In vivo single cell transcriptomics reveals Klebsiella pneumoniae-governed polarization of ontogenetically distinct macrophage lineages by targeting an evolutionary conserved type I IFN-IL10-STAT6 axis to promote infection.

Mice were infected with Klebsiella pneumoniae and interstitial and alveolar macrophages isolated and subjected to single cell RNA sequencing to investigate the transcriptomes of Klebseilla-associated and uninfected bystander cells. C57BL/6 age and sex matched animals were infected intranasally with K. pneumoniae Kp52145 expressing mCherry After 24 h, lungs were homogenized, pooled and red cells lysed before pre-sorting using CD11c Microbeads, UltraPure (Miltenyi Biotech) according to manufacturer’s instructions. Cell suspensions were incubated with combinations of the following rat anti-mouse antibodies: against cell surface markers Ly6C APC/Cy7 (clone HK1.4, Ref: 128026), CD11b APC (Ref: 101212), CD11c Pacific Blue (Ref: 117322), SiglecF FITC (Ref: 155504, BioLegend). Ly6C+CD11b+CD11c+SIGLECF- Interstitial Macrophages (IMs) and Ly6C+CD11b-CD11c+Siglec F+ Alveoar macrophages (AMs) were sorted from PBS control mice. From infected animals, four separate populations were retrieved namely IMs associated with Kp52145 (Ly6C+CD11b+CD11c+SIGLECF-Kp52145+), bystander IMs (Ly6C+CD11b+CD11c+SIGLECF-Kp52145-), Kp52145-associated AMs (Ly6C+CD11b-CD11c+Siglec F+Kp52145+) and bystander AMs (Ly6C+CD11b-CD11c+Siglec F+Kp52145-) populations.