Influence of vasoactive intestinal polypeptide on growth performance, nutrient digestibility, nitrogen balance, and digestive enzyme activity in lambs.

This study evaluated if vasoactive intestinal polypeptide (VIP) influences growth performance, nutrient digestibility, nitrogen balance, and digestive enzyme activity. Sixteen wether lambs (69.6 ±â€…1.9 kg) were housed in individual pens, adapted to a corn grain-based diet, and randomly assigned to 2 treatment groups. Lambs were injected intraperitoneally every other day for 28 d with saline (0.9% NaCl) containing no VIP (n = 8; control) or containing VIP (n = 8; 1.3 nmol/kg body weight [BW]). All lambs were transferred to individual metabolic crates for the final 7 d of the experiment to measure nitrogen balance and nutrient digestibility. At the end of the treatment period, lambs were slaughtered, and pancreatic tissue, small intestinal tissue, and rumen fluid were collected for protein, digestive enzymes, ruminal pH, and volatile fatty acid (VFA) analyses. Lambs treated with VIP had greater final BW, average daily gain, and gain:feed (P = 0.01, 0.05, 0.03, respectively). No differences between treatment groups were observed (P ≥ 0.25) for nutrient intake, digestibility, nitrogen retention, ruminal pH, and VFA concentrations. Moreover, VIP treatment did not influence (P ≥ 0.19) plasma glucose, urea N, and insulin concentrations. Treatment with VIP increased (P = 0.03) relative cecum weight (g/kg BW) and decreased (P = 0.05) relative brain weight. Pancreatic and intestinal digestive enzyme activities, except for duodenal maltase (P = 0.02), were not influenced (P ≥ 0.09) by VIP treatment. These data suggest that the administration of VIP may have potential to improve average daily gain and gain:feed in lambs fed grain-based diets.

This research explored the influence of vasoactive intestinal polypeptide (VIP), an anti-inflammatory mediator, in lambs fed a high-concentrate finishing diet on growth performance, nutrient digestibility, nitrogen balance, and digestive enzyme activity. Wether lambs were fed a whole corn grain-based diet containing no added forage and randomly assigned to either the VIP or control group. Lambs received intraperitoneal saline injections with or without VIP every second day over a 28-d treatment period. Average daily gain and gain:feed ratio was positively influenced by VIP. However, treatment did not affect dry matter intake, nitrogen balance, nutrient digestibility, and digestive enzyme activity. These data indicate exogenous VIP treatment may influence growth in lambs fed a high-concentrate diet.

Sex-related differences in the response of anti-platelet drug therapies targeting purinergic signaling pathways in sepsis.

Sepsis, a complex clinical syndrome resulting from a serious infection, is a major healthcare problem associated with high mortality. Sex-related differences in the immune response to sepsis have been proposed but the mechanism is still unknown. Purinergic signaling is a sex-specific regulatory mechanism in immune cell physiology. Our studies have shown that blocking the ADP-receptor P2Y12 but not P2Y1 receptor was protective in male mice during sepsis, but not female. We now hypothesize that there are sex-related differences in modulating P2Y12 or P2Y1 signaling pathways during sepsis. Male and female wild-type (WT), P2Y12 knock-out (KO), and P2Y1 KO mice underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. The P2Y12 antagonist ticagrelor or the P2Y1 antagonist MRS2279 were administered intra-peritoneally after surgery to septic male and female mice. Blood, lungs and kidneys were collected 24 hours post-surgery. Sepsis-induced changes in platelet activation, secretion and platelet interaction with immune cells were measured by flow cytometry. Neutrophil infiltration in the lung and kidney was determined by a myeloperoxidase (MPO) colorimetric assay kit. Sepsis-induced platelet activation, secretion and aggregate formation were reduced in male CLP P2Y12 KO and in female CLP P2Y1 KO mice compared with their CLP WT counterpart. Sepsis-induced MPO activity was reduced in male CLP P2Y12 KO and CLP P2Y1 KO female mice. CLP males treated with ticagrelor or MRS2279 showed a decrease in sepsis-induced MPO levels in lung and kidneys, aggregate formation, and platelet activation as compared to untreated male CLP mice. There were no differences in platelet activation, aggregate formation, and neutrophil infiltration in lung and kidney between female CLP mice and female CLP mice treated with ticagrelor or MRS2279. In human T lymphocytes, blocking P2Y1 or P2Y12 alters cell growth and secretion in vitro in a sex-dependent manner, supporting the data obtained in mice. In conclusion, targeting purinergic signaling represents a promising therapy for sepsis but drug targeting purinergic signaling is sex-specific and needs to be investigated to determine sex-related targeted therapies in sepsis.

