TNF-?-induced Tumor-associated Inflammation Triggers Immunosuppression Of Dendritic Cell In Lung Adenocarcinoma

Objective: Lung cancer is currently one of the highest mortality of malignant tumor.Chronic inflammation is now being recognized as a major driving force in the development of lung cancer.In addition,chronic inflammation induces the expression of various molecules from the tumors themselves or surrounding cells,and promotes the development of tumor-associated immune-suppressive microenvironment at the beginning and promotion stage of lung tumorigenesis.In pro-tumor stage,tumor-associated inflammatory microenvironment consists of bone marrow-derived inhibitory cells(MDSC),regulatory T cells(Treg),tumor-associated macrophages(TAM),and stromal cells as well as cytokines.They inhibit anti-tumor immunity and immune surveillance,and then promote the development of lung cancer at the stage of tumor initiation.While in the promotion stage of lung adenocarcinoma,the tumor-associated immune-suppressive microenvironment also consist of tumor cells as well as their cytokines,which works with the surrounding cells to inhibit anti-tumor immunity and promote tumor cells metastasis and invasion.In recent years,numbers of researches have reported that tumor infiltrating DCs are found in tumor-associated inflammatory microenvironments in many different types of cancers(e.g.,breast cancer,colorectal cancer,lung cancer,kidney cancer,head and neck cancer,bladder cancer,gastric cancer and ovarian cancer),which is called tumor associated DCs(TADC).Dendritic cells are the key cell to professional antigen presenting cell(APC)that not only present antigens but also provide many other necessary signals(such as: costimulatory molecules and cytokines)for T cell activation.Then,DCs are known to play a key role in linking the innate and adaptive arms of the immune system.TADCs capture antigens released from tumor cells and cross-presents antigens to CD8~+T cells,driving tumor-specific cytotoxic T lymphocyte expansion.However,at the later stage of tumor formation,TADCs triggered by the tumor microenvironment can induce Treg expansion,which show immunosuppressive functions in antitumor immunity.In tumor-associated inflammatory environment,TADCs show phenotypic and functional alterations as regulatory DCs(DCreg),such as: upregulation of CD11 b,arginase I(arginase I),IDO,or downregulation of MHC-?,costimulatory molecules,CD11 c,etc;They also increase the secretion of negative inflammatory cytokines such as TGF-?,IL-10 and PGE-2.In recent years,there has been great focus on determining whether tumor cells could regulate TADCs to change their phenotype and induce Treg expansion in tumor-associated inflammatory environment.However,it is still unclear whether and how tumor-associated inflammation educates DC cells immunosuppression in pro-tumor stage or early stage of tumor formation.It is well accepted that urethane is applied to induce inflammationassociated lung adenocarcinoma in mice.Karabela showed that TNF neutralization by twice-weekly intraperitoneal soluble TNF-? receptor s TNFR: Fc(etanercept)treatment could prevent urethane-induced lung oncogenesis.In current experiment,we treated Balb/c mice with urethane to set up inflammation-associated lung adenocarcinoma model,and explored the filtration and phenotypic alterations of TADC in urethane-induced lung adenocarcinoma.We also explored the effects of TNF-? blockers s TNFR: Fc in the development of urethane-induced adenocarcinoma,as well as cell infiltration and phenotypic alterations of TADCs in tumor environment.To further investigate whether TNF-?-mediated lung inflammatory responses could induce DC immunosuppression in pro-tumor stage,we explored the effect of TNF-? blockers on DCs filtrations and phenotypic alteration as well as DCs functions in urethane-induced lung inflammatory environment.Methods:We treated the mice with urethane for 8 weeks,and then the mice were allowed to live for another 4 months to observe the development of lung cancer.We measured the filtration of TADC and Treg in tumor-associated environment by FCM.We applied soluble TNF-? receptors s TNFR: Fc and urethane for 8 weeks,and allowed the mice live for another 4 months.We measured the filtration of TADC and Treg by FCM.The phenotypic alterations of TADC,such as CD80,CD86,CD11 b,CD274,MHC-?,were measured by FCM.TADCs from urethane-induced adenocarcinoma were isolated and co-cultured with spleen CD4~+CD25~+ T cells for 3 days,and percentage of CD4~+ CD25 + cells and Foxp3 expression in vitro were detected by FCM.To further investigate whether TNF-?