Vasoactive Intestinal Peptide Production Research Articles

Vasoactive Intestinal Peptide Signaling in Transplant Recipients Modulates Gut Microbiome Diversity and Regulates Graft-Versus-Host Disease

Introduction: Allogeneic bone marrow transplantation (allo-BMT) presents a significant challenge in the form of graft-versus-host disease (GvHD), characterized by donor T cell-mediated attacks on recipient organs, leading to considerable morbidity and mortality. Vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, plays a crucial role in shaping T cell responses towards the Th2 phenotype while inhibiting proinflammatory cytokine production. Furthermore, VIP-producing enteric neurons contribute to enteric mucosal barrier homeostasis, maintaining optimal gut pH, and orchestrating the delicate balance between beneficial bacteria colonization and resistance against opportunistic pathogens. The potential impact of VIP production by host cells on GvHD regulation remains unclear. Our hypothesis proposes that the absence of VIP production by transplant recipients may exacerbate GvHD, either through increased T cell allo-reactivity, or via secondary modulation of the gut microbiome resulting from submucosal inflammation. Methods and Results: We investigated how endogenous production of VIP by host tissues on GvHD modulation in three murine allo-BMT models, where C57BL/6 wildtype (WT) and VIP-knockout (VIPKO) mice were subjected to 11Gy irradiation on day 0 and subsequently transplanted intravenously with 5x10 6 T cell depleted (TCD) bone marrow (BM) in combination with 0, 1x10 6, or 3x10 6 T cells from MHC mismatched B10.BR, Balb/c or FvB donors. In all three models, WT recipients had better survival than VIPKO recipients. Specifically, in B10BR B6, Balb/c B6 and FvB B6 model, the addition of 3 million donor T lymphocytes to the graft resulted in significantly more GvHD-related mortality in VIP-KO recipients compared with WT recipients, with 0% vs 82%, 18% vs 96%, and 0% vs 78% survival (p<0.01) among WT vs VIP-KO recipients. To elucidate the role of host cell-derived VIP production in T cell alloreactivity and GvHD exacerbation, we performed cytokine profile analysis using both serum and donor-derived lymphocytes from the spleen of transplant recipients. For serum cytokine analysis, VIP-KO recipients had significantly higher levels of IFNγ (p<0.05), TNFα (p<0.05) and IL-1beta (p<0.05) on day 7 post allo-BMT, and significantly higher levels of and IL-6 (p<0.01) and IL-10 (p<0.05) on day 14 post allo-BMT compared with WT recipients. For donor-derived lymphocytes cytokine analysis, we observed significantly increased Th1 and Th17 cytokines in VIP-KO recipient, with significantly higher frequencies of CD4+ IL2 (p<0.01) and CD4+ IFNγ (p<0.001) on day 4, significantly higher levels of CD4+ TNFα (p<0.05) and CD8+ TNFα (p<0.001) on day 19, and significantly increased CD8+ TNFα (p<0.05) on day 25 post allo-BMT compared to WT recipients. Immunofluorescence whole-mount imaging section staining demonstrated detectable VIP expression in enteric neurons innervating the entirety of the intestine in FvB VIP-GFP transplant recipients. Notably, VIP immunofluorescent signal was much higher in allo-BMT recipient intestines compared to syngeneic transplant recipient intestines, demonstrating immunomodulation of VIP-producing enteric neurons in response to local (GVHD-targeted organ) and systemic inflammation (Fig 1). To investigate the gut microbiome profile, we performed 16S ribosomal RNA sequencing and analyzed stool samples from three distinct groups: 1) naïve WT and VIP KO mice, 2) WT and VIPKO mice after co-housing, and 3) WT and VIPKO mice after co-housing followed by allo-BMT. Uniform Manifold Approximation and Projection high-dimensional cluster analysis revealed that VIP-KO mice exhibited a distinct gut microbiome compared to WT mice, regardless of their co-housing or allo-BMT conditioning (Fig 2A, 2B). The heatmap analysis demonstrated that VIP-KO mice had increased lactobacillus species (Fig 2C) and significantly decreased intestinal pH levels (p<0.01) compared to WT mice. Moreover, VIP-KO mice displayed significantly decreased gut microbiome diversity, indicating an inflammatory effect in the gut that influences changes in the microbiome composition. Conclusion: Overall, our findings highlight the crucial role of VIP in regulating immune responses and gut microbiome homeostasis, which could have implications for developing strategies to mitigate GvHD in allo-BMT recipients.

