Deficiency Of IL-18 Aggravates Esophageal Carcinoma Through Inhibiting IFN-? Production By CD8~+T Cells And NK Cells

Esophageal cancer is the eighth most common malignant tumor in the world for morbidity and mortality.China is the highest incidence area,and half of the esophageal cancer patients live in this area.A retrospective study has shown that the prognosis of the disease is closely related to the stage of the disease and the application of the treatment.However,due to the cumbersome diagnosis method and the low attention of the Chinese people to their own health status,the disease is already in the middle and late stage at the time of diagnosis.The overall prognosis of the patients is poor,and the five-year survival rate is low for a long time,which seriously threatens the health of the people in China.At present,the conventional treatment methods for esophageal cancer are extremely limited,and there are three kinds of conventional treatment methods including surgery,radiation therapy and chemical drug treatment,and these methods still have inherent defects.For example,radical surgery requires patients to be in the early and middle stages of esophageal cancer and the body can tolerate the surgery.Radiation therapy may cause serious complications such as tracheoesophageal fistula and radiation-induced lung injury,and the tumor-inducing effect of the radiation itself is also a great threat to the long-term survival of patients.The side effects of chemical drug treatment are obvious.Many patients are intolerable before they complete the treatment,and they are forced to give up chemotherapy.In order to reverse the unfavorable situation in the treatment of esophageal cancer,the government invests huge sums each year to support and encourage scientists,in exploring new effective methods while implementing large-scale esophageal cancer screening and routine treatment improvement.A lot of fruitful research results have been achieved in recent years.Among the research results,anti-tumor immunotherapy has attracted much attention of the academic community because of its small side effects and similarity with the natural immune state of the human body.In particular,biomacromolecules with anti-tumor immunity such as interleukin have been the focus of scholars'research in recent years.In this study,we selected interleukin-18 as the research object,which is a pro-inflammatory factor with anti-tumor and tumor-promoting effects,and an important member of the interleukin-1 superfamily.Using IL-18-KO and IL-18-WT esophageal cancer-bearing mice as the researching tools to explore the immunomodulatory effects of IL-18 as an anti-tumor inflammatory factor in the development of esophageal cancer,and to elucidate its intrinsic molecular mechanism and provide a basis for the anti-tumor clinical application of interleukin-18.Part one Lack of interleukin-18 promotes the development andprogression of 4 NQO-induced esophageal cancer in interleukin-18-KO mouseObjective:To explore a stable method of inducing esophageal cancer in mice by 4NQO,and to observe the effect of interleulin-18 deficiency on the occurrence of 4NQO-induced esophageal carcinoma in mice and the survival status of mice.Methods:Using PCR to confirm that the interleukin-18 gene of interleukin-18-KO mice has been substantially knocked out compared to interleukin-18-WT mice.Using the 4NQO to induce the occurrence of esophageal cancer in mice.In the preliminary experiment,we found that the mice that drank the 4NQO solution had a tumor formation rate of 100%at the24^th week and the mice with tumor at the early stage of experiment have very poor living conditions.Therefore,we killed the animals for subsequent experiments at the 22^nd week.We dissected the mouse and took its esophageal tissue,spleen and peripheral blood under sterile conditions.After the esophageal tissue was embedded in paraffin,the ultrathin section was used for HE staining,and the tumor was confirmed by the pathological diagnosis.Body weight,toxicity,disease status,and any abnormalities of all experimental mice were regularly checked during the modeling experiment and recorded in detail.Results:1.It was confirmed by PCR experiments that the IL-18 gene chain was lost in interleukin-18-KO mice compared with interleukin-18-WT mice,and IL-18-KO mice had lost IL-18 activity.2.Body weights were recorded starting in the fifth week after the start of the modeling experiment.In the first five weeks of the weight record,the weight changes of the four groups of mice was basically the same.The tenth week was the dividing point of change.The weight of the two groups of mice using the cancer-inducing agent began to decrease,and the weight of the IL-18-KO group decreased faster.At the 22nd week of the experiment,the IL-18-KO mice were in very poor physical condition.3.Gross pathological outcomes:The two groups of mice fed by a blank control solution,had no lesions in the esophageal tissue.The esophagus of the two groups of mice fed by 4NQO was thickened,and some wild-type mice had neoplasms in the esophageal mucosa with no obvious necrotic hemorrhage,and the boundary was clear.The esophagus of interleukin-18-KO mice became thick obviously,and all of the esophageal mucosa developed neoplasms.Most of the lesions were necrotic and hemorrhagic,and the boundary was unclear.Microscopic pathological outcomes:Part of the cells in the cancerous cell mass were keratinized and form keratinized beads.There were significant differences between cancerous cells and normal tissue