Sex Differences in the Impact of Dietary Fiber on Pulmonary Responses to Ozone.

Ozone causes airway hyperresponsiveness, a defining feature of asthma. We have reported that the gut microbiome contributes to sex differences in ozone-induced airway hyperresponsiveness. Altering dietary fiber affects the gut microbiome. The purpose of this study was to determine the effects of dietary fiber on pulmonary responses to ozone and whether these effects differ by sex. We fed male and female mice fiber-free diets or diets enriched in one of two types of dietary fiber, cellulose and pectin, for 3 days before ozone exposure. Compared with control diets or pectin-enriched diets, cellulose-enriched diets attenuated ozone-induced airway hyperresponsiveness in male but not female mice. In contrast, fiber-free diets augmented responses to ozone in female but not male mice. Analysis of 16S rRNA sequencing of fecal DNA also indicated sex differences in the impact of dietary fiber on the gut microbiome and identified bacterial taxa that were associated with ozone-induced airway hyperresponsiveness. Our data suggest that microbiome-based therapies such as prebiotics may provide an alternative therapeutic strategy for air pollution-triggered asthma, but they indicate that such therapeutics may need to be tailored differently for males and females.

IL-33, diet-induced obesity, and pulmonary responses to ozone.

BACKGROUND: Obesity augments pulmonary responses to ozone. We have reported that IL-33 contributes to these effects of obesity in db/db mice. The purpose of this study was to determine whether IL-33 also contributes to obesity-related changes in the response to ozone in mice with diet-induced obesity. METHODS: Male wildtype C57BL/6 mice and mice deficient in ST2, the IL-33 receptor, were placed on chow or high fat diets for 12 weeks from weaning. Because the microbiome has been implicated in obesity-related changes in the pulmonary response to ozone, mice were either housed with other mice of the same genotype (same housed) or with mice of the opposite genotype (cohoused). Cohousing transfers the gut microbiome from one mouse to its cagemates. RESULTS: Diet-induced increases in body mass were not affected by ST2 deficiency or cohousing. In same housed mice, ST2 deficiency reduced ozone-induced airway hyperresponsiveness and neutrophil recruitment in chow-fed but not HFD-fed mice even though ST2 deficiency reduced bronchoalveolar lavage IL-5 in both diet groups. In chow-fed mice, cohousing abolished ST2-related reductions in ozone-induced airway hyperresponsiveness and neutrophil recruitment, but in HFD-fed mice, no effect of cohousing on these responses to ozone was observed. In chow-fed mice, ST2 deficiency and cohousing caused changes in the gut microbiome. High fat diet-feeding caused marked changes in the gut microbiome and overrode both ST2-related and cohousing-related differences in the gut microbiome observed in chow-fed mice. CONCLUSION: Our data indicate a role for IL-33 in pulmonary responses to ozone in chow-fed but not high fat diet-fed mice and are consistent with the hypothesis that these diet-related differences in the role of IL-33 are the result of changes in the gut microbiome.

Sex Differences in Pulmonary Responses to Ozone in Mice. Role of the Microbiome.

We have previously reported that the mouse gut microbiome contributes to pulmonary responses to ozone, a common asthma trigger, and that short-chain fatty acids, end products of bacterial fermentation, likely contribute to this role of the microbiome. A growing body of evidence indicates that there are sex-related differences in gut microbiota and these differences can have important functional consequences. The purpose of this study was to determine whether there are sex-related differences in the impact of the gut microbiota on pulmonary responses to ozone. After acute exposure to ozone, male mice developed greater airway hyperresponsiveness than female mice. This difference was abolished after antibiotic ablation of the gut microbiome. Moreover, weanling female pups housed in cages conditioned by adult male mice developed greater ozone-induced airway hyperresponsiveness than weanling female pups raised in cages conditioned by adult females. Finally, ad libitum oral administration via drinking water of the short-chain fatty acid propionate resulted in augmented ozone-induced airway hyperresponsiveness in male, but not female, mice. Overall, these data are consistent with the hypothesis that the microbiome contributes to sex differences in ozone-induced airway hyperresponsiveness, likely as a result of sex differences in the response to short-chain fatty acids.

Microbiota Contribute to Obesity-related Increases in the Pulmonary Response to Ozone.

