Substance P ameliorates collagen II-induced arthritis in mice via suppression of the inflammatory response

Current rheumatoid arthritis (RA) therapies such as biologics inhibiting pathogenic cytokines substantially delay RA progression. However, patient responses to these agents are not always complete and long lasting. This study explored whether substance P (SP), an 11 amino acids long endogenous neuropeptide with the novel ability to mobilize mesenchymal stem cells (MSC) and modulate injury-mediated inflammation, can inhibit RA progression. SP efficacy was evaluated by paw swelling, clinical arthritis scoring, radiological analysis, histological analysis of cartilage destruction, and blood levels of tumor necrosis factor-alpha (TNF-α) interleukin (IL)-10, and IL-17 in vivo. SP treatment significantly reduced local inflammatory signs, mean arthritis scores, degradation of joint cartilage, and invasion of inflammatory cells into the synovial tissues. Moreover, the SP treatment markedly reduced the size of spleens enlarged by excessive inflammation in CIA, increased IL-10 levels, and decreased TNF-α and IL-17 levels. Mobilization of stem cells and induction of T_reg and M2 type macrophages in the circulation were also increased by the SP treatment. These effect of SP might be associated with the suppression of inflammatory responses in RA and, furthermore, blockade of RA progression. Our results propose SP as a potential therapeutic for autoimmune-related inflammatory diseases.     

Inhibition of Inflammatory Arthritis Using Fullerene Nanomaterials

Inflammatory arthritis (e.g. rheumatoid arthritis; RA) is a complex disease driven by the interplay of multiple cellular lineages. Fullerene derivatives have previously been shown to have anti-inflammatory capabilities mediated, in part, by their ability to prevent inflammatory mediator release by mast cells (MC). Recognizing that MC can serve as a cellular link between autoantibodies, soluble mediators, and other effector populations in inflammatory arthritis, it was hypothesized that fullerene derivatives might be used to target this inflammatory disease. A panel of fullerene derivatives was tested for their ability to affect the function of human skin-derived MC as well as other lineages implicated in arthritis, synovial fibroblasts and osteoclasts. It is shown that certain fullerene derivatives blocked FcγR- and TNF-α-induced mediator release from MC; TNF-α-induced mediator release from RA synovial fibroblasts; and maturation of human osteoclasts. MC inhibition by fullerene derivatives was mediated through the reduction of mitochondrial membrane potential and FcγR-mediated increases in cellular reactive oxygen species and NF-κB activation. Based on these in vitro data, two fullerene derivatives (ALM and TGA) were selected for in vivo studies using K/BxN serum transfer arthritis in C57BL/6 mice and collagen-induced arthritis (CIA) in DBA/1 mice. Dye-conjugated fullerenes confirmed localization to affected joints in arthritic animals but not in healthy controls. In the K/BxN moldel, fullerenes attenuated arthritis, an effect accompanied by reduced histologic inflammation, cartilage/bone erosion, and serum levels of TNF-α. Fullerenes remained capable of attenuating K/BxN arthritis in mast cell-deficient mice Cre-Master mice, suggesting that lineages beyond the MC represent relevant targets in this system. These studies suggest that fullerene derivatives may hold promise both as an assessment tool and as anti-inflammatory therapy of arthritis.     

Defining Immunological Impact and Therapeutic Benefit of Mild Heating in a Murine Model of Arthritis

Traditional treatments, including a variety of thermal therapies have been known since ancient times to provide relief from rheumatoid arthritis (RA) symptoms. However, a general absence of information on how heating affects molecular or immunological targets relevant to RA has limited heat treatment (HT) to the category of treatments known as “alternative therapies”. In this study, we evaluated the effectiveness of mild HT in a collagen-induced arthritis (CIA) model which has been used in many previous studies to evaluate newer pharmacological approaches for the treatment of RA, and tested whether inflammatory immune activity was altered. We also compared the effect of HT to methotrexate, a well characterized pharmacological treatment for RA. CIA mice were treated with either a single HT for several hours or daily 30 minute HT. Disease progression and macrophage infiltration were evaluated. We found that both HT regimens significantly reduced arthritis disease severity and macrophage infiltration into inflamed joints. Surprisingly, HT was as efficient as methotrexate in controlling disease progression. At the molecular level, HT suppressed TNF-α while increasing production of IL-10. We also observed an induction of HSP70 and a reduction in both NF-κB and HIF-1α in inflamed tissues. Additionally, using activated macrophages in vitro, we found that HT reduced production of pro-inflammatory cytokines, an effect which is correlated to induction of HSF-1 and HSP70 and inhibition of NF-κB and STAT activation. Our findings demonstrate a significant therapeutic benefit of HT in controlling arthritis progression in a clinically relevant mouse model, with an efficacy similar to methotrexate. Mechanistically, HT targets highly relevant anti-inflammatory pathways which strongly support its increased study for use in clinical trials for RA.     

