Mechanisms By Which Tim-3 Regulates The Polarizaition Of Macrophages

Background:Immune homeostasis is a balance state established and maintained with interactions between the immune system and the outside or among the internal components and a key factor in maintaining the body’s health. The developments of infectious diseases, autoimmune diseases, cancers and other diseases are closely associated with the imbalance of immune homeostasis. In recent years, the relationship among micro-environmental changes, immune homeostasis imbalance and the developments of diseases, having drawn more and more attentions, has become a focus in life science. Macrogphages are critical innate immune and antigen presenting cells, and polarizations are important machnisms for them to be involved in the maintance of immune homeostasis and the regulation of micro-environments. Macrophages can be categorized as classically(M1, pro-inflammatory) activated or alternatively(M2, anti-inflammatory) activated, which have different functional capabilities. Accumulating studies indicate that the imbalant polarization of macrophages is a kay pathological factor in many diseases. Signals controlling intestinal macrophages polarization are not well de?ned and represent a fundamental gap. Investigating these questions is of great value for understanding the mechanisms by which macrophages control the local microenvironment and affect intestinal homeostasis. It is known that the polarization state of macrophages is regulated in part by local concentrations of cytokines and chemokines, as well as varied interactions of macrophages with normal and degraded components of the extracellular matrix. As for The intracellular mechanisms, recent studies have unveiled a role for some transcriptional factors in regulating the macrophages differentiation and polarization. In particular, the signal transducer and activator of transcription(STAT) family member including STAT1, 3, 6 and Suppressor of cytokine signaling1(SOCS1), Interferon regulationg factor 4,5, have been reported as important regulators of macrophages polarization. In addition, dynamically modulated expression of specific micro RNAs also influences cell polarization and many other biological processes. For example, mi R-155, mi R-146 have been reported to regulate the physiological differentiation of macrophages. Targeting molecular pathways regulating macrophages polarization holds great promise for immunotherapy. T cell-Ig mucin-3(Tim-3) is an immune regulator newly identified on activated T effector cells including Th1, Th17 and Tc1 cells. And by inducing T cell tolerance or exhaustion, Tim-3 negatively regulates the response of above T effector cells. The fact that dysregulated Tim-3 expressions are associated with many clinical diseases including autoimmune diseases, tumor and chronic virus infections showed that Tim-3 contributes to immune homeostasis and can be used as an immunotherapy target for immune disorders. Recent findings including ours demonstrated that Tim-3 also acts as a negative regulator for innate immune cells such as macrophages and dendritic cells. In sepsis, we found that Tim-3 is involved in the negative regulation of macrophage in vivo, suggesting that Tim-3 can be used as a new regulator of macrophages. However, up to now, we still do not know the molecular mechainsms by which Tim-3 negatively regulate the function of macrophages. Molecular mechanisms by which Tim-3 maintains the homeostasis of innate immune cells and the clinical significance remain largely unclear.Objects:Based on our previous findings, the goals of this project include the following aspects: 1. To make clear of the effects that Tim-3 regulates the polarization of macrophages; 2. To confirm whether Tim-3 affected the progresses of diseases through affecting the polarization of macrophages, and evaluate the feasibility of taking Tim-3 as a new target for intervention via some intervention experiments. 3. To clarify the molecular mechanism of Tim-3 regulating the polarization of macrophages, and provide the theoretical basis for the application of Tim-3.Methods and results:First, it has been discovered that Tim3 can significantly affect the polarization of macrophages in vitro. In order to discuss the effects of Tim-3 regulating the polarization of macrophages, we use RAW264.7 macrophage cell line and primary peritoneal macrophages(derived from wild-type or Tim-3 transgenic mice) as the research objects, and take advantage of ELISA, Real-time PCR and other methods to disuss the regulating effects of Tim-3 on the polarization of macrophages, by means of Tim-3 knockdown, overexpression and functional bokade by fusion protein and other experimental tools. The results are as follows:(1) Tim-3 can inhibit the expression of inflammatory cytokines in macrophages.