The severity of experimental arthritis is independent of IL-36 receptor signaling

Introduction

Interleukin (IL)-36 refers to three related IL-1 family cytokines, IL-36α, IL-36β, and IL-36γ, that bind to the IL-36 receptor (IL-36R). IL-36 exerts proinflammatory effects in skin and lung and stimulates T cell responses. In the present study, we examined the expression and function of IL-36R and its ligands in experimental arthritis.

Methods

Collagen-induced arthritis (CIA), antigen-induced arthritis (AIA), and K/BxN serum transfer-induced arthritis were induced according to standard protocols. Messenger RNA levels for IL-36R and its ligands in the joints of mice with CIA were determined by RT-qPCR. Mice with CIA were injected with a blocking monoclonal anti-IL-36R, a blocking anti-IL-1RI, or their isotype-matched control antibodies at the time of arthritis onset. Anti-IL-36R or control antibodies were also injected at the time of AIA induction. Finally, IL-36R-deficient mice were examined in AIA and serum transfer-induced arthritis. The development and severity of arthritis were assessed by clinical and histological scoring.

Results

IL-36R, IL-36Ra and IL-36γ mRNA were detected in the joints of mice with CIA, but their levels did not correlate with arthritis severity. As opposed to anti-IL-1RI antibody treatment, the injection of an anti-IL-36R antibody was devoid of effect on the development and severity of CIA. The severity of joint inflammation and structural damage in AIA was also unaltered by anti-IL-36R antibody treatment. Finally, the severity of AIA and K/BxN serum transfer-induced arthritis was similar in IL-36R-deficient and wild-type mice.

Conclusions

The development and severity of experimental arthritis are independent of IL-36R signaling.     

Blockade but Not Overexpression of the Junctional Adhesion Molecule C Influences Virus-Induced Type 1 Diabetes in Mice

Type 1 diabetes (T1D) results from the autoimmune destruction of insulin-producing beta-cells in the pancreas. Recruitment of inflammatory cells is prerequisite to beta-cell-injury. The junctional adhesion molecule (JAM) family proteins JAM-B and JAM–C are involved in polarized leukocyte transendothelial migration and are expressed by vascular endothelial cells of peripheral tissue and high endothelial venules in lympoid organs. Blocking of JAM-C efficiently attenuated cerulean-induced pancreatitis, rheumatoid arthritis or inflammation induced by ischemia and reperfusion in mice. In order to investigate the influence of JAM-C on trafficking and transmigration of antigen-specific, autoaggressive T-cells, we used transgenic mice that express a protein of the lymphocytic choriomeningitis virus (LCMV) as a target autoantigen in the β-cells of the islets of Langerhans under the rat insulin promoter (RIP). Such RIP-LCMV mice turn diabetic after infection with LCMV. We found that upon LCMV-infection JAM-C protein was upregulated around the islets in RIP-LCMV mice. JAM-C expression correlated with islet infiltration and functional beta-cell impairment. Blockade with a neutralizing anti-JAM-C antibody reduced the T1D incidence. However, JAM-C overexpression on endothelial cells did not accelerate diabetes in the RIP-LCMV model. In summary, our data suggest that JAM-C might be involved in the final steps of trafficking and transmigration of antigen-specific autoaggressive T-cells to the islets of Langerhans.     

Ezrin regulates synovial angiogenesis in rheumatoid arthritis through YAP and Akt signalling

This study aimed to investigate the role and regulatory mechanisms of Ezrin in synovial vessels in rheumatoid arthritis (RA). Synovial tissues were obtained from people with osteoarthritis people and patients with RA patients. We also used an antigen-induced arthritis (AIA) mice model by using Freund's adjuvant injections. Ezrin expression was analysed by immunofluorescence and immunohistochemical staining in synovial vessels of patients with RA and AIA mice. We investigated the role of Ezrin on vascular endothelial cells and its regulatory mechanism in vivo and in vitro by adenoviral transfection technology. Our results suggest a role for the Ezrin protein in proliferation, migration and angiogenesis of vascular endothelial cells in RA. We also demonstrate that Ezrin plays an important role in vascular endothelial cell migration and tube formation through regulation of the Hippo-yes-associated protein 1 (YAP) pathway. YAP, as a key protein, can further regulate the activity of PI3K/Akt signalling pathway in vascular endothelial cells. In AIA mice experiments, we observed that the inhibition of Ezrin or of its downstream YAP pathway can affect synovial angiogenesis and may lead to progression of RA. In conclusion, Ezrin plays an important role in angiogenesis in the RA synovium by regulating YAP nuclear translocation and interacting with the PI3K/Akt signalling pathway.     

