Validation of CSF-1 receptor (CD115) staining for analysis of murine monocytes by flow cytometry.

CD115, the receptor for colony stimulating factor 1, is essential for survival and differentiation of monocytes and macrophages and is therefore frequently used to define monocyte subsets and their progenitors in immunological assays. However, CD115 surface expression and detection by flow cytometry is greatly influenced by cell isolation and processing methods, organ source, and disease context. In a systematic analysis of murine monocytes, we define experimental conditions that preserve or limit CD115 surface expression and staining by flow cytometry. We also find that, independent of conditions, CD115 surface levels are consistently lower in Ly6Clo monocytes than in Ly6Chi monocytes, with the exception of Ly6Clo monocytes in the bone marrow. Furthermore, in contrast to IL-34, the presence of colony stimulating factor 1 impairs CD115 antibody staining in a dose-dependent manner, which, in a model of ischemic kidney injury with elevated levels of colony stimulating factor 1, influenced quantification of kidney monocytes. Thus, staining and experimental conditions affect quantitative and qualitative analysis of monocytes and may influence experimental conclusions.

CSF-1 and Notch signaling cooperate in macrophage instruction and tissue repair during peripheral limb ischemia.

Ischemia causes an inflammatory response featuring monocyte-derived macrophages (MF) involved in angiogenesis and tissue repair. Angiogenesis and ischemic macrophage differentiation are regulated by Notch signaling via Notch ligand Delta-like 1 (Dll1). Colony stimulating factor 1 (CSF-1) is an essential MF lineage factor, but its role in ischemic macrophage development and the interaction with Notch signaling is so far unclear. Using a mouse model of hind limb ischemia with CSF-1 inhibitor studies and Dll1 heterozygous mice we show that CSF-1 is induced in the ischemic niche by a subpopulation of stromal cells expressing podoplanin, which was paralleled by the development of ischemic macrophages. Inhibition of CSF-1 signaling with small molecules or blocking antibodies impaired macrophage differentiation but prolonged the inflammatory response, resulting in impaired perfusion recovery and tissue regeneration. Yet, despite high levels of CSF-1, macrophage maturation and perfusion recovery were impaired in mice with Dll1 haploinsufficiency, while inflammation was exaggerated. In vitro, CSF-1 was not sufficient to induce full MF differentiation from donor monocytes in the absence of recombinant DLL1, while the presence of DLL1 in a dose-dependent manner stimulated MF differentiation in combination with CSF-1. Thus, CSF-1 is an ischemic niche factor that cooperates with Notch signaling in a non-redundant fashion to instruct macrophage cell fate and maturation, which is required for ischemic perfusion recovery and tissue repair.