Vasoactive Intestinal Peptide Signaling Research Articles

Abstract 1205: Targeting vasoactive intestinal peptide signaling to enhance pancreatic cancer responsiveness to immunotherapy

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer related death in the U.S., outranking breast and colorectal cancer by 2030. Being completely refractory to checkpoint therapies, management of PDAC remains an unmet clinical need. Intriguingly, the cancer genome atlas shows elevated mRNA expression of vasoactive intestinal peptide (VIP), a neurotransmitter in exocrine pancreatic cancers. Interestingly, VIP also has immunosuppressive properties leading to decreased T cell proliferation and enhances expansion of myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs). Thus, we are investigating the role of VIP in PDAC progression and the effect of inhibiting VIP signaling for improved responsiveness to immunotherapy. Methods: Level of secreted VIP in 1) serum of healthy volunteers, 2) untreated PDAC patients, 3) cell culture supernatant from murine melanoma, breast and PDAC cells and 4) serum from mice bearing these cell lines were tested via VIP specific enzyme immunoassay. Effect of inhibiting VIP signaling in-vivo was tested using VIPhyb, an antagonist for VIP receptor. Mice were subcutaneously inoculated with MT5 or luciferase transfected KPC (KPC luc), PDAC cell lines and treated with VIPhyb and/or anti-PD1 treatment after the tumors were palpable. The treatment regimen involved administering 10µg VIPhyb subcutaneously, every day and 200µg anti-PD1 or IgG2a, intraperitoneally, every three days, for 10 days. The tumors were measured using calipers or imaged via IVIS imaging system. The mice were sacrificed when tumors ulcerated or when they reached IACUC endpoint. Immunohistochemistry was performed on tumor tissue sections harvested at the time of sacrifice for expression of VIP, CD8 and DAPI. Results: Consistent with the human genome atlas, cell culture supernatants collected from murine PDAC cell lines had significantly higher levels of VIP as compared to melanoma and breast cancer cells. Similarly, elevated levels of VIP were observed in mice bearing PDAC tumors, and in plasma from untreated human PDAC patients, when compared to naïve mice and healthy volunteer samples, respectively. Treatment of immune competent mice bearing MT5 or KPC luc PDAC cells with the combination of VIPhyb and anti-PD1 produced complete and durable regression of tumors in 20% of the mice and delayed tumor progression in both tumor models. Further, upon analysis of the tumor tissues sections, significant infiltration of CD8+ T cells was observed in mice treated with the combination of VIPhyb and anti-PD1. Conclusion: VIP is a novel immune checkpoint pathway in PDAC. Blocking VIP signaling during anti-PD1 therapy, shows improved survival and delayed tumor progression in mouse models of PDAC. Experiments are underway to understand the mechanism of action, as well as, improving the potency of VIPhyb for enhanced clinical outcome. Citation Format: Sruthi Ravindranathan, Yiwen Li, Shuhua Wang, Mohammad Y. Zaidi, Gregory B. Lesinski, Bassel El-Rayes, Edmund K. Waller. Targeting vasoactive intestinal peptide signaling to enhance pancreatic cancer responsiveness to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1205.

Vasoactive intestinal peptide signaling – a novel immune checkpoint in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer related death in the U.S. by 2030. Being completely refractory to checkpoint therapies, management of PDAC remains an unmet clinical need. Intriguingly, the human protein atlas (THPA) shows elevated mRNA expression of vasoactive intestinal peptide (VIP), a neurotransmitter in exocrine pancreatic cancers. Interestingly, VIP also has immunosuppressive properties leading to decreased T cell proliferation and enhanced expansion of myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs). Thus, we are investigating the role of VIP in PDAC progression and the effect of inhibiting VIP signaling for improved responsiveness to immunotherapy. We found that consistent with THPA, cell culture supernatants collected from murine PDAC cell lines had significantly higher levels of VIP as compared to melanoma and breast cancer cells. Similarly, elevated levels of VIP were observed in mice bearing PDAC tumors, and in plasma from human PDAC patients, when compared to naïve mice and healthy volunteer samples, respectively. Treatment of immune competent mice bearing PDAC tumors (MT5 or KPC cell lines) with the combination of a VIP antagonist (VIPhyb) and anti-PD1, produced complete and durable regression of tumors in 20% of the mice and an overall delayed tumor progression in both tumor models. Upon analysis of the tumor tissues sections via immunohistochemistry, significant infiltration of CD8+ T cells was observed in mice treated with the combination of VIPhyb and anti-PD1. Further experiments are underway to understand the mechanism of action of VIP antagonist and to devise methods to improve its efficacy in-vivo.

