KIF1C and new Huntingtin-interacting protein 1 binding proteins regulate rheumatoid arthritis fibroblast-like synoviocytes' phenotypes.

Background: Huntingtin-interacting protein-1 (HIP1) is a new arthritis severity gene implicated in the regulation of the invasive properties of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). These invasive properties of FLS strongly correlate with radiographic and histology damage in patients with RA and rodent models of arthritis. While HIP1 has several intracellular functions, little is known about its binding proteins, and identifying them has the potential to expand our understanding of its role in cell invasion and other disease-contributing phenotypes, and potentially identify new targets for therapy. Methods: FLS cell lines from arthritic DA (highly invasive) and from arthritis-protected congenic rats R6 (minimally invasive), which differ in an amino-acid changing HIP1 SNP, were cultured and lysed, and proteins were immunoprecipitated with an anti-HIP1 antibody. Immunoprecipitates were analyzed by mass spectrometry. Differentially detected (bound) proteins were selected for functional experiments using siRNA knockdown in human RA FLS to examine their effect in cell invasiveness, adhesion, cell migration and proliferation, and immunofluorescence microscopy. Results: Proteins detected included a few known HIP1-binding proteins and several new ones. Forty-five proteins differed in levels detected in the DA versus R6 congenic mass spectrometry analyses. Thirty-two of these proteins were knocked down and studied in vitro, with 10 inducing significant changes in RA FLS phenotypes. Specifically, knockdown of five HIP1-binding protein genes (CHMP4BL1, COPE, KIF1C, YWHAG, and YWHAH) significantly decreased FLS invasiveness. Knockdown of KIF1C also reduced RA FLS migration. The binding of four selected proteins to human HIP1 was confirmed. KIF1C colocalized with lamellipodia, and its knockdown prevented RA FLS from developing an elongated morphology with thick linearized actin fibers or forming polarized lamellipodia, all required for cell mobility and invasion. Unlike HIP1, KIF1C knockdown did not affect Rac1 signaling. Conclusion: We have identified new HIP1-binding proteins and demonstrate that 10 of them regulate key FLS phenotypes. These HIP1-binding proteins have the potential to become new therapeutic targets and help better understand the RA FLS pathogenic behavior. KIF1C knockdown recapitulated the morphologic changes previously seen in the absence of HIP1, but did not affect the same cell signaling pathway, suggesting involvement in the regulation of different processes.

Regulation of cytokine and chemokine expression by histone lysine methyltransferase MLL1 in rheumatoid arthritis synovial fibroblasts.

Histone lysine methylation is thought to play a role in the pathogenesis of rheumatoid arthritis (RA). We previously reported aberrant expression of the gene encoding mixed-lineage leukemia 1 (MLL1), which catalyzes methylation of histone H3 lysine 4 (H3K4), in RA synovial fibroblasts (SFs). The aim of this study was to elucidate the involvement of MLL1 in the activated phenotype of RASFs. SFs were isolated from synovial tissues obtained from patients with RA or osteoarthritis (OA) during total knee joint replacement. MLL1 mRNA and protein levels were determined after stimulation with tumor necrosis factor α (TNFα). We also examined changes in trimethylation of H3K4 (H3K4me3) levels in the promoters of RA-associated genes (matrix-degrading enzymes, cytokines, and chemokines) and the mRNA levels upon small interfering RNA-mediated depletion of MLL1 in RASFs. We then determined the levels of H3K4me3 and mRNAs following treatment with the WD repeat domain 5 (WDR5)/MLL1 inhibitor MM-102. H3K4me3 levels in the gene promoters were also compared between RASFs and OASFs. After TNFα stimulation, MLL1 mRNA and protein levels were higher in RASFs than OASFs. Silencing of MLL1 significantly reduced H3K4me3 levels in the promoters of several cytokine (interleukin-6 [IL-6], IL-15) and chemokine (C-C motif chemokine ligand 2 [CCL2], CCL5, C-X-C motif chemokine ligand 9 [CXCL9], CXCL10, CXCL11, and C-X3-C motif chemokine ligand 1 [CX3CL1]) genes in RASFs. Correspondingly, the mRNA levels of these genes were significantly decreased. MM-102 significantly reduced the promoter H3K4me3 and mRNA levels of the CCL5, CXCL9, CXCL10, and CXCL11 genes in RASFs. In addition, H3K4me3 levels in the promoters of the IL-6, IL-15, CCL2, CCL5, CXCL9, CXCL10, CXCL11, and CX3CL1 genes were significantly higher in RASFs than OASFs. Our findings suggest that MLL1 regulates the expression of particular cytokines and chemokines in RASFs and is associated with the pathogenesis of RA. These results could lead to new therapies for RA.

