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

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

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

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

B₁R inhibition affects the peripheral and central MOG-specific immune responses during EAE pathology.

<p>Splenocytes (A, B) and drained lymph node (C) were isolated from immunized mice (EAE group) and B₁R^−/− and B₂R^−/− mice on days 14 and 25 p.i. The cells (1×10⁶/well) were cultured in the presence or absence of MOG_35–55 (10 µg/ml) for 72 h to assess proliferation by [³H] thymidine incorporation. Total RNA was extracted from the lumbar spinal cord of mice in the naive group, the control group (EAE), DALBK (50 nmol/kg) mice, HOE-140 (50 nmol/kg) mice, B₁R^−/− mice and B₂R^−/− mice on day 25 p.i. The mRNA levels of IL-17 (D), ROR-γT (E), TNF-α (F), IFN- γ (G), T-bet (H) and B₁R (I) were measured. GAPDH mRNA was used to normalize the relative amounts of mRNA. 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, **P<0.001 versus the EAE group (one-way ANOVA with the Newmann-Keuls post-hoc test).</p

Kinin B₁R inhibition or its deletion reduced the production of inflammatory cytokines and the activation of isolated CD4⁺ T lymphocytes.

<p>Lymphocytes (1×10⁶/well) obtained 25 days after immunization from the naive group, the control group (EAE), from mice pre-treated with the B₁R antagonist DALBK (50 nmol/kg), from mice pre-treated with the B₂R antagonist HOE-140 (50 nmol/kg), from B₁R^−/−mice and from B₂R^−/− mice were cultured in the presence or absence of MOG_35–55 (10 µg/ml) for 3 days; the supernatants were collected and measured for the concentrations of TNF-α (A, B), IFN-γ (C, D), IL-17 (E, F) and IL-4 (G, H) using ELISA assays. After culture supernatants were collected, the cells were analyzed by flow cytometry for CD4⁺CD69⁺ (I, J) and CD8⁺CD69⁺ (K, L) T cells. Each column represents the mean ± SEM of six to nine mice per group and is representative of three independent experiments. ^#P<0.05, ^##P<0.001 versus the naive group, *P<0.05, **P<0.001 versus the EAE group (one-way ANOVA with the Newmann-Keuls post-hoc test).</p

Pro and anti-inflammatory molecule expression in B1KO and wild type animals.

<p>All molecule expressions were measured by real-time PCR at 24 hours of reperfusion. B1KO group had lower pro-inflammatory molecule expression (T-bet and IL-1β) (A and B) and higher anti-inflammatory response (GATA-3, IL-4 and IL-10) (C, D and E). Statistical analyses were performed using the t-test.* B1KO <i>versus</i> B1B2WT, p<0.05.</p

Oligonucleotides sequence.

<p>Oligonucleotides sequence.</p