The Role of Kinin Receptors in Preventing Neuroinflammation and Its Clinical Severity during Experimental Autoimmune Encephalomyelitis in Mice

Background: Multiple sclerosis (MS) is a demyelinating and neuroinflammatory disease of the human central nervous system (CNS). the expression of kinins is increased in MS patients, but the underlying mechanisms by which the kinin receptor regulates MS development have not been elucidated.Methodology/Principal Findings: Experimental autoimmune encephalomyelitis (EAE) was induced in female C57BL/6 mice by immunization with MOG(35-55) peptide emulsified in complete Freund's adjuvant and injected with pertussis toxin on day 0 and day 2. Here, we report that blockade of the B(1)R in the induction phase of EAE markedly suppressed its progression by interfering with the onset of the immune response. Furthermore, B(1)R antagonist suppressed the production/expression of antigen-specific T(H)1 and T(H)17 cytokines and transcription factors, both in the periphery and in the CNS. in the chronic phase of EAE, the blockade of B(1)R consistently impaired the clinical progression of EAE. Conversely, administration of the B(1)R agonist in the acute phase of EAE suppressed disease progression and inhibited the increase in permeability of the blood-brain barrier (BBB) and any further CNS inflammation. of note, blockade of the B(2)R only showed a moderate impact on all of the studied parameters of EAE progression.Conclusions/Significance: Our results strongly suggest that kinin receptors, mainly the B(1)R subtype, play a dual role in EAE progression depending on the phase of treatment through the lymphocytes and glial cell-dependent pathways.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Programa de Apoio aos Nucleos de Excelencia (PRONEX), BrazilFundacaode Apoio a Pesquisa Cientifica Tecnologica do Estado de Santa Catarina (FAPESC), BrazilUniv Fed Santa Catarina, Dept Pharmacol, Ctr Biol Sci, Florianopolis, SC, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilWeb of Scienc

The role of kinin B-1 and B-2 receptors in the persistent pain induced by experimental autoimmune encephalomyelitis (EAE) in mice: Evidence for the involvement of astrocytes

Multiple sclerosis (MS) is a progressive, demyelinating inflammatory disease of the human central nervous system (CNS). While the primary symptoms of MS affect motor function, it is now recognized that chronic pain is a relevant symptom that affects both animals and MS patients. There is evidence that glial cells, such as astrocytes, play an important role in the development and maintenance of chronic pain. Kinins, notably bradykinin (BK) acting through B-1 (B1R) and B-2 (B2R) receptors, play a central role in pain and inflammatory processes. However, it remains unclear whether kinin receptors are involved in neuropathic pain in MS. Here we investigated by genetic and pharmacological approaches the role of kinin receptors in neuropathic pain behaviors induced in the experimental autoimmune encephalomyelitis (EAE) mouse model. Our results showed that gene deletion or antagonism of kinin receptors, especially B1R, significantly inhibited both tactile and thermal hypersensitivity in EAE animals. By contrast, animals with EAE and treated with a B1R selective agonist displayed a significant increase in tactile hypersensitivity. We also observed a marked increase in B1R mRNA and protein level in the mouse spinal cord 14 days after EAE immunization. Blockade of B1R significantly suppressed the levels of mRNAs for IL-17, IFN-gamma, IL-6, CXCL-1/KC, COX-2 and NOS2, as well as glial activation in the spinal cord. of note, the selective B-1 antagonist DALBK consistently prevented IFN-induced up-regulation of TNF-alpha and IL-6 release in astrocyte culture. Finally, both B1R and B2R antagonists significantly inhibited COX-2 and NOS2 expression in primary astrocyte culture. the B1R was co-localized with immunomarker of astrocytes in the spinal cord of EAE-treated animals. the above data constitute convincing experimental evidence indicating that both kinin receptors, especially the B-1 subtype, exert a critical role in the establishment of persistent hypersensitivity observed in the EAE model, an action that seems to involve a central inflammatory process, possibly acting on astrocytes. Thus, B-1 selective antagonists or drugs that reduce kinin release may have the potential to treat neuropathic pain in patients suffering from MS. (C) 2013 Elsevier Inc. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Programa de Apoio aos Nucleos de Excelencia (PRONEX) of BrazilFundacao de Apoio a Pesquisa do Estado de Santa Catarina (FAPESC) of BrazilUniv Fed Santa Catarina, Lab Autoimunidade & Imunofarmacol, BR-88900000 Ararangua, SC, BrazilUniv Fed Santa Catarina, Ctr Biol Sci, Dept Pharmacol, BR-88040970 Florianopolis, SC, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04023062 São Paulo, BrazilWeb of Scienc

