Co-expression of IL-18 binding protein and IL-4 regulates Th1/Th2 cytokine response in murine collagen-induced arthritis

We constructed a recombinant adenoviral vector containing a murine interleukin （IL）-18 binding protein （mlL-18BP） and murine IL-4 （mIL-4） fusion gene （AdmIL-18BP/mIL.4） and used a gene therapy approach to investigate the role of IL-18BP and IL-4 in modulating the T-helperl and T-helper2 （Th1/Th2） balance in mice with collagen-induced arthritis （CIA）. Mice with CIA were intra-articularly injected with 107 pfu/6 μl ofeitherAdmIL.18BP/mIL-4, or a controladenovirus, or with the control vehicle （phosphate-buffered saline）. After intra-articular gene therapy with AdmIL-18BP/mIL-4, the serum levels of tumor necrosis factor-α （TNF-α）, T-interferon （IFN-γ）, IL-4, IL-10, and IL-18 in mice with CIA were assessed by ELISA. IFN-T-expressing and IL-4-expressing CD4^＋ T cells from mice splenocytes were monitored by flow cytometry. Mice with CIA at weeks 1, 2, and 4 after intraarticular injection of AdmIL-18BP/mIL-4 showed significantly increased serum concentrations of IL-4 and IL-10 （P〈0.01 at all time points） but greatly decreased serum concentrations ofIFN-γ, TNF-α and IL-β （P〈0.01 at all time points ） compared to both the con trol adenovirus and phospha tebuffered saline control groups. The percentage of LFN-γ- producing CD4^＋ T cells was significantly decreased in response to local AdmIL-18BP/mIL-4 treatment. The percentage of IL-4-producing CD4^＋ T cells increased significantly at 1 week after local injection of AdmIL-18BP/ mIL-4 then returned to normal by week 4. These data indicated the significant modifying effects on the Th1/Th2 imbalance in murine CIA produced by local overexpression of IL-18BP and IL-4. Combination treatment with IL-18BP and IL-4 is a promising potential therapy for rheumatoid arthritis.     

IL-17B and IL-17C are associated with TNF-alpha production and contribute to the exacerbation of inflammatory arthritis

IL-17A is a T cell-derived proinflammatory cytokine that contributes to the pathogenesis of rheumatoid arthritis. Recently, six related molecules have been identified to form the IL-17 family, as follows: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. Whereas IL-17A and IL-17F up-regulate IL-6 in synovial fibroblasts, IL-17B and IL-17C are reported to stimulate the release of TNF-alpha and IL-1beta from the monocytic cell line, THP-1 cell. However, their detailed function remains to be elucidated. We report in this study the effects of IL-17 family on the collagen-induced arthritis (CIA) progression by T cell gene transfer and bone marrow chimeric mice. The mRNA expressions of IL-17 family (IL-17A, IL-17B, IL-17C, and IL-17F) and their receptor (IL-17R and IL-17Rh1) genes in the arthritic paws of CIA mice were elevated compared with controls. Although IL-17A and IL-17F were expressed in CD4(+) T cells, IL-17B and IL-17C were expressed in the cartilage and in various cell populations in the CIA arthritic paws, respectively. In vitro, IL-17A, IL-17B, IL-17C, and IL-17F induced TNF-alpha production in mouse peritoneal exudate cells. In vivo, adoptive transfer of IL-17B- and IL-17C-transduced CD4(+) T cells evidently exacerbated arthritis. Bone marrow chimeric mice of IL-17B and IL-17C exhibited elevated serum TNF-alpha concentration and the high arthritis score upon CIA induction. Moreover, neutralization of IL-17B significantly suppressed the progression of arthritis and bone destruction in CIA mice. Therefore, not only IL-17A, but also IL-17B and IL-17C play an important role in the pathogenesis of inflammatory arthritis.     

IL-10-deficient B10.Q mice develop more severe collagen-induced arthritis, but are protected from arthritis induced with anti-type II collagen antibodies.