Analysis of common differential gene expression in synovial cells of osteoarthritis and rheumatoid arthritis.

OBJECTIVE: To elucidate potential molecular mechanisms differentiating osteoarthritis (OA) and rheumatoid arthritis (RA) through a bioinformatics analysis of differentially expressed genes (DEGs) in patient synovial cells, aiming to provide new insights for clinical treatment strategies. MATERIALS AND METHODS: Gene expression datasets GSE1919, GSE82107, and GSE77298 were downloaded from the Gene Expression Omnibus (GEO) database to serve as the training groups, with GSE55235 being used as the validation dataset. The OA and RA data from the GSE1919 dataset were merged with the standardized data from GSE82107 and GSE77298, followed by batch effect removal to obtain the merged datasets of differential expressed genes (DEGs) for OA and RA. Intersection analysis was conducted on the DEGs between the two conditions to identify commonly upregulated and downregulated DEGs. Enrichment analysis was then performed on these common co-expressed DEGs, and a protein-protein interaction (PPI) network was constructed to identify hub genes. These hub genes were further analyzed using the GENEMANIA online platform and subjected to enrichment analysis. Subsequent validation analysis was conducted using the GSE55235 dataset. RESULTS: The analysis of differentially expressed genes in the synovial cells from patients with Osteoarthritis (OA) and Rheumatoid Arthritis (RA), compared to a control group (individuals without OA or RA), revealed significant changes in gene expression patterns. Specifically, the genes APOD, FASN, and SCD were observed to have lower expression levels in the synovial cells of both OA and RA patients, indicating downregulation within the pathological context of these diseases. In contrast, the SDC1 gene was found to be upregulated, displaying higher expression levels in the synovial cells of OA and RA patients compared to normal controls.Additionally, a noteworthy observation was the downregulation of the transcription factor PPARG in the synovial cells of patients with OA and RA. The decrease in expression levels of PPARG further validates the alteration in lipid metabolism and inflammatory processes associated with the pathogenesis of OA and RA. These findings underscore the significance of these genes and the transcription factor not only as biomarkers for differential diagnosis between OA and RA but also as potential targets for therapeutic interventions aimed at modulating their expression to counteract disease progression. CONCLUSION: The outcomes of this investigation reveal the existence of potentially shared molecular mechanisms within Osteoarthritis (OA) and Rheumatoid Arthritis (RA). The identification of APOD, FASN, SDC1, TNFSF11 as key target genes, along with their downstream transcription factor PPARG, highlights common potential factors implicated in both diseases. A deeper examination and exploration of these findings could pave the way for new candidate targets and directions in therapeutic research aimed at treating both OA and RA. This study underscores the significance of leveraging bioinformatics approaches to unravel complex disease mechanisms, offering a promising avenue for the development of more effective and targeted treatments.

Gut microbial metabolite targets HDAC3-FOXK1-interferon axis in fibroblast-like synoviocytes to ameliorate rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease. Early studies hold an opinion that gut microbiota is environmentally acquired and associated with RA susceptibility. However, accumulating evidence demonstrates that genetics also shape the gut microbiota. It is known that some strains of inbred laboratory mice are highly susceptible to collagen-induced arthritis (CIA), while the others are resistant to CIA. Here, we show that transplantation of fecal microbiota of CIA-resistant C57BL/6J mice to CIA-susceptible DBA/1J mice confer CIA resistance in DBA/1J mice. C57BL/6J mice and healthy human individuals have enriched B. fragilis than DBA/1J mice and RA patients. Transplantation of B. fragilis prevents CIA in DBA/1J mice. We identify that B. fragilis mainly produces propionate and C57BL/6J mice and healthy human individuals have higher level of propionate. Fibroblast-like synoviocytes (FLSs) in RA are activated to undergo tumor-like transformation. Propionate disrupts HDAC3-FOXK1 interaction to increase acetylation of FOXK1, resulting in reduced FOXK1 stability, blocked interferon signaling and deactivation of RA-FLSs. We treat CIA mice with propionate and show that propionate attenuates CIA. Moreover, a combination of propionate with anti-TNF etanercept synergistically relieves CIA. These results suggest that B. fragilis or propionate could be an alternative or complementary approach to the current therapies.