Abstract C007: Targeting vasoactive intestinal peptide receptor signaling enhances T-cell mediated anti-tumor response to immune checkpoint therapy in pancreatic ductal adenocarcinoma

Abstract Current immune checkpoint therapies including anti-CTLA-4 and anti-PD1 have limited efficacy in the treatment of pancreatic ductal adenocarcinoma (PDAC), largely due to a paucity of effector T cells within the tumor microenvironment (TME). In our study, we show that vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, is over-expressed in human PDAC tumors. Thus, we hypothesized that paracrine production of VIP by tumor cells within the PDAC TME constitutes an immune checkpoint pathway that limits the antitumor activity of VIP-receptor (VIP-R) expressing T cells. We first evaluated whether inhibiting VIP-R signaling by peptide-based VIP-R antagonist affects the growth of PDAC cells in vitro. Treatment of PDAC cells with increasing concentrations of VIP-R antagonist did not affect the viability of PDAC cell lines in vitro, except for a transient effect in MT5 albeit not significant. Furthermore, depletion of VIP receptor 2 (VPAC2) in a PDAC cell line, Panc02, by CRISPR-Cas9 KO had similar growth rates in vitro with a slight growth delay in vivo compared to the parental cell line. These data suggest that the expression of VPAC2 on PDAC cells may not confer a significant autocrine effect on tumor growth impacted by VIP signaling. On the contrary, human T cells cultured with VIP-R antagonists had enhanced activation, decreased proportions of Tregs, and exhausted T cells co-expressing PD-1, Tim-3, and Lag-3. Next, we performed pharmacological inhibition with VIP-R antagonists in tumor-bearing mice to model T cell homing, activation, and antitumor responses in preclinical murine models of PDAC. Daily subcutaneous injections of VIP-R antagonists had synergistic antitumor activity when combined with anti-PD-1 therapy with decreased tumor burden and improved survival. In contrast to the limited single-agent activity of anti-PD1 or VIP-R antagonists, combination therapy eliminated tumors in up to 40% of animals. Anti-tumor responses were T cell-dependent, as the combination therapy failed to improve survival in CD4 or CD8 deficient mice using knock-out strains and antibody depletion. In addition, tumor-free mice were resistant to tumor re-challenge, indicating the generation of anti-cancer immunological memory. In correspondent to these data, combination therapy led to enhanced T cell activation in vivo with increased antigen-specific T cells, decreased frequencies of Tregs as well as enhanced T cell infiltration to the tumor site as observed by infiltration of adoptively transferred GFP+ T cells into PDAC tumors. Taken together, our findings show that VIP-VIPR signaling is a targetable mechanism of immune escape in PDAC. Inhibiting VIP-R signaling improves T cell effector properties and enhances the responsiveness of PDAC to immune checkpoint therapy. Further pharmacokinetic and toxicology studies are underway. Citation Format: Tenzin Passang, Sruthi Ravindranathan, Jian-Ming Li, Shuhua Wang, Michael Brandon Ware, Hanwen Zhang, Gregory B. Lesinski, Edmund K. Waller. Targeting vasoactive intestinal peptide receptor signaling enhances T-cell mediated anti-tumor response to immune checkpoint therapy in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C007.

Donor Plasmacytoid Dendritic Cells Regulate GvHD in a VIP Dependent Manner in Allogeneic BMT Recipients

