Tumor Cell-Released Autophagosomes (TRAP) Induce Apoptosis And Biological Functions Of Neutrophils

Our previous studies have confirmed that tumor cell-released autophagosomes?TRAP?could induce the differentiation of B cells into IL-10⁺regulatory B cells?IL-10⁺Bregs?with suppressive activities on T lymphocytes via the TLR2-MyD88-NF-?B pathway.TRAP could also induce the differentiation of monocytes/macrophages into PD-L1^high M2 like macrophages with suppressive activities on T lymphocytes via the TLR4-MyD88-p38pathway.However,whether TRAP can regulate the immunomodulatory effect on human neutrophils is still largely unclear.ObjectivesTo investigate whether TRAP can regulate neutrophil cell biological behavior;to study the molecular mechanism of neutrophil apoptosis induced by TRAP;to preliminary discuss whether TRAP-induced neutrophils can regulate the functions of T cells and tumor cells and their mechanism.Methods1.Investigation of regulating neutrophil cell biological behavior by TRAPHuman neutrophils were cultured with different concentrations of CFSE-labeledTRAPs for 3 h;or CFSE-labeled TRAPs?3?g/mL?for different time;or withCFSE-labeled TRAPs?3?g/mL?at 4 or 37°C for 3 h.Flow cytometric analysis wasperformed to identify the phagocytizing of TRAPs by neutrophils.Human neutrophilswere cultured with different concentrations of TRAPs for 6 h;or TRAPs?3?g/mL?fordifferent time;or with autophagosomes,enriched from malignant effusions of lung canceror breast cancer patients or ascites of ovarian cancer patients for 6 h.The apoptosis ofneutrophils was assessed by flow cytometry.2.Study on the molecular mechanism of TRAP phagocytosed by neutrophil and induceneutrophil apoptosisNeutrophils were pre-treated with CD?10?g/mL?,CPZ?4?g/mL?,Filipin III?12?g/mL?or EIPA?20?M?for 30 min,and then co-cultured with TRAPs?3?g/mL?for 3 h.The phagocytizing of TRAPs by neutrophils was detected by flow cytometry.Neutrophils were pre-treated with CD?10?g/mL?,CPZ?4?g/mL?,Filipin III?12?g/mL?or EIPA?20?M?for 30 min,and then co-cultured with TRAPs?10?g/mL?for 6 h.The apoptosis of neutrophils was assessed by flow cytometry.Neutrophils were cultured with different concentrations of TRAPs for 30 min;or pre-treated with DPI?20?M?or NAC?7.5 mM?for 30 min and co-incubated with or without TRAPs?10?g/mL?for 30 min.The intracellular ROS and apoptosis of neutrophils were detected by flow cytometry.Neutrophils were pre-treated with CD?10?g/mL?,EIPA?20?M?or DPI?20?M?for 30min and then co-incubated with TRAPs?10?g/mL?for 24 h.Caspase-3 activities were analyzed.Neutrophils were pre-treated with zVAD-fmk?50?M?for 30 min and co-incubated with TRAPs?10?g/mL?for 6 h.The apoptosis of neutrophils was detected by flow cytometry.3.Investigation of TRAP-induced neutrophils regulate the functions of T cells andtumor cells and their mechanism3.1 Investigation of TRAP-induced neutrophils regulate the functions of T cells and their mechanismCFSE-labeled PBMCs were stimulated in 48 well plate pre-coated with anti-CD3/anti-CD28 and then they were co-cultured with or without neutrophilspre-incubated with different concentrations of TRAPs at ratio of 1:2 for six days.CFSEdilution was determined by flow cytometry.Neutrophils were co-incubated with TRAPs?30?g/mL?for 30 min.PBMCs were co-incubated with plates pre-coatedanti-CD3/anti-CD28 and then they were co-cultured with or without neutrophils at ratio of1:2 for three days.The percentage of IFN-?⁺T cells was determined by flow cytometry,the IFN-?in the supernatant was detected by ELISA and the activation markers?CD69and CD137?and exhaustion markers?PD1 and CTLA4?of CD4⁺T cells and CD8⁺T cellswere determined by flow cytometry.Neutrophils were pre-treated with NAC?7.5 mM?for30 min and then co-incubated with TRAPs?30?g/mL?for 30 min.CFSE-labeled PBMCswere co-incubated with plates pre-coated anti-CD3/anti-CD28 and then they wereco-cultured with or without neutrophils at ratio of 1:2 for six days.CFSE dilution wasdetermined by flow cytometry.In the Transwell experiment,neutrophils andCFSE-labeled PBMCs were added in the upper and lower chambers,respectively.Sixdays later,CFSE dilution was determined by flow cytometry.3.2 Investigation of TRAP-induced neutrophils regulate the functions of tumor cells and their mechanism3.2.1 Investigation of TRAP-induced human neutrophils regulate the functions of tumor cells and their mechanismHuman neutrophils were cultured with TRAPs?10?g/mL?for 12 h,the culture supernatant was collected.MDA-MB-231 cells were cultured with different culture supernatant and then the migration and invasion of MDA-MB-231 cells were examined using Transwell and Wound healing assay respectively.Neutrophils were cultured with TRAPs?3?g/mL?for 6 h,the expression of molecules such as CCL2 and MMP9 mRNA was detected by qRT-PCR.Neutrophils were cultured with TRAPs?10?g/mL?for 12 h,the production of MMP9 was determined by gelatin zymography.MDA-MB-231 cells were cultured with different culture supernatant adding MMP9 neutralizing antibody or isotype control antibody and then the migration and invasion of MDA-MB-231 cells were examined using Transwell assay.3.2.2 Investigation of TRAP-induced murine neutrophils regulate