novel Fth1-mScarlet reporter mouse supports quantification of monocyte-derived macrophage inflammatory state in vivo

Abstract Monocyte-derived macrophages (mo-macs) acquire a spectrum of pro- and anti-inflammatory functions during infection-associated inflammation and resolution. Quantification of mo-mac functional state is critical to elucidate mechanisms leading to dysregulation of inflammatory control and how these processes can be manipulated therapeutically. Given that binary M1/M2 cell markers often fail to explain macrophage function in vivo, we sought to identify genes predictive of inflammatory gene signatures. We analyzed published single cell RNAseq of macrophages during lung inflammation, using Lasso (L1-penalized linear regression) to weight genes according to their ability to predict all other genes, and identified Fth1 (Ferritin Heavy Chain 1) as the most predictive gene. We generated Fth1-mScarlet reporter mice using CRISPR/Cas9 technology, inserting mScarlet in-frame of Fth1, linked through a T2A self-cleaving peptide. Using live cell imaging in vitro, we found monocyte mScarlet expression peaked 24 hours after stimulation with S. aureus or TLR ligands. We are currently using monocyte adoptive transfer to track mo-mac fate during acute S. aureus lung infection. In this intra-nasal S. aureus model (strain FDA309), pro-inflammatory genes and infiltration of neutrophils and monocytes peak 1–2 days post infection. We transferred Fth1mScarlet bone marrow monocytes 6 hours after infection, analyzed their recruitment into the lungs, and quantified mScarlet expression over time. Increased mScarlet expression was associated with the transition of donor cells from Ly6C+CD11c-monocytes to Ly6C-CD11c+ mo-macs. This model will allow us to quantify the plasticity and persistence of mo-macs during inflammation and resolution. R35 GM146896 (PI - Gottschalk)