Dataset Information

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice.

ABSTRACT: MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a promising future of using miRNAs in the diagnosis and care of lung diseases. Here, we define a protocol for miRNA profiling to measure the relative abundance of a group of miRNAs predicted to regulate inflammatory genes in the lung tissue from of an ozone-induced airway inflammation mouse model. Because it has been shown that circulating sex hormone levels can affect the regulation of lung innate immunity in females, the purpose of this method is to describe an inflammatory miRNA profiling protocol in female mice, taking into consideration the estrous cycle stage of each animal at the time of ozone exposure. We also address applicable bioinformatics approaches to miRNA discovery and target identification methods using limma, an R/Bioconductor software, and functional analysis software to understand the biological context and pathways associated with differential miRNA expression.

SUBMITTER: Fuentes N

PROVIDER: S-EPMC6428684 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Publications

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice.

Fuentes Nathalie N Silveyra Patricia P

Journal of visualized experiments : JoVE 20190107 143

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a promising future of using miRNAs in the diagnosis and care of lung diseases. Here, we define a protocol for miRNA profiling to measure the relative abundance of a group of miRNAs predicted to regulate inflammatory genes in the lung tissue from of an ozone-induced airway inflammation mouse model. Because it has been shown that circulating sex hormone l ...[more]

PMID: 30663642

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Project description:Symbiotic microbial communities affect the host immune system and produce molecules contributing to the odor of an individual. In many mammalian species, saliva and vaginal fluids are important sources of chemical signals that originate from bacterial metabolism and may act as honest signals of health and reproductive status. In this study, we aimed to define oral and vaginal microbiomes and their dynamics throughout the estrous cycle in wild house mice. In addition, we analyzed a subset of vaginal proteomes and metabolomes to detect potential interactions with microbiomes. 16S rRNA sequencing revealed that both saliva and vagina are dominated by Firmicutes and Proteobacteria but differ at the genus level. The oral microbiome is more stable during the estrous cycle and most abundant bacteria belong to the genera Gemella and Streptococcus, while the vaginal microbiome shows higher bacterial diversity and dynamics during the reproductive cycle and is characterized by the dominance of Muribacter and Rodentibacter. These two genera cover around 50% of the bacterial community during estrus. Proteomic profiling of vaginal fluids revealed specific protein patterns associated with different estrous phases. Highly expressed proteins in estrus involve the keratinization process thus providing estrus markers (e.g., Hrnr) while some proteins are downregulated such as immune-related proteins that limit bacterial growth (Camp, Clu, Elane, Lyz2, and Ngp). The vaginal metabolome contains volatile compounds potentially involved in chemical communication, for example, ketones, aldehydes, and esters of carboxylic acids. Data integration of all three OMICs data sets revealed high correlations, thus providing evidence that microbiomes, host proteomes, and metabolomes may interact.IMPORTANCEOur data revealed dynamic changes in vaginal, but not salivary, microbiome composition during the reproductive cycle of wild mice. With multiple OMICs platforms, we provide evidence that changes in microbiota in the vaginal environment are accompanied by changes in the proteomic and metabolomics profiles of the host. This study describes the natural microbiota of wild mice and may contribute to a better understanding of microbiome-host immune system interactions during the hormonal and cellular changes in the female reproductive tract. Moreover, analysis of volatiles in the vaginal fluid shows particular substances that can be involved in chemical communication and reproductive behavior.

Project description:Lung epithelial lining fluid (ELF) harbors a variety of proteins that influence homeostatic and stress responses in the airspaces. Exosomes, nano-sized extracellular vesicles, contain many proteins that vary in abundance and composition based on the prevailing conditions. Ozone causes inflammatory responses in the airspaces of experimental animals and humans. However, the exosomal protein signatures contained within the ELF from ozone-exposed lung airspaces remain poorly characterized. To explore this, we hypothesized that ozone triggers the release of exosome-bound inflammatory proteins from various cells that reflect mucoobstructive lung disease. Accordingly, we repetitively exposed adult male and female C57BL/6 mice to HEPA-filtered air (air) or 0.8 ppm ozone (4 h per day) for 14 days (five consecutive days of exposure, 2 days of rest, five consecutive days of exposure, 2 days of rest, four consecutive days of exposure). Exosome-bound proteomic signatures, as well as the levels of soluble inflammatory mediators in the bronchoalveolar lavage fluid (BALF), were determined 12-16 h after the last exposure. Principal component analyses of the exosome-bound proteome revealed a clear distinction between air-exposed and ozone-exposed mice, as well as between ozone-exposed males and ozone-exposed females. In addition to 575 proteins that were enriched in both sexes upon ozone exposure, 243 and 326 proteins were enriched uniquely in ozone-exposed males and females, respectively. Ingenuity pathway analyses on enriched proteins between ozone- and air-exposed mice revealed enrichment of pro-inflammatory pathways. More specifically, macrophage activation-related proteins were enriched in exosomes from ozone-exposed mice. Cytokine analyses on the BALF revealed elevated levels of G-CSF, KC, IP-10, IL-6, and IL-5 in ozone-exposed mice. Finally, the histopathological assessment revealed significantly enhanced intracellular localization of mucoinflammatory proteins including MUC5B and FIZZ1 in ozone-exposed mice in a cell-specific manner indicating the cellular sources of the proteins that are ferried in the exosomes upon ozone-induced lung injury. Collectively, this study identified exosomal, secretory, and cell-specific proteins and biological pathways following repetitive exposure of mice to ozone.

Dataset Information

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice.

Publications

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice.

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