Examine the contribution of CD4+ cells to the elevations in BAL type 2 cytokines observed

Examine the contribution of CD4+ cells to the elevations in BAL type 2 cytokines observed in obese O 3-exposed mice, we depleted these cells with an anti-CD4 antibody. Flow cytometry indicated an approximate 75 reduction in lung CD4+ cells, confirming the efficacy of the depletion strategy (see Figure S2A). However, BAL type 2 cytokines were not significantly reduced in O3-exposed db/db mice treated with anti-CD4 versus isotype antibody (see Figure S2B ), suggesting that Th2 cells were not the source of the elevated BAL type 2 cytokines observed in these mice (Figure 2), nor were other ST2 dependent cytokines (CXCL1, IL-6, CCL4) affected (see Figure S2E ). In addition, depletion of CD4 cells had no effect on ozone-induced AHR or BAL neutrophils (see Figure S2G,H). Instead, ILC2 and/or T cells appear to account for IL-33 dependent changes in BAL type 2 cytokines observed in obese O3-exposed mice. Flow cytometry indicated no change with obesity or O3 in the total number of pulmonary ILC2s (Figure 4A). However, there was an increase in cytokine production by ILC2: O 3 increased the number of IL-5 + and IL-13 + ILC2s in obese db/db but not lean wildtype mice (Figure 4B,C; see also Figures S3 and S4), consistent with the observed changes inBAL IL-5 and IL-13 (Figure 2A,B). O 3 also increased the number of pulmonary IL-13+ T cells in db/db but not WT mice (Figure 4E). Total pulmonary T cells were not affected by obesity but were increased by O3 in both db/db and WT mice (Figure 4D). O3 also increased IL-13+ T cells in mice with dietary obesity but not in lean controls (Figure 4F). IL-5 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21187425 + T cells were not assessed. Importantly, after O 3 exposure, BAL IL-5 and IL-13 were lower in obese TCR??mice, which lack T cells, than in obese WT mice (Figure 5A,B), indicating that T cells likely contributed to increases in these cytokines observed in obese O3 exposed mice (Figure 2). Because type 2 cytokines were also dependent on ST2 (Figure 3), we determined whether T cells can respond to IL-33. Importantly, some pulmonary T cells expressed the ST2 receptor (see Figure S5) and the number of ST2+ T cells was greater in O3 than in air exposed mice (Figure 4G). To confirm that these ST2+ T cells could produce IL-13, we co-stained lung cells with antibodies to both ST2 and IL-13. In these experiments, only O3-exposed db/db mice were used, since we observed little or no IL-13 in air exposed mice or in WT mice exposed to O3 (Figure 2B). Our data indicate that approximately 5 ?1.5 (n = 3) of the T cells in O3-exposed db/db mice were IL-13+. Importantly, virtually all (> 85 ) of these IL-13+ T were also ST2+ (see Figure S5). Whereas T cell deficiency reduced BAL type 2 cytokines, it did not affect BAL CXCL1 or IL-6 in obese O3 exposedFigure 3. Anti-ST2 reduces BAL cytokines and chemokines in obese O3-exposed mice. Db/db mice were treated BIBN4096BS hydrochloride site intraperitoneal with ST2-blocking or isotype antibodies 24 hr prior to O3 exposure (2 ppm for 3 hr). BAL concentrations of (A) IL-5, (B) IL-13, (C) IL-6, (D) CXCL1, (E) CCL4, and (F) IL-9 were measured by ELISA or by multiplex using BAL that had been concentrated five times. Note: Results are the mean ?SE of 6?0 mice/group studied over 8 experimental days.*p < 0.05 versus isotype treated mice.Environmental Health Perspectives ?volume125 | number 2 | FebruaryMathews et al.mice (data not shown), indicating that other IL-33 target cells, perhaps epithelial cells or macrophages, are the source of these ST2-dependent.