View Abstract

A4483 - Macrophage Subpopulations in Murine Lungs Have Distinct Gene Expression Profiles and Show Differential Responses to Chronic Cigarette Smoke In Vivo
Author Block: M. Engle1, J. Dow2, L. T. Randall3, S. N. Ahmed3, M. Lemma3, H. Dang3, J. S. Parker4, C. M. Doerschuk3; 1Curriculum of Genetics and Molecular Biology, UNC-CH, Chapel Hill, NC, United States, 2UNC Flow Cytometry Core Facility, UNC-CH, Chapel Hill, NC, United States, 3Marsico Lung Institute, UNC-CH, Chapel Hill, NC, United States, 4Lineberger Comprehensive Cancer Center, UNC-CH, Chapel Hill, NC, United States.
RATIONALE
Cigarette smoke is a leading health hazard and causes an enormous impact on lung health. Males and females differ in their response to smoke. Macrophages have been shown to be dysregulated in chronic smokers. At least three subpopulations of macrophages have been identified, and the role of each macrophage subpopulation in response to smoke-induced lung injury and the contributions of sex and chronic inflammation are unclear. These studies tested the hypothesis that cigarette smoke exposure alters mRNA expression profiles differently in each macrophage subpopulation after 6 months of smoke exposure and that these profiles differ between sexes.
METHODS
Sex-matched 6-week old WT and Scnn1b-transgenic mice overexpressing βENaC, which results in mucus dehydration and chronic bronchitis, were used. Mice were exposed to cigarette or sham smoke for 6 months. Macrophages were isolated from single cell suspensions of the digested lung using FACS with the MoFlo Legacy. These subpopulations were identified as CD45+/Ly6G-/CD64+ and differentiated based on expression of Ly6C and Siglec F:
Alveolar macrophages (AM): Siglec F+/Ly6Clow-hi
Inflammatory macrophages (InfM): Siglec F-/Ly6Chi
Interstitial macrophages (IM): Siglec F-/Ly6C-
RNA from each subpopulation was pooled (n=2 mice per sample) and assessed using Affymetrix microarrays. The effects of exposure, genotype, and sex on gene expression were analyzed within each macrophage subpopulation using a linear model. Gene set analysis was done using the GSA method and the Canonical Pathways list (GSEA). Significant pathways were identified using a 5% FDR threshold.
RESULTS
The differences between the macrophage subpopulations are larger than those due to smoke exposure, sex, or genotype. Gene expression of each of the AM, InfM, and IM subpopulations revealed significantly distinct profiles. Within each subpopulation, smoke exposure responses and sex-specific variations were assessed. Of the three subpopulations, AMs showed the most changes in response to smoke exposure. AMs also had distinct biological pathway enrichment in response to smoke; extracellular matrix biology-related pathways were upregulated, while cell division pathways were downregulated. Interestingly, pathways of lysosome function showed the highest association, and AMs were the only subpopulation to become blackened due to uptake of smoke particulates. IMs and InfMs did not show significant changes due to exposure, but did show sex-specific gene expression patterns for certain genes. No subpopulation showed significant changes due to genotype.
CONCLUSION
AMs show significant changes in smoke-exposed compared to sham-exposed animals in gene expression and pathway analyses. This subpopulation reacts to smoke exposure differently than the IMs and InfMs.