Messenger RNA Gene Expression Screening of VIP and PACAP Neuropeptides and Their Endogenous Receptors in Ruminants.

Vasoactive Intestinal Peptide (VIP) and Pituitary Adenylate-Cyclase-Activating Peptide (PACAP) are anti-inflammatory neuropeptides that play important roles in human and rodent gut microbiota homeostasis and host immunity. Pharmacologically regulating these neuropeptides is expected to have significant health and feed efficiency benefits for agriculturally relevant animals. However, their expression profile in ruminant tissues is not well characterized. To this end, we screened for VIP and PACAP neuropeptides and their endogenous GPCRs using 15 different tissues from wethers and steers by RT-qPCR. Our results revealed relatively similar expression profiles for both VIP and PACAP neuropeptide ligands in the brain and intestinal tissue of both species. In contrast, the tissue expression profiles for VPAC1, VPAC2, and PAC1 were more widespread and disparate, with VPAC1 being the most diversely expressed receptor with mRNA detection in the brain and throughout the gastrointestinal tract. These data are an important first step to allow for future investigations regarding the VIP and PACAP signaling pathways in livestock ruminant species.

The G Protein-Coupled Receptor, VPAC1, Mediates Vasoactive Intestinal Peptide-Dependent Functional Homeostasis of the Gut Microbiota.

BACKGROUND AND AIMS: Vasoactive intestinal peptide (VIP) is a neuropeptide involved in the regulation of feeding behavior and circadian rhythms, metabolism, and immunity. Previous studies revealed the homeostatic effects of VIP signaling on the gut microbiota. VIP-deficient mice demonstrate a gut microbiota dysbiosis characterized by reduced α-diversity and decreased relative abundance (RA) of Gram-positive Firmicutes. However, the mechanism by which VIP signaling affects changes in the microbiota is unknown. METHODS: To investigate the role of the 2 cognate G protein-coupled receptors for VIP (VPAC1 and VPAC2) in VIP-mediated homeostasis of the microbiota, fecal samples from VPAC1- and VPAC2-deficient, heterozygous, and wild-type littermate mice were assessed via targeted amplicon sequencing. Their microbiota profiles were additionally compared with microbiota from VIP-deficient, heterozygous, and wild-type littermates, where genotype-dependent changes in the composition and predicted function of each cohort were compared. RESULTS: While wild-type mice in each line differed in α-diversity and ß-diversity, consistent changes in both metrics were observed in VIP-deficient and VPAC1-deficient mice. This includes a dramatic reduction in α-diversity, increased RA of Proteobacteria and Bacteroidetes, and decreased RA of Lachnospiraceae, Ruminococcaceae, Muribaculaceae, and Rikenellaceae. Specific amplicon sequence variants and predicted functions found to differ significantly based on VIP or VPAC1 genotype were concordant in their directions of change. Multiplatform predicted functional profiling suggested a defective VIP-VPAC1 axis was associated with reduced amino acid degradation along with reduced quinol and quinone biosynthesis. Furthermore, alterations in predicted functions include increased sugar degradation, nitrate reduction, and fatty acid biosynthetic pathways, among other changes. CONCLUSION: We conclude that VIP signaling through VPAC1 is critical for the maintenance of normal function of the gut microbiota.

mRNA-based CAR T-cells manufactured by miniaturized two-step electroporation produce selective cytotoxicity toward target cancer cells.

