The Role And Mechanism Of Tim4 And Mertk In Synergistically Regulating Efferocytosis In Inflammatory Bowel Disease And Related Colorectal Cancer

Background and objectives:Inflammatory bowel disease（IBD）is an autoimmune disease characterized by recurrent chronic inflammation of the gastrointestinal tract,which is mainly composed of two subtypes:ulcerative colitis（UC）and Crohn’s disease（CD）.IBD-related colorectal cancer（IBD-CRC）follows the pathogenesis of"inflammation-dysplasia-cancer"and is a major and serious complication in patients with long-standing IBD.However,the exact pathogenic mechanisms of IBD and IBD-CRC are still unknown,and the prevention and treatment of IBD and IBD-CRC remain a great challenge in clinical work.Efferocytosis refers to the biological process in which macrophages engulf and clear apoptotic cells that undergo programmed cell death.It is mainly composed of four parts:recognition,engulfment,digestion,and clearance of apoptotic cells,each of which is finely and intricately regulated by corresponding molecules and ligands.The success or failure of efferocytosis activates different downstream effector molecules,exerting either anti-inflammatory or pro-inflammatory effects.One of the intestinal characteristics of IBD and IBD-CRC is persistent inflammatory response.How does efferocytosis function in IBD and IBD-CRC?How do numerous efferocytosis-related molecules interact and interfere with each other to maintain the homeostasis of the body in IBD and IBD-CRC?It has not been reported yet.T cell immunoglobulin and mucin domain 4（Tim4）is an immunoregulatory molecule primarily expressed on the surface of antigen-presenting cells.It participates in the immune process and maintains the homeostasis of the body through various ways.Mertk is a member of the TAM receptor tyrosine kinase subfamily and plays an important role in inflammation,cell adhesion,migration,and proliferation.Research has shown that Tim4 on macrophages can bind to the phosphatidylserine（PS）receptor on the surface of apoptotic cells,anchoring them to the macrophages,and then signaling to Mertk to enhance efferocytosis.Tim4 alone does not support the transmission of efferocytosis signals,but can enhance TAM receptor-dependent efferocytosis.Therefore,Tim4 and Mertk need to coordinate with each other to ensure the completion of efferocytosis,thereby eliminating inflammation and promoting tissue repair.If there is an imbalance between Tim4 and Mertk,efferocytosis will be hindered,leading to an exacerbation of the inflammatory response.However,it is currently unclear how Tim4 and Mertk work synergistically to regulate efferocytosis in IBD and IBD-CRC.Based on the above research,the following research was conducted in this study:（1）Investigated the changes of Tim4,Mertk,and efferocytosis-related signaling molecules in clinical tissue samples and animal models,and analyzed their relationship with IBD and IBD-CRC;（2）Established a DSS-induced IBD model in mice treated with Tim4 antibodies to study the role and regulatory mechanism of efferocytosis,Tim4 and Mertk signaling in IBD;（3）Co-cultured Tim4 and Mertk-knockdown or overexpressing THP1 cells with normal/apoptotic Jurkat cells to study efferocytosis and the synergistic regulation mechanism of Tim4 and Mertk signaling;（4）Analyzed the biological functions of Tim4 in IBD and IBD-CRC based on transcriptomic analysis.Through the above study,we aim to reveal the pathogenesis of IBD and IBD-CRC from the new perspective that Tim4 and Mertk synergistically regulate efferocytosis and provide new targets for their treatment.Materials and Methods:I.Changes of efferocytosis in IBD and its related colorectal cancer1.Pathological changes in colonic tissues of each group of mice:（1）Study grouping:30 SPF-grade BALB/c mice were randomly divided into a normal control group,a DSS group,and a CAC group.