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

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.

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-1ß. 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-1ß production in the spinal cord. Our study, therefore, reveals an important role of oligodendrocyte-derived IL-33 in neuropathic pain.

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

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.

Therapeutic Treatment of Arthritic Mice with 15-Deoxy Δ12,14-Prostaglandin J2 (15d-PGJ2) Ameliorates Disease through the Suppression of Th17 Cells and the Induction of CD4+CD25-FOXP3+ Cells.

The prostaglandin, 15-deoxy Δ12,14-prostaglandin J2 (15d-PGJ2), is a lipid mediator that plays an important role in the control of chronic inflammatory disease. However, the role of prostanoid in rheumatoid arthritis (RA) is not well determined. We demonstrated the therapeutic effect of 15d-PGJ2 in an experimental model of arthritis. Daily administration of 15d-PGJ2 attenuated the severity of CIA, reducing the clinical score, pain, and edema. 15d-PGJ2 treatment was associated with a marked reduction in joint levels of proinflammatory cytokines. Although the mRNA expression of ROR-γt was profoundly reduced, FOXP3 was enhanced in draining lymph node cells from 15d-PGJ2-treated arthritic mice. The specific and polyclonal CD4+ Th17 cell responses were limited during the addition of prostaglandin to cell culture. Moreover, in vitro 15d-PGJ2 increased the expression of FOXP3, GITR, and CTLA-4 in the CD4+CD25- population, suggesting the induction of Tregs on conventional T cells. Prostanoid addition to CD4+CD25- cells selectively suppressed Th17 differentiation and promoted the enhancement of FOXP3 under polarization conditions. Thus, 15d-PGJ2 ameliorated symptoms of collagen-induced arthritis by regulating Th17 differentiation, concomitant with the induction of Tregs, and, consequently, protected mice from diseases aggravation. Altogether, these results indicate that 15d-PGJ2 may represent a potential therapeutic strategy in RA.

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-1ß, 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.

Endothelin-1 induces neutrophil recruitment in adaptive inflammation via TNFα and CXCL1/CXCR2 in mice.

Endothelin mediates neutrophil recruitment during innate inflammation. Herein we address whether endothelin-1 (ET-1) is involved in neutrophil recruitment in adaptive inflammation in mice, and its mechanisms. Pharmacological treatments were used to determine the role of endothelin in neutrophil recruitment to the peritoneal cavity of mice challenged with antigen (ovalbumin) or ET-1. Levels of ET-1, tumour necrosis factor α (TNFα), and CXC chemokine ligand 1 (CXCL1) were determined by enzyme-linked immunosorbent assay. Neutrophil migration and flow cytometry analyses were performed 4 h after the intraperitoneal stimulus. ET-1 induced dose-dependent neutrophil recruitment to the peritoneal cavity. Treatment with the non-selective ET(A)/ET(B) receptor antagonist bosentan, and selective ET(A) or ET(B) receptor antagonists BQ-123 or BQ-788, respectively, inhibited ET-1- and ovalbumin-induced neutrophil migration to the peritoneal cavity. In agreement with the above, the antigen challenge significantly increased levels of ET-1 in peritoneal exudates. The ET-1- and ovalbumin-induced neutrophil recruitment were reduced in TNFR1 deficient mice, and by treatments targeting CXCL1 or CXC chemokine receptor 2 (CXCR2); further, treatment with bosentan, BQ-123, or BQ-788 inhibited ET-1- and antigen-induced production of TNFα and CXCL1. Furthermore, ET-1 and ovalbumin challenge induced an increase in the number of cells expressing the Gr1(+) markers in the granulocyte gate, CD11c(+) markers in the monocyte gate, and CD4(+) and CD45(+) (B220) markers in the lymphocyte gate in an ET(A)- and ET(B)-dependent manner, as determined by flow cytometry analysis, suggesting that ET-1 might be involved in the recruitment of neutrophils and other cells in adaptive inflammation. Therefore, the present study demonstrates that ET-1 is an important mediator for neutrophil recruitment in adaptive inflammation via TNFα and CXCL1/CXCR2-dependent mechanism.