Vasoactive Intestinal Peptide Synthesis in Transplant Recipients Critically Regulate Donor T Cell Alloreactivity and Graft-Versus-Host Disease

Introduction: Vasoactive Intestinal peptide (VIP) is a neuropeptide expressed by neural and lymphoid cells with pleiotropic effects on brain, immune, pulmonary and GI systems. The potent immunomodulatory activity of VIP make it an attractive therapeutic target to suppresses immune responses in conditions of deleterious inflammation. In allogeneic bone marrow transplantation (allo-BMT), graft-versus-host disease (GvHD) is mediated by activated alloreactive T cells that also upregulate a variety of co-inhibitory pathway molecules, including VIP expression on T cells. It is unclear whether VIP production by host cells in the transplant recipient influence the severity of GvHD and donor T cell activation. We tested the hypothesis that lack of VIP production by recipient cells would enhance donor T cell activation and increase GvHD in allo-BMT recipients. Methods and Results: We examined the effect of endogenous VIP signaling in host tissues in modulating GvHD in a murine allo-BMT model, in which C57BL/6 wildtype (WT) and VIP-knock-out (VIP-KO) mice received 11Gy irradiation on day -1 and were transplanted i.v. on day 0 with 5x106 T cell depleted (TCD) bone marrow (BM) plus 0, 1x106 or 3x106 splenocytes (SP) from MHC mis-matched B10.BR donors. All three groups of WT recipients had better survival than VIP-KO recipients. VIP-KO mice transplanted with TCD BM alone had 33.3% survival at day 75 compared with 73.3% survival of WT recipients. Chimerism studies on day 20 post-BMT showed equivalent levels of donor chimerism within each group that received the same dose of donor SP. WT and VIP-KO recipients received BM only showed significantly lower donor chimerism compared to either WT or VIP-KO recipients received additional SP (p<0.001), indicating increased mortality in recipients transplanted with BM only was primarily due to early graft rejection. The addition of donor SP to the graft resulted in significantly more GvHD-related mortality in VIP-KO mice compared with WT recipients, with 80% vs 100% day 75 survival among recipients of 1x106 SP (p=NS) and 0% vs 40% survival (p<0.01) among recipients of 3x106 donor SP. WT and VIP-KO mice treated with lethal doses of irradiation without allo-BMT showed no survival difference, suggesting VIP-KO mice are not intrinsically more susceptible to irradiation. To determine how the absence of VIP synthesis by VIP-KO host cells modulates immune effector mechanisms that contributes to increased GvHD, we analyzed donor-derived lymphocytes in the spleen of transplant recipients received 5M TCD BM plus 3 × 106 SP from WT B10BR donors following a time course, with flow cytometry. WT recipients had significantly increased levels of Lag3+ CD4 (p<0.001) on D6, PD1+ CD8 (p<0.01) on D13, Tim3+ CD4 (p<0.05) on D19, and increased levels of Tim3+ CD4 (p<0.001), PD1+ CD4 (p<0.05), and PD1+ CD8 (p<0.001) on D25 post allo-BMT compared to VIP-KO recipients. In addition, VIP-KO recipients had significantly higher expression of selected Th1 and Th17 cytokines. We observed significantly higher levels of IFN-γ+ CD4 (p<0.001) and IL2+ CD4 (p<0.01) on D4, significantly higher levels of TNF-α+ CD4 (p<0.05) and TNF-α+ CD8 (p<0.001) on D19, and significantly increased TNF-α+ CD8 (p<0.05) on D25 post allo-BMT in VIP-KO compared to WT recipients. Consistent with the significant elevation of IL17 in CD4 and CD8 (p<0.001) donor T cells of VIP-KO mice on D19, increased transcriptional factor RORγt in VIP-KO mice were detected in both CD4 (p<0.0001) and CD8 (p<0.0001) donor T cells. Transplant experiments using radiation chimeric recipients in which either the hematopoietic or non-hematopoietic compartment lacked expression of VIP further demonstrated that VIP production in non-hematopoietic cells is the key factor in limiting the GvHD activity of donor T cells (Fig 1). Finally, immunofluorescent imaging of transgenic mice in which GFP is regulated by the VIP promoter showed VIP production is highly expressed in efferent neurons innervating the lungs, and that host expression of VIP in lung tissues continues for at least 15 days post-transplant (Fig 2). Conclusion: These data suggest local production of VIP in the lung may be a key factor in the control of alloreactive donor T cells and subsequent GVHD in epithelial target organs. Furthermore, local production of VIP by host neurons surrounding lung alveoli suggests a novel pathway for pharmacological modulation of allo-reactive T cells and control of GVHD. DisclosuresWaller:Pharmacyclics: Other: Travel Expenses, EHA, Research Funding; Kalytera: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celldex: Research Funding; Cambium Medical Technologies: Consultancy, Equity Ownership.

VIP is involved in peripheral CRF-induced stimulation of propulsive colonic motor function and diarrhea in male rats.

