Sex differences in lung inflammation and microbiome profiles in asthmatic mice exposed to ambient ozone

Abstract

Ambient ozone, a product of photochemical reactions between volatile organic compounds and nitrogen oxides, is known to be one of the most dangerous air pollutants. Ozone inhalation can aggravate preexisting inflammatory lung diseases such as asthma, which are more frequently diagnosed in females than males. Despite this, the molecular mechanisms underlying the effects of ozone in the male and female lung have yet to be discovered. We hypothesized that exposure to ozone exerts differential inflammatory and immune responses in the lungs of male and females with asthma. To examine this, we treated adult male and female C57BL/6J mice with an allergen (house dust mite extract) intranasally for 5 weeks to trigger asthma. We then exposed mice to 2 ppm of ozone or filtered air (FA) for 3 hours, and collected lung tissue 24 hours later. We extracted lung RNA, and measure the expression of 92 immune response associated genes by PCR with the TaqMan® Array 96‐well Mouse Immune Response Plate (ThermoFisher). We also extracted DNA to determined lung microbiome profiles via 16S rRNA amplicon sequencing using a MiSeq instrument. The resulting paired‐end reads were processed using QIIME 2 2018.11. Amplicon sequencing units from DADA2 were assigned taxonomic identifiers based on the GreenGenes database release 13_08 using the QIIME 2 q2‐featureclassifier. Results were summarized, visualized through Principal Coordinate Analysis, and significance was estimated as implemented in QIIME2. Our preliminary results show that, after exposure to ozone, asthmatic male mice had higher lung expression of immune response genes (cell surface receptors and signaling molecules) whereas asthmatic females had higher expression of pro‐inflammatory cytokines, transcription factors, and regulators of immunity than mice exposed to FA. We also identified differential microbiome profiles between male and female asthmatic mice and in response to ozone, including a higher content of Firmicutes and a higher Firmicutes/Bacteriodetes ratio in males than females. Moreover, asthmatic females displayed a more pronounced lung bacterial diversity and higher relative abundance of Proteobacteria when compared to males. We conclude that ozone exposure triggers differential inflammatory/immune mechanisms in the male and female lungs of asthmatic mice, and that these mechanisms could be mediated by lung resident microbes.