The tetraspan MS4A4A modulates macrophage activation

Background: Membrane-spanning 4A (MS4A) proteins are differentially expressed in different leukocytes subsets and evidence suggest that these proteins contribute to shaping immune cell activation by working as ion channels or by modulating the signaling of other immunoreceptors. We have focused our attention on MS4A4A protein that is overexpressed in alternative (IL4)-activated and tumor-associated macrophages and upon treatment with glucocorticoids (dexamethasone). However, the molecular mechanisms underlying MS4A4A activities and its biological function in macrophages remains largely undefined. Recent publications identified MS4A4A interaction with several partners (TREM2, MS4A6A, MS4A7, Dectin-1). The association of MS4A4A with Decin-1 is required for Dectin-1-dependent signaling in macrophages and for the consequent functional properties. Therefore, MS4A4A can control macrophage activation by modulating the signaling of its interacting proteins. Aims: Our aim is to characterize the biological function of MS4A4A and its involvement in the pathways of its partners, achieving a deeper mechanistic understanding of its role in macrophages to further understand its contribution in the immune response. Methods: In vitro experiments were performed using Bone Marrow Derived Macrophages (BMDM) from Wild Type (WT) and MS4A4A Knock Out (KO) mice that were compared in terms of gene expression (qPCR) and protein (Western Blot) levels. MS4A4A partners were identified by split-ubiquitin two‐hybrid and Co-Immunoprecipitation (Co-iP). Results: Based on our preliminary data, MS4A4A deficiency impairs expression of Arg1 and Cd206 (anti-inflammatory marker genes) in BMDM and Cox2 levels are increased, suggesting the acquisition of an inflammatory phenotype in MS4A4A-KO macrophages. Using a split-ubiquitin two-hybrid analysis, we identified the γ signalling chain of Fcγ Receptors (FcεRI) as a new MS4A4A partner. To validate this interaction, we set-up a Co-iP system and our data show no association between MS4A4A and FcεRI in not treated, Dex-treated and Agg-IgG-treated conditions. Nevertheless, we observed a reduction in Syk phosphorylation in MS4A4A-KO compared to WT. Furthermore, our preliminary data show that FcεRI protein is more expressed in resting macrophages from Ms4a4a-KO mice compared to WT and Dexamethasone down-regulates FcεRI protein levels both in WT and Ms4a4a-KO macrophages but the reduction is more significant in Ms4a4a-KO compared to WT. Conclusions: These results suggest that MS4A4A may be involved in FcεRI signaling, supporting Syk phosphorylation through a still unknown, likely indirect, mechanisms. Ongoing efforts will be helpful to dissect MS4A4A engagement in macrophage biology and in FcεRI pathway.