Faculty Bibliography

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes (1). Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension (2). Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases (3). Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention (4). The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system. Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery(5-13). The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues. The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography(5,14-17) and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

BACKGROUND: Hypomorphic mutations in the bone morphogenic protein receptor (BMPR2) confer a much greater risk for developing pulmonary arterial hypertension (PAH). However, not all carriers of a mutation in the BMPR2 gene suffer from PAH. We have previously shown that prolonged T helper 2 (Th2) responses in the lungs to a mild antigen delivered via the airways induce severe pulmonary arterial remodeling, but no pulmonary hypertension. The current studies were designed to test the idea that Th2 responses to a mild antigen together with the expression of a hypomorphic BMPR2 gene would trigger pulmonary hypertension. METHODOLOGY/PRINCIPAL FINDINGS: Mice that expressed a hypomorphic BMPR2 transgene (transgene-positive) and transgene-negative mice were either exposed to saline, or primed and exposed to a mild antigen (Ovalbumin) over a prolonged period of time. Only transgene-positive but not transgene-negative mice exposed to antigen developed significantly increased right ventricular systolic pressures, while both groups showed pulmonary artery remodeling with severe muscularization and airway inflammation to a similar degree. Antigen exposure resulted in a smaller increase in the percentage of Interleukin (IL)-13 positive T cells in the lymph nodes, and in a smaller increase in resistin-like-molecule (RELM)alpha expression and a decreased ratio of expression of IL-33 relative to its receptor (IL-1-receptor-like 1, IL1RL1-ST2) in the right ventricles of transgene-positive mice compared to transgene-negative animals. Furthermore, only antigen-challenged transgene-positive mice showed a significant increase in Interferon (IFN)gamma positive T cells over saline-exposed controls. CONCLUSIONS/SIGNIFICANCE: Our study suggests that exposure with a mild Th2 antigen can trigger pulmonary hypertension on the background of the expression of a hypomorphic BMPR2 gene and that conversely, the expression of the hypomorphic BMPR2 gene can alter the immune response to a mild, inhaled antigen.

RATIONALE: Hypomorphic mutations in the bone morphogenic protein receptor (BMPR2) confer a much greater risk for developing pulmonary arterial hypertension (PAH). However, not all carriers of a mutation in the BMPR2 gene suffer from PAH. We have previously shown that prolonged T helper 2 (Th2) responses in the lungs to a mild antigen delivered via the airways induce severe pulmonary arterial remodeling, but no pulmonary hypertension. The current studies were designed to test the idea that Th2 responses to a mild antigen together with the expression of a hypomorphic BMPR2 gene would trigger pulmonary hypertension. METHODS: Mice that expressed a hypomorphic BMPR2 transgene and littermate wild type mice were either exposed intranasally to saline, or primed and exposed intranasally to a mild antigen (Ovalbumin) over a prolonged period of time. RESULTS: When exposed to antigen, only mice that expressed the hypomorphic BMPR2 transgene but not wild type mice exposed to antigen developed significantly increased right ventricular systolic pressures, while both groups showed pulmonary artery remodeling with severe muscularization and airway inflammation to a similar degree. Compared with wild type, mice expressing the hypomorphic BMPR2 transgene had a significantly smaller percentage of T helper 2 (Th2) cytokine (Interleukin (IL)-13) positive cells and a significant increase in Th1 (IFNg) positive cells in the lymph nodes following antigen exposure. Correspondingly, antigen exposure resulted in a significantly reduced expression in the right heart of the Th2 related cytokine, resistin-like-molecule (RELM)a, and a decreased ratio of expression of IL-33 relative to its receptor (IL-1-receptor-like 1, IL1RL1-ST2) in the mice expressing the hypomorphic BMPR2 transgene relative to wild type. CONCLUSIONS: Our study suggests that exposure with a mild Th2 antigen can trigger the pulmonary hypertension phenotype on the background of the expression of a hypomorphic BMPR2 gene and that conversely, the expression of the hypomorphic BMPR2 gene can alter the immune response to a mild, inhaled antigen

BACKGROUND: Prenatal allergen exposure has been linked to both induction and protection of allergic sensitization in offspring. We hypothesized that prenatal exposure of mice (F0) to Aspergillus fumigatus (A. fumigatus) would be associated with decreased immunoglobulin (Ig) E and airway eosinophilia and alterations in CpG methylation of T-helper genes in third-generation mice (F2). METHODS: Female BALB/c mice were sensitized to A. fumigatus (62.5, 125, 1250 mug, or saline) and re-exposed to the same dose on days 7 and 14 (early) or days 12 and 17 (late) gestation. Grandoffspring were treated with A. fumigatus (62.5 mug) at 9 weeks. IgE, IgG(1) , and IgG(2a) levels and cell counts from bronchoalveolar lavage fluid were determined. Lung DNA was pyrosequenced at multiple sites in the interferon (IFN)-gamma and interleukin (IL)-4 promoters. RESULTS: Grandoffspring of mothers dosed with 1250 mug early during pregnancy developed increased airway eosinophilia (P < 0.05). Grandoffspring of mothers dosed late in pregnancy developed lower IgE (P < 0.05) and airway eosinophilia (P < 0.05). Grandoffspring of mothers dosed early had lower methylation at IL-4 CpG(-408) and CpG(-393) compared to late dosed mice (P < 0.005 across all doses). Few correlations were found between methylation levels and airway eosinophilia and IgE. CONCLUSION: Prenatal exposure to A. fumigatus late during pregnancy, but not early, was associated with lower IgE and airway eosinophilia in grandoffspring. Prenatal exposure to A. fumigatus was associated with changes in CpG methylation in the IFN-gamma and IL-4 promoters that did not correlate consistently with indicators of allergic sensitization.

