Experimental Inflammatory Arthritis Research Articles

Baroreflex activation in conscious rats modulates the joint inflammatory response via sympathetic function.

The baroreflex is a critical physiological mechanism controlling cardiovascular function by modulating both the sympathetic and parasympathetic activities. Here, we report that electrical activation of the baroreflex attenuates joint inflammation in experimental arthritis induced by the administration of zymosan into the femorotibial cavity. Baroreflex activation combined with lumbar sympathectomy, adrenalectomy, celiac subdiaphragmatic vagotomy or splenectomy dissected the mechanisms involved in the inflammatory modulation, highlighting the role played by sympathetic inhibition in the attenuation of joint inflammation. From the immunological standpoint, baroreflex activation attenuates neutrophil migration and the synovial levels of inflammatory cytokines including TNF, IL-1β and IL-6, but does not affect the levels of the anti-inflammatory cytokine IL-10. The anti-inflammatory effects of the baroreflex system are not mediated by IL-10, the vagus nerve, adrenal glands or the spleen, but by the inhibition of the sympathetic drive to the knee. These results reveal a novel physiological neuronal network controlling peripheral local inflammation.

Leucine-rich α2 -glycoprotein as a potential biomarker for joint inflammation during anti-interleukin-6 biologic therapy in rheumatoid arthritis.

To investigate whether leucine-rich α2 -glycoprotein (LRG) could be a biomarker for disease activity during interleukin-6 (IL-6) blockade treatment of rheumatoid arthritis (RA). In 59 RA patients who were treated with tocilizumab for 24 weeks, serum LRG levels were determined by enzyme-linked immunosorbent assay. RA disease activity was evaluated by the Clinical Disease Activity Index (CDAI). Receiver operating characteristic (ROC) curve analysis was used to examine the diagnostic performance of LRG and other biomarkers. In monkeys with experimental autoimmune arthritis, swollen joint counts, joint pathologic changes, and blood levels of C-reactive protein (CRP) and LRG were evaluated after treatment with anti-IL-6 receptor antibody. Among tocilizumab-treated RA patients, those with active disease (CDAI >2.8) had significantly higher serum LRG levels compared to those whose disease was in remission. ROC curve analysis suggested that the LRG level was more useful than the CRP or matrix metalloproteinase 3 level or the erythrocyte sedimentation rate in discriminating between remission and active disease during therapy with tocilizumab. In monkeys treated with IL-6 blockade, joint scores were more closely correlated with LRG levels than with CRP levels. Histologic analysis of joints revealed that LRG levels correlated significantly with granulomatous tissue formation, cartilage degeneration, and bone destruction in IL-6 blockade-treated monkeys with low levels of CRP. Under conditions of IL-6 inhibition, LRG was more useful than other biomarkers in discriminating between active and inactive disease in human RA and in detecting joint inflammation in experimental arthritis. LRG may serve as a convenient biomarker for RA disease activity during IL-6 blockade treatment.

Biased agonism as a novel strategy to harness the proresolving properties of melanocortin receptors without eliciting melanogenic effects.

There is a need for novel approaches to control pathologies with overexuberant inflammatory reactions. Targeting melanocortin (MC) receptors represents a promising therapy for obesity and chronic inflammation, but lack of selectivity and safety concerns limit development. A new way to increase selectivity of biological effects entails the identification of biased agonists. In this study, we characterize the small molecule AP1189 as a biased agonist at receptors MC1 and MC3. Although not provoking canonical cAMP generation, AP1189 addition to MC1 or MC3, but not empty vector, transfected HEK293 cells caused ERK1/2 phosphorylation, a signaling responsible for the proefferocytic effect evoked in mouse primary macrophages. Added to macrophage cultures, AP1189 reduced cytokine release, an effect reliant on both MC1 and MC3 as evident from the use of Mc1r(-/-) and Mc3r(-/-) macrophages. No melanogenesis was induced by AP1189 in B16-F10 melanocytes. In vivo, oral AP1189 elicited anti-inflammatory actions in peritonitis and, upon administration at the peak of inflammation, accelerated the resolution phase by ∼3-fold. Finally, given the clinical efficacy of adrenocorticotropin in joint diseases, AP1189 was tested in experimental inflammatory arthritis, where this biased agonist afforded significant reduction of macroscopic and histological parameters of joint disruption. These proof-of-concept analyses with AP1189, an active oral anti-inflammatory and resolution-promoting compound, indicate that biased agonism at MC receptors is an innovative, viable approach to yield novel anti-inflammatory molecules endowed with a more favorable safety profile.

