Tumor Cell-released Autophagosomes(TRAPs) Induce CCL3 Expression In Neutrophils And The Effect On Neutrophils Function

Our previous studies have found that tumor cells-released autophagosomes(TRAPs)could not only induce the differentiation of B cells into IL-10+ Breg type,but also induce the differentiation of Monocytes/Macrophages into PD-L1 high IL-10+ M2 type,which is a new mechanism for the formation of inhibitory immune cells in tumor microenvironment.Tumor associated neutrophils(TAN)can secrete cytokines,which directly affect the function of immune cells in tumor microenvironment and tumor cells metastasis.Previous study found that TRAPs could induce neutrophils to secrete ROS,which contribute to the inhibition of CD8+ T,CD4+ T cells proliferation and IFN-γ production.In this study,the effect of TRAPs on expressions of neutrophils chemokines and the chemotactic effect on immune cells were further investigated.Objectives: To investigate the effect of TRAPs on expressions of neutrophils chemokines,the corresponding mechanism and function,providing the experimental basis to understand the effect mechanism of TRAPs-induced neutrophils in tumor microenvironment.Methods: 1.Human peripheral blood neutrophils were isolated by density gradient centrifugation and the purity was determined by flow cytometry;mouse bone marrow neutrophils were isolated by magnetic bead separation.TRAPs in the supernatant of B16F10 and MDA-MB-231 tumor cell culture was collected by differential centrifugation,and its purity was measured by Western blot and flow cytometry.2.Mouse neutrophils were co-incubated with TRAPs of B16F10 cells origin(B16F10-TRAPs),and the expressions of mouse neutrophils chemotactic molecules before and after TRAPs treatment were analyzed by RNA sequence.Then,Human neutrophils were co-incubated with TRAPs of MDA-MB-231 cells origin(MDA-TRAPs),the m RNA expressions of human neutrophils CCL2,CCL3,CCL4,CXCL1 and CXCL16 before and after TRAPs treatment were measured by QRT-PCR.The expression of CCL3 was measured by QRT-PCR and ELISA after the co-incubation of MDA-TRAPs at different concentrationswith human neutrophils for different incubation periods.3.Human neutrophils were incubated with MDA-TRAPs for 0,15,30,60 and 120 minutes respectively,the expressions of p65,p-p65,IKBα,p-IKBα,IKKα/β,pIKKα/β and GAPDH proteins at different time points were measured by Western Blot.Human neutrophils were pretreated with NF-κB,p38 and Stat3 inhibitors for 1 hour and then co-incubated with MDA-TRAPs.The expression of CCL3 by human neutrophils before and after inhibitors treatment was measured by QRTPCR and ELISA respectively.4.10 μg/m L MDA-TRAPs was incubated with human neutrophils for 12 hours to obtain cell culture supernatants.THP-1 cells chemotaxis to the above human neutrophils culture supernatants before and after TRAPs treatment was analyzed by Transwell assay;TRAPs-induced human neutrophilic granulocyte culture supernatant was added with CCL3 blocked antibody or isotype control antibody,and then THP-1 cells chemotaxis to the above culture supernatants was analyzed by Transwell assay;then,human neutrophils and PBMC chemotaxis to the above human neutrophils culture supernatants before and after TRAPs treatment were analyzed by Transwell assay.Results: 1.The neutrophil purity was up to 98.9 % when isolated from human peripheral blood by density gradient centrifugation method.The purity of autophagosomes was up to 90.7 % when extracted from the culture supernatant of MDA-MB-231 tumor cells by differential centrifugation.2.After TRAPs treatment,14 mouse chemotatic molecules were upregulated,including CCL2,CCL3,CCL4,CCL5,CXCL1 and CXCL16,etc;8 mouse chemotatic molecules were downregulated,including CCL6,CCL9,etc.While CCL3,CCL4,CXCL1 and CXCL16 expression of TRAPs-treated human neutrophils were upregulated,among which,CCL3 had the biggest increase.However,m RNA expression of CCL2 was not statistically increased.Morever,the expression of CCL3 in human neutrophils induced by TRAPs was correlated with the TRAPs concentration and incubation time.3.TRAPs-induced significant phosphorylation of NF-κB signaling pathway of human neutrophils were observed by Western blot.The TRAPs-induced CCL3 upregulation of in human neutrophils were significantly decreased when the neutrophils were pretreated with NF-κB inhibitors,while pretreatment with p38 and Stat3 inhibitors did not have the decreasement effect.4.Culture supernatant of TRAPs-treated human neutrophils promoted the chemotaxis of THP-1 cells;However,when CCL3 blocked antibody was used,the number of chemotactic THP-1 cells was significantly reduced.Moreover,culture supernatant of TRAPs-treated human neutrophils promoted chemotaxis of human neutrophils and PBMC.Conclusions: 1.Through the NF-κB signaling pathway,TRAPs upregulated CCL3 expression in human neutrophils,which promoted the chemotaxis on THP-1 cells.2.The chemotactic effect of TRAPs-induced human neutrophils on human neutrophils and PBMC were enhanced.