RESUMO
Introduction:Neutrophil transmigration is multifactorial and primarily driven by selectins and ß2-integrins (CD11b/CD18), whose expression are dependent on the underlying stimulus. Ventilator-induced lung injury (VILI) results in a predominantly CD18-independent mechanism of neutrophil recruitment, while direct endotoxin-induced lung injury results from a CD18-dependent mechanism. We previously observed that lack of NADPH oxidases DUOX1 and DUOX2 resulted in reduced neutrophil influx in a VILI model of lung injury but had no influence on neutrophil influx after LPS exposure. Based on these observations, we hypothesized that DUOX1/DUOX2 are an important component of CD18-independent mechanisms of neutrophil recruitment in the lung. Methods:We exposed Duoxa -/- (KO) mice and Duoxa +/+ (WT) mice to either an intratracheal exposure of lipopolysaccharide (LPS/endotoxin)-or high tidal volume ventilation and compared expression of neutrophil markers between groups. WT mice (129S6/SvEvTac) were obtained from Taconic Biosciences (One Discovery Drive Suite 304; Rensselaer, NY 1244) and were allowed to acclimatize for one week prior to study enrollment. KO mice were generated as previously described [Grasberger 2012] and bred in-house on a 129S6 background. We provided positive-pressure ventilation at a tidal volume of 10 ml/kg with 2 cmH20 positive end-expiratory pressure (PEEP). Mice were assigned to groups consisting of KO (n = 5) and WT (n = 5) in each group and divided into non-ventilated, positive-pressure ventilation, or LPS IT exposure groups. Positive-pressure ventilation was instituted for 4-h using a FlexiVent (Flexiware 8.1, Scireq, Montreal, QC, Canada). Lipopolysaccharide (Salmonella enterica serotype tryphimurium L6143, Millipore Sigma) was administered via an intratracheal (IT) route at a dose of 0.1 mg/kg. Mice were humanely euthanized at 4-h post-injection consistent with the UC Davis IAUCAC-approved protocol. Results:As previously observed, neutrophilic influx into the airways was significantly impaired in the Duoxa -/- (KO) mice after VILI, but not after LPS exposure. LPS-induced lung injury resulted in upregulation of CD11b+ neutrophils and shedding of CD62L and CD162 regardless of DUOX expression, whereas VILI resulted in upregulation of CD49+ neutrophils in the Duoxa +/+ (WT) mice but not the Duoxa -/- (KO) mice. Conclusion:Our data suggest DUOX is required for CD18-independent mechanisms of neutrophil recruitment in the lung induced by acute lung injury, but not for canonical CD18depedent mechanisms after LPS exposure.
RESUMO
Obesity and accompanying metabolic disease is negatively correlated with lung health yet the exact mechanisms by which obesity affects the lung are not well characterized. Since obesity is associated with lung diseases as chronic bronchitis and asthma, we designed a series of experiments to measure changes in lung metabolism in mice fed obesogenic diets. Mice were fed either control or high fat/sugar diet (45%kcal fat/17%kcal sucrose), or very high fat diet (60%kcal fat/7% sucrose) for 150 days. We performed untargeted metabolomics by GC-TOFMS and HILIC-QTOFMS and lipidomics by RPLC-QTOFMS to reveal global changes in lung metabolism resulting from obesity and diet composition. From a total of 447 detected metabolites, we found 91 metabolite and lipid species significantly altered in mouse lung tissues upon dietary treatments. Significantly altered metabolites included complex lipids, free fatty acids, energy metabolites, amino acids and adenosine and NAD pathway members. While some metabolites were altered in both obese groups compared to control, others were different between obesogenic diet groups. Furthermore, a comparison of changes between lung, kidney and liver tissues indicated few metabolic changes were shared across organs, suggesting the lung is an independent metabolic organ. These results indicate obesity and diet composition have direct mechanistic effects on composition of the lung metabolome, which may contribute to disease progression by lung-specific pathways.