-mediated lung inflammatory responses could induce DC immunosuppression in pro-tumor stage,we treated the mice with soluble TNF-? receptors s TNFR: Fc and urethane for 8 weeks,and explored the effect of TNF-? blockers s TNFR: Fc on DCs filtrations and phenotypic alteration as well as DCs functions in urethane-induced lung inflammatory environment.The expression of TNF-?,NF-?B,COX-2 and Treg cell marker FOXP3 in inflammed lung tisssues were detected by immunohistochemical staining.The filtration of DCs and Treg as well as phenotypic alterations of DCs were measured by FCM.DCs from urethane-induced inflammed lung tisssues were isolated and co-cultured with spleen CD4~+ CD25~+ T cells for 3 days,and percentage of CD4~+ CD25 + cells and Foxp3 expression in vitro were detected by FCM.Results: 1 Cell infiltration and phenotypic alterations of TADCs in urethane-induced lung adenocarcinoma.1.1 Urethane induced inflammatory-associated lung adenocarcinoma with immunosuppressive environment.Representative H&E images shows urethane could induce lung adenocarcinoma in Balb/c mice.Immunohistochemistry results showed increased expression of Foxp3 in the infiltrated T lymphcyte,suggesting that urethane induced inflammatory-associated lung adenocarcinoma with immunosuppressive microenvironment.1.2 Cell infiltration and phenotypic alterations of TADCs in urethane-induced lung adenocarcinoma.FCM analysis showed the infiltration of CD11c+B220-cells and CD11c+CD11b+cells in urethane-tumor were higher than that in normol control.Phenotypic analysis by flow cytometry showed that downregulation of CD80 as well as upregulation of MHC-?,CD11 b and PD-L1 in TADC.The results indicate that increased infiltration of TADCs,which appear phenotypic alterations,in lung adenocarcinoma.1.3 Effect on the secretion of tumor-associated DC cytokines in inflammatory lung adenocarcinoma.RT-PCR result showed the secretion of IL-6,IL-10 and COX-2 cytokines were increased in urethane-tumor compared with the control group,and TNF-? secretion was decreased.The results suggest that the secretion of tumor-associated DC cytokines in inflammatory lung adenocarcinoma changes,which promotes the formation of immunosuppressive microenvironment.2 The effects of TNF-? blockers s TNFR: Fc in the development of urethane-induced adenocarcinoma,as well as cell infiltration and phenotypic alterations of TADCs.2.1 TNF-? blocker s TNFR: Fc inhibits the development of urethane-induced adenocarcinoma.TNF-? blocker s TNFR: Fc significantly inhibited the occurrence of lung adenocarcinoma,since we observed that the tumor nodules in lung surface were significantly reduced.The results indicate that TNF-?-mediated chronic inflammatory responses play a key role in the development of inflammation-associated lung adenocarcinoma induced by urethane.2.2 Inhibition of TNF-?-mediated chronic inflammatory responses reduced the infiltration of TADC and Treg in urethane-induced lung adenocarcinoma.FCM analysis showed the infiltration of CD11c+B220-cells and CD11c+CD11b+cells in urethane-tumor was reduced by s TNFR: Fc.The infiltration of CD4~+CD25~+Treg cells in urethane-tumor was higher than that in control group.However,s TNFR: Fc significant reduced the infiltration of CD4~+CD25~+Treg cells in urethane-induced lung adenocarcinoma.2.3 Inhibition of TNF-?-mediated chronic inflammatory responses affects the phenotypic alterations in urethane-induced lung adenocarcinoma.Downregulation of CD80 as well as upregulation of MHC-?,CD11 b and PD-L1 in TADC of urethane-tumor was reversed by s TNFR: Fc,which suggests that phenotypic alterations in urethane-induced lung adenocarcinoma were inhibited by inhibition of TNF-?