Silvio M. Vieira

IL-33 induces neutrophil migration in rheumatoid arthritis and is a target of anti-TNF therapy

Objectives Interleukin 33 (IL-33) is a new member of the IL-1 family of cytokines which signals via its receptor, ST2 (IL-33R), and has an important role in Th2 and mast cell responses. This study shows that IL-33 orchestrates neutrophil migration in arthritis. Methods and results Methylated bovine serum albumin (mBSA) challenge in the knee joint of mBSA-immunised mice induced local neutrophil migration accompanied by increased IL-33R and IL-33 mRNA expression. Cell migration was inhibited by systemic and local treatments with soluble (s)IL-33R, an IL-33 decoy receptor, and was not evident in IL-33R-deficient mice. IL-33 injection also induced IL-33R-dependent neutrophil migration. Antigen- and IL-33-induced neutrophil migration in the joint was dependent on CXCL1, CCL3, tumour necrosis factor α (TNFα) and IL-1β synthesis. Synovial tissue, macrophages and activated neutrophils expressed IL-33R. IL-33 induces neutrophil migration by activating macrophages to produce chemokines and cytokines and by directly acting on neutrophils. Importantly, neutrophils from patients with rheumatoid arthritis successfully treated with anti-TNFα antibody (infliximab) expressed significantly lower levels of IL-33R than patients treated with methotrexate alone. Only neutrophils from patients treated with methotrexate alone or from normal donors stimulated with TNFα responded to IL-33 in chemotaxis. Conclusions These results suggest that suppression of IL-33R expression in neutrophils, preventing IL-33-induced neutrophil migration, may be an important mechanism of anti-TNFα therapy of inflammation.

IL-27 acts on DCs to suppress the T cell response and autoimmunity by inducing expression of the immunoregulatory molecule CD39

Dendritic cells (DCs) control the balance between effector T cells and regulatory T cells in vivo. Hence, the study of DCs might identify mechanisms of disease pathogenesis and guide new therapeutic approaches for disorders mediated by the immune system. We found that interleukin 27 (IL-27) signaling in mouse DCs limited the generation of effector cells of the TH1 and TH17 subsets of helper T cells and the development of experimental autoimmune encephalomyelitis (EAE). The effects of IL-27 were mediated at least in part through induction of the immunoregulatory molecule CD39 in DCs. IL-27-induced CD39 decreased the extracellular concentration of ATP and downregulated nucleotide-dependent activation of the NLRP3 inflammasome. Finally, therapeutic vaccination with IL-27-conditioned DCs suppressed established relapsing-remitting EAE. Thus, IL-27 signaling in DCs limited pathogenic T cell responses and the development of autoimmunity.

IL-17 mediates articular hypernociception in antigen-induced arthritis in mice

&NA; IL‐17 is an important cytokine in the physiopathology of rheumatoid arthritis (RA). However, its participation in the genesis of nociception during RA remains undetermined. In this study, we evaluated the role of IL‐17 in the genesis of articular nociception in a model of antigen (mBSA)‐induced arthritis. We found that mBSA challenge in the femur–tibial joint of immunized mice induced a dose‐ and time‐dependent mechanical hypernociception. The local IL‐17 concentration within the mBSA‐injected joints increased significantly over time. Moreover, co‐treatment of mBSA challenged mice with an antibody against IL‐17 inhibited hypernociception and neutrophil recruitment. In agreement, intraarticular injection of IL‐17 induced hypernociception and neutrophil migration, which were reduced by the pre‐treatment with fucoidin, a leukocyte adhesion inhibitor. The hypernociceptive effect of IL‐17 was also reduced in TNFR1−/− mice and by pre‐treatment with infliximab (anti‐TNF antibody), a CXCR1/2 antagonist or by an IL‐1 receptor antagonist. Consistent with these findings, we found that IL‐17 injection into joints increased the production of TNF‐&agr;, IL‐1&bgr; and CXCL1/KC. Treatment with doxycycline (non‐specific MMPs inhibitor), bosentan (ETA/ETB antagonist), indomethacin (COX inhibitor) or guanethidine (sympathetic blocker) inhibited IL‐17‐induced hypernociception. IL‐17 injection also increased PGE2 production, MMP‐9 activity and COX‐2, MMP‐9 and PPET‐1 mRNA expression in synovial membrane. These results suggest that IL‐17 is a novel pro‐nociceptive cytokine in mBSA‐induced arthritis, whose effect depends on both neutrophil migration and various pro‐inflammatory mediators, as TNF‐&agr;, IL‐1&bgr;, CXCR1/2 chemokines ligands, MMPs, endothelins, prostaglandins and sympathetic amines. Therefore, it is reasonable to propose IL‐17 targeting therapies to control this important RA symptom.

