Assessing the impact of phagocytosis on mesenchymal-osteolineage cell function

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. Overall design: Following sacrifice, soft tissue was removed from the bilateral tibiae, femora, and pelvic bones and bones were each cut into 3-4 pieces. Bone pieces were crushed with a mortar and pestle to release bone marrow (BM) into PBS+2%FBS. Bone marrow was passed through a 16G needle to disassociate clumps and pelleted by centrifugation of 1200 RPM for 5 min. Red blood cells were removed via incubation in RBC lysis buffer (156mM NH4Cl, 127µM EDTA, and 12mM NaHC3) for 5 minutes. BM was digested in HBSS containing collagenase type IV (1 mg/mL; Sigma), dispase (1mg/mL, Gibco), and DNase (10 units/mL, New England Biolabs) for 35 min at 37°C. Digested BM was filtered through a 100 µM cell strainer (Corning) and washed with PBS+2%FBS. Cell numbers were determined using the TC20 Automated Cell Counter (Biorad) and Trypan Blue (Sigma-Aldrich) to exclude dead cells. A two-step approach was used to remove hematopoietic cells, first via magnetic-depletion and second via fluorescence-activated cell sorting (FACS). For magnetic-depletion of hematopoietic populations, BM was labeled with biotinylated antibodies against CD45 and lineage markers (Ter119, B220, CD3e, and Gr1) followed by secondary labeling with streptavidin-conjugated magnetic particles (IMag Streptavidin Particles Plus-DM, BD Biosciences). BM was incubated on the BD IMagnet to magnetically separate CD45+ and lineage+ hematopoietic cells from the non-hematopoietic fraction enriched for stromal cells. The stromal cell-enriched fraction was then labeled with PE-CF594 streptavidin, PerCP-Cy5.5 lineage antibodies, APC-Cy7 CD45, FITC CD31, and PE CD51. Cells were labeled with DAPI to exclude dead cells and FACS-purified using a FACSAria II (BD Biosciences) to remove residual hematopoietic cells (lineage+ and/or CD45+) and endothelial cells (CD31+) to obtain lineage- CD45- CD31- CD51+ marrow stromal cells. Sorted marrow stromal cells were seeded in 12-well plates at 1000 cells/cm2 in aMEM (ascorbic acid-free) +10%FBS+1%P/S and incubated in 2%O2/5%CO2/37°C. Media was changed on day 4 of culture initiation and every 3-4 days thereafter. Upon reaching confluence, cells were passaged and expanded in 6-well plates. For passaging, cultures were washed with PBS and treated with TrypLE Express (ThermoFisher Scientific) to detach cells. Equivalent volume of culture media was added and cells were re-plated at ratios ranging from 1:5 to 1:10. Marrow stromal cells were used at passage 2 or 3 for experiments. In all stromal cultures, flow cytometry was used to assess expression of hematopoietic and macrophage markers (lineage, CD45, CD11b, F4/80), endothelial markers (CD31), and stromal markers (CD51, Sca1, CD140a) and confirmed lack of contamination with macrophages and endothelial cells (data not shown). Marrow stromal cultures were grown in 6-well plates prepared as described above. Each well was pre-treated with 1 mL of media containing 5x106 apoptotic thymocytes/well. Primary murine apoptotic thymocytes were isolated and prepared as previously published (46), fluorescently labeled with 20nM efluro670 (ThermFischer) according to manufacturer's instructions. Culture plates were centrifuged for 40s at 100xg and incubated in 5%O2/5%CO2/37°C for 24hr. Control cultures received no target. Stromal cells were washed 3-6x with PBS to remove non-engulfed phagocytic targets. Treated and control cells were then washed 3x with PBS and then collected by FACS-isolated directly in RLT Plus buffer (Qiagen). Both populations were sorted and control as well as target+ cells were isolated.