LIN Daopo,SHAO Xiaoxiao,HU Dingyuan, et al. The role of Tim-3 in regulating macrophage polarization in the occurrence and development of Crohn’s disease[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2022, 52(12): 947-955.
Abstract：Objective: To explore the clinical effect and possible mechanism of T cells immunoglobulin domain and mucin domain protein 3, mucin 3 (Tim-3) in Crohn’s disease (CD) based on bioinformatics techniques. Methods: The Microarray data of expression profile (GSE112366, GSE75214, GSE16879 and GSE186582) were obtained from the common Gene Expression Omnibus (GEO). GEO2R online tool was used to analyze the expression level of Tim-3 in CD and healthy control. The mRNA expression of Tim-3 in normal tissues, intestinal tissues with active CD and inactive CD were observed. Then the relationship between Tim-3 mRNA expression and simplified endoscopy of Crohns’s disease score (SES-CD) was analyzed by Spearman rank correlation analysis. Subsequently, the expression level of Tim-3 in intestinal tissue of CD patients was analyzed before and after Infliximab (IFX) or Ustekinumab (UST) treatment. ROC curve analysis was performed to evaluate the mRNA expression of Tim-3 at week 0 in intestinal tissue of active CD in predicting the effect of biological agents. Cibersort online tool was used to estimate the proportion of M0, M1 and M2 macrophages in intestinal tissue of CD patients in each dataset, and analyze the correlation between Tim-3 expression and macrophage proportion in intestinal tissue. Next, the intestinal tissues of 12 patients with active CD and 15 age and gender-matched healthy controls were collected from the Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, and the expression levels of Tim-3, CD68 (macrophage marker) as well as the number of CD68+ Tim-3+ macrophages in intestinal tissues were measured by immunofluorescence method.Finally, the expression of Tim-3 and M1, M2-related cytokines at mRNA level in colon tissues of 6 patients with active CD and 8 healthy controls were determined by real time fluorescence quantitative PCR. Results: In all 4 datasets, it was found that compared with normal intestinal tissues, the mRNA expression level of Tim-3 in intestinal tissues of active CD patients was significantly higher. The expression level of Tim-3 in intestinal tissue of active CD patients was also significantly higher than of inactive CD patients. The expression level of Tim-3 in intestinal tissue of active CD patients was positively correlated with SES-CD score (r=0.39, P<0.001). After IFX/UST treatment, the expression of Tim-3 in intestinal tissue of CD patients was lower than before IFX/UST treatment. In addition, the expression level of Tim-3 in intestinal tissue of IFX-responsive group was lower than that of non-responsive group. At week 4 or 6 of Infliximab treatment, ROC curve analysis showed that the expression level of Tim-3 in intestinal tissue of patients with active CD at week 0 has predictive value for the efficacy of IFX (AUC=0.70, P=0.04). Cibersort analysis showed that the mRNA expression level of Tim-3 in intestinal tissue was positively correlated with the infiltration level of M0 and M1 macrophages in intestinal tissue of active CD patients. Immunofluorescence results showed that the expressions of CD68 and Tim-3 in inflamed intestinal mucosa of CD patients were higher than healthy controls (P<0.05). In addition, the proportion of CD68+ Tim-3+ double-positive macrophages was also significantly increased in the inflammatory sites of CD patients. The mRNA expression level of Tim-3 in intestinal tissue of active CD patients was significantly higher than healthy controls, and was positively correlated with the expression level of M1-related cytokines TNF-α and IL-1β. Conclusion: Tim-3 may play an important role in the development of CD by affecting the polarization of M1 macrophages. Meanwhile, it may predict the efficacy of early IFX.