The fibroid phenotype of biological naïve patients with rheumatoid arthritis are less likely to respond to anti-IL-6R treatment.

Type III collagen gene expression is upregulated in the synovium of patients with rheumatoid arthritis (RA) presenting the fibroid phenotype. The soluble type III collagen formation biomarker, PRO-C3, is known to measure fibrogenesis in fibrotic diseases. In this exploratory study, we aimed to investigate the association between fibrogenesis (PRO-C3) and the disease- and treatment response in patients with RA. We measured PRO-C3 in subsets of two clinical trials assessing the effect of the anti-interleukin-6 (IL-6) receptor treatment tocilizumab (TCZ) as monotherapy or polytherapy with methotrexate. PRO-C3 levels had weak or very weak correlations with the clinical parameters (Spearman's). However, when the patients were divided into Disease Activity Score-28 groups characterized by the erythrocyte sedimentation rate (DAS28-ESR), there was a statistical difference between the PRO-C3 levels of the different groups (p < 0.05). To determine the response in relation to PRO-C3, a cut-off based on PRO-C3 levels and patients in remission (DAS28-ESR ≤ 2.6) was identified. This showed that a reduction in PRO-C3 after treatment initiation was associated with decreased DAS28-ESR and a higher response rate in patients with low PRO-C3 levels than in those with high PRO-C3 levels. This indicates that a fibrotic component affects the responsiveness of patients.

Epstein-Barr Virus DNA Exacerbates Arthritis in a Mouse Model via Toll-like Receptor 9.

Epstein-Barr virus (EBV) DNA is known to be shed upon reactivation of latent EBV. Based on our previous findings linking Toll-like receptor-9 (TLR9) to an EBV DNA-driven surge in IL-17A production, we aimed to examine the therapeutic potential of TLR9 inhibition in EBV DNA-exacerbated arthritis in a collagen-induced arthritis (CIA) mouse model. C57BL/6J mice were administered either collagen, EBV DNA + collagen, EBV DNA + collagen + TLR9 inhibitor, or only the TLR9 inhibitor. After 70 days, paw thicknesses, clinical scores, and gripping strength were recorded. Moreover, affected joints, footpads, and colons were histologically scored. Furthermore, the number of cells co-expressing IL-17A, IFN-γ, and FOXP3 in joint sections was determined by immunofluorescence assays. Significantly decreased paw thicknesses, clinical scores, and histological scores with a significantly increased gripping strength were observed in the group receiving EBV DNA + collagen + TLR9 inhibitor, compared to those receiving EBV DNA + collagen. Similarly, this group showed decreased IL-17A+ IFN-γ+, IL-17A+ FOXP3+, and IL-17A+ IFN-γ+ FOXP3+ foci counts in joints. We show that inhibiting TLR9 limits the exacerbation of arthritis induced by EBV DNA in a CIA mouse model, suggesting that TLR9 could be a potential therapeutic target for rheumatoid arthritis management in EBV-infected individuals.

Gene Expression Profiling to Unfolded Proteins Response as a Risk Modulator of Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease. Despite new methods of diagnostics and treatment as well as extensive biological and immunosuppressive treatment, the etiology of RA is not fully understood. Moreover, the problem of diagnosis and treatment of RA patients is still current and affects a large group of patients. It is suggested that endoplasmic reticulum (ER)-related features may impair adaptation to chronic stress, inferring the risk of rheumatoid arthritis. The main goal in this study was evaluation of changes in mRNA translation to determine chronic ER stress conditions in rheumatoid arthritis patients. The study group consist of 86 individuals including a total of 56 rheumatoid arthritis patients and 30 healthy controls. The expression level of mRNA form blood samples of RA patients as well as controls of the unfolded protein response (UPR)-associated genes (p-eIF2, BCL-2, PERK, ATF4, and BAX) were investigated using real-time qPCR. GAPDH expression was used as a standard control. Considering the median, the expression levels of PERK, BCL-2, p-eIF2, ATF4, and BAX were found to be significantly increased in the blood of RA patients compared with the control group. The p-value for the PERK gene was 0.0000000036, the p-value for the BCL-2 gene was 0.000000014, the p-value for the p-eIF2 gene was 0.006948, the p-value for the ATF4 gene was 0.0000056, and the p-value for the BAX gene was 0.00019, respectively. Thus, it can be concluded that the targeting of the components of the PERK-dependent UPR signaling pathway via small-molecule PERK inhibitors may contribute to the development of novel, innovative treatment strategies against rheumatoid arthritis.

