Mechanisms Of Gal-9/Tim-3 Mediated Immune Regulation

suppression by binding to T cell Ig mucin-3 (Tim-3) expressed on activated Th1 and Th17 cells[1-3]. Due to its potent roles in T cell suppression and tolerance induction, galectin-9 has been considered as a therapeutic candidate for autoimmune diseases. However, recent studies also showed that Tim-3, the physiological receptor for Gal-9, is also constitutively expressed on innate immune cells . By activating innate immune cells, such as dendritic cells (DCs), monocytes, and macrophages, the Gal-9/Tim-3 signaling pathway can also promote a pro-in?ammatory response[4]. However, the mechanisms by which Gal-9 triggers these different effects are not well understood.Function is determined by structure. Structurally, Gal-9 belongs to the tandem repeat subfamily, with two carbohydrate recognition domains (CRDs) joined by a ?exible peptide linker . But there are many differences between the N-terminal and C-terminal CRDs (NCRD and CCRD) of Gal-9,such as their sequences, oligosaccharide binding affinitie[5]. The difference in the CRD sequences suggests that the two CRDs of Gal-9 may play different roles in mediating its multiple functions. In addition, many researches indicated that NCRD and CCRD of Gal-9 could mediate their functions independently. To test the mechanisms by which Gal-9 mediated different function,here we hypothesis that different domains of Gal-9 play different roles in mediating its functions in different cells. In order to test this hypothesis, at first, using homology modeling method, the 3-D theoretical structures of Gal-9-N, Gal-9-C and Tim-3 were constructed and the potential binding domains between the receptor and the ligand were predicted. Then, computer analysis suggested that Gal-9-N/Gal-9-C could bind to Tim-3 respectively with stable conformation. In spite of the similarity between NCRD and CCRD of Gal-9, such as their sequences,structures and their significance, there are different patterns and affinities for the binding of the Gal-9-N and Gal-9-C to their receptor, Tim-3. Further biological function experimentations are needed to testify it. So we cloned the gene of Gal-9-N,Gal-9-C,Gal-9-NC(removal of middle peptides).Because homodimer or heterodimer is necessary for active Gal-9[6].we constructed fusion proteins containing Gal-9-NC and a homodimer of the Gal-9-N or Gal-9-C.After identifing their purity quotient and specificity by SDS-PAGE and Western blot ,we Galectin-9 (Gal-9) belongs to the galectin family and mediates T cell death or 5 directly compared their ability in inducing T cell death or activating DC cells and found that N and C of Gal-9 contributes differently to these functions. The Gal-9-N is mainly responsible for activating innate immune cells , while Gal-9-C is mainly responsible for inducing T cell death.Subsequently, we investigated the roles of Gal-9/Tim-3 pathway in vivo in differenet diseases models by activating Gal-9/Tim-3 pathway by Gal-9-N, Gal-9-C or blocking Gal-9/Tim-3 pathway by Tim-3 recombination protein. We used NOD mice as autoimmune diabetes model to test the effects of Gal-9-N and Gal-9-C in inducing tolerance in vivo[7]. The result indicated that Gal-9-N and Gal-9-C both can prevent autoimmunity diabetes. However, the underlying mechanisms remain to be determined. Gal-9 extensively participates in the regulation of innate and adaptive immunity.Sepsis is a lethal diseases combined by complex innate and adaptive immune deregulation. To investigate whether Gal-9 is also involved in the regulation of sepsis, we established a CLP model in C57BL/6 mice. According to the published refferences, recombinant Tim-3 protein was used to block Gal-9/Tim-3 pathway in sepsis. After in vivo blocking ,we observed septicemic mice survival rate, detected apoptosis of thymocyte and cytokines level in peripheral blood, peritoneal lavage fluid, spleen and mesentery lymph node. The result showed that the apoptosis of thymocyte, and death rate were significantly increased after Tim-3 blocking. At the same time, we found up-regulation of inflammatory factor IL-6, and drawdown of protective factor IL-12 in peripheral blood, peritoneal lavage fluid, spleen and mesentery lymph node.All of these indicated activing this pathway may relieve pathogenetic condition of sepsis and protect organism.In conclusion, our data suggested that the Gal-9-N and Gal-9-C contribute differently to the multiple functions of Gal-9, Gal-9-N mainly being responsible for activing innate immune cells while Gal-9-C mainly being responsible for inducing T cell death. In vivo experiment suggested that Gal-9/Tim-3 pathway plays a very important role in inducing diabetic immunotolerance and maintaining homeostasis of sepsis in vivo.