Stromal Transcriptional Profiles Reveal Hierarchies of Anatomical Site,Serum Response and Disease and Identify Disease Specific Pathways

Synovial fibroblasts in persistent inflammatory arthritis have been suggested to have parallels with cancer growth and wound healing, both of which involve a stereotypical serum response programme. We tested the hypothesis that a serum response programme can be used to classify diseased tissues, and investigated the serum response programme in fibroblasts from multiple anatomical sites and two diseases. To test our hypothesis we utilized a bioinformatics approach to explore a publicly available microarray dataset including rheumatoid arthritis (RA), osteoarthritis (OA) and normal synovial tissue, then extended those findings in a new microarray dataset representing matched synovial, bone marrow and skin fibroblasts cultured from RA and OA patients undergoing arthroplasty. The classical fibroblast serum response programme discretely classified RA, OA and normal synovial tissues. Analysis of low and high serum treated fibroblast microarray data revealed a hierarchy of control, with anatomical site the most powerful classifier followed by response to serum and then disease. In contrast to skin and bone marrow fibroblasts, exposure of synovial fibroblasts to serum led to convergence of RA and OA expression profiles. Pathway analysis revealed three inter-linked gene networks characterising OA synovial fibroblasts: Cell remodelling through insulin-like growth factors, differentiation and angiogenesis through _3 integrin, and regulation of apoptosis through CD44. We have demonstrated that Fibroblast serum response signatures define disease at the tissue level, and that an OA specific, serum dependent repression of genes involved in cell adhesion, extracellular matrix remodelling and apoptosis is a critical discriminator between cultured OA and RA synovial fibroblasts.     

Deficiency of fibroblast activation protein alpha ameliorates cartilage destruction in inflammatory destructive arthritis

IntroductionInflammatory destructive arthritis, like rheumatoid arthritis (RA), is characterized by invasion of synovial fibroblasts (SF) into the articular cartilage and erosion of the underlying bone, leading to progressive joint destruction. Because fibroblast activation protein alpha (FAP) has been associated with cell migration and cell invasiveness, we studied the function of FAP in joint destruction in RA.MethodsExpression of FAP in synovial tissues and fibroblasts from patients with osteoarthritis (OA) and RA as well as from wild-type and arthritic mice was evaluated by immunohistochemistry, fluorescence microscopy and polymerase chain reaction (PCR). Fibroblast adhesion and migration capacity was assessed using cartilage attachment assays and wound-healing assays, respectively. For in vivo studies, FAP-deficient mice were crossed into the human tumor necrosis factor transgenic mice (hTNFtg), which develop a chronic inflammatory arthritis. Beside clinical assessment, inflammation, cartilage damage, and bone erosion were evaluated by histomorphometric analyses.ResultsRA synovial tissues demonstrated high expression of FAP whereas in OA samples only marginal expression was detectable. Consistently, a higher expression was detected in arthritis SF compared to non-arthritis OA SF in vitro. FAP-deficiency in hTNFtg mice led to less cartilage degradation despite unaltered inflammation and bone erosion. Accordingly, FAP^−/− hTNFtg SF demonstrated a lower cartilage adhesion capacity compared to hTNFtg SF in vitro.ConclusionsThese data point to a so far unknown role of FAP in the attachment of SF to cartilage, promoting proteoglycan loss and subsequently cartilage degradation in chronic inflammatory arthritis.     

c-Fms-mediated differentiation and priming of monocyte lineage cells play a central role in autoimmune arthritis

Introduction

Tyrosine kinases are key mediators of multiple signaling pathways implicated in rheumatoid arthritis (RA). We previously demonstrated that imatinib mesylate--a Food and Drug Administration (FDA)-approved, antineoplastic drug that potently inhibits the tyrosine kinases Abl, c-Kit, platelet-derived growth factor receptor (PDGFR), and c-Fms--ameliorates murine autoimmune arthritis. However, which of the imatinib-targeted kinases is the principal culprit in disease pathogenesis remains unknown. Here we examine the role of c-Fms in autoimmune arthritis.

