Long-term culture-expanded alveolar macrophages restore their full epigenetic identity after transfer in vivo.

Sethuraman Subramanian,Ridzky Anis Advent Yuda,Gregory Gimenez,Michael H Sieweke,Bérengère De Laval,Stephanie Vargas Aguilar,Sandrine Sarrazin,Michaela Burkon,Julien Maurizio,Clara Jana-Lui Busch,Jérémy Favret,Lena Alexopoulou,Sara Gholamhosseinian Najjar,Hassiba Belahbib,Kaaweh Molawi,Laufey Geirsdottir,Prashanth Kumar Kandalla

doi:10.1038/s41590-022-01146-w

Abstract

Alveolar macrophages (AMs) are lung tissue-resident macrophages that can be expanded in culture, but it is unknown to what extent culture affects their in vivo identity. Here we show that mouse long-term ex vivo expanded AMs (exAMs) maintained a core AM gene expression program, but showed culture adaptations related to adhesion, metabolism and proliferation. Upon transplantation into the lung, exAMs reacquired full transcriptional and epigenetic AM identity, even after several months in culture and could self-maintain long-term in the alveolar niche. Changes in open chromatin regions observed in culture were fully reversible in transplanted exAMs and resulted in a gene expression profile indistinguishable from resident AMs. Our results indicate that long-term proliferation of AMs in culture did not compromise cellular identity in vivo. The robustness of exAM identity provides new opportunities for mechanistic analysis and highlights the therapeutic potential of exAMs.

Highlights

Alveolar macrophages (AMs) are lung tissue-resident macrophages that can be expanded in culture, but it is unknown to what extent culture affects their in vivo identity
Mouse AMs from bronchoalveolar lavage (BAL) can be cultured[20] and expanded in granulocyte-macrophage colony-stimulating factor (GM-CSF)-containing medium[19]. We observed that these cells, hereafter referred to as expanded AMs (exAMs), could be kept in continuous culture for at least 10 months (Fig. 1a), resulting in 33 theoretical population doublings and an amplification factor of 1010. This correlated with a three-to-fourfold higher percentage of cells in S phase of the cell cycle in exAMs cultured for 4 months compared to fresh BAL (Fig. 1b), whereas the cell death rate determined by annexin V/7-AAD staining was low (Extended Data Fig. 1a)
Because the transfer from the in vivo environment to ex vivo culture results in major gene expression changes in mouse[9] or human[10] microglia and substantial changes in cytokine responsiveness and glucose metabolism in AMs11, we investigated whether the culture environment imposed changes in gene expression on exAMs

Summary

Introduction

Alveolar macrophages (AMs) are lung tissue-resident macrophages that can be expanded in culture, but it is unknown to what extent culture affects their in vivo identity. We have compared the transcriptional and epigenetic identity of mouse AMs in long-term culture before and after re-transplantation into the lung niche environment.

Results

Conclusion