Roles Of Tim-3 In Regulating The NLRP3 Inflammasome And The Underlying Mechanism

BackgroundRecently, the morbidity rate of auto-immunity and inflammatory diseases is increasing fast, which greatly threatens people’s health and life. Researches show that uncontrolled activation of NLRP3 inflammasome and excessive production of their cytokines such as IL-1βcontributes to the pathogenesis of inflammatory diseases. So, mechanisms of NLRP3 activation and negative regulators of NLRP3 are of great interest to be investigated.The activation of NLRP3 inflammasome needs both the “priming signal”(the first signal) and the “activation signal”(the second signal).The first signal is in charge of the transcription and translation of the components of NLRP3 inflammasome and effective protein. The second signal can promote independent components of NLRP3 inflammasome to assemble into a complex to cleave and activate pro-interleukin-1β(Pro-IL-1β)and pro-interleukin-18(Pro-IL-18) into a mature form. Potent release of IL-1βand IL-18 can lead to severe inflammatory responses.T-cell-Ig mucin-3, Tim-3, is a newly identified immune checkpoint inhibitor. Dysregulated Tim-3 on T cells is related to many immune disorders such as tumors and infectious diseases. It is now considered as a therapeutic target. Recently, Tim-3 has been found to be involved in the regulation of innate immune response, however, the underlying mechanisms remain largely unclear. Whether NLRP3 inflammasome, the important arm of innate immunity, can also be regulated by Tim-3, is totally unknown. Previous study in our lab found that Tim-3 can suppress TLR4 responses and then regulate inflammatory responses. At the same time, TLR4 responses are also involved in the process of NLRP3 inflammasome activation. So, here we will focus on exploring whether Tim-3 regulates the activation of NLRP3 inflammasome and, if so, what will be the underlying mechanisms and clinical significance..ObjectiveTo explore the effects of Tim-3 on NLRP3 inflammasome activation and try to find the underlying mechanisms.1. Effects of Tim-3 on NLRP3 inflammasome activation. Detect the activation of NLRP3 inflammasome in the macrophages isolated from Tim-3-TG and wild type mouse in the presence of LPS and ATP and in RAW264.7 macrophage cell lines in which Tim-3 signaling was blocked by sTim-3-Ig. 2. Mechanisms by which Tim-3 regulates NLRP3 inflammasome activation: effects of Tim-3 on NLRP3 inflammasome transcription and translation. Tim-3-Ig was used to block the Tim-3 signaling, and then to detect the expression of NLRP3 and IL-1βat the mRNA and protein levels in RAW264.7 and J774 A.1 cell lines in the presence or absence of NF-κB inhibitor. Determine the expression of NLRP3 and IL-1β in macrophages isolated from Tim-3-TG and wild type mouse in the presence of LPS. 3. Mechanisms by which Tim-3 regulates NLRP3 inflammasome activation: effects of Tim-3 on NLRP3 infalmmasome assembly and activation. Detect ATP release、potassium efflux and ROS production in RAW264.7 cell lines in the presence of sTim-3-Ig. Determine the activation of NLRP3 inflammasome in RAW264.7 cell lines when potassium efflux and ROS production were blocked. 4. Molecular mechanisms by which Tim-3 regulates NLRP3 inflammasome activation. Construct mutants of Tim-3 at the site of 256/263 tyrosine and determine the expression and activation of NLRP3 inflammasome under the signaling of Tim-3 or mutated Tim-3.5. Relationship between Tim-3 and NLRP3 inflammasome related diseases peritonitis. Collect the serum of peritonitis patients and detect the concentration of sTim-3.Detect the expression of NLRP3 and IL-1βin THP-1 and U937 when Tim-3 signaling was blocked.6. Effects of Tim-3 on the progression of peritonitis in mouse model. Establish the mouse peritonitis model and elevate its inflammatory responses when Tim-3 was over-expressed or blocked.Results1. Transgenic overexpression of Tim-3 inhibited the activation of NLRP3 induced by LPS plus ATP in macrophages isolated from mouse. NLRP3 inflammasome was activated when Tim-3 signaling was blocked by sTim-3-Ig. 2.sTim-3-Ig could increase the activity of NF-κB and promote the expression of NLRP3 and IL-1β,which was reversed by NF-κB inhibitor. 3. Tim-3-Ig promoted ATP release、potassium efflux and ROS production, which increased the release of IL-1β,but the increase of IL-1βinduced by sTim-3-Ig could be reversed when potassium efflux and ROS production were suppressed.4. Tim-3 lost its function on NLRP3 inflammasome when its tyrosine at the site of 256/263 were Methods mutated.5.The serum concentrations of sTim-3 are higher than healthy controls. sTim-3-Ig promoted the expression of NLRP3 and IL-1βat mRNA level in human macrophage cell lines THP-1 and U937.6. Tim-3 could protect mice from peritonitis.Conclusions1. Tim-3 negatively regulates the activation of NLRP3 inflammasome through inhibiting both the first and the second signals necessary for the activation of NLRP3 inflammsome.2. The tyrosine at the site of 256/263 in Tim-3 plays important roles in its regulation on the NLRP3 inflammasome.3. By negatively regulating the activation of NLRP3 inflammsome, Tim-3 protects against the peritonitis.