（2）Model construction:DSS group mice were given 5%DSS solution to drink.CAC group mice were injected intraperitoneally with 10mg/kg AOM and continuously drank 3%DSS solution for five days,followed by sterile drinking water from day 6 to 19.Three cycles of continuous drinking of 3%DSS solution and sterile drinking water were performed.（3）The histopathological changes in the colonic tissues of normal control group,DSS group,and CAC group mice were examined by H&E staining.2.Changes in efferocytosis in the colonic mucosa of each group of mice:Changes in efferocytosis in the colonic mucosa of normal control,DSS-treated,and CAC mouse models were detected by histological immunofluorescence and TUNEL staining.3.Pathological changes in colonic tissue of each group:The histopathological changes in colonic tissue of healthy individuals,active ulcerative colitis（AUC）,remission ulcerative colitis（RUC）,active Crohn’s disease（ACD）,remission Crohn’s disease（RCD）,and colorectal cancer（CRC）patients were detected by H&E staining.4.Changes in efferocytosis in the colonic mucosa of each group:Efferocytosis was assessed by histological immunofluorescence and TUNEL staining in the colonic mucosa of healthy individuals,AUC,RUC,ACD,RCD,and CRC patients.5.Macrophage efferocytic activity towards apoptotic cells:（1）Construction and verification of the apoptotic Jurkat cell model:Jurkat cells were induced to undergo apoptosis with 1μM staurosporine,and apoptotic cells were identified using Hoechst 33342 staining and flow cytometry.（2）Cell immunofluorescence was used to assess the efferocytic activity of THP1 cells towards apoptotic Jurkat cells.II.Expression changes of efferocytosis genes in IBD and its related colorectal cancer1.Expression of efferocytosis-related genes in the colonic mucosa of mice in each group:The expression of efferocytosis-related genes Tim4,Mertk,Gas6,MFGE8,Axl,Tyro3,Pros,and ERK5 in the colons of normal control group,DSS group,and CAC group mice were detected by Western blot,fluorescent quantitative PCR,and tissue immunofluorescence.2.Expression of efferocytosis-related genes in the colonic mucosa of human subjects in each group:The expression of efferocytosis-related genes Tim4,Mertk,Gas6,MFGE8,Axl,Tyro3,Pros,and ERK5 in the colons of healthy individuals,IBD patients,and CRC patients were detected by Western blot and fluorescent quantitative PCR.III.The role and mechanism of Tim4 and Mertk synergistically regulating efferocytosis in IBD and its related colorectal cancer1.Overall study（1）Establishment and intervention of DSS-induced colitis model in mice:（1）Grouping:40 SPF BALB/c mice were randomly divided into control group,DSS group,DSS+Anti-tim4 group,and Anti-tim4 group.（2）Model construction:DSS group mice were given 5%DSS solution to drink.12 hours before giving 5%DSS solution,anti-Fc antibody was used for blockade,and immunoglobulin G2b subtype antibody（Ig G2b antibody）was injected into the abdominal cavity of mice.DSS+Anti-tim4 group mice were given 5%DSS solution to drink.12 hours before giving 5%DSS solution,anti-Fc antibody was used for blockade,and Tim4 antibody inhibitor was injected into the abdominal cavity of mice.Anti-tim4 group mice were given normal drinking water.Before modeling,anti-Fc antibody was used for blockade,and Tim4 antibody inhibitor was injected into the abdominal cavity of mice.（3）Monitor changes in body weight,fecal viscosity,and fecal occult blood in each group of mice.（4）H&E staining was used to detect histopathological changes in the colon of each group of mice.（2）Tim4 expression in the colon mucosa of each group of mice:Immunohistochemistry was used to detect Tim4 expression and score it in the colon of the normal control group,DSS group,DSS+Anti-tim4 group,and Anti-tim4 group.