-mediated chronic inflammatory responses.3 The effects of TNF-? blockers s TNFR: Fc on TADC from urethane-induced adenocarcinoma to induce Treg expansion.TADCs from urethane-induced adenocarcinoma induced CD4~+CD25~+ Treg expansion evidenced by increased the percentage of CD4~+ CD25 + cells and Foxp3 expression in vitro.However,TNF-? blockers s TNFR: Fc could inhibit TADCs from urethane-induced adenocarcinoma to induce CD4~+CD25~+ Treg expansion.The result indicates that TNF-?-mediated chronic inflammatory responses may educate TADCs in lung adenocarcinoma to trigger Treg expansion.4 Tumor-associated TNF-?-mediated chronic inflammatory responses suppress DCs in pro-stage of lung adenocarcinoma.4.1 The effects of TNF-? blockers s TNFR: Fc on urethane-induced lung inflammation in mice.Compared with the control group,the expression of TNF-?,p-NF-?B and COX-2 protein was increased in the lung epithelium of the urethane-treated mice,which suggests that urethane induces lung chronic responses in pro-tumor stage.TNF-? blockers s TNFR: Fc could inhibit the urethane-induced lung inflammatory responses.The result shows that urethane also induces TNF-?-mediated lung inflammatory responses.4.2 The effects of TNF-? blockers s TNFR: Fc on cell infiltration of DCs and Treg in urethane-induced lung inflammatory environment.FCM analysis showed urethane promoted the infiltration of CD4~+CD25~+ Treg,CD11c+B220-cells and CD11c+CD11b+cells in lung inflammatory environment.Furthermore,the increased infiltration of CD4~+CD25~+ Treg,CD11c+B220-cells and CD11c+CD11b+cells in urethane-induced lung inflammatory environment was reduced by s TNFR: Fc.The results suggest that TNF-?-mediated lung inflammatory responses could promote DCs and Treg infiltration in pro-tumor stage.4.3 The effects of TNF-? blockers s TNFR: Fc on phenotypic alterations of DCs and Treg in urethane-induced lung inflammatory environment.FCM analysis showed that downregulation of CD80 as well as upregulation of MHC-?,CD11 b and PD-L1 could be observed in DCs of urethane-induced lung inflamed tissues,which was reversed by s TNFR: Fc treatment.The results suggest that TNF-?-mediated chronic inflammatory responses could induce DCs phenotypic alterations in pro-tumor stage,which may educate DCs to show immunosuppressive functions.4.4 The effects of TNF-? blockers s TNFR: Fc on DCs from urethane-induced lung inflammatory environment to induce Treg expansion.DCs from urethane-induced inflamed lung tissues induced CD4~+CD25~+ Treg expansion evidenced by increased the percentage of CD4~+ CD25 + cells and Foxp3 expression.However,TNF-? blockers s TNFR: Fc could inhibit DCs from urethane-induced lung inflammatory environment to induce CD4~+CD25~+ Treg expansion.The result indicates that TNF-?-mediated chronic inflammatory responses may educate DCs to trigger Treg expansion.Conclusions:1 Urethane induced inflammatory-associated lung adenocarcinoma associated with immunosuppressive microenvironment,where the infiltration of TADCs was increased,showing phenotypic alterations in lung adenocarcinoma.2 TNF-? blockers s TNFR: Fc inhibited the incidence of urethane-induced adenocarcinoma,reduced cell infiltration and phenotypic alterations of TADCs.TNF-? blockers s TNFR: Fc also inhibited TADCs from urethane-induced adenocarcinoma to induce CD4~+CD25~+ Treg expansion.The result indicates that TNF-?-mediated chronic inflammatory responses may educate TADCs in lung adenocarcinoma to trigger Treg expansion.3 In pro-stage of lung adenocarcinoma,urethane also induces chronic inflammatory responses,which promotes DCs infiltration as well as induces phenotypic alterations of DCs.4 TNF-? blocker s TNFR: Fc inhibited TNF-?-mediated chronic pulmonary inflammatory responses,and then reduced DCs and Treg infiltration as well as reversed phenotypic alterations of DCs.TNF-? blockers s TNFR: Fc also inhibited TADCs from inflammed lung tissues to induce CD4~+CD25~+ Treg expansion.Tumor-associated TNF-?-mediated chronic inflammatory responses could induce DCs to trigger Treg expansion in pro-stage of lung adenocarcinoma.Thus,TNF-?-mediated chronic inflammatory responses play an important role in development of immunosuppressive microenvironment in lung adenocarcinoma by educating TADCs immunosuppression.