We investigated whether vasoactive intestinal peptide (VIP) and/or prostaglandins contribute to peripheral corticotropin-releasing factor (CRF)-induced CRF1 receptor-mediated stimulation of colonic motor function and diarrhea in rats. The VIP antagonist, [4Cl-D-Phe6, Leu17]VIP injected intraperitoneally completely prevented CRF (10 µg/kg ip)-induced fecal output and diarrhea occurring within the first hour after injection, whereas pretreatment with the prostaglandins synthesis inhibitor, indomethacin, had no effect. In submucosal plexus neurons, CRF induced significant c-Fos expression most prominently in the terminal ileum compared with duodenum and jejunum, whereas no c-Fos was observed in the proximal colon. c-Fos expression in ileal submucosa was colocalized in 93.4% of VIP-positive neurons and 31.1% of non-VIP-labeled neurons. CRF1 receptor immunoreactivity was found on the VIP neurons. In myenteric neurons, CRF induced only a few c-Fos-positive neurons in the ileum and a robust expression in the proximal colon (17.5 ± 2.4 vs. 0.4 ± 0.3 cells/ganglion in vehicle). The VIP antagonist prevented intraperitoneal CRF-induced c-Fos induction in the ileal submucosal plexus and proximal colon myenteric plexus. At 60 min after injection, CRF decreased VIP levels in the terminal ileum compared with saline (0.8 ± 0.3 vs. 2.5 ± 0.7 ng/g), whereas VIP mRNA level detected by qPCR was not changed. These data indicate that intraperitoneal CRF activates intestinal submucosal VIP neurons most prominently in the ileum and myenteric neurons in the colon. It also implicates VIP signaling as part of underlying mechanisms driving the acute colonic secretomotor response to a peripheral injection of CRF, whereas prostaglandins do not play a role. NEW & NOTEWORTHY Corticotropin-releasing factor (CRF) in the gut plays a physiological role in the stimulation of lower gut secretomotor function induced by stress. We showed that vasoactive intestinal peptide (VIP)-immunoreactive neurons in the ileal submucosal plexus expressed CRF1 receptor and were prominently activated by CRF, unlike colonic submucosal neurons. VIP antagonist abrogated CRF-induced ileal submucosal and colonic myenteric activation along with functional responses (defecation and diarrhea). These data point to VIP signaling in ileum and colon as downstream effectors of CRF.

Activation of VIP signaling enhances immunosuppressive effect of MDSCs on CMV-induced adaptive immunity.

Vasoactive intestinal peptide (VIP) is recognized as a potent anti-inflammatory factor which affects both the innate and adaptive arms of the immune system. These effects include, but are not limited to, inhibition of T cell proliferation and disruption of immune homeostasis. Myeloid-derived suppressor cells (MDSC) are an immune regulatory cell type that has been described in settings of cancer and infectious disease._Here we demonstrate a reduced circulating monocytic MDSCs in the VIP -/- vs. wild type MCMV. VIP-/- MDSCs secretes less NO upon stimulation with LPS and interferon that relatively lose the ability to suppress T cells activation in vitro compared to wild type MDSCs._Considering the importance of VIP in immunomodulation, the possible effect of VIP in the suppressive function of MDSC populations following CMV infection remains unknown. We describe the possible role of VIP in the regulation of anti-CMV activity of T cells through the activation of MDSCs.

Deficiency in vasoactive intestinal peptide signaling causes compositional and functional changes in mouse fecal microbiota

Abstract By 2030, half the human population will be obese contributing to diabetes, cancer and cardiovascular disease. The gut peptide, vasoactive intestinal peptide (VIP) that acts as a regulator of innate and adaptive immunity, is elevated in obese humans. VIP mediates immunity changes through binding two receptors, called VPAC1 and VPAC2. Unfortunately, the pathways linking VIP signaling to obesity are not known. We hypothesized that defective VIP signaling would have an anti-obesity effect by lowering the gut firmicutes/bacteroidetes (f:b) ratio. To this end, knockout mice deficient for VIP, VPAC1 or VPAC2, were used to collect 16S rRNA reads from fecal samples. Operational taxonomic units for each strain were compared against each other to determine changes in bacterial taxa. Moreover, differences in gene functions were analyzed using PICRUSt. These data revealed a significant decrease in the f:b ratio for mice deficient in VIP and VPAC2 compared to wild type controls. Heterozygous and homozygous littermates for the VIP mutation showed the greatest reduction in fecal bacterial richness and clustered differently when compared phylogenetically to wild type or VIP receptor knockout strains. Metabolome predictions indicated that genes regulating the circulatory system and cardiovascular disease were consistently altered in mice deficient for VIP or its two receptors. In summary, deficiency in the VIP signaling axis significantly alters the gut microbiota and microbiome.

Vasoactive intestinal peptide induced epithelial to mesenchymal transition activities in PC3 prostate cancer cells

Abstract Epithelial to mesenchymal transition (EMT) is a fundamental cellular process preceding movement. Malignant transformation of epithelial cells also undergo EMT leading to metastasis and a worse prognosis for human cancer patients. Understanding molecular pathways that are linked to EMT may provide additional drug targets to treat epithelial cancers like prostate cancer. Vasoactive intestinal peptide signaling through one of its naturally occurring G-protein coupled receptors, termed VPAC1, induces non-tumor and tumorigenic epithelial cell lines (RWPE-1, LnCaP and PC3 cells) to undergo EMT and downregulate E-cadherin, a hallmark for EMT. Curcumin, an anti-inflammatory molecule, blocked these VIP EMT effects suggesting a role for NF-kB signaling. Our group has uncovered EMT-associated molecular mRNA targets induced by VIP treatment of primary, resting CD4 T cells, including the EMT master regulator transcription factor, SNAIL1. We hypothesize that EMT transition effects by VIP signaling may also involve the upregulation of SNAIL1 protein. To this end, we plan on treating PC3 cells with varying levels of VIP over time (0–24 hr) and measure by immunoblots the extent to which Snail1 protein, and up to 10 additional EMT-associated proteins, are differentially altered. Successful completion of this research is expected to uncover important VIP protein targets that is expected to better explain its epithelial tumorigenic potential.