Methods

We tested the therapeutic efficacy of orally administered imatinib or GW2580, a small molecule that specifically inhibits c-Fms, in three mouse models of RA: collagen-induced arthritis (CIA), anti-collagen antibody-induced arthritis (CAIA), and K/BxN serum transfer-induced arthritis (K/BxN). Efficacy was evaluated by visual scoring of arthritis severity, paw thickness measurements, and histological analysis. We assessed the in vivo effects of imatinib and GW2580 on macrophage infiltration of synovial joints in CIA, and their in vitro effects on macrophage and osteoclast differentiation, and on osteoclast-mediated bone resorption. Further, we determined the effects of imatinib and GW2580 on the ability of macrophage colony-stimulating factor (M-CSF; the ligand for c-Fms) to prime bone marrow-derived macrophages to produce tumor necrosis factor (TNF) upon subsequent Fc receptor ligation. Finally, we measured M-CSF levels in synovial fluid from patients with RA, osteoarthritis (OA), or psoriatic arthritis (PsA), and levels of total and phosphorylated c-Fms in synovial tissue from patients with RA.

Results

GW2580 was as efficacious as imatinib in reducing arthritis severity in CIA, CAIA, and K/BxN models of RA. Specific inhibition of c-Fms abrogated (i) infiltration of macrophages into synovial joints of arthritic mice; (ii) differentiation of monocytes into macrophages and osteoclasts; (iii) osteoclast-mediated bone resorption; and (iv) priming of macrophages to produce TNF upon Fc receptor stimulation, an important trigger of synovitis in RA. Expression and activation of c-Fms in RA synovium were high, and levels of M-CSF were higher in RA synovial fluid than in OA or PsA synovial fluid.

Conclusions

These results suggest that c-Fms plays a central role in the pathogenesis of RA by mediating the differentiation and priming of monocyte lineage cells. Therapeutic targeting of c-Fms could provide benefit in RA.     

CD97 neutralisation increases resistance to collagen-induced arthritis in mice

Synovial tissue of rheumatoid arthritis (RA) patients is characterised by an influx and retention of CD97-positive inflammatory cells. The ligands of CD97, CD55, chondroitin sulfate B, and α5β1 (very late antigen [VLA]-5) are expressed abundantly in the synovial tissue predominantly on fibroblast-like synoviocytes, endothelium, and extracellular matrix. Based upon this expression pattern, we hypothesise CD97 expression to result in accumulation of inflammatory cells in the synovial tissue of RA patients. To determine the therapeutic effect of blocking CD97 in an animal model of RA, collagen-induced arthritis was induced in a total of 124 DBA/J1 mice. Treatment was started on day 21 (early disease) or on day 35 (longstanding disease) with the blocking hamster anti-mouse CD97 monoclonal antibody (mAb) 1B2, control hamster immunoglobulin, or NaCl, applied intraperitoneally three times a week. The paws were evaluated for clinical signs of arthritis and, in addition, examined by radiological and histological analysis. Mice receiving 0.5 mg CD97 mAb starting from day 21 had significantly less arthritis activity and hind paw swelling. Furthermore, joint damage and inflammation were reduced and granulocyte infiltration was decreased. When treatment was started on day 35, CD97 mAb treatment had similar effects, albeit less pronounced. The results support the notion that CD97 contributes to synovial inflammation and joint destruction in arthritis.     