Suppression of experimental arthritis through AMP-activated protein kinase activation and autophagy modulation.

Autophagy plays a central role in various disease processes. However, its contribution to inflammatory arthritides such as rheumatoid arthritis (RA) is unclear. We observed that autophagy is engaged in the K/BxN serum transfer model of RA but autophagic flux is severely impaired. Metformin is an anti-diabetic drug that has been shown to stimulate autophagy. Induction of autophagic flux, through metformin-mediated AMP-activated protein kinase (AMPK) activation and interruption of mammalian target of rapamycin (mTOR) signaling mitigated the inflammation in experimental arthritis. Further investigation into the effects of metformin suggest that the drug directly activates AMPK and dose-dependently suppressed the release of TNF-α, IL-6, and MCP-1 by macrophages while enhancing the release of IL-10 in vitro. In vivo, metformin treatment significantly suppressed clinical arthritis and inflammatory cytokine production. Mechanistic studies suggest that metformin exerts its anti-inflammatory effects by correcting the impaired autophagic flux observed in the K/BxN arthritis model and suppressing NF-κB-mediated signaling through selective degradation of IκB kinase (IKK). These findings establish a central role for autophagy in inflammatory arthritis and argue that autophagy modulators such as metformin may represent potential therapeutic agents for the treatment of RA.

AB0066 Clinical Usefulness of Serum Soluble ST2 in Rheumatoid Arthritis with Anti-TNF-α Therapy

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory disease leading to synovial proliferation as well as cartilage and bone destruction. Cytokine-mediated immunity plays an important role in the pathogenesis of RA....

Protective Effects of C‐Type Natriuretic Peptide on Linear Growth and Articular Cartilage Integrity in a Mouse Model of Inflammatory Arthritis

ObjectiveThe C-type natriuretic peptide (CNP) signaling pathway is a major contributor to postnatal skeletal growth in humans. This study was undertaken to investigate whether CNP signaling could prevent growth delay and cartilage damage in an animal model of inflammatory arthritis.MethodsWe generated transgenic mice that overexpress CNP (B6.SJL-Col2a1-NPPC) in chondrocytes. We introduced the CNP transgene into mice with experimental systemic inflammatory arthritis (K/BxN T cell receptor [TCR]) and determined the effect of CNP overexpression in chondrocytes on the severity of arthritis, cartilage damage, and linear growth. We also examined primary chondrocyte cultures for changes in gene and protein expression resulting from CNP overexpression.ResultsK/BxN TCR mice exhibited linear growth delay (P < 0.01) compared to controls, and this growth delay was correlated with the severity of arthritis. Diminished chondrocyte proliferation and matrix production was also seen in K/BxN TCR mice. Compared to non–CNP-transgenic mice, K/BxN TCR mice with overexpressed CNP had milder arthritis, no growth delay, and less cartilage damage. Primary chondrocytes from mice overexpressing CNP were less sensitive to inflammatory cytokines than wild-type mouse chondrocytes.ConclusionCNP overexpression in chondrocytes can prevent endochondral growth delay and protect against cartilage damage in a mouse model of inflammatory arthritis. Pharmacologic or biologic modulation of the CNP signaling pathway may prevent growth retardation and protect cartilage in patients with inflammatory joint diseases, such as juvenile idiopathic arthritis.

IL-33 in rheumatoid arthritis: Potential role in pathogenesis and therapy

Rheumatoid arthritis (RA) is characterized by chronic inflammatory disease, including synovial proliferation and excessive pro-inflammatory cytokines production, leading to cartilage and bone destruction. Cytokine-mediated immunity plays an important role in the pathogenesis of various autoimmune diseases such as RA. Recently, the IL-1 family member IL-33, was recognized to perform as an inflammatory cytokine, exerted profound effects in human RA and experimental inflammatory arthritis. Furthermore, inhibition of IL-33 signaling proposed a potential therapeutic approach. In this review, we summarize recent advances on the pathological roles of IL-33 in RA and discuss the therapeutic significance of these new findings.

