The Role Of Tim-4 In The Pathogenesis Of Ulcerative Colitis And Its Clinical Value

Background and aims:Ulcerative colitis(UC)is a type of chronic,idiopathic,and immune-mediated inflammatory bowel disease.Due to the characteristic of repeated recurrence,the disease activity and severity of UC must be assessed and monitored repeatedly.Invasive endoscopy is the most primary tool for assessment of UC disease activity and severity in clinical practice,but the results are often inconsistent,thus reducing the frequency of evaluation.Digging out hematological biomarkers has become a focus in this area.C-reactive protein(CRP),currently used in clinical practice,can not accurately assess the degree of active mucosal inflammation for UC patients.Studies suggested that multiple factors such as dysbiosis of intestinal flora,genetic susceptibility and mucosal immunity may contribute to the development of UC.Some biologic therapeutic agents such as anti-TNF-?monoclonal antibodies have been approved,and it is crucial to investigate the mucosal immune mechanism of UC in order to develop new therapeutic targets.Previous studies have demonstrated that macrophage polarization,TLR4/NF-?B signaling pathway-mediated interactions with intestinal flora,and adaptive immune responses mediated by helper T cells(Th)such as Th2 and Treg may play an important role in mucosal immunity of UC.T cell immunoglobulin and mucin domain 4(Tim-4)is a novel immunomodulatory molecule involved in immune processes through different pathways,including regulation of macrophage polarization,TLR4/NF-?B signaling pathway and Th2 or Treg differentiation.Our previous study demonstrated that administration of recombinant Tim-4 protein exacerbated DSS-induced experimental colitis.Using anti-Tim-4 m Ab to block Tim-4 expression can ameliorate ischemia-reperfusion injury.Considering the above results,we infer that blocking Tim-4 may reduce the UC severity by modulating intestinal mucosal immune response.In this research,we analyzed the changes of Tim-4 in peripheral blood and its correlation with clinical data of UC patients to provide new non-invasive biomarkers for UC diagnosis and treatment,and analyzed the changes of Tim-4 in intestinal mucosal microenvironment and its correlation with histopathology,apoptosis,macrophage polarization markers,TLR4/NF-?B signaling,and transcription factors of Th2 and Treg to provide a more direct clue for exploring the role of Tim-4 in UC pathogenesis.The effects of Tim-4blockade on DSS-induced experimental colitis were also observed.Materials and methods:1.Expression and clinical significance of Tim-4 in peripheral blood of UC patientsIn total,36 patients with UC and 34 healthy controls were included in our study.The demographic data,clinical symptom data,imaging findings,gastrointestinal endoscopy findings and pathological histological findings were recorded,and laboratory data were collected.Flow cytometry was used to determine the frequencies of CD14⁺Tim-4⁺monocytes,Treg cells and CD14⁺HLA-DR^-/low cells.Fluorescence quantitative PCR was used to determine Tim-4 m RNA levels.And enzyme-linked immunosorbent assay was used to determine serum soluble Tim-4 concentration.2.Tim-4 expression in intestinal mucosal tissues of UC patientsUC data sets were screened from GEO database,and the difference in Tim-4gene expression between control and UC patients was analyzed.UC-associated infiltrating immune cells were analyzed using the CIBERSORT algorithm.Twenty-five UC patients with biopsy were selected.The intestinal mucosal tissue specimens of 25 UC patients and 12 healthy persons were collected.Hematoxylin-eosin staining(HE)was used to grade the histology,and immunohistochemical staining was used to determine the protein expression levels of Tim-4,TLR4,My D88,NF-?B,GATA3,Fox P3,CD206 and CD86 in mucosal tissues.Tunel stain was used to analyze the apoptosis of intestinal mucosal cells in UC patients.3.The effect and mechanism of blocking Tim-4 on DSS-induced experimental colitis in mice(1)Grouping:60 Balb/c mice were randomly divided into control group,DSS group,DSS+Anti-Tim-4 m Ab group,Anti-Tim-4 m Ab group,DSS+Mesalazine group and DSS+Gum Arabic group.