Faculty Bibliography

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

Introduction: Mouse RELMalpha is a member of the resistin family of adipokines and is expressed by many cell types including intestinal and airway epithelial cells, macrophages activated in the course of Th2 responses (alternatively activated macrophages), dendritic cells and white adipose tissue. This molecule is also known as Found in Inflammatory Zone (FIZZ) and Hypoxia Induced Mitogenic Factor (HIMF). The human homologue, RELMbeta, is expressed at increased levels in the pulmonary artery in pulmonary arterial hypertension and is a mitogen for human vascular smooth muscle cells. Methods: Our studies focused on the role of RELMalpha in severe pulmonary arterial remodeling induced by a T helper 2 (Th2) response to antigen. We studied wild type and RELMalpha deficient mice following immunization with Ovalbumin (OVA) complexed to Alum and exposure to soluble OVA alone or in combination with respirable urban particulate matter. The urban particulate matter (PM2.5) was collected from the air in New York City. Results: The study showed that the number of cells that stain positively for RELMalpha by immunohistochemistry was significantly correlated with the severity of pulmonary arterial remodeling. Furthermore, our studies demonstrated a significant exacerbation of pulmonary arterial remodeling in antigen primed wild type mice exposed to a combination of antigen and urban particulate matter. RELMalpha deficient mice, in contrast, failed to show the exacerbation and developed pulmonary arterial thickening to exposure with antigen and respirable urban particulate matter to a significantly smaller degree relative to wild type. The severity of inflammation and the type of the immune response in the RELMalpha deficient mice challenged with antigen or the combination of antigen and urban particulate matter was not significantly different from the responses in wild type mice. Conclusion: RELMalpha is known to have a critical role in hypoxia induced pulmonary arterial remodeling; and combustion of fossil fuels releases airborne metals that can mimic hypoxia induced cell signaling. Therefore, our data suggest the idea that hypoxia-mimetic metals contained within urban particulate matter exacerbate pulmonary arterial remodeling induced by a Th2 response to antigen via synergistic augmentation of RELMalpha expression

Rationale: 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 (mDC) maturation and promoted DC that support Th2 polarization via HBEC-derived thymic stromal lymphopoietin (TSLP). IL-33 is an IL-1 like cytokine that signals via a heterodimer (IL-1RL1, ST2 and IL-1R accessory protein; IL-1RAcP). Genetic variants of IL-33 and ST2 are associated with asthma. Since IL-33/ST2 signaling acts in synergy with TSLP to induce Th2 responses, we hypothesized that DEP-treated HBEC up-regulate expression and release of IL-33 and support mDC maturation. Methods: Primary culture HBEC (pHBEC) were treated with PBS (resting), PMA (5ng/ml), or DEP (3mug/cm²) and total RNA (6h) or supernatant (18h) isolated. Immature mDC isolated from peripheral blood (magnetic selection, BDCA-1) were exposed to resting or treated pHBEC (48h). mDC function was determined by the ability to support an allogeneic mixed lymphocyte reaction (aMLR) after rescue from pHBEC co-culture by FACS sorting. T cell proliferation was measured by BrdU-incorporation. Results: DEP-treated pHBEC upregulated IL-33 mRNA (2.5+/-0.5 fold induction, n=3; mean +/- SEM; p<0.05). Soluble IL-33 was undetected from resting pHBEC but was increased by DEP treatment (7.2+/-0.2pg/ml; n=3, p<0.05). mDC expressed ST2 and IL-1RacP (FACS analysis) but neither DEP nor DEP-treated pHBEC changed their expression. As previously reported, DEP-treated pHBEC upregulated mDC function (aMLR). Anti-IL-33 pAb reduced DEP-treated pHBEC-dependent functional maturation of mDC, (26+/-7% reduction; n=3; p<0.05). The reduction observed was less than that with anti-TSLP pAb (48+/-8% reduction; n=3; p<0.05). Conclusion: DEP-treated pHBEC upregulated IL-33 transcription and supernatant protein and upregulation was associated with functional mDC maturation. Our data suggest that DEP upregulated Il-33 and TSLP in pHBEC, and these may act in concert to induce mDC regulated Th2 immune-responses at mucosal sites