IL-32 with potential insights into rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease, characterized by chronic inflammation in synovial joints. Effective treatment for RA is lacking because the clear etiology and pathogenesis of RA have not been fully elucidated. Cytokine-mediated immunity has been found to play an important role in the pathogenesis of various autoimmune diseases such as RA. Recently, IL-32 is identified with high expression in RA patients and mice models of experimental inflammatory arthritis. IL-32 is recognized to play a crucial role in RA with pro-inflammatory effects. Furthermore, interventions for blocking IL-32 in RA seem possible and applicable. Therefore, targeting IL-32 may give therapeutic potential. In this article, we discuss the biological features of IL-32 and summarize recent advances in understanding the role of IL-32 in disease onset of and treatment for RA. Hopefully the information obtained will benefit for developing novel therapeutic strategies.

Combined treatment with hyaluronan inhibitor Pep-1 and a selective adenosine A2 receptor agonist reduces inflammation in experimental arthritis

Investigations have suggested degradation of native hyaluronan (HA) into small oligosaccharides as being involved in the development and progression of inflammatory diseases, particularly rheumatoid arthritis (RA). Inflammatory responses occur by modulating the TLR4 and 2, and the CD44 natural HA receptor. As reported recently, the adenosine A2 receptor (A2AR) plays an important anti-inflammatory role in arthritis. TLR4, TLR2 and CD44 stimulation activate NF-κB, which stimulates the production of pro-inflammatory cytokines and other mediators. In contrast, A2AR stimulation inhibits NF-κB activation. The aim of this study was to investigate the effect of combined treatment of HA inhibitor Pep-1 and a selective A2AR agonist (CV-1808) in collagen-induced arthritis (CIA) in mice. Arthritis was induced via intradermal injection of bovine collagen-II. Mice were treated with Pep-1 plus CV-1808 intraperitoneally daily for 20 d. CIA increased TLR4, TLR2, CD44 and A2AR mRNA expression and the related proteins in the joint cartilage of arthritic mice, where significantly increased concentrations were of TNF-α, IL-1-β, IL-17, matrix metalloprotease-13 and inducible nitric oxide synthase. Pep-1 with CV-1808 treatment significantly reduced CIA damage and all the up-regulated biochemical parameters. These reductions were supported by microscopic analysis and synovial fluid HA levels.

IL-18 Upregulates the Production of Key Regulators of Osteoclastogenesis from Fibroblast-Like Synoviocytes in Rheumatoid Arthritis

Recent data have demonstrated the importance of IL-18 in the induction and perpetuation of chronic inflammation in experimental arthritis. The aim of the present study was to elucidate whether IL-18 has any indirect effects on osteoclastogenesis by regulating the production of molecules from fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA). Human FLS were isolated from RA synovial tissue and cultured in vitro for three to five passages. The expression of IL-18 receptor was determined by RT-PCR. The levels of soluble receptor activator of nuclear factor κB ligand (RANKL), osteoprotegerin (OPG), macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) in culture supernatants were determined by ELISA. Membrane-bound RANKL expression was analyzed by flow cytometry. Both α and β chains of IL-18 receptor were confirmed in cultured FLS. IL-18 upregulated membrane-bound RANKL expression and soluble RANKL production by FLS in both time- and dose-dependent manners. In addition, IL-18 enhanced production of M-CSF, GM-CSF, and OPG from cultured FLS in a dose-dependent manner. IL-18 also increased the ratio of RANKL/OPG, suggesting that the net effect of IL-18 on FLS favors for the induction of osteoclast formation and bone resorption. In conclusion, IL-18 upregulates the production of key regulators of osteoclastogenesis from FLS in RA.