The Role of Kinin Receptors in Preventing Neuroinflammation and Its Clinical Severity during Experimental Autoimmune Encephalomyelitis in Mice

Background: Multiple sclerosis (MS) is a demyelinating and neuroinflammatory disease of the human central nervous system (CNS). The expression of kinins is increased in MS patients, but the underlying mechanisms by which the kinin receptor regulates MS development have not been elucidated. Methodology/Principal Findings: Experimental autoimmune encephalomyelitis (EAE) was induced in female C57BL/6 mice by immunization with MOG 35–55 peptide emulsified in complete Freund’s adjuvant and injected with pertussis toxin on day 0 and day 2. Here, we report that blockade of the B1R in the induction phase of EAE markedly suppressed its progression by interfering with the onset of the immune response. Furthermore, B1R antagonist suppressed the production/expression of antigen-specific TH1 and TH17 cytokines and transcription factors, both in the periphery and in the CNS. In the chronic phase of EAE, the blockade of B 1R consistently impaired the clinical progression of EAE. Conversely, administration of the B 1R agonist in the acute phase of EAE suppressed disease progression and inhibited the increase in permeability of the bloodbrain barrier (BBB) and any further CNS inflammation. Of note, blockade of the B2R only showed a moderate impact on all of the studied parameters of EAE progression. Conclusions/Significance: Our results strongly suggest that kinin receptors, mainly the B 1R subtype, play a dual role in EA

Schematic representation of the mechanism via which kinin regulates the physiopathology of the EAE model.

<p>The blockade of kinin B₁R in either the induction or the chronic phase of EAE suppressed disease progression with the concomitant suppression of T_H1 and T_H17-myelin-specific cell development in at least two different stages: (1) during onset of the peripheral immune response, through the modulation of differentiation and/or expansion of auto-aggressive T_H cells in the MOG_35–55-specific immune responses; and (2) during neuroinflammation by affecting the auto-aggressive function of T cells and astrocytes. However, the blockade of B₁R in the acute phase of EAE only had a slight effect, whereas that of the B₁ agonist also given at this time markedly reduced disease severity through the inhibition of increased BBB permeability and cell migration, and consequently blocked CNS inflammation. Altogether, we found that kinins, especially B₁R subtypes, have a dual role during the progression of EAE by distinct mechanisms of action at each stage of disease progression. APC: antigen-presenting cell; DALBK: des-Arg⁹-[Leu⁸]-BK; HOE-140: D-Arg-[Hyp³,Thi⁵,D-Tic⁷,Oic⁸]-BK; DABK: des-Arg⁹-BK; MOG: myelin oligodrendrocyte glycoprotein; B₁R: kinin B₁ receptor; B₂R: kinin B₂ receptor; T_HP: precursor T cell; BBB: blood-brain barrier; CXCL1/KC: keratinocyte-derived chemokine, TNF-α; tumour necrosis factor-alpha; IFN-γ: interferon-gamma; TGF-β: transforming growth factor beta; PMN: polymorphonuclear leukocytes; GFAP: glial fibrillary acidic protein; Iba1: ionized calcium binding adaptor molecule 1; CREB: cAMP response element-binding; NF-H: neurofilament-H. (), inhibition; ), stimulation.</p

Blockade of kinin B₁R in the chronic disease phase decreased the clinical symptoms and neuroinflammation in the EAE model.

