Tumor Necrosis Factor-α Dependent Lung Inflammation Promotes The Progression Of Lung Adenocarcinoma Originating From Alveolar Type Ⅱ Cells By Upregulating CD74

Background:Lung cancer is one of the most common malignant tumors in China,and lung adenocarcinoma is the most common type of lung cancer.Our recent research shows that Tumor necrosis factor-?（TNF-α）dependent chronic lung inflammation can induce the occurrence of inflammation-driven lung adenocarcinoma（IDLA）in mice,suggesting that chronic lung inflammation plays an important role in the progression of lung adenocarcinoma.Further studies find that the inflammation-driven lung adenocarcinoma originates from Alveolar typeâ…¡epithelial cells（AT-Ⅱ）,and TNF-αdependent chronic lung inflammation promotes the expansion of regulatory T cells（Treg）by up-regulating the expression of major histocompatibility complex（MHC）classâ…¡.AT-â…¡cell-derived lung adenocarcinoma may depend on MHC-â…¡to induce the formation of immun-osuppressive microenvironment,thereby,promoting the tumor genesis and progression.CD74（MHC II invariant chain,Ii）is a non-polymorphic type II transmembrane glycoprotein.The basic function of CD74 is to participate in antigen presentation as a MHC II chaperone protein.CD74 can be expressed in antigen-presenting cells such as dendritic cells and epithelial cells such as AT-â…¡cells,playing an immune regulatory role.Some studies have shown that CD74 not only participates in immune presentation,but also binds to macrophage migration inhibitory factor（MIF）to form MIF-CD74 pathway signals that participate in the regulation of proliferation and tumor progression of many kinds of malignant tumors,and are related to tumor prognosis.So far,there is no literature about the expression and biological significance of CD74in lung adenocarcinoma derived from AT-â…¡cells.Whether TNF-α-induced lung inflammation regulates the expression of MIF-CD74 to mediate the progression of lung adenocar-cinoma remains to be further studied.Objective:Using a urethane-induced TNF-α-dependent inflammation-mediated mouse IDLA model,we explored whether TNF-α-induced lung inflammatory response regulates the expression of CD74 in AT-â…¡cell derived lung adenocarcinoma and the expression of MIF in macrophages.Using human lung adenocarcinoma tissue samples and lung adenocarcinoma cells,we also further discussed the function of CD74 in the progression of lung adenocarcinoma.This study aims to provide new immune intervention strategies and ideas for the effective prevention and treatment of lung adenocarcinoma.Materials and Methods:1 44 samples of lung adenocarcinoma（LA）and 20 samples of normal lung tissue（NLT）Paraffin-embedded tissue specimens confirmed by pathological diagnosis were collected from Hebei Chest Hospital.Immunohistochemical staining（IHC）was used to analyze the expression of CD74 in tumor tissue and normal lung tissue.To investigate the relationship between CD74 and clinicpathological features,the relationship between CD74 expression and TNF-α,matrix metalloproteinase-9（MMP-9）,and Human leucocyte antigen（HLA）-DR expression were also analyzed.2 A549 and H1299 lung adenocarcinoma cells were cultured in vitro,and CD74 was knocked down or overexpressed by using small interfering RNA（si RNA）or plasmid pc DNA3.1-CD74.The roles of CD74 in the proliferation,migration,and invasion of lung adenocarcinoma cells were analyzed by using CCK-8,colony formation,wound healing,Transwell Assay,and western blot.3 The expression of CD74 and MIF in TNF-α-dependent inflammation-mediated lung adenocarcinoma3.1 We injected urethane intraperitoneally into mice for 8 weeks（2 months）to induce lung inflammation,and simultaneously administered TNF-αneutralizing antibody-etanercept（s TNFR:Fc）for 8 weeks（2 months）to block urethane induced lung inflammation and ensure their survival for another 4months to establish a lung adenocarcinoma IDLA model,total of 6 months.Experimental animals were divided into urethane treatment group,urethane+TNF-αblocker group and control group.Mice were euthanized during the inflammation phase at 8 weeks（2 months）and the tumor phase at 6months for the experiment.3.2 IHC and immunofluorescence staining（IF）were used to observe the expression of CD74 and the infiltration of CD68⁺macrophages in lung adenocarcinoma tissue.In addition,the expression of CD74 in AT-â…¡cells and the infiltration of CD11b^highF4/80⁺macrophages were measured by using flow cytometry fluorescence sorting（FACS）.The TNF-?-mediated inflammatory response was inhibited by intraperitoneal injection of etanercept,and the correlation between the expression of CD74 and the infiltration of CD68+macrophages and TNF-?