MRP8/14 Inhibits Macrophage Efferocytosis Via Gas6/MFG-E8 Or RAGE-Related Pathways

Efferocytosis means phagocytes engulf apoptotic cells.Removal of apoptotic cells from inflammatory sites by macrophages is an important step to reduce the number of apoptosis cells and avoid their secondary necrosis,which promotes the resolution of inflammation and tissue repair.MRP8/14 is released by immunocytes and plays an important role in inflammation as a key DAMP molecule.Actually,efferocytosis of macrophages is closely related to its phenotypes.M2 macrophages are anti-inflammatory and have a more affinity of efferocytosis,while efferocytosis will weaken and the ability to swallow the bacteria will strengthen if they polarize toward M1 macrophages.However,the effects of MRP8/14 on efferocytosis remains to be clarified.We added apoptotic thymocytes into mouse bone marrow-derived macrophages protreated with MRP8/14 and then the capacity of efferocytosis was detected.To determin the phenocype polarization of macrophages after the treatment of MRP8/14,the expression of surface markers(F4/80,CD11c and CD206),cytokines(TNF-α and IL-10)and phenotyperelated genes(iNOS,Argl,Chi3l3 and Mrc1)were tested.We also verified the effect of MRP8/14 on swallowing bacteria by macrophages.Using signal pathway inhibitors to test whether MRP8/14 regulates efferocytosis and phenotype polarization of macrophages via NF-κB or MAPKs signal pathway respectively.To test the function of RAGE,TLR4 and TLR2 in MRP8/14-induced macrophage efferocytosis and phenotype polarization,RAGE-/-,TLR4-/-and TLR2-/-mice were used.After being treated with MRP8/14,Gas6 and MFG-E8 expression of macrophages were detected.We also tested the effects of Gas6 and MFG-E8 on MRP8/14-induced macrophage efferocytosis.Lastly,we explored the related signal pathway in MRP8/14-mediated Gas6 and MFG-E8 expression.The results showed that MRP8/14 inhibited the efferocytosis of macrophages in timedependent and dose-dependent manners.We found that mouse bone marrow-derived macrophages were M2 macrophages(F480+ CD206+),and MRP8/14 could induce them polarize toward M1 macrophages(F480+CD11c+).The expression of signature Ml-marker genes(TNF-α and iNOS)incresased simultaneously while the expression of signature M2marker genes(Arg1,Chi3l3 and Mrc1).had decreased.However,when the NF-κB or MAPKs signal pathway were inhibited or TLR4 was kockout,we found that the ability of MRP8/14 to regulate macrophage phenotype polarization was inhibited while it had no effects on MRP8/14-regulated inhibition of efferocytosis.Conversely,when RAGE was kockout,we found that the ability of MRP8/14 to inhibit the macrophage efferocytosis was inhibited while it had no effects on MRP8/14-regulated phenotype polarization.It means that inhibition of macrophage efferocytosis was mediated through RAGE-related pathway and it was no related to MRP8/14-regulated phenotype polarization.With further exploration of the mechanism of MRP8/14-induced inhibition of efferocytosis,we found that MRP8/14 could significantly reduce the expression of bridging protein Gas6 and MFGE8 in macrophages,meanwhile we had verified the function of Gas6 and MFG-E8 in MRP8/14-induced inhibition of efferocytosis.Interestingly,we found that MRP8/14induced decrease of Gas6 and MFG-E8 gene expression was not relevant to RAGE,which means that there may exist two or more independent signal pathways that regulate MRP8/14-induced inhibition of efferocytosis.Therefore,we conclude that MRP8/14 can significantly inhibit macrophages efferocytosis,which is dependent on RAGE-related pathway or MRP8/14-induced reduction of the expression of Gas6 and MFG-E8.Although MRP8/14 can induce macrophage phenotype polarization via TLR4/NF-κB/p38 signal pathway,it has no influence on MRP8/14-induced inhibition of efferocytosis.