Methionine aminopeptidase-2 blockade reduces chronic collagen-induced arthritis: potential role for angiogenesis inhibition

The enzyme methionine aminopeptidase-2 (MetAP-2) is thought to play an important function in human endothelial cell proliferation, and as such provides a valuable target in both inflammation and cancer. Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with increased synovial vascularity, and hence is a potential therapeutic target for angiogenesis inhibitors. We examined the use of PPI-2458, a selective non-reversible inhibitor of MetAP-2, in disease models of RA, namely acute and chronic collagen-induced arthritis (CIA) in mice. Whilst acute CIA is a monophasic disease, CIA induced with murine collagen type II manifests as a chronic relapsing arthritis and mimics more closely the disease course of RA. Our study showed PPI-2458 was able to reduce clinical signs of arthritis in both acute and chronic CIA models. This reduction in arthritis was paralleled by decreased joint inflammation and destruction. Detailed mechanism of action studies demonstrated that PPI-2458 inhibited human endothelial cell proliferation and angiogenesis in vitro, without affecting production of inflammatory cytokines. Furthermore, we also investigated release of inflammatory cytokines and chemokines from human RA synovial cell cultures, and observed no effect of PPI-2458 on spontaneous expression of cytokines and chemokines, or indeed on the angiogenic molecule vascular endothelial growth factor (VEGF). These results highlight MetAP-2 as a good candidate for therapeutic intervention 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.     

CTRP3 plays an important role in the development of collagen-induced arthritis in mice

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease exhibited most commonly in joints. We found that the expression of C1qtnf3, which encodes C1q/TNF-related protein 3 (CTRP3), was highly increased in two mouse RA models with different etiology. To elucidate the pathogenic roles of CTRP3 in the development of arthritis, we generated C1qtnf3^−/− mice and examined the development of collagen-induced arthritis in these mice. We found that the incidence and severity score was higher in C1qtnf3^−/− mice compared with wild-type (WT) mice. Histopathology of the joints was also more severe in C1qtnf3^−/− mice. The levels of antibodies against type II collagen and pro-inflammatory cytokine mRNAs in C1qtnf3^−/− mice were higher than WT mice. These observations indicate that CTRP3 plays an important role in the development of autoimmune arthritis, suggesting CTRP3 as a possible medicine to treat RA.     

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.     

The involvement of Toll‐like receptor 9 in the pathogenesis of erosive autoimmune arthritis

Endogenous nucleic acids and their receptors may be involved in the initiation of systemic autoimmune diseases including rheumatoid arthritis (RA). As the role of the DNA sensing Toll‐like receptor (TLR) 9 in RA is unclear, we aimed to investigate its involvement in the pathogenesis of autoimmune arthritis using three different experimental models of RA. The data obtained revealed involvement of TLR9 in the T cell‐dependent phase of inflammatory arthritis. In rats with pristane‐induced arthritis (PIA), TLR9 inhibition before disease onset reduced arthritis significantly and almost completely abolished bone erosion. Accordingly, serum levels of IL‐6, α‐1‐acid‐glycoprotein and rheumatoid factor were reduced. Moreover, in TLR9^?/? mice, streptococcal cell wall (SCW)‐induced arthritis was reduced in the T cell‐dependent phase, whereas T cell‐independent serum‐transfer arthritis was not affected. Remarkably, while TLR7 expression did not change during in vitro osteoclastogenesis, TLR9 expression was higher in precursor cells than in mature osteoclasts and partial inhibition of osteoclastogenesis was achieved only by the TLR9 antagonist. These results demonstrate a pivotal role for TLR9 in the T cell‐dependent phases of inflammatory arthritis and additionally suggest some role during osteoclastogenesis. Hence, endogenous DNA seems to be crucially involved in the pathophysiology of inflammatory autoimmune arthritis.     

IL-17RA signaling amplifies antibody-induced arthritis

Objective

To investigate the role of IL-17RA signaling in the effector phase of inflammatory arthritis using the K/BxN serum-transfer model.

Methods

Wild-type and Il17ra^−/− mice were injected with serum isolated from arthritic K/BxN mice and their clinical score was recorded daily. Mice were also harvested on days 12 and 21 and ankles were analyzed for cytokine and chemokine mRNA expression by qPCR on day 12 and for bone and cartilage erosions by histology on day 21, respectively. The induction of cytokine and chemokine expression levels by IL-17A in synovial-like fibroblasts was also analyzed using qPCR.

