Single-cell RNA sequencing uncovers heterogenous transcriptional signatures in macrophages during efferocytosis

Efferocytosis triggers cellular reprogramming, including the induction of mRNA transcripts which encode anti-inflammatory cytokines that promote inflammation resolution. Our current understanding of this transcriptional response is largely informed from analysis of bulk phagocyte populations; however, this precludes the resolution of heterogeneity between individual macrophages and macrophage subsets. Moreover, phagocytes may contain so called “passenger” transcripts that originate from engulfed apoptotic bodies, thus obscuring the true transcriptional reprogramming of the phagocyte. To define the transcriptional diversity during efferocytosis, we utilized single-cell mRNA sequencing after co-cultivating macrophages with apoptotic cells. Importantly, transcriptomic analyses were performed after validating the disappearance of apoptotic cell-derived RNA sequences. Our findings reveal new heterogeneity of the efferocytic response at a single-cell resolution, particularly evident between F4/80⁺ MHCII^LO and F4/80⁻ MHCII^HI macrophage sub-populations. After exposure to apoptotic cells, the F4/80⁺ MHCII^LO subset significantly induced pathways associated with tissue and cellular homeostasis, while the F4/80⁻ MHCII^HI subset downregulated these putative signaling axes. Ablation of a canonical efferocytosis receptor, MerTK, blunted efferocytic signatures and led to the escalation of cell death-associated transcriptional signatures in F4/80⁺ MHCII^LO macrophages. Taken together, our results newly elucidate the heterogenous transcriptional response of single-cell peritoneal macrophages after exposure to apoptotic cells.