（3）Effect of Tim4 intervention on efferocytosis in mouse colonic mucosa:Immunofluorescence and TUNEL staining were used to detect changes in efferocytosis in the colons of each group of mice.Western blot and fluorescent quantitative PCR were used to detect the expression of efferocytosis-related genes Tim4,Mertk,Gas6,MFGE8,Axl,Tyro3,Pros,ICAM1 and ERK5 in the colons of each group of mice.（4）Effect of Tim4 intervention on macrophage polarization and pro-inflammatory cytokines in mouse colonic mucosa:Western blot and fluorescent quantitative PCR were used to detect the expression of M1 macrophage markers（CD86 and i NOS）,M2 macrophage marker（CD206）,and pro-inflammatory cytokines（IL-1β,TNF-α,and IL-6）in the colons of the normal control group,DSS group,DSS+Anti-tim4 group,and Anti-tim4 group.2.Cell biology study（1）Effects of Tim4 intervention on cell efferocytosis:（1）Construction of Tim4-o verexpressing cell line Tim4^highTHP1 and Tim4-knockdown cell line Tim4^lowTHP1.（2）Cell immunofluorescence detection of efferocytosis in THP1+Jurkat、THP1+Apo ptosis Jurkat、NC-Tim4^highTHP1+Jurkat、NC-Tim4^highTHP1+Apoptosis Jurkat、Ti m4^highTHP1+Jurkat、Tim4^highTHP1+Apoptosis Jurkat、NC-Tim4^lowTHP1+Jurkat、NC-Tim4^lowTHP1+Apoptosis Jurkat、Tim4^lowTHP1+Jurkat and Tim4^lowTHP1+Apop tosis Jurkat.（3）Western blot analysis was performed to assess the protein expression o f Tim4,Mertk,Gas6,Pros,and MFGE8 in THP1+Jurkat、THP1+Apoptosis Jurkat、NC-Tim4^highTHP1+Jurkat、NC-Tim4^highTHP1+Apoptosis Jurkat、Tim4^highTHP1+Ju rkat、Tim4^highTHP1+Apoptosis Jurkat、NC-Tim4^lowTHP1+Jurkat、NC-Tim4^lowTH P1+Apoptosis Jurkat、Tim4^lowTHP1+Jurkat and Tim4^lowTHP1+Apoptosis Jurkat.（2）Effect of Mertk intervention on efferocytosis:（1）Construction of Mertk overe xpression cell line Mertk^highTHP1 and Mertk knockdown cell line Mertk^lowTHP1.（2）Cell immunofluorescence assay to detect changes in the efferocytic activity of THP1+Jurkat、THP1+Apoptosis Jurkat、NC-Mertk^highTHP1+Jurkat、NC-Mertk^highTHP1+Apoptosis Jurkat、Mertk^highTHP1+Jurkat、Mertk^highTHP1+Apoptosis Jurkat、NC-Mertk^lowTHP1+Jurkat、NC-Mertk^lowTHP1+Apoptosis Jurkat、Mertk^lowTHP1+Jurk at and Mertk^lowTHP1+Apoptosis Jurkat.（3）Western blot analysis was performed to as sess the protein expression of Mertk,Tim4,Gas6,Pros,and MFGE8 in THP1+Jurka t、THP1+Apoptosis Jurkat、NC-Mertk^highTHP1+Jurkat、NC-Mertk^highTHP1+Apop tosis Jurkat、Mertk^highTHP1+Jurkat、Mertk^highTHP1+Apoptosis Jurkat、NC-Mertk^{l ow}THP1+Jurkat、NC-Mertk^lowTHP1+Apoptosis Jurkat、Mertk^lowTHP1+Jurkat and Mertk^lowTHP1+Apoptosis Jurkat.（3）Interaction between Tim4 and Mertk:Detection of the interaction between Tim4 and Mertk in THP1 cells by immunofluorescence and co-immunoprecipitation.IV.Transcriptome analysis of the role of Tim4 in IBD and its associated colorectal cancer1.Sample preparation:first extract total RNA from the colon tissue of mice in the normal control group,DSS group,DSS+Anti-tim4 group and Anti-tim4 group,and then perform enrichment,fragmentation,reverse transcription,fragment screening and library enrichment And so on,and finally use the Illumina Novaseq6000 platform for on-machine sequencing.2.Transcriptomics sequencing data analysis:（1）PCA analysis between samples;（2）Screening of differential genes:The differential gene analysis software is DEGseq,and the differential gene expression fold for screening is FC（Fold change）≧1,and the corrected P value（p-adjusted）<0.05;（3）GO enrichment analysis,KEGG enrichment analysis and Reactome enrichment analysis were performed on the differential genes.3.Correlation analysis between efferocytosis signaling pathways and transcriptome sequencing data:（1）Screening of differential genes:The expression fold of differential genes screened is FC（Fold change）>0.5,P<0.05;（2）GO enrichment analysis and analysis of differential genes KEGG enrichment analysis.4.Correlation between transcriptome sequencing data and immune metabolism-related genes:（1）Determination of immune metabolism-related genes