IL-10 is a pleiotropic cytokine with stimulatory and inhibitory properties, and is thought to have a protective role in rheumatoid arthritis and collagen-induced arthritis (CIA). In this study, we investigated how IL-10 deficiency affects CIA and anti-collagen type II (CII) Ab-transferred arthritis in C57BL/10.Q (B10.Q) mice. The B10.Q.IL-10(-/-) mice had an 8-cM 129/Ola fragment around the IL-10 gene. The mice were treated with antibiotics, appeared healthy, and had no colitis. T cells from IL-10(-/-) mice expressed similar levels of IFN-gamma, IL-2, and IL-4 after mitogen stimulation; however, macrophages showed a reduced TNF-alpha production compared with IL-10(+/-) littermates. IL-10(-/-) mice had an increased incidence, and a more severe CIA disease than the IL-10(+/-) littermates. To study the role of IL-10 in T cell tolerance, IL-10(-/-) were crossed into mice carrying the immunodominant epitope, CII(256-270), in cartilage (MMC) or in skin (TSC). Both IL-10(-/-) and IL-10(+/-) MMC and TSC mice were completely tolerized against CIA, indicating that lack of IL-10 in this context did not break tolerance. To investigate whether IL-10 was important in the effector phase of CIA, arthritis was induced with anti-CII Abs. Surprisingly, IL-10(-/-) were less susceptible to Ab-transferred arthritis, as only 30% showed signs of disease compared with 90% of the littermates. Therefore, IL-10 seemed to have a protective role in CIA, but seemed to exacerbate the arthritogenicity of anti-CII Abs. These data emphasize the importance of studying IL-10 in a defined genetic context in vivo, to understand its role in a complex disease like arthritis.     

A comparative study on intra-articular versus systemic gene electrotransfer in experimental arthritis

BACKGROUND: Electric pulse mediated gene transfer has been applied successfully in vivo for increasing naked DNA administration in various tissues. To achieve non-viral gene transfer into arthritic joint tissue, we investigated the use of electrotransfer (ET). Because anti-inflammatory cytokine strategies have proven efficient in experimental models of arthritis, we compared the therapeutic efficiency of local versus systemic delivery of the interleukin-10 (IL-10) using in vivo ET. METHODS: A plasmid vector expressing IL-10 was transferred into DBA/1 mouse knee joints by ET with 12 pulses of variable duration and voltage. The kinetics of transgene expression were analyzed by specific enzyme-linked immunosorbent assay (ELISA) in sera and knees. Optimal conditions were then used to deliver increasing amounts of IL-10 plasmid intra-articularly (i.a.) in the collagen-induced arthritis (CIA) mouse model. The therapeutic efficiency was compared with the potency of intra-muscular (i.m.) ET. RESULTS: Following i.a. ET, local IL-10 secretion peaked on day 7 and dropped 2 weeks after. A second ET produced the same kinetics without enhancing gene transfer efficiency, while transgene was still detected in injected muscles 4 weeks after ET. Only the i.m. ET of 25 microg of IL-10 significantly inhibited all the clinical and biological features of arthritis. The i.a. ET only showed mild improvement of arthritis when 100 microg of IL-10 plasmid were electrotransfered weekly from day 18 following arthritis induction. CONCLUSIONS: The present results suggest that gene transfer into arthritic joints by ET is an effective means to deliver anti-inflammatory cytokines. However, short duration of transgene expression impedes a significant effect for the treatment of arthritis, making i.m. ET more potent than i.a. ET for clinical benefit in CIA.     