Rationale Allergen exposure during gestation has been linked to both induction and protection of allergic sensitization in offspring. We previously showed that prenatal exposure to Aspergillus fumigatus (A. fumigatus) was associated with decreased IgE levels in offspring mice. Here we hypothesized that prenatal exposure of mice (F0) to A. fumigatus would be associated with decreased IgE that persists through the third generation of mice. We also hypothesized that exposure would be associated with decreased CpG methylation of T helper gene interferon (IFN)g in F2 offspring. Methods Female BALB/c mice were exposed intranasally to A. fumigatus (62.5 ug/ml, 125 ug/ml, or 1.25 mg/ml) or saline five times, four days apart, beginning 20 days prior to mating. Pregnant mice were treated again with A. fumigatus (62.5 ug/ml) or saline on days 7 and 14 (early dosing) or days 12 and 17 (late dosing). Adult offspring (F1) were mated with littermates. Their offspring (F2, n=62) were treated with five or six doses of A. fumigatus (62.5 ug/ml), four days apart, at age 9 weeks. Subsequently, serum IgE, IgG₁, and IgG_2a were measured using ELISA, cell counts from bronchoalveolar lavage fluid were determined, DNA was isolated from lung tissue, and PCR of the promoter region after bisulfite conversion was performed. Pyrosequencing was conducted to determine the percent of cytosine methylation (%_{5 m}C) at for CpG^-190, CpG^-170, and CpG^-53 within the promoter. Results Grandoffspring (F2) of mothers who received 1.25 mg/ml of A. fumigatus developed lower levels of total IgE following sensitization with A. fumigatus, compared to grandoffspring of mothers treated with saline or 125 ug/ml (p<0.01, Tukey). When stratified by timing of prenatal dose, only grandoffspring of mothers dosed late in pregnancy developed reduced IgE following sensitization (p<0.01, Tukey). Grandoffspring of mothers dosed later during pregnancy also developed lower levels of airway eosinophilia (p<0.01, Tukey). Only a nonsignificant decrease in DNA methylation at CpG^-190 (Mean % C 79.1, 79.7, 75.4, 74.6 for saline, 62.5 ug/ml, 125 ug/ml, and 1.25 5 m mg/ml, p=0.111, ANOVA) was found. Conclusions These findings suggest that maternal exposure during pregnancy to A. fumigatus is associated with reductions in IgE production and airway eosinophilia in grandoffspring (F2). Grandoffspring of mothers dosed late in pregnancy demonstrated the greatest effects. In these models, findings were not explained by alterations in CpG methylation in the IFN-gamma promoter. Further studies should explore other potential mechanisms and genes through which prenatal exposures could affect transgenerational inheritance of allergy

BACKGROUND: Interleukin (IL)-19 has been reported to enhance chronic inflammatory diseases such as asthma but the in vivo mechanism is incompletely understood. Because IL-19 is produced by and regulates cells of the monocyte lineage, our studies focused on in vivo responses of CD11c positive (CD11c+) alveolar macrophages and lung dendritic cells. METHODOLOGY/PRINCIPAL FINDINGS: IL-19-deficient (IL-19-/-) mice were studied at baseline (naive) and following intranasal challenge with microbial products, or recombinant cytokines. Naive IL-19-/- mixed background mice had a decreased percentage of CD11c+ cells in the bronchoalveolar-lavage (BAL) due to the deficiency in IL-19 and a trait inherited from the 129-mouse strain. BAL CD11c+ cells from fully backcrossed IL-19-/- BALB/c or C57BL/6 mice expressed significantly less Major Histocompatibility Complex class II (MHCII) in response to intranasal administration of lipopolysaccharide, Aspergillus antigen, or IL-13, a pro-allergic cytokine. Neurogenic-locus-notch-homolog-protein-2 (Notch2) expression by lung monocytes, the precursors of BAL CD11c+ cells, was dysregulated: extracellular Notch2 was significantly decreased, transmembrane/intracellular Notch2 was significantly increased in IL-19-/- mice relative to wild type. Instillation of recombinant IL-19 increased extracellular Notch2 expression and dendritic cells cultured from bone marrow cells in the presence of IL-19 showed upregulated extracellular Notch2. The CD205 positive subset among the CD11c+ cells was 3-5-fold decreased in the airways and lungs of naive IL-19-/- mice relative to wild type. Airway inflammation and histological changes in the lungs were ameliorated in IL-19-/- mice challenged with Aspergillus antigen that induces T lymphocyte-dependent allergic inflammation but not in IL-19-/- mice challenged with lipopolysaccharide or IL-13. CONCLUSIONS/SIGNIFICANCE: Because MHCII is the molecular platform that displays peptides to T lymphocytes and Notch2 determines cell fate decisions, our studies suggest that endogenous IL-19 is a constituent of the regulome that controls both processes in vivo