For histological analysis, lungs were fixed in formalin and embedded in paraffin. Four micrometer section containing the whole lobes of the lungs were stained with H&E. Images containing more than three lung lobes of each mouse were obtained using a BZ-9000 microscope (Keyence, Osaka, Japan) with low and high magnification views. For the quantification of injured lung areas, image processing and digital stitching were performed using BZ-X analyzer (Keyence) as described elsewhere [47, 48]. Briefly, partial lung images were stitched to the image of a whole lung lobe, and the percentage of the whole lobe composed of hypercellular areas associated with infiltration of inflammatory cells was determined using BZ-X analyzer software Three lobes were analyzed per animal, and the average percentage of the three lobes was calculated. For quantitative evaluation of neutrophilic inflammation, three high-power field images including terminal bronchioles were obtained from each slide stained with H&E. The number of infiltrated neutrophils found in each field were counted by the board-certificated pathologist (Y.S.) and the average number of neutrophil counts were calculated. Data were summarized as mean ± SEM of three mice per group.

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