Results

Il17ra^−/− mice were partially protected from clinical signs of arthritis and had markedly fewer cartilage and bone erosions. The expression of several pro-inflammatory mediators, including the chemokines KC/CXCL1, MIP-2/CXCL2, LIX/CXCL5 MIP-1γ/CCL9, MCP-3/CCL7, MIP-3α/CCL20, the cytokines IL-1β, IL-6, RANKL and the matrix metalloproteinases MMP2, MMP3, and MMP13 were decreased in the ankles of Il17ra^−/− mice compared to wild-type mice. Many of these proinflammatory genes attenuated in the ankles of Il17ra^−/− mice were shown to be directly induced by IL-17A in synovial fibroblasts in vitro.

Conclusions

IL-17RA signaling plays a role as an amplifier of the effector phase of inflammatory arthritis. This effect is likely mediated by direct activation of synovial fibroblasts by IL-17RA to produce multiple inflammatory mediators, including chemokines active on neutrophils. Therefore, interrupting IL-17RA signaling maybe a promising pharmacological target for the treatment of inflammatory arthritis.     

AMPK Activation by A-769662 Controls IL-6 Expression in Inflammatory Arthritis

Objective

AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase critically involved in the regulation of cellular energy homeostasis. It is a central regulator of both lipid and glucose metabolism. Many studies have suggested that AMPK activation exert significant anti-inflammatory and immunosuppressive effects. In this study, we assessed whether targeted activation of AMPK inhibits inflammatory arthritis in vivo.

Methods

We tested the effect of A-769662, a specific AMPK agonist (60mg/kg/bid) in mouse models of antigen-induced arthritis (AIA) and passive K/BxN serum-induced arthritis. The passive K/BxN serum-induced arthritis model was also applied to AMPKα1-deficient mice. Joints were harvested and subjected to histological analysis. IL-6 expression was measured in both joint tissues and sera by ELISA. The effect of A-769662 on bone marrow derived macrophage (BMDM) response to stimulation with TLR2 and TLR4 agonists was tested in vitro.

Results

AMPK activation by A-769662 reduced inflammatory infiltration and joint damage in both mouse models. IL-6 expression in serum and arthritic joints was significantly decreased in A-769662-treated mice. AMPKα1 deficient mice mildly elicited an increase of clinical arthritis. IL-6 expression at both mRNA and protein levels, phosphorylation of p65 NF-κB and MAPK phosphorylation were inhibited by A-769662 in BMDMs stimulated with either TLR2 or TLR4 agonists.

Conclusions

AMPK activation by specific AMPK agonist A-769662 suppressed inflammatory arthritis in mice as well as IL-6 expression in serum and arthritic joints. These data suggest that targeted activation of AMPK has a potential to be an effective therapeutic strategy for IL-6 dependent inflammatory 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.     

Fms-Like Tyrosine Kinase 3 Ligand Controls Formation of Regulatory T Cells in Autoimmune Arthritis

Fms-like tyrosine kinase 3 ligand (Flt3L) is known as the primary differentiation and survival factor for dendritic cells (DCs). Furthermore, Flt3L is involved in the homeostatic feedback loop between DCs and regulatory T cell (Treg). We have previously shown that Flt3L accumulates in the synovial fluid in rheumatoid arthritis (RA) and that local exposure to Flt3L aggravates arthritis in mice, suggesting a possible involvement in RA pathogenesis. In the present study we investigated the role of Flt3L on DC populations, Tregs as well as inflammatory responses in experimental antigen-induced arthritis. Arthritis was induced in mBSA-immunized mice by local knee injection of mBSA and Flt3L was provided by daily intraperitoneal injections. Flow cytometry analysis of spleen and lymph nodes revealed an increased formation of DCs and subsequently Tregs in mice treated with Flt3L. Flt3L-treatment was also associated with a reduced production of mBSA specific antibodies and reduced levels of the pro-inflammatory cytokines IL-6 and TNF-α. Morphological evaluation of mBSA injected joints revealed reduced joint destruction in Flt3L treated mice. The role of DCs in mBSA arthritis was further challenged in an adoptive transfer experiment. Transfer of DCs in combination with T-cells from mBSA immunized mice, predisposed naïve recipients for arthritis and production of mBSA specific antibodies. We provide experimental evidence that Flt3L has potent immunoregulatory properties. Flt3L facilitates formation of Treg cells and by this mechanism reduces severity of antigen-induced arthritis in mice. We suggest that high systemic levels of Flt3L have potential to modulate autoreactivity and autoimmunity.     

