The Effect And Mechanism Of TAM Receptor,A Negative Regulators Of Toll-like Receptor Signaling Pathway,on Inflammatory Bowel Diseases And Macrophage

Background and objectives:Inflammatory bowel disease（IBD）is a chronic inflammatory disease of the colon,including Ulcerative Colitis（UC）and Chron’s disease（CD）.The etiology of inflammatory bowel disease is not fully understood and may be related to the complex interactions between patients’genetic susceptibility,environment,intestinal flora,and immune system.Studies have shown that macrophages are a key player in inflammatory bowel disease.In the intestinal inflammatory response of IBD patients,the number of macrophages increased significantly,and various biologically active substances such as interleukins and tumor necrosis factor were released.Toll-like receptors（TLRs）are key to host defense and innate immunity.The excessive activation of TLRs signals is related to the occurrence of IBD,so it is particularly important to suppress the excessive activation of TLRs signals.TAM receptors（TAM-receptors）are a subfamily of receptor tyrosine kinases and one of the negative regulators of TLRs signaling pathways.They have a variety of biological functions including immune regulation.Our previous study found thatthe TAM receptor ligand Gas6 could inhibit the activation of TLR4 signaling pathway and the secretion of pro-inflammatory cytokines in macrophages after LPS stimulation.But how TAM receptors are regulated in this process is unclear.Therefore,this paper will conduct a preliminary study on the regulatory role and mechanism of TAM receptors in inflammatory bowel disease and macrophages.Methods:Ⅰ.Changes of TAM receptor signals and TLRs signaling pathway in IBD and their relationship with disease1.Changes of TAM receptor signal and ligand Gas6 in intestinal mucosa of IBD patients and its relationship with disease（1）Experimental grouping:（1）Normal patients（n=19）（2）Patients with inflammatory bowel disease（n=19）（2）Assessment:Immunohistochemical detection of mucosal TAM（Tyro3,Axl,Mertk）,Gas6,protein expression in each group of patients.2.Changes in TAM receptor signaling and TLRs signaling pathways in mouse experimental colitis models and their relationship with disease（1）Experimental groups:Twenty Balb/c mice were randomly divided into the following two groups as follows:（1）Control group（n=10）（2）DSS model group（n=10）（2）Establishment of experimental colitis models:Establishment of an experimental colitis model induced by DSS in mice:Balb/c mice aged 6-8 weeks were free to drink 5%DSS solution for 7 days,and the mice were sacrificed on the 8th day（This process has been completed by the research team in the early stage）.（3）Assessment:（1）Quantitative PCR was used to detect the expression of TAM（Tyro3,Axl,Mertk）,TLR2,TLR4andMyD88mRNA in the intestinal mucosa of each group（2）Wes automated western blotting system（ProteinSimple,San Jose,CA）to detect the expression of TAM（Tyro3,Axl,Mertk）,TLR2,TLR4and MyD88 protein in the intestinal mucosa of each group.Ⅱ.The role of TAM receptor signaling on macrophages and the mechanism of TLRs signaling pathway regulation（1）Experimental grouping:（1）Blank group:RAW264.7,Axl^LowRAW264.7,Tyro3^HighRAW264.7,Mertk^Highigh RAW264.7（2）LPSstimulationgroup:RAW264.7,Axl^LowRAW264.7+LPS,Tyro3^HighRAW264.7+LPS,Mertk^Highigh RAW264.7+LPS（3）LPS stimulation group and Gas6 treatment group:RAW264.7+LPS+Gas6,Axl^Lowow RAW264.7+LPS+Gas6（2）Construction of Tyro3,Axl,Mertk knockdown cell lines and overexpressed macrophages:By transfecting the lentiviral vector constructed by Shanghai Jikai Gene Co.,Ltd.,after puromycin drug screening,the transfected strain RAW264.7 cells were amplified to construct Axl knockdown cell lines,and two effective knockdown targets were screened And Tyro3 and Mertk overexpression stable transfectants.（3）Assessment:（1）Quantitative quantitative PCR to detect the expression of TLR2,TLR4 and MyD88 mRNA in each group of cells.（2）Wes automated western blotting system（ProteinSimple,San Jose,CA）to detect the protein expression of TLR4,MyD88 and pNF-κB p65 protein in each group of cells.Results:1.Changes in TAM receptor signaling and TLRs signaling pathways in IBD and their relationship with diseases（1）Expression of TAM receptor signal and ligand Gas6 in intestinal mucosa of IBD patientsIHC results showed that the number of positive cells in the intestinal mucosa tissue of IBD patients was significantly lower than that of the normal control group,and the expression of TAM（Tyro3,Axl,Mertk）and Gas6 protein were significantly lower than that of the normal control group（P<0.001）.（2）Expression of TAM receptor and TLRs signaling pathway related molecules in colon tissue of mice with experimental colitis induced by DSSRT-PCR results showed that compared with the control group,the expression of Axl mRNA in the colon tissue of mice with experimental colitis induced by DSS decreased,there was a significant difference（P<0.05）,the expression of Tyro3 and Mertk mRNA decreased,but the difference was not statistically significant Significance（P>0.05）;among molecules related to TLRs signaling pathway,TLR2mRNA expression increased,significantly higher than normal control group（P<0.05）,TLR4 and MyD88 mRNA expression increased compared with normal control group,but no difference Statistical significance（P>0.05）.WesTM protein results showed that the expression of Tyro3,Axl and Mertk protein in the colon tissue of experimental colitis mice induced by DSS decreased compared