(2)Establishment of DSS-induced colitis:5%(w/v)DSS was given to drink freely to establish acute colitis model.(3)Anti-Tim-4antibody intervention protocol:Anti-Tim-4 m Ab was given intraperitoneally at day 0,day 2,day 4 and day 6 of modeling.(4)Isotype control:Mice in DSS group,DSS+Mesalazine group,and DSS+Gum Arabic group were all given the same dose of Ig G 2b intraperitoneally as the Tim-4 antibody simultaneously,with injection time points of 0d,2d,4d,and 6d.(5)Mesalazine administration protocol:Mesalazine was given by gavage at day 2 to day 7 of modeling.(6)Fecal occult blood,body weight and DAI were monitored in each group.(7)Tunel staining was performed to determine the apoptosis in intestinal mucosa.(8)Fluorescence quantitative PCR was used to detect the relative m RNA expression of TLR4,My D88,Tim-4,GATA3,Fox P3,CD206,CD86,IL-6,IL-1?and TNF-?in colonic tissues.(9)Immunohistochemistry and western blot were used to determine the protein expression of TLR4,My D88,NF-?B,Tim-4,GATA3,Fox P3,CD206 and CD86.4.Effect of blocking Tim-4 on intestinal flora of mice with DSS-induced experimental colitisFecal specimens were collected from each group,and were immediately snap frozen in liquid nitrogen for 30 min,and were transferred to-80?refrigerator.Total DNA was extracted from the feces,and 16S genomic sequencing libraries were prepared through amplification of the 338F?806R region of 16S r DNA,and were clustered as operational taxonomic unit(OTUs).Then the OTUs were annotated with taxonomic units,and they were used to analyze the microbial abundance and community composition.Results:1.Expression and clinical significance of Tim-4 in peripheral blood of UC patients(1)Changes of Tim-4 expression in peripheral blood of UC patients:(1)The expression of Tim-4 on monocytes was significantly upregulated in UC patients(P<0.05).(2)The relative expression of Tim-4 m RNA in PBMC of UC patients was also significantly increased,while the difference between soluble Tim-4 levels in the two groups was not statistically significant(P>0.05).(2)The correlations between Tim-4 and clinical parameters of UC patients:(1)The frequency of CD14⁺Tim-4⁺cells in severe UC cases was significantly higher than that in mild or moderate UC patients,and the CD14⁺Tim-4⁺cells proportion in moderate patients was also higher than mild patients.Patients with?10 times of bowel movements had higher CD14⁺Tim-4⁺cells frequency when compared to patients with 1-5 times or 6-9 times of bowel movements.CD14⁺Tim-4⁺cells frequency in patients with type E3 was also higher than type E1,with a statistically significant difference(P<0.05).There was a significant correlation between Mayo score and CD14⁺Tim-4⁺cells frequency(r=0.5853,P=0.0002).(2)Tim-4 m RNA was significantly higher in moderate patients than mild patients,while the difference between severe group and mild group or moderate group was not statistically significant.A weak correlation between Mayo score and Tim-4 m RNA(r=0.3830,P=0.0211)was observed.(3)CD14⁺Tim-4⁺cells frequency was positively correlated with EOS(r=0.3344,P=0.0462),Ig G(r=0.4484,P=0.0061),Ig E(r=0.4160,P=0.0116)and IL-6(r=0.4061,P=0.0140),while negative correlations were found with ALB(r=-0.3496,P=0.0366)and 25(OH)D(r=-0.4830,P=0.0028).(4)CD14⁺Tim-4⁺cells frequency was positively correlated with TNF-?(r=0.6425,P<0.0001),IL-17(r=0.3533,P=0.0345),IL-1?(r=0.4381,P=0.0075),IL-13(r=0.3324,P=0.0476)and CD14⁺HLA-DR^-/lowMDSC(r=0.4094,P=0.0132),while was negatively correlated with frequency of Treg cells(r=-0.4958,P=0.0021)and serum IL-10(r=-0.6214,P<0.0001).(3)Comparison between CD14⁺Tim-4⁺cells frequency prior to and following the treatment in UC patients:CD14⁺Tim-4⁺cells frequency after treatment was lower than the levels before treatment,and the difference was statistically significant(P=0.028).2.Change of Tim-4 in intestinal mucosal tissues of UC patients(1)Results from the analysis of UC dataset in GEO database:(1)the expression level of Tim-4 in intestinal mucosal tissues of UC patients was significantly higher than that of healthy population(P<0.05).(2)There was a significant difference in M2-type macrophages counts between UC patients with high and low Tim-4 expression(P<0.05).Correlation analysis showed a significant negative correlation between Tim-4 expression in intestinal mucosa and counts of M2-type macrophages(P<0.05).