Obesity is a risk factor for asthma, especially nonatopic asthma, and attenuates the efficacy of standard asthma therapeutics. Obesity also augments pulmonary responses to ozone, a nonatopic asthma trigger. The purpose of this study was to determine whether obesity-related alterations in gut microbiota contribute to these augmented responses to ozone. Ozone-induced increases in airway responsiveness, a canonical feature of asthma, were greater in obese db/db mice than in lean wild-type control mice. Depletion of gut microbiota with a cocktail of antibiotics attenuated obesity-related increases in the response to ozone, indicating a role for microbiota. Moreover, ozone-induced airway hyperresponsiveness was greater in germ-free mice that had been reconstituted with colonic contents of db/db than in wild-type mice. In addition, compared with dietary supplementation with the nonfermentable fiber cellulose, dietary supplementation with the fermentable fiber pectin attenuated obesity-related increases in the pulmonary response to ozone, likely by reducing ozone-induced release of IL-17A. Our data indicate a role for microbiota in obesity-related increases in the response to an asthma trigger and suggest that microbiome-based therapies such as prebiotics may provide an alternative therapeutic strategy for obese patients with asthma.

The interleukin-33 receptor contributes to pulmonary responses to ozone in male mice: role of the microbiome.

BACKGROUND: Interleukin-33 is released in the airways following acute ozone exposure and has the ability to cause airway hyperresponsiveness, a defining feature of asthma. Ozone causes greater airway hyperresponsiveness in male than female mice. Moreover, sex differences in the gut microbiome account for sex differences in this response to ozone. The purpose of this study was to determine whether there were sex differences in the role of interleukin-33 in ozone-induced airway hyperresponsiveness and to examine the role of the microbiome in these events. METHODS: Wildtype mice and mice genetically deficient in ST2, the interleukin-33 receptor, were housed from weaning with either other mice of the same genotype and sex, or with mice of the same sex but opposite genotype. At 15 weeks of age, fecal pellets were harvested for 16S rRNA sequencing and the mice were then exposed to air or ozone. Airway responsiveness was measured and a bronchoalveolar lavage was performed 24 h after exposure. RESULTS: In same-housed mice, ozone-induced airway hyperresponsiveness was greater in male than female wildtype mice. ST2 deficiency reduced ozone-induced airway hyperresponsiveness in male but not female mice and abolished sex differences in the response to ozone. However, sex differences in the role of interleukin-33 were unrelated to type 2 cytokine release: ozone-induced increases in bronchoalveolar lavage interleukin-5 were greater in females than males and ST2 deficiency virtually abolished interleukin-5 in both sexes. Since gut microbiota contribute to sex differences in ozone-induced airway hyperresponsiveness, we examined the role of the microbiome in these ST2-dependent sex differences. To do so, we cohoused wildtype and ST2 deficient mice, a situation that allows for transfer of microbiota among cage-mates. Cohousing altered the gut microbial community structure, as indicated by 16S rRNA gene sequencing of fecal DNA and reversed the effect of ST2 deficiency on pulmonary responses to ozone in male mice. CONCLUSIONS: The data indicate that the interleukin-33 /ST2 pathway contributes to ozone-induced airway hyperresponsiveness in male mice and suggest that the role of interleukin-33 is mediated at the level of the gut microbiome.

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.

IL-13 Augments Compressive Stress-Induced Tissue Factor Expression in Human Airway Epithelial Cells.

Tissue factor (TF) is best known as a cellular initiator of coagulation, but it is also a multifunctional protein that has been implicated in multiple pathophysiologic conditions, including asthma. In the lung, airway epithelial cells express TF, but it is unknown how TF expression is regulated by asthma-associated mediators. We investigated the role of IL-13, a type 2 cytokine, alone and in combination with compressive stress, which mimics asthmatic bronchoconstriction, on TF expression and release of TF-positive extracellular vesicles from primary normal human bronchial epithelial cells. Well-differentiated normal human bronchial epithelial cells were treated with IL-13 and compressive stress, alone and in combination. TF mRNA, protein and activity were measured in the cells and conditioned media. TF was also measured in the bronchoalveolar lavage (BAL) fluid of allergen-challenged mice and patients with asthma. IL-13 and compressive stress increased TF expression, but only compressive stress induced TF-positive extracellular vesicle release. Pretreatment with IL-13 augmented compressive stress-induced TF expression and release. TF protein and activity in BAL fluid were increased in allergen-sensitized and -challenged mice. TF was elevated in the BAL fluid of patients with mild asthma after an allergen challenge. Our in vitro and in vivo data indicate close cooperation between mechanical and inflammatory stimuli on TF expression and release of TF-positive extracellular vesicles in the lungs, which may contribute to pathophysiology of asthma.

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.

