E role of CD4 cells in O 3-induced changes in type 2 cytokines, we depleted CD4 cells. Db/db mice were injected once with anti-CD4 (clone: GK1.5, Biolegend) (8 mg/kg) or isotype antibody (Rice and Bucy 1995). Mice were exposed to O3 6 days later and evaluated as described above. Confirmation of CD4 depletion in lung tissue was assessed by flow cytometry. To examine the role of T cells in obese mice, WT and TCR??mice were fed either an HFD or GPR120-IN-1 site normal chow, and then exposed to air or to O3, and evaluated as described above. Methods for BAL, measurement of cytokines and chemokines, RNA extraction and RT-qPCR, and flow cytometry were as previously described (Krishnamoorthy et al. 2015; Williams et al. 2013) and are found in an online supplement (see “Supplemental Methods” in the Supplemental Material).Environmental Health Perspectives ?volumeFigure 1. Role of IL-33 in pulmonary responses to O3 exposure in obese mice. (A) Bronchoalveolar lavage (BAL) IL-33 and (B) changes in pulmonary resistance (RL) induced by inhaled aerosolized methacholine in lean wildtype (WT) and obese db/db female mice exposed to air or ozone (O3) (2 ppm for 3 hr) and studied 24 hr after exposure. (C) Airway responsiveness to methacholine assessed using G, the coefficient of lung tissue damping, and (D) BAL neutrophils in a different cohort of WT and db/db treated with isotype or anti-ST2 antibody prior to O3 exposure. For panels A and B, results are mean ?SE of 4? mice/group studied over 16 experimental days. For panels C and D, results are mean ?SE of 4? mice/group studied over 8 experimental days.*p < 0.05 versus PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21187425 air. #p < 0.05 versus lean mice with same exposure. p < 0.05 versus isotype-treated mice of same genotype.125 | number 2 | FebruaryMathews et al.mice (PBS values in Figure 1B) consistent with the smaller lungs of the db/db mice (Lu et al. 2006). O3 increased baseline RL in db/db but not WT mice (Figure 1B). O3 also increased methacholine-induced changes in RL to a greater extent in db/db than WT mice (Figure 1B). Essentially similar results were observed for the coefficients of G and for elastance H, measures of the lung periphery and for Rn, a measure of the central airways (see Figure S1A ). However, the effect was greatest for G, suggesting that the effects of O3 are largely mediated in the lung periphery. Consequently, in subsequent analyses of airway responsiveness, methacholine-induced changes in G are presented. Effects of anti-ST2 treatment were assessed in a separate cohort of WT and db/db mice exposed to O3. Compared to isotype antibody, anti-ST2 treatment had no effect on airway responsiveness in O3-exposed WT mice (Figure 1C). However, in O3-exposeddb/db mice, anti-ST2 treatment significantly reduced baseline G, and significantly reduced airway responsiveness (Figure 1C). Similar results were obtained for RL (see Figure S1D). BAL neutrophils were greater in O3-exposed db/db versus WT mice (Figure 1D) treated with isotype antibody. Anti-ST2 significantly reduced BAL neutrophils in db/db but not in WT mice. Taken together, the results indicate a role for IL-33 in responses to O3 in obese mice. In contrast, we observed no significant effect of anti-ST2 versus isotype antibody treatment on airway responsiveness in air-exposed db/db mice (see Figure S1E).IL-33 Dependent BAL Cytokines and ChemokinesOthers have reported that exogenously administered IL-33 causes AHR and increases BAL neutrophils by inducing both type 2 cytokines like IL-13 and IL-5, c.
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