Introduction: Vasoactive intestinal peptide (VIP) is an anti-inflammatory neuropeptide known to induce differentiation of regulatory dendritic cells and regulatory T cells. Using allogeneic hematopoietic stem cell transplantation (allo-HSCT) models, we have shown that donor bone marrow (BM) plasmacytoid dendritic cells (pDCs) facilitate HSC engraftment and attenuate pathogenesis of graft vs. host disease (GvHD) through regulation of recipient T cells. However, the mechanism by which pDCs mitigate the GvHD activity of recipient T cells is not clearly understood. Here, we report that donor pDCs limit pathogenic T cell inflammation by VIP production.Methods: To study VIP production by pDCs, FACS-sorted pDCs from B6 mouse BM were cultured with or without PMA/ionomycin in-vitro. After activation and cytospin slide preparation, pDCs were labeled with anti-PDCA1 (pDC marker) and anti-VIP antibodies for confocal fluorescence microscopy. To investigate the effects of VIP production on T cell proliferation, an in-vitro co-culture assay was performed using R848 and CpG-activated WT or VIP-KO pDCs with anti-CD3-activated, CFSE-labeled syngeneic T cells. For GvHD experiments, irradiated B10.BR (H-2K k) mice received 5x10 3 HSCs, 5x10 4 pDCs and 1x10 6 T cells from WT B6 (H-2K b) or VIP-KO B6 (H-2K b) mice. H&E histology of intestine and colon was performed for GvHD scoring 7 days post-transplant. Graft vs. leukemia (GvL) effects were tested by inoculating recipient mice with 5x10 5 LBRM 33-5A4 cells in the same model. Recipient mice were monitored twice weekly using a 10-point GvHD scoring system. Gene expression analysis of FACS-sorted donor T-cells from recipient spleens was performed using the Nanostring Myeloid Innate Immunity Panel at days 8 and 15 post-transplant.Results: Confocal microscopic images of PMA/ionomycin stimulated or unstimulated sorted pDCs show that VIP is synthesized by pDCs (anti-VIP, green; anti-PCDA-1, red; DAPI counterstain, blue) (Fig 1). After in-vitro culture, VIP expression and frequencies of VIP + pDCs were similar in PMA/ionomycin treated or untreated cells (not shown). VIP-KO mice have significantly higher percentages of pDCs in BM compared to WT (Fig 2a). T cells co-cultured with VIP-KO pDCs showed higher proliferation than T cells co-cultured with WT pDCs, demonstrating that VIP secreted by pDCs reduces T cell proliferation (Fig 2b). Moreover, VIP-KO pDCs induce significantly greater proliferation of IFN-gamma + CD8 T cells compared to WT, indicating that pDCs lacking VIP promote Th1 polarization in-vitro (Fig 2c). The data are consistent with results from GvHD experiments showing increased frequencies of Th1 polarized T cells and fewer regulatory T cells in recipients of VIP-KO pDCs compared with recipients of WT pDCs. Intestinal GvHD scores and crypt apoptosis in the colon were higher in recipient groups transplanted without pDCs or with VIP-KO pDCs compared with recipients of WT pDCs (Fig 3a, b, c). These results indicate that VIP secreted from pDCs limits GvHD in the gut. In the GvL model, administration of pDCs lacking VIP did not alter the anti-tumor effect of donor T cells. Nanostring analysis of T cells recovered from VIP-KO pDC recipients had increased expression of the pro-inflammatory transcription factor Bhlhe40 during the first two weeks post-transplant, and higher transcription levels of the inflammatory mediator Cyclophilin A at day 15 post-transplant than T cells from recipients of WT pDCs.Conclusion: Data from in vitro and in vivo experiments suggest that VIP secreted by pDCs limits pathogenic T cell proliferation. In murine allo-BMT, increased gut GvHD scores and crypt apoptosis in recipients transplanted without pDCs or with VIP-KO pDCs indicates that VIP secreted by pDCs consolidates gut integrity without altering GvL. Gene expression analysis also supports a mechanism by which VIP-secreting donor pDCs reduce T cell inflammation through negative regulation of Bhlhe40. Our findings suggest paracrine VIP signaling is a novel immune checkpoint pathway by which donor pDCs limit T cell activation, Th1 polarization, and inflammation, and improve outcomes of allo-BMT by reducing GvHD activity. [Display omitted] DisclosuresWaller: Cambium Oncology: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Verastem Oncology: Consultancy, Research Funding.

Vasoactive Intestinal Peptide Derived From Liver Mesenchymal Cells Mediates Tight Junction Assembly in Mouse Intrahepatic Bile Ducts.

Formation of intrahepatic bile ducts (IHBDs) proceeds in accordance with their microenvironment. Particularly, mesenchymal cells around portal veins regulate the differentiation and ductular morphogenesis of cholangiocytes in the developing liver; however, further studies are needed to fully understand the arrangement of IHBDs into a continuous hierarchical network. This study aims to clarify the interaction between biliary and liver mesenchymal cells during IHBD formation. To identify candidate factors contributing to this cell–cell interaction, mesenchymal cells were isolated from embryonic day 16.5 matrix metalloproteinase 14 (MMP14)‐deficient (knockout [KO]) mice livers, in which IHBD formation is retarded, and compared with those of the wild type (WT). WT mesenchymal cells significantly facilitated the formation of luminal structures comprised of hepatoblast‐derived cholangiocytes (cholangiocytic cysts), whereas MMP14‐KO mesenchymal cells failed to promote cyst formation. Comprehensive analysis revealed that expression of vasoactive intestinal peptide (VIP) was significantly suppressed in MMP14‐KO mesenchymal cells. VIP and VIP receptor 1 (VIPR1) were mainly expressed in periportal mesenchymal cells and cholangiocytic progenitors during IHBD development, respectively, in vivo. VIP/VIPR1 signaling significantly encouraged cholangiocytic cyst formation and up‐regulated tight junction protein 1, cystic fibrosis transmembrane conductance regulator, and aquaporin 1, in vitro. VIP antagonist significantly suppressed the tight junction assembly and the up‐regulation of ion/water transporters during IHBD development in vivo. In a cholestatic injury model of adult mice, exogenous VIP administration promoted the restoration of damaged tight junctions in bile ducts and improved hyperbilirubinemia. Conclusion: VIP is produced by periportal mesenchymal cells during the perinatal stage. It supports bile duct development by establishing tight junctions and up‐regulating ion/water transporters in cholangiocytes. VIP contributes to prompt recovery from cholestatic damage through the establishment of tight junctions in the bile ducts.

