Distinct Roles of Interferon Alpha and Beta in Controlling Chikungunya Virus Replication and Modulating Neutrophil-Mediated Inflammation.

Type I interferons (IFNs) are key mediators of the innate immune response. Although members of this family of cytokines signal through a single shared receptor, biochemical and functional variation exists in response to different IFN subtypes. While previous work has demonstrated that type I IFNs are essential to control infection by chikungunya virus (CHIKV), a globally emerging alphavirus, the contributions of individual IFN subtypes remain undefined. To address this question, we evaluated CHIKV pathogenesis in mice lacking IFN-ß (IFN-ß knockout [IFN-ß-KO] mice or mice treated with an IFN-ß-blocking antibody) or IFN-α (IFN regulatory factor 7 knockout [IRF7-KO] mice or mice treated with a pan-IFN-α-blocking antibody). Mice lacking either IFN-α or IFN-ß developed severe clinical disease following infection with CHIKV, with a marked increase in foot swelling compared to wild-type mice. Virological analysis revealed that mice lacking IFN-α sustained elevated infection in the infected ankle and in distant tissues. In contrast, IFN-ß-KO mice displayed minimal differences in viral burdens within the ankle or at distal sites and instead had an altered cellular immune response. Mice lacking IFN-ß had increased neutrophil infiltration into musculoskeletal tissues, and depletion of neutrophils in IFN-ß-KO but not IRF7-KO mice mitigated musculoskeletal disease caused by CHIKV. Our findings suggest disparate roles for the IFN subtypes during CHIKV infection, with IFN-α limiting early viral replication and dissemination and IFN-ß modulating neutrophil-mediated inflammation.IMPORTANCE Type I interferons (IFNs) possess a range of biological activity and protect against a number of viruses, including alphaviruses. Despite signaling through a shared receptor, there are established biochemical and functional differences among the IFN subtypes. The significance of our research is in demonstrating that IFN-α and IFN-ß both have protective roles during acute chikungunya virus (CHIKV) infection but do so by distinct mechanisms. IFN-α limits CHIKV replication and dissemination, whereas IFN-ß protects from CHIKV pathogenesis by limiting inflammation mediated by neutrophils. Our findings support the premise that the IFN subtypes have distinct biological activities in the antiviral response.

FM0807 decelerates experimental arthritis progression by inhibiting inflammatory responses and joint destruction via modulating NF-κB and MAPK pathways.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic articular synovial inflammatory disease. The precise etiology underlying the pathogenesis of RA remains unknown. We aimed to investigate the inhibitory effect of curcumin analog FM0807 (curcumin salicylate monoester, 2-hydroxy-, 4-[(1E,6E)-7-(4-hydroxy-3-methoxyphenyl)-3,5-dioxo-1,6-heptadien-1-yl]-2-methoxyphenyl ester) on experimental RA and investigate its possible mechanisms of action. METHOD: Rats with Freund's complete adjuvant (FCA)-induced arthritis (AIA) were administered aspirin (0.1 mmol.kg-1), curcumin (0.1 mmol.kg-1), FM0807 (0.1, 0.2 mmol.kg-1) and vehicle via gastric gavage, from days 7 to 21, once daily. The hind paw volume and arthritis index (AI) were measured, and radiographic and histological examinations were performed. Twenty-one days later, the animals were killed and left ankle joints were removed to measure protein expression of the elements of the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathway by Western blot analysis. The enzyme-linked immunosorbent assay (ELISA) was employed to measure synovial fluid levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1ß and IL-10. RESULTS: Compared with AIA group, FM0807 reduced the AI and swelling of the injected hind paw in a dose-dependent manner, and inhibited increases in inflammatory cell infiltration, pannus formation and cartilage destruction. FM0807 also potently attenuated the increase in the expression of inflammatory factors TNF-α, IL-6 and IL-1ß in synovial fluid, while IL-10 levels were also elevated. FM0807 significantly suppressed phosphorylation of extracellular-signal-regulated kinase (ERK) 1/2 (ERK1/2), c-Jun-N-terminal kinase (JNK) 1/2 (JNK1/2), p38MAPK, inhibitor of NF-κB kinase (IKK), IκB and NF-κB p65 protein, (all P<0.05), which displayed more potential effects compared with those of the aspirin and curcumin groups. CONCLUSION: FM0807 exerts its therapeutic effects on RA by inhibiting cartilage degeneration. FM0807 treatment might be an effective therapeutic approach for RA.