Vasoactive intestinal peptide receptor - 1 deficiency causes an increase in the frequency of antibiotic producing bacteria in mouse fecal samples

Abstract New antibiotic discovery is critical because bacteria are becoming resistant to currently prescribed antibiotics. A mammalian gut peptide, vasoactive intestinal peptide (VIP), is highly expressed in the gut and possess antimicrobial activity. VIP binds two receptors, designated VPAC1 and VPAC2, and deficiency in all three have been shown to cause significant gut microbiota dysbiosis. We hypothesized that mice deficient for VIP signaling would be good candidates for exploring the existence of novel antibiotic-producing bacteria (APB). To this end, bacteria fecal samples from C57Bl/6 wild type (WT), and heterozygous (HET) and/or homozygous mutant (KO) VIP, VPAC1 and VPAC2 strains were used to test for zones of inhibition against escape pathogens. Our results showed that heterozygous VPAC1 mice resulted in the greatest increase in APB compared to WT controls, VIP or VPAC2 genetically altered strains. Initial taxonomic identification of APB from VPAC1 Het fecal samples reveled previously characterized aerobic bacteria. We conclude that reduction in VPAC1 receptor signaling in the gut, as opposed to loss of the VIP ligand or VPAC2 signaling, elevates the frequency of aerobic APB. Future studies will now focus on utilizing VPAC1 WT, HET and KO littermates to mine for the existence of novel anaerobic APB.

Modulation of Immune Checkpoints and Graft-versus-Leukemia in Allogeneic Transplants by Antagonizing Vasoactive Intestinal Peptide Signaling.

The goal of allogeneic bone marrow transplantation (allo-BMT) is elimination of leukemia cells through the graft-versus-leukemia (GvL) activity of donor cells, while limiting graft-versus-host disease (GvHD). Immune checkpoint pathways regulate GvL and GvHD activities, but blocking antibodies or genetic inactivation of these pathways can cause lethal GVHD. Vasoactive intestinal peptide (VIP) is an immunosuppressive neuropeptide that regulates coinhibitory pathways; its role in allo-BMT has not been studied. We found VIP transiently expressed in donor NK, NK-T, dendritic cells, and T cells after allo transplant, as well as host leukocytes. A peptide antagonist of VIP signaling (VIPhyb) increased T-cell proliferation in vitro and reduced IL10 expression in donor T cells. Treatment of allo-BMT recipients with VIPhyb, or transplanting donor grafts lacking VIP (VIP-KO), activated donor T-cells in lymphoid organs, reduced T-cell homing to GvHD target organs, and enhanced GvL without increasing GvHD in multiple allo-BMT models. Genetic or ex vivo depletion of donor NK cells or CD8+ T cells from allografts abrogated the VIPhyb-enhanced GvL activity. VIPhyb treatment led to downregulation of PD-1 and PD-L1 expression on donor immune cells, increased effector molecule expression, and expanded oligoclonal CD8+ T cells that protected secondary allo transplant recipients from leukemia. Blocking VIP signaling thus represents a novel pharmacologic approach to separate GvL from GvHD and enhance adaptive T-cell responses to leukemia-associated antigens in allo-BMT. Cancer Res; 76(23); 6802-15. ©2016 AACR.

Vasoactive intestinal peptide, whose receptor-mediated signalling may be defective in alopecia areata, provides protection from hair follicle immune privilege collapse.

Alopecia areata (AA) is an autoimmune disorder whose pathogenesis involves the collapse of the relative immune privilege (IP) of the hair follicle (HF). Given that vasoactive intestinal peptide (VIP) is an immunoinhibitory neuropeptide released by perifollicular sensory nerve fibres, which play a role in IP maintenance, it may modulate human HF-IP and thus be therapeutically relevant for AA. To answer the following questions: Do human HFs express VIP receptors, and does their stimulation protect from or restore experimentally induced HF-IP collapse? Is VIP signalling defective in AA HFs? Firstly, VIP and VIP receptor (VPAC1, VPAC2) expression in human scalp HFs and AA skin was assessed. In HF organ culture, we then explored whether VIP treatment can restore and/or protect from interferon-γ-induced HF-IP collapse, assessing the expression of the key IP markers by quantitative (immuno-)histomorphometry. Here we provide the first evidence that VIP receptors are expressed in the epithelium of healthy human HFs at the gene and protein level. Furthermore, VIP receptor protein expression, but not VIP(+) nerve fibres, is significantly downregulated in lesional hair bulbs of patients with AA, suggesting defects in VIP receptor-mediated signalling. Moreover, we show that VIP protects the HF from experimentally induced IP collapse invitro, but does not fully restore it once collapsed. These pilot data suggest that insufficient VIP receptor-mediated signalling may contribute to impairing HF-IP in patients with AA, and that VIP is a promising candidate 'HF-IP guardian' that may be therapeutically exploited to inhibit the progression of AA lesions.