There is a growing interest for viral vector-free chimeric antigen receptor (CAR) T-cells due to its ability to kill cancer cells without adverse side effects. A potential avenue for manufacturing viral-vector free CAR T-cells is to utilize mRNA electroporation. One of the major concerns with mRNA electroporated CAR T-cells is the shorter cytotoxic lifespan of a few days, which is insufficient or not ideal for therapy. To better understand this issue and develop a potential solution, this study focused on examining the translation of electroporated mRNA to CAR molecules, time dependent degradation of CAR molecules and cytotoxicity produced by CAR T-cells on cancer cells. It was found that the initial expression of CAR molecules dictates the cytotoxicity. Initial CAR expression could be controlled by the experimental parameters such as electroporation time and mRNA concentration in the electroporation buffer. Experiments were carried out using a novel two-step electroporation that allows for controlled and uniform transfection of T-cells. These technical advancements and subsequent findings could provide a viable path for producing CAR T-cells with longer cytotoxic lifespans.

Vasoactive Intestinal Peptide Deficiency Is Associated With Altered Gut Microbiota Communities in Male and Female C57BL/6 Mice.

Vasoactive intestinal peptide (VIP) is crucial for gastrointestinal tract (GIT) health. VIP sustains GIT homeostasis through maintenance of the intestinal epithelial barrier and acts as a potent anti-inflammatory mediator that contributes to gut bacterial tolerance. Based on these biological functions by VIP, we hypothesized that its deficiency would alter gut microbial ecology. To this end, fecal samples from male and female VIP+/+, VIP+/-, and VIP-/- littermates (n = 47) were collected and 16S rRNA sequencing was conducted. Our data revealed significant changes in bacterial composition, biodiversity, and weight loss from VIP-/- mice compared to VIP+/+ and VIP+/- littermates, irrespective of sex. The gut bacteria compositional changes observed in VIP-/- mice was consistent with gut microbial structure changes reported for certain inflammatory and autoimmune disorders. Moreover, predicted functional changes by PICRUSt software suggested an energy surplus within the altered microbiota from VIP-/- mice. These data support that VIP plays an important role in maintaining microbiota balance, biodiversity, and GIT function, and its genetic removal results in significant gut microbiota restructuring and weight loss.

Ikaros tumor suppressor function includes induction of active enhancers and super-enhancers along with pioneering activity.

Ikaros encodes a transcription factor that functions as a tumor suppressor in T-cell acute lymphoblastic leukemia (T-ALL). The mechanisms through which Ikaros regulates gene expression and cellular proliferation in T-ALL are unknown. Re-introduction of Ikaros into Ikaros-null T-ALL cells resulted in cessation of cellular proliferation and induction of T-cell differentiation. We performed dynamic, global, epigenomic, and gene expression analyses to determine the mechanisms of Ikaros tumor suppressor activity. Our results identified novel Ikaros functions in the epigenetic regulation of gene expression: Ikaros directly regulates de novo formation and depletion of enhancers, de novo formation of active enhancers and activation of poised enhancers; Ikaros directly induces the formation of super-enhancers; and Ikaros demonstrates pioneering activity by directly regulating chromatin accessibility. Dynamic analyses demonstrate the long-lasting effects of Ikaros DNA binding on enhancer activation, de novo formation of enhancers and super-enhancers, and chromatin accessibility. Our results establish that Ikaros' tumor suppressor function occurs via global regulation of the enhancer and super-enhancer landscape and through pioneering activity. Expression analysis identified a large number of novel signaling pathways that are directly regulated by Ikaros and Ikaros-induced enhancers, and that are responsible for the cessation of proliferation and induction of T-cell differentiation in T-ALL cells.

Transcriptional Regulation of JARID1B/KDM5B Histone Demethylase by Ikaros, Histone Deacetylase 1 (HDAC1), and Casein Kinase 2 (CK2) in B-cell Acute Lymphoblastic Leukemia.

Impaired function of the Ikaros (IKZF1) protein is associated with the development of high-risk B-cell precursor acute lymphoblastic leukemia (B-ALL). The mechanisms of Ikaros tumor suppressor activity in leukemia are unknown. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. Here, we report that Ikaros represses transcription of the histone H3K4 demethylase, JARID1B (KDM5B). Transcriptional repression of JARID1B is associated with increased global levels of H3K4 trimethylation. Ikaros-mediated repression of JARID1B is dependent on the activity of the histone deacetylase, HDAC1, which binds to the upstream regulatory element of JARID1B in complex with Ikaros. In leukemia, JARID1B is overexpressed, and its inhibition results in cellular growth arrest. Ikaros-mediated repression of JARID1B in leukemia is impaired by pro-oncogenic casein kinase 2 (CK2). Inhibition of CK2 results in increased binding of the Ikaros-HDAC1 complex to the promoter of JARID1B, with increased formation of trimethylated histone H3 lysine 27 and decreased histone H3 Lys-9 acetylation. In cases of high-risk B-ALL that carry deletion of one Ikaros (IKZF1) allele, targeted inhibition of CK2 restores Ikaros binding to the JARID1B promoter and repression of JARID1B. In summary, the presented data suggest a mechanism through which Ikaros and HDAC1 regulate the epigenetic signature in leukemia: via regulation of JARID1B transcription. The presented data identify JARID1B as a novel therapeutic target in B-ALL and provide a rationale for the use of CK2 inhibitors in the treatment of high-risk B-ALL.

