Inflammatory Arthritis In Mice Research Articles

Serum amyloid A expression in liver promotes synovial macrophage activation and chronic arthritis via NFAT5.

Nuclear factor of activated T-cells 5 (NFAT5), an osmo-sensitive transcription factor, can be activated by isotonic stimuli, such as infection. It remains unclear, however, whether NFAT5 is required for damage-associated molecular pattern-triggered (DAMP-triggered) inflammation and immunity. Here, we found that several DAMPs increased NFAT5 expression in macrophages. In particular, serum amyloid A (SAA), primarily generated by the liver, substantially upregulated NFAT5 expression and activity through TLR2/4-JNK signalling pathway. Moreover, the SAA-TLR2/4-NFAT5 axis promoted migration and chemotaxis of macrophages in an IL-6- and chemokine ligand 2-dependent (CCL2-dependent) manner in vitro. Intraarticular injection of SAA markedly accelerated macrophage infiltration and arthritis progression in mice. By contrast, genetic ablation of NFAT5 or TLR2/4 rescued the pathology induced by SAA, confirming the SAA-TLR2/4-NFAT5 axis in vivo. Myeloid-specific depletion of NFAT5 also attenuated SAA-accelerated arthritis. Of note, inflammatory arthritis in mice strikingly induced SAA overexpression in the liver. Conversely, forced overexpression of the SAA gene in the liver accelerated joint damage, indicating that the liver contributes to bolstering chronic inflammation at remote sites by secreting SAA. Collectively, this study underscores the importance of the SAA-TLR2/4-NFAT5 axis in innate immunity, suggesting that acute phase reactant SAA mediates mutual interactions between liver and joints and ultimately aggravates chronic arthritis by enhancing macrophage activation.

EPCR deficiency ameliorates inflammatory arthritis in mice by suppressing the activation and migration of T cells and dendritic cells.

Endothelial protein C receptor (EPCR) is highly expressed in synovial tissues of patients with RA, but the function of this receptor remains unknown in RA. This study investigated the effect of EPCR on the onset and development of inflammatory arthritis and its underlying mechanisms. CIA was induced in EPCR gene knockout (KO) and matched wild-type (WT) mice. The onset and development of arthritis was monitored clinically and histologically. T cells, dendritic cells (DCs), EPCR and cytokines from EPCR KO and WT mice, RA patients and healthy controls (HCs) were detected by flow cytometry and ELISA. EPCR KO mice displayed >40% lower arthritis incidence and 50% less disease severity than WT mice. EPCR KO mice also had significantly fewer Th1/Th17 cells in synovial tissues with more DCs in circulation. Lymph nodes and synovial CD4 T cells from EPCR KO mice expressed fewer chemokine receptors CXCR3, CXCR5 and CCR6 than WT mice. In vitro, EPCR KO spleen cells contained fewer Th1 and more Th2 and Th17 cells than WT and, in concordance, blocking EPCR in WT cells stimulated Th2 and Th17 cells. DCs generated from EPCR KO bone marrow were less mature and produced less MMP-9. Circulating T cells from RA patients expressed higher levels of EPCR than HC cells; blocking EPCR stimulated Th2 and Treg cells in vitro. Deficiency of EPCR ameliorates arthritis in CIA via inhibition of the activation and migration of pathogenic Th cells and DCs. Targeting EPCR may constitute a novel strategy for future RA treatment.

An orally active, small-molecule TNF inhibitor that disrupts the homotrimerization interface improves inflammatory arthritis in mice.

Excessive signaling by the proinflammatory cytokine TNF is involved in several autoimmune diseases, including rheumatoid arthritis (RA). However, unlike the approved biologics currently used to treat this and other conditions, commercially available small-molecule inhibitors of TNF trimerization are cytotoxic or exhibit low potency. Here, we report a TNF-inhibitory molecule (TIM) that reduced TNF signaling in vitro and was an effective treatment in a mouse model of RA. The initial lead compound, TIM1, attenuated TNF-induced apoptosis of human and mouse cells by delaying the induction of proinflammatory NF-κB and MAPK signaling and caspase 3- and caspase 8-dependent apoptosis. TIM1 inhibited the secretion of the proinflammatory cytokines IL-6 and IL-8 by disrupting TNF homotrimerization, thereby preventing its association with the TNF receptor. In a mouse model of collagen-induced polyarthritis, the more potent TIM1 analog TIM1c was orally bioavailable and reduced paw swelling, histological indicators of knee joint pathology, inflammatory infiltration of the joint, and the overall arthritis index. Orally delivered TIM1c showed immunological effects similar to those elicited by intraperitoneal injection of the FDA-approved TNF receptor decoy etanercept. Thus, TIM1c is a promising lead compound for the development of small-molecule therapies for the treatment of RA and other TNF-dependent systemic inflammation disorders.