Joint production of IL-22 participates in the initial phase of antigen-induced arthritis through IL-1β production

IntroductionRheumatoid arthritis (RA) is a chronic autoimmune disease characterized by neutrophil articular infiltration, joint pain and the progressive destruction of cartilage and bone. IL-22 is a key effector molecule that plays a critical role in autoimmune diseases. However, the function of IL-22 in the pathogenesis of RA remains controversial. In this study, we investigated the role of IL-22 in the early phase of antigen-induced arthritis (AIA) in mice.MethodsAIA was induced in C57BL/6, IL-22^−/−, ASC^−/− and IL-1R1^−/− immunized mice challenged intra-articularly with methylated bovine serum albumin (mBSA). Expression of IL-22 in synovial membranes was determined by RT-PCR. Articular hypernociception was evaluated using an electronic von Frey. Neutrophil recruitment and histopathological analyses were assessed in inflamed knee joint. Joint levels of inflammatory mediators and mBSA-specific IgG concentration in the serum were measured by ELISA.ResultsThe IL-22 mRNA expression and protein levels in synovial tissue were increased during the onset of AIA. In addition, pharmacological inhibition (anti-IL-22 antibody) and genetic deficiency (IL-22^−/− mice) reduced articular pain and neutrophil migration in arthritic mice. Consistent with these findings, recombinant IL-22 joint administration promoted articular inflammation per se in WT mice, restoring joint nociception and neutrophil infiltration in IL-22^−/− mice. Moreover, IL-22-deficient mice showed reduced synovitis (inflammatory cell influx) and lower joint IL-1β levels, whereas the production of IL-17, MCP-1/CCL2, and KC/CXCL1 and the humoral immune response were similar, compared with WT mice. Corroborating these results, the exogenous administration of IL-22 into the joints induced IL-1β production in WT mice and reestablished IL-1β production in IL-22^−/− mice challenged with mBSA. Additionally, IL-1R1^−/− mice showed attenuated inflammatory features induced by mBSA or IL-22 challenge. Articular nociception and neutrophil migration induced by IL-22 were also reduced in ASC^−/− mice.ConclusionsThese results suggest that IL-22 plays a pro-inflammatory/pathogenic role in the onset of AIA through an ASC-dependent stimulation of IL-1β production.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0759-2) contains supplementary material, which is available to authorized users.     

CXCL13/CXCR5 axis facilitates endothelial progenitor cell homing and angiogenesis during rheumatoid arthritis progression

Angiogenesis is a critical process in the formation of new capillaries and a key participant in rheumatoid arthritis (RA) pathogenesis. The chemokine (C-X-C motif) ligand 13 (CXCL13) plays important roles in several cellular functions such as infiltration, migration, and motility. We report significantly higher levels of CXCL13 expression in collagen-induced arthritis (CIA) mice compared with controls and also in synovial fluid from RA patients compared with human osteoarthritis (OA) samples. RA synovial fluid increased endothelial progenitor cell (EPC) homing and angiogenesis, which was blocked by the CXCL13 antibody. By interacting with the CXCR5 receptor, CXCL13 facilitated vascular endothelial growth factor (VEGF) expression and angiogenesis in EPC through the PLC, MEK, and AP-1 signaling pathways. Importantly, infection with CXCL13 short hairpin RNA (shRNA) mitigated EPC homing and angiogenesis, articular swelling, and cartilage erosion in ankle joints of mice with CIA. CXCL13 is therefore a novel therapeutic target for RA.Subject terms: miRNAs, Rheumatoid arthritis     

Pro-apoptotic Bid is required for the resolution of the effector phase of inflammatory arthritis

Rheumatoid arthritis is an autoimmune disease characterized by hyperplasia of the synovial lining and destruction of cartilage and bone. Recent studies have suggested that a lack of apoptosis contributes to the hyperplasia of the synovial lining and to the failure in eliminating autoreactive cells. Mice lacking Fas or Bim, two pro-apoptotic proteins that mediate the extrinsic and intrinsic death cascades, respectively, develop enhanced K/BxN serum transfer-induced arthritis. Since the pro-apoptotic protein Bid functions as an intermediate between the extrinsic and intrinsic apoptotic pathways, we examined the role that it plays in inflammatory arthritis. Mice deficient in Bid (Bid-/-) show a delay in the resolution of K/BxN serum transfer-induced arthritis. Bid-/- mice display increased inflammation, bone destruction, and pannus formation compared to wild-type mice. Furthermore, Bid-/- mice have elevated levels of CXC chemokine and IL-1β in serum, which are associated with more inflammatory cells throughout the arthritic joint. In addition, there are fewer apoptotic cells in the synovium of Bid-/- compared to Wt mice. These data suggest that extrinsic and intrinsic apoptotic pathways cooperate through Bid to limit development of inflammatory arthritis.     

