应用免疫耐受疫苗预防性治疗非酒精性脂肪性肝炎的实验研究
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摘要
非酒精性脂肪性肝炎（Nonalcoholic steatohepatitis，NASH）属于非酒精性脂肪性肝病（Nonalcoholic fatty liver disease，NAFLD），为现今社会的高发疾病。NASH不仅与动脉粥样硬化、糖尿病等肥胖症并发症紧密相关，而且约20%的NASH患者会发展为肝硬化，其中8%的肝硬化患者甚至可以发展为肝癌。另外，NASH的高发人群已呈现出由中老年为主逐渐向中青年过渡的趋势。虽然已有多个国家和地区的医学组织公布了NASH的治疗指南，但是目前针对NASH的治疗方法有限，尚无特异性治疗方案。
关于NASH发病机制的研究比较有限，其中被学者所广泛接收的即是“二次打击”学说。第一次打击是指各种原因导致的肝脏脂肪蓄积及肝细胞脂肪变性。二次打击即是指一次打击所触发的细胞毒素事件，最终导致肝脏炎症的发生。其中，细胞毒素事件包括众多致病因素如氧化应激、胰岛素抵抗和免疫细胞的激活等。新近研究表明NASH炎症进一步发展的重要环节可能为高脂饮食导致的体内抑制炎症作用的调节性T细胞（Regulatory T cells，Tregs）数量减少而不能建立起有效的免疫应答。同时，在体内脂类代谢异常所产生的新生抗原（如热休克蛋白60，HSP60等）的刺激下具有促进炎症作用的效应性T细胞（Effector T cells，Teffs）激活而启动连环打击诱发NASH。
免疫耐受疫苗（Tolerogenic vaccine, TV）即为一种新兴的通过使机体产生抗原特异性Tregs而诱导免疫耐受的治疗方法。其方法为在应用免疫抑制剂（例如糖皮质激素）作为免疫耐受佐剂的前提下，应用目标蛋白MHCII限制性抗原肽建立耐受性免疫应答。故此，我们推测如果能应用免疫耐受疫苗预先建立抗原特异性Tregs免疫应答即会在高脂饮食过程中改变体内Tregs与Teffs的不平衡应答状态，从而抑制NASH发生。
本实验首先选取C57BL/6和BALB/C小鼠作为研究对象，应用流式细胞术CD11c和CD40双染策略分析了在地塞米松作用下体内抗原提呈细胞（Antigen presenting cell，APC）的变化情况。结果显示脾脏、淋巴结及外周血中均有两个主要的CD11c~+CD40~+细胞亚群：CD11c~(low)与CD11c~(high)，并且同CD11c~(high)相比，CD11c~(low)（我们将其命名为Dex-APC）对地塞米松相对耐受。进一步研究显示给予地塞米松（4.5mg/kg，s.c.）24h后，在脾脏中，约75%的CD11c~(low)细胞存活，约有5%的CD11c~(high)细胞存活；在肝脏中，大部分MHCII~+细胞均被地塞米松清除，CD11c~(low)与CD11c~(high)细胞存活率均约为5%。地塞米松对抗原提呈细胞的筛选作用的最适浓度可能为4.5mg/kg，其作用可以维持近4天左右，峰值出现在注射后1-2天。
本研究应用流式细胞术进一步分析了CD11c~(low)（Dex-APC）细胞的表面谱系标志。结果示Dex-APC为Ly6C~(low)CD62L~(low)提示其为单核细胞来源；CD83~(low)F4/80~(high)CD68~(high)提示其为未成熟巨噬细胞。并且Dex-APC为MHCII类分子及CD86共刺激分子阳性提示其具备通过抗原性及共刺激信号受体激活T细胞增殖的能力。随后我们应用免疫磁珠细胞分选技术（MACS）将小鼠体内CD11c~(low)（Dex-APC）和CD11c~(high)细胞分别分离，应用实时荧光定量PCR技术检测不同细胞的糖皮质激素受体GR与GR的表达情况。