Emilie E. Vomhof-DeKrey

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).

Stimulatory and suppressive signal transduction regulates vasoactive intestinal peptide receptor-1 (VPAC-1) in primary mouse CD4 T cells

Vasoactive intestinal peptide receptor-1 (VPAC-1) is an anti-proliferative, G-protein coupled receptor that is highly expressed on naïve T cells, and has been reported to be downregulated upon T cell activation. The T cell signaling molecules involved in mediating low VPAC-1 levels have not been identified. Therefore, to gain a greater understanding into this regulation, this study investigated the signaling pathways that regulate (VPAC-1) in murine, primary CD4 T cells. To this end, murine, splenic CD4 T cells were pretreated separately with 10 different pharmacological inhibitors and incubated +/- anti-CD3 for 24h. Total RNA was isolated, and VPAC-1 mRNA levels were measured by qPCR. Our results support that JNK kinases, downstream from the protein kinase, Zap70, are involved in suppressive regulation of VPAC-1 steady-state mRNA levels after anti-CD3 treatment. In contrast, inhibitors against PKC, ERK, p38, Zap70 and Rac1 supported a stimulatory influence in VPAC-1 regulation in the absence of T cell signaling. By studying the signaling pathways that regulate VPAC-1 in T cells, we can gain greater insight into the role of this anti-inflammatory receptor in autoimmunity and infectious diseases.

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.

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.

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.

A transcriptionally permissive epigenetic landscape at the vasoactive intestinal peptide receptor-1 promoter suggests a euchromatin nuclear position in murine CD4 T cells.

T cells express receptors for neuropeptides that mediate immunological activities. Vasoactive intestinal peptide receptor-1 (VPAC1), the prototypical group II G protein coupled receptor, binds two neuropeptides with high-affinity, called vasoactive intestinal peptide and pituitary adenylate cyclase activating polypeptide. During T cell signaling, VPAC1 mRNA expression levels are significantly downregulated through a Src kinase dependent mechanism, thus altering the sensitivity for these neuropeptides during an immune reaction. Presently, it is unknown whether the mechanism that regulates VPAC1 during T cell signaling involves epigenetic changes. Therefore, we hypothesized that the epigenetic landscape consisting of diacetylation at H3K9/14 and trimethylation at H3K4, two transcriptionally permissive histone modifications, would parallel VPAC1 expression showing high enrichment in untreated T cells, but lower enrichment in alpha-CD3 treated T cells. To this end, quantitative chromatin immunoprecipitation (ChIP) analysis of H3K9/14ac and H3K4me3 was conducted using purified CD4(+) T cells, with CD45R(+) B cells as a negative control. Our data revealed that these histone modifications at the VPAC1 promoter did indeed parallel its mRNA levels between T and B lymphocytes, but did not decrease during T cell signaling. Collectively, these data strongly imply a euchromatin nuclear position for the VPAC1 locus irrespective of the activation status of T cells.

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.