ADTRP,ALDOB,APOBEC1,ASCL2,CEACAM7,CLCA1,CTXN1,ENO2,FLNA,GABBR1,NAT2,OLFM4,PEMT,PTPRU,SLC44A4 and SNCG;（2）Screening of differential genes:The expression fold of differential genes screened was FC（Fold change）>0.5,P<0.05.5.Correlation between efferocytosis signaling pathway and genes related to immune metabolism:（1）Determine 96 cell burying signaling pathway genes based on PCR array kit;（2）Use R software 4.0.5 for correlation analysis.Results:I.Changes of efferocytosis in IBD and its related colorectal cancer1.Pathological changes of colonic tissue in each group of mice:H&E staining showed that the colonic tissue structure of the control group mice was intact with parallel crypts and normal villi.The colonic tissue structure of the DSS group mice was incomplete,severely damaged,and had mucosal surface erosion and shallow ulcers formation;inflammatory cell infiltration and crypt abscess formation were observed in the lamina propria.The colonic tissue of CAC group mice lost normal glandular structure,and the cells showed pleomorphism,significant atypical nucleoli,and prominent nucleoli with high nuclear-cytoplasmic ratio.2.Changes of efferocytosis in colonic mucosa of each group of mice:Compared with the control group mice,the number of apoptotic cells in the colons of DSS group and CAC group mice was higher,and the overlapping yellow parts were more and denser,indicating that the efferocytosis was more significant.3.Pathological changes in colon tissues of different groups:H&E staining showed that the intestinal structure of healthy individuals was intact,with a smooth surface,mild inflammation,and no villi.The colon tissues of AUC patients showed severe destruction of intestinal structure,diffuse inflammation of the mucosa,infiltration of inflammatory cells such as lymphocytes and monocytes in the full layer of the lamina propria,obvious abnormalities in crypt structure,and a decrease in goblet cells.The colon tissues of RUC patients showed atrophic changes such as deformation,disordered arrangement,and reduced number of glandular structures,and disordered crypt structure.The colon tissues of ACD patients showed a large amount of lymphocyte aggregation in the lamina propria,focal enhanced inflammation in the colon mucosa accompanied by basal granuloma,fissure-like ulcers,an increase in goblet cells,and congestion,edema,lymphatic vessel dilation,and fibrous tissue hyperplasia.The colon mucosal structure of RCD patients was damaged,with deformed crypt structure and more obvious intestinal fibrosis and scar contraction.The colon tissues of CRC patients showed an irregular,serrated interface in the lamina propria,with cells exhibiting polymorphism and more prominent atypical nuclei.4.Changes in efferocytosis in colonic mucosa among different groups:AUC,RUC,ACD,RCD,and CRC patients had more apoptotic cells in the colon mucosa compared to healthy individuals,with more overlap and denser yellow areas,indicating a more significant efferocytic effect.AUC patients had more apoptotic cells in the colon than RUC patients,showing a more pronounced efferocytic effect.ACD patients had more apoptotic cells in the colon than RCD patients,exhibiting a more pronounced efferocytic effect.5.Efferocytic effect of macrophages on apoptotic cells:The STS group（polarized THP1 cells co-cultured with apoptotic Jurkat cells）had more overlap and denser yellow areas compared to the control group（polarized THP1 cells co-cultured with normal Jurkat cells）,indicating a more pronounced efferocytic effect.II.Expression changes of efferocytosis genes in IBD and its related colorectal cancer1.Expression of efferocytosis-related genes in the colonic mucosa of mice in each group:Tim4 expression in the colonic mucosa of DSS group mice was significantly higher than that in control group mice（P<0.001）,while the expression of Mertk,Gas6,MFGE8,Axl,Tyro3,Pros,and