Activation of kinin receptor B1 limits encephalitogenic T lymphocyte recruitment to the central nervous system

Abstract

Previous proteomic and transcriptional analyses of multiple sclerosis lesions revealed modulation of the renin-angiotensin and the opposing kallikrein-kinin pathways. Here we identify kinin receptor B1 (Bdkrb1) as a specific modulator of immune cell entry into the central nervous system (CNS). We demonstrate that the Bdkrb1 agonist R838 (Sar-[D-Phe]des-Arg(9)-bradykinin) markedly decreases the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in SJL mice, whereas the Bdkrb1 antagonist R715 (Ac-Lys-[D-betaNal(7), Ile(8)]des-Arg(9)-bradykinin) resulted in earlier onset and greater severity of the disease. Bdkrb1-deficient (Bdkrb1(-/-)) C57BL/6 mice immunized with a myelin oligodendrocyte glycoprotein fragment, MOG(35-55), showed more severe disease with enhanced CNS-immune cell infiltration. The same held true for mixed bone marrow-chimeric mice reconstituted with Bdkrb1(-/-) T lymphocytes, which showed enhanced T helper type 17 (T(H)17) cell invasion into the CNS. Pharmacological modulation of Bdkrb1 revealed that in vitro migration of human T(H)17 lymphocytes across blood-brain barrier endothelium is regulated by this receptor. Taken together, these results suggest that the kallikrein-kinin system is involved in the regulation of CNS inflammation, limiting encephalitogenic T lymphocyte infiltration into the CNS, and provide evidence that Bdkrb1 could be a new target for the treatment of chronic inflammatory diseases such as multiple sclerosis.

Figure 1. Morphological and functional evidence for the involvement of the kallikrein-kinin system in autoimmune CNS inflammation

(a) Histopathology for CD3 (green), kinin receptor B1 (Bdkrb1; red) and cell nuclei (Hoechst; blue), including overlay analysis in the CNS from a human with multiple sclerosis (top) or from a mouse with EAE (bottom). Asterisk marks the lumen of a blood vessel. Scale bars, 10 µm. (b) Pharmacological modulation of Bdkrb1 in adoptive transfer EAE in SJL mice. One of two experiments is shown. Mice were distributed into three groups and received intraperitoneal injections of either the Bdkrb1 agonist R838, the Bdkrb1 antagonist R715 or vehicle daily for days 0–10 (n = 6 per group). (c) Therapeutic treatment effect for the Bdkrb1 agonist R838, given after disease onset twice daily, is demonstrated in SJL mice immunized with PLP_139–151 (n = 6 per group). Representative results from two independent experiments are shown. (d) Bdkrb1 deficiency enhances autoimmune neuroinflammation. Clinical scores for wild-type (WT) EAE (n = 9) and Bdkrb1^−/− EAE (n = 9) mice after immunization with MOG_35–55. Representative results from three independent EAE experiments are shown. (e) Combined disruption of Bdkrb1 and Bdkrb2 results in a disease course similar to that seen with Bdkrb1 disruption alone. Bdkrb1^−/− ;Bdkrb2^−/− (n = 5) and Bdkrb1^−/− mice (n = 6) were immunized with MOG_35–55. Representative results from two independent EAE experiments are shown. For all EAE courses, mean disease scores ± s.e.m. are displayed; ^**P < 0.01, ^*P < 0.05, repeated-measures ANOVA.

Figure 2. Bdkrb1 deficiency leads to enhanced EAE pathology

(a–d) Histopathological analysis of three sections per mouse, comprising the assessment of inflammation by H&E staining (a), microglia and macrophage infiltration by immunohistochemistry for Iba-1 (b), demyelination by luxol fast blue staining (arrows indicate demyelinated areas) (c) and axonal damage by immunohistochemistry for APP (d). Representative images from spinal cord longitudinal sections and corresponding quantifications are shown. Scale bars, 50 µm. (e–h) Deficiency of Bdkrb1 has no impact on myelin-specific inflammatory responses in the periphery. Proliferation in response to MOG_35–55 in cells from draining lymph nodes (e,g,h) or spleens (f) from Bdkrb1^−/− or wild-type (WT) mice with EAE killed after immunization with MOG_35–55 but before the onset of disease. Shown are the results of [³H]thymidine incorporation assays in response to MOG_35–55 (e,f) and flow cytometric assessment of the expression of surface activation markers and cytokines (g,h). Values are means ± s.d.; ^**P < 0.01, ^***P < 0.001, Mann-Whitney U-test.