Inhibition of CREB signaling through antagonism of vasoactive intestinal peptide enhances CD8 T cell function in a murine model of AML

Abstract Signaling pathways that activate CREB may inhibit the potency of T cell responses following stimulation. One of the major mechanisms by which this occurs is the sequestration of CBP, which is also an NF-κB binding partner. One of the major molecules that signals through this pathway is vasoactive intestinal peptide (VIP). VIP is a 28 amino acid peptide that is secreted throughout the body by a variety of cell types including immune cells. VIP has shown to be a potent anti-inflammatory molecule with pleiotropic effects on T cells including cell cycle arrest and inhibition of cytokine secretion. The VIP signaling pathway represents a therapeutic target in disease settings in which a more robust T cell response would be beneficial. Acute myeloid leukemia (AML) is a hematological malignancy that has been explored as a candidate for a variety of T cell-based immunotherapies due to weak CD8 responses to AML blasts. We demonstrate here that administration of a hybrid peptide antagonist that blocks VIP signaling enhances CD8 T cell responses in a murine model of AML. CD8 T cells from treated mice showed enhanced secretion of IFN-γ and TNF-α as well as reduced expression of PD-1. These enhanced effector functions lead to a significant survival enhancement, which was lost when treated mice lacked T cells. Additionally, CD8 T cells from antagonist-treated survivors made up the majority of CD3+ cells following transfer to Rag1 knockout recipients. The phenotype of the cells was consistent with enhanced effector functions as transferred CD8+ T cells were predominately CD44+CD62L−. Taken together, these results demonstrate the potential for VIP antagonism as an immunotherapy strategy in settings of hematological malignancies.

Vasoactive intestinal peptide promotes differentiation and clock gene expression in granulosa cells from prehierarchal follicles.

Vasoactive intestinal peptide (VIP) signaling via cyclic adenosine monophosphate (cAMP) is reported to stimulate steroidogenesis in ovarian granulosa cells from a variety of vertebrate species, including the domestic hen. Prior to follicle selection in the hen ovary (i.e., cyclic recruitment) follicle-stimulating-hormone (FSH)-induced cAMP signaling is absent within the granulosa layer until immediately following follicle selection. As a consequence, granulosa cells remain in an undifferentiated state and are unable to initiate FSH-induced steroidogenesis. VIP receptors (VPAC1 and VPAC2), like the FSH receptor, are G protein-coupled receptors, so we predicted that VIP signaling in granulosa cells is also absent in follicles that have not yet been selected into the preovulatory hierarchy. Initial studies established that mRNA encoding VPAC1 and VPAC2 are expressed within the granulosa cells throughout follicle development. Nevertheless, undifferentiated granulosa cells from prehierarchal (6-8 mm) follicles do not accumulate cAMP in response to a 4-hr incubation with chicken VIP; the capacity for such receptor signaling is attained only following selection within actively differentiating granulosa cells. VIP treatment did, however, increase expression of mRNA encoding the Gallus circadian clock protein, BMAL1-but only in granulosa cells collected from selected follicles. These findings provide evidence that, at follicle selection, the acquisition of VIP-induced cAMP cell signaling helps initiate and promote the differentiation of of granulosa cells. Furthermore, we propose that VIP signaling may regulate BMAL1 expression and, thus, a daily rhythmicity within granulosa cells of preovulatory follicles. Mol. Reprod. Dev. 83: 455-463, 2016. © 2016 Wiley Periodicals, Inc.

VIP protects human retinal microvascular endothelial cells against high glucose-induced increases in TNF-α and enhances RvD1

PurposeThe purpose of our study was to evaluate the therapeutic effect of VIP on human retinal endothelial cells (HREC) under high glucose conditions. Diabetes affects almost 250 million people worldwide. Over 40% of diabetics are expected to develop diabetic retinopathy, which remains the leading cause of visual impairment/blindness. Currently, treatment is limited to late stages of retinopathy with no options available for early stages. To this end, the purpose of the current study is to evaluate the therapeutic effect of vasoactive intestinal peptide (VIP) on HREC under high glucose conditions. MethodsPrimary HREC were cultured in normal (5mM) or high (25mM) glucose medium +/− VIP treatment. Protein levels of TNF-α, resolvin D1 (RvD1), formyl peptide receptor 2 (FPR2), G protein-coupled receptor 32 (GPR32), VEGF, and VIP receptors, VPAC1 and VPAC2 were measured. ResultsHigh glucose-induced changes in TNF-α and RvD1 were restored to control levels with VIP treatment. RvD1 receptors, ALX/FPR2 and GPR32, were partially rescued with VIP treatment. VPAC2 expression appeared to be the major receptor involved in VIP signaling in HREC, as VPAC1 receptor was not detected. In addition, VIP did not induce HREC secretion of VEGF under high glucose conditions. ConclusionsOur results demonstrate that VIP’s therapeutic effect on HREC, occurs in part, through the balance between the pro-inflammatory cytokine, TNF-α, and the pro-resolving mediator, RvD1. Although VPAC1 is considered the major VIP receptor, VPAC2 is predominantly expressed on HREC under both normal and high glucose conditions.