<p>The clinical score (A) and locomotor activity (B) were analyzed 25 days after the animals were immunized with MOG_35–55 peptide/CFA. The inflammatory infiltrates (H&E staining; C, E), demyelination area (LFB staining; D, F), immunoreactivity of GFAP (G), Iba-1 (H), phospho-CREB (I) and neurofilament-H (J) were analyzed 25 days p.i. in the lumbar spinal cords of mice in the naive group, the control group (EAE) and in mice treated in the chronic phase (days 15–20) with B₁R (DALBK, 50 nmol/kg, i.p.) or B₂R (HOE-140, 150 nmol/kg, i.p.) antagonists. Specifically, four alternate 5-µm sections (six to nine animals/group) of the white matter (E–I) and grey matter (J) of the lumbar spinal cord were obtained between L4–L6. Scale bar corresponds to 25 µm and applies throughout. The data are presented as mean ± SEM of six to nine mice/group and are representative of three independent experiments. ^#P<0.05, ^##P<0.001 versus the naive group, *P<0.05 and **P<0.001 versus the EAE group (one-way ANOVA with the Newmann-Keuls post-hoc test).</p

Kinin B₁R inhibition or genetic deletion decreased the level of inflammatory cell infiltration and the demyelination area in experimental EAE.

<p>The lumbar spinal cords were histologically analyzed on day 25 p.i. in the different experimental groups for inflammation by H&E staining (A, B, E) and for demyelination by luxol fast blue staining (C, D, F). The degree of inflammatory infiltrates and demyelination was quantified from an average of four ocular field 5-µm sections of lumbar spinal cord white matter transverse sections per mouse for a total of six to nine mice/group in the naive group, the control group (EAE), in mice pre-treated with the B₁R antagonist DALBK (50 nmol/kg), in mice pre-treated with the B₂R antagonist HOE-140 (150 nmol/kg), in B₁R^−/− knockout mice and in B₂R^−/− knockout mice. Scale bar corresponds to 25 µm and applies throughout. Data are presented as mean ± SEM of six to nine mice/group and are representative of three independent experiments. ^#P<0.05 and ^##P<0.001 versus the naive group, *P<0.05 and **P<0.001 versus the EAE group (one-way ANOVA with the Newmann-Keuls post-hoc test).</p

Kinin B₁R inhibition or deletion in the disease induction phase attenuated the development of EAE and reduced the inflammatory response in the CNS 25 days post-immunization (p.i.).

<p>Animals were immunized with MOG_35–55 peptide/CFA and pertussis toxin. (A) Schematic representation of EAE progression. Day 0 to 7: induction phase; day 7 to 15: acute phase and day 15 to 25: chronic phase of the disease. EAE: experimental autoimmune encephalomyelitis; T_H: CD4⁺ T helper lymphocytes. The clinical score (B, E), area under the curve (AUC) (C, F) and locomotor activity (D, G) were analyzed in the naive group, the control group (EAE), in mice pre-treated with the selective kinin B₁R antagonist DALBK (50 nmol/kg), in mice pre-treated with the selective kinin B₂R antagonist HOE-140 (50 nmol/kg), in B₁R^−/− knockout mice and in B₂R^−/− knockout mice 25 days p.i. The antagonists were administered intraperitoneally (i.p.), twice a day (12/12 h), for 5 days (day 0–5). The results of clinical score are expressed as mean or as the AUC. Data are presented as mean ± SEM of six to nine mice/group and are representative of three independent experiments. ^#<i>P</i><0.05 and ^##<i>P</i><0.001 versus the naive group, *P<0.05 and **P<0.001 versus the EAE group, ^ΔP<0.05 and ^ΔΔP<0.001 versus the DALBK treatment or B₁R^−/− mice (one-way ANOVA with the Newmann-Keuls post-hoc test).</p

Mechanisms Underlying the Scratching Behavior Induced by the Activation of Proteinase-Activated Receptor-4 in Mice

