Cigarette smoke induces miR-132 in Th17 cells that enhance osteoclastogenesis in inflammatory arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by joint destruction and severe morbidity. Cigarette smoking (CS) can exacerbate the incidence and severity of RA. Although Th17 cells and the Aryl hydrocarbon receptor (AhR) have been implicated, the mechanism by which CS induces RA development remains unclear. Here, using transcriptomic analysis, we show that microRNA-132 is specifically induced in Th17 cells in the presence of either AhR agonist or CS-enriched medium. miRNA-132 thus induced is packaged into extracellular vesicles produced by Th17 and acts as a proinflammatory mediator increasing osteoclastogenesis through the down-regulation of COX2. In vivo, articular knockdown of miR-132 in murine arthritis models reduces the number of osteoclasts in the joints. Clinically, RA patients express higher levels of miR-132 than do healthy individuals. This increase is further elevated by cigarette smoking. Together, these results reveal a hitherto unrecognized mechanism by which CS could exacerbate RA and further advance understanding of the impact of environmental factors on the pathogenesis of chronic inflammatory diseases.

Smoking-induced aggravation of experimental arthritis is dependent of aryl hydrocarbon receptor activation in Th17 cells.

BACKGROUND: Epidemiologic studies have highlighted the association of environmental factors with the development and progression of autoimmune and chronic inflammatory diseases. Among the environmental factors, smoking has been associated with increased susceptibility and poor prognosis in rheumatoid arthritis (RA). However, the immune and molecular mechanism of smoking-induced arthritis aggravation remains unclear. The transcription factor aryl hydrocarbon receptor (AHR) regulates the generation of Th17 cells, CD4 T cells linked the development of autoimmune diseases. AHR is activated by organic compounds including polycyclic aromatic hydrocarbons (PAHs), which are environmental pollutants that are also present in cigarette smoke. In this study, we investigated the role of AHR activation in the aggravation of experiment arthritis induced by exposure to cigarette smoke. METHODS: Mice were exposed to cigarette smoke during the developmental phase of antigen-induced arthritis and collagen-induced arthritis to evaluate the effects of smoking on disease development. Aggravation of articular inflammation was assessed by measuring neutrophil migration to the joints, increase in articular hyperalgesia and changes in the frequencies of Th17 cells. In vitro studies were performed to evaluate the direct effects of cigarette smoke and PAH on Th17 differentiation. We also used mice genetically deficient for AHR (Ahr KO) and IL-17Ra (Il17ra KO) to determine the in vivo mechanism of smoking-induced arthritis aggravation. RESULTS: We found that smoking induces arthritis aggravation and increase in the frequencies of Th17 cells. The absence of IL-17 signaling (Il17ra KO) conferred protection to smoking-induced arthritis aggravation. Moreover, in vitro experiments showed that cigarette smoke can directly increase Th17 differentiation of T cells by inducing AHR activation. Indeed, Ahr KO mice were protected from cigarette smoke-induced arthritis aggravation and did not display increase in TH17 frequencies, suggesting that AHR activation is an important mechanism for cigarette smoke effects on arthritis. Finally, we demonstrate that PAHs are also able to induce arthritis aggravation. CONCLUSIONS: Our data demonstrate that the disease-exacerbating effects of cigarette smoking are AHR dependent and environmental pollutants with AHR agonist activity can induce arthritis aggravation by directly enhancing Th17 cell development.

TGF-ß signalling defect is linked to low CD39 expression on regulatory T cells and methotrexate resistance in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune arthropathy characterized by chronic articular inflammation. Methotrexate (MTX) remains the first-line therapy for RA and its anti-inflammatory effect is associated with the maintenance of high levels of extracellular adenosine (ADO). Nonetheless, up to 40% of RA patients are resistant to MTX treatment and this is linked to a reduction of CD39 expression, an ectoenzyme involved in the generation of extracellular ADO by ATP metabolism, on circulating regulatory T cells (Tregs). However, the mechanism mediating the reduction of CD39 expression on Tregs is unknown. Here we demonstrated that the impairment in TGF-ß signalling lead to the reduction of CD39 expression on Tregs that accounts for MTX resistance. TGF-ß increases CD39 expression on Tregs via the activation of TGFBRII/TGFBRI, SMAD2 and the transcription factor CREB, which is activated in a p38-dependent manner and induces CD39 expression by promoting ENTPD1 gene transcription. Importantly, unresponsive patients to MTX (UR-MTX) show reduced expression of TGFBR2 and CREB1 and decreased levels of p-SMAD2 and p-CREB in Tregs compared to MTX-responsive patients (R-MTX). Furthermore, RA patients carrying at least one mutant allele for rs1431131 (AT or AA) of the TGFBR2 gene are significantly (p = 0.0006) associated with UR-MTX. Therefore, we have uncovered a molecular mechanism for the reduced CD39 expression on Tregs, and revealed potential targets for therapeutic intervention for MTX resistance.

IL-33 signaling is essential to attenuate viral-induced encephalitis development by downregulating iNOS expression in the central nervous system.