the functions of tumor cellsMurine neutrophils were cultured with CFSE-labeled TRAPs?3?g/mL?for 3 h.Flow cytometric analysis was performed to identify the phagocytizing of TRAPs by murine neutrophils.Murine neutrophils were cultured with TRAPs?3?g/mL?for 6 h.The apoptosis of neutrophils was assessed by flow cytometry.Murine neutrophils were cultured with TRAPs?10?g/mL?for 12 h,the culture supernatant was collected.Hepa1-6cells were cultured with different culture supernatant and then the migration and invasion of Hepa1-6 cells were examined using Transwell assay.Mice were i.v.injected with B16F10 cells puls PBS,B16F10 cells puls neutrophils?CM?and B16F10 cells puls neutrophils?TRAP?.Two days later,mice were i.v.injected with PBS,neutrophils?CM?and neutrophils?TRAP?again.At day 21 after the first injection,the growth of B16F10tumors in the lung tissue of mice were continuously monitored and measured.Results1.Investigation of regulating neutrophil cell biological behavior by TRAPFlow cytometric analysis showed that there was a rapid and effective phagocytosis of TRAPs by human neutrophils,and TRAPs promote neutrophil apoptosis.Pretreatment of TRAPs by proteinase K,but not DNase I or RNase A,abolished the apoptosis-inducing activity of TRAPs.Furthermore,we found that sonication of TRAPs also impaired their ability to induce neutrophil apoptosis.2.Study on the molecular mechanism of TRAP phagocytosed by neutrophil andinduced neutrophil apoptosisPre-treatment with CD,but not CPZ or Filipin III,significantly inhibited the internalization of TRAPs and concomitantly decreased TRAP-induced apoptosis of neutrophils without affecting their spontaneous apoptosis.Meanwhile,pre-treatment with EIPA significantly inhibited the internalization of TRAPs and concomitantly decreased TRAP-induced apoptosis of neutrophils without affecting their spontaneous apoptosis.Following 30 min of incubation of neutrophils with various concentrations of TRAPs,there was a rapid and robust ROS production in neutrophils.When neutrophils werepre-treated with NADPH oxidase inhibitor DPI or the antioxidant NAC,TRAP-inducedneutrophil ROS production and neutrophil apoptosis were significantly diminished.Furthermore,pretreatment of neutrophils with CD,EIPA or DPI prior to TRAP incubationresulted in a significant inhibition of caspase-3 activity.Flow cytometric analysis showedthat pre-treatment with pan-caspase inhibitor zVAD-fmk significantly reducedTRAP-induced neutrophil apoptosis.3.Investigation of TRAP-induced neutrophils regulate the functions of T cells andtumor cells and their mechanism3.1 Investigation of TRAP-induced neutrophils regulate the functions of T cells and their mechanismTRAP-treated neutrophils significantly and dose-dependently suppressed anti-CD3/anti-CD28-induced proliferation of co-cultured CD4⁺T and CD8⁺T cells.Analysis of cytokines showed that TRAP-treated neutrophils diminished the percentage of IFN-?⁺T cells and IFN-?secretion into the supernatant.Meanwhile,TRAP-treated neutrophils suppressed the expression of CD69 and CTLA4,but not CD137 and PD1,of CD4⁺T and CD8⁺T cells.Pre-incubation of neutrophils with NAC before TRAP treatment reduced the inhibitory function of neutrophils on CD4⁺T and CD8⁺T cells proliferation.The suppressive activity of TRAP-treated neutrophils on T cell proliferation was also reduced when TRAP-treated neutrophils and PBMCs were seeded in Transwell that disrupted cell-cell contract.3.2 Investigation of TRAP-induced neutrophils regulate the functions of tumor cells and their mechanism3.2.1 Investigation of TRAP-induced human neutrophils regulate the functions of tumor cells and their mechanismTRAP-treated neutrophils significantly promoted migration and invasion of MDA-MB-231 cells.QRT-PCR analysis showed that there were obvious high expressions of CCL2,MMP9 and TNF-?mRNA in TRAP-treated neutrophils.Gelatin zymography analysis also showed that there was an augmented release of MMP9 in TRAP-treated neutrophils.The enhancement of TRAP-treated neutrophils on MDA-MB-231 cellsmigration and invasion was reduced when MMP9 neutralizing antibody was added to theculture supernatant.3.2.2 Investigation of TRAP-induced murine neutrophils regulate the functions of tumor cellsFlow cytometric analysis showed that there was a rapid phagocytosis of TRAPs by murine neutrophils,and TRAPs could promote murine neutrophil apoptosis.TRAP-treated murine neutrophils also significantly promote migration and invasion of Hepa1-6 cells.Moreover,TRAP-treated murine neutrophils increased the quantity and volume of B16F10 tumor nodules in pulmonary.Conclusions1.TRAPs can be macropinocytosed by human neutrophil rapidly and effectively andco-localized with lysosomes.2.TRAPs trigger NADPH oxidase-mediated ROS production and caspase-3 activation topromote neutrophil apoptosis in vivo and in vitro.3.TRAP-treated neutrophils suppress T cell proliferation and IFN-?secretion in a cellcontact-and ROS-dependent manner.4.TRAP-treated neutrophils promote MDA-MB-231 cells migration and invasion inMMP9-dependent manner.5.TRAP-treated murine neutrophils promote Hepa1-6 cells migration and invasion,and alsoaccelerate B16F10 tumor progression in vivo.