REGULATION AND FUNCTION OF THE TETRASPANIN-LIKE MOLECULE MS4A4A IN ALTERNATIVELY ACTIVATED AND TUMOR-ASSOCIATED MACROPHAGES

Macrophages are a heterogeneous population of the innate immune system operating at the boundary between health and disease. They represent the most plastic elements of the hematopoietic system, they are found in all tissues, and their main sake is the response to pathogens by processing and presenting the antigens to the adaptive immune mediators. Among their functions macrophages mediate generation and resolution of inflammation, tissue repair and homeostasis maintenance [1]. These cells undergo specific differentiation based on the local microenvironment [2] and the presence of cytokines [3]. In accordance with the Th1/Th2 polarization, two extreme states of macrophages polarization can be defined: the classically activated, pro-inflammatory M1 macrophage phenotype induced by IFNγ and LPS and the alternatively activated anti-inflammatory M2 macrophage phenotype [4, 5]. Among all the intermediate subpopulations of activating and suppressive macrophages [6], tumor associated macrophages (TAM) embody a peculiar M2-like subset favoring tumor progression and survival, angiogenesis and metastatization, making of these cells a negative prognostic index in many malignances. Membrane spanning 4-domains subfamily A (MS4A) is a newly described tetraspanin-like protein family, including 26 proteins in human and mice, whose members have been poorly characterized, with few exceptions [7, 8]. Most of them have been demonstrated playing a role in the immune system, either functioning as adaptor molecules for pivotal receptors (e.g. CD20/MS4A1-BCR; MS4A4B -TCR), or clustering complexes on the plasma membrane, or regulating the cell cycle. In the present study we identified and characterized a novel MS4A protein associated with alternative macrophage polarization, in two different contexts: TAMs association and fungal infections. MS4A4A is indeed a transmembrane molecule expressed by a special subset of M2 macrophages induced by glucocorticoids. The topology of the protein resembles the one of the other MS4A members, since it is re-localizes within lipid raft microdomains only upon macrophage activation and its surface expression is abolished upon cholesterol depletion. Also the murine homologue we identified as Ms4a4a shares the same features of the human one. The target has been found in human samples of Hodgkin’s lymphoma (HL) with a characteristic distribution, and its abundance varies among different types of HL. However, no or very faint staining resulted from healthy tissues, such as lymph nodes and spleen, suggesting the molecule might be implicated in both tumorgenesis and fibrosis. Evidences of MS4A4A association with TAM also derive from the murine models in vivo. Its distribution and topology makes MS4A4A an ideal target for antibody, potentially with a therapeutic significance. In the attempt of generating a human anti-human antibody directed against MS4A4A, we identified a clone able to recognize the human and cross-reacting with the murine protein, mediating cytotoxicity of the target bearing cells. Unfortunately, the tool turned out not being able to recognize the native protein in primary cells, given that the screening was originally performed against transfectants expressing the recombinant MS4A4A. Trying to uncover potential partners for the molecule, split-ubiquitin genetic screening was performed using MS4A4A as bait on a GC treated macrophages library. The screening showed interactions between MS4A4A and other two MS4A members, MS4A6A, MS4A7, and two tetraspanins, CD63 and CD9, suggesting the existence of platforms resembling the tetraspanin enriched microdomains, where these proteins might gather to facilitate the formation of signaling complexes. Furthermore, the molecule turned out interacting with the b-glucan receptor Dectin1, indicating a possible role in orchestrating the responses to fungal and yeast infections [9]. The relationship with Dectin1 was assessed making use of the Dectin1 ligand Zymosan, and revealed an inverse correlation between the two molecules. An increase in MS4A4A at the surface of the Zymosan treated macrophages corresponds in fact to an internalization of the b-glucan receptor, and occur only in the presence of the particulate polymer, not the soluble one, underlying the specificity of the interaction. The evidence that in resting conditions neither MS4A4A nor Dectin1 are localized within lipid raft, MS4A4A becomes co-localized upon GC, opposite to Dectin1, which instead becomes upon Zymosan engagement when the gathering of a receptor complex is required, suggest a modulatory role for the tetraspanin. This putative role became even more solid in vivo, where the response to the β-glucan in the KO macrophages was severely impaired both in terms of cytokines production and induction of the respiratory burst, and was observed along with a reduced internalization of Dectin1 upon stimulation. Although further investigations need to be done, our results strongly suggest MS4A4A may act as a regulatory adaptor for Dectin1 during the recognition of pathogens.