The Microbiome Regulates Pulmonary Responses to Ozone in Mice.

Previous reports demonstrate that the microbiome impacts allergic airway responses, including airway hyperresponsiveness, a characteristic feature of asthma. Here we examined the role of the microbiome in pulmonary responses to a nonallergic asthma trigger, ozone. We depleted the microbiota of conventional mice with either a single antibiotic (ampicillin, metronidazole, neomycin, or vancomycin) or a cocktail of all four antibiotics given via the drinking water. Mice were then exposed to room air or ozone. In air-exposed mice, airway responsiveness did not differ between antibiotic- and control water-treated mice. Ozone caused airway hyperresponsiveness, the magnitude of which was decreased in antibiotic cocktail-treated mice versus water-treated mice. Except for neomycin, single antibiotics had effects similar to those observed with the cocktail. Compared with conventional mice, germ-free mice also had attenuated airway responsiveness after ozone. 16S ribosomal RNA gene sequencing of fecal DNA to characterize the gut microbiome indicated that bacterial genera that were decreased in mice with reduced ozone-induced airway hyperresponsiveness after antibiotic treatment were short-chain fatty acid producers. Serum analysis indicated reduced concentrations of the short-chain fatty acid propionate in cocktail-treated mice but not in neomycin-treated mice. Dietary enrichment with pectin, which increased serum short-chain fatty acids, also augmented ozone-induced airway hyperresponsiveness. Furthermore, propionate supplementation of the drinking water augmented ozone-induced airway hyperresponsiveness in conventional mice. Our data indicate that the microbiome contributes to ozone-induced airway hyperresponsiveness, likely via its ability to produce short-chain fatty acids.

Augmented Responses to Ozone in Obese Mice Require IL-17A and Gastrin-Releasing Peptide.

Ozone and obesity both increase IL-17A in the lungs. In mice, obesity augments the airway hyperresponsiveness and neutrophil recruitment induced by acute ozone exposure. Therefore, we examined the role of IL-17A in obesity-related increases in the response to ozone observed in obese mice. Lean wild-type and obese db/db mice were pretreated with IL-17A-blocking or isotype antibodies, exposed to air or ozone (2 ppm for 3 h), and evaluated 24 hours later. Microarray analysis of lung tissue gene expression was used to examine the mechanistic basis for effects of anti-IL-17A. Compared with lean mice, ozone-exposed obese mice had greater concentrations of BAL IL-17A and greater numbers of pulmonary IL-17A+ cells. Ozone-induced increases in BAL IL-23 and CCL20, cytokines important for IL-17A+ cell recruitment and activation, were also greater in obese mice. Anti-IL-17A treatment reduced ozone-induced airway hyperresponsiveness toward levels observed in lean mice. Anti-IL-17A treatment also reduced BAL neutrophils in both lean and obese mice, possibly because of reductions in CXCL1. Microarray analysis identified gastrin-releasing peptide (GRP) receptor (Grpr) among those genes that were both elevated in the lungs of obese mice after ozone exposure and reduced after anti-IL-17A treatment. Furthermore, ozone exposure increased BAL GRP to a greater extent in obese than in lean mice, and GRP-neutralizing antibody treatment reduced obesity-related increases in ozone-induced airway hyperresponsiveness and neutrophil recruitment. Our data indicate that IL-17A contributes to augmented responses to ozone in db/db mice. Furthermore, IL-17A appears to act at least in part by inducing expression of Grpr.

Repeated Mouse Lung Exposures to Stachybotrys chartarum Shift Immune Response from Type 1 to Type 2.

After a single or multiple intratracheal instillations of Stachybotrys chartarum (S. chartarum or black mold) spores in BALB/c mice, we characterized cytokine production, metabolites, and inflammatory patterns by analyzing mouse bronchoalveolar lavage (BAL), lung tissue, and plasma. We found marked differences in BAL cell counts, especially large increases in lymphocytes and eosinophils in multiple-dosed mice. Formation of eosinophil-rich granulomas and airway goblet cell metaplasia were prevalent in the lungs of multiple-dosed mice but not in single- or saline-dosed groups. We detected changes in the cytokine expression profiles in both the BAL and plasma. Multiple pulmonary exposures to S. chartarum induced significant metabolic changes in the lungs but not in the plasma. These changes suggest a shift from type 1 inflammation after an acute exposure to type 2 inflammation after multiple exposures to S. chartarum. Eotaxin, vascular endothelial growth factor (VEGF), MIP-1α, MIP-1ß, TNF-α, and the IL-8 analogs macrophage inflammatory protein-2 (MIP-2) and keratinocyte chemoattractant (KC), had more dramatic changes in multiple- than in single-dosed mice, and parallel the cytokines that characterize humans with histories of mold exposures versus unexposed control subjects. This repeated exposure model allows us to more realistically characterize responses to mold, such as cytokine, metabolic, and cellular changes.

