Faculty Bibliography

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

INTRODUCTION: Particulate matter (PM), specifically nickel (Ni) found on or in PM, has been associated with an increased risk of mortality in human population studies and significant increases in vascular inflammation, generation of reactive oxygen species, altered vasomotor tone, and potentiated atherosclerosis in murine exposures. Recently, murine inhalation of Ni nanoparticles have been shown to cause pulmonary inflammation that affects cardiovascular tissue and potentiates atherosclerosis. These adverse cardiovascular outcomes may be due to the effects of Ni on endothelial progenitor cells (EPCs), endogenous semi-pluripotent stem cells that aid in endothelial repair. Thus, we hypothesize that Ni nanoparticle exposures decrease cell count and cause impairments in function that may ultimately have significant effects on various cardiovascular diseases, such as, atherosclerosis. METHODS: Experiments involving inhaled Ni nanoparticle exposures (2 days/5 h/day at approximately 1200 microg/m(3), 3 days/5 h/day at approximately 700 microg/m(3), and 5 days/5 h/day at approximately 100 microg/m(3)), were performed in order to quantify bone marrow resident EPCs using flow cytometry in C57BL/6 mice. Plasma levels of human stromal cell-derived factor 1alpha (SDF-1alpha) and vascular endothelial growth factor (VEGF) were assessed by enzyme-linked immunosorbent assay and in vitro functional assessments of cultured EPCs were conducted. RESULTS AND CONCLUSIONS: Significant EPC count differences between exposure and control groups for Ni nanoparticle exposures were observed. Differences in EPC tube formation and chemotaxis were also observed for the Ni nanoparticle exposed group. Plasma VEGF and SDF-1alpha differences were not statistically significant. In conclusion, this study shows that inhalation of Ni nanoparticles results in functionally impaired EPCs and reduced number in the bone marrow, which may lead to enhanced progression of atherosclerosis

ABSTRACT: BACKGROUND: Multiple studies have suggested that prenatal exposure to either allergens or air pollution may increase the risk for the development of allergic immune responses in young offspring. However, the effects of prenatal environmental exposures on adult offspring have not been well-studied. We hypothesized that combined prenatal exposure to Aspergillus fumigatus (A. fumigatus) allergen and diesel exhaust particles will be associated with altered IgE production, airway inflammation, airway hyperreactivity (AHR), and airway remodeling of adult offspring. METHODS: Following sensitization via the airway route to A. fumigatus and mating, pregnant BALB/c mice were exposed to additional A. fumigatus and/or diesel exhaust particles. At age 9-10 weeks, their offspring were sensitized and challenged with A. fumigatus. RESULTS: We found that adult offspring from mice that were exposed to A. fumigatus or diesel exhaust particles during pregnancy experienced decreases in IgE production. Adult offspring of mice that were exposed to both A. fumigatus and diesel exhaust particles during pregnancy experienced decreases in airway eosinophilia. CONCLUSION: These results suggest that, in this model, allergen and/or diesel administration during pregnancy may be associated with protection from developing systemic and airway allergic immune responses in the adult offspring

T cell-specific deletion of Blimp-1 causes abnormal T cell homeostasis and function, leading to spontaneous, fatal colitis in mice. Herein we explore the role of Blimp-1 in Th1/Th2 differentiation. Blimp-1 mRNA and protein are more highly expressed in Th2 cells compared with Th1 cells, and Blimp-1 attenuates IFN-gamma production in CD4 cells activated under nonpolarizing conditions. Although Blimp-1-deficient T cells differentiate normally to Th2 cytokines in vitro, Blimp-1 is required in vivo for normal Th2 humoral responses to NP-KLH (4-hydroxy-3-nitrophenylacetyl/keyhole lymphocyte hemocyanin) immunization. Lack of Blimp-1 in CD4 T cells causes increased IFN-gamma, T-bet, and Bcl-6 mRNA. By chromatin immunoprecipitation we show that Blimp-1 binds directly to a distal regulatory region in the ifng gene and at multiple sites in tbx21 and bcl6 genes. Our data provide evidence that Blimp-1 functions in Th2 cells to reinforce Th2 differentiation by repressing critical Th1 genes

