Epigenetic Memory of AM in vivo and ex vivo

Here we show that alveolar macrophage (AM) identity were conserved through culture expansion as determined by the analysis of epigenetic chromatin accessibility of core AM specific transcription markers and surface markers. Further, the adaptations to the culture environment were transient, reversible and fully restored to the in vivo status upon transplantation into the alveolar niche. BAL AM pooled from 3 mice were expanded ex vivo for 2 months and i.t transplanted into lungs of 2-3 WT CD45.1 mice. The lungs were collected at 4 months post transplantation and sorted for CD45.2+ exAM or CD45.1+ host AM. 3 mice each of BAL AM, exAM and 2-3 mice each for texAM or Host AM were pooled and used for ATAC-seq. The ATAC-seq was done in technical triplicate transposase reactions, from pools of 2-3 mice for each replicate, again controlling for biological variation. Triplicates were then further pooled bioinformatically for the analyses shown in Fig. 5.