Johnston RA, Theman TA, Lu FL, Terry RD, Williams ES, Shore SA. Diet-induced obesity causes innate airway hyperresponsiveness to methacholine and enhances ozone-induced pulmonary inflammation. J Appl Physiol 104: 1727-1735, 2008. First published March 6, 2008 doi:10.1152/japplphysiol.00075.2008.-We previously reported that genetically obese mice exhibit innate airway hyperresponsiveness (AHR) and enhanced ozone (O3)-induced pulmonary inflammation. Such genetic deficiencies in mice are rare in humans, and they may not be representative of human obesity. Thus the purpose of this study was to determine the pulmonary phenotype of mice with diet-induced obesity (DIO), which more closely mimics the cause of human obesity. Therefore, wild-type C57BL/6 mice were reared from the time of weaning until at least 30 wk of age on diets in which either 10 or 60% of the calories are derived from fat in the form of lard. Body mass was ϳ40% greater in mice fed 60 vs. 10% fat diets. Baseline airway responsiveness to intravenous methacholine, measured by forced oscillation, was greater in mice fed 60 vs. 10% fat diets. We also examined lung permeability and inflammation after exposure to room air or O3 (2 parts/million for 3 h), an asthma trigger. Four hours after the exposure ended, O3-induced increases in bronchoalveolar lavage fluid protein, interleukin-6, KC, macrophage inflammatory protein-2, interferon-␥-inducible protein-10, and eotaxin were greater in mice fed 60 vs. 10% fat diets. Innate AHR and augmented responses to O3 were not observed in mice raised from weaning until 20 -22 wk of age on a 60% fat diet. These results indicate that mice with DIO exhibit innate AHR and enhanced O3-induced pulmonary inflammation, similar to genetically obese mice. However, mice with DIO must remain obese for an extended period of time before this pulmonary phenotype is observed. bronchoalveolar lavage fluid; chemokine; leptin; lung elastance; resistance OBESITY IS AN IMPORTANT PUBLIC health problem that is associated with several respiratory diseases, including obesity-hypoventilation syndrome, obstructive sleep apnea, and asthma (14,15,53). Epidemiological studies indicate an increased incidence of asthma, wheezing, or airway hyperresponsiveness (AHR) in overweight or obese children, adolescents, and adults (14, 53). The relationship between obesity and asthma is likely to be a causal one, because longitudinal studies controlling for a number of potential confounders, including physical activity, indicate that the relative risk of incident asthma progressively increases with increasing body mass index and that obesity antedates asthma (6,7,18,41). Furthermore, morbidly obese asthmatic individuals examined after diet-or surgically induced weight loss report a decrease in both the severity and symptoms of asthma (38,42,56,57).Our laboratory has been utilizing murine models of obesity to explore the mechanistic basis for the relationship between obesity and asthma. Our laboratory has reported that obese mice exhibit innate AHR (29,30,37,48...

Obese mice have increased responses to acute ozone (O 3) exposure. T-cadherin is a binding protein for the high-molecular weight isoforms of adiponectin, an anti-inflammatory hormone that declines in obesity. The objective of the present study was to determine whether adiponectin affects pulmonary responses to O 3 , and whether these effects are mediated through T-cadherin. We performed bronchoalveolar lavage (BAL) and measured pulmonary responsiveness to methacholine after acute air or O 3 exposure (2 ppm for 3 h) in adiponectin-deficient (Adipo 2/2) or T-cadherindeficient (T-Cad 2/2) mice. O 3 increased pulmonary responses to methacholine and increased BAL neutrophils and protein to a greater extent in wild-type than in Adipo 2/2 mice, whereas T-cadherin deficiency had no effect. O 3-induced increases in BAL IL-6 and keratinocyte-derived chemokine (KC), which contribute to O 3induced pulmonary neutrophilia, were also greater in wild-type than in Adipo 2/2 mice. In contrast, responses to O 3 were not altered by transgenic overexpression of adiponectin. To determine which adiponectin isoforms are present in the lung, Western blotting was performed. The hexameric isoform of adiponectin dominated in serum, whereas BAL was dominated by the high-molecular weight isoform of adiponectin. Interestingly, serum adiponectin was greater in T-Cad 2/2 versus wild-type mice, whereas BAL adiponectin was lower in T-Cad 2/2 versus wild-type mice, suggesting that T-cadherin may be important for transit of high-molecular weight adiponectin from the blood to the lung. Our results indicate that adiponectin deficiency inhibits pulmonary inflammation induced by acute O 3 exposure, and that T-cadherin does not mediate the effects of adiponectin responsible for these events.

