The tetraspan MS4A4A modulates macropage activation

Background: Membrane-spanning 4A (MS4A) proteins are differentially expressed in different leukocytes subsets and evidence suggests that these proteins contribute to shape immune cell activation by working as ion channels or by modulating the signaling of other immunoreceptors. We showed that MS4A4A is selectively expressed by macrophages, in particular by alternatively (IL-4)-activated and tumor-associated macrophages, and that its expression is upregulated upon treatment with glucocorticoids (Dexamethasone, Dex). However, the molecular mechanisms by which MS4A4A exerts its biological function in macrophages remains largely undefined. Recent publications suggest MS4A4A interaction with several partners. In particular, we showed that the association of MS4A4A with the β-glucan receptor Dectin-1 is required for the Dectin-1-dependent signaling and for initiating macrophage inflammatory response. Therefore, MS4A4A can control macrophage activation by modulating the signaling of other immune receptors. Aims: Our aim is to characterize the biological function of MS4A4A and its contribution to the modulation of the signaling pathways of its partners, achieving a deeper mechanistic understanding of MS4A4A biology in macrophages and further disclose its contribution to immune responses. Methods: MS4A4A partners were identified by split-ubiquitin two‐hybrid analysis and confirmed by Co-Immunoprecipitation (Co-iP). In vitro experiments with Bone Marrow Derived Macrophages (BMDM) from Wild Type (WT) and MS4A4A Knock Out (KO) mice were performed to compare macrophage activation by the evaluation of macrophages gene (qPCR) and protein (Western Blot) expression. Results: Our preliminary data showed that MS4A4A deficiency impaired the expression of Arg1 and Cd206 (anti-inflammatory genes) in BMDM while Cox2 (inflammatory gene) levels were increased, suggesting the acquisition of an inflammatory phenotype in MS4A4A-KO macrophages. A split-ubiquitin two-hybrid analysis, identified the γ signalling chain of Fcγ Receptors (FcεRI) as a new potential MS4A4A partner. To validate this, we set-up a Co-iP system and our data showed no association of MS4A4A with FcεRI in resting BMDM and in BMDM treated with glucocorticoids (Dex) or with Fc receptor ligands (Agg-IgG). However, we observed a reduction of Syk phosphorylation in MS4A4A-KO compared to WT upon FcεRI engagment with Agg-IgG. Our preliminary data also showed that the expression of FcεRI protein was increased in Ms4a4a-KO BMDM compared to WT, whereas FcεRI expression was down regulated when both WT and Ms4a4a-KO BMDM were treated with Dex, although this downregulation was more pronounced in Ms4a4a-KO then WT control. Conclusions: These results suggest that MS4A4A may regulate the FcεRI signaling, supporting Syk phosphorylation through a still unknown, likely indirect, mechanisms. We are currently investigating MS4A4A role in macrophage’s biology and the possible mechanisms by which it could modulate FcεRI signaling pathway.