Peroxisome proliferator-activated receptor gamma gene variants modify human airway and systemic responses to indoor dibutyl phthalate exposure.

BACKGROUND: Single nucleotide polymorphisms (SNPs) of peroxisome proliferator-activated receptor gamma (PPAR-γ; gene: PPARG) and oxidative stress genes are associated with asthma risk. However, whether such variants modulate responses to dibutyl phthalate (DBP), a common plasticizer associated with increased asthma development, remains unknown. The purpose of this study is to investigate how SNPs in PPARG and oxidative stress genes, as represented by two separate genetic risk scores, modify the impact of DBP exposure on lung function and the airway and systemic response after an inhaled allergen challenge. METHODS: We conducted a double-blinded human crossover study with sixteen allergen-sensitized participants exposed for three hours to DBP and control air on distinct occasions separated by a 4-week washout. Each exposure was followed by an allergen inhalation challenge; subsequently, lung function was measured, and blood and bronchoalveolar lavage (BAL) were collected and analyzed for cell counts and allergen-specific immunoglobulin E (IgE). Genetic risk scores for PPAR-γ (P-GRS; weighted sum of PPARG SNPs rs10865710, rs709158, and rs3856806) and oxidative stress (OS-GRS; unweighted sum of 16 SNPs across multiple genes) were developed, and their ability to modify DBP effects were assessed using linear mixed-effects models. RESULTS: P-GRS and OS-GRS modified DBP effects on allergen-specific IgE in blood at 20 h (interaction effect [95% CI]: 1.43 [1.13 to 1.80], p = 0.005) and 3 h (0.99 [0.98 to 1], p = 0.03), respectively. P-GRS also modified DBP effects on Th2 cells in blood at 3 h (- 25.2 [- 47.7 to - 2.70], p = 0.03) and 20 h (- 39.1 [- 57.9 to - 20.3], p = 0.0005), and Th2 cells in BAL at 24 h (- 4.99 [- 8.97 to - 1.01], p = 0.02). An increasing P-GRS associated with reduced DBP effect on Th2 cells. Neither GRS significantly modified DBP effects on lung function parameters. CONCLUSIONS: PPAR-γ variants modulated several airway and systemic immune responses to the ubiquitous chemical plasticizer DBP. Our results suggest that PPAR-γ variants may play a greater role than those in oxidative stress-related genes in airway allergic responses to DBP. TRIAL REGISTRATION: This study reports results from The Phthalate-Allergen Immune Response Study that was registered on ClinicalTrials.gov with identification NCT02688478.

Dibutyl phthalate exposure alters T-cell subsets in blood from allergen-sensitized volunteers.

Phthalates are ubiquitous environmental contaminants associated with allergic disease in epidemiological and animal studies. This investigation aims to support these associations by interrogating systemic immune effects in allergen-sensitized volunteers after controlled indoor air exposure to a known concentration of dibutyl phthalate (DBP). The phthalate-allergen immune response (PAIR) study enrolled 16 allergen-sensitized participants to a double-blinded, randomized, crossover exposure to two conditions (DBP or control air for 3 hr), each followed immediately by inhaled allergen challenge. Peripheral blood immune cell composition and activation along with inflammatory mediators were measured before and after exposure. DBP exposure prior to the inhaled allergen challenge increased the percentage of CD4+ T helper cells and decreased the percentage of regulatory T cells (3 hr and 20 hr post-exposure), while only modest overall effects were observed for inflammatory mediators. The cells and mediators affected by the phthalate exposure were generally not overlapping with the endpoints affected by allergen inhalation alone. Thus, in distinction to our previously published effects on lung function, DBP appears to alter endpoints in peripheral blood that are not necessarily enhanced by allergen alone. Further studies are needed to clarify the role of phthalate-induced systemic effects in disease pathogenesis.

Dibutyl Phthalate Augments Allergen-induced Lung Function Decline and Alters Human Airway Immunology. A Randomized Crossover Study.

Rationale: Phthalates are a group of chemicals used in common commercial products. Epidemiological studies suggest that phthalate exposure is associated with development or worsening of allergic diseases such as asthma. However, effects of dibutyl phthalate (DBP) or other phthalates found in high concentrations in indoor air have never been examined in allergic individuals in a controlled exposure setting.Objectives: To investigate the airway effects in humans caused by inhalation of a known concentration of a single phthalate, DBP.Methods: In a randomized crossover study, 16 allergen-sensitized participants were exposed to control air or DBP for 3 hours in an environmental chamber followed immediately by an allergen inhalation challenge. Bronchoalveolar wash and lavage were obtained 24 hours after exposure. Lung function, early allergic response, airway responsiveness, inflammation, immune mediators, and immune cell phenotypes were assessed after DBP exposure.Measurements and Main Results: DBP exposure increased the early allergic response (21.4% decline in FEV1 area under the curve, P = 0.03). Airway responsiveness was increased by 48.1% after DBP exposure in participants without baseline hyperresponsiveness (P = 0.01). DBP increased the recruitment of BAL total macrophages by 4.6% (P = 0.07), whereas the M2 macrophage phenotype increased by 46.9% (P = 0.04). Airway immune mediator levels were modestly affected by DBP.Conclusions: DBP exposure augmented allergen-induced lung function decline, particularly in those without baseline hyperresponsiveness, and exhibited immunomodulatory effects in the airways of allergic individuals. This is the first controlled human exposure study providing biological evidence for phthalate-induced effects in the airways.Clinical trial registered with www.clinicaltrials.gov (NCT02688478).