结果示CD11c~(low)(Dex-APC)细胞GR：GR约为CD11c~(high)细胞7倍左右，可能为其对地塞米松耐受的原因。随后我们应用过继性细胞转移技术将已结合OVA_(323-339)的抗原提呈细胞作为供体植入OVA致敏的Foxp3-eGFP小鼠，然后分离出受体小鼠的脾脏细胞应用5-溴脱氧尿嘧啶核苷（BrdU）技术来分析CD4~+T细胞的增殖情况。结果示供体小鼠未经Dex处理而分离出的已结合OVA_(323-339)的抗原提呈细胞刺激小鼠体内的效应性T细胞（Teffs）增殖（P<0.05）；而经过Dex处理而分离出的已结合OVA_(323-339)的抗原提呈细胞（Dex-APC）刺激调节性T细胞（Tregs）增殖（P<0.05）。
本实验根据生物信息学新方法预测了热休克蛋白60（HSP60）的T细胞表位抗原肽片段HSP-1和HSP-2，并验证其免疫原性。随后应用MACS技术分离HSP60致敏的效应性T细胞（Teffs）和调节性T细胞（Tregs）并分别与肝脏Kupffer细胞联合培养。结果示Teffs被HSP-1激活时可导致Kupffer细胞活化，包括表面功能标志表达的增强（CD86与MHCII类分子），炎性细胞因子分泌的增加（TNF-和IL-12）；而Tregs被HSP-1激活时可导致Kupffer细胞功能抑制。
随后本实验进一步分析接种了以HSP60为靶点的免疫耐受疫苗（即在地塞米松的作用前提下给予HSP-1刺激）的C57BL/6小鼠体内免疫应答情况。结果给予HSP-1再刺激时外周血中， Tregs（CD4~+CD25~+Foxp3~+）占CD4~+辅助性T细胞的比率在各组中分别为：NT组（无处理组）1.41±0.09%，Dex组（单用地塞米松组）1.19±0.13%，HSP-1组（单用HSP-1组）1.01±0.11%及TV组（免疫耐受疫苗组）8.76±1.31%；肝脏中的结果为NT7.31±0.69%，Dex9.18±0.72%，HSP-19.87.±0.79%及TV17.96±3.83%。故相比于其他对照组，TV处理组抗原刺激后Tregs比率显著升高（p<0.01）。同时，本实验应用MACS技术将上述各组小鼠脾脏CD4~+T细胞分离并用CFSE标记，并给予不同抗原肽刺激，应用流式细胞术检测不同T细胞的增殖情况。结果示在HSP-1的刺激下，Tregs的明显增殖情况出现在TV处理组，其增殖部分细胞比率占CD4~+T细胞的26.48±4.32%，相比其他处理组均<8%增殖比率具有显著性差异（p<0.01）。该增殖具有HSP-1特异性，因TV处理组在无关抗原肽MOG的刺激下，增值比率<4%（p<0.01）。
本实验为最终观察TV在NASH发病中的作用，给予C57BL/6小鼠预防性接种以HSP60为靶点的免疫耐受疫苗以建立针对HSP60的免疫耐受，然后给予高脂饮食以模拟NASH的发病过程。结果示：1、TV处理组（免疫耐受疫苗组）小鼠肝脏的肝脏指数（肝重/体重100%）为3.64±0.82%，NT组（无处理组）为8.34±1.17%，Dex组（单用地塞米松组）为7.56±0.67%，HSP-1组（单用HSP-1组）为9.08±1.23%。在6个月的高脂饮食下，TV处理组的肝脏指数明显优于其他处理组（p<0.01）。2、TV处理组血清总胆固醇（TC）为118.27±15.18mg/dL，NT组为229.57±21.23mg/dL，Dex组为225.18±16.43mg/dL，HSP-1为304.39±38.32mg/dL。与其他高脂饮食组相比，TV组的血清TC水平显著性降低（p<0.01）。3、TV处理组的血清谷丙转氨酶（ALT）为65.15±11.09IU/L，NT组为157.18±21.26IU/L，Dex组为156.36±15.37IU/L，HSP-1为164.25±14.31IU/L。