Alpha 4/beta 1 integrin (VLA-4) ligands in arthritis. Vascular cell adhesion molecule-1 expression in synovium and on fibroblast-like synoviocytes.

Expression of vascular cell adhesion molecule-1 (VCAM-1) in synovial tissue was determined using the immunoperoxidase technique. Normal, rheumatoid arthritis (RA), and osteoarthritis (OA) synovia bound VCAM-1 antibodies in the intimal lining as well as blood vessels. The amount of VCAM-1 was significantly greater in the synovial lining of RA and OA tissues compared with normal synovium (p less than 0.002). There was also a trend toward greater levels of VCAM-1 staining in blood vessels of arthritic tissue (RA greater than OA greater than normal). Because VCAM-1 staining was especially intense in the synovial lining, VCAM-1 expression and regulation was studied on cultured fibroblast-like synoviocytes (FLS) derived from this region. Both VCAM-1 and intercellular adhesion molecule 1 were constitutively expressed on FLS. VCAM-1 expression was further increased by exposure to IL-1 beta, TNF-alpha, IL-4, and IFN-gamma. These cytokines (except for IL-4) also induced intercellular adhesion molecule 1 expression on FLS. ELAM was not detected on resting or cytokine-stimulated FLS. The specificity of VCAM-1 for FLS was demonstrated by the fact that only trace amounts were detected on normal and RA dermal fibroblasts. Cytokines induced intercellular adhesion molecule 1 display on dermal fibroblasts but had minimal effect on VCAM-1 expression. Finally, in adherence assays, Jurkat cell binding to resting FLS monolayers was inhibited by antibody to alpha 4/beta 1 integrin (VLA-4), CS-1 peptide from alternatively spliced fibronectin (which is another VLA-4 ligand), and, to a lesser extent, anti-VCAM-1 antibody. After cytokine stimulation of FLS, Jurkat-binding significantly increased, and this increase was blocked by anti-VCAM-1 antibody. Therefore, both CS-1 and VCAM-1 participate in VLA-4-mediated adherence to resting FLS in vitro, and VCAM-1 is responsible for the increase in Jurkat binding mediated by cytokines.     

Hypoxia-Inducible Factor-1α and Interleukin 33 Form a Regulatory Circuit to Perpetuate the Inflammation in Rheumatoid Arthritis

Hyperplasia of synovial fibroblasts, infiltration with inflammatory cytokines, and tissue hypoxia are the major characteristics of rheumatoid arthritis (RA). Interleukin 33 (IL-33) is a newly identified inflammatory cytokine exacerbating the disease severity of RA. Hypoxia-inducible factor-1α (HIF-1α) showed increased expression in RA synovium and could regulate a number of inflammatory cytokine productions. Nevertheless, its correlation with IL-33 remains largely unknown. Here, we showed that elevated levels of IL-33 were demonstrated in RA patient synovial fluids, with upregulated expression of HIF-1α and IL-33 in the synovial fibroblasts. Knocking down HIF-1α compromised IL-33 expression in rheumatoid arthritis synovial fibroblasts (RASF), while enforcing HIF-1α expression in RASF substantially upregulated IL-33 levels. HIF-1α promoted the activation of the signalling pathways controlling IL-33 production, particularly the p38 and ERK pathways. Moreover, we showed for the first time that IL-33 in turn could induce more HIF-1α expression in RASF, thus forming a HIF-1α/IL-33 regulatory circuit that would perpetuate the inflammatory process in RA. Targeting this pathological pathway and HIF-1α may provide new therapeutic strategies for overcoming the persistent and chronic inflammatory disease.     

The role of an epithelial neutrophil-activating peptide-78-like protein in rat adjuvant-induced arthritis.