ERK5 was significantly lower than that in control group mice（P<0.001）.Tim4 expression in the colonic mucosa of CAC group mice was significantly higher than that in control group mice（P<0.01）,while the expression of Mertk,Gas6,MFGE8,Axl,Tyro3,Pros,and ERK5 was significantly lower than that in control group mice（P<0.05）.2.Expression of efferocytosis-related genes in the colonic mucosa of human subjects in each group:Tim4 expression in the colonic mucosa of IBD patients was significantly higher than that in healthy individuals（P<0.001）,while the expression of Mertk,Gas6,MFGE8,Axl,Tyro3,Pros,and ERK5 was significantly lower than that in healthy individuals（P<0.05）.Tim4 expression in the colonic mucosa of CRC patients was significantly higher than that in healthy individuals（P<0.05）,while the expression of Mertk,Gas6,MFGE8,Axl,Tyro3,Pros,and ERK5 was significantly lower than that in healthy individuals（P<0.05）.III.The role and mechanism of Tim4 and Mertk synergistically regulating efferocytosis in IBD and its related colorectal cancer1.Overall study（1）Body weight,DAI,colon length,and histopathological changes of mice in each group:（1）Body weight:The weight of mice in the control group was higher than that in the DSS group（P<0.001）.The body weight of mice in DSS+Anti-tim4 group was higher than that in DSS group（P<0.05）.The body weight of the mice in the Anti-tim4 group was higher than that in the DSS+Anti-tim4 group（P<0.01）.（2）DAI score:The DAI score of the mice in the DSS group was higher than that in the control group（P<0.001）.The DAI score of mice in DSS group was higher than that in DSS+Anti-tim4 group（P<0.05）.The DAI score of mice in Anti-tim4 group was lower than that in DSS+Anti-tim4 group（P<0.001）.（3）Length of the colon:The length of the colon of the mice in the DSS group was lower than that in the control group（P<0.0001）.The colon length of mice in DSS group was lower than that in DSS+Anti-tim4 group（P<0.05）.The colon length of Anti-tim4 mice was higher than that of DSS+Anti-tim4group（P<0.0001）.（4）Colon histopathology:The mice in control group and Anti-tim4group had complete colon structure,mild inflammation,normal intestinal villi,and crypts covered with goblet cells,endocrine cells and precursor cells.In the DSS group,the colon tissue structure was incomplete,the damage was severe,the mucosal surface was eroded,and shallow ulcers were formed;the lamina propria showed acute and diffuse infiltration of neutrophils and lymphocytes.The colonic histological score of mice in DSS group was higher than that in control group（P<0.001）.The histology of DSS+Anti-tim4 group was lower than that of DSS group（P<0.01）.The histological score of DSS+Anti-tim4 group was significantly higher than that of Anti-tim4 group（P<0.01）.（2）Expression of Tim4 in the colonic mucosa of mice in each group:the expression of Tim4 in the colon of mice in the DSS group was higher than that in the normal control group（P<0.05）.The expression of Tim4 in colon of mice in DSS+Anti-tim4 group was lower than that in DSS group（P<0.05）.The expression of Tim4 in colon of mice in Anti-tim4 group was lower than that in DSS+Anti-tim4group（P<0.01）.（3）The effect of interfering with Tim4 on colonic mucosal efferocytosis in mice:the number of apoptotic cells in the colon of mice in the DSS group was more than that in the control group,and the overlapping yellow parts were more and dense,that is,the efferocytosis effect was more obvious.The number of apoptotic cells in the colon of mice in the DSS group was more than that in the DSS+Anti-tim4 group,and the overlapping yellow parts were more and denser,that is,the cell encroachment effect was more obvious.The number of apoptotic cells in the colon of mice in the DSS+Anti-tim4 group was more than that in the Anti-tim4 group,and the overlapping yellow parts were more and denser,that is,the cell