Hyaluronan stimulates ex vivo B lymphocyte chemotaxis and cytokine production in a murine model of fungal allergic asthma.

Allergic asthma is a chronic inflammatory disease of the airways characterized by excessive eosinophilic and lymphocytic inflammation with associated changes in the extracellular matrix (ECM) resulting in airway wall remodeling. Hyaluronan (HA) is a nonsulfated glycosaminoglycan ECM component that functions as a structural cushion in its high molecular mass (HMM) but has been implicated in metastasis and other disease processes when it is degraded to smaller fragments. However, relatively little is known about the role HA in mediating inflammatory responses in allergy and asthma. In the present study, we used a murine Aspergillus fumigatus inhalational model to mimic human disease. After observing in vivo that a robust B cell recruitment followed a massive eosinophilic egress to the lumen of the allergic lung and corresponded with the detection of low molecular mass HA (LMM HA), we examined the effect of HA on B cell chemotaxis and cytokine production in the ex vivo studies. We found that LMM HA functioned through a CD44-mediated mechanism to elicit chemotaxis of B lymphocytes, while high molecular mass HA (HMM HA) had little effect. LMM HA, but not HMM HA, also elicited the production of IL-10 and TGF-ß1 in these cells. Taken together, these findings demonstrate a critical role for ECM components in mediating leukocyte migration and function which are critical to the maintenance of allergic inflammatory responses.

Hyaluronan fragments as mediators of inflammation in allergic pulmonary disease.

Asthma is frequently caused and/or exacerbated by sensitization to allergens, which are ubiquitous in many indoor and outdoor environments. Severe asthma is characterized by airway hyperresponsiveness and bronchial constriction in response to an inhaled allergen, leading to a disease course that is often very difficult to treat with standard asthma therapies. As a result of interactions among inflammatory cells, structural cells, and the intercellular matrix of the allergic lung, patients with sensitization to allergens may experience a greater degree of tissue injury followed by airway wall remodeling and progressive, accumulated pulmonary dysfunction as part of the disease sequela. In addition, turnover of extracellular matrix (ECM) components is a hallmark of tissue injury and repair. This review focuses on the role of the glycosaminoglycan hyaluronan (HA), a component of the ECM, in pulmonary injury and repair with an emphasis on allergic asthma. Both the synthesis and degradation of the ECM are critical contributors to tissue repair and remodeling. Fragmented HA accumulates during tissue injury and functions in ways distinct from the larger native polymer. There is gathering evidence that HA degradation products are active participants in stimulating the expression of inflammatory genes in a variety of immune cells at the injury site. In this review, we will consider recent advances in the understanding of the mechanisms that are associated with HA accumulation and inflammatory cell recruitment in the asthmatic lung.

B lymphocytes regulate airway granulocytic inflammation and cytokine production in a murine model of fungal allergic asthma.