Efficacy of krill oil in the treatment of knee osteoarthritis: a 24-week multicentre randomised double-blind controlled trial

Purpose: Elevated levels of systemic inflammation are common in people with osteoarthritis and predict both pain and structural outcomes. Krill oil has anti-inflammatory properties and reduces severity of inflammatory arthritis in mice by 50% compared to controls (Ierna BMC MSD 2010;11:136). In humans, krill oil reduced knee pain and function in two short, moderate quality randomised controlled trials (RCTs) in people with osteoarthritis. However, evidence from longer trials is lacking. Therefore, the aim of this study was to compare the efficacy of krill oil (2 g/day) versus placebo for treating knee pain in patient with clinical knee osteoarthritis who have significant knee pain and effusion-synovitis.

Lymphatic muscle cells contribute to dysfunction of the synovial lymphatic system in inflammatory arthritis in mice

BackgroundOur previous studies reveal that impaired draining function of the synovial lymphatic vessel (LV) contributes to the pathogenesis of inflammatory arthritis, but the cellular and molecular mechanisms involved are not fully understood.ObjectiveTo investigate the involvement of lymphatic muscle cells (LMCs) in mediating impaired LV function in inflammatory arthritis.MethodsTNF transgenic (TNF-Tg) arthritic mice were used. The structure and function of the LVs that drained the hind limbs were examined by whole-mount immunofluorescence staining, electron microscopy, and near-infrared lymphatic imaging. Primary LMCs were treated with TNF, and the changes in proliferation, apoptosis, and functional gene expression were assessed. The roles of the herbal drug, Panax notoginseng saponins (PNS), in arthritis and LVs were studied.ResultsTNF-Tg mice developed ankle arthritis with age, which was associated with abnormalities of LVs: (1) dilated capillary LVs with few branch points, (2) mature LVs with reduced LMC coverage and draining function, and (3) degenerative and apoptotic appearance of LMCs. TNF caused LMC apoptosis, reduced expression of muscle functional genes, and promoted the production of nitric oxide (NO) by lymphatic endothelial cells (LECs). PNS attenuated arthritis, restored LMC coverage and draining function of mature LVs, inhibited TNF-mediated NO expression, and reduced LMC apoptosis.ConclusionThe impaired draining function of LVs in TNF-Tg mice involves LMC apoptosis. TNF promotes LMC death directly and indirectly via NO production by LECs. PNS attenuates arthritis, improves LVs, and prevents TNF-induced LMC apoptosis by inhibiting NO production of LECs. LMCs contribute to the dysfunction of synovial LVs in inflammatory arthritis.

CD4+ CCR6+ Tcells, but not γδ Tcells, are important for the IL-23R-dependent progression of antigen-induced inflammatory arthritis in mice.

IL‐23 plays an important role in the development of arthritis and the IL‐23 receptor (IL‐23R) is expressed on different types of T cells. However, it is not fully clear which IL‐23R+ T cells are critical in driving T cell‐mediated synovitis. We demonstrate, using knock‐in IL‐23R‐GFP reporter (IL‐23RGFP/+) mice, that CD4+CCR6+ T cells and γδ T cells, but not CD8+ T cells, express the IL‐23R(GFP). During early arthritis, IL‐23R(GFP)+CD4+CCR6+ T cells, but not IL‐23R(GFP)+ γδ T cells, were present in the inflamed joints. IL‐23RGFP/+ mice were bred as homozygotes to obtain IL‐23RGFP/GFP (IL‐23R deficient/IL‐23R−/−) mice, which express GFP under the IL‐23R promotor. Arthritis progression and joint damage were significantly milder in IL‐23R−/− mice, which revealed less IL‐17A+ cells in their lymphoid tissues. Surprisingly, IL‐23R−/− mice had increased numbers of IL‐23R(GFP)+CD4+CCR6+ and CCR7+CD4+CCR6+ T cells in their spleen compared to WT, and IL‐23 suppressed CCR7 expression in vitro. However, IL‐23R(GFP)+CD4+CCR6+ T cells were present in the synovium of IL‐23R−/− mice at day 4. Finally, adoptive transfer experiments revealed that CD4+CCR6+ T cells and not γδ T cells drive arthritis progression. These data suggest that IL‐23R‐dependent T cell‐mediated synovitis is dependent on CD4+CCR6+ T cells and not on γδ T cells.