Blockade of bradykinin receptors or angiotensin II type 2 receptor prevents paclitaxel-associated acute pain syndrome in mice.

BACKGROUND: Paclitaxel (PCX) is the first-line choice for the treatment of several types of cancer, including breast, ovarian, and lung cancers. However, patients who receive even a single dose with PCX commonly develop mechanical and cold allodynia, a symptom known as PCX-associated acute pain syndrome (P-APS). Here, we assessed possible involvement of kinin-kallikrein and renin-angiotensin systems in P-APS in mice. METHODS: Male mice C57Bl/6 wild type (WT) and knockouts for bradykinin receptors, B1 (B1-/- ) and B2 (B2-/- ), were used. Mechanical and cold allodynia were evaluated by using von Frey filaments and acetone test, respectively. P-APS was induced by administration of PCX 4 mg/kg, i.v.. ACE inhibitors (captopril and enalapril), antagonists for angiotensin II type 1 (losartan) and type 2 ([AT2R]; PD123319 and EMA 401) receptors were administrated prior the treatment with PCX. RT-PCR was used to analyse the expression of mRNA for B1, B2 and AT2R receptors. RESULTS: Administration of PCX in B1-/- and B2-/- mice induced lower mechanical and cold allodynia compared to the WT. However, the pre-treatment with ACE inhibitors reduced the development of mechanical and cold allodynia in P-APS. Surprisingly, we found that mice pre-treatment with the PD123319 or EMA401, but not losartan, prevented the development of mechanical and cold allodynia induced by PCX. CONCLUSION: Our results demonstrated the involvement of bradykinin receptors B1 and B2 as well as AT2R in the induction of P-APS in mice, and suggest the use of AT2R antagonists as a potential therapy for the prevention of P-APS in humans. SIGNIFICANCE: Kinin-kallikrein and renin-angiotensin systems, through B1, B2 and AT2 receptors, potentiates paclitaxel-associated acute pain syndrome (P-APS) in mice. Antagonists for AT2R are potential alternatives to prevent P-APS.

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

BACKGROUND: Caspase-1 is a cysteine protease responsible for the processing and secretion of IL-1ß 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-/-). RESULTS: Mechanical 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)-1ß 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-1ß 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-1ß 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. CONCLUSION: These results suggest that caspase-1 plays a critical role in the cascade of events involved in the genesis of inflammatory hypernociception by promoting IL-1ß 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.

Non-Peptidergic Nociceptive Neurons Are Essential for Mechanical Inflammatory Hypersensitivity in Mice.

Small nerve fibers that bind the isolectin B4 (IB4+ C-fibers) are a subpopulation of primary afferent neurons that are involved in nociceptive sensory transduction and do not express the neuropeptides substance P and calcitonin-gene related peptide (CGRP). Several studies have attempted to elucidate the functional role of IB4+-nociceptors in different models of pain. However, a functional characterization of the non-peptidergic nociceptors in mediating mechanical inflammatory hypersensitivity in mice is still lacking. To this end, in the present study, the neurotoxin IB4-Saporin (IB4-Sap) was employed to ablate non-peptidergic C-fibers. Firstly, we showed that intrathecal (i.t.) administration of IB4-Sap in mice depleted non-peptidergic C-fibers, since it decreased the expression of purinoceptor 3 (P2X3) and transient receptor potential cation channel subfamily V member 1 (TRPV1) in the dorsal root ganglia (DRGs) as well as IB4 labelling in the spinal cord. Non-peptidergic C-fibers depletion did not alter the mechanical nociceptive threshold, but it inhibited the mechanical inflammatory hypersensitivity induced by glial cell-derived neurotrophic factor (GDNF), but not nerve growth factor (NGF). Depletion of non-peptidergic C-fibers abrogated mechanical inflammatory hypersensitivity induced by carrageenan. Finally, it was found that the inflammatory mediators PGE2 and epinephrine produced a mechanical inflammatory hypersensitivity that was also blocked by depletion of non-peptidergic C-fibers. These data suggest that IB4-positive nociceptive nerve fibers are not involved in normal mechanical nociception but are sensitised by inflammatory stimuli and play a crucial role in mediating mechanical inflammatory hypersensitivity.