(3)The level of Tim-4 in UC patients treated with TNF-?inhibitor decreased significantly(P<0.05).(2)Change of Tim-4 in intestinal mucosal tissues of UC patients:Tim-4 was significantly higher in intestinal mucosal tissues of both active patients and patients in remission than in healthy controls(P<0.05 for both).The expression of Tim-4 in active patients was also significantly higher than that in patients in remission(P<0.05).(3)Changes of GATA3,Fox P3,TLR4,My D88,NF-?B,CD86,CD206 and the number of apoptotic cells in intestinal mucosal tissues of UC patients:(1)No statistically significant difference in GATA3 levels was detected between healthy controls and UC patients in remission(P>0.05).GATA3 level was higher in active patients than in healthy subjects and patients in remission(P<0.05 for both).(2)The expression of Fox P3 was significantly higher in UC patients than in healthy controls(P<0.05).(3)The expression levels of TLR4,My D88 and NF-?B in UC patients were all higher than those in the control group,and they were also higher in active patients than in patients in remission(P<0.05 for all).(4)The expression level of CD86 was significantly higher in UC patients than control group,and were higher in active patients than in patients in remission(P<0.05).In contrast,CD206,a marker of M2-type macrophages,was significantly decreased in UC patients,and was significantly lower in active patients than in patients in remission(P<0.05).(5)Compared to healthy subjects,the number of apoptotic cells in intestinal mucosa was significantly higher in UC patients,and was higher in active patients than in patients in remission.(4)Associations between Tim-4 and GATA3,Fox P3,TLR4/NF-?B,markers of M1-type macrophage and M2-type macrophage and apoptotic indexes in intestinal mucosal tissues of UC patients:Patients with high levels of Tim-4expression also had high levels of Fox P3,TLR4,NF-?B and apoptotic cells,suggesting there were positive correlations between Tim-4 and these indicators.In addition,patients with high Tim-4 expression had lower levels of CD206 compared to those with low Tim-4 expression,suggesting there was a negative correlation between Tim-4 and CD206.3.The effect of blocking Tim-4 on DSS-induced experimental colitis in mice and the underlying mechanism(1)Changes in weight,DAI and colon length in each group:(1)Mice in DSS group started to lose weight on the 2^nd day of modeling,and the decreasing trend was significant after the 4^th day.In contrast,mice in DSS+Anti-Tim-4 m Ab group began to lose weight on day 3^th day.The weight was higher than DSS group but lower than control during 4^th to 7^th day(P<0.05).(2)DAI of DSS group started to increase on the 1^st day of modeling,while DAI of DSS+Anti-Tim-4 m Ab group,although showing the same trend,was significantly lower than that of DSS group during 5^th to7^th day(P<0.05).(3)The colon length of mice in DSS group was significantly lower than control group,and the difference was statistically significant(P<0.05).The colon length of mice in DSS+Anti-Tim-4 m Ab group was also significantly lower than control group,but higher than DSS group(P<0.05).(2)Changes in the histology of colon pathology of experimental colitis mice and the intervention effect of blocking Tim-4:(1)Mice in DSS group had the highest histological scores.Mice in DSS+Anti-Tim-4 m Ab group had significantly lower histological score than DSS group(P<0.05).In addition,the histological score of mice in DSS+Mesalazine group was lower than that of DSS+Anti-Tim-4 m Ab group(P<0.05).(2)The frequency of apoptotic cells was significantly increased in mice in DSS+Anti-Tim-4 m Ab group and DSS+Mesalazine group compared to control group,but was significantly lower than that in DSS group(P<0.05 for both).