Increased ILC3s associated with higher levels of IL-1β aggravates inflammatory arthritis in mice lacking phagocytic NADPH oxidase.

The role of redox regulation in immune-mediated arthritis has been previously described. However, the relationship between innate immune cells, including innate lymphoid cells (ILCs) and phagocyte-derived ROS, in this process remains unclear. Here, we characterize ILCs and measure the IL-1 family cytokines along with other cytokines relevant to ILC functions and development in serum-induced arthritic joints in wild type and phagocytic NADPH oxidase (NOX2)-deficient Ncf1-/- mice. We found more severe serum-induced joint inflammation and increased NCR+ ILC3s in inflamed joints of Ncf1-/- mice. Furthermore, in vitro stimulation with IL-1β on Tbet+ ILC1s from joints facilitated their differentiation into ROR-γt+ ILC3s. Moreover, treatment with IL-1 antagonists effectively lowered the proportions of NCR+ ILC3s and IL-17A producing ILC3s in Ncf1-/- arthritic mice and ameliorated the joint inflammation. These results suggest that NOX2 is an essential regulator of ILC transdifferentiation and may mediate this process in a redox-dependent manner through IL-1β production in the inflammatory joint. Our findings shed important light on the role of ILCs in the initiation and progression in tissue inflammation and delineate a novel innate immune cell-mediated pathogenic mechanism through which redox regulation may determine the direction of immune responses in joints.

A novel NOX2 inhibitor attenuates human neutrophil oxidative stress and ameliorates inflammatory arthritis in mice

Neutrophil infiltration plays a significant pathological role in inflammatory diseases. NADPH oxidase type 2 (NOX2) is a respiratory burst oxidase that generates large amounts of superoxide anion (O2•−) and subsequent other reactive oxygen species (ROS). NOX2 is an emerging therapeutic target for treating neutrophilic inflammatory diseases. Herein, we show that 4-[(4-(dimethylamino)butoxy)imino]-1-methyl-1H-benzo[f]indol-9(4H)-one (CYR5099) acts as a NOX2 inhibitor and exerts a protective effect against complete Freund's adjuvant (CFA)-induced inflammatory arthritis in mice. CYR5099 restricted the production of O2•− and ROS, but not the elastase release, in human neutrophils activated with various stimulators. The upstream signaling pathways of NOX2 were not inhibited by CYR5099. Significantly, CYR5099 inhibited NOX2 activity in activated human neutrophils and in reconstituted subcellular assays. In addition, CYR5099 reduced ROS production, neutrophil infiltration, and edema in CFA-induced arthritis in mice. Our findings suggest that CYR5099 is a NOX2 inhibitor and has therapeutic potential for treating neutrophil-dominant oxidative inflammatory disorders.

A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis through its ZnF7 ubiquitin-binding domain.

Deficiency in the deubiquitinating enzyme A20 causes severe inflammation in mice, and impaired A20 function is associated with human inflammatory diseases. A20 has been implicated in negatively regulating NF-κB signalling, cell death and inflammasome activation; however, the mechanisms by which A20 inhibits inflammation in vivo remain poorly understood. Genetic studies in mice revealed that its deubiquitinase activity is not essential for A20 anti-inflammatory function. Here we show that A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis and that this function depends on its zinc finger 7 (ZnF7). We provide genetic evidence that RIPK1 kinase-dependent, RIPK3-MLKL-mediated necroptosis drives inflammasome activation in A20-deficient macrophages and causes inflammatory arthritis in mice. Single-cell imaging revealed that RIPK3-dependent death caused inflammasome-dependent IL-1β release from lipopolysaccharide-stimulated A20-deficient macrophages. Importantly, mutation of the A20 ZnF7 ubiquitin binding domain caused arthritis in mice, arguing that ZnF7-dependent inhibition of necroptosis is critical for A20 anti-inflammatory function in vivo.

Activated protein C targets immune cells and rheumatoid synovial fibroblasts to prevent inflammatory arthritis in mice.

