Fibroblast orchestration of inflammaging via NF-kB activation (Lung Resident CD45+ cell and fibroblast analysis)

In this study we used scRNA-sequencing to compare lung resident CD45+ cells and bulk RNA-sequencing to compare lung PDGFRa+ fibroblasts from 2-month-old mice in which the NF-kB inhibitor Tnfaip3 has been deleted from fibroblasts (D1cA20) with control mice (A20control). We identified enrichment of Gzmk+CD8+ T cells in the D1cA20 relative to control mice. As a follow-up to this initial study we used bulk RNA sequencing to compare the transcriptomes of lung adventitial fibroblasts from 4-month C57BL/6 (Young B6), 21-month C57BL/6 (Aged B6), 4-month Tnfaip3 f/f (Tnfaip3 Control) and 4-month Dermo1-cre;Tnfaip3f/f (Tnfaip3 CKO/D1cA20) mice. We identified that aged B6 adventitial fibroblasts share transcriptional similarity with those from young Tnfaip3 CKO mice. Pathway analysis revealed NF-kB to be significantly upregulated in both Aged B6 and Tnfaip3 CKO relative to Young B6 or Tnfaip3 control. Overall design: Cells were sorted from 1) 2-month-old mice in which Tnfaip3 had been deleted from fibroblasts (D1cA20) and 2) 2-month-old controls (A20control). All mice were female and on a C57BL/6 background. To identify resident vs circulating immune cells mice were injected with a fluorescently-labeled anti-CD45 antibody 3 minutes prior to sacrifice. Resident CD45+ T cells were identified as ivCD45-CD45+. For each sample cells from 3 independent mice were combined. Fibroblasts for bulk RNA sequencing were sorted from the same samples and identified as CD45-Epcam-CD31-PDGFRa+. For bulk sequencing each mouse was submitted as a separate sample. For the subsequent lung adventitial fibroblast analysis, adventitial fibroblasts were isolated and sorted from Young B6, Aged B6, Tnfaip3 Control, and Tnfaip3 CKO mice and submitted for bulk RNA-sequencing. All mice were female and on a C57BL/6 background. 4 biological replicates were submitted per group. Adventitial fibroblasts were sorted as Lin- (CD45-Epcam-PECAM-Mcam-)Sca1+.