TNF-α-dependent CXCR2 Expressing Alveolar Macrophages Are Involved In Inflammation-induced Lung Adenocarcinoma

Objective:TNF-α-dependent pulmonary inflammatory microenvironment,including immune cells,stroma cells and cytokines,contributes to the lung tumorigenesis.Our recent study showed that TNF-α-dependent inflammatory response regulated the expression of MHC-II in alveolar epithelial cells（AT-II）through CXCR2,which may play a role in inflammation-induced lung adenocarcinoma.CXC receptor 2（CXCR2）belongs to the family of G protein-coupled receptors,which could be activated by binding with its ligands inflammatory chemokines such as CXCL1 and CXCL2.CXCR2pathway mostly contributes to bone marrow-derived monocytes recruitment and differentiation.In a tumor-bearing mice model,CXCR2 regulates monocytes to differentiate into M2 tumor-associated macrophages（tumor-associated macrophage,TAM）,and then promotes tumor growth.However,it is unknown whether and how CXCR2 as well as its downstream signaling pathway contributes to alveolar macrophage infiltration and differentiate into TAM.Alveolar Macrophage（AM）may include two populations by distinct origins,tissue resident alveolar macrophages（TRAM）and recruited monocyte-derived macrophages（Mo M）.Malignant tumor cells play an important role in reprograming AM into immunosuppressive phenotype,which in turn contributes to carcinogenesis.However,at pro-tumor inflammatory stage,whether and how tumor-associated TNF-α-mediated inflammation induces AM recruitment and reprograms AM differentiation through CXCR2 is not clear.In this study,using a urethane-induced inflammation-driven lung adenocarcinoma（IDLA）mice model,we sought to investigate whether TNF-α-mediated inflammation upregulates CXCR2expression on TRAMs or Mo Ms.Then,we explore the effect of CXCR2 on the infiltration and phenotypic alteration of alveolar macrophages in CXCR2^-/-mice and in vitro bone marrow monocyte-derived macrophage model.The study will provide a better understanding of the role of CXCR2 expressing alveolar macrophage in TNF-α-mediated inflammation-induced lung tumorigenesis,and may help developing new therapeutic strategies for lung adenocarcinoma.Methods:1.IDLA model was established:Balb/c mice were injected with urethane for 8 weeks to induce inflammation in the lungs;some experimental mice continued to be reared for 4 months to induce inflammation-driven lung adenocarcinoma（IDLA）.Western blot and immunohistochemical methods were used to measure the expression of CXCR2 in inflamed lung tissues and lung adenocarcinoma tissues.Immunofluorescence staining was used to detect the expression of CXCR2 on CD68⁺macrophages in inflamed lung tissues and lung adenocarcinoma tissues.Flow cytometry（FCM）was used to measure the infiltration of macrophages and the expression of CXCR2.2.We employed Etanercept（s TNFR:Fc,10 mg/kg body weight）,an antagonist of TNF-α,to treat the urethane-induced mice with s TNFR:Fc for 8weeks.Immunohistochemical and immunofluorescence staining of CXCR2expression on CD68⁺macrophage was observed in inflammatory lung tissues and lung adenocarcinoma tissues.FCM was used to detect the expression of CXCR2 in macrophages in inflammatory lung tissues and lung adenocarcinoma tissues.3.CXCR2 knockout mice were injected with urethane for 8 weeks to induce inflammation in the lungs,and the expression of CXCR2,PD-L1,TNF-α,IL-6 and other cytokines as well as macrophage infiltration in the inflamed lung tissues were measured.4.Bone-marrow derived macrophages（BM-M0）were cultured under M-CSF treatment,and CXCL1 and CXCL2 were applied to stimulate the cells migration with or without anti-CXCR2.The expression of CXCR2 was measured when M0 differentiate into M1 or M2.Results:1.The expression of CXCR2 up-regulation