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. 2015 Jul 2;10(7):e0131236.
doi: 10.1371/journal.pone.0131236. eCollection 2015.

γδ T Cells Are Required for M2 Macrophage Polarization and Resolution of Ozone-Induced Pulmonary Inflammation in Mice

Joel A Mathews  1 , David I Kasahara  1 , Luiza Ribeiro  1 , Allison P Wurmbrand  1 , Fernanda M C Ninin  1 , Stephanie A Shore  1
Affiliations

γδ T Cells Are Required for M2 Macrophage Polarization and Resolution of Ozone-Induced Pulmonary Inflammation in Mice

Joel A Mathews et al. PLoS One. .

Abstract

We examined the role of γδ T cells in the induction of alternatively activated M2 macrophages and the resolution of inflammation after ozone exposure. Wildtype (WT) mice and mice deficient in γδ T cells (TCRδ-/- mice) were exposed to air or to ozone (0.3 ppm for up to 72h) and euthanized immediately or 1, 3, or 5 days after cessation of exposure. In WT mice, M2 macrophages accumulated in the lungs over the course of ozone exposure. Pulmonary mRNA abundance of the M2 genes, Arg1, Retnla, and Clec10a, also increased after ozone. In contrast, no evidence of M2 polarization was observed in TCRδ-/- mice. WT but not TCRδ-/- mice expressed the M2c polarizing cytokine, IL-17A, after ozone exposure and WT mice treated with an IL-17A neutralizing antibody exhibited attenuated ozone-induced M2 gene expression. In WT mice, ozone-induced increases in bronchoalveolar lavage neutrophils and macrophages resolved quickly after cessation of ozone exposure returning to air exposed levels within 3 days. However, lack of M2 macrophages in TCRδ-/- mice was associated with delayed clearance of inflammatory cells after cessation of ozone and increased accumulation of apoptotic macrophages in the lungs. Delayed restoration of normal lung architecture was also observed in TCRδ-/- mice. In summary, our data indicate that γδ T cells are required for the resolution of ozone-induced inflammation, likely because γδ T cells, through their secretion of IL-17A, contribute to changes in macrophage polarization that promote clearance of apoptotic cells.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Induction of M2 macrophage by subacute O3 exposure is reduced in TCRδ-/- mice.
WT mice were exposed to either air or O3 (0.3 ppm) for 24, 48 or 72 hours and euthanized immediately after exposure. (A) Total lung macrophages (F4/80+ cells) and (B) total lung M2 macrophages (F4/80+CD80-CD206+ cells) were measured by flow cytometry. The pulmonary mRNA abundance of M2 markers (C) Arg1 (D) Clec10a and (E) Retnla were also assessed by RT-qPCR in WT and TCRδ-/- mice exposed to room air or O3. Total macrophages (F), M1 macrophages (G) and M2 macrophages (H) were also assessed in WT and TCRδ-/- mice exposed to air or O3 (0.3 ppm for 72 h). (I) BAL TNFα was measured in the BAL by ELISA. Results are mean ± SE of 4–8 air exposed mice and 6–14 O3 exposed mice in each group. * p<0.05 versus air; # p<0.05 versus WT mice.
Fig 2
Fig 2. Pulmonary M2 gene expression after cessation of O3 exposure.
Pulmonary (A) Clec10a, (B) Arg1, (C) Retnla, (D) Il13, and (E) Il17a mRNA abundance in WT and TCRδ-/- mice exposed to room air or to ozone (O3, 0.3 ppm for 72 h) and then euthanized either immediately or 1 or 3 days after cessation of O3 exposure. (F) IL-17A+γδ were determined by flow cytometry. Note that data from the air and immediately post mice have been previously published [28] Results are mean ± SE of 4–8 air exposed mice and 6–14 O3 exposed mice in each group. * p<0.05 versus air; # p<0.05 versus 72 hour O3; $ p<0.05 versus WT mice.
Fig 3
Fig 3. Blocking IL-17A reduces pulmonary expression of Arg1 and Clec10a.
Pulmonary mRNA abundance of (A) Clec10a and (B) Arg1 measured as changes in Ct values in lungs from mice treated with IL-17A neutralizing versus isotype control antibody injected i.p. prior to O3 exposure. Note that an increase in Ct indicates a decrease in expression. Mice were exposed to O3 for either 48 or 72 h and euthanized immediately after cessation of exposure. Other data from these mice has been previously published [27,28]. (C) As a marker of M1 activation, TNFα was measured in the BAL by ELISA. Results are mean ± SE 5–7 mice in each group. % p<0.05 versus isotype control, as assessed by factorial ANOVA.
Fig 4
Fig 4. O3-induced inflammation in WT and γδ T cell deficient mice after cessation of O3 exposure.
Bronchoalveolar lavage (BAL) neutrophils (A), macrophages (B), G-CSF (C), MCP-1 in wildtype (WT) and γδ T cell deficient (TCRδ-/-) mice exposed to room air or to ozone (O3, 0.3 ppm for 72 h) and then euthanized either immediately or 1, 3, or 5 days after cessation of O3 exposure. Data for the air and immediately post O3 time points have been previously published [28]. Results are mean ± SE of 4–8 air exposed mice and 6–14 O3 exposed mice in each group. * p<0.05 versus air; # p<0.05 versus immediately post O3; $ p<0.05 versus WT mice.
Fig 5
Fig 5. Lung apoptotic macrophages are elevated after O3 exposure.
WT mice were exposed to either air or O3 (0.3 ppm for 72 h) and lungs were harvested either immediately or 1 or 3 days after cessation of O3 exposure. (A) Total macrophages, (B) Alveolar Macrophages, and (C) Interstitial macrophages assessed by flow cytometry. (D) Representative gating for apoptotic macrophages in a WT mouse studied 1 day after cessation of O3 exposure. (E) Early apoptotic interstitial macrophages and (F) late apoptotic interstitial macrophages in WT mice at various times after cessation of O3 exposure. Results are mean ± SEM for 4–6 mice per group. * p<0.05 versus air; # p<0.05 versus immediate post.
Fig 6
Fig 6. Macrophages accumulate in the lungs of TCRδ-/- mice.
(A) Total interstitial macrophages, (B) alive macrophages, (C) early apoptotic interstitial macrophages, and (D) late apoptotic interstitial macrophages in lungs of WT and TCRδ-/- mice exposed to O3 for 72 h, and then transferred to room air and studied 3 days later. Results are mean ± SEM for 4–6 mice per group. $ p<0.05 versus WT mice.
Fig 7
Fig 7. O3 induced injury.
(A) pulmonary Cldn4 mRNA abundance, (B) BAL protein, and (C) terminal bronchiolar lesions, scored as explained in the methods. Results are mean ± SE of 4–8 air exposed mice and 6–14 O3 exposed mice in each group. * p<0.05 versus air; # p<0.05 versus immediate post O3; $ p<0.05 versus WT mice.
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