MS4A6D Promotes Acute Inflammation Of Macrophages Through SYK

Backgroud:Inflammation is a defense response of the body’s immune system to harmful stimuli,which can remove invasive pathogens,promote tissue repair and maintain homeostasis.However,excessive or persistent inflammation can also cause acute injury or suffer from some chronic diseases.As one of the important members of the immune system,macrophages play a key role in inflammatory response.Due to their diversity and plasticity,macrophages can differentiate into pro-inflammatory M1-type macrophages or anti-inflammatory M2-type macrophages under external environmental stimulation,in which M1-type macrophages can be activated by LPS alone or in conjunction with IFN-γ.Under normal circumstances,there is a dynamic balance between M1 and M2 macrophages type polarization in the human,when this balance is broken by a variety of external stimuli(such as infection,injury,stress),activatory macrophages will result in systemic inflammation and multiple organ failure to damage human body seriously.Therefore,to decipher the molecular mechanism how to regulate macrophage inflammation will help us to deal with the negative effect caused by inflammation.MS4A6D,a member of the MS4 A protein family,consists of four highly hydrophobic transmembrane domains,two extracellular rings,and the N-and C-terminus of the cytoplasm.Our previous study found that VSIG4 can influence the expression of NLRP3 and IL-1β by binding to MS4A6 D on the surface of macrophage membrane,thus playing a crucial role in regulating macrophage inflammation.However,it has been reported that the expression of VSIG4 is significantly down-regulated after macrophage activation under inflammatory conditions,also some researchers have pointed out that,the transcriptional level of Ms4a6 d was significantly increased in mice with experimental autoimmune encephalomyelitis or viral infection.These clues suggest that MS4A6 D may have a unique biological role in macrophage inflammation.In this study,we first identified that MS4A6 D is mainly expressed in macrophages,and constructed MS4A6 D knockout mice to study the function of MS4A6 D.We found that Ms4a6 d knockdown did not affect macrophage development in mice,but made mice significantly resistant to acute inflammatory injury(lipopolysaccharide induced sepsis model and carrageenan-induced paw edema Model).Furthermore,we found that MS4A6 D promotes macrophage-mediated inflammation through hem ITAM motif activating SYK signal.Moreover,to mutate the tyrosine site of hem ITAM motif in MS4A6 D could result in a phenotype identical to that of Ms4a6 d knockout mice in acute inflammatory models.Finally,we screened the small molecule anti-inflammatory compound C1632 against MS4A6D-SYK signal,and confirmed its potential as a clinical anti-inflammatory drug by constructing animal models.Method:The distribution and localization of Ms4a6 d in tissues and organs of C57BL/6 mice were determined by immunohistochemistry and immunofluorescence.Ms4a6 d knockout mice were constructed,and the frequency and number of macrophages and dendritic cells,reactive oxygen species of macrophages and markers of activated macrophage were detected by flow cytometry.Sepsis model was established by intraperitoneal injection of lipopolysaccharide.Serum inflammatory factors were detected by ELISA.Macrophages derived from bone marrow of mice were induced in vitro and the gene transcription level was detected and fluorescence quantitative PCR after Lipopolysaccharide stimulation.Western blot determined the expression of inflammatory factors and phosphorylation of signaling molecules.Co-Immunoprecipitation and Proximity Ligation Assay were used to explicit interaction between proteins.Results:1.MS4A6 D widely exists in tissues and organs of mice and is mainly located in macrophages.Deletion of Ms4a6 d does not affect the development of macrophages,but MS4A6 D deficiency can make mice significantly resistant to LPS-induced sepsis and CGN-induced paw edema;2.MS4A6 D deficiency can reduce the inflammatory level of primary macrophages of mice by binding to the SH2 domain of SYK through the intracellular carboxyl-terminal hem ITAM motif;The point mutation of MS4A6 D at tyrosine site 241 made mice have the same phenotype as Ms4a6 d knockout;3.C1632 can inhibit macrophage inflammation and M1-type polarization,possibly by suppressing MS4A6D-SYK signaling,and significantly alleviates the survival rate in a mouse model of lipopolysaccharide-induced sepsis.Conclusion:During the inflammatory response of macrophages,MS4A6 D can recruit,bind and activate intracellular SYK through hem ITAM motif at the carboxyl terminal of its intracellular segment,thus initiating downstream signal cascade.Small molecule compound C1632 can inhibit macrophage inflammation through MS4A6D-SYK signaling and effectively improve the mortality of LPS-induced sepsis in mice.