Project description:Macrophages can adjust their phenotype and function in response to environmental cues, such as encountering apoptotic cells or pathogens. After tissue injury or inflammation, macrophages must successively engulf and process multiple apoptotic corpses (via efferocytosis) to achieve tissue homeostasis. How macrophages may rapidly adapt their transcription to achieve continued corpse uptake is incompletely understood. Transcriptional pause/release is an evolutionarily conserved process wherein RNA polymerase II (Pol II), after initiating transcription for 20-60 nucleotides, gets ‘paused’ for seconds to hours; paused Pol II then gets ‘released’ to complete transcription to make full-length mRNA. Here we show that macrophages, within minutes of corpse encounter, use transcriptional pause/release as a mechanism to unleash a rapid transcriptional response. For human and murine macrophages, the Pol II pause/release was crucial for continued efferocytosis of corpses in vitro and in vivo. Interestingly, blocking Pol II pause/release did not impede FcR-mediated phagocytosis, yeast uptake, or bacterial phagocytosis. Integrating data from three complementary genomic approaches of PRO-seq, RNA-seq, and ATAC-seq on efferocytic macrophages at different time points, we found that Pol II pause/release controls expression of select transcription factors and downstream target genes. Further mechanistic studies on transcription factor Egr3, one of the genes prominently affected by pause/release, uncovered Egr3-related reprogramming of macrophage genes involved in cytoskeleton and corpse processing. Via lysosomal probes and a newly designed genetic fluorescent reporter, we identify a key role for pause/release in phagosome acidification during efferocytosis. Further, microglia from egr3-deficient zebrafish embryos displayed reduced phagocytosis of apoptotic neurons and fewer maturing endosomes, supporting a defect in corpse processing. Collectively, these data advance a novel concept that macrophages use Polymerase II pause/release as a mechanism to rapidly alter their transcriptional programs for efficient processing of the ingested apoptotic corpse and for successive efferocytosis.

Project description:The efficient clearance of dead and dying cells, also known as efferocytosis, is critical to maintain tissue homeostasis. In the bone marrow microenvironment (BMME), this role is primarily fulfilled by professional bone marrow macrophages, but recent work has shown that mesenchymal stromal cells (MSCs) act as a non-professional phagocyte within the BMME. However, little is known about the mechanism and impact of efferocytosis on MSCs in the BMME and on their function. To investigate this, we performed flow cytometric analysis of neutrophil uptake by ST2 cells, a murine bone marrow-derived stromal cell line, and in murine primary bone marrow-derived stromal cells. Transcriptional analysis showed that MSCs possess the necessary receptors and internal processing machinery to conduct efferocytosis, with Axl and Tyro3 serving as the main receptors, while MerTK was not expressed. Moreover, the expression of these receptors was modulated by efferocytic behavior, regardless of apoptotic target. MSCs derived from human bone marrow also demonstrated efferocytic behavior, showing that MSC efferocytosis is conserved. In all MSCs, efferocytosis impaired osteoblastic differentiation. Transcriptional analysis and functional assays identified downregulation in MSC mitochondrial function upon efferocytosis. Experimentally, efferocytosis induced mitochondrial fission in MSCs. Pharmacologic inhibition of mitochondrial fission in MSCs not only decreased efferocytic activity but also rescued osteoblastic differentiation, demonstrating that efferocytosis-mediated mitochondrial remodeling plays a critical role in regulating MSC differentiation. This work describes a novel function of MSCs as non-professional phagocytes within the BMME and demonstrates that efferocytosis by MSCs plays a key role in directing mitochondrial remodeling and MSC differentiation. Efferocytosis by MSCs may therefore be a novel mechanism of dysfunction and senescence. Since our data in human MSCs show that MSC efferocytosis is conserved, the consequences of MSC efferocytosis may impact the behavior of these cells in the human skeleton, including osteoporosis in aging.

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Interpreting an apoptotic corpse as anti-inflammatory involves a chloride sensing pathway

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