Background: Vasoactive intestinal peptide (VIP) is a 3.3 kDa pleiotropic protein with a broad expression profile that displays immune modulating activities. VIP binds two G protein-coupled receptors called Vasoactive Intestinal Pepide/Pituitary Adenylate Cyclase activating polypeptide receptor (VPAC) 1 and 2 that elicit multiple downstream signaling molecules, including adenylate cyclase. VIP differentially regulates over 300 genes in resting and activated murine CD4 T cells, and is chemotactic to resting and Th2 effector T cells. Keywords: cAMP, chemotaxis, gene expression, signaling, T cells, vasoactive intestinal peptide.

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

Although fungi are pervasive in many environments, few cause disease in humans. Of these, Aspergillus fumigatus is particularly well suited to be a pathogen of the human lung. Its physical and biological characteristics combine to provide an organism that can cause tremendous morbidity and high mortality if left unchecked. Luckily, that is rarely the case. However, repeated exposure to inhaled A. fumigatus spores often results in an immune response that carries significant immunopathology, exacerbating asthma and changing the structure of the lung with chronic impacts to pulmonary function. This review focuses on the current understanding of the mechanisms that are associated with fungal exposure, sensitization, and infection in asthmatics, as well as the function of various inflammatory cells associated with severe asthma with fungal sensitization.

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-β1 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.

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.

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. JH−/− 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, JH−/− 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 JH−/− 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 JH−/− mice relative to the BALB/c controls. By contrast, levels of the inflammatory cytokines IL-17A and IL-6 were significantly elevated in the JH−/− 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.

Asthma was the most common comorbidity in hospitalized patients during the 2009 influenza pandemic. For unknown reasons, hospitalized asthmatics had less severe outcomes and were less likely to die from pandemic influenza. Our data with primary human bronchial cells indicate that changes intrinsic to epithelial cells in asthma may protect against cytopathology induced by influenza virus. To further study influenza virus pathogenesis in allergic hosts, we aimed to develop and characterize murine models of asthma and influenza comorbidity to determine structural, physiological and immunological changes induced by influenza in the context of asthma. Aspergillus fumigatus-sensitized and -challenged C57BL/6 mice were infected with pandemic H1N1 influenza virus, either during peak allergic inflammation or during airway remodeling to gain insight into disease pathogenesis. Mice infected with the influenza virus during peak allergic inflammation did not lose body weight and cleared the virus rapidly. These mice exhibited high eosinophilia, preserved airway epithelial cell integrity, increased mucus, reduced interferon response and increased insulin-like growth factor-1. In contrast, weight loss and viral replication kinetics in the mice that were infected during the late airway remodeling phase were equivalent to flu-only controls. These mice had neutrophils in the airways, damaged airway epithelial cells, less mucus production, increased interferons and decreased insulin-like growth factor-1. The state of the allergic airways at the time of influenza virus infection alters host responses against the virus. These murine models of asthma and influenza comorbidity may improve our understanding of the epidemiology and pathogenesis of viral infections in humans with asthma.

Asthma was the most common comorbidity in hospitalized patients during the 2009 influenza pandemic. For unknown reasons, hospitalized asthmatics had less severe outcomes and were less likely to die from pandemic influenza. Our data with primary human bronchial cells indicate that changes intrinsic to epithelial cells in asthma may protect against cytopathology induced by influenza virus. To further study influenza virus pathogenesis in allergic hosts, we aimed to develop and characterize murine models of asthma and influenza comorbidity to determine structural, physiological and immunological changes induced by influenza in the context of asthma. Aspergillus fumigatus -sensitized and -challenged C57BL/6 mice were infected with pandemic H1N1 influenza virus, either during peak allergic inflammation or during airway remodeling to gain insight into disease pathogenesis. Mice infected with the influenza virus during peak allergic inflammation did not lose body weight and cleared the virus rapidly. These mice exhibited high eosinophilia, preserved airway epithelial cell integrity, increased mucus, reduced interferon response and increased insulin-like growth factor-1. In contrast, weight loss and viral replication kinetics in the mice that were infected during the late airway remodeling phase were equivalent to flu-only controls. These mice had neutrophils in the airways, damaged airway epithelial cells, less mucus production, increased interferons and decreased insulin-like growth factor-1. The state of the allergic airways at the time of influenza virus infection alters host responses against the virus. These murine models of asthma and influenza comorbidity may improve our understanding of the epidemiology and pathogenesis of viral infections in humans with asthma.

