Fth1-mScarlet reports monocyte state during lipopolysaccharide-induced lung inflammation

Monocytes and macrophages are central to host defense but also contribute to inflammation-associated pathology. Efforts to manipulate monocyte and macrophage function are limited by our ability to effectively quantify the functional programs of these cells. We identified the gene Fth1, which encodes the Ferritin heavy chain, as highly predictive of alveolar macrophage transcriptomic states during LPS-induced lung inflammation and developed a novel Fth1-mScarlet reporter mouse. In the steady state lung, high Fth1-mScarlet expression is restricted to alveolar macrophages. In response to LPS-induced lung inflammation, Fth1 reporter activity is robustly increased in monocytes, with its expression reporting genes that are differentially expressed in monocytes versus macrophages. Consistent with this reporter associated gene profile, within the Lyz2-GFP+CD11b+Ly6C+ gate the highest Fth1 reporter expression was observed CD11c+ cells, indicative of monocyte-to-macrophage differentiation. While Fth1-mScarlet was induced in monocytes responding to either TLR4 ligation or M-CSF induced macrophage differentiation in vitro, TLR4-dependent expression occurred with greater speed and magnitude. Considering this, we suggest that Fth1-mScarlet expression reports monocyte-to-macrophage differentiation, with increased expression in pro-inflammatory states. Dissecting macrophage differentiation from inflammatory programs will be enhanced when combining Fth1-mScarlet with other reporter systems. Thus, the Fth1-mScarlet model addresses an important lack of tools to report the diverse spectrum of monocyte and macrophage states in vivo. Overall design: LPS (20 µg in 50 µl; E. coli 0111:B4 from InvivoGen) was instilled directly into the tracheas of Fth1-mScarlet x Lyz2GFP mice sedated with isoflurane. Three days later, mice were sacrificed using CO2 euthanasia. Lungs were cut into 1-mm3-sized pieces and digested in DMEM containing Liberase TM (10 mg/ml) and deoxyribonuclease I (10 U/ml) in a shaker for 30 minutes at 37°C. Lungs were then further dissociated using a gentleMACS Dissociator (Miltenyi Biotec) and red blood cells were lysed with ACK Lysing Buffer (Lonza). Lung homogenate was stained and sorted for Lyz2GFP+CD11b+Ly6C+Ly6G- cells gated on low versus high Fth1-mScarlet expression, as well as low Fth1-mScarlet cells from untreated mice. These samples were submitted for RNA-seq analysis.