Abrogation of airway hyperresponsiveness but not inflammation by rho kinase insufficiency.

BACKGROUND: Major features of allergic asthma include airway hyperresponsiveness (AHR), eosinophilic inflammation, and goblet cell metaplasia. Rho kinase (ROCK) is a serine/threonine protein kinase that regulates the actin cytoskeleton. By doing so, it can modulate airway smooth muscle cell contraction and leucocyte migration and proliferation. This study was designed to determine the contributions of the two ROCK isoforms, ROCK1 and ROCK2, to AHR, inflammation and goblet cell metaplasia in a mast cell-dependent model of allergic airways disease. METHODS AND RESULTS: Repeated intranasal challenges with OVA caused AHR, eosinophilic inflammation, and goblet cell hyperplasia in wild-type (WT) mice. OVA-induced AHR was partially or completely abrogated in mice haploinsufficient for ROCK2 (ROCK2(+/-) ) or ROCK1 (ROCK1(+/-) ), respectively. In contrast, there was no effect of ROCK insufficiency on allergic airways inflammation, although both ROCK1 and ROCK2 insufficiency attenuated mast cell degranulation. Goblet cell hyperplasia, as indicated by PAS staining, was not different in ROCK1(+/-) vs. WT mice. However, in ROCK2(+/-) mice, goblet cell hyperplasia was reduced in medium but not large airways. Maximal acetylcholine-induced force generation was reduced in tracheal rings from ROCK1(+/-) and ROCK2(+/-) vs. WT mice. The ROCK inhibitor, fasudil, also reduced airway responsiveness in OVA-challenged mice, without affecting inflammatory responses. CONCLUSION: In a mast cell model of allergic airways disease, ROCK1 and ROCK2 both contribute to AHR, likely through direct effects on smooth muscle cell and effects on mast cell degranulation. In addition, ROCK2 but not ROCK1 plays a role in allergen-induced goblet cell hyperplasia.

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.

Pulmonary inflammation induced by subacute ozone is augmented in adiponectin-deficient mice: role of IL-17A.

Pulmonary responses to ozone, a common air pollutant, are augmented in obese individuals. Adiponectin, an adipose-derived hormone that declines in obesity, has regulatory effects on the immune system. To determine the role of adiponectin in the pulmonary inflammation induced by extended (48-72 h) low-dose (0.3 parts per million) exposure to ozone, adiponectin-deficient (Adipo(-/-)) and wild-type mice were exposed to ozone or to room air. In wild-type mice, ozone exposure increased total bronchoalveolar lavage (BAL) adiponectin. Ozone-induced lung inflammation, including increases in BAL neutrophils, protein (an index of lung injury), IL-6, keratinocyte-derived chemokine, LPS-induced CXC chemokine, and G-CSF were augmented in Adipo(-/-) versus wild-type mice. Ozone also increased IL-17A mRNA expression to a greater extent in Adipo(-/-) versus wild-type mice. Moreover, compared with control Ab, anti-IL-17A Ab attenuated ozone-induced increases in BAL neutrophils and G-CSF in Adipo(-/-) but not in wild-type mice, suggesting that IL-17A, by promoting G-CSF release, contributed to augmented neutrophilia in Adipo(-/-) mice. Flow cytometric analysis of lung cells revealed that the number of CD45(+)/F4/80(+)/IL-17A(+) macrophages and γδ T cells expressing IL-17A increased after ozone exposure in wild-type mice and further increased in Adipo(-/-) mice. The IL-17(+) macrophages were CD11c(-) (interstitial macrophages), whereas CD11c(+) macrophages (alveolar macrophages) did not express IL-17A. Taken together, the data are consistent with the hypothesis that adiponectin protects against neutrophil recruitment induced by extended low-dose ozone exposure by inhibiting the induction and/or recruitment of IL-17A in interstitial macrophages and/or γδ T cells.

T-cell immunoglobulin and mucin domain 1 deficiency eliminates airway hyperreactivity triggered by the recognition of airway cell death.

