Dissecting structure and function of the macrophage tetraspan MS4A4A

Membrane-spanning 4A (MS4A) proteins are a new class of immune response regulators, but the molecular mechanisms underpinning their biological functions are poorly understood. MS4A4A is expressed by mast cells and macrophages, in human and mice. MS4A4A is induced during macrophage differentiation, increased in alternative (IL4/dexamethasone) activated macrophages (M2), associated with and regulates membrane distribution and signaling properties of M2/M2-like molecules in tissue-resident macrophages, infiltrating macrophages from inflamed synovium, and tumor-associated macrophages. To achieve a deeper mechanistic understanding of MS4A4A biology in macrophages, we used bone marrow-derived macrophage (BMDM) from Ms4a4a knock-out (KO) animals. Preliminary data showed no difference in BMDM differentiation from wild-type (WT) and KO animals. However, impaired expression of anti-inflammatory genes has been observed in IL4-treated KO BMDM, suggesting a possible role of MS4A4A in fine-tuning macrophage polarization towards a M2-phenotype. To figure out MS4A4A contribution in macrophage polarization, RNA-sequencing has been applied to WT and KO BMDM. At the same time, analysis of cytokines production, extracellular vesicles secretion/uptake, and calcium fluxes has been carried out to assess the biological relevance of the M2-defective phenotype in KO BMDM. Furthermore, to gain insight into MS4A4A partners’ profile, split-ubiquitin two-hybrid approach and mass spectrometry-based immunoprecipitation proteomic has been applied to WT and KO BMDM and identified the γ-signaling chain of Fcγ receptors, Toll-Like Receptor2 (TLR2), and PhosphoDiEsterase1A (PDE1A) as new possible partners. Finally, the structure of MS4A4A has been characterized via machine learning, docking and molecular dynamics to provide further information about its role in macrophage’s biology.