α-Lactose Improves The Survival Of Septic Mice By Blockade Of Tim-3 Signaling To Prevent NKT Cell Apoptosis And Attenuate Cytokines Storm

Background:Sepsis is a common and frequently fatal clinical condition and remains one of the leading causes of death in intensive care units.Previous studies have provided evidence that the dysregulated immune response in sepsis consists of the strong initial proinflammatory response and the subsequent development of an immunosuppressed state,which appears to be associated with inability to clear the infection,increased susceptibility to secondary infections,multiple organ failure and septic morbidity.The immunoregulatory function of NKT cells is their ability to promptly secrete large amounts of Thl and Th2 cytokines and these rapid responses to a variety of pathogens and subsequently activate other cell types indicates a critical role for NKT cells in bridging innate and acquired immunity.Cumulative evidences demonstrate that the potential of NKT cells to produce pro-and anti-inflammatory cytokines provides them with the capacity to promote or inhibit immune responses following sepsis.Although there are many strong correlations have been made between septic mortality and NKT cell activation and cytokine production,the exact mechanism by which NKT cells participate in the septic immune response,and how their activity is regulated in general remain unclear.T cell immunoglobulin and mucin domain 3 is a type-1 transmembrane receptor and Tim-3/Gal-9 interaction plays highly complex roles in immune regulation depend on the cell type on which it is expressed We previously reported that Tim-3/Gal-9 signaling plays an important role in hepatic NKT cell regulation.Activation of NKT cells led to a upregulation of Tim-3 expression,and Tim-3+ NKT cells have the ability to proliferate and to produce more cytokines upon sTimulation,however,Tim-3 +NKT cells were also more prone to apoptosis.Recent evidence have provided compelling evidence for a role of Tim-3 in sepsis.Therefore,it is important to know whether the Tim-3 pathway is also involved in the regulation of NKT cells in sepsis,and,if so,play an important role in regulating the immune disorders following sepsis.Methods:Polymicrobial sepsis was induced in mice by cecal ligation and puncture(CLP)or LPS injection。For α-Lactose treatment,CLP-mice were administrated with either 200μL 5%or 10%a-Lactose(Sigma-Aldrich,St.Louis,MO)or 200μL PBS by hypodermic injection at different Timepoint post-CLP.For the survival study,mice were constantly monitored for 28 days.For cell surface marker staining、intracellular cytokine staining and apoptosis detecting,the single-cell suspensions were stained with different anti-mouse antibodies。NKT cells and DCs were cultured in the presence or absence of recombination mouse IL-12、anti-IL-12mAb、LPS、Galectin-9 or a-Lactose.Tim-3 expression in NKT cells was determined by Flow cytometry,the culture supernatants were collected and IL-12 production was determined by ELISA.Results:Herein,we showed Tim-3 expression in NKT cells is elevated in experimental mice during sepsis.Tim-3 expression was positively correlated with NKT cell activation and apoptosis.In sepsis,IL-12 secreted by DCs exposure to LPS increased the expression of Tim-3 in NKT cells.Administration of a-Lactose to blockade Tim-3 signaling pathway significantly improved the survival of septic mice,concomitant with reduced IL-12 production by dendritic cells,reduced Tim-3 expression and prevented NKT cells apoptosis,attenuated production of inflammatory cytokines.Conclusion:Collectively,Tim-3 signaling in NKT cells plays a critical role in the immunopathogenesis of sepsis.Thus,a-Lactose could be a promising immunomodulatory agent in the treatment of sepsis.