Fetal Liver–Derived Alveolar-like Macrophages: A Self-Replicating Ex Vivo Model of Alveolar Macrophages for Functional Genetic Studies

Abstract

Alveolar macrophages (AMs) are tissue-resident cells in the lungs derived from the fetal liver that maintain lung homeostasis and respond to inhaled stimuli. Although the importance of AMs is undisputed, they remain refractory to standard experimental approaches and high-throughput functional genetics, as they are challenging to isolate and rapidly lose AM properties in standard culture. This limitation hinders our understanding of key regulatory mechanisms that control AM maintenance and function. In this study, we describe the development of a new model, fetal liver-derived alveolar-like macrophages (FLAMs), which maintains cellular morphologies, expression profiles, and functional mechanisms similar to murine AMs. FLAMs combine treatment with two key cytokines for AM maintenance, GM-CSF and TGF-b. We leveraged the long-term stability of FLAMs to develop functional genetic tools using CRISPR-Cas9-mediated gene editing. Targeted editing confirmed the role of AM-specific gene Marco and the IL-1 receptor Il1r1 in modulating the AM response to crystalline silica. Furthermore, a genome-wide knockout library using FLAMs identified novel genes required for surface expression of the AM marker Siglec-F, most notably those related to the peroxisome. Taken together, our results suggest that FLAMs are a stable, self-replicating model of AM function that enables previously impossible global genetic approaches to define the underlying mechanisms of AM maintenance and function.

Single cell analysis of docosahexaenoic acid suppression of sequential LPS-induced proinflammatory and interferon-regulated gene expression in the macrophage

“…Finally, although the in vitro macrophage model used here is similar to an inflammatory monocyte-derived macrophage, it might not precisely mimic tissue-specific macrophage populations observed in vivo . Thus, many of the pathways identified here need to be confirmed in tissue-relevant models such as alveolar-like macrophages derived by culturing fetal liver cells with GM-CSF and TGF-β ( 82 ).…”

GSK3α/β Restrain IFN-γ–Inducible Costimulatory Molecule Expression in Alveolar Macrophages, Limiting CD4+ T Cell Activation

“…J2 virus–immortalized Cas9 + BMDMs (iBMDMs) were isolated and immortalized from C57BL/6J mice as previously described ( 14 , 18 ). FLAMs were isolated from C57BL/6J mice or Ifnar1 −/− mice as previously described ( 23 , 25 ). Briefly, fetal livers were extracted from euthanized dams immediately after sacrifice.…”

“…Until recently, the availability of ex vivo models for AMs posed a challenge because AMs are notoriously difficult to maintain and isolate ( 23 , 24 ). Thus, this technical hurdle has limited our understanding of regulatory networks that control AM functionality.…”

“…It is important to understand how these lung-resident cells uniquely respond to inflammatory signals compared with other macrophages to develop lung-specific therapies that protect against infection while maintaining pulmonary function. To address this gap, several groups developed approaches to culture AM-like cells ex vivo that maintain AM functions ( 23 , 25 – 27 ). Although the details of these approaches differ, they all leverage lung-specific cytokine cues from GM-CSF and TGF-β that are required to maintain AM populations in the lung environment.…”

“…We previously developed an ex vivo AM model known as fetal liver–derived alveolar-like macrophages (FLAMs) that uses fetal liver cells, which are AM progenitors, to interrogate AM function. Our previous work showed that FLAMs maintain high expression of the AM surface marker SiglecF and the key transcription factor Pparγ ( 23 ). FLAMs are easy to isolate, culture, and expand, which, along with their genetic tractability, are strengths that allow mechanisms underlying AM functions to be interrogated.…”