Targeting pathogenic fibroblast-like synoviocyte subsets in rheumatoid arthritis.

Fibroblast-like synoviocytes (FLSs) play a central role in RA pathogenesis and are the main cellular component in the inflamed synovium of patients with rheumatoid arthritis (RA). FLSs are emerging as promising new therapeutic targets in RA. However, fibroblasts perform many essential functions that are required for sustaining tissue homeostasis. Direct targeting of general fibroblast markers on FLSs is challenging because fibroblasts in other tissues might be altered and side effects such as reduced wound healing or fibrosis can occur. To date, no FLS-specific targeted therapies have been applied in the clinical management of RA. With the help of high-throughput technologies such as scRNA-seq in recent years, several specific pathogenic FLS subsets in RA have been identified. Understanding the characteristics of these pathogenic FLS clusters and the mechanisms that drive their differentiation can provide new insights into the development of novel FLS-targeting strategies for RA. Here, we discuss the pathogenic FLS subsets in RA that have been elucidated in recent years and potential strategies for targeting pathogenic FLSs.

JAK2 inhibitors improve RA combined with pulmonary fibrosis in rats by downregulating SMAD3 phosphorylation.

BACKGROUND: JAK inhibitors are well known for the treatment of rheumatoid arthritis (RA), but whether they can be used to treat pulmonary fibrosis, a common extra-articular disease of RA, remains to be clarified. METHODS: A jak2 inhibitor, CEP33779 (CEP), was administered to a rat model of RA-associated interstitial lung disease to observe the degree of improvement in both joint swelling and pulmonary fibrosis. HFL1 cells were stimulated with TGF-ß1 to observe the expression of p-JAK2. Then, different concentrations of related gene inhibitors (JAK2, TGFß-R1/2, and p-STAT3) or silencers (STAT3, JAK2) were administered to HFL1 cells, and the expression levels of related proteins were detected to explore the underlying mechanisms of action. RESULTS: CEP not only reduced the degree of joint swelling and inflammation in rats but also improved lung function, inhibited the pro-inflammatory factors IL-1ß and IL-6, reduced lung inflammation and collagen deposition, and alleviated lung fibrosis. CEP decreased the expression levels of TGFß-R2, p-SMAD, p-STAT3, and ECM proteins in rat lung tissues. TGF-ß1 induced HFL1 cells to highly express p-JAK2, with the most pronounced expression at 48 h. The levels of p-STAT3, p-SMAD3, and ECM-related proteins were significantly reduced after inhibition of either JAK2 or STAT3. CONCLUSION: JAK2 inhibitors may be an important and novel immunotherapeutic drug that can improve RA symptoms while also delaying or blocking the development of associated pulmonary fibrotic disease. The mechanism may be related to the downregulation of p-STAT3 protein via inhibition of the JAK2/STAT signaling pathway, which affects the phosphorylation of SMAD3.

The role of YAP/TAZ on joint and arthritis.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are two common forms of arthritis with undefined etiology and pathogenesis. Yes-associated protein (YAP) and its homolog transcriptional coactivator with PDZ-binding motif (TAZ), which act as sensors for cellular mechanical and inflammatory cues, have been identified as crucial players in the regulation of joint homeostasis. Current studies also reveal a significant association between YAP/TAZ and the pathogenesis of OA and RA. The objective of this review is to elucidate the impact of YAP/TAZ on different joint tissues and to provide inspiration for further studying the potential therapeutic implications of YAP/TAZ on arthritis. Databases, such as PubMed, Cochran Library, and Embase, were searched for all available studies during the past two decades, with keywords "YAP," "TAZ," "OA," and "RA."

Discovery of novel diaryl substituted isoquinolin-1(2H)-one derivatives as hypoxia-inducible factor-1 signaling inhibitors for the treatment of rheumatoid arthritis.