To investigate whether activated protein C (APC), a physiological anticoagulant can inhibit the inflammatory/invasive properties of immune cells and rheumatoid arthritis synovial fibroblasts (RASFs) in vitro and prevent inflammatory arthritis in murine antigen-induced arthritis (AIA) and CIA models. RASFs isolated from synovial tissues of patients with RA, human peripheral blood mononuclear cells (PBMCs) and mouse thymus cells were treated with APC or TNF-α/IL-17 and the following assays were performed: RASF proliferation and invasion by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell invasion assays, respectively; cytokines and signalling molecules using ELISA or western blot; Th1 and Th17 phenotypes in human PBMCs or mouse thymus cells by flow cytometry. The in vivo effect of APC was evaluated in AIA and CIA models. In vitro, APC inhibited IL-1β, IL-17 and TNF-α production, IL-17-stimulated cell proliferation and invasion and p21 and nuclear factor κB activation in RASFs. In mouse thymus cells and human PBMCs, APC suppressed Th1 and Th17 phenotypes. In vivo, APC inhibited pannus formation, cartilage destruction and arthritis incidence/severity in both CIA and AIA models. In CIA, serum levels of IL-1β, IL-6, IL-17, TNF-α and soluble endothelial protein C receptor were significantly reduced by APC treatment. Blocking endothelial protein C receptor, the specific receptor for APC, abolished the early or preventative effect of APC in AIA. APC prevents the onset and development of arthritis in CIA and AIA models via suppressing inflammation, Th1/Th17 phenotypes and RASF invasion, which is likely mediated via endothelial protein C receptor.

Sodium Chloride Aggravates Arthritis via Th17 Polarization.

PurposeSodium chloride (NaCl) has been proposed as a driving factor in autoimmune diseases through the induction of pathogenic CD4+ T helper cells that produce interleukin-17 (Th17 cells). This study investigated the effects of NaCl on inflammatory arthritis in mice and humans.Materials and MethodsCollagen-induced arthritis (CIA) mice were fed a normal or high-salt diet ad libitum, and clinical and histologic features of arthritis were evaluated. The proportion of Th17 cells in the spleens of CIA mice fed a normal or high-salt diet was evaluated by flow cytometry, and the expression of IL-17 in joints and intestines was determined by immunohistochemical staining. We also analyzed the effect of NaCl on Th17 differentiation from peripheral blood monocytes of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and evaluated the contents of sodium and IL-17 in the synovial fluid of RA and OA patients.ResultsNaCl increased murine and human Th17 cell differentiation in a dose-dependent manner. Clinical and histological arthritis was more severe in the high-salt-fed CIA mice, compared to control CIA mice. The proportion of Th17 cells among splenocytes was higher in CIA mice fed a high-salt diet. Expression of synovial and intestinal IL-17 was also higher in high-salt-fed CIA mice. Comparison of synovial fluid between RA patients and OA patients revealed that Na+ and IL-17 were more abundant in RA synovial fluid.ConclusionThis study suggests that NaCl can aggravate arthritis by affecting Th17 differentiation. Accordingly, limiting salt intake may be helpful for treating inflammatory arthritis, such as RA.

GDF11 antagonizes TNF-α-induced inflammation and protects against the development of inflammatory arthritis in mice.

Growth differentiation factor 11 (GDF11), a key member of the TGF-β superfamily, plays critical roles in various medical conditions. Recently, GDF11 was found to suppress the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway and protect against inflammation. This study aimed to investigate the role of GDF11 in the development of rheumatoid arthritis (RA). We demonstrated that GDF11 treatment antagonized TNF-α-induced inflammation in macrophages. Moreover, GDF11 inhibited the development of arthritis in the collagen-induced arthritis and collagen antibody-induced arthritis models. Local gene transfer of GDF11 via adeno-associated virus exerted therapeutic effects, while local knockdown of GDF11 exaggerated inflammation in our collagen-induced arthritis model, as detected by expression levels of inflammatory biomarkers and the destruction of joint structures. Additionally, the results from both in vitro experiments and luciferase reporter gene mouse experiments implied that the NF-κB pathway might play a critical role in the therapeutic effect of GDF11 in RA. This study presents GDF11 as a potential target for the treatment of inflammatory arthritis, including RA.-Li, W., Wang, W., Liu, L., Qu, R., Chen, X., Qiu, C., Li, J., Hayball, J., Liu, L., Chen, J., Wang, X., Pan, X., Zhao, Y. GDF11 antagonizes TNF-α-induced inflammation and protects against the development of inflammatory arthritis in mice.