Electrotransfer of human IL-1Ra into skeletal muscles reduces the incidence of murine collagen-induced arthritis

BACKGROUND: It has previously been demonstrated that high levels of gene expression in skeletal muscles can be achieved after direct in vivo electrotransfer of naked plasmid DNA. The purpose of this study is to examine the potential of in vivo electroporation of plasmid DNA encoding human IL-1Ra for the prevention of murine collagen-induced arthritis (CIA). METHODS: DBA/1 mice were injected in gastrocnemius muscles with plasmid DNA followed by in vivo electroporation. To uncover the optimum conditions of gene transfer, various electric field strengths and different amounts of plasmid DNA were applied. Calf muscles around the injected areas were investigated with histological methods for damage to muscle tissue. The levels of human IL-1Ra expression in the injected area and also in the serum were determined with ELISA for human IL-1Ra. Based on these data, the effects of electrotransfer of plasmid DNA were tested using the murine CIA model. DBA/1 mice were immunized with bovine collagen type II at the base of the tail. On day 21, mice were given a booster injection with the same antigen. Mice were divided into two groups on day 26. One group of mice received plasmid containing the IL-1Ra cDNA sequence, while control mice were given plasmid lacking the IL-1Ra coding sequence. The incidence of arthritis was evaluated by macroscopic analysis, histological analysis, and the levels of inflammatory cytokines. RESULTS: IL-1Ra expression increased as a function of the electrical field strength and the amount of DNA. 200 V/cm (eight pulses; 20 ms per pulse; 1 Hz) and 15 microg of plasmid DNA per mouse were found to be optimum for gene transfer. After in vivo electroporation, gene expression in both muscle and serum increased gradually, reaching a peak value on day 10. Significant levels of human IL-1Ra expression were maintained for 20 days. Macroscopic analysis showed that the onset of CIA was significantly inhibited by direct electrotransfer of plasmid DNA encoding human IL-1Ra. Histological analysis of knee joints showed that the incidence of arthritis in knee joints was also prevented. The levels of mouse IL-1beta and IL-12 in paws were significantly lower in the group treated with IL-1Ra than those in the control group. CONCLUSIONS: These results demonstrate that direct electrotransfer of plasmid containing the human IL-1Ra cDNA sequence to skeletal muscle can reduce the incidence of CIA in mice.     

IL-1 alpha beta blockade prevents cartilage and bone destruction in murine type II collagen-induced arthritis, whereas TNF-alpha blockade only ameliorates joint inflammation.

Anti-TNF-alpha treatment of rheumatoid arthritis patients markedly suppresses inflammatory disease activity, but so far no tissue-protective effects have been reported. In contrast, blockade of IL-1 in rheumatoid arthritis patients, by an IL-1 receptor antagonist, was only moderately effective in suppressing inflammatory symptoms but appeared to reduce the rate of progression of joint destruction. We therefore used an established collagen II murine arthritis model (collagen-induced arthritis(CIA)) to study effects on joint structures of neutralization of either TNF-alpha or IL-1. Both soluble TNF binding protein and anti-IL-1 treatment ameliorated disease activity when applied shortly after onset of CIA. Serum analysis revealed that early anti-TNF-alpha treatment of CIA did not decrease the process in the cartilage, as indicated by the elevated COMP levels. In contrast, anti-IL-1 treatment of established CIA normalized COMP levels, apparently alleviating the process in the tissue. Histology of knee and ankle joints corroborated the finding and showed that cartilage and joint destruction was significantly decreased after anti-IL-1 treatment but was hardly affected by anti-TNF-alpha treatment. Radiographic analysis of knee and ankle joints revealed that bone erosions were prevented by anti-IL-1 treatment, whereas the anti-TNF-alpha-treated animals exhibited changes comparable to the controls. In line with these findings, metalloproteinase activity, visualized by VDIPEN production, was almost absent throughout the cartilage layers in anti-IL-1-treated animals, whereas massive VDIPEN appearance was found in control and sTNFbp-treated mice. These results indicate that blocking of IL-1 is a cartilage- and bone-protective therapy in destructive arthritis, whereas the TNF-alpha antagonist has little effect on tissue destruction.     

