Functional characterization of a self-replicating and genetically tractable murine alveolar macrophage model

Abstract

Alveolar macrophages (AMs) are a critical element of the innate immune response to inhaled agents, yet functional and genetic studies of this unique macrophage population are lacking. Current strategies to obtain large quantities of AMs are cumbersome and inefficient. This is due largely to both the high cost of time and resources involved in the extraction of AMs and the inability to effectively culture AMs ex vivo for extended periods of time. While bone marrow derived macrophages (BMDMs) are modeled in numerous immortalized cell lines, AMs currently lack an acceptable model that can be used in vitro. Recently, self-replicating cells derived from the fetal mouse liver, termed “MPI” cells, have been shown to possess AM-like characteristics. Here, we show that early after isolation, these cells are SiglecFhi, Cd11chi, and Cd14low, while also expressing high levels of Pparg, Marco, Itgax, and Car4, akin to AMs. Additionally, like AMs, MPI cells effectively efferocytose dead cell debris and phagocytose silica particles. While these cells lose their “AM-likeness” over time, addition of the cytokine TGF-β dramatically delays this shift away from the AM-like phenotype. Gene expression analysis shows that in contrast to cells treated with TGF-β, untreated MPI cells cease expressing Tgfbr1, the receptor for TGF-β, concurrent with the shift away from the AM-phenotype. Further, these cells are amenable to viral transduction, and we have successfully employed CRISPR/Cas9 targeted genetic editing in MPI cells. These findings further our understanding of MPI cells as an accessible and genetically tractable model for AMs that allow for long-term and large-scale studies that are not possible with AMs isolated ex vivo.