-mediated inflammation was analyzed.In addition,the expression of MIF in CD68⁺macrophages was detected in the lung tissue of the IDLA model,the changes of MIF transcription levels in inflammatory and tumor tissues were detected by RT-PCR,and analyzed the relationship between MIF expression in macrophages and TNF-?-dependent chronic inflammation.4 50ng/m L PMA was applied to induce human monocyte THP-1 cells（3.5×10⁵cells/well）for 48 hours to differentiate into M0 macrophages（THP-1-M0）.The conditioned medium from A549 or H1299 cells was used to stimulate THP1-M0 cells after blocked with 15μg/m L of anti-TNF-?for 4hours,and the cells were cultured for 24 hours.MIF expression in macrophages was measured.In additional,bone marrow mononuclear cells were isolated from the femora of mice and treated with M-CSF to differentiate into bone marrow derived macrophages（BM-M0）.The conditioned medium from activated AT-â…¡cells was used to stimulate BM-M0 cells after blocked with 15μg/m L of anti-TNF-?for 4 hours,and the cells were cultured for 24 hours.MIF expression in macrophages was measured using Western Blot and RT-PCR.Results:1 The correlation between the increased CD74 expression in human lung adenocarcinoma cells and clinicopathological features1.1 Immunohistochemical staining（IHC）showed that the CD74 immuno-positive response was localized in the cytoplasm of lung adenocarcinoma cells and alveolar epithelial cells,with brown-yellow granular staining.The positive expression rate of CD74 in human lung adenocarcinoma was significantly higher than that in control group.CD74 expression in lung adenocarcinoma was positively correlated with lymph node metastasis and TNM stage,but not with age and sex.1.2 IHC staining showed that CD74,TNF-α,MMP-9 and HLA-DR were highly expressed in lung adenocarcinoma tissue.Spearman correlation analysis showed that high expression of CD74 was positively correlated with the expression levels of TNF-α,MMP-9 and HLA-DR in human lung adenocarcarcinoma,indicates that TNF-α-mediated inflammation may regulate the expression of CD74.MMP-9 is a marker of epithelial interstitium transformation-EMT,which can promote the invasion and migration of tumor cells,and also involved in the regulation of tumor cell proliferation.The expression of CD74 is positively correlated with the expression of MMP-9suggestes that CD74 may be involved in tumor progression.2 The relationship between the expression of CD74 and proliferation,migration and invasion of lung adenocarcinoma cellsWe found that the blockade of CD74 by using si RNA significantly inhibited the proliferation,migration and invasion of A549 and H1299 cells.The western blot analysis suggested that the inhibition of CD74 significantly downregulated the expression of Cyclin D1 in A549 and H1299 cells.To further explore the effect of CD74 on invasion and metastasis of lung adenocarcinoma cells,the overexpression of CD74 by transfecting with plasmid pc DNA3.1-CD74.The results showed that overexpression of CD74upregulated Cyclin D1 and MMP-9 expression,and significantly increased the proliferation ability of A549 cells,and promoted the invasion and migration of A549 cells.3 Expression of CD74 in IDLA3.1 Gross observation and HE staining of tissue sections confirmed that the urethane-induced lung adenocarcinoma model was successfully established.Immunohistochemical staining suggested the increasing expressions of CD74in both inflammatory lung tissue and lung adenocarcinoma tissue,and the expression of CD74 in the lung adenocarcinoma was significantly higher than that in the inflammatory group.3.2 AT-II derived lung adenocarcinoma cells were labeled using the thyroid transcription factor TTF-1.Immunofluore-scence staining suggested that CD74 co-expressed with TTF-1,CD74 was highly expressed in AT-II derived lung adenocarcinoma cells.FACS results suggested that CD74 was expressed in AT-II cells both in inflammatory lung tissue and lung adenocarcinoma tissue,and the expression of CD74 in AT-II gradually increased during the progression from chronic inflammation to tumor.3.3 Immunofluorescence staining showed that CD74 and CD68-labeled macrophages localized in the same area of tumor cells in lung adenocarcinoma tissue,suggesting that CD74 is also expressed on CD68⁺macrophages.TNF-?expression was upregulated in CD68⁺macrophages in inflammatory lung tissue and lung adenocarcinoma tissue.The number of TNF-?⁺CD68⁺cells was significantly higher than that of the control group,and the number in cancer tissue was significantly higher than that in inflammatory tissue,suggesting that TNF-?may involved in regulating the expression of CD74.4 TNF-?