Yossan-Var Tan

Pituitary adenylyl cyclase-activating polypeptide is an intrinsic regulator of Treg abundance and protects against experimental autoimmune encephalomyelitis.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a widely expressed neuropeptide originally discovered in the hypothalamus. It closely resembles vasoactive intestinal peptide (VIP), a neuropeptide well known to inhibit macrophage activity, promote Th2-type responses, and enhance regulatory T cell (Treg) production. Recent studies have shown that administration of PACAP, like VIP, can attenuate dramatically the clinical and pathological features of murine models of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis. However, specific roles (if any) of endogenous VIP and PACAP in the protection against autoimmune diseases have not been explored. Here, we subjected PACAP-deficient mice to myelin oligodendrocyte glycoprotein (MOG35–55)-induced EAE. MOG immunization of PACAP-deficient mice triggered heightened clinical and pathological manifestations of EAE compared to wild-type mice. The increased sensitivity was accompanied by enhanced mRNA expression of proinflammatory cytokines (TNFα, IL-6, IFN-γ, IL-12p35, IL-23p19, and IL-17), chemokines (MCP-1/CCL2, MIP-1α/CCL3, and RANTES/CCL5), and chemotactic factor receptors (CCR1, CCR2, and CCR5), but downregulation of the anti-inflammatory cytokines (IL-4, IL-10, and TGF-β) in the spinal cord. Moreover, the abundance of CD4+CD25+FoxP3+ Tregs in lymph nodes and levels of FoxP3 mRNA in the spinal cord were also diminished. The reduction in Tregs was associated with increased proliferation and decreased TGF-β secretion in lymph node cultures stimulated with MOG. These results demonstrate that endogenous PACAP provides protection in EAE and identify PACAP as an intrinsic regulator of Treg abundance after inflammation.

Vasoactive intestinal peptide loss leads to impaired CNS parenchymal T-cell infiltration and resistance to experimental autoimmune encephalomyelitis

The neuropeptide vasoactive intestinal peptide (VIP) has been shown to inhibit macrophage proinflammatory actions, promote a positive Th2/Th1 balance, and stimulate regulatory T-cell production. The fact that this peptide is highly efficacious in animal models of inflammatory diseases such as collagen-induced arthritis and experimental autoimmune encephalomyelitis (EAE) suggests that the endogenous peptide might normally provide protection against such pathologies. We thus studied the response of VIP-deficient (i.e., VIP KO) mice to myelin oligodendrocyte protein-induced EAE. Surprisingly, VIP KO mice were almost completely resistant to EAE, with delayed onset and mild or absent clinical profile. Despite this, flow cytometric analyses and antigen-rechallenge experiments indicated that myelin oligodendrocyte protein-treated VIP KO mice exhibited robust Th1/Th17 cell inductions and antigen-specific proliferation and cytokine responses. Moreover, adoptive transfer of lymphocytes from immunized VIP KO mice to WT recipients resulted in full-blown EAE, supporting their encephalitogenic potential. In contrast, transfer of encephalitogenic WT cells to VIP KO hosts did not produce EAE, suggesting that loss of VIP specifically affected the effector phase of the disease. Histological analyses indicated that CD4 T cells entered the meningeal and perivascular areas of VIP-deficient mice, but that parenchymal infiltration was strongly impaired. Finally, VIP pretreatment of VIP KO mice before immunization was able to restore their sensitivity to EAE. These results indicate that VIP plays an unanticipated permissive and/or proinflammatory role in the propagation of the inflammatory response in the CNS, a finding with potential therapeutic relevance in autoimmune neuroinflammatory diseases such as multiple sclerosis.

Calcium Signaling via Orai1 Is Essential for Induction of the Nuclear Orphan Receptor Pathway To Drive Th17 Differentiation

Orai1 is the pore subunit of Ca2+ release–activated Ca2+ (CRAC) channels that stimulate downstream signaling pathways crucial for T cell activation. CRAC channels are an attractive therapeutic target for alleviation of autoimmune diseases. Using high-throughput chemical library screening targeting Orai1, we identified a novel class of small molecules that inhibit CRAC channel activity. One of these molecules, compound 5D, inhibited CRAC channel activity by blocking ion permeation. When included during differentiation, Th17 cells showed higher sensitivity to compound 5D than Th1 and Th2 cells. The selectivity was attributable to high dependence of promoters of retinoic-acid-receptor-related orphan receptors on the Ca2+-NFAT pathway. Blocking of CRAC channels drastically decreased recruitment of NFAT and histone modifications within key gene loci involved in Th17 differentiation. The impairment in Th17 differentiation by treatment with CRAC channel blocker was recapitulated in Orai1-deficient T cells, which could be rescued by exogenous expression of retinoic-acid-receptor-related orphan receptors or a constitutive active mutant of NFAT. In vivo administration of CRAC channel blockers effectively reduced the severity of experimental autoimmune encephalomyelitis by suppression of differentiation of inflammatory T cells. These results suggest that CRAC channel blockers can be considered as chemical templates for the development of therapeutic agents to suppress inflammatory responses.