Sex Dimorphism of Allergen-Induced Secreted Proteins in Murine and Human Lungs.

Biological sex influences disease severity, prevalence and response to therapy in allergic asthma. However, allergen-mediated sex-specific changes in lung protein biomarkers remain undefined. Here, we report sex-related differences in specific proteins secreted in the lungs of both mice and humans, in response to inhaled allergens. Female and male BALB/c mice (7-8 weeks) were intranasally challenged with the allergen house dust mite (HDM) for 2 weeks. Bronchoalveolar lavage fluid (BALF) was collected 24 hour after the last HDM challenge from allergen-naïve and HDM-challenged mice (N=10 per group, each sex). In a human study, adult participants were exposed to nebulized (2 min) allergens (based on individual sensitivity), BALF was obtained after 24 hour (N=5 each female and male). The BALF samples were examined in immunoblots for the abundance of 10 proteins shown to increase in response to allergen in both murine and human BALF, selected from proteomics studies. We showed significant sex-bias in allergen-driven increase in five out of the 10 selected proteins. Of these, increase in eosinophil peroxidase (EPX) was significantly higher in females compared to males, in both mice and human BALF. We also showed specific sex-related differences between murine and human samples. For example, allergen-driven increase in S100A8 and S100A9 was significantly higher in BALF of females compared to males in mice, but significantly higher in males compared to females in humans. Overall, this study provides sex-specific protein biomarkers that are enhanced in response to allergen in murine and human lungs, informing and motivating translational research in allergic asthma.

Dibutyl phthalate modulates phenotype of granulocytes in human blood in response to inflammatory stimuli.

BACKGROUND: Phthalates are plasticizers used in many common commercial products. They are ubiquitous environmental contaminants and epidemiological studies suggest that phthalate exposure is associated with development or worsening of airway diseases. Dibutyl phthalate (DBP) is a type of phthalate, found in high concentrations in indoor air, which appears to have a high inflammatory potential. In vitro studies on innate immune cells like macrophages have shown a reduction in phagocytic and antigen-presenting capacity and decreased production of stimuli-induced cytokines after DBP exposure. OBJECTIVE: We aimed to assess how DBP may alter the in vitro cellular and humoral innate immune response to inflammatory stimuli in blood innate immune cells. METHODS: Human whole blood was stimulated with inflammatory stimuli (lipopolysaccharide (LPS), resiquimod (R848) and phorbol 12-myristate 13-acetate (PMA)) in the presence or absence of DBP. The expression of surface markers CD16, CD24, CD69 and CD14 on granulocytes and monocytes was quantified by flow cytometry analysis. The release of TNFα, IFNγ, IL8 and IL10 cytokines was measured by ELISA. RESULTS: The presence of DBP reduced the inflammatory stimuli-induced expression of CD24 on neutrophils and eosinophils and CD69 on activated eosinophils, whereas expression of CD16 on neutrophils was increased. DBP also had a dampening effect on the release of pro-inflammatory mediators TNFα and IFNγ in response to the inflammatory stimuli. CONCLUSIONS: These responses may reflect an immunosuppressive effect of DBP through impairment of immune cell function.

Novel flow cytometry approach to identify bronchial epithelial cells from healthy human airways.

Sampling various compartments within the lower airways to examine human bronchial epithelial cells (HBEC) is essential for understanding numerous lung diseases. Conventional methods to identify HBEC in bronchoalveolar lavage (BAL) and wash (BW) have throughput limitations in terms of efficiency and ensuring adequate cell numbers for quantification. Flow cytometry can provide high-throughput quantification of cell number and function in BAL and BW samples, while requiring low cell numbers. To date, a flow cytometric method to identify HBEC recovered from lower human airway samples is unavailable. In this study we present a flow cytometric method identifying HBEC as CD45 negative, EpCAM/pan-cytokeratin (pan-CK) double-positive population after excluding debris, doublets and dead cells from the analysis. For validation, the HBEC panel was applied to primary HBEC resulting in 98.6% of live cells. In healthy volunteers, HBEC recovered from BAL (2.3% of live cells), BW (32.5%) and bronchial brushing samples (88.9%) correlated significantly (p = 0.0001) with the manual microscopy counts with an overall Pearson correlation of 0.96 across the three sample types. We therefore have developed, validated, and applied a flow cytometric method that will be useful to interrogate the role of the respiratory epithelium in multiple lung diseases.