A complement C3 inhibitor specifically targeted to sites of complement activation effectively ameliorates collagen-induced arthritis in DBA/1J mice

Collagen-induced arthritis (CIA) represents an animal model of autoimmune polyarthritis with similarities to human rheumatoid arthritis, and therapy with various systemic complement-inhibitory proteins has been investigated in this model with varying results. We investigated the use of complement receptor 2 (CR2)-Crry, a complement inhibitor with the ability to target C3 breakdown products deposited in a rheumatic joint. Following induction of CIA in DBA/1J mice, animals were treated with either PBS or CR2-Crry (every other day, every 4 days, or with a single injection). The severity of clinical disease was significantly reduced in all CR2-Crry-treated groups compared with controls. Joints from mice receiving multiple doses of CR2-Crry showed significantly decreased inflammatory cell infiltrate, cartilage damage, pannus formation, and bone damage. CR2-Crry treatment also significantly decreased production of anti-collagen IgG and the inflammatory cytokines TNF-alpha and IL-1beta. IL-10 and IL-1Ra levels were increased in CR2-Crry-treated mice. CR2-Crry localized preferentially in the joints of mice with CIA. Analysis of IgG and C3 deposition in the joints of treated animals indicated that both complement regulation and the modulation of anti-collagen Ab production contributed to the protective effects of CR2-Crry. Of interest, a previous study reported that Crry-Ig, an untargeted counterpart of CR2-Crry, had minimal effect on disease, even when administered at a sufficiently high dose to maintain chronic complement inhibition.     

A proinflammatory role for Fas in joints of mice with collagen-induced arthritis

Collagen-induced arthritis (CIA) is a chronic inflammatory disease bearing all the hallmarks of rheumatoid arthritis, e.g. polyarthritis, synovitis, and subsequent cartilage/bone erosions. One feature of the disease contributing to joint damage is synovial hyperplasia. The factors responsible for the hyperplasia are unknown; however, an imbalance between rates of cell proliferation and cell death (apoptosis) has been suggested. To evaluate the role of a major pathway of cell death – Fas (CD95)/FasL – in the pathogenesis of CIA, DBA/1J mice with a mutation of the Fas gene (lpr) were generated. The susceptibility of the mutant DBA-lpr/lpr mice to arthritis induced by collagen type II was evaluated. Contrary to expectations, the DBA-lpr/lpr mice developed significantly milder disease than the control littermates. The incidence of disease was also significantly lower in the lpr/lpr mice than in the controls (40% versus 81%; P < 0.05). However DBA-lpr/lpr mice mounted a robust immune response to collagen, and the expression of local proinflammatory cytokines such as, e.g., tumor necrosis factor α (TNF-α) and IL-6 were increased at the onset of disease. Since the contribution of synovial fibroblasts to inflammation and joint destruction is crucial, the potential activating effect of Fas on mouse fibroblast cell line NIH3T3 was investigated. On treatment with anti-Fas in vitro, the cell death of NIH3T3 fibroblasts was reduced and the expression of proinflammatory cytokines TNF-α and IL-6 was increased. These findings suggest that impairment of immune tolerance by increased T-cell reactivity does not lead to enhanced susceptibility to CIA and point to a role of Fas in joint destruction.     

Interleukin-35 (IL-35) inhibits proliferation and promotes apoptosis of fibroblast-like synoviocytes isolated from mice with collagen-induced arthritis

Rheumatoid arthritis (RA) is an inflammatory disorder of the joints that affects 0.5–1 % of adults. Excessive growth of the fibroblast-like synoviocytes (FLS) promotes hyperplasia of synovial tissues and causes its invasion into the bone and cartilage, which eventually causes deformity and dysfunction of affected joints. Interleukin 35 (IL-35) was shown to suppress the inflammatory responses to collagen-induced arthritis (CIA) via upregulation of T regulatory cells and suppression of T helper type 17 cells in a mouse model. To study the effects of IL-35 on the proliferation and apoptosis frequency of cultured FLS isolated from mice with CIA as well as to examine the effects of IL-35 on CIA in vivo. Thirty DBA/1 J mice, which are used as an animal model for RA, were divided randomly (ten mice per group) to a CIA group (collagen treatment), a CIA + IL-35 group (collagen and IL-35 treatments), and a control group (no treatment). Starting on the 24th day after collagen administration, IL-35 was injected intraperitoneally into mice of the CIA + IL-35 group once per day for 10 days. An arthritis index was calculated, and pathological analysis of synovial tissue was performed. FLS isolated from CIA mice were treated with various concentrations of IL-35 (12.5–100 ng/ml). The MTT assay was used to examine FLS proliferation, and apoptosis frequency of FLS was detected by flow cytometry. On day 24, the CIA mice began to exhibit arthritis symptoms, and the symptoms rapidly progressed with time. Treatment with IL-35 significantly alleviated arthritis symptoms and reduced the synovial tissue inflammation. In addition, IL-35 treatment inhibited proliferation and promoted apoptosis in cultured FLS from CIA mice in a dose-dependent manner. IL-35 could ameliorate the symptoms of arthritis in the CIA mouse model in vivo and inhibited FLS proliferation while promoting FLS apoptosis in vitro, thereby exhibited the potential in inhibiting the progression of RA.     

