To investigate the role of CXCL4 in activation of macrophages in vitro and in vivo

CXCL4 activates myeloid cells and augments endosomal TLR responses contributing to the pathogenesis of inflammatory and fibrotic diseases. However, the underlying mechanisms are not well defined because the receptor(s) that mediates CXCL4 inflammatory responses is not known and downstream signaling pathways leading to gene induction are not well characterized. Here we report that CXCL4 activates inflammatory and tissue repair/fibrotic gene expression in human primary macrophages ex vivo and regulates macrophage skin infiltration and promotes skin wound healing in vivo. Overall design: In vitro experiment, human CD14+ monocytes were harvested from PBMCs and cultured with 20 ng/ml M-CSF overnight. The cells were then stimulated with 10 ug/ml CXCL4 for 1 h and 3 h. The cells were then harvested for RNA-seq. In vivo experiment, skin wound from WT littermates and Cxcl4-/- mice were harvested. Two to three wounds in each genotype were pooled together and digested. Then macrophages in the cell suspension were sorted based on Ly6C and F4/80 expression. The sorted cells were used for RNA-seq to investigate how CXCL4 affect macrophage activation in vivo.