Immune responses of lung mesenchymal cells during pneumonia

Mesenchymal cell roles during respiratory infection are not well defined, including whether, which, and how the different types of mesenchymal cells respond. We collected all mesenchymal cells from lung single cell suspensions of mice that were naïve (after receiving only saline vehicle), infected (after intratracheal instillation of pneumococcus 24 hours previously), or recovered from infection (after non-lethal pneumococcal infections 6 weeks previously) and performed single-cell RNA sequencing. Cells clustered into five well-separated groups based on their transcriptomes: matrix fibroblasts, myofibroblasts, pericytes, smooth muscle cells, and mesothelial cells. Fibroblasts were the most abundant and could be further segregated into Pdgfra+Npnt+Ces1d+Col13a1+ alveolar fibroblasts and Cd9+Pi16+Sca1+Col14a1+ adventitial fibroblasts. The cells from naïve and recovered groups overlapped in dimension reduction plots, suggesting mesenchymal cells return to baseline transcriptomes after resolution. During pneumonia, all mesenchymal cells responded with altered transcriptomes, revealing a core response conserved across cell-types as well as distinct mesenchymal cell type-specific responses. The different subsets of fibroblasts induced similar gene sets, but the alveolar fibroblasts responded more strongly than the adventitial fibroblasts. These data demonstrate diverse and specialized immune activities of lung mesenchymal cells during pneumonia. We used flow cytometry to isolate all cells from the lung that were not leukocytes, epithelial cells, or endothelial cells (i.e., CD45-CD326-CD31-), to compare cell-types and transcriptomes between young adult mice that were naïve, experiencing acute pneumonia, or fully recovered with a remodeled lung immune system (10, 12). All mice across these three groups received the same types of surgeries and intratracheal instillations, but the instillates differed. The control group, labeled as “Naïve,” received only the sterile saline vehicle. The active infection group, labeled as “Pneumonic,” received pneumococcus 24 hours prior to euthanasia. The recovered group, labeled as “Resolved”, had pneumococcus infections a month previously, yielding lungs with no ongoing infection or inflammation but a remodeled resident immune system that provides improved protection against further infections. Cells were barcoded using 10x Chromium Genomics, and all three groups were sequenced together using Next Generation Sequencing, before cell-specific transcriptomes were analyzed using CeLDA, Seurat, and SPRING (27-30).