VPAC1 receptor (Vipr1)-deficient mice exhibit ameliorated experimental autoimmune encephalomyelitis, with specific deficits in the effector stage

Abstract

Background: Vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating polypeptide (PACAP) are two highly homologous neuropeptides. In vitro and ex vivo experiments repeatedly demonstrate that these peptides exert pronounced immunomodulatory (primarily anti-inflammatory) actions which are mediated by common VPAC1 and VPAC2 G protein-coupled receptors. In agreement, we have shown that mice deficient in PACAP ligand or VPAC2 receptors exhibit exacerbated experimental autoimmune encephalomyelitis (EAE). However, we observed that VIP-deficient mice are unexpectedly resistant to EAE, suggesting a requirement for this peptide at some stage of disease development. Here, we investigated the involvement of VPAC1 in the development of EAE using a VPAC1-deficient mouse model.

Methods: EAE was induced in wild-type (WT) and VPAC1 knockout (KO) mice using myelin oligodendrocyte glycoprotein 35-55 (MOG35-55), and clinical scores were assessed continuously over 30 days. Immune responses in the spinal cords were determined by histology, real-time PCR and immunofluorescence, and in the draining lymph nodes by antigen-recall assays. The contribution of VPAC1 expression in the immune system to the development of EAE was evaluated by means of adoptive transfer and bone marrow chimera experiments. In other experiments, VPAC1 receptor analogs were given to WT mice.

Results: MOG35-55-induced EAE was ameliorated in VPAC1 KO mice compared to WT mice. The EAE-resistant phenotype of VPAC1 KO mice correlated with reduced central nervous system (CNS) histopathology and cytokine expression in the spinal cord. The immunization phase of EAE appeared to be unimpaired because lymph node cells from EAE-induced VPAC1 KO mice stimulated in vitro with MOG exhibited robust proliferative and Th1/Th17 responses. Moreover, lymph node and spleen cells from KO mice were fully capable of inducing EAE upon transfer to WT recipients. In contrast, WT cells from MOG-immunized mice did not transfer the disease when administered to VPAC1 KO recipients, implicating a defect in the effector phase of the disease. Bone marrow chimera studies suggested that the resistance of VPAC1-deficient mice was only minimally dependent on the expression of this receptor in the immunogenic/hematopoietic compartment. Consistent with this, impaired spinal cord inductions of several chemokine mRNAs were observed in VPAC1 KO mice. Finally, treatment of WT mice with the VPAC1 receptor antagonist PG97-269 before, but not after, EAE induction mimicked the clinical phenotype of VPAC1 KO mice.

Conclusions: VPAC1 gene loss impairs the development of EAE in part by preventing an upregulation of CNS chemokines and invasion of inflammatory cells into the CNS. Use of VPAC1 antagonists in WT mice prior to EAE induction also support a critical role for VPAC1 signaling for the development of EAE.

Keywords: Experimental autoimmune encephalomyelitis; Multiple sclerosis; Neuroimmunomodulation; Neuropeptide; VPAC1.

Fig. 1

Reduced clinical EAE and histopathology in VPAC1 KO vs. WT mice. EAE was induced by immunizing mice subcutaneously with 100 μg of MOG_35–55 in CFA supplemented with Mycobacterium tuberculosis. EAE clinical scores were monitored daily on a scale of 0 to 4 as described in the “Methods” section. Spinal cord microsections were stained with luxol fast blue (for demyelination) and hematoxylin-eosin (for immune cell infiltration), and histopathology was scored from 0 to 3 as described in the “Methods” section. a Clinical curve displaying mean clinical scores ± SEM of immunized WT vs. VPAC1 KO mice, representing the summation of three experiments (n = 18 mice per genotype). b Clinical curve displaying mean clinical scores ± SEM of immunized WT mice pretreated with PBS or a VPAC1 antagonist, representing the summation of two experiments (n = 15 for WT-PBS and n = 11 for WT-VPAC1 antagonist). c Mean histopathological score at day 30 of three experiments ± SEM (n = 12 in each group). ***p < 0.001 Student’s t test. ND = not determined. d Low- (×4) and e high- (×20) magnification photomicrographs of day 30 WT and VPAC1 KO mice transverse thoracolumbar spinal cord sections

Fig. 2

Cytokine expression in the CNS of EAE-immunized mice is reduced in VPAC1 KO mice. EAE was induced in WT- and VPAC1-deficient mice, and the spinal cords were collected and fresh-frozen in liquid nitrogen 30 days later. RNA was extracted and retrotranscribed to cDNA, and the levels of expression of TNFα, IL-6, IFNγ, IL-17, IL-23p19, IL-4, IL-10, and Foxp3 determined by real-time RT-PCR as described in the “Methods” sections. Results shown are representative of three independent experiments of n = 8 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant; Student’s t test