The chemokine, epithelial neutrophil-activating peptide-78 (ENA-78), is a potent neutrophil chemotaxin whose expression is increased in inflamed synovial tissue and fluid in human rheumatoid arthritis compared with osteoarthritis. Since ENA-78 has been implicated in the pathogenesis of RA, we examined the expression of an ENA-78-like protein during the development of rat adjuvant-induced arthritis (AIA). Using an ELISA assay, we found increased levels of antigenic ENA-78-like protein in the sera of AIA animals compared with control normal animals by day 7 postadjuvant injection. ENA-78-like protein levels continued to increase as AIA developed. ENA-78-like protein levels in joint homogenates were increased in AIA animals later in the development of the disease, by day 18 during maximal arthritis, compared with control animals. Expression of ENA-78-like protein in both the AIA serum and joint correlated with the progression of inflammation of the joints. Anti-human ENA-78 administered before disease onset modified the severity of AIA, while administration of anti-ENA-78 after clinical onset of AIA did not modify the disease. These data support a role for an ENA-78-like protein as an important chemokine in the progression and maintenance of AIA.     

A possible role for TSLP in inflammatory arthritis

Thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine that triggers dendritic cell-mediated Th2-type inflammatory responses and is considered as a master switch for allergic inflammation. In this study, we found increased levels of TSLP and, also TNF-alpha as previously reported, in synovial fluid specimens derived from patients with rheumatoid arthritis (RA) when compared with those from patients with osteoarthritis (OA). In addition, TNF-alpha up-regulated TSLP expression in RA- and OA-derived synovial fibroblasts, which was inhibited by IFN-gamma. Furthermore, anti-TSLP neutralizing antibody ameliorated a TNF-alpha-dependent experimental arthritis induced by anti-type II collagen antibody in mice. Collectively, these results suggest that TSLP, as a downstream molecule of TNF-alpha, may be involved in the pathophysiology of inflammatory arthritis. TSLP might thus play a role not only in allergic diseases but also in inflammatory arthritis such as RA.     

Decrease of CD68 Synovial Macrophages in Celastrol Treated Arthritic Rats

Background

Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease characterized by cellular infiltration into the joints, hyperproliferation of synovial cells and bone damage. Available treatments for RA only induce remission in around 30% of the patients, have important adverse effects and its use is limited by their high cost. Therefore, compounds that can control arthritis, with an acceptable safety profile and low production costs are still an unmet need. We have shown, in vitro, that celastrol inhibits both IL-1β and TNF, which play an important role in RA, and, in vivo, that celastrol has significant anti-inflammatory properties. Our main goal in this work was to test the effect of celastrol in the number of sublining CD68 macrophages (a biomarker of therapeutic response for novel RA treatments) and on the overall synovial tissue cellularity and joint structure in the adjuvant-induced rat model of arthritis (AIA).

Methods

Celastrol was administered to AIA rats both in the early (4 days after disease induction) and late (11 days after disease induction) phases of arthritis development. The inflammatory score, ankle perimeter and body weight were evaluated during treatment period. Rats were sacrificed after 22 days of disease progression and blood, internal organs and paw samples were collected for toxicological blood parameters and serum proinflammatory cytokine quantification, as well as histopathological and immunohistochemical evaluation, respectively.

Results

Here we report that celastrol significantly decreases the number of sublining CD68 macrophages and the overall synovial inflammatory cellularity, and halted joint destruction without side effects.

Conclusions

Our results validate celastrol as a promising compound for the treatment of arthritis.     

Salvia plebeia extract inhibits the inflammatory response in human rheumatoid synovial fibroblasts and a murine model of arthritis

Salvia plebeia R. Br. has been used to treat a variety of inflammatory diseases and as an antioxidant in many countries, including Korea and China. In this study, we investigated the effects of S. plebeia extract (SPE) on inflammatory arthritis and the underlying mechanisms of action. We used a collagen-induced arthritis (CIA) mouse model. TNF-α-stimulated rheumatoid arthritis (RA) synovial fibroblasts were used to elucidate the underlying mechanisms of action. Oral administration of SPE improved the clinical arthritis score, footpad thickness, and histologic changes, as well as serum IgG1 and IgG2a levels. SPE administration inhibited Th1/Th2/Th17 phenotype CD4⁺ T lymphocyte expansion in inguinal lymph node and expression of inflammatory mediators such as cytokines, MMP-1, and MMP-3 in the ankle joint tissue. SPE significantly suppressed the expression of cytokines and MMP-1 by down-regulating NF-κB, Akt, and mitogen-activated protein kinases in RA synovial fibroblasts. Taken together, these results indicate that SPE is therapeutically efficacious against chronic inflammatory arthritis, suggesting that SPE is a candidate for treating RA.