Role of ADAMTS-12 in Protecting Against Inflammatory Arthritis in Mice By Interacting With and Inactivating Proinflammatory Connective Tissue Growth Factor.

It has been reported that ADAMTS-12 is a susceptibility gene for rheumatoid arthritis (RA) development, and its level is significantly increased in RA patients. In addition, ADAMTS-12 is reported to be required for inflammation in otherwise healthy subjects. This study was undertaken to determine the role of ADAMTS-12 and the underlying mechanisms in the pathogenesis of inflammatory arthritis. The collagen-induced arthritis (CIA) model was established in ADAMTS-12-deficient mice and their control littermates to determine the role of ADAMTS-12 invivo. Micro-computed tomography scanning was used to demonstrate the destruction of the ankle joint; histologic analysis illustrated synovitis, pannus formation, and bone and cartilage destruction; enzyme-linked immunosorbent assay was performed to measure serum levels of inflammatory cytokines; and protein-protein interaction assays were performed to detect the interactions of ADAMTS-12 and its various deletion mutants with connective tissue growth factor (CTGF). Deficiency of ADAMTS-12 led to accelerated inflammatory arthritis in the CIA mouse model. Loss of ADAMTS-12 caused enhanced osteoclastogenesis. In vitro and invivo protein-protein interaction assays demonstrated that ADAMTS-12 bound and processed CTGF, a previously unrecognized substrate of ADAMTS-12. In addition, deletion of ADAMTS-12 enhanced, while overexpression of ADMATS-12 reduced, CTGF-mediated inflammation. Furthermore, ADAMTS-12 regulation of inflammation was largely lost in CTGF-deficient macrophages. Importantly, blocking of CTGF attenuated elevated inflammatory arthritis seen in the ADAMTS-12-deficient CIA mouse model. This study provides evidence that ADAMTS-12 is a critical regulator of inflammatory arthritis and that this is mediated, at least in part, through control of CTGF turnover.

Modulation of Inflammatory Arthritis in Mice by Gut Microbiota Through Mucosal Inflammation and Autoantibody Generation.

Observations of microbial dysbiosis in patients with rheumatoid arthritis (RA) have raised interest in studying microbial-mucosal interactions as a potential trigger of RA. Using the murine collagen-induced arthritis (CIA) model, we undertook this study to test our hypothesis that microbiota modulate immune responses leading to autoimmune arthritis. CIA was induced by immunization of mice with type II collagen (CII) in adjuvant on days 0 and 21, with arthritis appearing on days 23 and 24. Intestinal microbiota were profiled by 16S ribosomal RNA sequencing every 7 days during the course of CIA, and intestinal mucosal changes were evaluated on days 14 and 35. Then, microbiota were depleted either early (7 days before immunization) or late (day 21 after immunization) by administration of broad-spectrum antibiotics. Disease severity, autoantibody and systemic cytokine production, and intestinal mucosal responses were monitored in the setting of microbial reduction. Significant dysbiosis and mucosal inflammation occurred early in CIA, prior to visible arthritis, and continued to evolve during the course of disease. Depletion of the microbiota prior to the induction of CIA resulted in an ~40% reduction in disease severity and in significantly reduced levels of serum inflammatory cytokines and anti-CII antibodies. In intestinal tissue, production of interleukin-17A (IL-17A) and IL-22 was delayed. Unexpectedly, microbial depletion during the late phase of CIA resulted in a >50% decrease in disease severity. Anti-CII antibodies were mildly reduced but were significantly impaired in their ability to activate complement, likely due to altered glycosylation profiles. These data support a model in which intestinal dysbiosis triggers mucosal immune responses that stimulate T and B cells that are key for the development of inflammatory arthritis.

RIgG1 Fc Hexamer Inhibits Antibody-Mediated Autoimmune Disease via Effects on Complement and FcγRs.