Peripheral inflammatory disease associated with centrally activated IL-1 system in humans and mice

During peripheral immune activation caused by an infection or an inflammatory condition, the innate immune response signals to the brain and causes an up-regulation of central nervous system (CNS) cytokine production. Central actions of proinflammatory cytokines, in particular IL-1β, are pivotal for the induction of fever and fatigue. In the present study, the influence of peripheral chronic joint inflammatory disease in rheumatoid arthritis (RA) on CNS inflammation was investigated. Intrathecal interleukin (IL)-1β concentrations were markedly elevated in RA patients compared with controls or with patients with multiple sclerosis. Conversely, the anti-inflammatory IL-1 receptor antagonist and IL-4 were decreased in RA cerebrospinal fluid (CSF). Tumor necrosis factor and IL-6 levels in the CSF did not differ between patients and controls. Concerning IL-1β, CSF concentrations in RA patients were higher than in serum, indicating local production in the CNS, and there was a positive correlation between CSF IL-1β and fatigue assessments. Next, spinal inflammation in experimental arthritis was investigated. A marked increase of IL-1β, IL-18, and tumor necrosis factor, but not IL-6 mRNA production, in the spinal cord was observed, coinciding with increased arthritis scores in the KBxN serum transfer model. These data provide evidence that peripheral inflammation such as arthritis is associated with an immunological activation in the CNS in both humans and mice, suggesting a possible therapeutic target for centrally affecting conditions as fatigue in chronic inflammatory diseases, for which to date there are no specific treatments.

Sialylation‐independent mechanism involved in the amelioration of murine immune thrombocytopenia using intravenous gammaglobulin

Sialylation of the N-linked glycan on asparagine-297 within the Fc region of intravenous gammaglobulins (IVIgs) was shown to be necessary for efficacy of IVIg in the amelioration of experimental inflammatory arthritis. To test the role for Fc sialylation of IVIg in immune modulating therapies beyond the K/BxN arthritis model, we examined the efficacy of sialylated compared to nonsialylated IVIg for the ability to attenuate immune thrombocytopenia (ITP) in a mouse model that approximates the clinical setting of human ITP. We used a published, passive anti-platelet (PLT) dose-escalation mouse model of ITP that approximates clinical ITP. PLT counts were followed over time before and after IVIg treatment. IVIg from two different manufacturers was used to prepare untreated and neuraminidase-treated IVIg. Sambucus nigra agglutinin (SNA) affinity chromatography was used to obtain sialic acid-enriched and -depleted IVIg. Sialic acid content was determined using Western blot, enzyme-linked immunosorbent assay, and high-performance liquid chromatography. Results were the same using sialylated and desialylated (neuraminidase-treated) IVIg from two different manufacturers. No differences were observed between sialic acid-enriched and -depleted IVIg compared to normal IVIg in their efficacy to alleviate ITP. Using quantitative reverse transcription-polymerase chain reaction, no increase in the spleen FcγRIIB mRNA was detectable, but a pronounced increase of FcγRIIB mRNA in the marrow was seen after IVIg administration. We conclude that IVIg ameliorates experimental ITP by a mechanism that is independent of sialylation either in the Fc or the Fab region of IVIg.

Cyclin‐dependent kinase inhibitor p21, via its C‐terminal domain, is essential for resolution of murine inflammatory arthritis

The mechanism responsible for persistent synovial inflammation in rheumatoid arthritis (RA) is unknown. Previously, we demonstrated that expression of the cyclin-dependent kinase inhibitor p21 is reduced in synovial tissue from RA patients compared to osteoarthritis patients and that p21 is a novel suppressor of the inflammatory response in macrophages. The present study was undertaken to investigate the role and mechanism of p21-mediated suppression of experimental inflammatory arthritis. Experimental arthritis was induced in wild-type or p21-/- (C57BL/6) mice, using the K/BxN serum-transfer model. Mice were administered p21 peptide mimetics as a prophylactic for arthritis development. Lipopolysaccharide-induced cytokine and signal transduction pathways in macrophages that were treated with p21 peptide mimetics were examined by Luminex-based assay, flow cytometry, or enzyme-linked immunosorbent assay. Enhanced and sustained development of experimental inflammatory arthritis, associated with markedly increased numbers of macrophages and severe articular destruction, was observed in p21-/- mice. Administration of a p21 peptide mimetic suppressed activation of macrophages and reduced the severity of experimental arthritis in p21-intact mice only. Mechanistically, treatment with the p21 peptide mimetic led to activation of the serine/threonine kinase Akt and subsequent reduction of the activated isoform of p38 MAPK in macrophages. These are the first reported data to reveal that p21 has a key role in limiting the activation response of macrophages in an inflammatory disease such as RA. Thus, targeting p21 in macrophages may be crucial for suppressing the development and persistence of RA.

