Tumor Cell-released Autophagosomes (TRAP) Induce CCL2 Production Of Mouse Neutrophils And The Influence On Macrophages Chemotaxis

Backgound&Aims:Tumor-associated neutrophils（TAN）play an important role in cancer biology,which can secrete various chemokines and cytokines to affect the chemotaxis and function of other immune cells in tumor microenvironment.Depending on the tumor microenvironment,neutrophils can either promote or inhibit tumor progression.However,whether TRAP can effect the phenotype and function of neutrophils have not been reported yet.Our previous studies have confirmed that tumor cell-released autophagosomes（TRAP）could induce the differentiation of B cells into IL-10⁺regulatory B cells（Bregs）and polarization of macrophages into M2.Herein,the regulation of TRAP on the expression of mouse neutrophil related molecules was investigated.The mechanism that TRAP induced CCL2 production of mouse neutrophils and the influence on macrophages chemotaxis was researched.Methods:1.To detect that TRAP induce CCL2 and IL-10 production of mouse neutrophils1)Mouse neutrophils were isolated from bone marrow by magnetic bead sorting method andthe purity was identified using flow cytometry.TRAP was extracted by differentialcentrifugation and was identified using flow cytometry.2)Mouse neutrophils were co-incubated with CFSE-labeled TRAPs and the phagocytosisratio was detected by flow cytometry.Mouse neutrophils were co-incubated with differentconcentrations of TRAPs for 12 hours or with 3μg/ml TRAPs for different time,thepercentage of apoptotic neutrophils was assessed by flow cytometry.3)After co-incubation of mouse neutrophils with 10μg/ml TRAPs,the expression of relatedmolecules were detected by QRT-PCR;mouse neutrophils were co-incubated with differentconcentrations of TRAPs and the expression of CCL2 and IL-10 was tested respectivelyby QRT-PCR,ELISA or flow cytometry.2.To research the mechanism that TRAP induced CCL2 production of mouseneutrophils and the influence on macrophages chemotaxis1)Neutrophils isolated from WT,TLR2^-/-or TLR4^-/-mice were co-incubated with TRAPs,then the production of CCL2 was assessed using QRT-PCR and ELISA.Neutrophils werepretreated with PI3K,ERK or p38 inhibitors for 1 hours respectively and co-incubated withTRAPs,then the production of CCL2 was assessed using QRT-PCR and ELISA.Neutrophils were co-incubated with TRAPs for 0,15,30,60,120 minutes,then theexpression of AKT,P-AKT,ERK,P-ERK and GAPDH was detected by Western blot.2)RAW264.7 cells were cultured with different culture supernatant of neutrophils and thenthe chemotaxis of RAW264.7 cells were examined using Transwell assay.Anti-CCL2antibody and the isotype antibody were added to culture supernatant of TRAP-inducedneutrophils respectively,the chemotaxis of macrophages was detected by Transwell assay.After constructing lung tumor model of B16F10 cells,the infiltration of macrophages inlung tissue was measured after 3 weeks.Bone marrow-derived macrophages were culturedwith different culture supernatant of neutrophils,and the expression of CD206 and PD-L1of macrophages was tested by flow cytometry.Results:1.The detection that TRAP induce CCL2 and IL-10 production of mouse neutrophils1)The purity of neutrophils isolated from bone marrow by bead sorting method is 99.4%.TRAP（more than 84%purity）can be extracted from hepa1-6 cells culture supernatant bydifferential centrifugation.2)Mouse neutrophils can phagocytized TRAPs and phagocytosis ratio is more than 77.3%after 3 hours co-incubation;TRAP accelerated mouse bone marrow neutrophil apoptosis ina dose-and time-dependent manner.TRAPs led to a greater and faster neutrophil apoptosiscompared with spontaneous apoptosis.3)TRAP induce the expression of IL-10,CCL2,IL-12,ICAM-1,TNF-and IL-6,and reducethe expression of MMP9 and IFN-β.With the TRAP concentration increased,the mRNAexpression and protein levels of CCL2 and IL-10 all increase gradually.The flow cytometryresult shows that the proportion of IL-10⁺neutrophils is increased from 2%to 19.3%.2.The research about mechanism that TRAP induced CCL2 production of mouseneutrophils and the influence on macrophages chemotaxis1)TRAP can promote CCL2 expression of WT or TLR4^-/-mice neutrophils,but can notinduce CCL2 production of TLR2^-/-mouse neutrophils.When neutrophils pretreated withPI3K and ERK inhibitors,the mRNA expression and protein levels of CCL2 aresignificantly reduced.However,when neutrophils pretreated with P38 inhibitor,there is nosignificant change of CCL2 production.TRAP also induce the activation of AKT and ERKsignaling pathway in mouse neutrophils.2)TRAP-induced neutrophils can promote chemotaxis and CD206 expression of macrophagein vitro.The enhancement of TRAP-treated neutrophils on macrophages chemotaxis wasreduced when the culture supernatant was added CCL2 neutralizing antibody.TRAP-induced neutrophils increase macrophage infiltration in lung tissue significantly.Conclusions:1.TRAP can regulate the expression of mouse neutrophils related molecules and promote theproduction of CCL2 and IL-10 significantly.2.TRAP induces CCL2 production depending on binding to TLR2 receptors on mouseneutrophils and activation of PI3K/AKT and ERK signaling pathways.TRAP-inducedneutrophils promote macrophage chemotaxis in vitro and in vivo.