encapsulation effect was more obvious.The expression of Tim4 and ICAM1 in colon of mice in DSS group was higher than that in control group（P<0.001）.The expression of Tim4 and ICAM1 in colon of mice in control group was higher than that in Anti-tim4 group（P<0.05）.The expression of Tim4 and ICAM1 in colon of mice in DSS group was higher than that in DSS+Anti-tim4 group（P<0.05）.The expression of Tim4 and ICAM1 in colon of mice in Anti-tim4 group was lower than that in DSS+Anti-tim4 group（P<0.001）.The expressions of Mertk,Gas6,MFGE8,Axl,Tyro3,Pros and ERK5 in the DSS group were lower than those in the control group（P<0.05）.The expressions of Mertk,MFGE8,Axl,Tyro3,Pros and ERK5 in colon of mice in control group were lower than those in Anti-tim4 group（P<0.05）.The expressions of Mertk,MFGE8,Axl,Tyro3,Pros and ERK5 in colon of mice in DSS group were lower than those in DSS+Anti-tim4 group（P<0.05）.The expression of Mertk,MFGE8,Axl,Tyro3,Pros and ERK5 in colon of mice in Anti-tim4 group was higher than that in DSS+Anti-tim4 group（P<0.05）.（4）Effects of Tim4 intervention on macrophage polarization and pro-inflammatory cytokines in the colonic mucosa of mice:the expressions of CD86,i NOS and CD206 in the colonic mucosa of mice in the DSS group were significantly higher than those in the normal control group（P<0.05）.The expressions of CD86,i NOS and CD206 in colonic mucosa of mice in DSS+Anti-tim4 group were significantly lower than those in DSS group（P<0.05）.The expressions of IL-1β,TNF-αand IL-6 in colonic mucosa of mice in DSS group were significantly higher than those in normal control group（P<0.001）.The expressions of IL-1β,TNF-αand IL-6 in colonic mucosa of mice in DSS+Anti-tim4 group were significantly lower than those in DSS group（P<0.01）.2.Cytological research（1）The effect of intervening Tim4 on efferocytosis:（1）Compared with THP1+Jurkat group,the efferocytosis effect in THP1+Apoptosis Jurkat group was more obvious.Compared with the NC-Tim4^highTHP1+Jurkat group,the NC-Tim4^highTHP1+Apoptosis Jurkat group had a more obvious efferocytosis effect.Compared with the Tim4^highTHP1+Jurkat group,the Tim4^highTHP1+Apoptosis Jurkat group had a more obvious efferocytosis effect.Compared with the NC-Tim4^lowTHP1+Jurkat group,the NC-Tim4^lowTHP1+Apoptosis Jurkat group had a more obvious efferocytosis effect.In addition,compared with the Tim4^lowTHP1+Jurkat group,the Tim4^lowTHP1+Apoptosis Jurkat group had a more pronounced efferocytosis effect.Compared with the THP1+Apoptosis Jurkat group,the Tim4^highTHP1+Apoptosis Jurkat group had a more obvious efferocytosis effect.Compared with the NC-Tim4^highTHP1+Apoptosis Jurkat group,the Tim4^highTHP1+Apoptosis Jurkat group had a more obvious efferocytosis effect.Compared with the THP1+Apoptosis Jurkat group,the Tim4^lowTHP1+Apoptosis Jurkat group had inhibited the efferocytosis.Compared with the NC-Tim4^lowTHP1+Apoptosis Jurkat group,the Tim4^lowTHP1+Apoptosis Jurkat group was inhibited in efferocytosis.（2）Compared to the THP1+Jurkat group,the THP1+Apoptosis Jurkat group exhibited increased protein expression of Tim4,Mertk,Gas6,Pros,and MFGE8.In comparison to the NC-Tim4^highTHP1+Apoptosis Jurkat group,the Tim4^highTHP1+Apoptosis Jurkat group demonstrated decreased protein expression of Mertk,Gas6,Pros,and MFGE8,while Tim4 expression was increased.When compared to the NC-Tim4^lowTHP1+Apoptosis Jurkat group,the Tim4^lowTHP1+Apoptosis Jurkat group exhibited increased protein expression of Mertk,Gas6,Pros,and MFGE8,whereas Tim4 expression was decreased.