Activation of Fc receptors and complement by immune complexes is a common important pathogenic trigger in many autoimmune diseases and so blockade of these innate immune pathways may be an attractive target for treatment of immune complex-mediated pathomechanisms. High-dose IVIG is used to treat autoimmune and inflammatory diseases, and several studies demonstrate that the therapeutic effects of IVIG can be recapitulated with the Fc portion. Further, recent data indicate that recombinant multimerized Fc molecules exhibit potent anti-inflammatory properties. In this study, we investigated the biochemical and biological properties of an rFc hexamer (termed Fc-μTP-L309C) generated by fusion of the IgM μ-tailpiece to the C terminus of human IgG1 Fc. Fc-μTP-L309C bound FcγRs with high avidity and inhibited FcγR-mediated effector functions (Ab-dependent cell-mediated cytotoxicity, phagocytosis, respiratory burst) in vitro. In addition, Fc-μTP-L309C prevented full activation of the classical complement pathway by blocking C2 cleavage, avoiding generation of inflammatory downstream products (C5a or sC5b-9). In vivo, Fc-μTP-L309C suppressed inflammatory arthritis in mice when given therapeutically at approximately a 10-fold lower dose than IVIG, which was associated with reduced inflammatory cytokine production and complement activation. Likewise, administration of Fc-μTP-L309C restored platelet counts in a mouse model of immune thrombocytopenia. Our data demonstrate a potent anti-inflammatory effect of Fc-μTP-L309C in vitro and in vivo, likely mediated by blockade of FcγRs and its unique inhibition of complement activation.

A unique role for galectin-9 in angiogenesis and inflammatory arthritis

BackgroundGalectin-9 (Gal-9) is a mammalian lectin secreted by endothelial cells that is highly expressed in rheumatoid arthritis synovial tissues and synovial fluid. Roles have been proposed for galectins in the regulation of inflammation and angiogenesis. Therefore, we examined the contribution of Gal-9 to angiogenesis and inflammation in arthritis.MethodsTo determine the role of Gal-9 in angiogenesis, we performed human dermal microvascular endothelial cell (HMVEC) chemotaxis, Matrigel tube formation, and mouse Matrigel plug angiogenesis assays. We also examined the role of signaling molecules in Gal-9-induced angiogenesis by using signaling inhibitors and small interfering RNA (siRNA). We performed monocyte (MN) migration assays in a modified Boyden chamber and assessed the arthritogenicity of Gal-9 by injecting Gal-9 into mouse knees.ResultsGal-9 significantly increased HMVEC migration, which was decreased by inhibitors of extracellular signal-regulating kinases 1/2 (Erk1/2), p38, Janus kinase (Jnk), and phosphatidylinositol 3-kinase. Gal-9 HMVEC-induced tube formation was reduced by Erk1/2, p38, and Jnk inhibitors, and this was confirmed by siRNA knockdown. In mouse Matrigel plug assays, plugs containing Gal-9 induced significantly higher angiogenesis, which was attenuated by a Jnk inhibitor. Gal-9 also induced MN migration, and there was a marked increase in MN ingress when C57BL/6 mouse knees were injected with Gal-9 compared with the control, pointing to a proinflammatory role for Gal-9.ConclusionsGal-9 mediates angiogenesis, increases MN migration in vitro, and induces acute inflammatory arthritis in mice, suggesting a novel role for Gal-9 in angiogenesis, joint inflammation, and possibly other inflammatory diseases.

Alteration of the intestinal microbiome characterizes preclinical inflammatory arthritis in mice and its modulation attenuates established arthritis

Perturbations of the intestinal microbiome have been observed in patients with new-onset and chronic autoimmune inflammatory arthritis. However, it is currently unknown whether these alterations precede the development of arthritis or are rather a consequence of disease. Modulation of intestinal microbiota by oral antibiotics or germ-free condition can prevent arthritis in mice. Yet, the therapeutic potential of modulation of the microbiota after the onset of arthritis is not well characterized. We here show that the intestinal microbial community undergoes marked changes in the preclinical phase of collagen induced arthritis (CIA). The abundance of the phylum Bacteroidetes, specifically families S24-7 and Bacteroidaceae was reduced, whereas Firmicutes and Proteobacteria, such as Ruminococcaceae, Lachnospiraceae and Desulfovibrinocaceae, were expanded during the immune-priming phase of arthritis. In addition, we found that the abundance of lamina propria Th17, but not Th1, cells is highly correlated with the severity of arthritis. Elimination of the intestinal microbiota during established arthritis specifically reduced intestinal Th17 cells and attenuated arthritis. These effects were associated with reduced serum amyloid A expression in ileum and synovial tissue. Our observations suggest that intestinal microbiota perturbations precede arthritis, and that modulation of the intestinal microbiota after the onset of arthritis may offer therapeutic opportunities.