BACKGROUND: Studies of asthma have been limited by a poor understanding of how nonallergic environmental exposures, such as air pollution and infection, are translated in the lung into inflammation and wheezing. OBJECTIVE: Our goal was to understand the mechanism of nonallergic asthma that leads to airway hyperreactivity (AHR), a cardinal feature of asthma independent of adaptive immunity. METHOD: We examined mouse models of experimental asthma in which AHR was induced by respiratory syncytial virus infection or ozone exposure using mice deficient in T-cell immunoglobulin and mucin domain 1 (TIM1/HAVCR1), an important asthma susceptibility gene. RESULTS: TIM1(-/-) mice did not have airways disease when infected with RSV or when repeatedly exposed to ozone, a major component of air pollution. On the other hand, the TIM1(-/-) mice had allergen-induced experimental asthma, as previously shown. The RSV- and ozone-induced pathways were blocked by treatment with caspase inhibitors, indicating an absolute requirement for programmed cell death and apoptosis. TIM-1-expressing, but not TIM-1-deficient, natural killer T cells responded to apoptotic airway epithelial cells by secreting cytokines, which mediated the development of AHR. CONCLUSION: We defined a novel pathway in which TIM-1, a receptor for phosphatidylserine expressed by apoptotic cells, drives the development of asthma by sensing and responding to injured and apoptotic airway epithelial cells.

Human neutrophil elastase-mediated goblet cell metaplasia is attenuated in TACE-deficient mice.

Neutrophilic inflammation is associated with chronic airway diseases. It has been observed that human neutrophil elastase (HNE), which is secreted by active neutrophils during inflammation, induces both mucin overproduction and goblet cell metaplasia. Several in vitro studies suggest that tumor necrosis factor-α converting enzyme (TACE) regulates the signaling axis that mediates HNE-induced mucin overproduction; however, it is unknown whether TACE performs a similar function in HNE-induced goblet cell metaplasia in vivo. We conducted this study to determine whether the inactivation of Tace gene expression attenuates HNE-induced goblet cell metaplasia in mice. Deletion of Tace is lethal shortly after birth in mice; therefore, we utilized Tace(flox/flox)R26CreER(+/-) mice and induced conditional deletion of Tace using a tamoxifen injection. Wild-type mice were given tamoxifen to control for its effect. Tace conditional deletion mice and wild-type mice were exposed to HNE via nasal instillation three times at 3-day intervals, and the lungs were harvested on day 11 after initial HNE exposure. Using periodic acid-Schiff staining and MUC5AC immunohistochemical staining to visualize goblet cells in the lungs, we found that HNE induced goblet cell metaplasia in the wild-type mice and that HNE-induced goblet cell metaplasia was significantly attenuated in the Tace conditional deletion mice. These findings suggest that TACE could be a potential target in the treatment of goblet cell metaplasia in patients with chronic airway diseases.

Obesity and airway responsiveness: role of TNFR2.

Obese mice exhibit innate airway hyperresponsiveness (AHR), a feature of asthma. Tumor necrosis factor alpha (TNFα) is implicated in the disease progression and chronic inflammatory status of both obesity and asthma. TNF acts via two TNF receptors, TNFR1 and TNFR2. To examine the role of TNFR2 in the AHR observed in obese mice, we generated obese Cpe(fat) mice that were either sufficient or deficient in TNFR2 (Cpe(fat) and Cpe(fat)/TNFR2(-/-) mice, respectively) and compared them with their lean controls (WT and TNFR2(-/-) mice). Compared to WT mice, Cpe(fat) mice exhibited AHR to aerosolized methacholine (measured using the forced oscillation technique) which was ablated in Cpe(fat)/TNFR2(-/-) mice. Bioplex or ELISA assay indicated significant increases in serum leptin, G-CSF, IL-7, IL-17A, TNFα, and KC in obese versus lean mice, as well as significant obesity-related increases in bronchoalveolar lavage fluid (BALF) G-CSF and IP-10, regardless of TNFR2 status. Importantly, BALF IL-17A was significantly increased over lean controls in Cpe(fat) but not Cpe(fat)/TNFR2(-/-) mice. Functional annotation clustering of significantly affected genes identified from microarray analysis comparing gene expression in lungs of Cpe(fat) and WT mice, identified blood vessel morphogenesis as the gene ontology category most affected by obesity. This category included several genes associated with AHR, including endothelin and trkB. Obesity increased pulmonary mRNA expression of endothelin and trkB in TNFR2 sufficient but not deficient mice. Our results indicate that TNFR2 signaling is required for the innate AHR that develops in obese mice, and suggest that TNFR2 may act by promoting IL-17A, endothelin, and/or trkB expression.

Antagonizing cholecystokinin A receptor in the lung attenuates obesity-induced airway hyperresponsiveness.

