γδ T Cells Are Required for Pulmonary IL-17A Expression after Ozone Exposure in Mice: Role of TNFα

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

Primers used for real time PCR.

<p>Primers used for real time PCR.</p

Effect of anti-IL-17A on O₃-induced pulmonary inflammation and injury.

<p>WT mice were injected with anti-IL-17A or isotype 24 h prior to O₃ (0.3 ppm O₃ for 72 h). (A) BAL macrophages and neutrophils; (B) BAL protein; (C) BAL cytokines determined by multiplex assay. Results are mean±SEM of 5–7 mice per group. ^#p<0.05 versus isotype control.</p

Effect of O₃ exposure on IL-17A positive lung cells assessed by flow cytometry.

<p>(A) lung IL-17A⁺CD45⁺; (B) lung IL-17A⁺ γδ T cells; (C) total lung γδ T cells. Results are mean±SEM for 3–6 air-exposed and 4–11 O₃-exposed mice. *p<0.05 versus genotype-matched air-exposed mice. ^#p<0.05 versus WT mice with same exposure.</p

Role of TNFα for IL-17A expression in γδ T cells.

<p>Total number of (A) lung IL-17A⁺CD45⁺ cells; (B) lung IL-17A⁺ γδ T cells; and (C) total lung γδ T cells. Results are mean±SE of data from 5–6 mice in each group. ^#p<0.05 compared to lean mice with same TNFR2 genotype; & p<0.05 compared to TNFR2^+/+ Cpe genotype matched mice.</p

Impact of TNFR2 deficiency (A–C) or etanercept (D–F) on O₃-induced inflammation in obese (<i>Cpe^fat</i>) and lean (WT) mice.

<p>(A, D) BAL neutrophils; (B, E) <i>Il17a</i> mRNA expression; (C, F) BAL G-CSF. Results are mean±SE of data from 3–11 mice in each group.*p<0.05 versus air-exposed mice of same genotype and treatment; ^#p<0.05 versus exposure matched lean mice with same TNFR2 genotype or treatment; & p<0.05 versus TNFR2 sufficient (A–C) or vehicle treated mice (D–F) with same exposure and Cpe genotype.</p