Larissa G. Pinto

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.

Low expression of CD39 on regulatory T cells as a biomarker for resistance to methotrexate therapy in rheumatoid arthritis

Significance Methotrexate (MTX) is the first-line therapy for rheumatoid arthritis (RA). However, about 40% of patients are resistant to MTX. Furthermore, MTX resistance is only apparent after a prolonged continuous MTX treatment (>3 mo), by which time the disease of the nonresponders would have aggravated. Thus, there is a considerable unmet need for a biomarker to select MTX-resistant patients and place them immediately on alternative therapy. We found here that the low density of CD39 on peripheral regulatory T cells in RA patients is a rapid, convenient, and reliable (P < 0.01) biomarker for MTX resistance. Our findings also provide previously unrecognized information on aspects of immune regulation in RA and the mechanism of action of MTX. Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by joint destruction and severe morbidity. Methotrexate (MTX) is the standard first-line therapy of RA. However, about 40% of RA patients are unresponsive to MTX treatment. Regulatory T cells (Tregs, CD4+CD25+FoxP3+) are thought to play an important role in attenuating RA. To investigate the role of Tregs in MTX resistance, we recruited 122 RA patients (53 responsive, R-MTX; 69 unresponsive, UR-MTX) and 33 healthy controls. Three months after MTX treatment, R-MTX but not UR-MTX showed higher frequency of peripheral blood CD39+CD4+CD25+FoxP3+ Tregs than the healthy controls. Tregs produce adenosine (ADO) through ATP degradation by sequential actions of two cell surface ectonucleotidases: CD39 and CD73. Tregs from UR-MTX expressed a lower density of CD39, produced less ADO, and had reduced suppressive activity than Tregs from R-MTX. In a prospective study, before MTX treatment, UR-MTX expressed a lower density of CD39 on Tregs than those of R-MTX or control (P < 0.01). In a murine model of arthritis, CD39 blockade reversed the antiarthritic effects of MTX treatment. Our results demonstrate that MTX unresponsiveness in RA is associated with low expression of CD39 on Tregs and the decreased suppressive activity of these cells through reduced ADO production. Our findings thus provide hitherto unrecognized mechanism of immune regulation in RA and on mode of action of MTX. Furthermore, our data suggest that low expression of CD39 on Tregs could be a noninvasive biomarker for identifying MTX-resistant RA patients.

IL-33/ST2 signalling contributes to carrageenin-induced innate inflammation and inflammatory pain: role of cytokines, endothelin-1 and prostaglandin E2

IL‐33 signals through ST2 receptors and induces adaptive and innate inflammation. IL‐33/ST2 is involved in adaptive inflammation‐induced pain. Here, we have investigated the contribution of IL‐33/ST2‐triggered mechanisms to carrageenin‐induced innate inflammation.

Spinal cord oligodendrocyte-derived alarmin IL-33 mediates neuropathic pain

Neuropathic pain from injury to the peripheral and CNS represents a major health care issue. We have investigated the role of IL‐33/IL‐33 receptor (ST2) signaling in experimental models of neuropathic pain in mice. Chronic constriction injury (CCI) of the sciatic nerve induced IL‐33 production in the spinal cord. IL‐33/citrine reporter mice revealed that oligodendrocytes are the main cells expressing IL‐33 within the spinal cord together with a minor expression by neurons, microglia, and astrocytes. CCI‐induced mechanical hyperalgesia was reduced in IL‐33R (ST2)‐/‐ mice compared with wild‐type (WT) mice. Intrathecal treatment of WT mice with soluble IL‐33 receptor (IL‐33 decoy receptor) markedly reduced CCI‐induced hyperalgesia. Consistent with these observations, intrathecal injection of IL‐33 enhanced CCI hyperalgesia and induced hyperalgesia in naive mice. IL‐33‐mediated hyperalgesia during CCI was dependent on a reciprocal relationship with TNF‐α and IL‐1β. IL‐33‐induced hyperalgesia was markedly attenuated by inhibitors of PI3K, mammalian target of rapamycin, MAPKs (p38, ERK, and JNK), NF‐κB, and also by the inhibitors of glial cells (microglia and astrocytes). Furthermore, targeting these signaling pathways and cells inhibited IL‐33‐induced TNF‐α and IL‐1β production in the spinal cord. Our study, therefore, reveals an important role of oligodendrocyte‐derived IL‐33 in neuropathic pain.— Zarpelon, A. C., Rodrigues, F. C., Lopes, A. H., Souza, G. R., Carvalho, T. T., Pinto, L. G., Xu, D., Ferreira, S. H., Alves‐Filho, J. C., McInnes, I. B., Ryffel, B., Quesniaux, V. F. J., Reverchon, F., Mortaud, S., Menuet, A., Liew, F. Y., Cunha, F. Q., Cunha, T. M., Verri, Jr., W. A. Spinal cord oligodendrocyte‐derived alarmin IL‐33 mediates neuropathic pain. FASEB J. 30, 54‐65 (2016). www.fasebj.org

CCR2 Expression in Neutrophils Plays a Critical Role in Their Migration Into the Joints in Rheumatoid Arthritis

Infiltration of neutrophils into the joints plays an important role in bone erosion and articular destruction in rheumatoid arthritis (RA). Neutrophil trafficking during inflammation is a process that involves activation of chemotactic receptors. Recent findings suggest that changes in chemotactic receptor patterns could occur in neutrophils under certain inflammatory conditions. The aim of this study was to evaluate the gain of responsiveness of neutrophils to CCL2 in RA patients and to assess the role of CCL2 in driving neutrophil infiltration into the joints.