A asthma is a disease of the airways that affects over 25 million people in the United States incurring over $50 billion in direct and indirect costs per year. Although we understand the symptoms associated with allergic asthma, the exact causes and sequence of events that follow allergen exposure warrants further investigation. Animal models that effectively recapitulate the hallmarks of human disease are important to delineate the pathways that lead to the immunological, architectural and physiological changes that occur. We have identified that the neuroimmunological axis plays a role in the development of allergic asthma using a novel murine model induced by Aspergillus fumigatus conidia. Vasoactive intestinal peptide (VIP) is a neuropeptide with cytokine properties which has been demonstrated to have an anti-inflammatory role in the lungs when acting through its receptor VPAC2. We showed that VIP localization in the columnar epithelium of the airways was dynamically regulated following allergen provocation with levels decreasing early during the allergic cascade and increasing after the influx of lymphocytes into the airways. The VIP/VPAC2 axis, previously shown to promote TH2 immunity, was demonstrated herein not to play a role in the development or maintenance of allergic asthma. VPAC2 null mice have elevated levels of IgG2a and IgA in response to allergen challenge, indicating its novel role in regulating the humoral immune response.

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.

Abstract B lymphocytes are well recognized in the development of IgE responses, which exacerbates asthma symptoms, but may also be important as a protective response against inhaled fungi. Further, these lymphocytes may play a direct role in the maintenance of airway wall fibrosis. Jh-/- mice have been used extensively to study the role of B lymphocytes in immune responses. These mice lack the Jh gene, which codes for the antibody heavy chain and is thought to be critical for B cell survival and function. In this study, we examined the role of B lymphocytes using a murine fungal aeroallergen model to mimic human fungal allergic airway disease. We compared disease progression in the BALB/c and Jh-/- mice and found no differences in airway hyperresponsiveness, collagen deposition, or overall pulmonary inflammation. However, the Jh-/- mice had a significantly higher number of neutrophils and eosinophils when compared to the BALB/c mice. IL-17A and IL-6 cytokines, which play a role in driving eosinophilia and neutrophilia, were also significantly higher in the Jh-/- mice after fungal challenge. We plan to characterize temporal activation and the relative contribution of these B lymphocytes in the maintenance of Tregs and Th17 lymphocytes within the pulmonary compartment. These studies will help expand our knowledge of how B lymphocytes regulate T lymphocyte responses in allergic asthma.

Vasoactive intestinal peptide (VIP) is a potent chemoattractant for immune cells that is delivered by the peripheral nervous system to immune organs. However, the molecular mechanism regulating VIP’s chemoattractant properties is not known. Previously, our group published the VIP transcriptome in resting CD4 T cells, which identified a chemoattractant gene set (e.g. EGFR, Snail and syt XIII) that was coordinately upregulated. We hypothesize that these gene products control the chemoattractant activity of VIP in immune cells. Moreover, we propose that this chemoattractant gene set is transcriptionally upregulated by the transactivation of EGFR by VIP as is observed in non‐immune cells, including breast and colon cancer cells. This research will utilize primary mouse splenocytes treated +/‐ VIP over various time intervals in an attempt to identify which FACS‐sorted splenocyte subpopulations are sensitive to VIP and the extent to which the chemoattractant gene set is upregulated. Optimal conditions will be repeated +/‐ EGFR neutralizing antibody or a specific EGFR kinase inhibitor to assess the contribution of EGFR enzymatic activity for the chemoattractant gene expression changes. Lastly, Boyden‐chamber chemotaxis assays will be employed to test whether pharmacological inhibition against EGFR kinase activity and/or other chemoattractant gene products suppresses VIP’s chemoattractant activity using identified splenocyte subpopulations. The significance of the proposed research is to better understand the molecular control of lymphocyte trafficking mediated by the nervous system.Grant Funding Source: This research was supported by NIH/NIAID (R15 to GD).

Vasoactive intestinal peptide (VIP) is a potent immune regulator and secretagogue delivered by the peripheral nervous system to immune organs. VIP inhibits pro‐inflammatory, but increases anti‐inflammatory, cytokine secretion, deactivates macrophages and induces regulatory T cells. However, little is known about how VIP affects resting immune cells. Our laboratory discovered that VIP upregulated synaptotagmin (syt) XIII in resting CD4 T cells. Syt XIII is a calcium‐independent transmembrane protein that plays a vital role in vesicle exocytosis. This research is significant as the upregulation of syt XIII by VIP would maintain an important “exocytosis program” in resting immune cells. We hypothesize that VIP induced upregulation of syt XIII is dependent on its receptor VIP1, but not VIP2, through a Gαs→AC→PKA signaling mechanism in splenocytes. This research will utilize primary mouse splenocytes treated +/‐ VIP to identify peak expression of syt XIII by qPCR and western analysis. Optimal conditions will be repeated with VIP2 KO animals, VIP1 receptor antagonists and small molecule inhibitors of PKA (H89) and assessed by qPCR and/or western analysis for syt XIII expression. The significance of the proposed research is to better delineate how the nervous system regulates immunity.Grant Funding Source: This research will be supported by NIH/NIAID (R15 to GD).