Increased ILC3s associated with higher levels of IL-1ß 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-1ß 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-1ß 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.

Tyro3/Axl/Mertk-deficient mice develop bone marrow edema which is an early pathological marker in rheumatoid arthritis.

Rheumatoid arthritis is an auto-immune disease of the synovial joints, hallmarked by chronic inflammation and subsequent progressive tissue destruction. TYRO3, AXL and MER (gene name Mertk) (TAM) receptors are part of a negative feedback signaling system in the immune reaction and mediate efferocytosis thereby tempering the inflammatory process. We have shown that Axl-/- and Mertk-/- mice develop more severe arthritis whereas activating these receptors by overexpressing their ligands Pros1 and Gas6 ameliorates arthritis. Mice genetically ablated for the three genes of the TAM receptor family Tyro3/Axl/Mertk (TAM triple knock-out or TKO) have been described to spontaneously develop macroscopic signs of arthritis. In this study we aimed to analyze arthritis development in TAM TKO mice histologically to determine the extent and sequence of pathological changes in the joint. Ankle joints of three different age groups, adolescence (14 weeks), mature adult (34 weeks) and middle-age (52 weeks), of TAM TKO or wild-type mice were examined macroscopically, histologically and immunohistochemically. Surprisingly, until the age of 52 weeks, none of the mice examined developed spontaneous macroscopic signs of arthritis. There was no synovial inflammation nor any signs of damage to the cartilage or bone. However, bone marrow edema was observed in TAM TKO mice in the two latter age groups. The infiltrate in the bone marrow was characterized by both myeloid cells and lymphocytes. This study showed that TAM TKO mice developed a pre-stage (pre-clinical phase) of arthritis marked by bone marrow edema.

Berberis aristata Ameliorates Adjuvant-Induced Arthritis by Inhibition of NF-κB and Activating Nuclear Factor-E2-related Factor 2/hem Oxygenase (HO)-1 Signaling Pathway.

The present study was carried out to investigate the anti-arthritic activity of Berberis aristata hydroalcoholic extract (BAHE) in formaldehyde-induced arthritis and adjuvant-induced arthritis (AIA) model. Arthritis was induced by administration of either formaldehyde (2% v/v) or CFA into the subplantar surface of the hind paw of the animal. In formaldehyde-induced arthritis and AIA, treatment of BAHE at doses 50, 100 and 200 mg/kg orally significantly decreased joint inflammation as evidenced by decrease in joint diameter and reduced inflammatory cell infiltration in histopathological examination. BAHE treatment demonstrated dose-dependent improvement in the redox status of synovium (decrease in GSH, MDA, and NO levels and increase in SOD and CAT activities). The beneficial effect of BAHE was substantiated with decreased expression of inflammatory markers such as IL-1ß, IL-6, TNF-R1, and VEGF by immunohistochemistry analysis in AIA model. BAHE increased HO-1/Nrf-2 and suppressed NF-κB mRNA and protein expression in adjuvant immunized joint. Additionally, BAHE abrogated degrading enzymes, as there was decreased protein expression of MMP-3 and -9 in AIA. In conclusion, we demonstrated the anti-arthritic activity of Berberis aristata hydroalcoholic extract via the mechanism of inhibition of NF-κB and activation of Nrf-2/HO-1.

Effects of an antagonist of the bombesin/gastrin-releasing peptide receptor on complete Freund's adjuvant-induced arthritis in rats.