（2）The effect of intervening Mertk on efferocytosis:（1）Compared with THP1+Jurkat group,the efferocytosis effect of THP1+Apoptosis Jurkat group was more obvious.Compared with NC-Mertk^highTHP1+Jurkat group,NC-Mertk^highTHP1+Apoptosis Jurkat group had a more obvious efferocytosis effect.Compared with the Mertk^highTHP1+Jurkat group,the Mertk^highTHP1+Apoptosis Jurkat group had a more obvious efferocytosis effect.Compared with NC-Mertk^lowTHP1+Jurkat group,NC-Mertk^lowTHP1+Apoptosis Jurkat group had more obvious efferocytosis effect.In addition,compared with the Mertk^lowTHP1+Jurkat group,the Mertk^lowTHP1+Apoptosis Jurkat group had a more obvious efferocytosis effect.Compared with the THP1+Apoptosis Jurkat group,the Mertk^highTHP1+Apoptosis Jurkat group had a more obvious efferocytosis effect.Compared with the NC-Mertk^highTHP1+Apoptosis Jurkat group,the Mertk^highTHP1+Apoptosis Jurkat group had a more obvious efferocytosis effect.Compared with the THP1+Apoptosis Jurkat group,the Mertk^lowTHP1+Apoptosis Jurkat group inhibited the efferocytosis.Compared with the NC-Mertk^lowTHP1+Apoptosis Jurkat group,the Mertk^lowTHP1+Apoptosis Jurkat group was inhibited in efferocytosis.（2）Compared to the THP1+Jurkat group,the THP1+Apoptosis Jurkat group showed increased protein expression of Mertk,Tim4,Gas6,Pros,and MFGE8.In comparison to the NC-Mertk^highTHP1+Apoptosis Jurkat group,the Mertk^highTHP1+Apoptosis Jurkat group exhibited increased protein expression of Mertk,Gas6,Pros,and MFGE8,while Tim4 expression was decreased.When compared to the NC-Mertk^lowTHP1+Apoptosis Jurkat group,the Mertk^lowTHP1+Apoptosis Jurkat group demonstrated decreased protein expression of Mertk,Gas6,Pros,and MFGE8,whereas Tim4 expression was increased.（3）Interaction between Tim4 and Mertk:The results of immunofluorescence,forward,and reverse immunoprecipitation experiments all indicate the presence of an interaction between Tim4 and Mertk in THP1 cells.IV.Transcriptome analysis of the role of Tim4 in IBD and its associated colorectal cancer1.The effect of Tim4 and DSS on the transcription level of mouse colon tissue（1）The total number of differential genes between the normal control group and the Anti-tim4 group was 1757,including 795 up-regulated genes and 962 down-regulated genes.The GO pathways with differential gene enrichment mainly included regulation of inflammatory response,positive regulation of interferon-γproduction,negative regulation of cell activation,leukocyte-mediated immune regulation,and immune response activation of cell surface receptors.The KEGG pathways with differential gene enrichment mainly include Fc gamma R-mediated phagocytosis signaling pathway,Toll-like receptor signaling pathway,AMPK signaling pathway,PPAR signaling pathway and NF-κB signaling pathway.（2）The total number of differential genes between DSS group and DSS+Anti-tim4 group was 1126,including 653 up-regulated genes and 473 down-regulated genes.The differentially enriched GO pathways mainly included positive regulation of inflammatory response,positive regulation of leukotriene production involved in inflammatory response,negative regulation of vascular wound healing,interleukin 33binding,and cellular aging.The KEGG pathways with differential gene enrichment mainly included HIF-1 signaling pathway,AMPK signaling pathway,PPAR signaling pathway and cytokine-cytokine receptor interaction signaling pathway.（3）The total number of differential genes between the normal control group and the DSS group was 2592,including 727 up-regulated genes and 1865 down-regulated genes.The GO pathways with differential gene enrichment mainly included recognition and phagocytosis,neutrophil chemotaxis,response to tumor necrosis factor,cell membrane invagination,and chemokine activity.The KEGG pathways with differential gene enrichment mainly included interleukin-17 signaling pathway,NOD-like receptor signaling pathway,TNF signaling pathway,Toll-like receptor signaling pathway and NF-κB signaling pathway.