与其他高脂饮食组相比，TV组的血清ALT水平显著性降低（p<0.01）。4、HOMA-IR的检测结果：TV组为3.72±1.49，NT组为8.49±1.02，Dex组为8.91±0.72，HSP-1为9.61±1.21。与其他高脂饮食组相比，TV组的HOMA-IR水平显著性降低（p<0.01）。5、石蜡切片行HE染色结果为TV组肝小叶结构基本正常，汇管区可见少许炎细胞侵润，少数肝细胞核周围出现小脂肪空泡。而其他高脂饮食组NT、Dex及HSP-1可见肝细胞排列松散，出现弥漫性的肝细胞脂肪变性，遍及整个肝小叶，某些部分可见较大空泡样脂肪变；冰冻切片的脂肪特异性油红O染色结果为相比于TV处理组，NT、Dex及HSP-1组均出现大量肝细胞脂肪变性、细胞肿大、肝索排列紊乱，并且肝脏切片可见广泛、大片的红色脂滴沉着。
同时，本研究为了解以HSP60为靶点的免疫耐受疫苗预防性治疗NASH的机理。在高脂饮食过程每2个月检测一次外周血辅助性T细胞的应答变化水平；每个月检测一次血清C反应蛋白（CRP）的浓度。结果示：1、相比于其他对照组，TV组在0-2月其外周血刺激后Tregs（CD4~+CD25+FoxP3+）检测得到的比率明显高升高（p<0.01），至第4个月时仍升高（p<0.05）。2、与其他组相比，TV处理组血清C反应蛋白（CRP）在6个月高脂饮食期间持续保持较低水平。
本实验发现了地塞米松对体内抗原提呈细胞的功能筛选作用，即保留了相对耐受的Dex-APC，并且发现其具备向调节性T细胞（Tregs）提呈特异性抗原并诱导其增殖的能力，导致抗原特异性免疫耐受。进一步完善和丰富了地塞米松免疫耐受的细胞学机理。随后本实验应用以HSP60为靶点的免疫耐受疫苗预防性治疗非酒精性脂肪性肝炎（NASH）并取得一定疗效，证明了预先建立针对脂类代谢异常所释放新抗原（如HSP60）的调节性T细胞耐受性免疫应答可以缓解NASH的炎症程度。同时，本研究进一步解释了NASH的发病机理，并且免疫耐受疫苗的应用与研究为治疗慢性炎症性疾病提供新的策略。
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~(low)cells 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.
关于NASH发病机制的研究比较有限，其中被学者所广泛接收的即是“二次打击”学说。第一次打击是指各种原因导致的肝脏脂肪蓄积及肝细胞脂肪变性。二次打击即是指一次打击所触发的细胞毒素事件，最终导致肝脏炎症的发生。其中，细胞毒素事件包括众多致病因素如氧化应激、胰岛素抵抗和免疫细胞的激活等。新近研究表明NASH炎症进一步发展的重要环节可能为高脂饮食导致的体内抑制炎症作用的调节性T细胞（Regulatory T cells，Tregs）数量减少而不能建立起有效的免疫应答。同时，在体内脂类代谢异常所产生的新生抗原（如热休克蛋白60，HSP60等）的刺激下具有促进炎症作用的效应性T细胞（Effector T cells，Teffs）激活而启动连环打击诱发NASH。
免疫耐受疫苗（Tolerogenic vaccine, TV）即为一种新兴的通过使机体产生抗原特异性Tregs而诱导免疫耐受的治疗方法。其方法为在应用免疫抑制剂（例如糖皮质激素）作为免疫耐受佐剂的前提下，应用目标蛋白MHCII限制性抗原肽建立耐受性免疫应答。故此，我们推测如果能应用免疫耐受疫苗预先建立抗原特异性Tregs免疫应答即会在高脂饮食过程中改变体内Tregs与Teffs的不平衡应答状态，从而抑制NASH发生。