OBJECTIVE: To determine the effects of RC-3095 in clinical and histopathologic parameters and inflammatory mediators on complete Freund's adjuvant-induced arthritis (CFA). METHODS: The arthritis was induced by injection of CFA into the left hind footpad. The animals were divided into control, vehicle injected control, placebo group (saline subcutaneously 50ml/kg, once daily for 8 days after modeling), treatment group (0.3mg/kg of RC-3095 subcutaneously, once daily for 8 days after induction). Clinical evaluation was accomplished daily, through scoring of the paw edema. The animals were sacrificed 15 days after induction for collection of hind foot joints for histology. We used a histological scoring system which was previously described, and interferon (INF)-gamma, interleukin (IL)-1beta, tumor necrosis factor (TNF), interleukin (IL)-6 and interleukin (IL)-10 were measured by ELISA. RESULTS: There was a significant inhibition of joint histological findings in the RC-3095 treated group, including synovial inflammatory infiltration and hyperplasia, cartilage and bone erosion. IFN-gamma, IL-1beta, TNF, IL-6 and IL-10 serum levels were significantly lower in the treated group. Paw swelling and subcutaneous inflammation, evaluated clinically, were not different between CFA-induced groups. CONCLUSIONS: RC-3095 was able to improve experimental arthritis, attenuate joint damage and decrease serum levels of IFN-gamma, IL-1beta, TNF, IL-6 and IL-10. These data indicate that interference with GRP pathway is a potential new strategy for the treatment of RA that needs further investigational studies.

Fractalkine, a novel chemokine in rheumatoid arthritis and in rat adjuvant-induced arthritis.

OBJECTIVE: To examine the expression of the novel CX3C chemokine fractalkine (Fkn) and its receptor (CX3CR1) in rheumatoid arthritis (RA) and rat adjuvant-induced arthritis (AIA), a model of RA. METHODS: Immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR), and chemotaxis assays were used. RESULTS: In rat AIA, synovial tissue (ST) macrophages, fibroblasts, endothelial cells, and dendritic cells were Fkn immunopositive, whereas lymphocytes did not significantly express Fkn. Significant staining for CX3CR1 was found in ST macrophages, fibroblasts, and dendritic cells, whereas only a small percentage of endothelial cells stained for CX3CR1 in rat AIA. We immunolocalized Fkn to RA ST macrophages, fibroblasts, endothelial cells, and dendritic cells. We also found intense ST macrophage and dendritic cell staining for CX3CR1 in RA ST. Flow cytometry analysis of RA synovial fluid (SF) and peripheral blood revealed a greater percentage of monocytes expressing Fkn and CX3CR1 compared with T cells. By ELISA, we found significantly elevated soluble Fkn (sFkn) levels in RA SF compared with SF from patients with osteoarthritis or other forms of arthritis. By RT-PCR, we found enhanced expression of Fkn and CX3CR1 mRNA on day 18 in rat AIA, a time of pronounced inflammation in the rat joint. Soluble Fkn-depleted RA SF showed significantly decreased chemotactic activity for monocytes compared with sham-depleted RA SF. CONCLUSION: These results indicate that Fkn and its receptor are both expressed in RA and in rat AIA, and that sFkn is up-regulated in RA SF. Furthermore, our data suggest a new role for Fkn in monocyte chemotaxis in the inflamed RA joint.

Adenoviral transfer of the viral IL-10 gene periarticularly to mouse paws suppresses development of collagen-induced arthritis in both injected and uninjected paws.

Gene therapy is a promising new approach in the treatment of rheumatoid arthritis. Gene delivery to diseased joints offers the prospect of achieving high, local concentrations of a therapeutic gene product in a sustained manner, while minimizing exposure of nontarget organs. We report that a single administration of a modified adenovirus encoding the Epstein-Barr-derived homologue of IL-10 can suppress the development of disease for extended periods of time when injected locally within the periarticular tissue surrounding the ankle joints of mice with collagen type II-induced arthritis. Furthermore, we show that injection of an adenoviral vector carrying the IL-10 gene into a single paw can suppress development of arthritis in other, noninjected paws of the same individual. The systemic protection resulting from local gene therapy occurred in the absence of detectable levels of viral IL-10 in the serum. Circulating Ab levels to heterologous collagen were unaffected; however, treatment with viral IL-10 significantly suppressed the development of Abs to autologous mouse type II collagen. Thus, the treatment of a single joint by local delivery of the vIL-10 gene may protect multiple joints of the same individual while avoiding deleterious side effects often associated with systemic therapy.

Intra-articular IL-4 gene therapy in arthritis: anti-inflammatory effect and enhanced th2activity.