Gastrin-releasing peptide receptor (GRPR) mediates chemotaxis in neutrophils

Neutrophil migration to inflamed sites is crucial for both the initiation of inflammation and resolution of infection, yet these cells are involved in perpetuation of different chronic inflammatory diseases. Gastrin-releasing peptide (GRP) is a neuropeptide that acts through G protein coupled receptors (GPCRs) involved in signal transmission in both central and peripheral nervous systems. Its receptor, gastrin-releasing peptide receptor (GRPR), is expressed by various cell types, and it is overexpressed in cancer cells. RC-3095 is a selective GRPR antagonist, recently found to have antiinflammatory properties in arthritis and sepsis models. Here we demonstrate that i.p. injection of GRP attracts neutrophils in 4 h, and attraction is blocked by RC-3095. Macrophage depletion or neutralization of TNF abrogates GRP-induced neutrophil recruitment to the peritoneum. In vitro, GRP-induced neutrophil migration was dependent on PLC-β2, PI3K, ERK, p38 and independent of Gαi protein, and neutrophil migration toward synovial fluid of arthritis patients was inhibited by treatment with RC-3095. We propose that GRPR is an alternative chemotactic receptor that may play a role in the pathogenesis of inflammatory disorders.

Joint NOD2/RIPK2 Signaling Regulates IL-17 Axis and Contributes to the Development of Experimental Arthritis

Intracellular pattern recognition receptors such as the nucleotide-binding oligomerization domain (NOD)-like receptors family members are key for innate immune recognition of microbial infection and may play important roles in the development of inflammatory diseases, including rheumatic diseases. In this study, we evaluated the role of NOD1 and NOD2 on development of experimental arthritis. Ag-induced arthritis was generated in wild-type, NOD1−/−, NOD2−/−, or receptor-interacting serine-threonine kinase 2−/− (RIPK2−/−) immunized mice challenged intra-articularly with methylated BSA. Nociception was determined by electronic Von Frey test. Neutrophil recruitment and histopathological analysis of proteoglycan lost was evaluated in inflamed joints. Joint levels of inflammatory cytokine/chemokine were measured by ELISA. Cytokine (IL-6 and IL-23) and NOD2 expressions were determined in mice synovial tissue by RT-PCR. The NOD2−/− and RIPK2−/−, but not NOD1−/−, mice are protected from Ag-induced arthritis, which was characterized by a reduction in neutrophil recruitment, nociception, and cartilage degradation. NOD2/RIPK2 signaling impairment was associated with a reduction in proinflammatory cytokines and chemokines (TNF, IL-1β, and CXCL1/KC). IL-17 and IL-17 triggering cytokines (IL-6 and IL-23) were also reduced in the joint, but there is no difference in the percentage of CD4+ IL-17+ cells in the lymph node between arthritic wild-type and NOD2−/− mice. Altogether, these findings point to a pivotal role of the NOD2/RIPK2 signaling in the onset of experimental arthritis by triggering an IL-17–dependent joint immune response. Therefore, we could propose that NOD2 signaling is a target for the development of new therapies for the control of rheumatoid arthritis.

Targeting the minor pocket of C5aR for the rational design of an oral allosteric inhibitor for inflammatory and neuropathic pain relief