Blocking VIP Signaling during Allogeneic Bone Marrow Transplantation Separates the Graft Versus Leukemia from Graft Versus Host Disease Activity of Donor CD8 T-Cells

Background: Separating graft versus leukemia (GvL) from graft versus host disease (GvHD) is a critical issue in allogeneic bone marrow transplantation (allo BMT) for malignant hematopoietic cancers. Vasoactive intestinal peptide (VIP) is capable of inducing tolerogenic dendritic cells (DC) from bone marrow progenitors that limit GvHD (Blood 2006 107:3787). We have published that use of donor mice deficient in VIP or pharmacological blockade of VIP-signaling both increased cellular anti-viral activity in murine allo BMT models of CMV infection (JI 2011 187:1057, Blood 2013 121:2347). We hypothesized that blockade of VIP could enhance GvL activity without increasing GvHD in recipients of allo BMT.Methods: We used the B10BR to B6 and B6 to B10BR murine allo BMT models, with luciferase-expressing C1498 acute myeloid leukemia and LBRM acute T-lymphoblastic lymphoma tumor cells, respectively, to study the effect of VIP-signaling on GvL and GvHD. Murine recipients of WT TCD-BM plus splenic allografts (5 x106 TCD-BM plus 0, 0.5, 1.0 and 3.0 x106 splenocytes) were treated with 7 daily s.c. injections of VIPhyb, a VIP antagonist, on d-1 to d+5 (early treatment) or d+6 to d+12 (late treatment), or PBS. Tumor cells were administered by i.v. injection one day before transplant (d-1), and tumor burden was monitored by bioluminescence imaging (BLI). Expression of activation immune cell markers, intracellular cytokines, and ex vivo cytolytic activity against tumor were measured by flow cytometry. TCR-β clono-types were analyzed by TCR-β CDR3 deep sequencing.Results: Recipients of VIP-KO donor cells and VIPhyb-treated recipients of WT donor cells did not have more GvHD than untreated recipients of equivalent numbers of splenocytes from WT donors. VIPhyb-treatment significantly increased survival of transplant tumor recipients in the B10.BR to B6 model with C1498 (2 replicate experiments, n= 10 in VIPhyb early-treatment, p<0.001; n=15 in VIPhyb late-treatment, p=0.005 vs. control, n=14; Figure 1A) and in the B6 to B10.BR model with LBRM (3 replicate experiments in 0.5 x 106 splenocyte dose, n=10, p =0.001 in VIP-KO, n=20, p=0.01 in VIPhyb-treatment, n=40 in control; Figure 1B). All recipients of TCD-BM alone died of tumors within 60 days post-BMT. VIPhyb-treatment, and transplantation of VIP-KO donor cells, led to significant decreases in tumor-burden as measured by BLI. Tumor-free recipients following VIPhyb treatment re-challenged with a 10x dose of LBRM had 100% survival compared with 40% survival among saline-treated recipients (p=0.009). Secondary transplantation of splenocytes from tumor-free VIPhyb-treated recipients resulted in 100% tumor-free survival compared with 30% survival among recipients of naive donor T cells (p=0.001). Splenic T cells, and cDC and pDC from VIPhyb-treated mice expressed lower levels of PD-1, and PD-L1, respectively, compared with saline-treated control (Figure 2). Donor T cells from leukemia-bearing mice treated with VIPhyb expressed higher-levels of ICOS and IFN-γ compared with T cells from saline-treated mice. Both cDC and pDC from VIPhyb-treated mice had increased expression of ICOS-L and MHC-II. Donor T-cells recovered from mice treated with VIPhyb had increased levels of granzyme B and enhanced cytolytic activity against tumor targets but not against non-tumor recipient-type splenocytes or third party splenocytes. Genetic or ex vivo depletion of donor NK cells or CD8+ T-cells abrogated the enhanced GvL activity seen with VIP antagonist-treatment. VIPhyb had no direct anti-proliferative activity against LBRM in vitro using a range of concentrations that exceeded predicted in vivo concentration. VIPhyb-treated transplant recipients had expansion of oligo-clonal T-cell subsets that conferred leukemia protection to secondary recipients and expressed distinct TCR-β sequences compared with T cells from donor or GvHD mice.Conclusions: Blocking VIP signaling enhanced GvL activity without increasing GvHD. Donor CD8+ T-cells and NK cells were indispensable for the enhanced GvL activity. The ability of VIP antagonist treatment to down-regulate PD-1/PD-L1 signaling and enhance adaptive cellular immunity is a novel approach to treat and prevent relapse in allo BMT. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Absence of Vasoactive Intestinal Peptide Signaling Significantly Enhances T Cell Survival Following MCMV Infection Via Increased Expression of Bcl-2 and Bcl-XL

