Dissecting structure and function of the macrophage testraspan MS4A4A

Membrane-spanning 4A (MS4A) proteins are emerging as a new class of immune cell regulators, but the molecular mechanisms underpinning their biological functions are currently poorly characterized. In the recent years, we focused our attention on MS4A4A, which shows a pattern of expression restricted to mast cells and macrophages, both in human and mice. MS4A4A expression is induced during macrophage differentiation and is detected in tissue resident macrophages and in some body districts (CNS, colon, lungs, skin). In vitro MS4A4A expression is increased in “alternative activated” macrophages (i.e. IL-4, glucocorticoids) and is found highly expressed in some pathological settings (rheumatoid arthritis) and in tumor-associated macrophages (colon carcinoma, lung adenocarcinoma, melanoma). Here, we aim to reveal the role of MS4A4A in macrophage biology, achieving a deeper mechanistic understanding of its function in macrophages and disclosing its contribution to immune responses. Our preliminary data showed no difference during bone marrow-derived macrophage (BMDM) generation from wild-type (WT) and Ms4a4a knock-out (KO) animals. However, MS4A4A deficiency impaired the expression of anti-inflammatory genes in BMDM after stimulation with IL-4, suggesting that this protein is involved in the acquisition of an alternative phenotype. To define MS4A4A contribution in macrophage polarization, RNA sequencing of WT and KO BMDM has been performed. To deeper investigate the biological relevance of the defect in “alternative activation” observed in Ms4a4a-KO macrophages, cytokines production, extracellular vesicles secretion and uptake and calcium fluxes upon different stimuli has been evaluated. As other tetraspan proteins, MS4A4A forms omo- and ethero-complexes. Indeed, recent publications identified several partners for MS4A4A, including MS4A4A itself, MS4A6A, MS4A7, TREM2 and Dectin-1 and they showed that MS4A4A influences macrophage functions by regulating membrane distribution and signaling properties of its partners. Interestingly, using a split-ubiquitin two-hybrid approach and mass spectrometry-based immunoprecipitation proteomics, we identified new possible partners for MS4A4A, including the γ signaling chain of Fcγ receptors, Toll-Like Receptor 2, and calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1A. Furthermore, we are computationally characterizing MS4A4A structure and dynamics aiming to understand its function in macrophages, also by means of an extensive molecular docking calculations campaign with some families of endogenous and exogenous bioactive compounds.