ROCK insufficiency attenuates ozone-induced airway hyperresponsiveness in mice.

Ozone causes airway hyperresponsiveness (AHR) and pulmonary inflammation. Rho kinase (ROCK) is a key regulator of smooth muscle cell contraction and inflammatory cell migration. To determine the contribution of the two ROCK isoforms ROCK1 and ROCK2 to ozone-induced AHR, we exposed wild-type, ROCK1(+/-), and ROCK2(+/-) mice to air or ozone (2 ppm for 3 h) and evaluated mice 24 h later. ROCK1 or ROCK2 haploinsufficiency did not affect airway responsiveness in air-exposed mice but significantly reduced ozone-induced AHR, with a greater reduction in ROCK2(+/-) mice despite increased bronchoalveolar lavage (BAL) inflammatory cells in ROCK2(+/-) mice. Compared with wild-type mice, ozone-induced increases in BAL hyaluronan, a matrix protein implicated in ozone-induced AHR, were lower in ROCK1(+/-) but not ROCK2(+/-) mice. Ozone-induced increases in other inflammatory moieties reported to contribute to ozone-induced AHR (IL-17A, osteopontin, TNFα) were not different in wild-type vs. ROCK1(+/-) or ROCK2(+/-) mice. We also observed a dose-dependent reduction in ozone-induced AHR after treatment with the ROCK1/ROCK2 inhibitor fasudil, even though fasudil was administered after induction of inflammation. Ozone increased pulmonary expression of ROCK2 but not ROCK1 or RhoA. A ROCK2 inhibitor, SR3677, reduced contractile forces in primary human airway smooth muscle cells, confirming a role for ROCK2 in airway smooth muscle contraction. Our results demonstrate that ozone-induced AHR requires ROCK. Whereas ROCK1-dependent changes in hyaluronan may contribute to ROCK1's role in O3-induced AHR, the role of ROCK2 is downstream of inflammation, likely at the level of airway smooth muscle contraction.

Pivotal role of IL-6 in the hyperinflammatory responses to subacute ozone in adiponectin-deficient mice.

Adiponectin is an adipose-derived hormone with anti-inflammatory activity. Following subacute ozone exposure (0.3 ppm for 24-72 h), neutrophilic inflammation and IL-6 are augmented in adiponectin-deficient (Adipo(-/-)) mice. The IL-17/granulocyte colony-stimulating factor (G-CSF) axis is required for this increased neutrophilia. We hypothesized that elevated IL-6 in Adipo(-/-) mice contributes to their augmented responses to ozone via effects on IL-17A expression. Therefore, we generated mice deficient in both adiponectin and IL-6 (Adipo(-/-)/IL-6(-/-)) and exposed them to ozone or air. In ozone-exposed mice, bronchoalveolar lavage (BAL) neutrophils, IL-6, and G-CSF, and pulmonary Il17a mRNA expression were greater in Adipo(-/-) vs. wild-type mice, but reduced in Adipo(-/-)/IL-6(-/-) vs. Adipo(-/-) mice. IL-17A(+) F4/80(+) cells and IL-17A(+) γδ T cells were also reduced in Adipo(-/-)/IL-6(-/-) vs. Adipo(-/-) mice exposed to ozone. Only BAL neutrophils were reduced in IL-6(-/-) vs. wild-type mice. In wild-type mice, IL-6 was expressed in Gr-1(+)F4/80(-)CD11c(-) cells, whereas in Adipo(-/-) mice F4/80(+)CD11c(+) cells also expressed IL-6, suggesting that IL-6 is regulated by adiponectin in these alveolar macrophages. Transcriptomic analysis identified serum amyloid A3 (Saa3), which promotes IL-17A expression, as the gene most differentially augmented by ozone in Adipo(-/-) vs. wild-type mice. After ozone, Saa3 mRNA expression was markedly greater in Adipo(-/-) vs. wild-type mice but reduced in Adipo(-/-)/IL-6(-/-) vs. Adipo(-/-) mice. In conclusion, our data support a pivotal role of IL-6 in the hyperinflammatory condition observed in Adipo(-/-) mice after ozone exposure and suggest that this role of IL-6 involves its ability to induce Saa3, IL-17A, and G-CSF.

CD23 Sheddase A disintegrin and metalloproteinase 10 (ADAM10) is also required for CD23 sorting into B cell-derived exosomes.