本实验首先选取C57BL/6和BALB/C小鼠作为研究对象，应用流式细胞术CD11c和CD40双染策略分析了在地塞米松作用下体内抗原提呈细胞（Antigen presenting cell，APC）的变化情况。结果显示脾脏、淋巴结及外周血中均有两个主要的CD11c~+CD40~+细胞亚群：CD11c~(low)与CD11c~(high)，并且同CD11c~(high)相比，CD11c~(low)（我们将其命名为Dex-APC）对地塞米松相对耐受。进一步研究显示给予地塞米松（4.5mg/kg，s.c.）24h后，在脾脏中，约75%的CD11c~(low)细胞存活，约有5%的CD11c~(high)细胞存活；在肝脏中，大部分MHCII~+细胞均被地塞米松清除，CD11c~(low)与CD11c~(high)细胞存活率均约为5%。地塞米松对抗原提呈细胞的筛选作用的最适浓度可能为4.5mg/kg，其作用可以维持近4天左右，峰值出现在注射后1-2天。
本研究应用流式细胞术进一步分析了CD11c~(low)（Dex-APC）细胞的表面谱系标志。结果示Dex-APC为Ly6C~(low)CD62L~(low)提示其为单核细胞来源；CD83~(low)F4/80~(high)CD68~(high)提示其为未成熟巨噬细胞。并且Dex-APC为MHCII类分子及CD86共刺激分子阳性提示其具备通过抗原性及共刺激信号受体激活T细胞增殖的能力。随后我们应用免疫磁珠细胞分选技术（MACS）将小鼠体内CD11c~(low)（Dex-APC）和CD11c~(high)细胞分别分离，应用实时荧光定量PCR技术检测不同细胞的糖皮质激素受体GR与GR的表达情况。结果示CD11c~(low)(Dex-APC)细胞GR：GR约为CD11c~(high)细胞7倍左右，可能为其对地塞米松耐受的原因。随后我们应用过继性细胞转移技术将已结合OVA_(323-339)的抗原提呈细胞作为供体植入OVA致敏的Foxp3-eGFP小鼠，然后分离出受体小鼠的脾脏细胞应用5-溴脱氧尿嘧啶核苷（BrdU）技术来分析CD4~+T细胞的增殖情况。结果示供体小鼠未经Dex处理而分离出的已结合OVA_(323-339)的抗原提呈细胞刺激小鼠体内的效应性T细胞（Teffs）增殖（P<0.05）；而经过Dex处理而分离出的已结合OVA_(323-339)的抗原提呈细胞（Dex-APC）刺激调节性T细胞（Tregs）增殖（P<0.05）。
本实验根据生物信息学新方法预测了热休克蛋白60（HSP60）的T细胞表位抗原肽片段HSP-1和HSP-2，并验证其免疫原性。随后应用MACS技术分离HSP60致敏的效应性T细胞（Teffs）和调节性T细胞（Tregs）并分别与肝脏Kupffer细胞联合培养。结果示Teffs被HSP-1激活时可导致Kupffer细胞活化，包括表面功能标志表达的增强（CD86与MHCII类分子），炎性细胞因子分泌的增加（TNF-和IL-12）；而Tregs被HSP-1激活时可导致Kupffer细胞功能抑制。
随后本实验进一步分析接种了以HSP60为靶点的免疫耐受疫苗（即在地塞米松的作用前提下给予HSP-1刺激）的C57BL/6小鼠体内免疫应答情况。结果给予HSP-1再刺激时外周血中， Tregs（CD4~+CD25~+Foxp3~+）占CD4~+辅助性T细胞的比率在各组中分别为：NT组（无处理组）1.41±0.09%，Dex组（单用地塞米松组）1.19±0.13%，HSP-1组（单用HSP-1组）1.01±0.11%及TV组（免疫耐受疫苗组）8.76±1.31%；肝脏中的结果为NT7.31±0.69%，Dex9.18±0.72%，HSP-19.87.±0.79%及TV17.96±3.83%。故相比于其他对照组，TV处理组抗原刺激后Tregs比率显著升高（p<0.01）。同时，本实验应用MACS技术将上述各组小鼠脾脏CD4~+T细胞分离并用CFSE标记，并给予不同抗原肽刺激，应用流式细胞术检测不同T细胞的增殖情况。结果示在HSP-1的刺激下，Tregs的明显增殖情况出现在TV处理组，其增殖部分细胞比率占CD4~+T细胞的26.48±4.32%，相比其他处理组均<8%增殖比率具有显著性差异（p<0.01）。该增殖具有HSP-1特异性，因TV处理组在无关抗原肽MOG的刺激下，增值比率<4%（p<0.01）。
