Date Published: April 16, 2013
Publisher: Public Library of Science
Author(s): Yossan-Var Tan, Catalina Abad, Yuqi Wang, Robert Lopez, James A. Waschek, Alexandre Salgado Basso. http://doi.org/10.1371/journal.pone.0061200
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.
It has been shown that the nervous system and the immune system interact with each other during health and disease. In this regard, there is robust evidence that neurological mediators, including multiple neurotransmitters and neuropeptides, exert modulatory effects on immune cells such as myeloid cells or lymphocytes, which are key players of innate and adaptive immunity. Understanding how the nervous system regulates the immune system is crucial to comprehend the complex pathogenesis of autoimmune diseases, and to develop new therapeutic tools. A neuropeptide with well-described modulatory actions in the nervous, endocrine and immune systems is pituitary adenylate cyclase-activating polypeptide (PACAP, gene name ADCYAP1) . It binds to three receptors of the G-protein coupled receptor (GPCR) family, VPAC1, VPAC2 and PAC1, which are present in the surface of various cell types including many immune cell types , . The names of these receptors refer to their ligand affinities: whereas the VPAC receptors bind both PACAP and the highly-homologous polypeptide vasoactive intestinal peptide (VIP) with similar affinity, PAC1 is a PACAP-preferring receptor. These receptors stimulate a canonical adenylate cyclase (AC)/cyclic AMP (cAMP)/protein kinase A (PKA) signalling pathway, but can in a variety of contexts activate inositol triphosphate/PLC/PKC and other signalling pathways , . Although PACAP exerts diverse actions in the immune system, it is primarily recognized as an anti-inflammatory peptide. In this respect, PACAP strongly inhibits the release of proinflammatory cytokines such as TNFα, IL-6 and IL-12 and chemokines such as RANTES, KC, MIP-1α, MIP-1β and MCP-1 from macrophages and primary microglia stimulated in vitro with lipopolysaccharide (LPS) , . Evidence suggest that these effects are mediated at least in part by activation of PKA, but also by inhibiton of NF-kB and/or MEKK1/MEK4/JNK pathways, and by induction of CREB phosphorylation . In addition, it has been shown that PACAP modulates T cell function, promoting Th2 over Th1/Th17 cytokine profiles , . In this respect, PACAP acts on antigen presenting dendritic cells and macrophages by promoting the production of purported Th2-recruiting chemokines (CCL11 and CCL22), modifying the expression of co-stimulatory molecules (B7.1 and B7.2), and promoting the generation of Th2 vs Th1 memory cells –. The anti-inflammatory and Th2-promoting actions of PACAP have been corroborated by studies using in vivo models of acute and chronic inflammation, including experimental autoimmune encephalomyelitis (EAE), which exhibits many of the clinical and molecular features of multiple sclerosis (MS) . Moreover, PACAP and/or PACAP mRNA have been shown to be strongly induced in neurons in several models of inflammation –, and have been found to be also expressed by lymphocytes in naïve animals , .
Inflammation is a coordinated process designed by evolution to eliminate pathogens and enable healing. However, this is carefully orchestrated in the sense that when it is no longer necessary, it must be actively terminated to avoid damage. Moreover, tolerogenic mechanisms must be in place to prevent the action of autoreactive T cells that escape negative selection. Antiinflammatory cytokines like IL-10 or TGFβ subserve some of these purposes, and are secreted by Tregs and other cells. Our prior study showing that PACAP KO mice exhibit enhanced EAE with a reduction of Tregs in the lymph nodes provided evidence that PACAP might belong to this group of intrinsic immunomodulatory molecules . The current studies provide additional information on how endogenous PACAP may regulate specific T cells in this context.