Pulmonary arterial remodeling characterized by increased vascular smooth muscle density is a common lesion seen in pulmonary arterial hypertension (PAH), a deadly condition. Clinical correlation studies have suggested an immune pathogenesis of pulmonary arterial remodeling, but experimental proof has been lacking. We show that immunization and prolonged intermittent challenge via the airways with either of two different soluble antigens induced severe muscularization in small- to medium-sized pulmonary arteries. Depletion of CD4(+) T cells, antigen-specific T helper type 2 (Th2) response, or the pathogenic Th2 cytokine interleukin 13 significantly ameliorated pulmonary arterial muscularization. The severity of pulmonary arterial muscularization was associated with increased numbers of epithelial cells and macrophages that expressed a smooth muscle cell mitogen, resistin-like molecule alpha, but surprisingly, there was no correlation with pulmonary hypertension. Our data are the first to provide experimental proof that the adaptive immune response to a soluble antigen is sufficient to cause severe pulmonary arterial muscularization, and support the clinical observations in pediatric patients and in companion animals that muscularization represents one of several injurious events to the pulmonary artery that may collectively contribute to PAH

OBJECTIVE: To evaluate time-dependent alterations in gene expression of chemokines in bronchial epithelium of recurrent airway obstruction (RAO)-affected horses and whether alterations resulted from increases in gene expression of interleukin (IL)-17 in cells isolated from bronchoalveolar lavage fluid (BALF). ANIMALS: 8 RAO-susceptible horses and 9 control horses. PROCEDURE: In 2 experiments, both groups of horses were evaluated after being maintained on pasture and after being stabled and fed dusty hay for 1, 14, 35, and 49 days (experiment 1) or 14 and 28 days (experiment 2). In experiment 1, gene expression of IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and Toll-like receptor 4 (TLR4) in epithelium and IL-8, IL-17, and TLR4 in BALF cells was measured. In experiment 2, bronchial biopsy specimens were evaluated for IL-8 immunoreactivity. RESULTS: In RAO-susceptible horses after 14 days of challenge exposure, there was a 3- and 10-fold increase in gene expression of IL-8 for epithelial and BALF cells and an increase in IL-8 immunoreactivity in epithelial cells. Challenge exposure failed to alter gene expression of CXCL1, GM-CSF, G-CSF, and TLR4 in epithelial cells of any horses at any time point. During challenge exposure, gene expression of BALF cell IL-17 was downregulated in control horses (day 1) and upregulated in RAO-affected horses (day 35). CONCLUSIONS AND CLINICAL RELEVANCE: Epithelial-derived IL-8 may promote airway neutrophilia, but the inciting stimulus is unlikely to be IL-17 because upregulation of this gene is subsequent to that of IL-8 in epithelial cells

RATIONALE: Acid aspiration causes acute lung injury (ALI). Recently, we showed that a brief intravascular infusion of hyperosmolar sucrose, given concurrently with airway acid instillation, effectively blocks the ensuing ALI. OBJECTIVES: The objective of the present study was to determine the extent to which intravascular infusion of hyperosmolar sucrose might protect against acid-induced ALI when given either before or after acid instillation. METHODS: Our studies were conducted in anesthetized rats and in isolated, blood-perfused rat lungs. We instilled HCl through the airway, and we quantified lung injury in terms of the extravascular lung water (EVLW) content, filtration coefficient (Kfc), and cell counts and protein concentration in the bronchoalveolar lavage. We infused hyperosmolar sucrose via the femoral vein. RESULTS: In anesthetized rats, airway HCl instillation induced ALI as indicated by a 52% increase of EVLW and a threefold increase in Kfc. However, a 15-min intravenous infusion of hyperosmolar sucrose given up to 1 h before or 30 min after acid instillation markedly blunted the increases in EVLW, as well as the increases in cell count, and in protein concentration in the bronchoalveolar lavage. Hyperosmolar pretreatment also blocked the acid-induced increase of Kfc. Studies in isolated perfused lungs indicated that the protective effect of hyperosmolar sucrose was leukocyte independent. CONCLUSIONS: We conclude that a brief period of vascular hyperosmolarity protects against acid-induced ALI when the infusion is administered shortly before, or shortly after, acid instillation in the airway. The potential applicability of hyperosmolar sucrose in therapy for ALI requires consideration