CaMKK2 regulates mechanically-induced macrophage fate

Macrophages play crucial roles in immunity and tissue homeostasis, exhibiting diverse phenotypes influenced by their microenvironment. While matrix stiffness is recognized as a key regulator of macrophage fate, the underlying mechanism remains elusive. Here, we investigate primary bone marrow-derived macrophages (BMDMs) in a physiologically relevant 3D environment using a dynamic stiffening collagen-based hydrogel system. We show that increasing matrix stiffness promotes BMDM polarization towards a pro-regenerative phenotype, characterized by elevated CD206 levels, and M2-like gene signature. Mechanistically, we identify the calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) as an important component of molecular machinery controlling BMDM response to stiff microenvironment. Surprisingly, the blocking of CaMKK2 does not interfere with the IL-4-induced pro-regenerative programs. On the contrary, the inhibition of this kinase abolishes phenotypic and transcriptional changes induced by stiffness, highlighting the specific function of the CaMKK2-STAT6 axis in macrophage mechanosensory signaling. Functionally, matrix stiffening promotes M2-like/pro-tumoral polarization of BMDM in a CaMKK2-dependent manner, confirming the critical role of this kinase in the tumor microenvironment and identifying a direct pro-tumoral effect mediated by Camkk2-signaling in macrophages. In a murine wound healing model, stiffened matrices drive immune cell recruitment and macrophage polarization towards a pro-regenerative phenotype, facilitating vascularization, while deletion of Camkk2 impairs this phenomenon. Our findings elucidate the mechanistic basis of matrix stiffness-mediated macrophage fate determination, identifying CaMKK2 as a druggable target to selectively modulate the mechanoresponsiveness of macrophages in malignant and injured tissues.