Transplanted Vasoactive Intestinal Peptide-Secreting Donor Plasmacytoid Dendritic Cells Migrate to Lymphoid Organs and GvHD Target Organs

Introduction: Donor plasmacytoid dendritic cells (pDCs) have important roles in the pathogenesis of graft versus host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Vasoactive intestinal peptide (VIP), a potent anti-inflammatory neuropeptide with pleiotropic effects, has shown promise in managing GvHD through induction of regulatory dendritic cells (Chorny, Blood 2006) and regulatory T cells (Delgado, JLB 2005). Our previous data showed that transplantation of hematopoietic stem cells (HSC) lacking VIP expression in enhanced Th1 polarization and antiviral immunity (Li, JI 2011). Whether VIP produced by pDCs plays a key role in preventing GvHD is the focus of the current study. Method: A reductive allo-HSCT model was established by transplanting purified populations of 5 x 103 hematopoietic stem cells (HSC), 5 x 104 pDCs and 106 T cells from B6 (H-2Kb) to MHC mismatched B10.BR (H-2Kk) recipients. Mice received either VIP knockout pDCs (experimental group) or wild-type pDCs (control). Survival and clinical GvHD scores were monitored for at least 60 days. Serum pro-inflammatory cytokines were measured by luminex on day 3 and day 8. Additional recipients were transplanted for interim analyses of GvHD pathology in histological sections of small intestine and colon on day 8 and day 15. To investigate pDC migration and proliferation status, 5 x 104 luciferase-expressing pDCs, together with 5 x 103 WT B6 HSC, were transplanted to MHC mismatched Balb/c (H2kd) recipients. Bioluminescent imaging was performed on day 14. We used mice carrying an eGFP reporter linked to a VIP promoter to measure VIP production by donor cells in vivo. The construct has the eGFP reporter gene, followed by a polyadenylation sequence, inserted into BAC clone RP23-25A8 at the initiating ATG codon of the first coding exon of the VIP gene, so that eGFP expression is driven by the regulatory sequences of the BAC gene. The BAC is inserted randomly in the genome. We used serial breeding to establish the eGFP-VIP reporter in B6 mice. Bone marrow pDCs were sorted from transgenic eGFP-VIP B6 mice and used in transplant experiments, allowing VIP gene activity from donor-derived pDCs to be detected as GFP expression using confocal microscopy. Results: Compared to recipients of WT pDCs, recipients of VIP-KO pDCs have a significantly lower survival rate with increased GvHD clinical score (Figure 1; *p<0.05). Transplant recipients of VIP-KO donor pDC had higher serum pro-inflammatory cytokine levels on day 3 and day 8 respectively. (Figure 2). At 2 weeks post-transplant donor pDCs migrated primarily to the spleen, with limited presence of donor pDCs in the gastrointestinal tract and lung (Figure 3). Compared to mice transplanted with WT pDCs, recipients of VIP-KO pDCs had increased donor T cell migration to the lung and paw, which are chronic GvHD target organs after two weeks. (Figure 4; *p<0.05). When stimulated with IFN-g, eGFP-VIP transgenic pDCs have activation of the VIP gene promoter in vitro, as detected using eGFP (Figure 5). Confocal microscopy data showed that pDCs have activated the VIP promoter and secreted more VIP following stimulation by IFN-g, consistent with VIP up-regulation by the inflammatory mileu in allo-transplant recipients. Conclusions: These data show that lack of VIP secretion by donor pDCs resulted in lower survival rates and higher GvHD scores in allo-BMT recipients. Recipients of VIP KO donor pDC had increased donor T cell proliferation in GvHD target organs and increased GvHD clinical signs. The major site of donor pDCs migration in recipients is spleen, suggesting that pDCs may interact with other lymphoid cells, such as T cells, inducing a more tolerogenic environment. Donor pDCs were also found, to a lesser extent, in the lung and gastrointestinal tract, which are both GvHD target organs. Thus, pDCs may protect recipients from GvHD by limiting the activity of alloreactive T cells at these sites. The mechanism by which VIP-secreting donor pDCs regulate GvHD will be discussed. Disclosures Waller: Amgen: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Other: Travel expenses, Research Funding; Cerus Corporation: Other: Stock, Patents & Royalties; Chimerix: Other: Stock; Cambium Oncology: Patents & Royalties: Patents, royalties or other intellectual property ; Kalytera: Consultancy.

Vasoactive intestinal polypeptide synthesis by somatic tissues in transplant recipients limits GVHD in allo-BMT