Sensitization to fungi often leads to a severe form of asthma that is particularly difficult to manage clinically, resulting in increased morbidity and hospitalizations in these patients. Although B lymphocytes might exacerbate asthma symptoms through the production of IgE, these cells might also be important in the protective response against inhaled fungi. Through cytokine release and T-cell interactions, these lymphocytes might also influence the development and maintenance of airway wall fibrosis. J(H)(-/-) mice lack the JH gene for the heavy chain component of antibodies, which is critical for B-cell function and survival. These animals have facilitated the elucidation of the role of B lymphocytes in a number of immune responses; however, J(H)(-/-) mice have not been used to study fungal allergy. In this study, we examined the role of B lymphocytes using an Aspergillus fumigatus murine fungal aeroallergen model that mimics human airway disease that is triggered by environmental fungal exposure. We compared disease progression in sensitized wild-type BALB/c and J(H)(-/-) mice that were exposed to repeated fungal exposure and found no differences in airway hyperresponsiveness, overall pulmonary inflammation or collagen deposition around the large airways. However, the levels of the Th2-type cytokines IL-4 and IL-13 were significantly attenuated in the airways of J(H)(-/-) mice relative to the BALB/c controls. By contrast, levels of the inflammatory cytokines IL-17A and IL-6 were significantly elevated in the J(H)(-/-) animals, and there was significantly more robust airway eosinophilia and neutrophilia than in control animals. Taken together, these findings demonstrate that B lymphocytes help to regulate granulocytic responses to fungal exposure in the pulmonary compartment.

Hyaluronan deposition and co-localization with inflammatory cells and collagen in a murine model of fungal allergic asthma.

OBJECTIVE: Allergic asthma is a chronic inflammatory disease of the airways characterized by excessive inflammation and remodeling of the extracellular matrix (ECM) and associated cells of the airway wall. Under inflammatory conditions, hyaluronan (HA), a major component of the ECM, undergoes dynamic changes, which may in turn affect the recruitment and activation of inflammatory cells leading to acute and chronic immunopathology of allergic asthma. METHODS: In the present study, we measured the changes in HA levels generated at sites of inflammation, and examined its effect on inflammatory responses and collagen deposition in an Aspergillus fumigatus murine inhalational model of allergic asthma. RESULTS: We found that HA levels are elevated in allergic animals and that the increase correlated with the influx of inflammatory cells 5 days after the second allergen challenge. This increase in HA levels appeared largely due to upregulation of hyaluronidase-1 (HYAL1) and hyaluronidase-2 (HYAL2). Furthermore, HA co-localizes with areas of new collagen synthesis and deposition. CONCLUSIONS: Overall, our findings contribute to the growing literature that focuses on the components of ECM as inflammatory mediators rather than mere structural support products. The evidence of HA localization in fungal allergic asthma provides the impetus to study HA more closely with allergic leukocytes in murine models. Further studies examining HA's role in mediating cellular responses may help to develop targets for treatment in patients with severe asthma due to fungal sensitization.

Eosinophils in fungus-associated allergic pulmonary disease.

Asthma is frequently caused and/or exacerbated by sensitization to fungal allergens, which are ubiquitous in many indoor and outdoor environments. Severe asthma with fungal sensitization is characterized by airway hyperresponsiveness and bronchial constriction in response to an inhaled allergen that is worsened by environmental exposure to airborne fungi and which leads to a disease course that is often very difficult to treat with standard asthma therapies. As a result of complex interactions among inflammatory cells, structural cells, and the intercellular matrix of the allergic lung, patients with sensitization to fungal allergens may experience a greater degree of airway wall remodeling and progressive, accumulated pulmonary dysfunction as part of the disease sequela. From their development in the bone marrow to their recruitment to the lung via chemokine and cytokine networks, eosinophils form an important component of the inflammatory milieu that is associated with this syndrome. Eosinophils are recognized as complex multi-factorial leukocytes with diverse functions in the context of allergic fungal asthma. In this review, we will consider recent advances in our understanding of the molecular mechanisms that are associated with eosinophil development and migration to the allergic lung in response to fungal inhalation, along with the eosinophil's function in the immune response to and the immunopathology attributed to fungus-associated allergic pulmonary disease.

Characterization and use of a rabbit-anti-mouse VPAC1 antibody by flow cytometry.

