Efferocytosis by bone marrow mesenchymal stromal cells disrupts osteoblastic differentiation via mitochondrial remodeling

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. ST2 cells, a bone marrow derived mesenchymal stromal cell line, were plated at 2x10^4 per cm2 in αMEM without ascorbic acid (Gibco)+ 10% FBS+1% pen-strep and incubated in normoxia at 37°C until 80% confluent. Neutrophils were isolated from human peripheral blood via Mono-Poly resolving medium (MP Biomedicals, Inc) according to manufacturer’s instructions and incubated at -80°C in FBS+ 10% DMSO for a minimum of 18hrs. End-stage neutrophils were washed with PBS and fluorescently labeled with 20μM eFluor670 dye (eBioscience) in PBS at 37°C for 20 min and then washed with RPMI+10% FBS+10mM HEPES to bind free dye. End-stage neutrophils were then given in excess (10:1) to plated MSCs for 3 or 24hrs. Cells were then washed 3x with PBS and collected for isolation via sorting flow cytometry analysis (BD FACSAriaII). ST2 cells and isolated neutrophils were FACS-isolated directly in RLT Plus buffer (Qiagen). RNA extraction was performed with Qiagen RNeasy PLUS Micro kit following standard operating procedures of the URMC Genomic Core. RNA quality was assessed using Agilent Bioanalyzer 2100. One nanogram of high-quality (RNA integrity number >8.0) total RNA from each sample was reverse-transcribed into cDNA using the Clontech SMART-Seq v4 Ultra Low Input RNA Kit. Final Illumina libraries were constructed using 150 pg of cDNA with the Illumina Nextera XT DNA Library Preparation Kit.