This is a concise review on Interleukin (IL)-13 and the evolution of asthma therapy, from discovery of the molecule, the identification of its pathogenic role in animal models of asthma, to the development of clinically successful neutralizing agents. The translational path from basic research to clinical application was not sequential as expected but random with respect to the tools (molecular & cell biology, animal models, human studies) used and to the application of academic versus industry research. The experiences with the development of neutralizing anti-IL-13 reagents emphasize the need for inclusion of a biomarker assay in the clinical trials that both identifies individuals that actually have aberrant expression of the pathway of interest and allows determining whether the target of interest is neutralized.

Introduction: Ambient pollutants upregulate cytokines involved in mucosal immune responses. We recently demonstrated that diesel exhaust particle (DEP)-treated human bronchial epithelial cells (HBEC) upregulated myeloid dendritic cell maturation and Th2 polarization via HBEC-derived thymic stromal lymphopoietin (TSLP), suggesting that TSLP links environmental exposures and airway immune responses. In airway epithelial cells, TSLP is regulated in part by nuclear factor (NF)-kappaB activation, but additional regulatory signals remain unclear. Since a correlation of miR-375 and TSLP expression has been described in the murine gut, we hypothesized that hsa-miR-375 regulated TSLP expression in human primary HBEC. Methods: Primary human bronchial epithelial cells (pHBEC) were treated with defined stimuli including DEP (3mug/cm2) and total RNA was isolated (6h). Levels of mRNA or microRNA were measured using qRT-PCR and normalized against housekeeping genes (GAP-DH and RNU6-2 respectively). For transfection studies, pHBEC were seeded (60% confluence) and transfected with synthetic miRNA, anti-miRNA, and respective control species (HiPerFect transfection reagent). Data were normalized to housekeeping control and expressed as fold increase compared to resting controls. Results: Exposure of pHBEC to DEP, but not to TNFalpha upregulated hsa-miR-375 (4.2+/-1.1and 1.6+/-1.2 fold, respectively). In the same samples, TSLP was induced (5.0+/-1.4 fold) by DEP and 12.1+/-2.6 fold by TNFalpha. To confirm the role of hsa-miR-375, pHBEC were transfected with synthetic miR-375, anti-miR-375, or respective miRNA controls. Transfection with hsa-miR-375 but not control, upregulated TSLP expression (1.8+/-0.4 fold). Treatment of pHBEC with DEP in the presence of transfected anti-miR-375, but not control, reduced TSLP expression (53.0+/-15.0 % reduction compared to mock transfected DEP). In contrast, transfection with anti-miR-375 failed to reduce TNFalpha induced TSLP expression. Because hsa-miR-375 was upregulated by DEP, we performed an in silico analysis for putative targets and identified the aryl hydrocarbon receptor (AhR) (Targetscan 5.1). Both DEP and ambient PM reduced AhR transcript but increased transcriptional activity (CYP1A1 expression) in pHBEC. Transfection of pHBEC with synthetic miR-375 reduced AhR transcript (80+/-35 % reduction) and upregulated TSLP. Conclusion: These data suggest a selective mechanism of TSLP regulation by DEP in pHBEC via hsa-miR-375. Since the AhR has been described to interfere with NF-kappaB activation via stabilization of the inhibitory RelB subunit, its transcript reduction by miR-375 may be a potential pathway for DEP regulation of TSLP

Background: Autoantibody responses have long been associated with the severity of pulmonary arterial hypertension. Previous studies in our lab have shown that a prolonged T helper 2 (Th2) response to inhaled antigen induces severe pulmonary arterial remodeling. We have also shown that urban particulate matter (PM) from air pollution exacerbates antigen induced pulmonary arterial remodeling and pulmonary hypertension. The present study was designed to identify the role of B cells (antibody producing lymphocytes) for pulmonary hypertension induced by antigen and urban PM. Methods: The respirable fraction of urban airborne PM (urban PM2.5) was collected in New York City. Mice were primed for a Th2 response with antigen adsorbed to Alum and then challenged with soluble antigen (Ovalbumin) combined with urban PM2.5 intranasally. We determined pulmonary arterial remodeling by histology, right heart hypertrophy by measuring heart weights and right ventricular systolic pressures by heart catheterization in anaesthetized, spontaneously breathing mice. Results: In contrast to wild type, Th2 primed B cell KO mice had no significantly increased right heart weights, or right heart systolic pressures in response to intranasal antigen and urban PM2.5. Reconstitution with anti-antigen antibody restored the development of pulmonary hypertension in antigen-urban PM2.5 challenged B cell KO mice. Like wild type mice, B cell KO mice had significant pulmonary arterial remodeling that was slightly increased by reconstitution with antibody. Conclusions: Our studies suggest that antigen-specific antibody is necessary for the development of pulmonary hypertension induced by the exposure to a Th2 antigen combined with urban PM2.5. Current studies are aimed at identifying mRNA and microRNA species that are differentially expressed in the right hearts of antibody reconstituted B cell KO mice that were exposed to antigen and urban PM2.5