BackgroundThe nervous system's influence on immune responses has become increasingly appreciated following the identification of a variety of immunomodulatory products secreted from neurons. One such product, vasoactive intestinal peptide (VIP), has been identified as an anti-inflammatory mediator exerting effects on both the innate and adaptive arms of the immune system. Interference with this pathway through blocking the VIP receptors VPAC1 and VPAC2 has been demonstrated to enhance T cell responses to viral infection as well as cancer. VIP antagonism results in increased numbers of antigen-specific cells in the peripheral blood and a shift in the balance of co-stimulatory and co-inhibitory molecules towards a pro-inflammatory phenotype. The consequences of such effects are a reduction in viral load in a model of MCMV infection and a reduced tumor burden and increased survival in a model of lymphoma.MethodsIn order to better understand the molecular mechanisms underlying the effects of VIP absence or antagonism, we used the MCMV infection model. Male VIP WT and VIP KO mice were infected with 105 pfu MCMV ip and sacrificed at various time points. Spleens were harvested and T cells were FACS sorted based on the expression of activation markers. Expression of pro and anti-apoptotic molecules was assessed via Western blot. Expression of VIP and its receptor VPAC1 was determined using qRT-PCR of extracted mRNA. In vitro T cell proliferation was assessed using CFSE dilution of splenocytes stimulated with CD3/CD28 beads and IL-2.ResultsWe first examined levels of VIP and VPAC1 over the course of MCMV infection via qRT-PCR of extracted mRNA. Expression of VIP and VPAC1 was found to change in response to infection with an initial decrease in the levels of VPAC1 followed by an increase back to baseline levels. The pattern of VIP expression was opposite with an initial increase followed by a decrease. In order to gain more mechanistic insight into the effects of VIP signaling in T cells during MCMV infection, activated splenic CD8 T cells from WT mice and mice lacking VIP were FACS sorted at various time points. Activated CD69+CD8+ T cells from KO mice expressed higher levels of the anti-apoptotic protein Bcl-XL both during the initial response and at the peak by Western blot (Figure 1). Intracellular staining revealed an increased frequency of Bcl-2-expressing CD8 T cells in KO mice at the peak of the response (Figure 1). Proliferation of CFSE-labeled CD8 T cells in response to in vitro CD3/CD28 stimulation was found to be identical in wild type and VIP knockout cells suggesting modified T cell survival rather than proliferation. The negative influence of VIP on T cell survival resulted in significantly higher T cell frequency and a significant increase in the absolute numbers of antigen-specific splenic CD8 T cells in knockout mice (Figure 2). These results provide further support for VIP antagonism as a viable option for th e enhancement of CD8 T cell-mediated immunity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Administration of a vasoactive intestinal peptide antagonist significantly enhances the autologous anti-leukemia T cell response in a murine model of AML

Products secreted from nerve terminals can have profound effects on the phenotype and functioning of T cells. One such product, vasoactive intestinal peptide (VIP) is a small neuroendocrine peptide hormone with potent anti-inflammatory activities. Binding to either of its type B G protein coupled receptors VPAC1 and VPAC2 initiates a signaling cascade that ultimately inhibits the secretion of pro-inflammatory cytokines and reduces proliferative capacity via inhibition of NF-κB[1]. The anti-inflammatory effects of VIP signaling in immune cells have demonstrated to be inhibitable with the use of one of several small molecule antagonists. VIPhyb is a hybrid peptide antagonist consisting of the VIP peptide with a six amino acid substitution at the N-terminus. We demonstrate here that subcutaneous administration of small doses of VIPhyb enhanced the autologous T cell response in mice bearing C1498 acute myeloid leukemia. Secretion of both IFN-γ and TNF-α was significantly increased in VIPhyb-treated mice. Additionally, the frequency of PD-1-expressing CD4 and CD8 T cells was significantly lower in mice treated with the VIP antagonist. This was determined to be of relevance in this model as PD-L1 was found to be inducible on C1498 cells. This expression was not affected by VIPhyb treatment. Each of these effects contributed to a reduction in tumor burden in VIPhyb-treated mice as evidenced by bioluminescent imaging. No direct cytotoxic effect of VIPhyb on the tumor cells themselves was observed despite the expression of VPAC2. Overall survival of mice that received 1, 3, or 7 doses was significantly higher than vehicle-treated controls as a result of enhanced immunological function. Importantly, VIPhyb-treated survivors were found to be resistant to further tumor development following re-challenge with C1498 cells. This observation highlights a contribution of VIP signaling blockade in the development of anti-leukemia T cell memory. The data heretofore described highlight the contribution of VIP to the inhibition of anti-leukemia T cell responses and thus present rationale for considering this pathway in the design of therapeutic strategies for AML. VIP signaling blockade is thus a promising candidate for the enhancement of immunotherapies that seek to strengthen the anti-leukemia T cell response. Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

Electroacupuncture attenuates collagen-induced arthritis in rats through vasoactive intestinal peptide signalling-dependent re-establishment of the regulatory T cell/T-helper 17 cell balance.

Imbalance between T-helper 17 (Th17) cells and regulatory T (Treg) cells is causally linked to the development of rheumatoid arthritis (RA). In this study, we tested the hypothesis that electroacupuncture (EA) confers therapeutic benefits in RA through activation of vasoactive intestinal peptide (VIP)-dependent signalling and restoration of the Th17/Treg cell balance. A collagen-induced arthritis (CIA) model was induced in Sprague-Dawley rats by injection of bovine type II collagen in incomplete Freund's adjuvant on day 0 and day 7. Three days after the second injection, EA was given at acupuncture points GB39 and ST36 three times per week for 4 weeks. To block VIP signalling, [D-P-Cl-Phe(6)-Leu(17)]-VIP, a VIP receptor antagonist, was administered intraperitoneally 30 min before EA. Inflammatory and pathological responses in the joint were assessed. Synovial VIP receptor mRNA levels and Treg and Th17 cell frequencies in the spleen were determined. EA significantly reduced the severity of CIA, as evidenced by reduced paw volumes, arthritis scores and inflammation scores. EA significantly increased mRNA expression of the VIP receptor VPAC1 and led to an elevation in CD4(+)FOXP3(+) Treg cell frequency and a reduction in CD4(+)IL17(+) Th17 cell frequency. Pre-injection of a VIP receptor antagonist significantly reversed EA-induced expansion of Treg cells, but did not alter the frequencies of Th17 cells. EA exerts anti-inflammatory effects in a collagen-induced rat model of arthritis. These effects appear to be mediated through activation of VIP signalling and re-establishment of the Th17/Treg cell balance.

