Caspase-9 driven murine model of selective cell apoptosis and efferocytosis

Abstract

Apoptosis and efficient efferocytosis are integral to growth, development, and homeostasis. The heterogeneity of these mechanisms in different cells across distinct tissues renders it difficult to develop broadly applicable in vivo technologies. Here, we introduced a novel inducible caspase-9 (iCasp9) mouse model which allowed targeted cell apoptosis and further facilitated investigation of concomitant efferocytosis. We generated iCasp9+/+ mice with conditional expression of chemically inducible caspase-9 protein that is triggered in the presence of Cre recombinase. In vitro, bone marrow cells from iCasp9+/+ mice showed expression of the iCasp9 protein when transduced with Cre-expressing adenovirus. Treatment of these cells with the chemical dimerizer (AP20187/AP) resulted in iCasp9 processing and cleaved caspase-3 upregulation, indicating successful apoptosis induction. The in vivo functionality and versatility of this model was demonstrated by crossing iCasp9+/+ mice with CD19-Cre and Osteocalcin (OCN)-Cre mice to target CD19+ B cells or OCN+ bone-lining osteoblasts. Immunofluorescence and/or immunohistochemical staining in combination with histomorphometric analysis of EGFP, CD19/OCN, and cleaved caspase-3 expression demonstrated that a single dose of AP effectively induced apoptosis in CD19+ B cells or OCN+ osteoblasts. Examination of the known efferocytes in the target tissues showed that CD19+ cell apoptosis was associated with infiltration of dendritic cells into splenic B cell follicles. In the bone, where efferocytosis remains under-explored, the use of iCasp9 provided direct in vivo evidence that macrophages are important mediators of apoptotic osteoblast clearance. Collectively, this study presented the first mouse model of iCasp9 which achieved selective apoptosis, allowing examination of subsequent efferocytosis. Given its unique feature of being controlled by any Cre-expressing mouse lines, the potential applications of this model are extensive and will bring forth more insights into the diversity of mechanisms and cellular effects induced by apoptosis including the physiologically important efferocytic process that follows.