Pulmonary responses to ozone, a common air pollutant, are augmented in obese individuals. Adiponectin, an adipose-derived hormone that declines in obesity, has regulatory effects on the immune system. To determine the role of adiponectin in the pulmonary inflammation induced by extended (48–72 h) low-dose (0.3 parts per million) exposure to ozone, adiponectin-deficient (Adipo−/−) and wild-type mice were exposed to ozone or to room air. In wild-type mice, ozone exposure increased total bronchoalveolar lavage (BAL) adiponectin. Ozone-induced lung inflammation, including increases in BAL neutrophils, protein (an index of lung injury), IL-6, keratinocyte-derived chemokine, LPS-induced CXC chemokine, and G-CSF were augmented in Adipo−/− versus wild-type mice. Ozone also increased IL-17A mRNA expression to a greater extent in Adipo−/− versus wild-type mice. Moreover, compared with control Ab, anti–IL-17A Ab attenuated ozone-induced increases in BAL neutrophils and G-CSF in Adipo−/− but not in wild-type mice, suggesting that IL-17A, by promoting G-CSF release, contributed to augmented neutrophilia in Adipo−/− mice. Flow cytometric analysis of lung cells revealed that the number of CD45+/F4/80+/IL-17A+ macrophages and γδ T cells expressing IL-17A increased after ozone exposure in wild-type mice and further increased in Adipo−/− mice. The IL-17+ macrophages were CD11c− (interstitial macrophages), whereas CD11c+ macrophages (alveolar macrophages) did not express IL-17A. Taken together, the data are consistent with the hypothesis that adiponectin protects against neutrophil recruitment induced by extended low-dose ozone exposure by inhibiting the induction and/or recruitment of IL-17A in interstitial macrophages and/or γδ T cells.

Background:Ozone increases IL-33 in the lungs, and obesity augments the pulmonary effects of acute ozone exposure.Objectives:We assessed the role of IL-33 in the augmented effects of ozone observed in obese mice.Methods:Lean wildtype and obese db/db mice were pretreated with antibodies blocking the IL-33 receptor, ST2, and then exposed to ozone (2 ppm for 3 hr). Airway responsiveness was assessed, bronchoalveolar lavage (BAL) was performed, and lung cells harvested for flow cytometry 24 hr later. Effects of ozone were also assessed in obese and lean mice deficient in γδ T cells and their wildtype controls.Resultsand Discussion: Ozone caused greater increases in BAL IL-33, neutrophils, and airway responsiveness in obese than lean mice. Anti-ST2 reduced ozone-induced airway hyperresponsiveness and inflammation in obese mice but had no effect in lean mice. Obesity also augmented ozone-induced increases in BAL CXCL1 and IL-6, and in BAL type 2 cytokines, whereas anti-ST2 treatment reduced these cytokines. In obese mice, ozone increased lung IL-13+ innate lymphoid cells type 2 (ILC2) and IL-13+ γδ T cells. Ozone increased ST2+ γδ T cells, indicating that these cells can be targets of IL-33, and γδ T cell deficiency reduced obesity-related increases in the response to ozone, including increases in type 2 cytokines.Conclusions:Our data indicate that IL-33 contributes to augmented responses to ozone in obese mice. Obesity and ozone also interacted to promote type 2 cytokine production in γδ T cells and ILC2 in the lungs, which may contribute to the observed effects of IL-33.Citation:Mathews JA, Krishnamoorthy N, Kasahara DI, Cho Y, Wurmbrand AP, Ribeiro L, Smith D, Umetsu D, Levy BD, Shore SA. 2017. IL-33 drives augmented responses to ozone in obese mice. Environ Health Perspect 125:246–253; http://dx.doi.org/10.1289/EHP272