Vasoactive intestinal peptide receptor-1 signaling in lymphocytes has been shown to regulate chemotaxis, proliferation, apoptosis and differentiation. During T cell activation, VPAC1 mRNA is downregulated, but the effect on its protein levels is less clear. A small number of studies have reported measurement of human VPAC1 by flow cytometry, but murine VPAC1 reagents are unavailable. Therefore, we set out to generate a reliable and highly specific α-mouse VPAC1 polyclonal antibody for use with flow cytometry. After successfully generating a rabbit α-VPAC1 polyclonal antibody (α-mVPAC1 pAb), we characterized its cross-reactivity and showed that it does not recognize other family receptors (mouse VPAC2 and PAC1, and human VPAC1, VPAC2 and PAC1) by flow cytometry. Partial purification of the rabbit α-VPAC1 sera increased the specific-activity of the α-mVPAC1 pAb by 20-fold, and immunofluorescence microscopy (IF) confirmed a plasma membrane subcellular localization for mouse VPAC1 protein. To test the usefulness of this specific α-mVPAC1 pAb, we showed that primary, resting mouse T cells express detectable levels of VPAC1 protein, with little detectable signal from activated T cells, or CD19 B cells. These data support our previously published data showing a downregulation of VPAC1 mRNA during T cell activation. Collectively, we have established a well-characterized, and highly species specific α-mVPAC1 pAb for VPAC1 surface measurement by IF and flow cytometry.

Radical reversal of vasoactive intestinal peptide (VIP) receptors during early lymphopoiesis.

Successful thymocyte maturation is essential for normal, peripheral T cell function. Vasoactive intestinal peptide (VIP) is a neuropeptide which is highly expressed in the thymus that has been shown to modulate thymocyte development. VIP predominantly binds two G protein coupled receptors, termed vasoactive intestinal peptide receptor 1 (VPAC1) and VPAC2, but their expression profiles in CD4(-)/CD8(-) (double negative, DN) thymocyte subsets, termed DN1-4, have yet to be identified. We hypothesized that a high VPAC1:VPAC2 ratio in the earliest thymocyte progenitors (ETP cells) would be reversed during early lymphopoiesis as observed in activated, peripheral Th(2) cells, as the thymus is rich in Th(2) cytokines. In support of this hypothesis, high VPAC1 mRNA levels decreased 1000-fold, accompanied with a simultaneous increase in VPAC2 mRNA expression during early thymocyte progenitor (ETP/DN1)→DN3 differentiation. Moreover, arrested DN3 cells derived from an Ikaros null mouse (JE-131 cells) failed to completely reverse the VIP receptor ratio compared to wild type DN3 thymocytes. Surprisingly, VPAC2(-/-) mice did not show significant changes in relative thymocyte subset numbers. These data support the notion that both VPAC1 and VPAC2 receptors are dynamically regulated by Ikaros, a master transcriptional regulator for thymocyte differentiation, during early thymic development. Moreover, high VPAC1 mRNA is a novel marker for the ETP population making it enticing to speculate that the chemotactic VIP/VPAC1 signaling axis may play a role in thymocyte movement. Also, despite the results that VPAC2 deficiency did not affect thymic subset numbers, future studies are necessary to determine whether downstream T cell phenotypic changes manifest themselves, such as a propensity for a Th(1) versus Th(2) polarization.

Vasoactive intestinal peptide signaling axis in human leukemia.

The vasoactive intestinal peptide (VIP) signaling axis constitutes a master "communication coordinator" between cells of the nervous and immune systems. To date, VIP and its two main receptors expressed in T lymphocytes, vasoactive intestinal peptide receptor (VPAC)1 and VPAC2, mediate critical cellular functions regulating adaptive immunity, including arresting CD4 T cells in G(1) of the cell cycle, protection from apoptosis and a potent chemotactic recruiter of T cells to the mucosa associated lymphoid compartment of the gastrointestinal tissues. Since the discovery of VIP in 1970, followed by the cloning of VPAC1 and VPAC2 in the early 1990s, this signaling axis has been associated with common human cancers, including leukemia. This review highlights the present day knowledge of the VIP ligand and its receptor expression profile in T cell leukemia and cell lines. Also, there will be a discussion describing how the anti-leukemic DNA binding transcription factor, Ikaros, regulates VIP receptor expression in primary human CD4 T lymphocytes and T cell lymphoblastic cell lines (e.g. Hut-78). Lastly, future goals will be mentioned that are expected to uncover the role of how the VIP signaling axis contributes to human leukemogenesis, and to establish whether the VIP receptor signature expressed by leukemic blasts can provide therapeutic and/or diagnostic information.

Vasoactive intestinal peptide receptor 1 is downregulated during expansion of antigen-specific CD8 T cells following primary and secondary Listeria monocytogenes infections.