Viral IL-10 and soluble TNF receptor act synergistically to inhibit collagen-induced arthritis following adenovirus-mediated gene transfer

Viral IL-10 (vIL-10) and soluble TNF receptor (sTNFR) are anti-inflammatory proteins that can suppress collagen-induced arthritis (CIA). These and related proteins have shown efficacy in the treatment of human rheumatoid arthritis; however, neither alone is able to completely suppress disease. Furthermore, they have short half-lives, necessitating frequent administration. To determine the ability of these proteins to act synergistically following gene transfer, arthritis was induced in DBA/1 male mice by immunization with type II collagen on days 0 and 21. Mice were injected i.v. either before disease onset (day 20) or after disease onset (day 28) with 1010 particles of adenovirus encoding vIL-10, a soluble TNF receptor-IgG1 fusion protein (sTNFR-Ig), a combination of both vectors, or a control vector lacking a transgene. Significant synergism was observed with the combination of vIL-10 and sTNFR-Ig, with a substantial reduction in both the incidence and severity of disease as well as inhibition of progression of established disease. sTNFR-Ig alone had no effect on CIA. vIL-10 alone inhibited disease when given before disease onset, but had minimal effect on established disease. Both proteins inhibited spleen cell proliferation and IFN-gamma secretion in response to stimulation with type II collagen, but only vIL-10 reduced the synovial mRNA levels of the proinflammatory cytokines IL-1beta, TNF-alpha, and IL-6. These findings demonstrate that vIL-10 and sTNFR-Ig act synergistically in suppressing CIA and suggest that gene transfer offers a potential therapeutic modality for the treatment of arthritis.     

Bone-Targeting Endogenous Secretory Receptor for Advanced Glycation End Products Rescues Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory synovitis that leads to the destruction of bone and cartilage. The receptor for advanced glycation end products (RAGE) is a multiligand membrane-bound receptor for high-mobility group box-1 (HMGB1) associated with development of RA by inducing production of proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1 and IL-6. We developed a bone-targeting therapeutic agent by tagging acidic oligopeptide to a nonmembrane-bound form of RAGE (endogenous secretory RAGE [esRAGE]) functioning as a decoy receptor. We assessed its tissue distribution and therapeutic effectiveness in a murine model of collagen-induced arthritis (CIA). Acidic oligopeptide–tagged esRAGE (D₆-esRAGE) was localized to mineralized region in bone, resulting in the prolonged retention of more than 1 wk. Weekly administration of D₆-esRAGE with a dose of 1 mg/kg to RA model mice significantly ameliorated inflammatory arthritis, synovial hyperplasia, cartilage destruction and bone destruction, while untagged esRAGE showed little effectiveness. Moreover, D₆-esRAGE reduced plasma levels of proinflammatory cytokines including TNF-α, IL-1 and IL-6, while esRAGE reduced the levels of IL-1 and IL-6 to a lesser extent, suggesting that production of IL-1 and IL-6 reduced along the blockade of HMGB1 receptor downstream signals by D₆-esRAGE could be attributed to remission of CIA. These findings indicate that D₆-esRAGE enhances drug delivery to bone, leading to rescue of clinical and pathological lesions in murine CIA.     

