The Mechanism Of Flagellin Stimulated-hepatocytes Mediated Modulation On T Cell Function

Hepatic parenchymal cells,the target cells of hepadnavirus like HBV,parasites,and gastrointestinal bacteria invasion,are one of immunological agents in liver.During intrahepatic acute or chronic infection,CD4⁺T and CD8⁺T cells are predominant effector cells against the pathogens.However,hepatocytes usually down-regulate the activation of naive T cells and the function of activated T cells in order to maintain the physiological function and avoid pathologic injury,which inhibits intrahepatic immune response and prolongs infection.Because innate immunity helps to modulate adapted immunity,activation of Toll-like receptors may regulate the effects of hepatocytes to T cells.Hepatocytes express certain TLRs like TLR5 which will be activated by its agonist,flagellin,resulting in innate immune response,followed by production of IL-6,TNF-?and other immune modulator molecules indicating flagellin can modulate immune function of hepatocytes.Based on previous research in our lab,separated primary hepatocytes instead of LSEC and Kupffer cells from wild type C57BL/6 mice received hydrodynamic injection of recombinant-Salmonella Typhi flagellin?SF?which is activating agent of TLR5 and intracellular NLRC4 pathway,after 7 days of injection,significantly promoted activation and function of co-cultured naive mice T cells.However,it is still unclear that the molecule mechanism about the modulation of T cells function by hepatocytes stimulated with SF.Therefore,this thesis used in vitro hepatocytes-T cells co-culture system to identify the effect that SF stimulation can indirectly elevate T cells activation,and further investigated the molecules produced by SF-stimulated hepatocytes that may be involved in the regulation on T cell function.1.To confirm SF modulates hepatocytes immune condition and the dependent pathway,wild type mice hepatocytes were stimulated with SF and then co-cultured with TLR5^-/-splenocytes.The results show hepatocytes suppress T cell function while SF-stimulated hepatocytes reduce the inhibition.In SF group,IFN-?secretion of T cells is raised,accompanied with elevated expression of CD8⁺T cells transcription factors T-bet and Eomes,and activation molecule CD44.Since SF has intracellular NLRC4-signaling pathway activity also,using different engineered proteins without certain pathway activity shows SF lack of TLR5 signaling pathway activation domain can not modulate T cells,namely TLR5 signaling pathway activation of hepatocytes by flagellin plays key role in modulation towards T cells.2.To detect expression of immune regulatory molecules produced by hepatocytes under SF stimulation and needed by T cells activation,some hepatocytes-gene expression profiles were searched for candidate molecules in this research as reference.Under multifaceted detection,the activation of TLR5 on hepatocytes in vitro increases expression of ICAM-1,IL-6 and GM-CSF.After 7 days of SF injection into mice,the expression of ICAM-1 and IL-6 at protein level increases on primary hepatocytes.3.To explore key effector molecule produced by hepatocytes in the modulation,hepatocytes-splenocytes separate culture and hepatocytes-supernatant transfer system were used.Firstly,secreted component of hepatocytes can modulate T cell function.Secondly,blockade of ICAM-1,IL-6 and GM-CSF with specific antibodies in co-culture or supernatant transfer shows they involve in T cells activation at different degree,but the immune regulation of SF-stimulated hepatocytes on T cells remains.This research confirms SF can activate hepatocytes and then modulate hepatocytes immune condition resulting in positive modulation towards T cells,which is through hepatocytes TLR5 signaling pathway activation.Based on that,SF promotes hepatocytes produce immune regulatory molecules ICAM-1,IL-6 and GM-CSF,but in vitro molecule-block experiment can't reveal which is the key immune molecule produced by SF-stimulated hepatocytes to T cell function.The role of upregulated expression of ICAM-1,IL-6 and GM-CSF on hepatocytes by flgellin in vivo deserves more exploration.This research promotes further discuss on flagellin intrahepatic modulation and design of new vaccine or immune modulator.