Significance Persistent pain in inflammatory and neuropathic conditions is often refractory to conventional analgesic therapy, with most patients suffering with unrelieved pain and serious treatment-related side effects. There is still a tremendous need to identify novel therapeutics for pain control with innovative biological mechanisms and minimal side effects. In this paper we challenge the hypothesis that a conserved structural motif across the G protein-coupled receptor family plays a regulatory role in the negative modulation of receptor activation and use a multidisciplinary approach to the rational drug design and characterization of a novel potent allosteric inhibitor of the C5a anaphylatoxin receptor (C5aR), thus providing a new promising avenue for the improvement of pharmacotherapy of chronic pain. Chronic pain resulting from inflammatory and neuropathic disorders causes considerable economic and social burden. Pharmacological therapies currently available for certain types of pain are only partially effective and may cause severe adverse side effects. The C5a anaphylatoxin acting on its cognate G protein-coupled receptor (GPCR), C5aR, is a potent pronociceptive mediator in several models of inflammatory and neuropathic pain. Although there has long been interest in the identification of C5aR inhibitors, their development has been complicated, as for many peptidomimetic drugs, mostly by poor drug-like properties. Herein, we report the de novo design of a potent and selective C5aR noncompetitive allosteric inhibitor, DF2593A, guided by the hypothesis that an allosteric site, the “minor pocket,” previously characterized in CXC chemokine receptors-1 and -2, is functionally conserved in the GPCR class. In vitro, DF2593A potently inhibited C5a-induced migration of human and rodent neutrophils. In vivo, oral administration of DF2593A effectively reduced mechanical hyperalgesia in several models of acute and chronic inflammatory and neuropathic pain, without any apparent side effects. Mechanical hyperalgesia after spared nerve injury was also reduced in C5aR−/− mice compared with WT mice. Furthermore, treatment of C5aR−/− mice with DF2593A did not produce any further antinociceptive effect compared with C5aR−/− mice treated with vehicle. The successful medicinal chemistry strategy confirms that a conserved minor pocket is amenable for the rational design of selective inhibitors and the pharmacological results support that the allosteric blockade of the C5aR represents a highly promising therapeutic approach to control chronic inflammatory and neuropathic pain.

Caspase-1 is involved in the genesis of inflammatory hypernociception by contributing to peripheral IL-1β maturation

BackgroundCaspase-1 is a cysteine protease responsible for the processing and secretion of IL-1β and IL-18, which are closely related to the induction of inflammation. However, limited evidence addresses the participation of caspase-1 in inflammatory pain. Here, we investigated the role of caspase-1 in inflammatory hypernociception (a decrease in the nociceptive threshold) using caspase-1 deficient mice (casp1-/-).ResultsMechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. The production of cytokines, PGE2 and neutrophil migration were evaluated by ELISA, radioimmunoassay and myeloperoxidase activity, respectively. The interleukin (IL)-1β and cyclooxygenase (COX)-2 protein expression were evaluated by western blotting. The mechanical hypernociception induced by intraplantar injection of carrageenin, tumour necrosis factor (TNF)α and CXCL1/KC was reduced in casp1-/- mice compared with WT mice. However, the hypernociception induced by IL-1β and PGE2 did not differ in WT and casp1-/- mice. Carrageenin-induced TNF-α and CXCL1/KC production and neutrophil recruitment in the paws of WT mice were not different from casp1-/- mice, while the maturation of IL-1β was reduced in casp1-/- mice. Furthermore, carrageenin induced an increase in the expression of COX-2 and PGE2 production in the paw of WT mice, but was reduced in casp1-/- mice.ConclusionThese results suggest that caspase-1 plays a critical role in the cascade of events involved in the genesis of inflammatory hypernociception by promoting IL-1β maturation. Because caspase-1 is involved in the induction of COX-2 expression and PGE2 production, our data support the assertion that caspase-1 is a key target to control inflammatory pain.

Protective Effect of RC-3095, an Antagonist of the Gastrin-Releasing Peptide Receptor, in Experimental Arthritis

OBJECTIVE To evaluate the antiinflammatory effects of RC-3095 in 2 experimental models of arthritis, collagen-induced arthritis (CIA) and antigen-induced arthritis (AIA), and to determine the mechanisms of action involved. METHODS RC-3095 was administered daily to mice with CIA and mice with AIA, after induction of disease with methylated bovine serum albumin. Disease incidence and severity were assessed using a clinical index and evaluation of histologic features, respectively. In mice with CIA, gastrin-releasing peptide receptor (GRPR) was detected by immunohistochemical analysis, while in mice with AIA, migration of neutrophils, presence of glycosaminoglycans, and lymphocyte proliferation, determined using the MTT assay, were assessed. Expression of cytokines interleukin-17 (IL-17), IL-1β, and tumor necrosis factor α (TNFα) was evaluated in all mouse knees using enzyme-linked immunosorbent assay. Treg cell production was assessed by flow cytometry in the joints of mice with AIA. RESULTS In mice with AIA, administration of RC-3095 reduced neutrophil migration, mechanical hypernociception, and proteoglycan loss. These findings were associated with inhibition of the levels of all 3 proinflammatory cytokines, decreased lymphocyte proliferation, and increased Treg cell numbers. In the CIA model, treatment with RC-3095 led to a significant reduction in arthritis clinical scores and the severity of disease determined histologically. Synovial inflammation, synovial hyperplasia, pannus formation, and extensive erosive changes were all dramatically reduced in the arthritic mice treated with RC-3095. Furthermore, arthritic mice treated with RC-3095 showed a significant reduction in the concentrations of IL-17, IL-1β, and TNFα, and showed a diminished expression of GRPR. CONCLUSION These findings suggest that the GRP pathway has a significant role in chronic arthritis, and its inhibition can be explored as a possible therapeutic strategy in rheumatoid arthritis.