本实验为最终观察TV在NASH发病中的作用，给予C57BL/6小鼠预防性接种以HSP60为靶点的免疫耐受疫苗以建立针对HSP60的免疫耐受，然后给予高脂饮食以模拟NASH的发病过程。结果示：1、TV处理组（免疫耐受疫苗组）小鼠肝脏的肝脏指数（肝重/体重100%）为3.64±0.82%，NT组（无处理组）为8.34±1.17%，Dex组（单用地塞米松组）为7.56±0.67%，HSP-1组（单用HSP-1组）为9.08±1.23%。在6个月的高脂饮食下，TV处理组的肝脏指数明显优于其他处理组（p<0.01）。2、TV处理组血清总胆固醇（TC）为118.27±15.18mg/dL，NT组为229.57±21.23mg/dL，Dex组为225.18±16.43mg/dL，HSP-1为304.39±38.32mg/dL。与其他高脂饮食组相比，TV组的血清TC水平显著性降低（p<0.01）。3、TV处理组的血清谷丙转氨酶（ALT）为65.15±11.09IU/L，NT组为157.18±21.26IU/L，Dex组为156.36±15.37IU/L，HSP-1为164.25±14.31IU/L。与其他高脂饮食组相比，TV组的血清ALT水平显著性降低（p<0.01）。4、HOMA-IR的检测结果：TV组为3.72±1.49，NT组为8.49±1.02，Dex组为8.91±0.72，HSP-1为9.61±1.21。与其他高脂饮食组相比，TV组的HOMA-IR水平显著性降低（p<0.01）。5、石蜡切片行HE染色结果为TV组肝小叶结构基本正常，汇管区可见少许炎细胞侵润，少数肝细胞核周围出现小脂肪空泡。而其他高脂饮食组NT、Dex及HSP-1可见肝细胞排列松散，出现弥漫性的肝细胞脂肪变性，遍及整个肝小叶，某些部分可见较大空泡样脂肪变；冰冻切片的脂肪特异性油红O染色结果为相比于TV处理组，NT、Dex及HSP-1组均出现大量肝细胞脂肪变性、细胞肿大、肝索排列紊乱，并且肝脏切片可见广泛、大片的红色脂滴沉着。
同时，本研究为了解以HSP60为靶点的免疫耐受疫苗预防性治疗NASH的机理。在高脂饮食过程每2个月检测一次外周血辅助性T细胞的应答变化水平；每个月检测一次血清C反应蛋白（CRP）的浓度。结果示：1、相比于其他对照组，TV组在0-2月其外周血刺激后Tregs（CD4~+CD25+FoxP3+）检测得到的比率明显高升高（p<0.01），至第4个月时仍升高（p<0.05）。2、与其他组相比，TV处理组血清C反应蛋白（CRP）在6个月高脂饮食期间持续保持较低水平。
本实验发现了地塞米松对体内抗原提呈细胞的功能筛选作用，即保留了相对耐受的Dex-APC，并且发现其具备向调节性T细胞（Tregs）提呈特异性抗原并诱导其增殖的能力，导致抗原特异性免疫耐受。进一步完善和丰富了地塞米松免疫耐受的细胞学机理。随后本实验应用以HSP60为靶点的免疫耐受疫苗预防性治疗非酒精性脂肪性肝炎（NASH）并取得一定疗效，证明了预先建立针对脂类代谢异常所释放新抗原（如HSP60）的调节性T细胞耐受性免疫应答可以缓解NASH的炎症程度。同时，本研究进一步解释了NASH的发病机理，并且免疫耐受疫苗的应用与研究为治疗慢性炎症性疾病提供新的策略。
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~(low)cells 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.
