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. To investigate the effect of matrix stiffness on the regulation of macrophages activation programs, we leveraged 3D dynamics hydrogels cultures. Immunoprofiling, transcriptomics, and phospho-proteins analysis were used to determine the effects of matrix stiffness on BMDM. WT and Camkk2-/- BMDM were used to determine the role on CaMKK2 in mechanosensory signaling.