The Role And Mechanism Of IL-32? In Regulating Macrophage Polarization And Immunosuppressive Function In Multiple Myeloma

BackgroundMultiple myeloma(MM)is a B-lymphocyte tumor characterized by the accumulation of malignant plasma cells in bone marrow.MM cells are highly dependent on the bone marrow microenvironment and can modify surrounding immune cells by secreting cytokines or directly contacting,creating an immunosuppressive microenvironment that is conducive to tumor growth.Macrophages(M?s)are abundant in bone marrow of MM patients,which can promote the growth,proliferation and drug resistance of tumor cells,and participate in the formation of immunosuppressive microenvironment.Our previous study found that the expression of interleukin 32(IL-32)in MM patients was significantly higher than that in healthy controls,and could promote the proliferation of MM cells through bone marrow stromal cells.However,there is no related research on the effect of IL-32 on M?s in MM microenvironment.The purpose of this study was to explore the regulation and molecular mechanism of IL-32 on the phenotype and function of M?s,as well as the final effect on the proliferation and drug resistance of MM cells.Objective1.To clarify the expression of IL-32 in patients with MM and its clinical significance.2.To explore the effect of IL-32 on the phenotype and function of M?s and its mechanism,as well as its effect on drug resistance of MM.3.To explore the effect of IL-32 on the immunosuppressive function of M?s,and then on the proliferation and function of CD4+T cells.Methods1.The expression of IL-32 in bone marrow of patients with MM was detected by immunohistochemistry,and the expression and activity of IL-32 subtypes in MM cells were detected by qRT-PCR and western blot.2.Flow cytometry,cytokine microarray,qRT-PCR and western blot were used to detect the expression of M1 and M2 characteristic surface molecules,cytokines and metabolic enzymes in M?s stimulated by IL-32?.3.IL-32?-educated M?s was co-cultured with MM cells,the apoptosis of MM cells was detected by flow cytometry,and the apoptotic protein in MM cells was detected by western blot.The role of IL-32?-educated M?s in MM drug resistance was verified in NSG mice.4.Western blot,qRT-PCR and immunofluorescence were used to detect the expression of indoleamine 2,3 dioxygenase(IDO)in M?s after the treatment of MM cells or IL-32?,and the changes of the above phenomena were observed after knocking down IL-32 in MM and Proteinase 3(PR3)in M?s with short hairpin RNA(shRNA)and small interference RNA(siRNA).5.CFSE tracing was used to detect the proliferation of CD4+T cells,and flow cytometry was used to detect the vitality of CD4+T cells and the expression of cytokines.Results1.IL-32 was highly expressed in MM patients,which was positively correlated with ISS stage and serum ?2-microglobulin.IL-32? was a higher content and the most active subtype in MM cells.2.IL-32? can induce polarization of M2 M?s,enhance the protective effect of M?s on MM cells and induce drug resistance,which can be weakened by CSF-1R inhibitors.3.In the tumor model of NSG mice,IL-32?-educated M?s could reduce the drug sensitivity of MM cells to Bor and increase the tumor burden.4.IL-32? can promote the expression of immunosuppressive molecule IDO in M?s in a concentration-and time-dependent manner.5.MM cells can up-regulate the IDO expression in M?s,and this effect was weakened when IL-32 in the MM cells was knocked down by shRNA.6.PR3 is generally expressed on the surface of M?s and can bind to IL-32.After PR3 in M?s is knocked down by siRNA,the expression of IDO induced by IL-32? will decrease.7.IL-32? could obviously activate STAT3 and NF-?B pathways in M?s.After using inhibitors of the corresponding pathways,the ability of IL-32? to induce IDO expression is significantly reduced.8.IL-32?-educated M?s could inhibit the proliferation of CD4+T cells and the expression of IL-2,IFN-? and TNF-?.ConclusionIn this study,we found that IL-32 was highly expressed in patients with MM,and it was significantly correlated with ISS stage and serum ?2-microglobulin.In microenvironment,IL-32? can induce polarization of M2 M?s by up-regulating the expression of M-CSF,and then enhance the protective effect of M?s on MM cells and induce drug resistance of MM.In addition,MM cell-derived IL-32? up-regulates the expression of IDO in M?s by binding to PR3 receptor and activating STAT3 and NF-?B signal pathways,thereby inhibiting the proliferation and function of CD4+T cells and promoting the formation of immunosuppressive bone marrow microenvironment.