Computational protocol: Augmented Pulmonary Responses to Acute Ozone Exposure in Obese Mice: Roles of TNFR2 and IL-13

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[…] Animals. This study was approved by the Harvard Medical Area Standing Committee on Animals. Animals were treated humanely and with regard for alleviation of suffering. We bred Cpefat/TNFR2–/–, TNFR2–/–, Cpefat, and WT mice as previously described (). Female mice were on a C57BL/6 background, were fed standard mouse chow diets, and were 10–12 weeks old.Protocol. We exposed mice to 2 ppm O3 for 3 hr. Twenty-four hours after exposure, we measured pulmonary mechanics and airway responsiveness, performed bronchoalveolar lavage (BAL), harvested blood by cardiac puncture, and collected lungs for preparation of RNA. Controls for these mice were not exposed to O3 but were otherwise treated identically and studied simultaneously. Pulmonary mechanics and airway responsiveness for these unexposed mice have been previously reported (). We treated other mice with anti–IL-13 antibody (2 μg/g body weight intraperitoneally) 24 hr before O3 exposure. In a final cohort of O3-exposed mice, we harvested lungs at 24 hr, enzymatically digested the lungs, and isolated lung cells for flow cytometry to quantitate IL-13–expressing CD4+ cells.Measurement of pulmonary mechanics and airway responsiveness. We generated quasi static pressure volume (PV) curves, assessed baseline pulmonary mechanics using the forced oscillation technique, and measured airway responsiveness to aerosolized methacholine as previously described (). We assessed Newtonian resistance (Rn), which largely reflects the resistance of the conducting airways, and the coefficients of lung tissue damping (G) and lung tissue elastance (H), which reflect changes in the small airways and pulmonary parenchyma.Bronchoalveolar lavage. We lavaged lungs and counted BAL cells as previously described (). We measured BAL cytokines, chemokines, and hyaluronan by ELISA (R&D Systems Inc., Minneapolis, MN; eBioscience, San Diego, CA; and Echelon Biosciences, Salt Lake City, UT). We measured BAL protein by Bradford assay (BioRad, Hercules, CA).RNA extraction and real-time polymerase chain reaction (PCR). We used real-time PCR to quantitate IL-13 and IL-17A mRNA expression as described (). We subtracted Ct values for a housekeeping gene, 36B4 (rplp0) (which codes for a ribosomal protein), from Ct values for IL-13 or IL-17A to obtain ΔCt values. We expressed changes in mRNA relative to values from the WT unexposed mice, using the ΔΔCt method.Flow cytometry. We flushed the lungs to remove blood cells, and then excised, minced, and digested lung tissue as previously described (). We cultured lung cells either with or without PMA (phorbol myristate acetate) and ionomycin, in the presence of Golgi Stop (BD Bioscience, Franklin Lakes, NJ), for 5 hr before staining for flow cytometry. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, incubated with anti-Fcγ blocking mAb (clone 93; Biolegend, San Diego, CA), and washed. We stained the cells with Alexa Fluor 488-conjugated CD4 mAb (clone GK1.5; Biolegend) and Alexa Fluor 647-conjugated anti-mouse IL-13 (clone eBio13A; eBioscience). We passed the cells through a BD Canto flow cytometer (BD Bioscience), and analyzed the data with FlowJo software (Tree Star Inc., Ashland, OR).Statistics. We used factorial analysis of variance to assess the significance of differences in outcome indicators, as previously described (). We performed analyses using Statistica version 6 (StatSoft, Tulsa, OK). […]

Pipeline specifications

Software tools FlowJo, Statistica
Applications Miscellaneous, Flow cytometry
Organisms Mus musculus
Diseases Airway Obstruction, Hamartoma Syndrome, Multiple, Neoplasms, Pneumonia, Machado-Joseph Disease
Chemicals Ozone