引文
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[35] Ma X, Hua J, Mohamood AR, Hamad AR, Ravi R, et al.(2007) A high-fat diet andregulatory T cells influence susceptibility to endotoxin-induced liver injury. Hepatology46:1519-1529.
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[38] Ratziu V, Bellentani S, Cortez-Pinto H, Day C, Marchesini G (2010) A positionstatement on NAFLD/NASH based on the EASL2009special conference. J Hepatol53:372-384.
[39] Newsome PN, Allison ME, Andrews PA, Auzinger G, Day CP, et al.(2012) Guidelinesfor liver transplantation for patients with non-alcoholic steatohepatitis. Gut61:484-500.
[40] Xu W, Lan Q, Chen M, Chen H, Zhu N, et al.(2012) Adoptive transfer of induced-Tregcells effectively attenuates murine airway allergic inflammation. PLoS One7: e40314.
[41] Jeon EJ, Yoon BY, Lim JY, Oh HJ, Park HS, et al.(2012) Adoptive transfer of all-trans-retinal-induced regulatory T cells ameliorates experimental autoimmune arthritisin an interferon-gamma knockout model. Autoimmunity45:460-469.
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[43] van Puijvelde GH, Hauer AD, de Vos P, van den Heuvel R, van Herwijnen MJ, et al.(2006) Induction of oral tolerance to oxidized low-density lipoprotein amelioratesatherosclerosis. Circulation114:1968-1976.
[44] van Puijvelde GH, van Es T, van Wanrooij EJ, Habets KL, de Vos P, et al.(2007)Induction of oral tolerance to HSP60or an HSP60-peptide activates T cell regulationand reduces atherosclerosis. Arterioscler Thromb Vasc Biol27:2677-2683.
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[46] Kang Y, Xu L, Wang B, Chen A, Zheng G (2008) Cutting edge: Immunosuppressant asadjuvant for tolerogenic immunization. J Immunol180:5172-5176.
[47] Nilsson J, Bjorkbacka H, Fredrikson GN (2012) Apolipoprotein B100autoimmunity andatherosclerosis-disease mechanisms and therapeutic potential. Curr Opin Lipidol23:422-428.
[48] Yang H, Mohamed AS, Zhou SH (2012) Oxidized low density lipoprotein, stem cells,and atherosclerosis. Lipids Health Dis11:85.
[49] Yan M, Mehta JL, Zhang W, Hu C (2011) LOX-1, oxidative stress and inflammation: anovel mechanism for diabetic cardiovascular complications. Cardiovasc Drugs Ther25:451-459.
[50] Mitra S, Deshmukh A, Sachdeva R, Lu J, Mehta JL (2011) Oxidized low-densitylipoprotein and atherosclerosis implications in antioxidant therapy. Am J Med Sci342:135-142.
[51] Tamura Y, Torigoe T, Kukita K, Saito K, Okuya K, et al.(2012) Heat-shock proteins asendogenous ligands building a bridge between innate and adaptive immunity.Immunotherapy4:841-852.
[52] van Noort JM, Bsibsi M, Nacken P, Gerritsen WH, Amor S (2012) The link betweensmall heat shock proteins and the immune system. Int J Biochem Cell Biol44:1670-1679.
[53] Vidal Magalhaes W, Gouveia Nogueira MF, Kaneko TM (2012) Heat shock proteins(HSP): dermatological implications and perspectives. Eur J Dermatol22:8-13.
[54] Pisetsky D (2011) Cell death in the pathogenesis of immune-mediated diseases: the roleof HMGB1and DAMP-PAMP complexes. Swiss Med Wkly141: w13256.
[55] Henderson B, Pockley AG (2012) Proteotoxic stress and circulating cell stress proteinsin the cardiovascular diseases. Cell Stress Chaperones17:303-311.
[56] Quintana FJ, Cohen IR (2011) The HSP60immune system network. Trends Immunol32:89-95.
[57] Grundtman C, Wick G (2011) The autoimmune concept of atherosclerosis. Curr OpinLipidol22:327-334.