Obesity increases asthma prevalence and severity. However, the underlying mechanisms are poorly understood, and consequently, therapeutic options for asthma patients with obesity remain limited. Here we report that cholecystokinin-a metabolic hormone best known for its role in signaling satiation and fat metabolism-is increased in the lungs of obese mice and that pharmacological blockade of cholecystokinin A receptor signaling reduces obesity-associated airway hyperresponsiveness. Activation of cholecystokinin A receptor by the hormone induces contraction of airway smooth muscle cells. In vivo, cholecystokinin level is elevated in the lungs of both genetically and diet-induced obese mice. Importantly, intranasal administration of cholecystokinin A receptor antagonists (proglumide and devazepide) suppresses the airway hyperresponsiveness in the obese mice. Together, our results reveal an unexpected role for cholecystokinin in the lung and support the repurposing of cholecystokinin A receptor antagonists as a potential therapy for asthma patients with obesity.

The tobacco smoke component, acrolein, suppresses innate macrophage responses by direct alkylation of c-Jun N-terminal kinase.

The respiratory innate immune system is often compromised by tobacco smoke exposure, and previous studies have indicated that acrolein, a reactive electrophile in tobacco smoke, may contribute to the immunosuppressive effects of smoking. Exposure of mice to acrolein at concentrations similar to those in cigarette smoke (5 ppm, 4 h) significantly suppressed alveolar macrophage responses to bacterial LPS, indicated by reduced induction of nitric oxide synthase 2, TNF-α, and IL-12p40. Mechanistic studies with bone marrow-derived macrophages or MH-S macrophages demonstrated that acrolein (1-30 µM) attenuated these LPS-mediated innate responses in association with depletion of cellular glutathione, although glutathione depletion itself was not fully responsible for these immunosuppressive effects. Inhibitory actions of acrolein were most prominent after acute exposure (<2 h), indicating the involvement of direct and reversible interactions of acrolein with critical signaling pathways. Among the key signaling pathways involved in innate macrophage responses, acrolein marginally affected LPS-mediated activation of nuclear factor (NF)-κB, and significantly suppressed phosphorylation of c-Jun N-terminal kinase (JNK) and activation of c-Jun. Using biotin hydrazide labeling, NF-κB RelA and p50, as well as JNK2, a critical mediator of innate macrophage responses, were revealed as direct targets for alkylation by acrolein. Mass spectrometry analysis of acrolein-modified recombinant JNK2 indicated adduction to Cys(41) and Cys(177), putative important sites involved in mitogen-activated protein kinase (MAPK) kinase (MEK) binding and JNK2 phosphorylation. Our findings indicate that direct alkylation of JNK2 by electrophiles, such as acrolein, may be a prominent and hitherto unrecognized mechanism in their immunosuppressive effects, and may be a major factor in smoking-induced effects on the immune system.

Modulation of NF-κB and hypoxia-inducible factor--1 by S-nitrosoglutathione does not alter allergic airway inflammation in mice.

Induction of nitric oxide synthase (NOS)-2 and production of nitric oxide (NO) are common features of allergic airway disease. Conditions of severe asthma are associated with deficiency of airway S-nitrosothiols, a biological product of NO that can suppress inflammation by S-nitrosylation of the proinflammatory transcription factor, NF-κB. Therefore, restoration of airway S-nitrosothiols might have therapeutic benefit, and this was tested in a mouse model of ovalbumin (OVA)-induced allergic inflammation. Naive or OVA-sensitized animals were administered S-nitrosoglutathione (GSNO; 50 µl, 10 mM) intratracheally before OVA challenge and analyzed 48 hours later. GSNO administration enhanced lung tissue S-nitrosothiol levels and reduced NF-κB activity in OVA-challenged animals compared with control animals, but did not lead to significant changes in total bronchoalveolar lavage cell counts, differentials, or mucus metaplasia markers. Administration of GSNO also altered the activation of hypoxia-inducible factor (HIF)-1, leading to HIF-1 activation in naive mice, but suppressed HIF-1 activation in OVA-challenged mice. We assessed the contribution of endogenous NOS2 in regulating NF-κB and/or HIF-1 activation and allergic airway inflammation using NOS2(-/-) mice. Although OVA-induced NF-κB activation was slightly increased in NOS2(-/-) mice, associated with small increases in bronchoalveolar lavage neutrophils, other markers of allergic inflammation and HIF-1 activation were similar in NOS2(-/-) and wild-type mice. Collectively, our studies indicate that instillation of GSNO can suppress NF-κB activation during allergic airway inflammation, but does not significantly affect overall markers of inflammation or mucus metaplasia, thus potentially limiting its therapeutic potential due to effects on additional signaling pathways, such as HIF-1.