Since synovial hypoxic microenvironment significantly promotes the pathological progress of rheumatoid arthritis (RA), hypoxia-inducible factor 1 (HIF-1) has been emerged as a promising target for the development of novel therapeutic agents for RA treatment. In this study, we designed and synthesized a series of diaryl substituted isoquinolin-1(2H)-one derivatives as HIF-1 signaling inhibitors using scaffold-hopping strategy. By modifying the substituents on N-atom and 6-position of isoquinolin-1-one, we discovered compound 17q with the most potent activities against HIF-1 (IC50 = 0.55 µM) in a hypoxia-reactive element (HRE) luciferase reporter assay. Further pharmacological studies revealed that 17q concentration-dependently blocked hypoxia-induced HIF-1α protein accumulation, reduced inflammation response, inhibited cellular invasiveness and promoted VHL-dependent HIF-1α degradation in human RA synovial cell line. Moreover, 17q improved the pathological injury of ankle joints, decreased angiogenesis and attenuated inflammation response in the adjuvant-induced arthritis (AIA) rat model, indicating the promising therapeutic potential of compound 17q as an effective HIF-1 inhibitor for RA therapy.

TNF-α signals through ITK-Akt-mTOR to drive CD4+ T cell metabolic reprogramming, which is dysregulated in rheumatoid arthritis.

Upon activation, T cells undergo metabolic reprogramming to meet the bioenergetic demands of clonal expansion and effector function. Because dysregulated T cell cytokine production and metabolic phenotypes coexist in chronic inflammatory disease, including rheumatoid arthritis (RA), we investigated whether inflammatory cytokines released by differentiating T cells amplified their metabolic changes. We found that tumor necrosis factor-α (TNF-α) released by human naïve CD4+ T cells upon activation stimulated the expression of a metabolic transcriptome and increased glycolysis, amino acid uptake, mitochondrial oxidation of glutamine, and mitochondrial biogenesis. The effects of TNF-α were mediated by activation of Akt-mTOR signaling by the kinase ITK and did not require the NF-κB pathway. TNF-α stimulated the differentiation of naïve cells into proinflammatory T helper 1 (TH1) and TH17 cells, but not that of regulatory T cells. CD4+ T cells from patients with RA showed increased TNF-α production and consequent Akt phosphorylation upon activation. These cells also exhibited increased mitochondrial mass, particularly within proinflammatory T cell subsets implicated in disease. Together, these findings suggest that T cell-derived TNF-α drives their metabolic reprogramming by promoting signaling through ITK, Akt, and mTOR, which is dysregulated in autoinflammatory disease.

Phlorizin Regulates Synovial Hyperplasia and Inflammation in Rats With Rheumatoid Arthritis by Regulating the mTOR Pathway.

BACKGROUND/AIM: Rheumatoid arthritis (RA) is an inflammatory autoimmune disease, and management of it is still a challenge. The present investigation assessed the potential preventive effect of phlorizin on rats with RA. MATERIALS AND METHODS: A total of 40 healthy Wistar rats were used for this study. Bovine type II collagen and Freund's incomplete adjuvant (1:1 and 1 mg/ml) were administered on days 1 and 8 of the protocol to induce RA in rats; treatment with phlorizin at 60 or 120 mg/kg was started after the 4th week of the protocol, and its effect on inflammation, level of inflammatory cytokines, and expression of proteins were estimated in RA rats. Moreover, an in vitro study was performed on fibroblast-like synoviocytes (FLSs), and the effects of phlorizin on proliferation, apoptosis, and expression of the mechanistic target of rapamycin kinase pathway protein after stimulating these cells with tumor necrosis factor α (TNF-α) were estimated. RESULTS: The data obtained from the study indicate that phlorizin has the potential to mitigate inflammation and enhance weight management in rats with RA induced by bovine type II collagen (CII). The level of inflammatory cytokines in the serum and the expression of protein kinase B (AKT), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), and mechanistic target of rapamycin kinase (mTOR) proteins in the joint tissue were reduced in phlorizin-treated rats with RA. In this investigation, phlorizin was shown to reverse the histological abnormalities in the joint tissue of rats with RA. The in-vitro study showed that phlorizin reduced proliferation and had no apoptotic effect on TNF-α-stimulated FLSs. Expression of AKT, PI3K, and mTOR proteins was also down-regulated in phlorizin-treated TNF-α-stimulated FLSs. CONCLUSION: Phlorizin protects against inflammation and reduces injury to synovial tissues in RA by modulating the AKT/PI3K/mTOR pathway.

PTEN: an emerging target in rheumatoid arthritis?