Figure 3. Bdkrb1 controls the migratory capacities of T cells targeting the CNS

(a) Induction of EAE by MOG_35–55 immunization of lethally irradiated C57BL/6-CD45.1 recipients reconstituted with Bdkrb1-deficient (Bdkrb1^−/− → WT; n = 5) bone marrow or a mix of TCR-β-chain– and Bdkrb1-deficient bone marrow (Bdkrb1^−/−Tcrb^−/− → WT; n = 6). To control for the impact of the transplantation procedure, control mice received a mix of TCR-β-chain–deficient and C57BL/6 WT bone marrow (WTTcrb^−/− → WT, n = 7). (b) EAE course in Bdkrb1-deficient mice that received a mix of TCR-β-chain–deficient and C57BL/6-CD45.1 bone marrow (WT Tcrb^−/− → Bdkrb1^−/−, n = 5), as compared to that in Bdkrb1^−/− → WT mice (n = 4) and Bdkrb1^−/− → Bdkrb1^−/− (n = 5) chimeras. Mean clinical disease scores ± s.e.m. are given for all three groups. ^*P < 0.05, repeated-measures ANOVA. Representative results from three (a) and two (b) independent EAE experiments are shown. (c) C57BL/6 recipients were reconstituted with mixed Bdkrb1^−/− (C57BL/6-CD45.2) and WT (C57BL/6-CD45.1) bone marrow (1:1 ratio; n = 6), and CD4⁺ T cells were isolated from the CNS at disease peak. ^**P < 0.01, Mann-Whitney U-test. (d) Migratory capacity, toward a CXCL12 chemokine gradient in a Transwell system, of PLP_139–151-specific T cells (from immunized SJL mice) and MOG_35–55-specific CD4⁺ T cells (from immunized Bdkrb1^−/− mice) that were seeded on a mouse bEnd3 brain–derived endothelial cell monolayer. Bdkrb1 was modulated by incubating T cells with the Bdkrb1 agonist R838 or the Bdkrb1 antagonist R715 before application to the endothelial monolayer. (e) Decreased F-actin polymerization of CD4⁺ T cells upon Bdkrb1 activation. (f) Downregulation of the small GTPase RhoA in CD4⁺ T cells by activation of Bdkrb1. Shown is an immunoblot of a cell extract prepared from T cells after RhoA pulldown. GTPγS-loaded controls were used as a positive control for RhoA pulldown.

Figure 4. Bdkrb1 activation primarily targets the invasion of T_H17 cells

(a–c) Proportions of CD4⁺ cells in the CNS (a) and of IL-17⁺ and IFN-γ⁺ cells within the CD4⁺ T cell population (b,c) in C57BL/6 Rag1^−/− mice with adoptive transfer EAE induced by injection of Bdkrb1^−/− or WT T cells. Immune cells were isolated from the CNS of four mice per group at day 13 after cell transfer (which corresponds to the time of disease onset). (d–f) Proportions of IL-17⁺ cells (d), IFN-γ⁺ cells (e) and FoxP3⁺ CD25⁺ cells (f) within the CD4⁺ T cell population from CNS-invading immune cells recovered from bone marrow chimeric mice with T cells lacking Bdkrb1 or from control mice (see Fig. 3a). (g) Bdkrb1 activation primarily targets the migration of human memory CD45RO⁺ T_H17 rather than T_H1 cells across human brain-derived microvascular endothelium; ^*P < 0.05, Mann-Whitney U-test. (h) Increased expression of Bdkrb1 in human T_H17 cells analyzed by FACS. (i,j) Activation of Bdkrb1 decreased the average number of Celltracker Orange–labeled T_H17 cells after application to hippocampal slice cultures for multiphoton microscopy. The average number of T cells per minute and per defined volume (between 60–120 m depth) over time is shown, including quantification (i) (see also Supplementary Movies 1,2,3) and representative overviews (j). Data shown are means ± s.e.m.; ^*P < 0.05, Mann-Whitney U-test.