(3)Changes of Tim-4,GATA3,Fox P3,TLR4/NF-?B and markers of M1-type macrophage and M2-type macrophage in intestinal mucosal tissues of mice with experimental colitis and the intervention effect of Tim-4 blockade:(1)The results of fluorescence quantitative PCR showed that Tim-4 m RNA,Fox P3m RNA,TLR4 m RNA,My D88 m RNA,CD206 m RNA and CD86 m RNA were all significantly highly expressed in intestinal mucosa of mice in DSS group,while these m RNAs were significantly decreased after Tim-4 blockade or mesalazine treatment.GATA3 m RNA was significantly overexpressed in the intestinal mucosa of the DSS group.Its level decreased after Tim-4 blockade,while the difference was not statistically significant(P>0.05).(2)Results of immunohistochemical staining showed that Tim-4 was significantly overexpressed in the intestinal mucosa and Peyer patch(PP node)of DSS group,and the intestinal mucosal Tim-4 expression decreased after Tim-4 blockade or mesalazine treatment,and the differences were statistically significant(P<0.05).GATA3,TLR4,My D88,NF-?B,Fox P3,CD206 and CD86 were significantly upregulated in DSS group,while their levels were significantly decreased after Tim-4 blockade or mesalazine treatment,and the differences were all statistically significant(P<0.05 for all).(3)Western blot results showed that Tim-4,GATA3,TLR4,My D88,NF-?B,Fox P3,CD206 and CD86 were significantly upregulated in the intestinal mucosa of DSS mice compared with control group(P<0.05).Their levels were significantly decreased after Tim-4 blockade or mesalazine treatment,and the differences were all statistically significant(P<0.05 for all).(4)Changes in m RNA levels of IL-6,TNF-?and IL-1?in the intestinal mucosa of mice with experimental colitis and the effect of Tim-4 blockade:Compared with control group,the m RNA levels of IL-6 m RNA,TNF-?m RNA and IL-1?m RNA in the intestinal mucosa of mice in DSS group were significantly increased(P<0.05),while decreased significantly after Tim-4 blockade,and the differences were statistically significant(P<0.05).The relative m RNA expression of these cytokines also decreased significantly after mesalazine treatment,and were significantly lower than thoes in mice with Tim-4 blockade(P<0.05 for all).4.Effect of blocking Tim-4 on intestinal flora of mice with DSS-induced experimental colitis(1)The mean count of OTUs in DSS group was significantly lower than that in normal control(P<0.05),while the count was significantly upregulated in mice in Tim-4 blockade group(P=0.025).(2)The sobs index and the PD index of DSS group were lower than control group(P<0.05),and they were significantly upregulated in DSS+Anti-Tim-4 m Ab groups compared to DSS group(P<0.05).(3)The results of ANOSIM/Adonis analysis showed a significant improvement in the abundance of intestinal flora in mice after Tim-4 antibody intervention.(4)Bacteroidetes and Firmicutes were the dominated intestinal microbiota in all mice.There were significant decreases in Firmicutes and Desulfobacterota,and significant increases in Proteobacteria,Campilobacterota,Deferribacterota and Verrucomicrobia in mice in DSS group.In DSS+Anti-Tim-4 m Ab groups,although there were still some differences with control group,an increase in Firmicutes and a significant decrease in Proteobacteria were observed when compared to DSS group.Conclusions:1.Upregulated circulating CD14⁺Tim-4⁺cells frequency in UC patients correlated to disease severity and activity,indicating it could be an objective and non-invasive biomarker to evaluate the severity and activity of UC.2.There were significant correlations between CD14⁺Tim-4⁺cells frequency and Treg cells,MDSC cells and inflammatory indexes in peripheral blood of UC patients.3.Tim-4 was increased in intestinal mucosal tissues of UC patients and correlated with the histopathological activity of UC and expression levels of Fox P3,TLR-4/NF-?B pathway and CD206.4.Tim-4 blockade can significantly alleviate the clinical symptoms,DAI and pathological histological inflammation levels for mice with DSS-induced colitis.And blocking Tim-4 also can downregulated TLR4/NF-?B signaling pathway and normalized Treg cells and M1/M2 balance.5.Mice with DSS-induced experimental colitis showed a significant decrease in intestinal flora abundance and significant change in flora structure,mainly in the reduction of Firmicutes and the increases of Bacteroidetes,Proteobacteria and Campilobacterota.Tim-4 blockade could increase the abundance of intestinal microorganisms and improve the colony structure of intestinal flora.