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a critical tumor suppressor protein that regulates various biological processes such as cell proliferation, apoptosis, and inflammatory responses by controlling the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PI3K/AKT) signaling pathway. PTEN plays a crucial role in the pathogenesis of rheumatoid arthritis (RA). Loss of PTEN may contribute to survival, proliferation, and pro-inflammatory cytokine release of fibroblast-like synoviocytes (FLS). Also, persistent PI3K signaling increases myeloid cells' osteoclastic potential, enhancing localized bone destruction. Recent studies have shown that the expression of PTEN protein in the synovial lining of RA patients with aggressive FLS is minimal. Experimental upregulation of PTEN protein expression could reduce the damage caused by RA. Nonetheless, a complete comprehension of aberrant PTEN drives RA progression and its interactions with other crucial molecules remains elusive. This review is dedicated to promoting a thorough understanding of the signaling mechanisms of aberrant PTEN in RA and aims to furnish pertinent theoretical support for forthcoming endeavors in both basic and clinical research within this domain.

Macrophage-derived mir-100-5p orchestrates synovial proliferation and inflammation in rheumatoid arthritis through mTOR signaling.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by synovial inflammation, causing substantial disability and reducing life quality. While macrophages are widely appreciated as a master regulator in the inflammatory response of RA, the precise mechanisms underlying the regulation of proliferation and inflammation in RA-derived fibroblast-like synoviocytes (RA-FLS) remain elusive. Here, we provide extensive evidence to demonstrate that macrophage contributes to RA microenvironment remodeling by extracellular vesicles (sEVs) and downstream miR-100-5p/ mammalian target of rapamycin (mTOR) axis. RESULTS: We showed that bone marrow derived macrophage (BMDM) derived-sEVs (BMDM-sEVs) from collagen-induced arthritis (CIA) mice (cBMDM-sEVs) exhibited a notable increase in abundance compared with BMDM-sEVs from normal mice (nBMDM-sEVs). cBMDM-sEVs induced significant RA-FLS proliferation and potent inflammatory responses. Mechanistically, decreased levels of miR-100-5p were detected in cBMDM-sEVs compared with nBMDM-sEVs. miR-100-5p overexpression ameliorated RA-FLS proliferation and inflammation by targeting the mTOR pathway. Partial attenuation of the inflammatory effects induced by cBMDM-sEVs on RA-FLS was achieved through the introduction of an overexpression of miR-100-5p. CONCLUSIONS: Our work reveals the critical role of macrophages in exacerbating RA by facilitating the transfer of miR-100-5p-deficient sEVs to RA-FLS, and sheds light on novel disease mechanisms and provides potential therapeutic targets for RA interventions.

Periplogenin inhibits pyroptosis of fibroblastic synoviocytes in rheumatoid arthritis through the NLRP3/Caspase-1/GSDMD signaling pathway.

Although the pathogenesis of rheumatoid arthritis (RA) remains unclear, an increasing number of studies have confirmed that pyroptosis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) is an important factor affecting the progression of RA. Periplogenin (PPN) is a natural cardiac glycoside; reportedly, it exerts anti-inflammatory and analgesic effects in diseases by inhibiting cell growth and migration. This study aimed to determine the effect of PPN on the growth, migration, and invasion of RA-FLS and the potential mechanism of pyroptosis regulation. We discovered that PPN could inhibit the migration and invasion abilities of RA-FLS and block their growth cycle, down-regulate the secretion and activation of NLRP3, Caspase-1, GSDMD, IL-1ß, and IL-18, and reduce the number of pyroptosis. In summary, PPN inhibited pyroptosis, reduced the release of inflammatory factors, and improved RA-FLS inflammation by regulating the NLRP3/Caspase-1/GSDMD signaling pathway.

The immune cells in modulating osteoclast formation and bone metabolism.

Osteoclasts are pivotal in regulating bone metabolism, with immune cells significantly influencing both physiological and pathological processes by modulating osteoclast functions. This is particularly evident in conditions of inflammatory bone resorption, such as rheumatoid arthritis and periodontitis. This review summarizes and comprehensively analyzes the research progress on the regulation of osteoclast formation by immune cells, aiming to unveil the underlying mechanisms and pathways through which diseases, such as rheumatoid arthritis and periodontitis, impact bone metabolism.

Epstein-barr virus infections induce aberrant osteoclastogenesis in immune system-humanized NOD/Shi-scid/IL-2RγCnull mice.