(2) Tim-3 promotes the M2 polarization of macrophages. Second, the relationship, among the expression or function changes of Tim-3, the polarization abnormality of macrophages and the developments of diseases, has been clarified in some immune-related diseases including sepsis, ulcercollitis and intestinal tumors. In order to explore the relationship between the regulation of Tim-3 on macrophage polarization and diseases, three models for research have been estabilished with wild-type and Tim-3 transgenic mice, namely sepsis model by means of cecal ligation and puncture, ulcercolitis model induced with dextran sulfate sodium, and colitis associated colorectal cancer model co-induced with azoxymethane and dextran sulfate sodium, and some clinical samples have been collected. The main contents of this study include 1) to analyze the relationship among the expression of Tim-3, the polarization of macrophages and the developments of diseases through FACS, Real-time PCR and other mehods; 2) to discuss the effects of Tim-3 intervention on macrophage polarization and disease progress through transgene and adoptive therapy. The results are as follows:(1).Tim-3 can ameliorate disease by promoting the M2 polarization to resume the polarization balance of macrophages and the immune homeostasis in sepsis.(2).The deceased expression of Tim-3 and the biased M1 polarizaton of macrophages have been observed in ulcercolitis, which can be aggravated by adoptive therapy with Tim-3 knockdown macrophages.(3). It has been found that the decreased expression of Tim-3 is associated with the enhanced M2 polarization of macrophages in colorectal cancer. And on this basis, Tim-3 transgene will further exacerbate the polarization imbalances of macrophages and worsen the conditions of colorectal cancer. Third, the molecular mechanisms for Tim-3 to regulate the polarization of macrophages have been clarified. In order to investigate the molecular mechanisms for Tim-3 to regulate the polarization of macrophages, we use cell lines including RAW264.7, CHO, 293 T, and U2 OS as the main models and take advantage of m RNA array, micro RNA array, western blot, Real-time PCR, point mutantion, co-immuoprecipitaton, high-content analysis and other methods to dicuss the crucial signaling pathways through which Tim-3 regulates the polarization of macrophages on emphasis, by means of Tim-3 knockdown, overexpression, fusion protein functional blockade and other experimental tools. Some new regulating mechnisms have been discovered as follows: 1. It has been discovered that Tim-3 can inhibit the activation of NF-κB induced by LPS/TLR4 through upregulating the expression level of A20 and enhancing the activity of PI3K-AKT. In addition, Tim-3 also participates in the regulation of IRF3/7 by inhibiting the activation of IRF3 and the expression of IRF7. 2. Stat1 has been identified as a key cellular adaptor of Tim-3 in regulating the polarization of macrophages. It is confirmed that Tim-3 can regulate the expression of SOCS1, promote the expression of IL-10 and Arg-1, and enhance the M2 polarization of macrophages through inhibiting Stat1-mi-155 axis.Conclusion:Tim-3 is a recent-year-identified immune regulation protein, and it is considered as a new target for immune regulation following CTLA-4 and PD-1 by medical workers. Many studies have focused on the role of Tim-3 regulating T cells. In this study, we have confirmed Tim-3’s regulation on the polarization of macrophages, a kind of critical innate immune cell, and clarified the molecular mechanism, which provides a new experimental basis for the comprehensive understanding of the molecular regulation feature. Macrophages are widely distributed in the body, and play a key role in the maintenance of immune homeostasis and health. Our study provide a new means for the immune regulation strategies targeting macrophages, through illustrating the functions and signaling pathways of Tim-3 involving in the regulation on the polarization of macrophages. It is more important that our researches further confirm the feasibility of the intervention of immune-related diseases targeting Tim-3 and provide some new strategies and new ideas for the clinical intervention of televant diseases, through clarifying the relationship among the expression and function of Tim-3, the polarization of macrophages and the development of diseases in vivo.