A role for proteinase-activated receptor-4 (PAR-4) was recently suggested in itch sensation. Here, we investigated the mechanisms underlying the pruriceptive actions of the selective PAR-4 agonist AYPGKF-NH2 (AYP) in mice. Dorsal intradermal (i.d.) administration of AYP elicited intense scratching behavior in mice, which was prevented by the selective PAR-4 antagonist (pepducin P4pal-10). PAR-4 was found to be coexpressed in 32% of tryptase-positive skin mast cells, and AYP caused a 2-fold increase in mast cell degranulation. However, neither the treatment with cromolyn nor the deficiency of mast cells (WBB6F1-KitW/Wv mice) was able to affect AYP-induced itch. PAR-4 was also found on gastrin-releasing peptide (GRP)-positive neurons (pruriceptive fibers), and AYP-induced itch was reduced by the selective GRP receptor antagonist RC-3095. In addition, AYP evoked calcium influx in ∼1.5% of cultured DRG neurons also sensitive to TRPV1 (capsaicin) and/or TRPA1 (AITC) agonists. Importantly, AYP-induced itch was reduced by treatment with either the selective TRPV1 (SB366791), TRPA1 (HC-030031), or NK1 (FK888) receptor antagonists. However, genetic loss of TRPV1, but not of TRPA1, diminished AYP-induced calcium influx in DRG neurons and the scratching behavior in mice. These findings provide evidence that PAR-4 activation by AYP causes pruriceptive itch in mice via a TRPV1/TRPA1-dependent mechanism

The blockade of kinin B₁R in the disease induction phase by pharmacological treatment or genetic deletion ameliorated EAE pathology.

<p>The spinal lumbar cords obtained on the 25^th day after immunization from the different experimental groups were processed for immunohistochemistry assays: T cell infiltration by CD3 immunoreactivity (A–F); astrocytes activation by GFAP immunoreactivity (G–L); and CREB phosphorylation (M–R). Specifically, four 5-µm sections of lumbar spinal cord white matter (six to nine mice/group)≈150 µm apart were obtained between L4 and L6 from the naive group (A, G and M), the EAE group (B, H and N), from mice pre-treated with the B₁R antagonist DALBK (50 nmol/kg) (C, I and O), from mice pre-treated with the B₂R antagonist HOE-140 (150 nmol/kg) (D, J and P) and from mice deficient in B₁R (E, K and Q) and B₂R (F, L and R). The antagonists were administered i.p. twice a day (12/12 h), during day 0–5 p.i. Representative sections from three independent experiments are shown. Scale bar corresponds to 25 µm and applies throughout.</p

Activation of kinin B₁R in the acute disease phase improved the pathology of EAE.

<p>The clinical score (A, C and I) and area under the curve (AUC) (B, D and J) were analyzed in the induction (day 0–5), acute (day 7–17) and chronic (day 15–20) phases of EAE, respectively. The animals were separated into different groups: naive group, EAE group, mice treated with the B₁R agonist DABK (300 nmol/kg, i.p.) and mice treated with the B₁R antagonist DALBK (50 nmol/kg, i.p.). The agonists/antagonists were administered intraperitoneally (i.p.), twice a day (12/12 h), during different time-points of EAE. In the acute phase of EAE, the pharmacological activation of B₁R (DABK treatment: 300 nmol/kg, i.p.) significantly decreased Evan's blue extravasations in the spinal cord on day 21 compared to the EAE control group (E) and the mRNA levels of IL-17 (F), IFN-γ (G) and TNF-α (H), as measured by RT-PCR. The GAPDH mRNA was used as an endogenous control in the RT-PCR assay. The data are presented as mean ± SEM of six to nine mice/group and are representative of four independent experiments. ^#P<0.05, ^##P<0.001 versus the naive group, *P<0.05 and **P<0.001 versus the EAE-treated group (one-way ANOVA with the Newmann-Keuls post-hoc test). N.D. not detected.</p