Abstract Both donor and recipient hematopoietic cells regulate GvHD in allo-BMT, but the role of somatic host tissues in the induction and maintenance of GvHD is less clear. Vasoactive Intestinal peptide (VIP) is a neuropeptide with potent immune-regulatory activity. Our goal was to examine the role of VIP synthesis by recipient tissues on donor T cell activation and GvHD in allo-BMT. We used MHC mis-matched allo-BMT models, B10.BR-->B6 and Balb/c-->B6, irradiating WT and VIP-KO B6 mice with 11Gy on day −1 and transplanting with 5 × 10E6 T cell depleted (TCD) bone marrow (BM) plus 0, 1 × 10E6, or 3 × 10E6 T cells from MHC mis-matched donors. B6-->B6 radiation chimeras lacking VIP in either hematopoietic or non-hematopoietic cells were created by performing B6 WT-->VIP-KO and VIP-KO-->WT syngeneic transplants. Production of VIP in transplant recipients was visualized with immune-fluorescent staining of tissues using an anti-VIP antibody. Transplanting TCD BM plus 3 × 10E6 donor T cells from B10.BR or Balb/c donors in VIP KIO mice led significantly increased GVHD-mortality compared with WT recipients. Donor-derived T cells in VIP-KO recipients had significantly higher Th1 and Th17 polarization. B10.BR-->B6 second allogeneic transplantation of radiation chimeras caused lethal GVHD mortality in VIP-KO-- >VIP-KO and WT-->VIP-KO mice, but not in WT-->WT or VIP-KO-->WT B6 mice, demonstrating the protective effect of VIP was due to synthesis by somatic recipient cells. Immunofluorescent imaging of allo-BMT recipients showed up-regulation of VIP in lungs post-transplant and high VIP production within neurons innervating the lungs. Short-term administration of VIP from D0 to D10 prevented GvHD-mortality in VIP-KO recipients transplanted BM & T cells.

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.

Donor Plasmacytoid Dendritic Cells Home to the Thymus of the Transplant Recipient and Limit Graft Versus Host Diseases through Production of Vasoactive Intestinal Polypeptide

Introduction: In allogeneic hematopoietic stem cell transplant (AHSCT), donor T cells promote hematopoietic engraftment, reconstitute T-cell immunity and mediate potent beneficial antitumor effects, such as graft versus leukemia (GVL) as well as detrimental graft-versus-host disease (GVHD). We have shown that purified bone marrow pDC to a donor graft composed of purified HSC and T cells significantly improved long-term leukemia-free survival without increasing the risk of GVHD (Lu BLOOD 2012). Vasoactive intestinal peptide (VIP) is a neuropeptide/neurotransmitter, which acts as a major anti-inflammatory factor in animal models of inflammatory and autoimmune diseases. VIP is produced by T cells and dendritic cells under conditions of inflammation (Li Cancer Research 2016). VIP-signaling, thus represents a newly appreciated co-inhibitory pathway involved in T cell activation and expansion and persistence of antigen-specific T cells, but the role of VIP produced by donor dendritic cells in allo-BMT is unclear. Our previous data has shown that production of VIP in pDC improves survival in a murine allo-BMT model. Since the thymus plays a critical role in regenerating naive T cells in which allo-reactive donor T cells undergoes central deletion. We hypothesized that donor pDCs that home to thymus modulate the negative selection of allo-reactive T cells and iTreg production through VIP signaling.Methods and Results: Our previous data has shown that the mice receiving HSC, T cells and WT pDC had a significantly higher survival (71%) compared to those receiving VIP-KO pDC (31%). On day 15 after transplant, recipients of WT pDC, VIP-KO pDC and no pDC developed ~98% chimerism, without significant differences among the three groups. Local production of VIP in pDC inhibited activation and Th1 immune polarization of donor T cells.Recipient spleens were harvested on day 15 after transplant for analysis of cytokine production by donor T cells. The percentage of CD8+ donor T cells producing IL17 was significantly higher in recipients of VIP-KO pDC compared to recipients of WT-pDC (Fig 2.A-B). The ratio of foxp3+ CD4+ donor T-reg to IL17+ CD4+ T cells from recipients of WT pDC was almost 3 times higher than the recipients of VIP-KO pDC (Fig 2.C-D).To visualize production of VIP by donor pDC, B10.BR mice were transplanted with 5,000 stem cells, 1M T cells and 50,000 pDCs from VIP-GFP (VIP promoter and GFP reporter) or GFP mice. On day 7 post-transplant, the thymus was examined with confocal microscopy with GFP (green), anti-PDCA-1-Alexa Fluor 568 (red) and DAPI (blue). A superimposed profile of the thymus showed that donor GFP pDCs homed to thymus (Fig.2-A), and that donor pDC in the recipient thymus produced VIP (Fig.2-B).Conclusion: Expression of VIP in donor pDCs inhibited activation and Th17 immune polarization of donor CD8+ T cells after allo-BMT. Foxp3+ expression tended to be higher among CD4+ donor T cells from recipients of WT pDCs compared with recipients of VIP-KO pDCs. Thus, VIP-producing donor pDC in thymus could be very critical to contribute to negative selection of allo-reactive donor T cell or facilitate the generation of Foxp3+ nTreg. This data supported a new mechanism by which GvHD maybe regulated and central tolerance maintained. Ongoing experiments aim at defining the role of VIP production in the thymus by donor pDC in positive and negative T cell selection. [Display omitted] DisclosuresWaller:Celldex: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cambium Medical Technologies: Consultancy, Equity Ownership; Kalytera: Consultancy; Pharmacyclics: Other: Travel Expenses, EHA, Research Funding.