Objective Inhibition of Rho-associated coiled-coil forming kinases (ROCKs) reduces allergic airway responses in mice. The purpose of this study was to determine the roles of the two ROCK isoforms, ROCK1 and ROCK2, in these responses. Methods Wildtype mice and heterozygous ROCK1 and ROCK2 knockout mice (ROCK1+/− and ROCK2+/− respectively) were sensitized and challenged with ovalbumin. ROCK expression and activation were assessed by Western blotting. Airway responsiveness was measured by forced oscillation. Bronchoalveolar lavage was performed and the lungs were fixed for histological assessment. Results Compared to wildtype mice, ROCK1 and ROCK2 expression were 50% lower in lungs of ROCK1+/− and ROCK2+/− mice, respectively, without changes in the other isoform. In wildtype lungs, ROCK activation increased after ovalbumin challenge, was sustained for several hours, and was reduced in ROCK1+/− and ROCK2+/− lungs. Airway responsiveness was comparable in wildtype, ROCK1+/−, and ROCK2+/− mice challenged with PBS. Ovalbumin challenge caused airway hyperresponsiveness in wildtype, but not ROCK1+/− or ROCK2+/− mice. Lavage eosinophils and goblet cell hyperplasia were significantly reduced in ovalbumin-challenged ROCK1+/− and ROCK2+/− versus wildtype mice. Ovalbumin-induced changes in lavage interleukin-13, interleukin-5, and lymphocytes were also reduced in ROCK1+/− mice. Conclusions Both ROCK1 and ROCK2 are important in regulating allergic airway responses.

Interleukin (IL)-1, a proinflammatory cytokine, is expressed in the lung after ozone (O(3)) exposure. IL-1 mediates its effects through the type I IL-1 receptor (IL-1RI), the only signaling receptor for both IL-1alpha and IL-1beta. The purpose of this study was to determine the role of IL-1RI in pulmonary responses to O(3.) To that end, wild-type, C57BL/6 (IL-1RI(+/+)) mice and IL-1RI-deficient (IL-1RI(-/-)) mice were exposed to O(3) either subacutely (0.3 ppm for 72 h) or acutely (2 ppm for 3 h). Subacute O(3) exposure increased bronchoalveolar lavage fluid (BALF) protein, interferon-gamma-inducible protein (IP)-10, soluble tumor necrosis factor receptor 1 (sTNFR1), and neutrophils in IL-1RI(+/+) and IL-1RI(-/-) mice. With the exception of IP-10, all outcome indicators were reduced in IL-1RI(-/-) mice. Furthermore, subacute O(3) exposure increased IL-6 mRNA expression in IL-1RI(+/+), but not IL-1RI(-/-) mice. Acute (2 ppm) O(3) exposure increased BALF protein, IL-6, eotaxin, KC, macrophage inflammatory protein (MIP)-2, IP-10, monocyte chemotactic protein-1, sTNFR1, neutrophils, and epithelial cells in IL-1RI(+/+) and IL-1RI(-/-) mice. For IL-6, eotaxin, MIP-2, and sTNFR1, there were small but significant reductions of these outcome indicators in IL-1RI(-/-) versus IL-1RI(+/+) mice at 6 hours after exposure, but not at other time points, whereas other outcome indicators were unaffected by IL-1RI deficiency. These results suggest that IL-1RI is required for O(3)-induced pulmonary inflammation during subacute O(3) exposure, but plays a more minor role during acute O(3) exposure. In addition, these results suggest that the induction of IL-6 via IL-1RI may be important in mediating the effects of O(3) during subacute exposure.

The purpose of this study was to determine whether obesity affects pulmonary responses following a 3-day ozone exposure. Obese db/db and lean wild-type mice were exposed to ozone (0.3 ppm) for 72 h. In wild-type mice, ozone exposure caused pulmonary injury and inflammation, and these events were associated with reduced pulmonary compliance. In db/db mice, ozone-induced neutrophil recruitment to the lung was reduced and no reduction in compliance was observed. Similar results were obtained in obese Cpe(fat) mice, indicating that loss of leptin signaling in db/db mice does not account for these obesity-related changes. To examine the role of interleukin (IL)-6 in this obesity-related difference in ozone responsiveness, wild-type and IL-6-deficient mice were raised on 10% or 60% fat diets. Compared with 10% fat-fed mice, wild-type 60% fat-fed mice were obese and had reduced neutrophil recruitment following ozone. IL-6 deficiency reduced ozone-induced neutrophil recruitment in 10% fat-fed mice. In contrast, in obese mice, no effect of IL-6 deficiency on neutrophil recruitment was observed. Obesity-related differences in the effect of ozone on compliance were observed in both wild-type and IL-6-deficient mice. Obesity-related differences in serum IL-6 were observed and may account for obesity-related differences in the effect of IL-6 deficiency on neutrophil recruitment. In summary, the neutrophilic inflammation induced by prolonged low level ozone exposure was attenuated in obese mice and appeared to result from an absence of IL-6-dependent neutrophil recruitment in the obese mice.