Fig. 3

Antigen-recall assay demonstrates robust responses to MOG of lymph node cells from either WT or VPAC1 KO mice after EAE immunization. EAE was induced to WT and VPAC1 KO mice and draining lymph nodes were isolated at the peak of the WT disease (day 14). Cells were cultured for 48 h at 1 × 10⁶ cells/ml in 96-well plates in the presence of OVA or MOG (10 μg/ml). a Proliferation determined by measurement of [³H]-thymidine incorporation. b Cytokine responses in WT vs. VPAC1 KO lymph node cultures. The levels of IFNγ (Th1), IL-17 (Th17), and IL-10 (Th2/Treg) were measured in the supernatants by ELISA. The expression of IL-4 mRNA in the cultured cells was determined by real-time RT-PCR. Results shown are representative of three independent experiments of n = 8 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test

Fig. 4

VPAC1-deficient mice exhibit reduced immune cell infiltration into the CNS meninges and parenchyma after EAE induction. EAE was induced in WT and VPAC1 KO mice as described in the “Methods”, and the spinal cord tissues were obtained on day 14 post-immunization. Cryostat section were stained by immunofluorescence for CD4 (Alexa 594), laminin (FITC), and nuclei (DAPI). Representative photomicrographs at ×20 magnification are shown. Results shown are representative of two independent experiments of n = 3 mice/group

Fig. 5

The expression of VPAC1 in the immune/hematopoietic compartment is not critical for the development of full EAE. Adoptive transfer and bone marrow chimera studies were performed in order to investigate the contribution of VPAC1 receptor expression in immune/hematopoietic cells to the resistant phenotype observed in VPAC1 KO mice. a For adoptive transfer experiments, 5 × 10⁶ splenocytes and 5 × 10⁶ lymph node cells from EAE-immunized WT and VPAC1 KO mice restimulated with MOG in vitro for 3 days were injected i.v. into naïve WT or VPAC1 KO recipients. The EAE clinical curve of a representative experiment out of three with n = 5 for each group is shown. b In order to generate bone marrow (BM) chimeric mice, WT mice were γ-irradiated (n = 5 for WT→WT and n = 9 for VPAC1 KO→WT) and received 10⁷ BM cells from either VPAC1 receptor KO (CD45.2) or WT (CD45.2) mice. Six weeks later, mice were MOG_35–55-immunized for EAE induction. *p < 0.05, Student’s t test

Fig. 6

Chemokine and chemokine receptor but not adhesion molecule expressions in the CNS of EAE-immunized mice are reduced in VPAC1 KO mice. EAE was induced in WT- and VPAC1-deficient mice, and the spinal cords were collected and fresh-frozen in liquid nitrogen at the peak of EAE (14–15 days later). RNA was extracted and retrotranscribed to cDNA, and the levels of expression of a the lymphocyte chemokines RANTES/CCL5, MIG/CXCL9 (Th1), IP-10/CXCL10 (Th1), and MIP-3α/CCL20 (Th17); b the monocyte chemokines MCP-1/CCL2 and MCP-2/CCL8; c the chemokine receptors CCR1, CCR2, and CCR5; and d the adhesion molecules ICAM and VCAM were determined by real-time RT-PCR as described in the “Methods” sections. Results shown are representative of two independent experiments of n = 5 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant; Student’s t test

Fig. 7

Scheme suggesting potential roles of VPAC1 on EAE. Upon MOG administration, EAE has two phases: the induction or priming phase and the effector phase. In the induction phase, MOG drains to the local lymph nodes and T cells are activated and polarized towards Th1 and Th17 cells. In the effector phase, these cells migrate to the CNS, where they mount an inflammatory response amplified by immune cell recruitment. Our data suggests that VPAC1 signaling on non-immune cells or microglia may be required for the effector phase of EAE and may explain the lack of clinical disease in VPAC1 KO mice or upon pretreatment of WT mice with a VPAC1 antagonist (VPAC1 antago). VPAC1 anti-inflammatory actions would be prevalent only during ongoing disease, and thus a VPAC1 agonist (VPAC1 ago) administered during this time blocked the disease. A treatment with a VPAC1 antagonist during ongoing disease during the same time frame post-EAE induction did not prevent and may have exacerbated the disease. Mice deficient in one of the two VPAC1 ligands, VIP, are also resistant to EAE, whereas PACAP-deficient mice exhibit exacerbated EAE, implying a critical VIP/VPAC1 interaction that is required for the development of EAE