Obese mice exhibit innate airway hyperresponsiveness (AHR), a feature of asthma. Tumor necrosis factor alpha (TNFα) is implicated in the disease progression and chronic inflammatory status of both obesity and asthma. TNF acts via two TNF receptors, TNFR1 and TNFR2. To examine the role of TNFR2 in the AHR observed in obese mice, we generated obese Cpefat mice that were either sufficient or deficient in TNFR2 (Cpefat and Cpefat/TNFR2−/− mice, respectively) and compared them with their lean controls (WT and TNFR2−/− mice). Compared to WT mice, Cpefat mice exhibited AHR to aerosolized methacholine (measured using the forced oscillation technique) which was ablated in Cpefat/TNFR2−/− mice. Bioplex or ELISA assay indicated significant increases in serum leptin, G-CSF, IL-7, IL-17A, TNFα, and KC in obese versus lean mice, as well as significant obesity-related increases in bronchoalveolar lavage fluid (BALF) G-CSF and IP-10, regardless of TNFR2 status. Importantly, BALF IL-17A was significantly increased over lean controls in Cpefat but not Cpefat/TNFR2−/− mice. Functional annotation clustering of significantly affected genes identified from microarray analysis comparing gene expression in lungs of Cpefat and WT mice, identified blood vessel morphogenesis as the gene ontology category most affected by obesity. This category included several genes associated with AHR, including endothelin and trkB. Obesity increased pulmonary mRNA expression of endothelin and trkB in TNFR2 sufficient but not deficient mice. Our results indicate that TNFR2 signaling is required for the innate AHR that develops in obese mice, and suggest that TNFR2 may act by promoting IL-17A, endothelin, and/or trkB expression.

Background: Acute ozone (O3) exposure results in greater inflammation and airway hyperresponsiveness (AHR) in obese versus lean mice.Objectives: We examined the hypothesis that these augmented responses to O3 are the result of greater signaling through tumor necrosis factor receptor 2 (TNFR2) and/or interleukin (IL)-13.Methods: We exposed lean wild-type (WT) and TNFR2-deficient (TNFR2–/–) mice, and obese Cpefat and TNFR2-deficient Cpefat mice (Cpefat/TNFR2–/–), to O3 (2 ppm for 3 hr) either with or without treatment with anti–IL-13 or left them unexposed.Results: O3-induced increases in baseline pulmonary mechanics, airway responsiveness, and cellular inflammation were greater in Cpefat than in WT mice. In lean mice, TNFR2 deficiency ablated O3-induced AHR without affecting pulmonary inflammation; whereas in obese mice, TNFR2 deficiency augmented O3-induced AHR but reduced inflammatory cell recruitment. O3 increased pulmonary expression of IL-13 in Cpefat but not WT mice. Flow cytometry analysis of lung cells indicated greater IL-13–expressing CD4+ cells in Cpefat versus WT mice after O3 exposure. In Cpefat mice, anti–IL-13 treatment attenuated O3-induced increases in pulmonary mechanics and inflammatory cell recruitment, but did not affect AHR. These effects of anti–IL-13 treatment were not observed in Cpefat/TNFR2–/– mice. There was no effect of anti–IL-13 treatment in WT mice.Conclusions: Pulmonary responses to O3 are not just greater, but qualitatively different, in obese versus lean mice. In particular, in obese mice, O3 induces IL-13 and IL-13 synergizes with TNF via TNFR2 to exacerbate O3-induced changes in pulmonary mechanics and inflammatory cell recruitment but not AHR.

Ozone is an air pollutant that causes pulmonary symptoms. In mice, ozone exposure causes pulmonary injury and increases bronchoalveolar lavage macrophages and neutrophils. We have shown that IL-17A is important in the recruitment of neutrophils after subacute ozone exposure (0.3 ppm for 24–72 h). We hypothesized that γδ T cells are the main producers of IL-17A after subacute ozone. To explore this hypothesis we exposed wildtype mice and mice deficient in γδ T cells (TCRδ−/−) to ozone or room air. Ozone-induced increases in BAL macrophages and neutrophils were attenuated in TCRδ−/− mice. Ozone increased the number of γδ T cells in the lungs and increased pulmonary Il17a mRNA expression and the number of IL-17A+ CD45+ cells in the lungs and these effects were abolished in TCRδ−/− mice. Ozone-induced increases in factors downstream of IL-17A signaling, including G-CSF, IL-6, IP-10 and KC were also decreased in TCRδ−/− versus wildtype mice. Neutralization of IL-17A during ozone exposure in wildtype mice mimicked the effects of γδ T cell deficiency. TNFR2 deficiency and etanercept, a TNFα antagonist, also reduced ozone-induced increases in Il17a mRNA, IL-17A+ CD45+ cells and BAL G-CSF as well as BAL neutrophils. TNFR2 deficient mice also had decreased ozone-induced increases in Ccl20, a chemoattractant for IL-17A+ γδ T cells. Il17a mRNA and IL-17A+ γδ T cells were also lower in obese Cpefat versus lean WT mice exposed to subacute ozone, consistent with the reduced neutrophil recruitment observed in the obese mice. Taken together, our data indicate that pulmonary inflammation induced by subacute ozone requires γδ T cells and TNFα-dependent recruitment of IL-17A+ γδ T cells to the lung.