Induction of IL-17A Precedes Development of Airway Hyperresponsiveness during Diet-Induced Obesity and Correlates with Complement Factor D

Obesity is a risk factor for the development of asthma. Obese mice exhibit innate airway hyperresponsiveness (AHR), a characteristic feature of asthma, and IL-17A is required for development of AHR in obese mice. The purpose of this study was to examine the temporal association between the onset of AHR and changes in IL-17A during the development of obesity by high-fat feeding in mice. At weaning, C57BL/6J mice were placed either on mouse chow or on a high-fat diet (HFD) and examined 9, 12, 15, 18, or 24 weeks later. Airway responsiveness to aerosolized methacholine (assessed via the forced oscillation technique) was greater in mice fed HFD versus chow for 24 weeks but not at earlier time points. Bronchoalveolar lavage and serum IL-17A were not affected by either the type or duration of diet, but increased pulmonary IL17a mRNA abundance was observed in HFD versus chow fed mice after both 18 and 24 weeks. Flow cytometry also confirmed an increase in IL-17A+ γδ T cells and IL-17A+ CD4+ T (Th17) cells in lungs of HFD versus chow fed mice. Pulmonary expression of Cfd (complement factor D, adipsin), a gene whose expression can be reduced by IL-17A, decreased after both 18 and 24 weeks in HFD versus chow fed mice. Furthermore, pulmonary Cfd mRNA abundance correlated with elevations in pulmonary Il17a mRNA expression and with AHR. Serum levels of TNFα, MIP-1α, and MIP-1β, and classical markers of systemic inflammation of obesity were significantly greater in HFD than chow fed mice after 24 weeks, but not earlier. In conclusion, our data indicate that pulmonary rather than systemic IL-17A is important for obesity-related AHR and suggest that changes in pulmonary Cfd expression contribute to these effects of IL-17A. Further, the observation that increases in Il17a preceded the development of AHR by several weeks suggests that IL-17A interacts with other factors to promote AHR. The observation that the onset of the systemic inflammation of obesity coincided temporally with the development of AHR suggest that systemic inflammation may be one of these factors

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

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

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

Lung apoptotic macrophages are elevated after O₃ exposure.

<p>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.</p

Impact of Adiponectin Overexpression on Allergic Airways Responses in Mice

Obesity is an important risk factor for asthma. Obese individuals have decreased circulating adiponectin, an adipose-derived hormone with anti-inflammatory properties. We hypothesized that transgenic overexpression of adiponectin would attenuate allergic airways inflammation and mucous hyperplasia in mice. To test this hypothesis, we used mice overexpressing adiponectin (Adipo Tg). Adipo Tg mice had marked increases in both serum adiponectin and bronchoalveolar lavage (BAL) fluid adiponectin. Both acute and chronic ovalbumin (OVA) sensitization and challenge protocols were used. In both protocols, OVA-induced increases in total BAL cells were attenuated in Adipo Tg versus WT mice. In the acute protocol, OVA-induced increases in several IL-13 dependent genes were attenuated in Adipo Tg versus WT mice, even though IL-13 per se was not affected. With chronic exposure, though OVA-induced increases in goblet cells numbers per millimeter of basement membrane were greater in Adipo Tg versus WT mice, mRNA abundance of mucous genes in lungs was not different. Also, adiponectin overexpression did not induce M2 polarization in alveolar macrophages. Our results indicate that adiponectin protects against allergen-induced inflammatory cell recruitment to the airspaces, but not development of goblet cell hyperplasia

Pulmonary M2 gene expression after cessation of O₃ exposure.

<p>Pulmonary (A) <i>Clec10a</i>, (B) <i>Arg1</i>, (C) <i>Retnla</i>, (D) <i>Il13</i>, <i>and (E) Il17a</i> 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131236#pone.0131236.ref028" target="_blank">28</a>] 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.</p

Macrophages accumulate in the lungs of TCRδ^-/- mice.

<p>(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.</p

O₃ induced injury.

<p>(A) pulmonary <i>Cldn4</i> 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.</p

Blocking IL-17A reduces pulmonary expression of <i>Arg1 and Clec10a</i>.

<p>Pulmonary mRNA abundance of (A) <i>Clec10a</i> and (B) <i>Arg1</i> 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131236#pone.0131236.ref027" target="_blank">27</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131236#pone.0131236.ref028" target="_blank">28</a>]. (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.</p