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.

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.

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 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.

The transcriptome of the leukemogenic homeoprotein HOXA9 in human hematopoietic cells.

Hematopoietic defects in HOXA9(-/-) mice demonstrate a key role for this homeoprotein in blood cell development. Conversely, enforced HOXA9 expression is leukemogenic in mice, and HOXA9 is frequently activated in human acute myeloid leukemia (AML). Although HOXA9 is thought to function as a transcription factor, few downstream targets have been identified. We searched for early HOXA9 target genes by using a transient overexpression strategy in 3 hematopoietic cell lines (2 myeloid, 1 lymphoid). cDNA microarray analyses identified 220 genes whose expression was modulated at least 2-fold. Expression signatures in myeloid and lymphoid cells demonstrated that HOXA9 functions as both an activator and repressor of a variety of genes in cell-specific patterns suggesting that the transcriptional effects of HOXA9 are largely dependent on the cell context. Transient transcription assays and target gene expression patterns in HOXA9(-/-) marrow cells imply that we have identified direct physiologic targets. Many target genes are expressed in CD34+ stem cells or are members of gene families involved in proliferation or myeloid differentiation. Expression of 14 HOXA9 target genes correlated with high-level HOXA9 expression in primary AML. These data suggest that many genes identified in this survey may mediate the biologic effects of HOXA9 in normal and leukemic hematopoiesis.