As regulation of CD8 T cell homeostasis is incompletely understood, we investigated the expression profile of the vasoactive intestinal peptide (VIP) receptors, VPAC1 and VPAC2, on CD8 T cells throughout an in vivo immune response. Herein, we show that adoptively transferred CD8 T cells responding to a Listeria monocytogenes infection significantly downregulated, functionally active VPAC1 protein expression during primary and secondary expansion. VPAC1 mRNA expression was restored during contraction and regained naïve levels in primary, but remained low during secondary, memory generation. VIP co-administration with primary infection suppressed CD8 T cell expansion (≈ 50%). VPAC2 was not detected at any time points throughout primary and secondary infections. Collectively, our data demonstrate that functionally active VPAC1 is dynamically downregulated to render expanding CD8 T cells unresponsive to VIP.

Gene expression profiling and network analysis of peripheral blood monocytes in a chronic model of allergic asthma.

The Aspergillus fumigatus mouse model of asthma mimics the characteristics of human fungal asthma, including local and systemic inflammation. Monocyte/macrophage lineage cells direct innate immune responses and guide adaptive responses. To identify gene expression changes in peripheral blood monocytes in the context of fungal allergy, mice were exposed to systemic and intranasal inoculations of fungal antigen (sensitized), and naïve and sensitized animals were challenged intratracheally with live A. fumigatus conidia. Microarray analysis of blood monocytes from allergic versus non-allergic mice showed ≥ twofold modulation of 45 genes. Ingenuity pathway analysis revealed a network of these genes involved in antigen presentation, inflammation, and immune cell trafficking. These data show that allergen sensitization and challenge affects gene expression in peripheral monocytes.

Identification of the early VIP-regulated transcriptome and its associated, interactome in resting and activated murine CD4 T cells.

More than 40 years after the discovery of vasoactive intestinal peptide (VIP), its transcriptome in the immune system has still not been completely elucidated. In an attempt to understand the biological role of this neuropeptide in immunity, we chose CD4 T cells as a cellular system. Agilent Mouse Whole Genome microarrays were hybridized with fluorescently labeled total RNA isolated from resting CD4 T cells cultured +/-10(-7)M VIP for 5h or PMA/ionomycin activated CD4 T cells cultured +/-10(-7)M VIP for 5h. These VIP-regulated transcriptomes were analyzed by Significance Analysis of Microarrays (SAM) and Ingenuity Pathway Analysis (IPA) software to identify relevant signaling pathways modulated by VIP in the absence and presence of T cell activation. In resting CD4 T cells, VIP-modulated 368 genes, ranging from 3.49 to -4.78-fold. In the PMA/ionomycin activated CD4 T cells, 326 gene expression levels were changed by VIP, ranging from 2.94 to -1.66-fold. IPA analysis revealed that VIP exposure alters cellular function through EGFR signaling in resting CD4 T cells, and modulates immediate early genes, Fos and CREM/ICER, in activated CD4 T cells. These gene expression changes are suggested to explain at a molecular level how VIP can regulate T cell homing to the gut and induce regulatory T cell generation.

TCR signaling and environment affect vasoactive intestinal peptide receptor-1 (VPAC-1) expression in primary mouse CD4 T cells.

Strict regulation of T cell function is imperative to control adaptive immunity, and dysregulation of T cell activation can contribute to infectious and autoimmune diseases. Vasoactive intestinal peptide receptor-1 (VPAC-1), an anti-inflammatory G-protein coupled receptor, has been reported to be downregulated during T cell activation. However, the regulatory mechanisms controlling the expression of VPAC-1 in T cells are not well understood. Therefore, mouse splenic CD4 T cells were treated in complete media+/-anti-CD3 for 24h, total RNA isolated and VPAC-1 levels measured by qPCR. Surprisingly, we discovered that T cells incubated in complete media steadily upregulated VPAC-1 mRNA levels over time (24h). Importantly, CD4 T cells isolated from blood also showed elevated VPAC-1 expression compared to splenic T cells. Collectively, these data support that the vascular environment positively influences VPAC-1 mRNA expression that is negatively regulated by TCR signaling. This research was supported by a national service award (1KO1 DK064828) to G.D., the Center for Protease Research (2P20RR015566), and INBRE (P20 RR016741).