Epstein-Barr virus (EBV) and other viral infections are possible triggers of autoimmune diseases, such as rheumatoid arthritis (RA). To analyze the causative relationship between EBV infections and RA development, we performed experiment on humanized NOD/Shi-scid/IL-2RγCnull (hu-NOG) mice reconstituted human immune system components and infected with EBV. In EBV-infected hu-NOG mice, breakdown of knee joint bones was found to be accompanied by the accumulation of receptor activator of nuclear factor-κB (NF-κB) (RANK) ligand (RANKL), a key factor in osteoclastogenesis, human CD19 and EBV-encoded small RNA (EBER)-bearing cells. Accumulation of these cells expanded in the bone marrow adjacent to the bone breakage, showing a histological feature like to that in bone marrow edema. On the other hand, human RANK/human matrix metalloprotease-9 (MMP-9) positive, osteoclast-like cells were found at broken bone portion of EBV-infected mouse knee joint. In addition, human macrophage-colony stimulating factor (M-CSF), an essential factor in development of osteoclasts, evidently expressed in spleen and bone marrow of EBV-infected humanized mice. Furthermore, RANKL and M-CSF were identified at certain period of EBV-transformed B lymphoblastoid cells (BLBCs) derived from umbilical cord blood lymphocytes. Co-culturing bone marrow cells of hu-NOG mice with EBV-transformed BLBCs resulted in the induction of a multinucleated cell population positive for tartrate-resistant acid phosphatase and human MMP-9 which indicating human osteoclast-like cells. These findings suggest that EBV-infected BLBCs induce human aberrant osteoclastogenesis, which cause erosive arthritis in the joints.

miRNA-148a-containing GMSC-derived EVs modulate Treg/Th17 balance via IKKB/NF-κB pathway and treat a rheumatoid arthritis model.

Mesenchymal stem cells (MSCs) have demonstrated potent immunomodulatory properties that have shown promise in the treatment of autoimmune diseases, including rheumatoid arthritis (RA). However, the inherent heterogeneity of MSCs triggered conflicting therapeutic outcomes, raising safety concerns and limiting their clinical application. This study aimed to investigate the potential of extracellular vesicles derived from human gingival mesenchymal stem cells (GMSC-EVs) as a therapeutic strategy for RA. Through in vivo experiments using an experimental RA model, our results demonstrate that GMSC-EVs selectively homed to inflamed joints and recovered Treg and Th17 cell balance, resulting in the reduction of arthritis progression. Our investigations also uncovered miR-148a-3p as a critical contributor to the Treg/Th17 balance modulation via IKKB/NF-κB signaling orchestrated by GMSC-EVs, which was subsequently validated in a model of human xenograft versus host disease (xGvHD). Furthermore, we successfully developed a humanized animal model by utilizing synovial fibroblasts obtained from patients with RA (RASFs). We found that GMSC-EVs impeded the invasiveness of RASFs and minimized cartilage destruction, indicating their potential therapeutic efficacy in the context of patients with RA. Overall, the unique characteristics - including reduced immunogenicity, simplified administration, and inherent ability to target inflamed tissues - position GMSC-EVs as a viable alternative for RA and other autoimmune diseases.

Targeted delivery of NO donor and ROS scavenger for synergistic treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by high level of reactive oxygen species (ROS) and proinflammatory cytokines, which facilitate the activation of the inflammatory signaling such as NF-κB pathway and exacerbate the development of inflammation. Herein, we designed a nanodrug by encapsulating the NO donor S-nitrosoglutathione (GSNO) into an emulsion and coating the surface with a polydopamine (PDA) layer to yield GSNO@PDA, which simultaneously scavenged the extra ROS and suppressed NF-κB signaling for potent RA treatment. To enhance the cellular uptake and NO generation efficiency, dextran sulfate (DS) and Cu2+ were anchored on the surface of GSNO@PDA to obtain the final formulation GSNO@PDA@DS. Our results demonstrated that GSNO@PDA@DS were successfully prepared and the modification of DS effectively boosted the cellular uptake of GSNO@PDA@DS. Moreover, GSNO@PDA@DS lowered cellular ROS and elevated intracellular NO, resulting in a decrease of M1 phenotype, inhibition of NF-κB pathway and down-regulation of proinflammatory cytokine tumor necrosis factor-α (TNF-α). Further in vivo studies confirmed that GSNO@PDA@DS significantly relieved symptoms and bone erosion by regulating the microenvironment of RA, highlighting the potential of GSNO@PDA@DS for RA therapy through ROS scavenging and NO-mediated suppression of inflammatory signaling.