Experimental Research On The Effects Of The Tolerogenic Vaccine To Prevent Nonalcoholic Steatohepatitis

Nonalcoholic steatohepatitis （NASH） was characterized for the first time by Ludwig andcolleagues in1980as the inflammatory stage following reversible steatosis in the liver. It ispart of the nonalcoholic fatty liver disease （NAFLD）, which is estimated to affect at least25%of the Western population. There is also an evidence to suggest that hepatic steatosis maycontribute not only to NAFLD but also to an increased risk of cardiovascular disease anddiabetes. Further more, one study showed that20%of patients with NASH eventuallyprogressed to cirrhosis, and among them,8%would go on to develop a potentially fatal liverdisease, such as liver cancer. Thus, understanding the pathogenesis of NASH is of greatclinical importance and is critical for the prevention and treatment of the disease.To our knowledge, the pathogenesis of NASH is described by the “two-hit” hypothesis.The “first hit” is fat accumulation, while the “second hit” leads to the development ofsteatohepatitis and fibrosis. A variety of factors that may be involved in the “second hit”include cytokine overproduction, lipid peroxidation, and reactive oxygen species （ROS）. Allof these factors which contribute to insulin resistance and inflammation may finally cause theNASH. Recent studies show that adaptive immune response may be also involved in the“second hit” by reducing the count of regulatory T cells （Tregs）, a suppressive T cell subsetpivotal for governing peripheral tolerance, lead to a lowered suppression of infammatoryresponses. So, we presume that naive CD4+T cells preferentially differentiate into T effectorcells （Teffs） not Tregs in the NASH and these Teffs could recognize self-antigens such as heatshock protein60（HSP60） which might be a potential trigger of human adipocyteinflammation. Then, Activated Teffs could produce pro-inflammatory cytokines （such asIFN-and TNF-）, causing activation of macrophages and lymphocytes lead to the “secondhit”. According to these, the undesirable activation of immune system could be a particularlyimportant part of reason that may cause the NASH. We propose that the development of NASH is driven partially by an autoimmune processand also offers preventive measures. We previously showed that antigen immunization in thepresence of the immunosuppressant dexamethasone （a strategy we termed “tolerogenicvaccine, TV”） caused the suppression of recall responses of T effector cells （Teffs）, andexpansion of antigen-specific regulatory T cells （Tregs）. This prompted us to designanti-NASH therapy that aimed at resetting the balance between Tregs and Teffs via increasingantigen-specific Treg in vivo.We examined CD11c⁺cell subset in MHCII⁺cells of Dex treated mice after excluding Bcells both in spleen and liver. BALB/C mice were injected （s.c.） with a pharmacological doseof Dex （4.5mg/kg）. A day later, cells in the spleen and liver were analyzed by flow cytometry.We detected two major CD11c⁺CD40⁺cell populations both in spleen and liver whichconstitute more than90%of the MHC II⁺cells after excluding B cells. One is CD11c^low cellswhich about75%of them were retained after Dex treatment in spleen which named asDex-APCs; the other one is CD11c^hi cells which about95%of them were depleted by Dex inspleen. However, the situation in liver is different that most of CD11c⁺cells （nearly94%）including both CD11c^hi and CD11c^low cells were depleted after Dex treatment. Similar resultswere obtained both in BALB/c and C57BL/6mice. Further experiments showed that with asingle injection of Dex at4.5mg/kg, the above effect in spleen and liver could both last4days, peaking between1–2days.We examined the functions of Dex-APCs in vivo using a transferred delayed-typehypersensitivity （DTH） model. First, antigen primed control APCs or Dex-APCs wereisolated from spleen of OVA-sensitized wild-type C57BL/6mice via MACS. Then, theseAPCs were intravenously transferred into OVA-sensitized Foxp3-eGFP C57BL/6micerespectively. Proliferation of endogenous memory T cells in the spleen was then determinedby BrdU incorporation. While the control APCs preferentially stimulated Teffs, Dex-APCspreferentially stimulated Tregs. We could also found that when Dex-APCs were antigenprimed with an irrelevant control peptide （MOG）, there was no proliferation of Tregs. Thus,Dex-APCs could cause the proliferation of antigen-specific Tregs.Next, we analyzed human HSP60for T-cell epitopes using an online program named SVRMHC and two top-scored peptides were selected, one is HSP60294-308（named HSP-1）,KVGLQVVAVKAPGFG and the other one is HSP60_476-490（HSP-2）, IPAMTIAKNAGVEGS.The peptides show identical sequences in the human and mouse and are predicted to bindstrongly to both human HLA-DR and mouse I-Ab （in C57BL/6）. We thus sought to determinethe immune response evoked by these peptides in HSP60-immunized C57BL/6mice. IL-2andIFN-（T-cell-dependent cytokines） were increased in the both of supernatants upon HSP-1andHSP-2when compared with an irrelevant control peptide （MOG）（p<0.01）. Further, HSP-1could be more effective in inducing T cell proliferation when