Whole-body deletion of LPS-induced TNF-α factor (LITAF) markedly improves experimental endotoxic shock and inflammatory arthritis

LPS-induced TNF-α factor (LITAF) mediates cytokine expression in response to endotoxin challenge. Previously, we reported that macrophage-specific LITAF-deficient (macLITAF-/-) mice exposed to LPS have a delayed onset in the serum levels of proinflammatory cytokines and prolonged persistence of anti-inflammatory cytokines, but only partial protection from endotoxic shock. We postulated that greater protection might be achieved if LITAF were deleted from all LITAF-producing cells, including macrophages. Using a Cre-loxP system, we engineered a tamoxifen-induced recombination mouse [tamLITAF(i)-/-] that resulted in whole-body LITAF deficiency. Our findings demonstrate that (i) tamLITAF(i)-/- mice are more resistant to systemic Escherichia coli LPS-induced lethality than our previous macLITAF-/- mice, providing evidence that LITAF-producing cells other than LysMCre-positive cells play an important role in mediating endotoxic shock; (ii) tamLITAF(i)-/- mice show a similar pattern of cytokine expression with decreased proinflammatory and prolonged anti-inflammatory mediators compared with WT mice; and (iii) tamLITAF(i)-/- mice, compared with WT mice, display a significant reduction in bone resorption and inflammation associated with a local chronic inflammatory disease--namely, collagen antibody-induced arthritis. Our findings offer a unique model to study the role of LITAF in systemic and chronic local inflammatory processes, and pave the way for anti-LITAF therapeutic approaches for the treatment of TNF-mediated inflammatory diseases.

Regulation of systemic and local myeloid cell subpopulations by bone marrow cell–derived granulocyte–macrophage colony‐stimulating factor in experimental inflammatory arthritis

Even though there are clinical trials assessing granulocyte-macrophage colony-stimulating factor (GM-CSF) blockade in rheumatoid arthritis (RA), questions remain as to how GM-CSF acts as a proinflammatory cytokine. The aims of this study on the regulation of arthritis progression by GM-CSF were to determine the source of the GM-CSF, whether there are systemic effects, the changes in synovial tissue leukocyte populations, and the arthritis model dependence on GM-CSF. Bone marrow chimeras were used to determine the source of GM-CSF required for the development of collagen-induced arthritis (CIA). The K/BxN serum-transfer model of arthritis was tested in GM-CSF(-/-) mice and using anti-GM-CSF monoclonal antibodies. Cell populations from arthritic mice were assessed by differential staining and flow cytometry. In the CIA model, GM-CSF produced by bone marrow-derived cells was required for arthritis development. GM-CSF blockade, while ameliorating the development of CIA, was found to have systemic effects, limiting the increase in circulating Ly-6C(high) monocytes and neutrophils. GM-CSF blockade led to fewer synovial macrophages (both Ly-6C(high) and Ly-6C(low)), neutrophils, and lymphocytes. In the absence of GM-CSF, K/BxN serum-transfer arthritis initially developed normally; however, the numbers of Ly-6C(high) monocytes and synovial macrophages (both Ly-6C(high) and Ly-6C(low)) were again reduced, along with the peak disease severity and maintenance. GM-CSF is a key player in two arthritis models, participating in interactions between hemopoietic cells, both locally and systemically, to control myeloid cell numbers as well as presumably to "activate" them. These results could be useful for the analysis of current clinical trials targeting GM-CSF in patients with RA.