Circadian Control of the Female Reproductive Axis Through Gated Responsiveness of the RFRP-3 System to VIP Signaling.

Throughout most of the ovulatory cycle, estrogen negative feedback restrains the GnRH neuronal system. Just before ovulation, however, estrogen negative feedback is removed to permit stimulation of the preovulatory GnRH/LH surge (positive feedback) by the circadian clock in the suprachiasmatic nucleus (SCN). The mammalian ortholog of avian gonadotropin-inhibitory hormone, RFamide-related peptide 3 (RFRP-3), participates in the circadian-timed removal of estrogen negative feedback to permit the LH surge. The present study examined the specific neurochemical means by which the SCN controls RFRP-3 activity and explored whether the RFRP-3 system exhibits time-dependent responsiveness to SCN signaling to precisely time the LH surge. We found that RFRP-3 cells in female Syrian hamsters (Mesocricetus auratus) receive close appositions from SCN-derived vasopressin-ergic and vasoactive intestinal peptide (VIP)-ergic terminal fibers. Central VIP administration markedly suppressed RFRP-3 cellular activity in the evening, but not the morning, relative to saline controls, whereas vasopressin was without effect at either time point. Double-label in situ hybridization for Rfrp-3 and the VIP receptors VPAC1 and VPAC2 revealed that the majority of RFRP-3 cells do not coexpress either receptor in Syrian hamsters or mice, suggesting that SCN VIP-ergic signaling inhibits RFRP-3 cells indirectly. The timing of this VIP-mediated disinhibition is further coordinated via temporally gated responsiveness of RFRP-3 cells to circadian signaling. Together, these findings reveal a novel circadian hierarchy of control coordinating the preovulatory LH surge and ovulation.

VIP regulates CFTR membrane expression and function in Calu-3 cells by increasing its interaction with NHERF1 and P-ERM in a VPAC1- and PKCε-dependent manner.

Vasoactive intestinal peptide (VIP) is a topical airway gland secretagogue regulating fluid secretions, primarily by stimulating cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride secretion that contributes to the airways innate defense mechanism. We previously reported that prolonged VIP stimulation of pituitary adenylate cyclase-activating peptide receptors (VPAC1) in airway cells enhances CFTR function by increasing its membrane stability. In the present study, we identified the key effectors in the VIP signaling cascade in the human bronchial serous cell line Calu-3. Using immunocytochemistry and in situ proximity ligation assays, we found that VIP stimulation increased CFTR membrane localization by promoting its colocalization and interaction with the scaffolding protein Na(+)/H(+) exchange factor 1 (NHERF1), a PDZ protein known as a positive regulator for CFTR membrane localization. VIP stimulation also increased phosphorylation, by protein kinase Cε of the actin-binding protein complex ezrin/radixin/moesin (ERM) and its interaction with NHERF1 and CFTR complex. On the other hand, it reduced intracellular CFTR colocalization and interaction with CFTR associated ligand, another PDZ protein known to compete with NHERF1 for CFTR interaction, inducing cytoplasmic retention and lysosomal degradation. Reducing NHERF1 or ERM expression levels by specific siRNAs prevented the VIP effect on CFTR membrane stability. Furthermore, iodide efflux assays confirmed that NHERF1 and P-ERM are necessary for VIP regulation of the stability and sustained activity of membrane CFTR. This study shows the cellular mechanism by which prolonged VIP stimulation of airway epithelial cells regulates CFTR-dependent secretions.

Vasoactive Intestinal Peptide Modulation of the Steroid-Induced LH Surge Involves Kisspeptin Signaling in Young but Not in Middle-Aged Female Rats

Age-related LH surge dysfunction in middle-aged rats is characterized, in part, by reduced responsiveness to estradiol (E2)-positive feedback and reduced hypothalamic kisspeptin neurotransmission. Vasoactive intestinal peptide (VIP) neurons in the suprachiasmatic nucleus project to hypothalamic regions that house kisspeptin neurons. Additionally, middle-age females express less VIP mRNA in the suprachiasmatic nucleus on the day of the LH surge and intracerebroventricular (icv) VIP infusion restores LH surges. We tested the hypothesis that icv infusion of VIP modulates the LH surge through effects on the kisspeptin and RFamide-related peptide-3 (RFRP-3; an estradiol-regulated inhibitor of GnRH neurons) neurotransmitter systems. Brains were collected for in situ hybridization analyses from ovariectomized and ovarian hormone-primed young and middle-aged females infused with VIP or saline. The percentage of GnRH and Kiss1 cells coexpressing cfos and total Kiss1 mRNA were reduced in saline-infused middle-aged compared with young females. In young females, VIP reduced the percentage of GnRH and Kiss1 cells coexpressing cfos, suggesting that increased VIP signaling in young females adversely affected the function of Kiss1 and GnRH neurons. In middle-aged females, VIP increased the percentage of GnRH but not Kiss1 neurons coexpressing cfos, suggesting VIP affects LH release in middle-aged females through kisspeptin-independent effects on GnRH neurons. Neither reproductive age nor VIP affected Rfrp cell number, Rfrp mRNA levels per cell, or coexpression of cfos in Rfrp cells. These data suggest that VIP differentially affects activation of GnRH and kisspeptin neurons of female rats in an age-dependent manner.