Aspergillus fumigatusis a ubiquitously present respiratory pathogen. The outcome of a pulmonary disease may vary significantly with fungal viability and host immune status. Our objective in this study was (1) to assess the ability of inhaled irradiation-killed or liveA. fumigatusspores to induce allergic pulmonary disease and (2) to assess the extent to which inhaled dead or liveA. fumigatusspores influence pulmonary symptoms in a previously established allergic state. Our newly developed fungal delivery apparatus allowed us to recapitulate human exposure through repeated inhalation of dry fungal spores in an animal model. We found that liveA. fumigatusspore inhalation led to a significantly increased humoral response, pulmonary inflammation, and airway remodeling in naïve mice and is more likely to induce allergic asthma symptoms than the dead spores. In contrast, in allergic mice, inhalation of dead and live conidia recruited neutrophils and induced goblet cell metaplasia. This data suggests that asthma symptoms might be exacerbated by the inhalation of live or dead spores in individuals with established allergy to fungal antigens, although the extent of symptoms was less with dead spores. These results are likely to be important while considering fungal exposure assessment methods and for making informed therapeutic decisions for mold-associated diseases.

Agricultural workers and farming communities are repeatedly exposed to grain dust during harvest, transport, and storage. The recognition that this complex mixture of particulates can trigger acute respiratory distress is not new, but the ways in which it may exacerbate both immediate and long‐term outcomes of allergic asthma remain unclear. The objectives of this study are to investigate the impact of repeated grain dust inhalation on the immunologic mechanisms that drive acute and chronic changes in the allergic lung. Using an inhalation model of Aspergillus fumigatus‐induced allergic asthma in mouse, we are investigating the effect of repeated exposures to corn and soybean grain dusts (collected from the rafters of commercial grain elevators). Our early data has revealed a prominent, but short‐lived, neutrophilia in the airways of naïve mice subjected to a single, 20‐min dust exposure. Continuing, we will compare airway hyperresponsiveness and inflammatory responses after multiple grain dust exposures in naïve and allergic lungs, as well as the chronic architectural changes that account for much of the morbidity of long‐term asthma.This pilot project was funded through Central States‐Centre for Agricultural Safety and Health (CDC/NIOSH/OD).

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.

The ability to accurately mimic normal processes for sensitization and allergen challenge in an experimental animal model are useful in that they allow researchers to critically manipulate the complex interactions of multiple cell types. In the context of the allergic lung, multiple cell types form complex cellular networks and function to regulate a variety of temporal and spatial changes. Mouse models of allergic airway disease have proven to be highly useful for dissecting these complex interactions, particularly in addressing remodeling of the allergic airway in chronic asthma. Until we can better represent the normal processes that initiate and perpetuate asthma, our understanding of the mechanisms of tissue injury leading to chronic remodeling of the airways and effective therapeutic strategies to treat this disease will remain limited. It was with this goal in mind that we set about devising an inhalational model of Aspergillus fumigatus-induced fungal asthma in a murine experimental system.

Aspergillus fumigatus is a ubiquitously present respiratory pathogen. The outcome of a pulmonary disease may vary significantly with fungal viability and host immune status. Our objective in this study was (1) to assess the ability of inhaled irradiation-killed or live A. fumigatus spores to induce allergic pulmonary disease and (2) to assess the extent to which inhaled dead or live A. fumigatus spores influence pulmonary symptoms in a previously established allergic state. Our newly developed fungal delivery apparatus allowed us to recapitulate human exposure through repeated inhalation of dry fungal spores in an animal model. We found that live A. fumigatus spore inhalation led to a significantly increased humoral response, pulmonary inflammation, and airway remodeling in naïve mice and is more likely to induce allergic asthma symptoms than the dead spores. In contrast, in allergic mice, inhalation of dead and live conidia recruited neutrophils and induced goblet cell metaplasia. This data suggests that asthma symptoms might be exacerbated by the inhalation of live or dead spores in individuals with established allergy to fungal antigens, although the extent of symptoms was less with dead spores. These results are likely to be important while considering fungal exposure assessment methods and for making informed therapeutic decisions for mold-associated diseases.