The Effects Of TLR4/TLR9 Agonists Polarized Macrophages On The Biological Characteristics Of Hepatocellular Carcinoma

ObjectHepatocellular carcinoma(HCC)is one of the most serious diseases in humans,and it ranks the world's second most deadly cancer.There are about 750,000 new liver cancer patients each year,and the incidence of HCC is on the rise.Chronic hepatitis caused by HBV or HCV infection,alcoholic cirrhosis caused by excessive drinking,chronic liver disease caused by nonalcoholic steatohepatitis(NASH),are major risk factors for HCC production.Because the pathogenesis of hepatocellular carcinoma is extremely complex and heterogeneous,so far,clinical treatment did not take the relevant molecular biology information into account At present,the clinical treatment of HCC including liver tissue local ablation,liver resection,liver transplantation,and the use of chemotherapy or radiotherapy drugs for portal vein infiltration or extrahepatic proliferation of patients with systemic treatment.However,most of the above treatment methods are lack of specificity and targeting,damage to the function of tissue cells in patients,so that the patient's immune system has been destroyed,bringing great toxic effects.How to break the body immune tolerance and activate the killing function to the tumor cells are the main directions of the current immunotherapy to HCC treatment.In recent years,the use of immune cells on the HCC cell immunotherapy treatment has become a hot spot.More and more studies are based on target therapy of dendritic cells(DCs)and natural killer(NK)cells.Adoptively transferred DC or NK cells to patients and make them activate in vitro.Besides,amplification of the inherent anti-tumor activity of autologous immune cells,such as lymphokine activated killer cells(LAK),tumor infiltrating lymphocytes(TIL),cytokine-induced killer cells(CIK),Etc.,to play a role in resisting the tumor.Macrophage is an important component of the innate immune system with potent phagocyte and antigen presentation ability.Macrophages can recognize and kill pathogenic microorganisms,also they can treat,remove or kill the cells through the PRRs,constitute the first line of the immune system to recognizes "non-self' In addition,macrophages swallow antigens and present antigens to the T cells,mediated adaptive immune response.Thus macrophage is known as a bridge that link to innate immune response and adaptive immune response.It is noteworthy that macrophages are heterogeneous cells,they can polarized into Ml and M2 subtypes in different cytokine microenvironments,mediating inflammatory response,anti-tumor or immune regulation and other corresponding biological functions.More and more studies have shown that the imbalance of macrophage polarization leads to a variety of immune-related diseases such as autoimmune diseases and cancer.In recent years,the study of target molecules for macrophage polarization has become a new hotspot.Toll-like receptors(TLRs)belong to the PRR family,which can specifically recognize the pathogen associated molecular patterns(PAMPs)on the surface of pathogenic microorganisms,further initiating the innate immune response,TLRs family is widely expressed in a variety of cells and rich in macrophages.Among them,TLR4 expressed on the cell surface mainly recognizes lipopolysaccharide(LPS)from bacteria,activates downstream NF-?B signaling pathway,releases inflammatory factors such as IL-1? and IL-6,and mediates the inflammatory response.Endosomal TLR9,which can identify CpG DNA from bacteria or viruses,activate downstream MAPK pathway,to produce a large number of interferon,participate in anti-virus and anti-tumor response Because of the presence of gut/liver axis,HCC patients often accompanied with intestinal disorders,bacteria or viruses from the intestinal tract could further aggravating the patient's liver inflammatory response.Therefore,we.used LPS and CpG-ODN to stimulate macrophages.The polarization direction of macrophages stimulated by agonists was observed in vitro.In this study,we detected the effect of cytokines secreted by macrophages on tumor cells and the ability of macrophages to engulf tumor cells,to further explore the effect of TLR4 and TLR9 agonist polarized macrophages on the biological function of hepatocellular carcinoma.Methods1.The expression levels of TLR1?TLR9 in mouse macrophage cell line RAW264.7 and mouse primary peritoneal macrophages were detected by RT-PCR.LPS could induce the expression of TLR1,TLR6,TLR9 in macrophages.2.Western blot was used to detect the activation of NF-kappa B signaling pathway in macrophages after the stimulation with LPS or CpG-ODN.3.The expression of inflammatory cytokines in macrophages stimulated by LPS or CpG-ODN was detected by Real Time PCR.4.The mRNA levels of macrophage polarization-related cytokines in mouse macrophages stimulated by LPS or CpG-ODN were detected by Real