Regulation of IL-17A expression in mice following subacute ozone exposure

Abstract

Exposure to subacute ozone (O3) causes pulmonary neutrophil recruitment. In mice, this recruitment requires IL-17A. Ozone also causes expression of IL-23 and IL-1, which can induce IL-17A. The purpose of this study was to examine the hypothesis that IL-23 and IL-1 contribute to IL-17A expression and subsequent neutrophil recruitment after subacute O3 exposure. Wild-type, IL-23(-/-), and Flt3l(-/-) mice were exposed to air or 0.3 ppm O3 for 72 h. Flt3l(-/-) mice lack conventional dendritic cells (cDC) that can express IL-23 and IL-1. Other wild-type mice were pre-treated with saline or the IL-1R1 antagonist anakinra prior to O3 exposure. After exposure, bronchoalveolar lavage (BAL) was performed and lung tissue harvested. The results indicated that pulmonary Il17a mRNA abundance and IL-17A(+) F4/80(+) cells were significantly reduced in O3-exposed IL-23(-/-) vs in wild-type mice. In contrast, anakinra had no effect on Il23a or Il17a pulmonary mRNA abundance or on BAL concentrations of the neutrophil survival factor G-CSF, but anakinra did reduce BAL neutrophil numbers, likely because anakinra also reduced BAL IL-6. Compared to air, O3 caused a significant increase in DC numbers in wild-type, but not in Flt3(-/-) mice. However, there was no significant difference in Il23a or Il17a mRNA abundance or in BAL neutrophil count in O3-exposed Flt3(-/-) vs in wild-type mice. From these results, it was concluded that IL-23 but not IL-1 contributes to the IL-17A expression induced by subacute O3 exposure. Induction of IL-23 by O3 does not appear to require cDC.

Keywords: IL-1; IL-23; IL-6; dendritic cells; neutrophils.

Figure 1

(A) Pulmonary Il23a and (B) Il17a mRNA abundance, determined by qRT-PCR, in WT mice exposed to room air or ozone (0.3 ppm) for 72 hr. Results shown are means [± SE] from 5–6 mice/group and are expressed relative to the air exposed mice. *p < 0.05 vs. air.

Figure 2

(A) Pulmonary Il17a mRNA abundance (expressed relative to in WT mice). (B) Pulmonary IL-17A⁺ γδ T-cells. (C) Pulmonary IL-17A⁺ interstitial macrophages measured by flow cytometry in WT and IL-23^−/− mice exposed to ozone (0.3 ppm for 72 hr). Results shown are means ± SEM of 8–9 mice/group for qRT-PCR and 4 mice/group for flow cytometry. *p < 0.05 vs. WT mice.

Figure 3

Bronchoalveolar lavage (BAL) (A) neutrophils and (B) macrophages in WT and IL-23^−/− mice exposed to ozone (0.3 ppm for 72 hr). Results shown are means ± SE of 8–9 mice/group. (C) Log %BAL neutrophils plotted vs. ΔΔCt values for Il17a. All mice were ozone-exposed. The regression line shown was calculated from the combined data from the WT and IL-23^−/− mice. Note: increase in ΔΔCt indicates reduced Il17a expression.

Figure 4

Pulmonary (A) Il17a and (B) Il23a mRNA abundance (expressed relative to WT mice), and BAL (C) neutrophils, (D) macrophages, and (E) total protein in WT mice exposed to ozone (0.3 ppm for 72 hr) that had been treated with anakinra or saline. Results shown are means ± SE of 6–12 mice/group. *p < 0.05 vs. saline-treated mice.

Figure 5

BAL (A) LIF, (B) IL-6, and (C) GM-CSF assayed by multiplex from WT (C57BL/6J) mice exposed to ozone (0.3 ppm for 72 hr) that had been treated with anakinra or saline. Results shown are means ± SE of 8 mice/group. *p < 0.05 vs. saline-treated mice.

Figure 6

(A) Gating strategy used to assess lung DC. Cells were first gated based on forward scatter (FSC) and side-scatter (SSC) characteristics. Cells positive for MCH-II and CD11c were determined to be either dendritic cells (DC, SiglecF⁻) or macrophages (MØ, SiglecF⁺). (B) Total number of MHC-II⁺/CD11c⁺/SiglecF⁻ DC in WT mice exposed to air or ozone (0.3 ppm) for 24, 48 or 72 hr. (C) Total number of MHC-II⁺/CD11c⁺/SiglecF⁻ DC in WT and Flt3l^−/− mice exposed to air or ozone (0.3 ppm) for 72 hr. Results shown are means ± SE of 7–8 mice/group. *p < 0.05 vs. air-exposed mice with same genotype; ^#p < 0.05 vs. WT mice with same exposure.

Figure 7

Pulmonary (A) Il23a and (B) Il17a mRNA abundance (expressed relative to WT air mice), and (C) BAL neutrophil numbers in WT and Flt3l^−/− mice exposed to room air or ozone (0.3 ppm) for 72 hr. Results shown are means ± SE of 7–8 mice/group. *p < 0.05 vs. air-exposed mice with same genotype; ^#p < 0.05 vs. WT mice with same exposure.