cells in morphology.The cell bodies were enlarged,the shape and arrangement were irregular,the mitotic phase was increased,and the nucleoplasmic ratio was imbalanced.4.At the end of the 22nd week,the success rate of the model was 18%in the interleukin-18-WT group and 86.7%in the interleukin-18-KO group fed by 4NQO.There was significant statistical difference between the two groups.Conclusion:1.The loss of interleukin-18 has little effect on the normal survival of mice.2.Deletion of interleukin-18 makes it easier for mice to develop esophageal cancer under the action of a carcinogen.3.The deletion of interleukin-18 has a great influence on the mice in the state of malignant tumor disease after the action of the carcinogen,and there is a critical time point for the carcinogen to accumulate in the body and the time of action with the body.Part two Deletion of interleukin-18 inhibits anti-tumor immunity of thebodyObjective:Exploring the cellular immunological mechanism of the loss of interleukin-18 in living experimental animal models with esophageal cancer promoting the development of esophageal cancer.Method:The mice were aseptically dissected,and the esophageal cancer tissues were taken out to prepare a single cell suspension after being confirmed by the pathological examination.The spleen tissue of the tumor-bearing mice was taken out to prepare a single cell suspension,and the lymphocytes in the mouse spleen were preliminarily separated by density gradient centrifugation and nylon hair column.The single-cell suspension was taken to labeling CD4-PE,CD8-APC and CD69-FITC as the antibody-to cell ratio recommended in the manual.The samples to be tested were placed in a cell culture incubator with 37�C and 5%carbon dioxide for 4 hours and detected by FCM.We added the appropriate amount of PMA,ionomycin and BFA to the preliminary isolated lymphocyte suspension.And then,the sample was incubated for 4 hours in the dark with 37�C and 5%carbon dioxide.CD8-APC,CD3-FITC and NK1.1-PE were labeled on the cells in an appropriate ratio and stained in the dark at 4�C for 30 minutes,then cells were fixed in 4%paraformaldehyde,and cell membrane permeabilization was performed with 1%saponin.Intracellular staining was performed using IFN-?-PerCPCy5.5 antibody,and the sample was detected by flow cytometry.Mouse peripheral blood was used to detect the levels of interleukin-4,interleukin-6,IFN-?and tumor necrosis factor?TNF?in peripheral blood of tumor-bearing mice by cytokines flow cytometry microarray.Results:1.The number of activated CD8⁺T cells or CD4⁺T cells in the tumor-bearing IL-18-KO mice was significantly reduced.2.The fluorescence intensity of intracellular staining of IFN-?antibody in NK cells and CD8⁺T cells in spleen and esophageal tissues of interleukin-18-deficient tumor-bearing mice was significantly lower than that in wild-type tumor-bearing mice.3.The trend of tumor necrosis factor and IFN-?in serum of two groups of tumor-bearing mice was similar to that of IFN-?in tissues,and the difference was statistically significant.Conclusion:1.Deletion of interleukin-18 inhibits T lymphocyte activity in esophageal carcinoma tissue.2.Deletion of interleukin-18 inhibits IFN-?production by CD8⁺T cells and NK cells.3.Deletion of interleukin-18 reduces the levels of tumor necrosis factor?TNF?and IFN-?in the peripheral blood of tumor-bearing mice.Part three The loss of interleukin-18 affects the proliferation andapoptosis of anti-tumor immune cells and inhibits their cytotoxicity in vitro experiments.Objective:To observe the effect of interleukin-18 deficiency on the ability of anti-tumor immune cells to kill standard cell lines of esophageal cancer in vitro,and to verify consistency with in vivo results.Methods:Using Miltenyi Separation Kit to purify the CD8⁺T cells and NK cells.The cytoplasmic lactate dehydrogenase assay kit was used to detect the killing effect of effector cells?CD8⁺T lymphocytes and NK cells?on target cells?TE-8?at 1:1,3:1 and 10:1,respectively.The Annexin-V/PI Apoptosis Detection Kit was used to stain for 15 minutes in the dark at room temperature.Flow cytometry was used to detect the percentage of dead cells.The purified spleen CD8⁺T lymphocytes of the tumor-bearing mice were used to detect the proliferation of CD8⁺T lymphocytes in the interleukin-18-KO group and the interleukin-18-WT group using CCK-8 test kit.Results:1.The deletion of interleukin-18 significantly reduced the cytotoxicity of spleen CD8⁺T cells and NK cells in tumor-bearing mice,and the results were statistically different.2.Compared with CTL cells derived from spleen of wild-type esophageal cancer-bearing mice,CTL cells derived from interleukin-18 knockout esophageal cancer-bearing mice showed a strong apoptosis under the same conventional cell culture conditions.3.The 72-hour cell proliferation assay determined by the CCK-8 kit showed that the proliferation of CTL cells derived from IL-18-KO esophageal cancer-bearing mice was higher than that of wild-type esophageal cancer-bearing mice under the same conventional cell culture conditions.Conclusion:1.Deletion of interleukin-18 significantly inhibits the direct cytotoxicity of anti-tumor immune effector cells--CD8⁺T cells and NK cells against target cells in esophageal cancer-bearing mice.2.Deletion of interleukin-18 is a clear signal for inducing apoptosis of cytotoxic lymphocytes in esophageal cancer-bearing mice.3.Deletion of interleukin-18 can significantly inhibit the proliferation of CD8⁺T cells in esophageal cancer-bearing mice.