Targeting VIP and PACAP receptor signalling: new therapeutic strategies in multiple sclerosis

MS (multiple sclerosis) is a chronic autoimmune and neurodegenerative pathology of the CNS (central nervous system) affecting approx. 2.5 million people worldwide. Current and emerging DMDs (disease-modifying drugs) predominantly target the immune system. These therapeutic agents slow progression and reduce severity at early stages of MS, but show little activity on the neurodegenerative component of the disease. As the latter determines permanent disability, there is a critical need to pursue alternative modalities. VIP (vasoactive intestinal peptide) and PACAP (pituitary adenylate cyclase-activating peptide) have potent anti-inflammatory and neuroprotective actions, and have shown significant activity in animal inflammatory disease models including the EAE (experimental autoimmune encephalomyelitis) MS model. Thus, their receptors have become candidate targets for inflammatory diseases. Here, we will discuss the immunomodulatory and neuroprotective actions of VIP and PACAP and their signalling pathways, and then extensively review the structure–activity relationship data and biophysical interaction studies of these peptides with their cognate receptors.

VPAC2 (vasoactive intestinal peptide receptor type 2) receptor deficient mice develop exacerbated experimental autoimmune encephalomyelitis with increased Th1/Th17 and reduced Th2/Treg responses.

Vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating polypeptide (PACAP) are two structurally-related neuropeptides with widespread expression in the central and peripheral nervous systems. Although these peptides have been repeatedly shown to exert potent anti-inflammatory actions when administered in animal models of inflammatory disease, mice deficient in VIP and PACAP were recently shown to exhibit different phenotypes (ameliorated and exacerbated, respectively) in response to experimental autoimmune encephalomyelitis (EAE). Therefore, elucidating what are the specific immunoregulatory roles played by each of their receptor subtypes (VPAC1, VPAC2, and PAC1) is critical. In this study, we found that mice with a genetic deletion of VIPR2, encoding the VPAC2 receptor, exhibited exacerbated (MOG35-55)-induced EAE compared to wild type mice, characterized by enhanced clinical and histopathological features, increased proinflammatory cytokines (TNF-α, IL-6, IFN-γ (Th1), and IL-17 (Th17)) and reduced anti-inflammatory cytokines (IL-10, TGFβ, and IL-4 (Th2)) in the CNS and lymph nodes. Moreover, the abundance and proliferative index of lymph node, thymus and CNS CD4(+)CD25(+)FoxP3(+) Tregs were strikingly reduced in VPAC2-deficient mice with EAE. Finally, the in vitro suppressive activity of lymph node and splenic Tregs from VPAC2-deficient mice was impaired. Overall, our results demonstrate critical protective roles for PACAP and the VPAC2 receptor against autoimmunity, promoting the expansion and maintenance of the Treg pool.

VIP deficient mice exhibit resistance to lipopolysaccharide induced endotoxemia with an intrinsic defect in proinflammatory cellular responses.

Vasoactive intestinal peptide (VIP) is a pleiotropic neuropeptide with immunomodulatory properties. The administration of this peptide has been shown to have beneficial effects in murine models of inflammatory diseases including septic shock, rheumatoid arthritis, multiple sclerosis (MS) and Crohns disease. However, the role of the endogenous peptide in inflammatory disease remains obscure because VIP-deficient mice were recently found to exhibit profound resistance in a model of MS. In the present study, we analyzed the response of female VIP deficient (KO) mice to intraperitoneal lipopolysaccharide (LPS) administration. We observed significant resistance to LPS in VIP KO mice, as evidenced by lower mortality and reduced tissue damage. The increased survival was associated with decreased levels of proinflammatory cytokines (TNFα, IL-6 and IL-12) in sera and peritoneal suspensions of these mice. Moreover, the expression of TNFα and IL-6 mRNA was reduced in peritoneal cells, spleens and lungs from LPS-treated VIP KO vs. WT mice, suggesting that the resistance might be mediated by an intrinsic defect in the responsiveness of immune cells to endotoxin. In agreement with this hypothesis, peritoneal cells isolated from VIP KO naive mice produced lower levels of proinflammatory cytokines in response to LPS in vitro. Finally, decreased NF-κB pathway activity in peritoneal cells was observed both in vivo and in vitro, as determined by assay of phosphorylated I-κB. The results demonstrate that female VIP KO mice exhibit resistance to LPS-induced shock, explainable in part by the presence of an intrinsic defect in the responsiveness of inflammatory cells to endotoxin.

Pituitary adenylate cyclase activating peptide deficient mice exhibit impaired thymic and extrathymic regulatory T cell proliferation during EAE.

