inhibitory effect of baricitinib on microglia and STAT3 in a region with a weak blood-brain barrier in a mouse model of rheumatoid arthritis.

In patients with RA, baricitinib not only improves arthritis symptom severity, but also patients' neuropsychological symptoms, such as depression and fatigue. However, the cellular mechanisms through which baricitinib can affect neural activity is unexplored. While the blood-brain barrier (BBB) permeability of this drug remains unclear, Janus kinase inhibitors (JAKi) might reach the area postrema, which is a unique brain region with a weak BBB function. Our recent study demonstrated microglial activation during experimental arthritis in the area postrema. Therefore, we sought to assess the effect of baricitinib on microglia in the area postrema using the CIA mouse model. Microglia number and morphology in the area postrema were assessed by immunostaining for ionized calcium-binding adaptor molecule-1 (Iba-1). Data were collected on post-immunization day 35 (early phase) and 84 (late phase), and compared between baricitinib- and vehicle-treated mice. The effect on signal transducers and activators of transcription (STAT3) in the area postrema was also immunohistochemically examined. Behavioural outcomes were assessed by examining feeding behaviours and sucrose preference tests. In the early phase, activated microglial levels in the area postrema were decreased by baricitinib, accompanied by the inhibition of phosphorylated-STAT3 and recovery of food intake and sucrose preference. On the other hand, baricitinib did not affect microglial morphology in the late phase. Our results demonstrate that baricitinib can affect brain cells, specifically microglia, in the brain region with a weak BBB and mitigate aberrant behaviours during autoimmune arthritis, pointing to the potential therapeutic effect of JAKi on brain pathologies underpinning RA.

Biomineralized Nanoscavenger Abrogates Proinflammatory Macrophage Polarization and Induces Neutrophil Clearance through Reverse Migration during Gouty Arthritis.

The deposition of monosodium urate (MSU) crystals induces the overexpression of reactive oxygen species (ROS) and proinflammatory cytokines in residential macrophages, further promoting the infiltration of inflammatory leukocytes in the joints of gouty arthritis. Herein, a peroxidase-mimicking nanoscavenger was developed by forming manganese dioxide over albumin nanoparticles loaded with an anti-inflammatory drug, indomethacin (BIM), to block the secretion of ROS and COX2-induced proinflammatory cytokines in the MSU-induced gouty arthritis model. In the MSU-induced arthritis mouse model, the BIM nanoparticles alleviated joint swelling, which is attributed to the abrogation of ROS and inflammatory cytokine secretions from proinflammatory macrophages that induces neutrophil infiltration and fluid building up in the inflammation site. Further, the BIM nanoparticle treatment reduced the influx of macrophages and neutrophils in the injured region by blocking migration and inducing reverse migration in the zebrafish larva tail amputation model as well as in MSU-induced peritonitis and air pouch mouse models. Overall, the current strategy of employing biomineralized nanoscavengers for arthritis demonstrates clinical significance in dual blocking of peroxides and COX2 to prevent influx of inflammatory cells into the sites of inflammation.

Genomic repertoires linked with pathogenic potency of arthritogenic Prevotella copri isolated from the gut of patients with rheumatoid arthritis

ObjectivesPrevotella copri is considered to be a contributing factor in rheumatoid arthritis (RA). However, in some non-Westernised countries, healthy individuals also harbour an abundance of P. copri in the intestine....

RNA sequencing analysis reveals the potential therapeutic mechanisms of Huzhang Tongfeng granule in MSU-induced acute gouty arthritis mouse model

BackgroundGout is one of the most prevailing inflammatory arthritis, in which joint pain and dysfunction may drastically reduce patients' quality of life, work efficiency, and increase the burden of medical treatment. The Huzhang Tongfeng granule (HZTFG) has been utilized clinically for more than 30 years with satisfactory effects and no significant adverse reactions. However, the mechanisms that underlie HZTFG's effect remain indeterminate. MethodsFirstly, the Liquid Chromatography-Mass Spectrometry (LC-MS) analyses were applied to clarify the multi-components of HZTFG to establish the quality control parameters. After the monosodium urate (MSU) crystals induced Acute gouty arthritis (AGA) model was established, behavioral testing, joint swelling, hematoxylin, and eosin (H&E) staining were applied to assess the effect of HZTFG on improving joint pain, swelling, synovial inflammation of the MSU crystals-injected mice. Moreover, to further understand the underlying mechanism of the action of HZTFG on AGA, RNA Sequencing (RNA-seq) analysis was performed to detect differentially expressed genes (DEGs) after HZTFG treatment. Thereafter, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were enriched via gene set enrichment analysis (GSEA) to assess the functions and pathways of DEGs. Furthermore, Reverse Transcription Polymerase Chain Reaction (RT-qPCR) analysis and enzyme-linked immunosorbent assay (ELISA) were applied for validation in vivo. ResultsAfter treatment with HZTFG, RNA-seq results revealed 44 DEGs, of which 20 genes were remarkably down-regulated and 24 genes were up-regulated. Besides, the expression of these assayed genes were noticeably decreased, including Cxcl1, Cxcl2, Cebpb, Lcn2, Ptgs2, Mmp13, Fos, Socs3, Sele, and Junb. Moreover, animal experiments also demonstrated that TNF signaling pathway and IL-17 signaling pathway are key pathways in HZTFG treated MSU-induced AGA mouse model. ConclusionHZTFG attenuated MSU crystals-induced inflammatory response through TNF and IL-17 signaling pathways by inhibiting the activation of related chemokines, inflammatory proteins, transcription factors, suppressor of cytokine signaling, and adhesion molecule in AGA. This study further provides the potential therapeutic targets for AGA treatment.