Heterogeneous effects of IL-2 on collagen-induced arthritis

IL-2 is generally considered a pro-inflammatory cytokine that exacerbates Th1-mediated disease states, such as autoimmune arthritis. Consistent with this role for IL-2, recent studies from our laboratory demonstrate that IL-2 mRNA is markedly increased during the acute stage of collagen-induced arthritis (CIA), an animal model of rheumatoid arthritis. To further define the role of IL-2 in CIA, the levels of IL-2 protein and its receptor and the effects of IL-2 administration were analyzed during CIA. IL-2 protein and IL-2R were preferentially expressed at disease onset, compared with later stages of disease. Administration of recombinant human IL-2 (rhIL-2) at, or just before, disease onset exacerbated disease; surprisingly, rhIL-2 given before disease onset inhibited CIA, associated with reduced cellular and humoral responses to type II collagen. Determination of in vivo serum levels of Th1 and Th2 cytokines in response to rhIL-2 treatment demonstrated that IFN-gamma, but not IL-4, was markedly up-regulated in response to IL-2. In mice treated with anti-IFN-gamma Ab, both early and late IL-2 administration exacerbated CIA. Thus, IL-2 can have two opposite effects on autoimmune arthritis, a direct stimulatory effect and an indirect suppressive effect that is mediated by IFN-gamma.     

Genome Edited Sirt1-Overexpressing Human Mesenchymal Stem Cells Exhibit Therapeutic Effects in Treating Collagen-Induced Arthritis

Even though mesenchymal stem cells (MSCs) are known for cartilage regeneration, their therapeutic efficacy needs to be enhanced. In the present study, we produced genome-edited silent information regulator 2 type 1 (Sirt1)-overexpressing MSCs, and evaluated their therapeutic potential in a damaged cartilage mouse liver fibrosis model. The Sirt1 gene was successfully inserted into a ‘safe harbor’ genomic locus in amniotic mesenchymal stem cells (AMMs), and the chondrogenic properties of the Sirt1 gene overexpressing AMMs (AMM/S) were characterized using quantitative PCR and histology. Therapeutic potentials were investigated in a collagen-induced arthritis (CIA) mouse model. Chondrocyte-differentiated AMM/S expressed cartilage-specific genes and were positive for Safranin O staining. Transplantation of AMM/S attenuated CIA progression and suppressed T helper (Th)-17 cell activation while increasing the Treg cell population in CIA mice. Pro-inflammatory factors, such as interleukin (IL)-1β, IL-6, monocyte chemoattractant protein (MCP)-1, and tumor necrosis factor (TNF)-α were significantly decreased in AMM/S-injected joint tissues. In conclusion, genome-edited AMM/S may represent a safe and alternative therapeutic option for the treatment and repair of damaged cartilage, or in inflammatory joint arthritis.     

IL-23 Dependent and Independent Stages of Experimental Arthritis: No Clinical Effect of Therapeutic IL-23p19 Inhibition in Collagen-induced Arthritis

IL-23p19 deficient mice have revealed a critical role of IL-23 in the development of experimental autoimmune diseases, such as collagen-induced arthritis (CIA). Neutralizing IL-23 after onset of CIA in rats has been shown to reduce paw volume, but the effect on synovial inflammation and the immunological autoimmune response is not clear. In this study, we examined the role of IL-23 at different stages of CIA and during T cell memory mediated flare-up arthritis with focus on changes in B cell activity and Th1/Th17 modulation. Anti-IL-23p19 antibody (anti-IL23p19) treatment, starting 15 days after the type II collagen (CII)-immunization but before clinical signs of disease onset, significantly suppressed the severity of CIA. This was accompanied with significantly lower CII-specific IgG1 levels and lower IgG2a levels in the anti-IL-23p19 treated mice compared to the control group. Importantly, neutralizing IL-23 after the first signs of CIA did not ameliorate the disease. This was in contrast to arthritic mice that underwent an arthritis flare-up since a significantly lower disease score was observed in the IL-23p19 treated mice compared to the control group, accompanied by lower synovial IL-17A and IL-22 expression in the knee joints of these mice. These data show IL-23-dependent and IL-23-independent stages during autoimmune CIA. Furthermore, the memory T cell mediated flare-up arthritis is IL-23-mediated. These data suggest that specific neutralization of IL-23p19 after onset of autoimmune arthritis may not be beneficial as a therapeutic therapy for patients with rheumatoid arthritis (RA). However, T cell mediated arthritis relapses in patients with RA might be controlled by anti-IL-23p19 treatment.     