Clinical study of cerebrospinal fluid neuropeptides in patients with primary trigeminal neuralgia

ObjectivesTo investigate the expression levels of calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal peptide (VIP), and β-endorphin in the cerebrospinal fluid (CSF) and peripheral blood of patients with primary trigeminal neuralgia (TN). Patients and methodsWe included 20 patients with primary TN who underwent percutaneous radiofrequency thermocoagulation and collected four types of samples from all of them: sample A: CSF samples; sample B: peripheral blood samples; sample C: peripheral blood samples collected one day before the operation; sample D: peripheral blood samples withdrawn one day after the operation. Another 20 CSF samples of patients with nervous system disease or gynecological disease were collected as a control (sample E). Samples A and B were obtained at the same time. We also evaluated the expression of CGRP, SP, β-endorphin, and VIP by visual analog scale (VAS) scores one day before and one day after the operation. In addition, heart rate (HR) at baseline and at the time of sample collection, mean arterial pressure (MAP), and all side effects of the procedure were recorded. ResultsSignificance were found concerning about CGRP, SP, β-endorphin, and VIP in TN patients and the controls (P<0.001). The expression of CGRP, SP, and VIP in sample A was higher than that in sample E. However, the β-endorphin level in sample A was lower than that in sample E. There was a positive correlation between sample A and B regarding the expression of CGRP, SP, β-endorphin, and VIP (P<0. 01). There was no relationship between the time of disease onset and the expression of CGRP, SP, β-endorphin, and VIP in sample A and sample B (P>0.05). No difference was detected between the neuropeptides levels in samples B and C (P>0.05). Notably, VAS in sample D was significantly lower than that in sample C (P<0.01). Finally, there was no difference between the intraoperative HR and MAP values in the studied samples. ConclusionIn primary TN patients, the blood levels of CGRP, SP, β-endorphin, and VIP were associated with those in CSF samples. There was a significant difference between the levels of the four neuropeptides in CSF and control samples. Our results also indicated that the levels of neuropeptides in blood samples can be tested for those in CSF. The disease onset and duration exerted insignificant effects on the production and release of CGRP, SP, β-endorphin, and VIP.

Human amniotic epithelial cells: Proliferation and apoptosis during their hepatic differentiation

Human amniotic epithelial cells: Proliferation and apoptosis during their hepatic differentiation

The neuropeptide vasoactive intestinal peptide: direct effects on immune cells and involvement in inflammatory and autoimmune diseases

Neuropeptides represent an important category of endogenous contributors to the establishment and maintenance of immune deviation in the immune-privileged organs such as the CNS and in the control of acute inflammation in the peripheral immune organs. Vasoactive intestinal peptide (VIP) is a major immunoregulatory neuropeptide widely distributed in the central and peripheral nervous system. In addition to neurones, VIP is synthesized by immune cells which also express VIP receptors. Here, we review the current information on VIP production and VIP-receptor-mediated effects in the immune system, the role of endogenous and exogenous VIP in inflammatory and autoimmune disorders and the present and future VIP therapeutic approaches.

VIP contribution to the decidualization program: regulatory T cell recruitment

During early pregnancy, the human uterus undergoes profound tissue remodeling characterized by leukocyte invasion and production of proinflammatory cytokines, followed by tissue repair and tolerance maintenance induction. Vasoactive intestinal peptide (VIP) is produced by trophoblast cells and modulates the maternal immune response toward a tolerogenic profile. Here, we evaluated the contribution of the VIP/VPAC to endometrial renewal, inducing decidualization and the recruitment of induced regulatory T cells (iTregs) that accompany the implantation period. For that purpose, we used an in vitro model of decidualization with a human endometrial stromal cell line (HESC) stimulated with progesterone (P4) and lipopolysaccharide (LPS) simulating the inflammatory response during implantation and human iTregs (CD4(+)CD25(+)FOXP3(+)) differentiated from naïve T cells obtained from peripheral blood mononuclear cells of fertile women. We observed that VIP and its receptor VPAC1 are constitutively expressed in HESCs and that P4 increased VIP expression. Moreover, in HESC VIP induced expression of RANTES (CCL5), one of the main chemokines involved in T cell recruitment, and this effect is enhanced by the presence of P4 and LPS. Finally, assays of the migration of iTregs toward conditioned media from HESCs revealed that endogenous VIP production induced by P4 and LPS and RANTES production were involved, as anti-RANTES neutralizing Ab or VIP antagonist prevented their migration. We conclude that VIP may have an active role in the decidualization process, thus contributing to recruitment of iTregs toward endometrial stromal cells by increasing RANTES expression in a P4-dependent manner.