Interaction of glycyrol with calcineurin A studied by spectroscopic methods and docking

We have shown previously that glycyrol has an inhibitory effect on the immune response in mice by reducing calcineurin activity (Li et al., 2010, Pharm Biol 48:1177–1184). Here, we investigated the interaction of glycyrol with calcineurin A (CNA, catalytic A subunit of calcineurin) by spectroscopic methods and docking. We showed that glycyrol binds to CNA via hydrophobic interactions in a ratio of 1:1, and the main binding site is in the catalytic domain of CNA close to the calcineurin B subunit-binding domain. Binding of glycyrol changes the secondary structure of CNA, and this effect may possibly inhibit CN activity.     

Inhibition of DNM1L and mitochondrial fission attenuates inflammatory response in fibroblast‐like synoviocytes of rheumatoid arthritis

Mitochondrial fission and fusion are important for mitochondrial function, and dynamin 1‐like protein (DNM1L) is a key regulator of mitochondrial fission. We investigated the effect of mitochondrial fission on mitochondrial function and inflammation in fibroblast‐like synoviocytes (FLSs) during rheumatoid arthritis (RA). DNM1L expression was determined in synovial tissues (STs) from RA and non‐RA patients. FLSs were isolated from STs and treated with a DNM1L inhibitor (mdivi‐1, mitochondrial division inhibitor 1) or transfected with DNM1L‐specific siRNA. Mitochondrial morphology, DNM1L expression, cell viability, mitochondrial membrane potential, reactive oxygen species (ROS), apoptosis, inflammatory cytokine expression and autophagy were examined. The impact of mdivi‐1 treatment on development and severity of collagen‐induced arthritis (CIA) was determined in mice. Up‐regulated DNM1L expression was associated with reduced mitochondrial length in STs from patients with RA and increased RA severity. Inhibition of DNM1L in FLSs triggered mitochondrial depolarization, mitochondrial elongation, decreased cell viability, production of ROS, IL‐8 and COX‐2, and increased apoptosis. DNM1L deficiency inhibited IL‐1β–mediated AKT/IKK activation, NF‐κBp65 nuclear translocation and LC3B‐related autophagy, but enhanced NFKBIA expression. Treatment of CIA mice with mdivi‐1 decreased disease severity by modulating inflammatory cytokine and ROS production. Our major results are that up‐regulated DNM1L and mitochondrial fission promoted survival, LC3B‐related autophagy and ROS production in FLSs, factors that lead to inflammation by regulating AKT/IKK/NFKBIA/NF‐κB signalling. Thus, inhibition of DNM1L may be a new strategy for treatment of RA.     

Equol suppresses inflammatory response and bone erosion due to rheumatoid arthritis in mice

Rheumatoid arthritis (RA) is a chronic and systemic autoimmune inflammatory disease. Typical pathological findings of RA include persistent synovitis and bone degradation in the peripheral joints. Equol, a metabolite of the major soybean isoflavone daidzein, shows superior bioactivity than other isoflavones. We investigated the effects of equol administration on inflammatory response and bone erosion in mice with collagen-induced arthritis (CIA). The severity of arthritis symptoms was significantly low in the equol-administered CIA mice. In addition, equol administration improved the CIA-induced bone mineral density decline. In the inflamed area of CIA mice, equol administration suppressed the expression of interleukin-6 and its receptor. Furthermore, equol reduced the expression of genes associated with bone formation inhibition, osteoclast and immature osteoblast specificity and cartilage destruction. These results suggest that equol suppresses RA development and RA-induced bone erosion by regulating inflammation and bone metabolism.     

Toll‐like receptor‐mediated IRE1α activation as a therapeutic target for inflammatory arthritis

In rheumatoid arthritis (RA), macrophage is one of the major sources of inflammatory mediators. Macrophages produce inflammatory cytokines through toll‐like receptor (TLR)‐mediated signalling during RA. Herein, we studied macrophages from the synovial fluid of RA patients and observed a significant increase in activation of inositol‐requiring enzyme 1α (IRE1α), a primary unfolded protein response (UPR) transducer. Myeloid‐specific deletion of the IRE1α gene protected mice from inflammatory arthritis, and treatment with the IRE1α‐specific inhibitor 4U8C attenuated joint inflammation in mice. IRE1α was required for optimal production of pro‐inflammatory cytokines as evidenced by impaired TLR‐induced cytokine production in IRE1α‐null macrophages and neutrophils. Further analyses demonstrated that tumour necrosis factor (TNF) receptor‐associated factor 6 (TRAF6) plays a key role in TLR‐mediated IRE1α activation by catalysing IRE1α ubiquitination and blocking the recruitment of protein phosphatase 2A (PP2A), a phosphatase that inhibits IRE1α phosphorylation. In summary, we discovered a novel regulatory axis through TRAF6‐mediated IRE1α ubiquitination in regulating TLR‐induced IRE1α activation in pro‐inflammatory cytokine production, and demonstrated that IRE1α is a potential therapeutic target for inflammatory arthritis.