in inflammation-driven lung adenocarcinoma（IDLA）In IDLA,Western blot results showed that the expression of CXCR2 at the protein level in lung adenocarcinoma tissue was significantly increased.Immunohistochemical results showed that the positive expression of CXCR2in inflammatory tissues and lung adenocarcinoma.CXCR2 was not only expressed in tumor cells in lung adenocarcinoma,but also in tumor-adjacent stroma cells including alveolar macrophages.2.Up-regulation of CXCR2 expression in bone marrow monocyte-derived alveolar macrophages（Mo M）in IDLAThe results of immunofluorescence staining showed that CXCR2 was expressed on CD68⁺macrophages in IDLA.Compared with the control group,the number of CXCR2⁺CD68⁺macrophages in the inflammation and lung adenocarcinoma group were significantly increased.FCM results showed that the expression of CXCR2 in the infiltrating CD11b^highF4/80⁺Mo M in the inflammatory tissues and lung adenocarcinoma was up-regulated,while the expression of CXCR2 in CD11b^lowF4/80⁺TRAM and CD11b⁺Gr-1⁺MDSC did not show any change compared with the control group.The results indicate that CXCR2 expressing Mo M may be involved in lung tumorigenesis.3.TNF-α-dependent lung inflammation regulates the expression of CXCR2 in Mo MTo further confirm whether TNF-α-dependent lung inflammation regulated CXCR2 expression on Mo M,we explored the role of TNF-α-neutralization on CXCR2 expression on macrophage in urethane-treated mice at pro-tumor inflammatory stage.TNF-α-neutralization reduced the number of CXCR2⁺cells in the inflamed lung tissues.Immunofluorescence results showed that TNF-αblocking reduced the number of CXCR2⁺CD68⁺cells in inflamed lung tissues.FCM results showed that TNF-α-neutralization inhibited the expression of CXCR2 in CD11b^highF4/80⁺Mo M in inflamed lung tissue.It suggests that TNF-α-dependent lung inflammation regulates the expression of CXCR2 on Mo M.4.Inhibition of CXCR2 reduces Mo M infiltration and its phenotypic alterationWe used urethane to induce lung inflammation in CXCR2-deficient mice,and explored whether lung inflammation induces Mo M infiltration and its phenotypic alteration through CXCR2 at pro-tumor stage.HE staining results showed that the inflammatory response in lung tissue of CXCR2^-/-treated by urethane was significantly reduced.Inhibition of CXCR2 also reduced the expression of IL-6,TNF-α,CXCL2,and CXCL1 in inflammatory lung tissues.FCM results showed that CXCR2 knockout inhibited the infiltration of Mo M in urethane-treated lung tissue.The immunohistochemical and immunofluorescence staining showed that CXCR2 knockout inhibited the expression of phenotypic molecules PD-L1 and TNF-αin CD68⁺macrophages of inflamed lung tissues.5.CXCR2 mediates the infiltration of bone marrow-derived macrophagesCXCL1 and CXCL2 could induce bone marrow-derived macrophages（BM-M0）migration,and administration of the CXCR2 antagonist SB225002can reduce the migration ability of macrophage.M1 and M2 stimulating factors were given to induce M0 to differentiate into M1 and M2,and then the expression of CXCR2 was measured by ICC immunofluorescence staing and RT-PCR.The results showed that the expression of CXCR2 on M2 type macrophages was significantly increased,suggesting that CXCR2 may be associated with M2 macrophages differentiation.Conclusions:1.In inflammation-driven lung adenocarcinoma,CXCR2 expression is increased on Monocyte-derived alveolar Macrophage.2.In inflammation-driven lung adenocarcinoma,TNF-α-dependent inflammatory response regulates CXCR2 expression on Monocyte-derived alveolar Macrophage.3.In inflammation-driven lung adenocarcinoma,CXCR2 regulates the recruitment of Monocyte-derived alveolar Macrophage to lung tissue and affects the expression of macrophage phenotypic molecules.