The low affinity receptor for IgE, CD23, is the natural regulator of IgE synthesis, and understanding both the synthesis and the catabolism of CD23 are, thus, important issues. Membrane CD23 is cleaved by a disintegrin and metalloproteinase 10 (ADAM10) and this cleavage influences the ability of CD23 to regulate IgE. In contrast to the belief that cleavage is a cell surface event, endosomal neutralization with NH(4)Cl was found to dramatically reduce CD23 cleavage, suggesting that the majority of CD23 cleavage occurred subsequent to internalization in the endosomal pathway and not at the cell surface. In line with this, full-length CD23 was shown to be sorted in an ADAM10-dependent manner into exosomes. Greatly increased ADAM10-mediated CD23 cleavage was seen at endosomal pH. Additionally, the stalk region of CD23 was found to interact with ADAM10 and ADAM10 binding of CD23 was found to be protease independent. SPR analysis of the interaction indicated about a 10-fold increase in the R(max) at endosomal pH (pH 5.8) compared with pH 7.4, whereas the affinity of the interaction was not significantly changed. The R(max) change, combined with the increased cleavage at endosomal pH, indicates greater accessibility of the CD23 stalk region for ADAM10 at the lower pH. These results indicate a model where CD23 internalization results in ADAM10-dependent incorporation into exosomes, followed by partial cleavage of CD23 by ADAM10 prior to being released from the cell. The increased cleavage at endosomal pH also has implications for other ADAM10 substrates.

Considerations for Soluble Protein Biomarker Blood Sample Matrix Selection.

Blood-based soluble protein biomarkers provide invaluable clinical information about patients and are used as diagnostic, prognostic, and pharmacodynamic markers. The most commonly used blood sample matrices are serum and different types of plasma. In drug development research, the impact of sample matrix selection on successful protein biomarker quantification is sometimes overlooked. The sample matrix for a specific analyte is often chosen based on prior experience or literature searches, without good understanding of the possible effects on analyte quantification. Using a data set of 32 different soluble protein markers measured in matched serum and plasma samples, we examined the differences between serum and plasma and discussed how platelet or immune cell activation can change the quantified concentration of the analyte. We have also reviewed the effect of anticoagulant on analyte quantification. Finally, we provide specific recommendations for biomarker sample matrix selection and propose a systematic and data-driven approach for sample matrix selection. This review is intended to raise awareness of the impact and considerations of sample matrix selection on biomarker quantification.

IL-33 Drives Augmented Responses to Ozone in Obese Mice.

BACKGROUND: Ozone increases IL-33 in the lungs, and obesity augments the pulmonary effects of acute ozone exposure. OBJECTIVES: We assessed the role of IL-33 in the augmented effects of ozone observed in obese mice. METHODS: Lean wildtype and obese db/db mice were pretreated with antibodies blocking the IL-33 receptor, ST2, and then exposed to ozone (2 ppm for 3 hr). Airway responsiveness was assessed, bronchoalveolar lavage (BAL) was performed, and lung cells harvested for flow cytometry 24 hr later. Effects of ozone were also assessed in obese and lean mice deficient in γδ T cells and their wildtype controls. RESULTS AND DISCUSSION: Ozone caused greater increases in BAL IL-33, neutrophils, and airway responsiveness in obese than lean mice. Anti-ST2 reduced ozone-induced airway hyperresponsiveness and inflammation in obese mice but had no effect in lean mice. Obesity also augmented ozone-induced increases in BAL CXCL1 and IL-6, and in BAL type 2 cytokines, whereas anti-ST2 treatment reduced these cytokines. In obese mice, ozone increased lung IL-13+ innate lymphoid cells type 2 (ILC2) and IL-13+ γδ T cells. Ozone increased ST2+ γδ T cells, indicating that these cells can be targets of IL-33, and γδ T cell deficiency reduced obesity-related increases in the response to ozone, including increases in type 2 cytokines. CONCLUSIONS: Our data indicate that IL-33 contributes to augmented responses to ozone in obese mice. Obesity and ozone also interacted to promote type 2 cytokine production in γδ T cells and ILC2 in the lungs, which may contribute to the observed effects of IL-33. Citation: Mathews JA, Krishnamoorthy N, Kasahara DI, Cho Y, Wurmbrand AP, Ribeiro L, Smith D, Umetsu D, Levy BD, Shore SA. 2017. IL-33 drives augmented responses to ozone in obese mice. Environ Health Perspect 125:246-253; http://dx.doi.org/10.1289/EHP272.

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

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.

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.

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-1ß, 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.