D101-03 Lung Interstitial Macrophage Remodeling After Pneumonia Recovery

Abstract Pneumococcal exposures profoundly change the immune landscape of the lung. In mice, alveolar macrophages become remodeled and tissue resident memory T and B cells are recruited. Lung interstitial macrophages (IMs) hold a variety of tissue-specific immune roles during and after respiratory infections. How these exposures alter IM phenotypes and responses to future insults remains unknown. We hypothesize IMs have the capacity to remodel after pneumococcal infections. Mice were infected with left lobe intratracheal instillations of pneumococcus (“Experienced”) or saline (“Naïve”) at days 0 and 7. At day 35, IMs were quantified by flow cytometry and multispectral fluorescence immunohistochemistry in Cx3cr1+/GFP reporter mice. IM numbers were also quantified in CCR2-/- mice and after depletion of B cells or T helper cells using antibodies against CD20 or CD4, respectively. To determine IM ontogeny, we used Flk/switch reporter mice, whereby embryonic cells express tdTomato and bone marrow derived cells express GFP. Lungs from experienced and naïve mice were prepared for CITE-seq to examine effects of experience on surface proteins and transcriptomes of IM subpopulations. Phagocytosis assays were carried out by instilling fluorescent pneumococcus or E. coli in naïve and experienced mice. Ontogeny experiments revealed the majority of lung IMs were bone marrow-derived, even in naïve adult mice. Experienced mice had increased numbers of IM subpopulations, outnumbering alveolar macrophages. This IM expansion was dependent on a second pneumococcal infection, but not on B cells or T cells or CCR2. Experienced CD206+ IMs, but not CD206- IMs, showed changes in several surface marker expression levels, including a decrease in MHCII that persisted up to day 98, suggesting a remodeled phenotype. Single cell analysis identified distinct naïve and experienced CD206+ IM clusters. Differential gene expression comparing naïve and experienced clusters included changes in cytokines, endothelial and extracellular matrix factors, and phagocytic receptors. Phagocytosis assays revealed experienced CD206+ IMs had enhanced phagocytic capacity of Gram negative and Gram positive bacteria, suggesting a remodeled functional phenotype in these cells. From these studies, we conclude that IMs increase in abundance after pneumonia recovery, and CD206+ IMs develop altered phenotypes that contribute to innate immune responses and lung defense. This abstract is funded by: NIH