-dependent inflammatory response regulated CD74 expression in pre-tumor inflammatory lung tissueIn order to further confirm the relationship between TNF-?and CD74 in the IDLA model,we used TNF-?neutralizing antibody-etanercept（s TNFR:Fc,10mg/kg）to block urethane-induced lung inflammation,and observed the expression of CD74.The increased number of CD74 and TNF-α-positive cells was significantly inhibited by s TNFR:Fc in the inflamed tissues.The m RNA expression of CD74 and TNF-αwas upregulated in the inflamed tissues.This effect was inhibited by blocking TNF-α.In the s TNFR:Fc-treated mice,the number of CD74⁺TTF-1⁺positive cells was significantly reduced in the inflamed lung tissues.The number of TNF-α⁺CD68⁺cells was higher in the inflamed lung tissues than in the control tissues,whereas the number of CD68⁺macrophages expressing TNF-αin the inflamed lung tissues was reduced by s TNFR:Fc.5 TNF-?-dependent inflammatory response regulated CD74 expression in lung adenocarcinomas TNFR:Fc treatment significantly reduced the number of tumor nodules on the lung surface in IDLA.RT-PCR and Western blot suggested that the expressions of CD74 and TNF-αwas increased in lung adenocarcinoma tissue,while the expression was significantly reduced by the adminstration of TNF-αneutralizing antibody.FACS analysis suggested that CD74 was upregulated in AT-â…¡derived lung adenocarcinoma cells,while administration of s TNFR:Fc inhibited the expression of CD74.IHC staining suggested that the TNF-?neutralizing antibody inhibited the expression of MMP-9 and CD74 in IDLA model.The above results indicate that TNF-αdependent inflammatory response up-regulate the expression of CD74 in in AT-â…¡derived tumor cells in lung adenocarcinoma tissue.Immunofluorescence staining suggested that t s TNFR:Fc significantly reduced the number of CD74⁺CD68⁺cells in IDLA.FACS results suggested that the expression of CD74 in CD11b^highF4/80⁺in lung adenocarcinoma tissue was up-regulated,and administration of s TNFR:Fc inhibited the expression of CD74.These results suggest that TNF-αdependent inflammatory response up-reguate the expression of CD74 in lung adenocarcinoma tissue.6 TNF-α-dependent inflammatory response regulated MIF expression in IDLAMIF was mostly expressed in CD68⁺macrophages and lung adenocarcinoma cells.The number of MIF⁺CD68⁺cells in the IDLA tissues was significantly higher than that in the control tissues.At the pro-tumor inflammatory stage,s TNFR:Fc reduced the expression of MIF m RNA in inflamed tissues.IHC staining indicated that the number of MIF-positive cells was significantly reduced by s TNFR:Fc in the inflamed tissues.The number of MIF⁺CD68⁺cells increased in the inflamed lung tissues,which was reduced by s TNFR:Fc treatment.We found that the neutralization of TNF-αin the inflammatory phase decreased the m RNA expression level of MIF and MIF⁺CD68⁺cells in IDLA,confirming that lung inflammation depends on TNF-αto regulate MIF expression in macrophages in IDLA.7 The relationship between AT-â…¡cells and MIF expression in macrophagesWestern blot showed that the conditioned medium from A549 or H1299cells up-regulated MIF expression in the THP1-M0 cells,and this effect was inhibited in the A549 cell medium treated with anti-TNF-α.RT-PCR and Western blot confirmed that the TNF-αactivated AT-â…¡cells medium of mouse up-regulated MIF expression in BM-M0.Blocking TNF-?inhibited the increased expression of MIF in BM-M0.These results suggest that AT-â…¡cells of mouse induce MIF up-regulation in macrophages by secreting TNF-α.Conclusions:1.The positive expression rate of CD74 in human lung adenocarcinoma is significantly higher than that in normal lung tissue.The high expression of CD74 is positively correlated with lymph node metastasis,TNM stage,TNF-αand MMP-9 expressions.CD74 plays an oncogenic role in promoting the proliferation,migration,and invasion of lung adenocarci-noma cells.2.In the urethane-induced IDLA mouse model,there is an overex-pression of CD74 in the tumor cells derived from AT-â…¡cells.TNF-α-dependent inflammatory response up-regulates the expression of CD74 in inflammatory lung tissues and lung adenocarcinoma tissues.3.TNF-α-dependent inflammatory response upregulates the MIF expres-sion in macrophages in IDLA mouse model.4.AT-â…¡cells upregulats MIF expression in macrophages by secreting TNF-α.5.TNF-α-dependent inflammatory response upregulates the CD74 expres-sion on macrophages and tumor cells in the IDLA model and also upregulates the MIF expression in macrophages.TNF-αdependent lung inflammation promotes the progression of lung adenocarcinoma originating from alveolar typeâ…¡Cells by up-regulating CD74.