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.

Spinal mechanisms of antinociceptive action caused by diphenyl diselenide.

The present study was designed to investigate further the mechanisms involved in the antinociception caused by diphenyl diselenide in behavioral model of pain in mice. Diphenyl diselenide (1-100 mg/kg), given orally, produced significant inhibition of the biting behavior induced by intrathecal (i.t.) injection of glutamate (175 nmol/site) and N-methyl-d-aspartate (NMDA; 450 pmol/site), with mean ID(50) values of 45.92 (39.74-60.4) and 55.77 (36.52-77.5) mg/kg respectively. However, diphenyl diselenide completely failed to affect the nociception induced by alpha-amino-3-hydroxy-5-mehtyl-4-isoxazolepropionic acid (AMPA; 135 pmol/site), (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD; 50 nmol/site) and kainate (110 pmol/site). This compound also reduced the nociceptive response induced by substance P (SP) (135 ng/site, i.t.), interleukin 1beta (IL-1beta; 1 pg/site), tumor necrosis factor-alpha (TNF-alpha; 0.1 pg/site), bradykinin (BK; 0.1 microg/site) and capsaicin (30 ng/site) with mean ID(50) values of 16.22, 7.06, 6.06, 4.18 and 7.90 mg/kg, respectively. Together, these results indicate that diphenyl diselenide produced antinociception at spinal sites, with a possible interaction with glutamatergic pathways, more specifically via interaction with NMDA receptors, peptidergic or vanilloid systems.

Diphenyl diselenide attenuates acute thermal hyperalgesia and persistent inflammatory and neuropathic pain behavior in mice.

Experiments were designed to address whether diphenyl diselenide (PhSe)(2) has antiallodynic and antihyperalgesic properties. The neuropathic pain was caused by a partial tying (2/3) of sciatic nerve and the inflammatory pain was induced by an intraplantar (i.pl.) injection of 20 microl of Freund's Complete Adjuvant (CFA) in mice. Seven days after sciatic nerve constriction and 24 h after CFA intraplantar (i.pl.) injection, mouse pain threshold was evaluated through tactile allodynia, using Von Frey Hair (VHF) filaments. The acute thermal hyperalgesia was induced by intrathecal (i.t.) injection of glutamate, N-methyl-d-aspartate (NMDA), bradykinin (BK) and prostaglandin E(2) (PGE(2)), and the nociceptive response was assessed using hot-plate test. (PhSe)(2) administered by oral route (p.o.) (10 mg/kg) decreased the paw withdrawal response on the ipsilateral side of the partial sciatic nerve ligation 30 min after drug administration (64+/-7%) and this effect was kept for 1 h after treatment. (PhSe)(2) (10 mg/kg, p.o.) produced a reduction in mechanical allodynia induced by CFA started 30 min after (PhSe)(2) administration (71+/-5%) and this effect was maintained for up 4 h. (PhSe)(2) (0.1-50 mg/kg, p.o.) caused a significant inhibition of glutamate-, NMDA- and BK-(PGE(2))-induced acute thermal hyperalgesia in mice. Together, the present results indicate that (PhSe)(2) produces systemic antiallodynic action when assessed in mechanical stimulus (VHF) in the hindpaw and also attenuates acute thermal hyperalgesia. Thus, this compound might be potentially interesting in the development of new clinically relevant drugs for the management of pain.

