Next generation sequencing of IL-10 stimulated (M2c) macrophages. Homo sapiens

Alternatively activated “M2” macrophages are commonly believed to function at late stages of wound healing, behaving in an anti-inflammatory manner to mediate the resolution of the pro-inflammatory response caused by “M1” macrophages. However, the differences between two main subtypes of M2 macrophages, namely interleukin-4 (IL4)-stimulated “M2a” macrophages and IL10-stimulated “M2c” macrophages, are not well understood. M2a macrophages are characterized by the secretion of anti-inflammatory cytokines and the stabilization of angiogenesis. However, the role and temporal profile of M2c macrophages in wound healing are not known. Therefore, we performed next generation sequencing (RNAseq) to identify biological functions and gene expression signatures of M2c macrophages compared to M1 and M2a macrophages plus an unactivated control (M0). Following validation by Nanostring, 18 genes were found to be upregulated by M2c macrophages compared to the other phenotypes (using an adjusted p-value cutoff of 0.05 and fold change of 1.5). Many of these genes are associated with angiogenesis and matrix remodeling, including CD163, TIMP1, SERPINA1, VCAN, and MMP8. Analysis of the macrophage-conditioned media for secretion of matrix-remodeling proteins showed that M2c macrophages secreted higher levels of MMP7, MMP8, and TIMP1 compared to the other phenotypes. Surprisingly, temporal gene expression analysis of a publicly available microarray data set of human wound healing showed that M2c-related genes were upregulated at early times after injury, similar to M1-related genes, while M2a-related genes appeared later or were downregulated. While further studies are required to confirm the timing and role of M2c macrophages in vivo, these results suggest M2c macrophages function at early stages of wound healing. Identification of markers of the M2c phenotype will allow more detailed investigations into the role of M2c macrophages in vivo.