Polyubiquitination Events Mediate Polymethylmethacrylate (PMMA) Particle Activation of NF-κB Pathway

The pathologic response to implant wear-debris constitutes a major component of inflammatory osteolysis and remains under intense investigation. Polymethylmethacrylate (PMMA) particles, which are released during implant wear and loosening, constitute a major culprit by virtue of inducing inflammatory and osteolytic responses by macrophages and osteoclasts, respectively. Recent work by several groups has identified important cellular entities and secreted factors that contribute to inflammatory osteolysis. In previous work, we have shown that PMMA particles contribute to inflammatory osteolysis through stimulation of major pathways in monocytes/macrophages, primarily NF-κB and MAP kinases. The former pathway requires assembly of large IKK complex encompassing IKK1, IKK2, and IKKγ/NEMO. We have shown recently that interfering with the NF-κB and MAPK activation pathways, through introduction of inhibitors and decoy molecules, impedes PMMA-induced inflammation and osteolysis in mouse models of experimental calvarial osteolysis and inflammatory arthritis. In this study, we report that PMMA particles activate the upstream transforming growth factor β-activated kinase-1 (TAK1), which is a key regulator of signal transduction cascades leading to activation of NF-κB and AP-1 factors. More importantly, we found that PMMA particles induce TAK1 binding to NEMO and UBC13. In addition, we show that PMMA particles induce TRAF6 and UBC13 binding to NEMO and that lack of TRAF6 significantly attenuates NEMO ubiquitination. Altogether, these observations suggest that PMMA particles induce ubiquitination of NEMO, an event likely mediated by TRAF6, TAK1, and UBC13. Our findings provide important information for better understanding of the mechanisms underlying PMMA particle-induced inflammatory responses.

Hyaluronan reduces inflammation in experimental arthritis by modulating TLR-2 and TLR-4 cartilage expression

Previous studies have reported that low molecular mass HA and highly polymerized HA respectively elicited pro- and anti-inflammatory responses by modulating the toll-like receptor 4 (TLR-4) and the TLR-2. The activation of TLR-4 and TLR-2 mediated by collagen-induced arthritis (CIA) induces the myeloid differentiation primary response protein (MyD88) and the tumor necrosis factor receptor-associated factor 6 (TRAF6), and ends with the liberation of NF-kB which, in turn, stimulates pro-inflammatory cytokine production. The aim of this study was to investigate the influence of high molecular weight HA at different concentrations on TLR-4 and TLR-2 modulation in CIA in mice. Arthritis was induced in mice via intradermal injection of an emulsion containing bovine type II collagen in complete Freund's adjuvant. Mice were treated with HA intraperitoneally daily for 30 days. CIA increased TLR-4, TLR-2, MyD88 and TRAF6 mRNA expression and the related protein in the cartilage of arthritic joints. High levels of both mRNA and related protein were also detected for tumor necrosis factor alpha (TNF-α), interleukin 1-beta (IL-1-β), interleukin-17 (IL-17), matrix metalloprotease-13 (MMP-13) and inducible nitric oxide synthase (iNOS) in the joint of arthritic mice. HA treatment significantly limited CIA incidence and decreased all the parameters up-regulated by CIA. The improvement of biochemical parameters was also supported by histological analysis, plasma and synovial fluid HA levels. These results suggest that the TLR-4 and TLR-2 play an important role in the arthritis mechanism and the interaction/block of HA at high molecular mass may reduce inflammation and cartilage injury.

Skin fibroblasts are potent suppressors of inflammation in experimental arthritis

ObjectivesMesenchymal stromal cells (MSC) are characterised by their capacity to suppress immune reactions. This function was reported to be shared in vitro by fibroblasts but their role has been poorly...

A novel Saa3-promoter reporter distinguishes inflammatory subtypes in experimental arthritis and human synovial fibroblasts

ObjectiveTo evaluate the applicability of a lentiviral (LV) serum amyloid A3 (Saa3)-promoter luciferase (Luc) reporter for assessing inflammation in experimental arthritis, synovial fibroblasts (SF) from osteoarthritis (OA) and rheumatoid arthritis...

Skin fibroblasts are potent suppressors of inflammation in experimental arthritis

Background and objectivesFibroblasts possess in vitro immunomodulatory properties that are similar to those of multipotent mesenchymal stromal cells (MSC) but their role has been poorly investigated in vivo. Here, we...