The active metabolite of leflunomide, A77 1726, attenuates inflammatory arthritis in mice with spontaneous arthritis via induction of heme oxygenase-1

BackgroundLeflunomide is a low-molecular-weight compound that is widely used in the treatment of rheumatoid arthritis. Although leflunomide is thought to act through the inhibition of the de novo pyrimidine synthesis, the molecular mechanism of the drug remains largely unknown. We investigated the antiarthritis effects and mechanisms of action of the active metabolite of leflunomide, A77 1726, in interleukin-1 receptor antagonist-knockout (IL-1Ra-KO) mice.Methods14- to 15-week-old male IL-1Ra-KO mice were treated with 10 or 30 mg/kg A77 1726 via intraperitoneal injection three times per week for 6 weeks. The effects of A77 1726 on arthritis severities were assessed by clinical scoring and histological analysis. The serum concentrations of IL-1β, tumor necrosis factor-α (TNF-α), and malondialdehyde were measured by enzyme-linked immunosorbent assay. Histologic analysis of the joints was performed using Safranin O, and immunohistochemical staining. The frequencies of interleukin-17-producing CD4+ T (Th17) cells were analyzed by flow cytometry. Heme oxygenase-1 (HO-1) expression in splenic CD4+ T cells isolated from A77 1726-treated arthritis mice were assessed by western blotting.ResultsA77 1726 treatment induced heme oxygenase-1 (HO-1) in Jurkat cells and primary mouse T cells. Interestingly, A77 1726 inhibited Th17 cell differentiation. In vivo, A77 1726 reduced the clinical arthritis severity of histological inflammation and cartilage destruction. The joints isolated from A77 1726-treated mice showed decreased expression of inducible nitric oxide synthase, nitrotyrosine, TNF-α, and IL-1β. The serum levels of TNF-α, IL-1β, and malondialdehyde were also decreased in A77 1726-treated mice. Whereas the number of Th17 cells in spleens was decreased in A77 1726-treated arthritis mice, a significant increase in the number of Treg cells in spleens was observed. Interestingly, HO-1 expression was significantly higher in splenic CD4+ T cells isolated from A77 1726-treated mice compared with those from vehicle-treated mice, whereas HO-1 expression of splenic non-CD4+ T cells did not differ between groups.ConclusionThe inhibitory effects of A77 1726 on joint inflammation and oxidative stress in autoimmune arthritis may be associated with HO-1 induction in CD4+ T cells.

Treatment of inflammatory arthritis via targeting of tristetraprolin, a master regulator of pro-inflammatory gene expression

ObjectivesTristetraprolin (TTP), a negative regulator of many pro-inflammatory genes, is strongly expressed in rheumatoid synovial cells. The mitogen-activated protein kinase (MAPK) p38 pathway mediates the inactivation of TTP via phosphorylation...

Deletion of calponin 2 in macrophages attenuates the severity of inflammatory arthritis in mice.

Calponin is an actin cytoskeleton-associated protein that regulates motility-based cellular functions. Three isoforms of calponin are present in vertebrates, among which calponin 2 encoded by the Cnn2 gene is expressed in multiple types of cells, including blood cells from the myeloid lineage. Our previous studies demonstrated that macrophages from Cnn2 knockout (KO) mice exhibit increased migration and phagocytosis. Intrigued by an observation that monocytes and macrophages from patients with rheumatoid arthritis had increased calponin 2, we investigated anti-glucose-6-phosphate isomerase serum-induced arthritis in Cnn2-KO mice for the effect of calponin 2 deletion on the pathogenesis and pathology of inflammatory arthritis. The results showed that the development of arthritis was attenuated in systemic Cnn2-KO mice with significantly reduced inflammation and bone erosion than that in age- and stain background-matched C57BL/6 wild-type mice. In vitro differentiation of calponin 2-null mouse bone marrow cells produced fewer osteoclasts with decreased bone resorption. The attenuation of inflammatory arthritis was confirmed in conditional myeloid cell-specific Cnn2-KO mice. The increased phagocytotic activity of calponin 2-null macrophages may facilitate the clearance of autoimmune complexes and the resolution of inflammation, whereas the decreased substrate adhesion may reduce osteoclastogenesis and bone resorption. The data suggest that calponin 2 regulation of cytoskeleton function plays a novel role in the pathogenesis of inflammatory arthritis, implicating a potentially therapeutic target.