Gene therapy has been explored as a potential method for treating chronic inflammatory diseases such as rheumatoid arthritis. To determine the efficacy of intra-articular IL-4 gene therapy in an animal model of arthritis using a retroviral vector, a retrovirus encoding rat IL-4 (DA-IL-4) was engineered, purified and concentrated to high titer (>/=109 CFU/ml). Infectivity and expression levels were demonstrated in vitro using cultured fibroblast-like synoviocytes. Efficacy was evaluated in the rat adjuvant arthritis model. DA-IL-4 or DA-beta-gal retrovirus was injected into the intra-articular joint space of the right ankle on day 12 after immunization. Three days after joint injection, the injected paw contained increased levels of IL-4 compared with control or with the contralateral uninjected paw, demonstrating successful transgene expression. Surprisingly, 8 days after treatment IL-4 levels continued to increase in the injected and contralateral paw compared with DA-beta-gal-treated animals. Serum IL-4 levels were also elevated in DA-IL-4-treated rats. RT-PCR studies demonstrated that the transgene was expressed in the injected ankle but not in the contralateral joint. IL-4 gene therapy resulted in a significant reduction in paw swelling and decreased radiographic evidence of bone destruction. This is the first demonstration of successful intra-articular retroviral gene treatment using a therapeutic gene. In addition to its anti-inflammatory effect, this study supports the potential application of intra-articular gene therapy as a method for enhancing systemic Th2 function.

Immunohistochemical detection of lymphocyte subpopulations in the tarsal joints of chickens with experimental viral arthritis.

We characterized the lymphocytes in the tarsal joint synovium of chickens inoculated with an arthrotropic strain of avian reovirus. Cryostat sections of whole joints taken from 2 days to 35 days postinoculation were analyzed using monoclonal antibodies directed against B lymphocytes, T lymphocytes, and chicken Ia antigen. Plasma cells were morphologically identified using stained sections of whole joints. Time-dependent changes were found in the type and number of positively staining cells. Synoviocytes and cells with a dendritic morphology stained positive for Ia in normal joint sections. T cells, mostly CD8 positive, were present in low numbers in acute phase arthritis (2-6 days postinfection) in the perivascular and superficial regions of the synovium. Subacute arthritis (8-14 days postinfection) was characterized by increased numbers of CD4 and Cd8 T cells in the perivascular and superficial regions. The perivascular T cells began to organize into aggregates, with IgM-positive B cells and plasma cells on the periphery of these aggregates. Some CD8-positive cells were detected on the surface of the articular cartilage. Cells staining positively for Ia were not lymphocytes. Chronic arthritis ( > 14 days postinfection) was characterized by large numbers of T cells in the perivascular and superficial regions, with the CD4-positive T cells found primarily in the lymphoid aggregates of the perivascular regions. IgM-positive B cells were fewer, but more plasma cells, few of which stained positive for IgM, were present. Lymphocytes in chronic arthritis stained positively for Ia. These data suggest that the types, numbers, and activation level of lymphocytes present in the tarsal joints are similar but not identical to those seen in rheumatoid arthritis.

Effects of psychological stress on joint inflammation and adrenal function during induction of arthritis in the Lewis rat.

Glucocorticoids are effective immunosuppressive and anti-inflammatory agents, but some aspects of stress appear to be proinflammatory. This study investigates this apparent paradox as it applies to stress exposure and the development of arthritis in a rat strain that has subnormal hypothalamic-pituitary-adrenal (HPA) responsiveness. Female Lewis rats were subjected to 1 week of rotating, psychological stressors for 5 h daily, beginning 7 days following inoculation with type II collagen. The collagen-induced arthritis (CIA) group exposed to stress showed reduced ankle width increase (p < 0.001) and decreased hindlimb severity scores (p < 0.001). At sacrifice, 2 days following stress termination, no differences in either measure remained and there was no difference in hind paw volume. However, the area of the tibia invaded by stroma, as quantitated by image analysis, was reduced in the stressed rats (p < 0.05). In animals exposed to stress, adrenal weights were increased (p < 0.005) and plasma corticosterone levels were elevated at sacrifice (p < 0.02). Both injected groups had significantly larger adrenal (p < 0.005) and lower thymus weights (p < 0.05) than did uninjected controls. Likewise, both CIA groups had reduced glucocorticoid receptor immunoreactivity in synovial membranes compared to controls (p < 0.001), suggesting that the Lewis rat's HPA deficiency may be intensified by glucocorticoid receptor downregulation during the induction of CIA. These data indicate that the responsiveness of the HPA axis to psychological stress in this strain is sufficient to alter disease progression.