DISSECTING THE ROLE OF THE TETRASPANIN MS4A4A IN MODULATING MACROPHAGE PLASTICITY AND FUNCTION

MS4A4A belongs to the membrane-spanning 4A (MS4A) family, whose proteins show preferential expression in distinct cell types and structural similarities to tetraspanins, including the ability to interact with and modulate the activity of various immunoreceptors, such as pattern recognition receptors (PRRs) and Ig receptors. MS4A4A is selectively expressed by macrophages, and its expression is upregulated upon treatment with Interleukin-4 (IL-4) and glucocorticoids (Dexamethasone, Dex). However, the molecular mechanisms by which MS4A4A exerts its biological function in macrophages remains largely uncharacterised. MS4A4A has been increasingly studied in relation to several diseases with inflammatory components. Specifically, it has been implicated in Alzheimer’s disease, potentially affecting microglial activation. In the context of cancer, we have previously reported that MS4A4A is required for the macrophage-dependent activation of NK cell anti-metastatic functions. Recently, we have shown that MS4A4A+ macrophages are detected in the synovial tissue of rheumatoid arthritis (RA) patients. Although less extensively studied, MS4A4A expression was associated with inflammatory diseases such as sepsis, atherosclerosis, and inflammatory bowel disease. Macrophages are key players in the pathogenesis of these diseases due to their ability to shift between pro-inflammatory and anti-inflammatory phenotypes, allowing them to drive tissue damage or promote repair. In this context, the present study aimed to characterize the biological function of MS4A4A in macrophages and its involvement in macrophage activation and function. Firstly, the structure of MS4A4A was investigated and characterised via artificial intelligence analysis and molecular dynamics simulation, to clarify its role in cellular signaling and potentially linking its structure to specific biological outcomes. Then, having observed only minimal differences in the transcriptional profiles between WT and KO macrophages, further investigation focused on the role of MS4A4A in macrophage activation. This led to the identification of two potential partners: the γ-chain of Fc gamma receptors (FcγRs) and Toll-like receptor 2 (TLR2), suggesting a possible involvement of MS4A4A in their inflammatory signaling. In the context of RA, where macrophages show altered FcγRIII expression, MS4A4A expression was found to be upregulated and positively correlated with disease pathogenesis, as well as with FcγRIII and the FcγR γ-chain expression in RA patients. Notably, both MS4A4A and FcγRIII are induced by Dex. MS4A4A was shown to limit the anti-inflammatory effects of Dex during FcγR-mediated inflammation by affecting the release of TNF-α and CXCL2. This modulatory role of MS4A4A appears to be independent on direct influence on FcγR signaling, and the precise mechanism remains to be defined. To dissect the role of MS4A4A in macrophage activation mediated by TLR-2, we found that MS4A4A regulates the surface expression of TLR2 and affects the release of the anti-inflammatory cytokine IL-10. This impairment in IL-10 release is not limited to TLR2 stimulation but is also observed with other TLR agonists. Notably, during LPS stimulation, the reduced IL-10 release persists over time, starting as early as 2 hours post-stimulation. Further analysis showed that LPS induces MS4A4A expression and led to the identification of an IL-10 storage pool in resting macrophages. The defect in IL-10 release may result from a role of MS4A4A in regulating IL-10 de novo synthesis and/or the mobilization of this intracellular IL-10 pool. This study provides a valuable contribution to the field, expanding the current knowledge of MS4A4A’s role in macrophage activation and its impact on inflammatory response.