with the normal control group,and was significantly lower than that of the normal control group（P<0.01,P<0.05）;The protein expressionof TLR2 increased,significantly higher than the normal control group（P<0.05）.2.The effect of TAM receptor on the TLRs signaling pathway of macrophages（1）Construction and verification of Tyro3,Axl,Mertk knockdown cell lines and overexpressed stable macrophage cell linesBy commissioning Shanghai Jikai Gene to constructvirus,transfection of Tyro3,Axl,Mertk knockdown and overexpression macrophages,4 days after transfection,puromycin drug screening,screening of knockdown cell lines and overexpression stable transfected cells Strains,and the cell transfection efficiency was observed to be more than 90%through an inverted fluorescence microscope.Subsequently,the mRNA and protein levels were verified,and the mRNA and protein expressions were detected by RT-PCR and Western Blot to verify the successful construction of Tyro3,Axl,Mertk knockdown cell lines and over-expression stable transfected macrophage cell lines and select the best knockdown target point.（2）The effect of Axl on TLRs signaling pathway of macrophages（1）Knock down the effect of TLRs signaling pathway in AXL macrophageCompared with Axl^Lowow RAW264.7 and WT-RAW264.7,at the mRNA level,the expression of TLR2 increased significantly（P<0.01）,the expression of TLR4 and MyD88 mRNA also increased,but the difference was not statistically significant（P>0.05）;At the protein level,the expression of pNF-κB p65 increased significantly（P<0.05）,and the expression of TLR4 and MyD88 proteins also increased,but the difference was not statistically significant（P>0.05）.After LPS stimulation,Axl^Lowow RAW264.7 compared with WT-RAW264.7,at the mRNA level,TLR2 and TLR4 mRNA expression was significantly increased（p<0.01）,MyD88 mRNA expression was also increased,but the difference was not statistically significant（P>0.05）;at the protein level,pNF-κB p65 protein expression was significantly increased（P<0.01）,TLR4 and MyD88 protein expression also increased,but the difference was not statistically significant（P>0.05）.（2）The effect of Gas6 on TLRs signaling pathway in AXL knockdown macrophagesAfter LPS stimulated Axl ^LowRAW264.7,TLR2 and MyD88 mRNA expression were significantly higher than those without LPS stimulation（P<0.01）,and TLR4mRNA expression was also higher than those without LPS stimulation,but the difference was not statistically significant（P>0.05）.The protein expressions of TLR4and MyD88 were significantly higher than those without LPS stimulation（P<0.01）.After LPS and Gas6 treatments,the expression of TLR2,TLR4 and MyD88mRNA decreased significantly and was lower than that without the Gas6 treatment group（P<0.01）,and the protein expression of TLR4 and MyD88 also decreased significantly and was lower than that without the Gas6 treatment group（P<0.01）.（3）The effect of Tyro3 on TLRs signaling pathway of macrophagesCompared with Tyro3^HighRAW264.7 and WT-RAW264.7,at the mRNA level,TLR2 mRNA expression decreased significantly（P<0.01）,TLR4 and MyD88 mRNA expression also decreased,but the difference was not statistically significant（P>0.05）;at the protein level,MyD88 and pNF-κB p65 protein expression decreased significantly（P<0.05）,TLR4 protein expression also decreased,but the difference was not statistically significant（P>0.05）.After LPS stimulation,Tyro3^HighRAW264.7 compared with WT-RAW264.7,at the mRNA level,TLR4 mRNA expression decreased,significantly lower than the wild-type macrophage group（P<0.01）,TLR2 and MyD88 mRNA expression also decreased,But the difference was not statistically significant（P>0.05）;at the protein level,the expression of TLR4 and MyD88 protein decreased,both were significantly lower than that of wild-type macrophage group（P<0.01,P<0.05）,the expression of pNF-κB p65 protein Decreased,but the difference was not statistically significant（P>0.05）.（4）The effect of Mertk on TLRs signaling pathway of macrophagesCompared with Mertk ^Highigh RAW264.7 and WT-RAW264.7,at the mRNA level,TLR2 and MyD88 mRNA expression decreased significantly（P<0.01,P<0.05）,TLR4 mRNA expression also decreased,but the difference was not statistically significant（P>0.05）;At the protein level,the expression of TLR4 and MyD88protein decreased significantly（P<0.01,P<0.05）,the expression of pNF-κB p65protein also decreased,but the difference was not statistically significant（P>0.05）.After LPS stimulation,Mertk^Highigh RAW264.7 compared with WT-RAW264.7,at the mRNA level,TLR4 and MyD88 mRNA expression decreased,significantly lower than the wild-type macrophage group（P<0.01,P<0.05）,TLR2 mRNA The expression also decreased,but the difference was not statistically significant（P>0.05）;at the protein level,the expression of TLR4 and MyD88 protein decreased significantly（P<0.01,P<0.05）,the expression of pNF-κB p65 protein also decreased,but the difference was not Statistical significance（P>0.05）.Conclusions:1.Intestinal inflammation in colonic mucosa of IBD patients andDSS-induced excessive activation of TLRs in the intestinal mucosa of mice with experimental colitis may be related to the inhibition of the negative regulator TAM（Tyro3,Axl,Mertk）/Gas6 signal.2.TAM receptor can inhibit the activation of TLRs signaling pathway in macrophages after LPS stimulation,and its pathway may involve both MyD88dependent and independent pathways.3.In macrophages,in addition to Axl,Gas6 may also negatively regulate the TLRs signaling pathway through Tyro3 and Mertk or non-TAM pathways.