Contribution of Vasoactive Intestinal Peptide to Immune Homeostasis in Trophoblast-Maternal Leukocyte Interaction under LPS Stimulation

Background/Aims: The maternal-fetal interface is a unique immunological site that generates an adequate microenvironment during pregnancy, recognizing and eliminating infections and tolerating the trophoblast/placenta unit. For that purpose, trophoblast cells display several tolerogenic mechanisms to allow fetal survival, such as production of the neuropeptide vasoactive intestinal peptide (VIP). Here we investigated the contribution of VIP to maintain homeostasis at the maternal-placental interface under lipopolysaccharide (LPS) stimulation. Methods: We performed cocultures between trophoblast cells (Swan-71 cell line) and maternal leukocytes obtained from fertile women as an in vitro model of maternal-placental interaction, and we focused on the effects of LPS on the modulation of VIP and their receptors (VPAC₁ and VPAC₂). Results: VIP could prevent the upregulation of IL-6, MCP-1, and nitrite production and maintain the production of IL-10 and TGF-β under LPS (10 µg/ml) stimulation after 48 h of coculture. To gain deeper insight into the mechanisms of how VIP could contribute to a tolerogenic microenvironment even in the presence of LPS, we investigated VIP production by maternal leukocytes and observed a significant increase in the frequency of CD4+VIP+ cells after interaction with Swan-71 cells in the presence of LPS. LPS increased VIP and inducible receptor VPAC₂ expression directly on trophoblast cells in a dose- and time-dependent manner. Conclusions: The present results suggest that VIP might act as an additional homeostatic mechanism during early stages at the maternal-placental interface to control exacerbated inflammatory responses such as the ones observed in intrauterine infections.

Epithelial Expression of Vasoactive Intestinal Peptide in Ulcerative Colitis: Down-Regulation in Markedly Inflamed Colon

Vasoactive intestinal peptide (VIP) has a number of important effects in intestinal physiology and pathology, including in ulcerative colitis (UC). The expression patterns of the predominant VIP receptor in the mucosa (the VPAC1 receptor) are unknown for the mucosa in UC. It is assumed that the sources of VIP in the intestine are the innervation and the inflammatory cells. The VIP and VPAC1 receptor expression patterns in the epithelial layer of UC and non-UC patients were examined in the present study. The influence of marked inflammation of the mucosa was evaluated. Specimens of the human colon, including the colon of UC patients, were examined concerning expressions of VIP and VPAC1 receptor, focusing on the epithelial layer. Immunohistochemistry and in situ hybridization were utilized. There were VIP mRNA reactions and also marked VPAC1 receptor immunoreactions in the normal and slightly/moderately affected epithelium. VIP mRNA reactions were not detected and VPAC1 immunoreactions were minimal in response to marked mucosal derangement. The findings suggest that there is a local production of VIP in the epithelial cells in normal and slightly/moderately inflamed mucosa but not in severely inflamed mucosa. Furthermore, a marked downregulation in VPAC1 receptor expressions occurs in the epithelium in severe UC. Based on the knowledge that VIP can have trophic, healing and anti-inflammatory effects, it is likely that the decrease in VIP mRNA and VPAC1 receptor reactions seen in severely affected mucosa in UC may be associated with adverse effects on intestinal function.

Vasoactive intestinal peptide: a neuropeptide with pleiotropic immune functions

Vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide/neurotransmitter, is widely distributed in both the central and peripheral nervous system. VIP is released by both neurons and immune cells. Various cell types, including immune cells, express VIP receptors. VIP has pleiotropic effects as a neurotransmitter, immune regulator, vasodilator and secretagogue. This review is focused on VIP production and effects on immune cells, VIP receptor signaling as related to immune functions, and the involvement of VIP in inflammatory and autoimmune disorders. The review addresses present clinical use of VIP and future therapeutic directions.

VIP, From Gene to Behavior and Back: Summarizing my 25 Years of Research

Vasoactive intestinal peptide (VIP) is an interesting example of a 28-amino acid neuropeptide that is abundantly expressed in discrete brain regions/neurons and hence may contribute to brain function. This short review summarizes my own point of view and encompasses 25 years of work and over 100 publications targeting the understanding of VIP production and biological activity. The review starts with our original cloning of the VIP gene, it then continues to discoveries of regulation of VIP synthesis and the establishment of the first VIP transgenic mice. The review ends with the identification of novel VIP analogs that helped decipher VIP's important role during development, in regulation of the biological clock(s) and diurnal rhythms, sexual activity, learning and memory as well as social behavior, and cancer. This review cites only articles that I have coauthored and gives my own perspective of this exciting ever-growing field.