Antinociceptive properties of diphenyl diselenide: evidences for the mechanism of action.

The present study examined the acute toxicity and antinociceptive effects of diphenyl diselenide (PhSe)2, given orally (p.o.), in chemical and thermical models of pain in mice. Diphenyl diselenide (7.8-312 mg/kg, p.o.) did not cause mortality. This compound did not change plasma AST (aspartate aminotransferase) and ALT (alanine aminotransferase) activities as well as urea and creatinine levels in mice after 72 h of exposure. Diphenyl diselenide (1-100 mg/kg, p.o.) inhibited acetic acid-, capsaicin-, glutamate-, bradykinin(BK)- and phorbol myristate acetate (PMA)-induced pain. Diphenyl diselenide also reduced glutamate-, bradykinin-, PMA-induced paw oedema formation. Moreover, diphenyl diselenide caused a significant increase in tail-immersion response latency time. Diphenyl diselenide co-injected subplantarly in association with glutamate-induced a significant reduction of the licking and in the paw oedema formation induced by glutamate. The local pre-treatment of mice with l-arginine, intraplantarly, restored antinociception caused by diphenyl diselenide or N(G)-nitro-L-arginine methyl ester (L-NAME) when analyzed against glutamate-induced nociception. The pre-treatment of mice with dithiothreitol (DTT) intraplantarly restored local antinociception caused by diphenyl diselenide or 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) when analyzed against glutamate-induced nociception. These results indicate that diphenyl diselenide produced antinociception in several models of pain through mechanisms that involve an interaction with not only nitrergic system but also via interaction with redox modulatory sites of glutamate receptors.

Monoaminergic agents modulate antidepressant-like effect caused by diphenyl diselenide in rats.

In this study, the antidepressant-like effect caused by diphenyl diselenide on rat forced swimming test (FST) was investigated. The involvement of the monoaminergic system in the antidepressant-like effect was also evaluated. Diphenyl diselenide (0.1-30 mg/kg), given by oral route (p.o.), 30 min earlier, reduced the immobility time in the FST, without accompanying changes in ambulation when assessed in an open field. The anti-immobility effect of diphenyl diselenide (1 mg/kg, p.o.) on the FST was prevented by pretreatment of rats with p-chlorophenylalanine methyl ester (PCPA; 100 mg/kg, i.p., an inhibitor of serotonin synthesis, given once a day, for 3 consecutive days), WAY100635 (0.1 mg/kg, s.c., a selective 5-HT(1A) receptor antagonist), ketanserin (1 mg/kg, i.p., a 5-HT(2A)/(2C) receptor antagonist), ondasentron (1 mg/kg, i.p., a 5-HT(3) receptor antagonist), haloperidol (1 mg/kg, i.p., a D(1), D(2) and D(3) receptor antagonist), SCH233390 (0.05 mg/kg, s.c., a D(1) receptor antagonist), sulpiride (50 mg/kg, i.p., a D(2) receptor antagonist), prazosin (1 mg/kg, i.p., an alpha(1)-adrenoceptor antagonist), yohimbine (1 mg/kg, i.p., an alpha(2)-adrenoceptor antagonist). However, the anti-immobility effect caused by diphenyl diselenide (1 mg/kg, p.o.) on the FST was not affected by pretreatment with propanolol (2 mg/kg, i.p., a beta-adrenoceptor antagonist). Furthermore, monoamine oxidase (MAO) activity was inhibited (39%) in the animals treated with diphenyl diselenide (30 mg/kg, p.o.) when compared to the control group. Taken together these data demonstrated that the antidepressant-like effect caused by diphenyl diselenide seems to be mediated by involvement of the central monoaminergic system.