Time-PCR.M2-macrophages were labeled with CD206.Flow cytometry was used to detect type M2 macrophages after LPS or CpG-ODN-stimulation;flow cytometry was used to detect the expression of MHC class ? molecules in macrophages stimulated by LPS or CpG-ODN.5.Flow cytometry and immunofluorescence were used to detect the phagocytosis of Hepal-6 cells by LPS or CpG-ODN-stimulated macrophages.6.The expression of chemokine was detected by real time-PCR in mouse macrophages stimulated by LPS or CpG-ODN.7.The macrophages were stimulated by TLR4 and TLR9 agonists for 24 hours.The macrophages were washed and cultured for 48 hours.The macrophage conditioned medium was collected and placed in the lower chamber.Transwell migration assay was performed on Hepal-6 cells.Western Blot was used to detect the activation of NF-?B and Erk signaling pathways in Hepal-6 cells which incubated with macrophage conditioned supernatant for 12 hours.8.The macrophages were stimulated with TLR4 and TLR9 agonists for 24 hours,and the macrophages were washed and cultured for 48 hours.The macrophage conditioned medium was collected and placed in the lower chamber The transwell invasion assay was performed on Hepal-6 cells.Western Blot was used to detect the activation of stat3 signaling pathway in Hepal-6 cells which incubated with macrophage conditioned supernatant for 12 hours.9.Hepal-6 cells were incubated with LPS,CpG-ODN-stimulated macrophage condition supernatants,the proliferation,apoptosis and cell cycle of hepatocarcinoma cells were detected by RTCA and flow cytometry.10.The cytotoxicity of spleen lymphocytes to hepatocellular carcinoma cells after incubation with macrophages was assayed by MTT assay.11.Transwell migration assay was used to detect the recruitment of spleen lymphocytes by Hepal-6 after incubation with macrophages.12.Flow cytometry was used to detect the activation of CD8+T,CD4+T and NK1.1+ cells in spleen lymphocytes.13.Flow cytometry was used to detect the expression of PD-L1 in Hepal-6,which was incubated by supernatant of macrophages.Results1.LPS stimulated the expression of TLR4 in primary peritoneal macrophages,and the expression of TLR9 was increased after the stimulation with CpG-ODN;2.LPS and CpG-ODN could activate NF-?B signaling pathway in PEMs.3.The IL-1?,IL-18,IL-36?,and IL-37mRNA levels were increased in mouse macrophages stimulated by LPS and CpG-ODN,but LPS exerted a significantly stronge activityr than CpG-ODN;4.IL-10 mRNA level was reduced after treatment by LPS inmacrophage;but the IL-10 mRNA level was increased in CpG-ODN-stimulated macrophages;CD206 fluorescence intensity was decreased in LPS-stimulated macrophages,MHC class ? molecules were up-regulated and the fluorescence intensity of CD 206 increased in CpG-ODN-stimulated macrophages,while MHC class ?molecules were down-regulated.5.CpG-ODN-stimulated macrophages exerted a strong phagocytosis against Hepal-6 cells;6.The expression of CCL3/4/5/12 and CXCL1/10 mRNA was increased in LPS or CpG-ODN-stimulated macrophages,7.The migration ability of hepatocarcinoma cells incubated with the supernatant of LPS-stimulated macrophages was enhanced.8.The invasion ability of hepatocarcinoma cells incubated with both LPS and CpG-ODN-stimulated macrophage condition supernatant was enhanced.9.Condition supernatant from LPS and CpG-ODN-stimulated macrophage restrained the proliferation of hepatoma cells and promoted the apoptosis of hepatocellular carcinoma cells;10.Condition supernatant from LPS and CpG-ODN-stimulated macrophage had on effect on the spleen cell killing to Hepal-6 cells;11.Hepal-6 cells have a strong ability to recruit splenic monocytes after incubation with LPS-stimulated macrophage condition supernatants;12.There was no significant change in the activation of CD8+T,CD4+T,NK1.1+NK cells in spleen lymphocytes recruited by hepatocellular carcinoma cells after incubation with LPS and CpG-ODN macrophages.:13.The expression of PD-L1 was not affected in HCC after incubation with LPS and CpG-ODN-stimulated macrophages.Conclusion1.In this study;we found that the ratio of macrophages M1/M2 was increased after LPS stimulation and the percentage of macrophages M1/M2 decreased after CpG-ODN stimulation,The results showed that LPS and CpG-stimulated macrophages are polarized in different directions.2.The ability of LPS to induce macrophages to express inflammatory factors was significantly stronger than that of CpG-ODN,and LPS and CpG-ODN could promote the migration of macrophages.3.In this study,it was found that the supernatant of macrophages stimulated by LPS and CpG-ODN could inhibit the proliferation of hepatocarcinoma cells,and the macrophage supernatant could promote the apoptosis of hepatocellular carcinoma cells.4.The effect of macrophages on the development of HCC was further elucidated,and this provided evidence for the clinical application of TLR agonists in the treatment of HCC.