Altered expression of matrix metalloproteinase‐2 correlates with increased vasoactive intestinal peptide in a murine model of asthma

Vasoactive intestinal peptide (VIP) is a pleuripotent neuropeptide and cytokine that we hypothesize may regulate the production of matrix metalloproteinase (MMP)‐2 in asthma. METHODS: As the first step of this investigation, mice were sensitized and challenged with Aspergillus fumigatus to elicit allergic airways disease. Expression of VIP mRNA was determined with real‐time PCR. Total MMP‐2 expression in serum was determined by enzyme‐inked immunosorbent assay (ELISA), and immunohistochemistry was used to identify the spatial expression of VIP and MMP‐2 in the allergic lungs. RESULTS: The mRNA for VIP was markedly increased 14 days post challenge in comparison to naïve controls. Serum MMP‐2 peaked at day 7, and was most prominent in the columnar epithelium. While naïve animals showed constitutive VIP production in the airway columnar epithelium and smooth muscle surrounding arterioles, experimental animals produced more VIP in these regions as well as the alveolar lining. CONCLUSION: VIP production spatially and temporally correlates with MMP‐2 expression, suggesting its role as a regulator of this important matrix proteinase. Research supported by Grant Number 2P20RR015566 from the National Center for Research Resources (NCRR).

Pulmonary Expression of Vasoactive Intestinal Peptide in a Murine Model of Allergic Asthma (B107)

Abstract Chronic allergic asthma is maintained by a Th2-type immune response and is characterized by persistent inflammation of the airways, goblet cell metaplasia, smooth muscle cell hyperplasia, and airway remodeling. Among its many roles, Vasoactive Intestinal Peptide (VIP) is a cytokine known to regulate lymphocytes, macrophages, mast cells, and natural killer cells. We investigated the distribution of VIP in naïve versus allergic mice that had been sensitized and challenged with live Aspergillus fumigatus fungal spores. In situ staining revealed that, although naïve animals produced VIP in the vascular endothelium, the presence of VIP-positive cells in and around the airways is minimal. Allergic mice, however, displayed exaggerated VIP expression in the bronchial epithelium of large and small airways, Type II alveolar cells, and resident macrophages. Recruited macrophages, eosinophils, and lymphocytes were also VIP-positive. Expression of VIP in allergic mice was maximal at days one and three after antigen challenge. Airborne delivery of spores increased the VIP production in distal airways above that seen when the challenge was delivered intratracheally in liquid. The basal epithelial cell layer was conspicuously and persistently negative for VIP expression in all samples. We hypothesize that cells in this layer secrete neutral endopeptidase (NEP) which is responsible for cleaving VIP in the healthy lung.

Ileal VIP submucous neurons: confocal study of the area enlargement induced by myenteric denervation in weanling rats

Vasoactive Intestinal Peptide (VIP) neurons are maturing during suckling and weaning periods and the neuropeptide VIP is thought to be neurotrophic during ontogenesis. We have previously demonstrated that suckling rats with myenteric ablation have significantly higher mitotic index and an increase on villus height and crypt depth 15 days after treatment. In the current study, we measured the area of VIP neurons of submucous plexus in the ileum of weanling rats, in which myenteric neurons were ablated by serosal application of benzalkonium chloride (BAC). The area of VIP immunoreactive cell bodies, reconstructed under confocal microscope, was significantly increased in response to denervation. This result suggests that the myenteric plexus may have an inhibitory role over submucous plexus in the normal intestine. The enhanced production of VIP may be correlated with the increased epithelial proliferation induced by denervation in a critical period of life, from suckling to weaning time.

VIP and PACAP are autocrine factors that protect the androgen-independent prostate cancer cell line PC-3 from apoptosis induced by serum withdrawal.

1. In the present study, we describe the expression of the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) as well as their receptors in PC-3 cells, a human prostate cancer cell line. In addition, we have investigated their role in apoptosis induced by serum starvation. 2. By RT-PCR and immunocytochemistry assays, we have demonstrated the production of VIP and PACAP in PC-3 cells. 3. We have demonstrated by RT-PCR and binding assays the expression of common PACAP/VIP (VPAC(1) and VPAC(2)) receptors, but not PACAP-specific (PAC(1)) receptors. The pharmacological profile of [(125)I]-VIP binding assays was as follows: VPAC(1) antagonist=VPAC(1) agonist>VIP>VPAC(2) agonist (IC(50)=1.2, 1.5, 2.3 and 30 nM, respectively). In addition, both receptor subtypes are functional since VIP, PACAP-27 or VPAC(1) and VPAC(2) agonists all increased the intracellular levels of cAMP. 4. The expression of both peptides and their receptors is similar in serum-cultured and serum-deprived PC-3 cells. The treatment of serum-deprived PC-3 cells with exogenous VIP or PACAP-27 increases cell number and viability in a dose-dependent manner, as demonstrated by cellular counting and MTT assays. The increased cell survival is exerted through the VPAC(1) receptor, since a VPAC(1), but not VPAC(2), receptor agonist, mimics the effects and a VPAC(1) receptor antagonist blocks it. Moreover, VIP and PACAP-27 inhibit genomic DNA fragmentation in PC-3 cells triggered by serum starvation, and increase the immunoreactivity of the antiapoptotic protein bcl-2. 5. Our results suggest that VIP and PACAP are autocrine/paracrine factors that protect PC-3 cells from apoptosis through VPAC1 receptors.