The Sesquiterpene Lactone, Budlein A, Inhibits Antigen-Induced Arthritis in Mice: Role of NF-κB and Cytokines.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by debilitating pain, cartilage destruction, and loss of joint function. Management of RA includes drugs that target NF-κB and downstream cytokine production. Therefore, molecules that act by inhibiting this signaling pathway without the severe side effects of, for instance, corticoids would be suitable therapeutic strategies. Budlein A is a sesquiterpene lactone with antinociceptive and anti-inflammatory properties related to the inhibition of pro-inflammatory cytokines and neutrophil recruitment. In this study, the effect of budlein A was evaluated in antigen-induced arthritis (AIA) in mice. At the 26th day, leukocyte recruitment to the knee joint, knee contents of proteoglycans, blood levels of ALT and AST, stomach tissue myeloperoxidase activity, and RT-qPCR for pro-inflammatory gene mRNA expression in knee joint samples was performed. NF-κB luciferase activity was evaluated in RAW 264.7 macrophages. Budlein A treatment dose-dependently inhibited AIA-induced mechanical hyperalgesia, edema, total leukocytes and neutrophil recruitment, and proteoglycan degradation. Budlein A did not induce gastric or liver damage. Budlein also inhibited AIA-induced Il-33, Tnf, Il-1ß, preproET-1, and Cox-2 mRNA expression. In vitro, budlein reduced TNF- and IL-1ß-induced NF-κB activity in RAW 264.7 macrophages. Altogether, we demonstrate that budlein A ameliorates AIA-induced inflammation and pain by targeting NF-κB. Importantly, budlein A does not induce in vivo side effects, suggesting that it possesses a favorable pre-clinical profile as analgesic and it is a prosperous molecule to be further investigated for the treatment of RA.

Mechanisms underlying the hyperalgesic responses triggered by joint activation of TLR4.

BACKGROUND: Toll-like receptors (TLRs) including TLR4 and their signal pathways contribute to the pathogenesis of arthritis. Herein, we evaluated the mechanisms underlying the hyperalgesic response caused by TLR4 activation in the tibio-tarsal joint in mice. METHODS: Joint inflammatory hyperalgesia was induced by intra-articular (ia) injection of LPS (lipopolysaccharide- TLR4 agonist) in C57BL/6, TLR4, TLR2, MyD88, TRIF, TNFR1/2 and IL-1R1 knockout (-/-) mice. Joint hyperalgesia was evaluated using an electronic von Frey. Neutrophil recruitment was assessed by MPO activity. Joint levels of cytokines were measured by ELISA. RESULTS: Firstly, it was shown that LPS injected into the joints causes a dose- and time-dependent reduction in the mechanical nociceptive threshold. The TLR4 activation in the joint triggers mechanical hyperalgesia and neutrophil migration, which was abolished in TLR4 -/- and MyD88-/-, but not in TLR2-/- and TRIF-/- mice. Besides, joint administration of LPS increased the release of TNF-α, IL-1ß, and KC/CXCL1, which were reduced in TLR4-/- and MyD88-/-, but not in TRIF-/- mice. In agreement, the LPS-induced joint nociceptive effect was decreased in TNFR1/2-/- and IL-1R1-/- mice or in mice pre-treated with a CXCR1/2 selective antagonist (DF2156A). CONCLUSIONS: These results suggest that TLR4 activation in the joint produces articular hyperalgesia via MyD88 signaling pathway. Moreover, this pathway is involved in the cascade of events of articular hyperalgesia through mechanisms dependent on cytokines and chemokines production. Thus, TLR4/MyD88 signaling pathway inhibitors might be useful for the treatment of inflammatory joint pain.

Neutrophil Recruitment and Articular Hyperalgesia in Antigen-Induced Arthritis are Modulated by the Cholinergic Anti-Inflammatory Pathway.