Prostaglandin mediates IL-23/IL-17-induced neutrophil migration in inflammation by inhibiting IL-12 and IFNγ production

IL-23/IL-17-induced neutrophil recruitment plays a pivotal role in rheumatoid arthritis (RA). However, the mechanism of the neutrophil recruitment is obscure. Here we report that prostaglandin enhances the IL-23/IL-17-induced neutrophil migration in a murine model of RA by inhibiting IL-12 and IFN γ production. Methylated BSA (mBSA) and IL-23-induced neutrophil migration was inhibited by anti-IL-23 and anti-IL-17 antibodies, COX inhibitors, IL-12, or IFNγ but was enhanced by prostaglandin E2 (PGE2). IL-23-induced IL-17 production was increased by PGE2 and suppressed by COX-inhibition or IL-12. Furthermore, COX inhibition failed to reduce IL-23-induced neutrophil migration in IL-12- or IFNγ-deficient mice. IL-17-induced neutrophil migration was not affected by COX inhibitors, IL-12, or IFNγ but was inhibited by MK886 (a leukotriene synthesis inhibitor), anti-TNFα, anti-CXCL1, and anti-CXCL5 antibodies and by repertaxin (a CXCR1/2 antagonist). These treatments all inhibited mBSA- or IL-23-induced neutrophil migration. IL-17 induced neutrophil chemotaxis through a CXC chemokines-dependent pathway. Our results suggest that prostaglandin plays an important role in IL-23-induced neutrophil migration in arthritis by enhancing IL-17 synthesis and by inhibiting IL-12 and IFNγ production. We thus provide a mechanism for the pathogenic role of the IL-23/IL-17 axis in RA and also suggest an additional mechanism of action for nonsteroidal anti-inflammatory drugs.

CXCR2-Specific Chemokines Mediate Leukotriene B4-Dependent Recruitment of Neutrophils to Inflamed Joints in Mice With Antigen-Induced Arthritis

OBJECTIVE To investigate the mechanism underlying neutrophil migration into the articular cavity in experimental arthritis and, by extension, human inflammatory synovitis. METHODS Antigen-induced arthritis (AIA) was generated in mice with methylated bovine serum albumin (mBSA). Migration assays and histologic analysis were used to evaluate neutrophil recruitment to knee joints. Levels of inflammatory mediators were measured by enzyme-linked immunosorbent assay. Antibodies and pharmacologic inhibitors were used in vivo to determine the role of specific disease mediators. Samples of synovial tissue and synovial fluid from rheumatoid arthritis (RA) or osteoarthritis patients were evaluated for CXCL1 and CXCL5 expression. RESULTS High levels of CXCL1, CXCL5, and leukotriene B4 (LTB4) were expressed in the joints of arthritic mice. Confirming their respective functional roles, repertaxin (a CXCR1/CXCR2 receptor antagonist), anti-CXCL1 antibody, anti-CXCL5 antibody, and MK886 (a leukotriene synthesis inhibitor) reduced mBSA-induced neutrophil migration to knee joints. Repertaxin reduced LTB4 production in joint tissue, and neutrophil recruitment induced by CXCL1 or CXCL5 was inhibited by MK886, suggesting a sequential mechanism. Levels of both CXCL1 and CXCL5 were elevated in synovial fluid and were released in vitro by RA synovial tissues. Moreover, RA synovial fluid neutrophils stimulated with CXCL1 or CXCL5 released significant amounts of LTB4. CONCLUSION Our data implicate CXCL1, CXCL5, and LTB4, acting sequentially, in neutrophil migration in AIA. Elevated levels of CXCL1 and CXCL5 in the synovial compartment of RA patients provide robust comparative data indicating that this mechanism plays a role in inflammatory joint disease. Together, these results suggest that inhibition of CXCL1, CXCL5, or LTB4 may represent a potential therapeutic strategy in RA.

Quercetin Reduces Inflammatory Pain: Inhibition of Oxidative Stress and Cytokine Production

Quercetin (1) is known to have both antioxidant and antinociceptive effects. However, the mechanism involved in its antinociceptive effect is not fully elucidated. Cytokines and reactive oxygen species have been implicated in the cascade of events resulting in inflammatory pain. Therefore, we evaluated the antinociceptive mechanism of 1 focusing on the role of cytokines and oxidative stress. Intraperitoneal and oral treatments with 1 dose-dependently inhibited inflammatory nociception induced by acetic acid and phenyl-p-benzoquinone and also the second phase of formalin- and carrageenin-induced mechanical hypernociception. Compound 1 also inhibited the hypernociception induced by cytokines (e.g., TNFalpha and CXCL1), but not by inflammatory mediators that directly sensitize the nociceptor such as PGE2 and dopamine. On the other hand, 1 reduced carrageenin-induced IL-1beta production as well as carrageenin-induced decrease of reduced glutathione (GSH) levels. These results suggest that 1 exerts its analgesic effect by inhibiting pro-nociceptive cytokine production and the oxidative imbalance mediation of inflammatory pain.

A crucial role for TNF‐α in mediating neutrophil influx induced by endogenously generated or exogenous chemokines, KC/CXCL1 and LIX/CXCL5

Background and purpose:  Chemokines orchestrate neutrophil recruitment to inflammatory foci. In the present study, we evaluated the participation of three chemokines, KC/CXCL1, MIP‐2/CXCL2 and LIX/CXCL5, which are ligands for chemokine receptor 2 (CXCR2), in mediating neutrophil recruitment in immune inflammation induced by antigen in immunized mice.

