Signalling pathways governing the transition from monocytes to a lipid-associated macrophage-like state

Background Lipid-associated macrophages (LAMs) are a recently established hallmark of metabolic diseases and the tumour microenvironment. They are identified by expression of specific marker genes and proteins, including GPNMB, SPP1, and TREM2. In vivo cell fate tracing demonstrated that LAMs are recruited to diseased lesions from circulating monocyte pools. However, due to challenges in isolating LAMs and a lack of in vitro models, the intracellular signalling pathways and transcriptional regulators that govern the transition of monocytes into LAMs remain elusive. Methods LAM marker expression was measured in monocytic cell lines (THP-1 and U937) and primary monocytes (mouse bone marrow-derived and human PBMC-derived). THP-1 and U937 cells were treated with phorbol 12-myristate 14-acetate (PMA). Analyses of transcriptomic data sets and pharmacological activators and inhibitors were used to dissect the underlying pathways. Lysosomal acidification, phagocytosis, and lipid handling was assessed with biochemical assays. Public datasets, a reporter cell line, and specific inhibitors were utilised to evaluate the role of nuclear factor-kappa B (NF-κB) and peroxisome proliferator-activated receptor gamma (PPARG). Results We show that induction of monocyte cell lines with the potent protein kinase C (PKC) activator PMA leads to a strong upregulation of LAM marker genes on the mRNA and protein level, mirroring the LAM marker expression observed in vivo in a mouse model of steatohepatitis. Pharmacological PKC inhibition completely repressed cell adhesion and LAM marker expression. Functionally, PKC is necessary for triglyceride storage upon lipid loading, effective lysosomal function, and phagocytosis. Downstream of PKC, focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signalling regulated adhesion and sustained LAM marker expression. On the transcriptional level, NF-κB is crucial for general macrophage differentiation and PPARG as one player in the transcriptional network regulating the LAM program. In primary mouse and human monocytes, FAK-dependent adherence to culture plates is sufficient for strong upregulation of LAM markers. Conclusion Thus, we report a simple model to study LAM biology in vitro and shed light on the intracellular signalling pathways and downstream transcriptional regulation required for a LAM program.