BACKGROUND: Viral encephalitis is a common cause of lethal infections in humans, and several different viruses are documented to be responsible. Rocio virus is a flavivirus that causes a severe lethal encephalitis syndrome in humans and also mice, providing an interesting model to study the CNS compartmentalized immune response. Interleukin 33 (IL-33), a member of the IL-1 family, is an immunomodulatory cytokine that is highly expressed in the CNS. However, the role of IL-33 on viral encephalitis remains unclear. Therefore, we aimed to explore how the IL-33/ST2 axis regulates the local immune response during Rocio virus infection. METHODS: Wild-type (WT), ST2 (ST2(-/-)), and nitric oxide synthase-deficient mice (iNOS(-/-)) and Stat6 (Stat6(-/-))-deficient mice were infected with different concentrations of the Rocio virus by intraperitoneal route, the cytokine mRNA level in CNS was analyzed by qPCR, and cellular immunophenotyping was performed on infected mice by the flow cytometry of isolated CNS mononuclear cells. RESULTS: We have shown that the mRNA expression of IL-33 and ST2 receptors is increased in the CNS of Rocio virus-infected WT mice and that ST2(-/-) mice showed increased susceptibility to infection. ST2 deficiency was correlated with increased tissue pathology, cellular infiltration, and tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ) mRNA levels and higher viral load in the CNS, compared with wild-type mice. The increased Th1 cytokine levels released in the CNS acted on infiltrating macrophages, as evidenced by flow cytometry characterization of cellular infiltrates, inducing the expression of iNOS, contributing to brain injury. Moreover, iNOS(-/-) mice were more resistant to Rocio virus encephalitis, presenting a lower clinical score and reduced mortality rate, despite the increased tissue pathology. CONCLUSIONS: We provide evidences of a specific role for IL-33 receptor signaling in nitric oxide induction through local IFN-γ modulation, suggesting that nitric oxide overproduction might have an important role in the progression of experimental viral encephalitis.

Fructose 1,6-bisphosphate, a high-energy intermediate of glycolysis, attenuates experimental arthritis by activating anti-inflammatory adenosinergic pathway.

Fructose 1,6-bisphosphate (FBP) is an endogenous intermediate of the glycolytic pathway. Exogenous administration of FBP has been shown to exert protective effects in a variety of ischemic injury models, which are attributed to its ability to sustain glycolysis and increase ATP production. Here, we demonstrated that a single treatment with FBP markedly attenuated arthritis, assessed by reduction of articular hyperalgesia, joint swelling, neutrophil infiltration and production of inflammatory cytokines, TNF and IL-6, while enhancing IL-10 production in two mouse models of arthritis. Our mechanistic studies showed that FBP reduces joint inflammation through the systemic generation of extracellular adenosine and subsequent activation of adenosine receptor A2a (A2aR). Moreover, we showed that FBP-induced adenosine generation requires hydrolysis of extracellular ATP through the activity of the ectonucleosides triphosphate diphosphohydrolase-1 (ENTPD1, also known as CD39) and ecto-5'-nucleotidase (E5NT, also known as CD73). In accordance, inhibition of CD39 and CD73 abolished anti-arthritic effects of FBP. Taken together, our findings provide a new insight into the molecular mechanism underlying the anti-inflammatory effect of FBP, showing that it effectively attenuates experimental arthritis by activating the anti-inflammatory adenosinergic pathway. Therefore, FBP may represent a new therapeutic strategy for treatment of rheumatoid arthritis (RA).

NLRP3 inflammasome-mediated neutrophil recruitment and hypernociception depend on leukotriene B(4) in a murine model of gout.

OBJECTIVE: Deposition of monosodium urate monohydrate (MSU) crystals in the joints promotes an intense inflammatory response and joint dysfunction. This study evaluated the role of the NLRP3 inflammasome and 5-lipoxygenase (5-LOX)-derived leukotriene B(4) (LTB(4) ) in driving tissue inflammation and hypernociception in a murine model of gout. METHODS: Gout was induced by injecting MSU crystals into the joints of mice. Wild-type mice and mice deficient in NLRP3, ASC, caspase 1, interleukin-1ß (IL-1ß), IL-1 receptor type I (IL-1RI), IL-18R, myeloid differentiation factor 88 (MyD88), or 5-LOX were used. Evaluations were performed to assess neutrophil influx, LTB(4) activity, cytokine (IL-1ß, CXCL1) production (by enzyme-linked immunosorbent assay), synovial microvasculature cell adhesion (by intravital microscopy), and hypernociception. Cleaved caspase 1 and production of reactive oxygen species (ROS) were analyzed in macrophages by Western blotting and fluorometric assay, respectively. RESULTS: Injection of MSU crystals into the knee joints of mice induced neutrophil influx and neutrophil-dependent hypernociception. MSU crystal-induced neutrophil influx was CXCR2-dependent and relied on the induction of CXCL1 in an NLRP3/ASC/caspase 1/IL-1ß/MyD88-dependent manner. LTB(4) was produced rapidly after injection of MSU crystals, and this was necessary for caspase 1-dependent IL-1ß production and consequent release of CXCR2-acting chemokines in vivo. In vitro, macrophages produced LTB(4) after MSU crystal injection, and LTB(4) was relevant in the MSU crystal-induced maturation of IL-1ß. Mechanistically, LTB(4) drove MSU crystal-induced production of ROS and ROS-dependent activation of the NLRP3 inflammasome. CONCLUSION: These results reveal the role of the NLRP3 inflammasome in mediating MSU crystal-induced inflammation and dysfunction of the joints, and highlight a previously unrecognized role of LTB(4) in driving NLRP3 inflammasome activation in response to MSU crystals, both in vitro and in vivo.