Inhibition of synovitis and joint destruction by a new single domain antibody specific for cyclophilin A in two different mouse models of rheumatoid arthritis

Introduction

Cyclophilin A (CypA) is implicated in rheumatoid arthritis (RA) pathogenesis. We studied whether a novel anti-CypA single domain antibody (sdAb) treatment would modulate the severity of the disease in two different animal models of RA.

Methods

A novel sdAb, named sdAbA1, was screened from an immunized camel sdAb library and found to have a high binding affinity (K_D = 6.9 × 10^-9 M) for CypA. The SCID-HuRAg model and the collagen-induced arthritis (CIA) in mice were used to evaluate the effects of sdAbA1 treatment on inflammation and joint destruction. For in vitro analysis, monocytes/macrophages were purified from synovial fluid and peripheral blood of patients with RA and were tested for the effect of anti-CypA sdAb on metalloproteinase (MMP) production. Human monocyte cell line THP-1 cells were selected and western blot analyses were performed to examine the potential signaling pathways.

Results

In the CIA model of RA, the sdAbA1 treatment resulted in a significant decrease in clinical symptoms as well as of joint damage (P <0.05). In the SCID-HuRAg model, treatment with anti-CypA antibody sdAbA1 significantly reduced cartilage erosion, inflammatory cell numbers and MMP-9 production in the implanted tissues (P <0.05). It also significantly reduced the levels of human inflammatory cytokines IL-6 and IL-8 in mouse serum (P <0.05). No toxic effects were observed in the two animal models. In vitro results showed that sdAbA1 could counteract CypA-dependent MMP-9 secretion and IL-8 production by interfering with the ERK-NF-κB pathway.

Conclusions

Blockade of CypA significantly inhibited synovitis and cartilage/bone erosion in the two tested animal models of RA. Our findings provide evidence that sdAbA1 may be a potential therapeutic agent for RA.     

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.     

Eosinophils attenuate arthritis by inducing M2 macrophage polarization via inhibiting the IκB/P38 MAPK signaling pathway

Rheumatoid arthritis (RA) represents a type of autoimmune disease that mainly affect the joints due to persistent synovitis. Eosinophils were Th2 effector cells that have been shown to have anti-inflammatory role recently. In this study, we aimed to investigate the effects of eosinophils transfer on arthritis and underlying mechanisms. DBA/1 mice were induced with collagen-induced arthritis (CIA) and treated with purified eosinophils at different time points. We showed that eosinophils transfer attenuated arthritis in CIA mice. Meanwhile, TNF-α, IL-6, IL-12 and iNOS levels were decreased whereas TGF-β, IL-10, IL-13 and Arg1 levels were increased after eosinophil transfer. In vitro stimulation of bone marrow-derived macrophage (BMDM) with LPS and IFN-γ induced high expression of CD68, iNOS, TNF-α, IL-6, and IL-12, while treatment with eosinophils downregulated their expression levels. Furthermore, high levels of p-IκB and p-P38 expression in BMDM induced by LPS and IFN-γ could be suppressed by eosinophil treatment, and a P38 or IκB inhibitor accelerated the effect of eosinophils on macrophage polarization. Our results demonstrate that eosinophils exert anti-inflammatory effects in arthritis by inducing M2 macrophage polarization via inhibiting the IκB/P38 MAPK signaling pathway.