compared with HSP-2（p<0.05）.Base on these results, we consider choosing HSP-1as immunogen in tolerogenic vaccine ofthis study.We further investigate whether the Tregs may exert its immune suppressive functions onKCs. Tregs （CD4⁺CD25⁺T cells） and Teffs（CD4⁺CD25-T cells） were isolated from HSP60（whole protein） immuned C57BL/6mice and cultured with syngeneic KCs respectively for48h. Culture medium contains HSP-1（10μg/ml） and KCs cultured alone were chosen as thecontrol. The expression of CD86and MHCII in KCs was determined by Flow cytometry.Tregs treated KCs significantly decreased CD86and MHCII expression compared with that inno T cells and in Teffs. At the same time, the co-cultured mediums were collected fordetection of cytokines such as IL-10, IL-12, and TNF-. Our data showed that, afterco-culture with Tregs, the KCs displayed a decrease in their capacity to producepro-inflammatory cytokines （TNF-, IL-12）（p<0.01）. In contrast, the production of theanti-inflammatory cytokines （IL-10） was enhanced in Tregs treated cultures （p<0.01）.We next determined whether tolerogenic vaccine using the designed peptides togetherwith Dex could raise antigen-specific Tregs in vivo. To investigate the feasibility of ourproposal, male C57BL/6mice （8-week-old） were received different treatment and wererestimulated with HSP-1two weeks later. Blood and liver were taken for analyzing the countof Tregs. At the same time, CD4+T cells which isolated from the spleen via MACS werelabeled with CFSE and restimulated for another2days with HSP-1for test of antigenspecificity. TV treatment selectively enriches Tregs in the blood. The results in liver wereconsistent with what we found in blood; the difference is that the majority of CD4⁺CD25⁺T cells in the livers were found to express Foxp3. This means that Tregs from the blood andliver may have the similar reactions after Dex treatment. The results from spleen indicatedthat TV treatment showed Tregs （Foxp3⁺） proliferation while rarely Teffs （Foxp3-）proliferated when restimulated with HSP-1. As controls, mice treated with either Dex orHSP-1did not show selective expansion of Tregs. The proliferation of Tregs is HSP60-specific, as Tregs did not proliferate when restimulated with control peptide （MOG） all thetime （TV+MOG）.To examine the effect of tolerogenic vaccine on hepatic steatosis,8-week old maleC57BL/6mice were received different treatment （TV treatment, Dex or HSP-1alone and notreatment） and fed on high fat diet for6months. TV treatment decreased the liver/bodyweight ratio significantly as compared to the other groups which fed on high fat diet （p<0.01）,without changing food and water intake. Further, Serum alanine aminotransferase （ALT） andtotal cholesterol（TC） have been tested at the end of6^th month. Both of serum ALT and TC inTV treated group have statistical difference when compare to other high fat group （p<0.01）.TV treatment improved insulin sensitivity of animals which fed with high fat diet （p<0.01）.We further evaluated the effect of tolerogenic vaccine on hepatic cholesterol and lipidmetabolism. H&E stained liver sections showed that there was extensive hepatocytevacuolation in animals fed with a high-fat diet, reflecting intrahepatic fat accumulation inhigh-fat diet-fed mice. In contrast, TV treatment efficiently ameliorated lipids accumulation inhepatocytes. Oil Red-O staining of liver sections confirmed that a massive accumulation offatty components in the livers of mice on a high fat diet groups including NT, Dex and HSP-1treated group and significantly less oil accumulation in the TV-treated high fat diet group.Next, we focus on the inflammatory process which caused by high fat diet and attempt tofind how tolerogenic vaccine functions. During the6months of high fat feeding, mice fromdifferent group were restimulated with HSP-1every two months. Blood was taken foranalyzing the count of Tregs. The Tregs counts in TV treated group were significant higherthan other groups during the first4months. We monitor the C-reactive protein （CRP） in theserum during our experiment. The CRP of TV treatment was keeping lower when comparedto other high fat group （p<0.05or p<0.01）. In this study, we confirmed that Dex alters the balance between CD11c^hi and CD11c^lowcells by depleting the former in spleen. We further determined how Dex-APCs potentiateT-helper cells response during immunization. Normally, APCs preferentially stimulated Teffswhich produce proinflammatory cytokines, causing further activation of immune system. Incontrast, Dex-APCs preferentially stimulated Tregs which function in the suppressedimmunization.In summary, our present research points to the need for an expanded definition of vaccinealso include "tolerogenic vaccine", which means that antigenic immunization, when performedunder the influence of an immunosuppressant （serving as tolerogenic adjuvant）, may lead not toimmunity, but rather to tolerance. These remind us to searching for the treatment for thediseases which might be caused by undesirable activation of immune system. Proper generationand manipulation of Tregs by tolerogenic vaccine might provide a promise for the treatment ofinflammatory diseases.