We have shown that mice deficient in pituitary adenylate cyclase-activating polypeptide (PACAP, gene name ADCYAP1) manifest enhanced sensitivity to experimental autoimmune encephalomyelitis (EAE), supporting the anti-inflammatory actions described for this neuropeptide. In addition to an increased proinflammatory cytokine response in these mice, a reduction in regulatory T cell (Treg) abundance in the lymph nodes (LN) was observed, suggesting altered Treg kinetics. In the present study, we compared in PACAP deficient (KO) vs. wild type mice the abundances and rates of proliferation FoxP3+ Tregs in three sites, the LN, central nervous system (CNS) and thymus and the relative proportions of Th1, Th2, and Th17 effector subsets in the LN and CNS. Flow cytometry analyses revealed a decrease in Treg proliferation and an increased T effector/Tregs ratio in the LN and CNS of PACAP KO mice. In the thymus, the primary site of do novo natural Treg production, the total numbers and proliferative rates of FoxP3+ Tregs were significantly reduced. Moreover, the expression of IL-7, a cytokine implicated in thymic Treg expansion during EAE, failed to increase at the peak of the disease in the thymus and LN of PACAP KO mice. In addition to these Treg alterations, a specific reduction of Th2 cells (about 4-fold) was observed in the lymph nodes in PACAP KO mice, with no effects on Th1 and Th17 subsets, whereas in the CNS, Th1 and Th17 cells were increased and Th2 decreased. Our results suggest that endogenous production of the neuropeptide PACAP protects against EAE by modulating Treg expansion and Th subsets at multiple sites.

Reductions in synaptic proteins and selective alteration of prepulse inhibition in male C57BL/6 mice after postnatal administration of a VIP receptor (VIPR2) agonist

RationaleAn abundance of genetic and epidemiologic evidence as well as longitudinal neuroimaging data point to developmental origins for schizophrenia and other mental health disorders. Recent clinical studies indicate that microduplications of VIPR2, encoding the vasoactive intestinal peptide (VIP) receptor VPAC2, confer significant risk for schizophrenia and autism spectrum disorder. Lymphocytes from patients with these mutations exhibited higher VIPR2 gene expression and VIP responsiveness (cAMP induction), but mechanisms by which overactive VPAC2 signaling may lead to these psychiatric disorders are unknown.ObjectivesWe subcutaneously administered the highly selective VPAC2 receptor agonist Ro 25-1553 to C57BL/6 mice from postnatal day 1 (P1) to P14 to determine if overactivation of VPAC2 receptor signaling during postnatal brain maturation affects synaptogenesis and selected behaviors.ResultsWestern blot analyses on P21 revealed significant reductions of synaptophysin and postsynaptic density protein 95 (PSD-95) in the prefrontal cortex, but not in the hippocampus in Ro 25-1553-treated mice. The same postnatally restricted treatment resulted in a disruption in prepulse inhibition of the acoustic startle measured in adult mice. No effects were observed in open-field locomotor activity, sociability in the three-chamber social interaction test, or fear conditioning or extinction.ConclusionOveractivation of the VPAC2 receptor in the postnatal mouse results in a reduction in synaptic proteins in the prefrontal cortex and selective alterations in prepulse inhibition. These findings suggest that the VIPR2-linkage to mental health disorders may be due in part to overactive VPAC2 receptor signaling during a critical time of synaptic maturation.

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

BackgroundVasoactive 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.MethodsEAE 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 30xa0days. 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.ResultsMOG35–55-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,xa0treatment of WT mice with the VPAC1 receptor antagonist PG97-269 before,xa0but not after, EAE induction mimicked the clinical phenotype of VPAC1 KO mice.ConclusionsVPAC1 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.

Immunomodulatory Roles of PACAP and VIP: Lessons from Knockout Mice

A bidirectional cross-talk is established between the nervous and immune systems through common mediators including neuropeptides, neurotransmitters, and cytokines. Among these, PACAP and VIP are two highly related neuropeptides widely distributed in the organism with purported immunomodulatory actions. Due to their well-known anti-inflammatory properties, administration of these peptides has proven to be beneficial in models of acute and chronic inflammatory diseases. Nevertheless, the relevance of the endogenous source of these peptides in the modulation of immune responses remains to be elucidated. The development of transgenic mice with specific deletions in the genes coding for these neuropeptides (Vip and Adcyap1) or for their G-protein-coupled receptors VPAC1, VPAC2, and PAC1 (Vipr1, Vipr2, Adcyap1r1) has allowed to address this question, underscoring the complexity of the immunoregulatory properties of PACAP and VIP. The goal of this review is to integrate the existing information on the immune phenotypes of mice deficient for PACAP, VIP, or their receptors, to provide a global view on the roles of these endogenous neuropeptides during immunological health and disease.