The cholinergic anti-inflammatory pathway (CAP) is a complex neuroimmune mechanism triggered by the central nervous system to regulate peripheral inflammatory responses. Understanding the role of CAP in the pathogenesis of rheumatoid arthritis (RA) could help develop new therapeutic strategies for this disease. Therefore, we investigated the participation of this neuroimmune pathway on the progression of experimental arthritis. Using antigen-induced arthritis (AIA) model, we investigated in mice the effects of vagotomy or the pharmacological treatments with hexamethonium (peripheral nicotinic receptor antagonist), methylatropine (peripheral muscarinic receptor antagonist) or neostigmine (peripheral acetylcholinesterase inhibitor) on AIA progression. Unilateral cervical vagotomy was performed 1 week before the immunization protocol with methylated bovine serum albumin (mBSA), while drug administration was conducted during the period of immunization. On day 21, 6 hr after the challenge with mBSA injection in the femur-tibial joint, the local neutrophil migration and articular mechanical hyperalgesia were assessed. Herein, we observed that vagotomy or blockade of peripheral nicotinic (but not muscarinic) receptors exacerbated the clinical parameters of this disease. Moreover, peripheral acetylcholinesterase inhibition by neostigmine treatment promoted a reduction of neutrophil recruitment in the knee joint and articular hyperalgesia. Our results demonstrated that peripheral activation of CAP modulates experimental arthritis, providing a pre-clinical evidence of a potential therapeutic strategy for RA.

Pharmacokinetic properties, in vitro metabolism and plasma protein binding of govaniadine an alkaloid isolated from Corydalis govaniana Wall.

Govaniadine (GOV) is an alkaloid isolated from Corydalis govaniana Wall. It has been reported to show a different number of biological activities including anti-urease, leishmanicidal and antinociceptive. The present study aims to characterize the GOV in vitro metabolism after incubation with rat and human liver microsomes (RLM and HLM, respectively) and to evaluate its pharmacokinetic properties. The identification of GOV metabolites was conducted by different mass analyzers: a micrOTOF II-ESI-ToF Bruker Daltonics® and an amaZon-SL ion trap (IT) Bruker Daltonics®. For the pharmacokinetic study of GOV in rats after intravenous administration, a LC-MS/MS method was developed and applied to. The analyses were performed using an Acquity UPLC® coupled to an Acquity TQD detector equipped with an ESI interface. The liver microsomal incubation resulted in new O-demethylated, di-hydroxylated and mono-hydroxylated compounds. Regarding the method validation, the calibration curve was linear over the concentration range of 2.5-3150.0ngmL-1, with a lower limit of quantitation (LLOQ) of 2.5ngmL-1. This method was successfully applied to a pharmacokinetic study. The profile was best fitted to a two-compartment model, the first phase with a high distribution rate constant (α) 0.139±0.086min-1, reflected by the short distribution half-life (t1/2α) 9.2±8.9min and the later one, with an elimination half-life (t1/2ß) 55.1±37.9min. The main plasma protein binding was 96.1%. This is a first report in this field and it will be useful for further development of govaniadine as a drug candidate.

Dynamic weight bearing is an efficient and predictable method for evaluation of arthritic nociception and its pathophysiological mechanisms in mice.

The assessment of articular nociception in experimental animals is a challenge because available methods are limited and subject to investigator influence. In an attempt to solve this problem, the purpose of this study was to establish the use of dynamic weight bearing (DWB) as a new device for evaluating joint nociception in an experimental model of antigen-induced arthritis (AIA) in mice. AIA was induced in Balb/c and C57BL/6 mice, and joint nociception was evaluated by DWB. Western Blotting and real-time PCR were used to determine protein and mRNA expression, respectively. DWB detected a dose- and time-dependent increase in joint nociception during AIA and was able to detect the dose-response effects of different classes of analgesics. Using DWB, it was possible to evaluate the participation of spinal glial cells (microglia and astrocytes) and cytokines (IL-1ß and TNFα) for the genesis of joint nociception during AIA. In conclusion, the present results indicated that DWB is an effective, objective and predictable test to study both the pathophysiological mechanisms involved in arthritic nociception in mice and for evaluating novel analgesic drugs against arthritis.