（4）The total number of differential genes between DSS+Anti-tim4 group and Anti-tim4 group was 2573,including 1691 up-regulated genes and 882 down-regulated genes.The GO pathways with differential gene enrichment mainly included inflammatory response,cell chemotaxis,signaling receptor binding,growth factor activity and cytokine receptor binding.The KEGG pathways with differential gene enrichment mainly included IL-17 signaling pathway,JAK-STAT signaling pathway,TNF signaling pathway,NF-κB signaling pathway and cytokine-cytokine receptor interaction signaling pathway.2.Correlation analysis between efferocytosis signaling pathway and transcriptome sequencing data（1）The differential genes of the normal control group and the Anti-tim4 group were intersected with the genes of the efferocytosis signaling pathway,and the GO pathways enriched by differential genes mainly included the regulation of vesicle-mediated transport,endocytic vesicles,and receptor-mediated signaling pathways.Regulation of endocytosis,tissue homeostasis,and transmembrane trafficking.The KEGG pathways with differential gene enrichment mainly included Fc gamma R mediated phagocytosis signaling pathway,neutrophil extracellular trap formation signaling pathway,adherens junction,Toll-like receptor cascade signaling pathway and Fc epsilon RI signaling pathway.（2）The differential genes of the DSS group and the DSS+Anti-tim4 group were intersected with the genes of the efferocytosis signaling pathway,and the GO pathways enriched by differential genes mainly included phagocytosis,phagocytosis and recognition,regulation of phagocytosis,and intercellular adhesion.Consistent with positive regulation of interleukin-8 production.The KEGG pathways with differential gene enrichment are mainly phagosomes.（3）The differential genes of the normal control group and the DSS group were intersected with the genes of the cell-buried signaling pathway,and the GO pathways enriched by differential genes mainly included phagocytosis,endocytic vesicles,regulation of vesicle-mediated transport,and motility.Protein cytoskeletal organization and extrinsic apoptotic signaling pathways.The KEGG pathways with differential gene enrichment mainly included phagosome,Fc gamma R mediated phagocytosis,Toll-like receptor cascade signaling pathway,neutrophil degranulation and adherens junction.（4）The differential genes of the DSS+Anti-tim4 group and the Anti-tim4 group were intersected with the genes of the efferocytosis signaling pathway,and the GO pathways enriched in differential genes mainly included phagocytosis,regulation of vesicle-mediated transport,and endocytosis.Upregulation of action,modulation of defense responses,and modulation of macrophage activation.The KEGG pathways with differential gene enrichment mainly included phagosome signaling pathway,Fc epsilon RI signaling pathway,HIF-1 signaling pathway,neutrophil degranulation signaling pathway and interleukin 1 family signaling pathway.3.Correlation between efferocytosis signaling pathways and genes related to immune metabolism:There are 3 core genes in the prognosis model with correlations of>0.4 or-0.4 with efferocytosis signaling pathways,namely FLNA,OLFM4 and NAT2.Conclusion:1.In IBD and its related CRC colonic mucosa,efferocytosis is enhanced,but the expression of efferocytosis ligand Tim4 and other efferocytosis ligands and bridging molecules（Mertk,etc.）is unbalanced,resulting in the blockage of efferocytosis,which in turn promotes the inflammatory response.2.Blocking Tim4 can attenuate DSS-induced colonic inflammatory injury in mice.3.There is an interaction between Tim4 and Mertk,and blocking Tim4 can up-regulate the expression of TAM receptors and efferocytosis bridging molecules,which may correct the imbalance in the expression of Tim4 and other efferocytosis ligands and bridging molecules in IBD and related CRC,thereby alleviating the inflammation.4.Tim4 may be involved in the pathogenesis of IBD and its related CRC by regulating phagocytosis,inflammatory response,immune metabolism and other signaling pathways.