Diet, microbiota and autoimmune diseases

There is growing evidence that the commensal bacteria in the gastrointestinal tract (the gut microbiota) influence the development of autoimmunity in rodent models. Since humans have co-evolved with commensals for millennia, it is likely that people, who are genetically predisposed to autoimmunity, harbor gut microbial communities that similarly influence the onset and/or severity of disease. Beyond the current efforts to identify such disease-promoting or -preventing commensals (“pathobionts” or “symbionts”), it will be important to determine what factors modulate them. Dietary changes are known to affect both the composition and function of the gut microbial communities, which in turn can alter the innate and adaptive immune system. In this review, we focus on the relationships between diet, microbiota, and autoimmune diseases. We hypothesize that the beneficial and life-prolonging effects of caloric restriction on a variety of autoimmune models including lupus might partly be mediated by its effects on the gut microbiome and associated virome, the collection of all viruses in the gut. We give recent examples of the immunomodulatory potential of select gut commensals and their products or diet-derived metabolites in murine models of arthritis, multiple sclerosis, and type 1 diabetes. Lastly, we summarize the published phenotypes of germ-free mouse models of lupus and speculate on any role of the diet-sensitive microbiome and virome in systemic lupus and the related antiphospholipid syndrome.

Anti-inflammatory effects of red pepper (Capsicum baccatum) on carrageenan- and antigen-induced inflammation.

Inflammation is a pivotal component of a variety of diseases, such as atherosclerosis and tumour progression. Various naturally occurring phytochemicals exhibit anti‐inflammatory activity and are considered to be potential drug candidates against inflammation‐related pathological processes. Capsicum baccatum L. var. pendulum (Willd.) Eshbaugh (Solanaceae) is the most consumed species in Brazil, and its compounds, such as capsaicinoids, have been found to inhibit the inflammatory process. However, the anti‐inflammatory effects of C. baccatum have not been characterized. Thus, this study was designed to evaluate the effects of C. baccatum juice in animal models of acute inflammation induced by carrageenan and immune inflammation induced by methylated bovine serum albumin. Pretreatment (30 min) of rats with pepper juice (0.25–2.0 g kg−1) significantly decreased leucocyte and neutrophil migration, exudate volume and protein and LDH concentration in pleural exudates of a pleurisy model. This juice also inhibited neutrophil migration and reduced the vascular permeability on carrageenan‐induced peritonitis in mice. C. baccatum juice also reduced neutrophil recruitment and exudate levels of pro‐inflammatory cytokines TNF‐α and IL‐1β in mouse inflammatory immune peritonitis. Furthermore, we demonstrated that the main constituent of C. baccatum juice, as extracted with chloroform, is capsaicin. In agreement with this, capsaicin was able to inhibit the neutrophil migration towards the inflammatory focus. To our knowledge, this is the first demonstration of the anti‐inflammatory effect of C. baccatum juice and our data suggest that this effect may be induced by capsaicin. Moreover, the anti‐inflammatory effect induced by red pepper may be by inhibition of pro‐inflammatory cytokine production at the inflammatory site.

Role of cytokines in mediating mechanical hypernociception in a model of delayed-type hypersensitivity in mice

In the present study, we used the electronic version of the von Frey test to investigate the role of cytokines (TNF‐α and IL‐1β) and chemokines (KC/CXCL‐1) in the genesis of mechanical hypernociception during antigen‐induced inflammation in mice. The nociceptive test consisted of evoking a hindpaw flexion reflex with a hand‐held force transducer (electronic anesthesiometer) adapted with a 0.5mm2 polypropylene tip. The intraplantar administration of methylated bovine serum albumin (mBSA) in previously immunized (IM), but not in sham‐immunized (SI) mice, induced mechanical hypernociception in a dose‐dependent manner. Hypernociception induced by antigen was reduced in animals pretreated with IL‐1ra and reparixin (a non‐competitive allosteric inhibitor of CXCR2), and in TNF receptor type 1 deficient (TNFR1−/−) mice. Consistently, antigen challenge induced a time‐dependent release of TNF‐α, IL‐1β and KC/CXCL‐1 in IM, but not in SI, mice. The increase in TNF‐α levels preceded the increase in IL‐1β and KC/CXCL1. Antigen‐induced release of IL‐1β and KC/CXCL1 was reduced in TNFR1−/− mice, and TNF‐α‐induced hypernociception was inhibited by IL‐1ra and reparixin. Hypernociception induced by IL‐1β in immunized mice was inhibited by indomethacin, whereas KC/CXCL1‐induced hypernociception was inhibited by indomethacin and guanethidine. Antigen‐induced hypernociception was reduced by indomethacin and guanethidine and abolished by the two drugs combined. Together, these results suggest that inflammation associated with an adaptive immune response induces hypernociception that is mediated by an initial release of TNF‐α, which triggers the subsequent release of IL‐1β and KC/CXCL1. The latter cytokines in turn stimulate the release of the direct‐acting final mediators, prostanoids and sympathetic amines.