Resident macrophages populate all tissues of the body, where they perform homeostatic, immune, and repair functions. Their phenotypic specialization is strongly dependent on their microenvironment, to the extent that the role of the tissue niche in shaping their identity remains the subject of intense debate. Alveolar macrophages, which reside in the lung, display a remarkable feature: unlike most primary macrophages, whose proliferative capacity in culture is very limited and whose populations are heterogeneous, they can expand in large numbers and over long periods. This property is linked to the constitutive expression of low levels of the antiproliferative transcription factors MafB and cMaf, which keeps accessible a network of self-renewal genes that is normally repressed in other macrophage lineages. However, it had been established that these cells, like microglia, lose part of their tissue-specific transcriptional signature when placed in culture, most likely owing to the absence of in vivo niche signals. It remained an open question whether these adaptations were permanent or reversible, and in particular whether they were accompanied by irreversible changes in chromatin accessibility.
To address this, the team compared the transcriptional and epigenetic identity of murine alveolar macrophages after prolonged expansion in culture, before and after their retransplantation into the pulmonary niche. The cultured cells, obtained from bronchoalveolar lavages, were expanded for two months and then transferred intratracheally into recipient mice, with the allotypic markers CD45.1 and CD45.2 used to distinguish transplanted cells from host macrophages. The analysis combined RNA sequencing (RNA-seq) and assessment of chromatin accessibility by ATAC-seq, four months after engraftment.
The results show that in vitro–expanded macrophages retain a core lineage-specific expression program while displaying adaptations related to adhesion, metabolism, and proliferation. These changes proved to be transient: after transplantation, the cells reacquired a transcriptional and epigenetic identity virtually indistinguishable from that of resident alveolar macrophages that had never been cultured, even after several months of expansion. The open chromatin regions altered in culture returned to full concordance with those of resident cells, and the engrafted macrophages self-maintained durably within the alveolar niche. These cells therefore retain, across long periods and multiple divisions, their responsiveness to the environmental signals that define the pulmonary niche.
This work indicates that the prolonged proliferation of alveolar macrophages in culture does not compromise their cellular identity in vivo. The robustness of this identity makes these cells a high-fidelity culture system, capable of moving back and forth between ex vivo experimental manipulation and in vivo validation, opening new possibilities for mechanistic analysis and underscoring their therapeutic potential.