[58] Hassan GA, Sliem HA, Ellethy AT, Salama Mel S (2012) Role of immune systemmodulation in prevention of type1diabetes mellitus. Indian J Endocrinol Metab16:904-909.
[59] Coelho V, Faria AM (2011) HSP60: issues and insights on its therapeutic use as animmunoregulatory agent. Front Immunol2:97.
[60] Marker T, Sell H, Zillessen P, Glode A, Kriebel J, et al.(2012) Heat shock protein60asa mediator of adipose tissue inflammation and insulin resistance. Diabetes61:615-625.
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[63] Kerzerho J, Wunsch D, Szely N, Meyer HA, Lurz L, et al.(2012) Effects of systemicversus local administration of corticosteroids on mucosal tolerance. J Immunol188:470-476.
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[73] Lehner B, Sandner B, Marschallinger J, Lehner C, Furtner T, et al.(2011) The dark sideof BrdU in neural stem cell biology: detrimental effects on cell cycle, differentiation andsurvival. Cell Tissue Res345:313-328.
[74] Schlichting CL, Schareck WD, Nickel T, Weis M (2005) Dendritic cells aspharmacological targets for the generation of regulatory immunosuppressive effectors.New implications for allo-transplantation. Curr Med Chem12:1921-1930.
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[80] Schaaf MJ, Cidlowski JA (2002) AUUUA motifs in the3'UTR of human glucocort-icoid receptor alpha and beta mRNA destabilize mRNA and decrease receptor proteinexpression. Steroids67:627-636.
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[34] Suzuki J, Ricordi C, Chen Z (2010) Immune tolerance induction by integrating innateand adaptive immune regulators. Cell Transplant19:253-268.
[35] Ma X, Hua J, Mohamood AR, Hamad AR, Ravi R, et al.(2007) A high-fat diet andregulatory T cells influence susceptibility to endotoxin-induced liver injury. Hepatology46:1519-1529.
[36] Fan JG, Jia JD, Li YM, Wang BY, Lu LG, et al.(2011) Guidelines for the diagnosis andmanagement of nonalcoholic fatty liver disease: update2010:(published in Chinese onChinese Journal of Hepatology2010;18:163-166). J Dig Dis12:38-44.
[37] Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, et al.(2012) The diagnosisand management of non-alcoholic fatty liver disease: practice guideline by the AmericanGastroenterological Association, American Association for the Study of Liver Diseases,and American College of Gastroenterology. Gastroenterology142:1592-1609.
[38] Ratziu V, Bellentani S, Cortez-Pinto H, Day C, Marchesini G (2010) A positionstatement on NAFLD/NASH based on the EASL2009special conference. J Hepatol53:372-384.
[39] Newsome PN, Allison ME, Andrews PA, Auzinger G, Day CP, et al.(2012) Guidelinesfor liver transplantation for patients with non-alcoholic steatohepatitis. Gut61:484-500.
[40] Xu W, Lan Q, Chen M, Chen H, Zhu N, et al.(2012) Adoptive transfer of induced-Tregcells effectively attenuates murine airway allergic inflammation. PLoS One7: e40314.
[41] Jeon EJ, Yoon BY, Lim JY, Oh HJ, Park HS, et al.(2012) Adoptive transfer of all-trans-retinal-induced regulatory T cells ameliorates experimental autoimmune arthritisin an interferon-gamma knockout model. Autoimmunity45:460-469.
[42] Steffens S, Burger F, Pelli G, Dean Y, Elson G, et al.(2006) Short-term treatment withanti-CD3antibody reduces the development and progression of atherosclerosis in mice.Circulation114:1977-1984.
[43] van Puijvelde GH, Hauer AD, de Vos P, van den Heuvel R, van Herwijnen MJ, et al.(2006) Induction of oral tolerance to oxidized low-density lipoprotein amelioratesatherosclerosis. Circulation114:1968-1976.
[44] van Puijvelde GH, van Es T, van Wanrooij EJ, Habets KL, de Vos P, et al.(2007)Induction of oral tolerance to HSP60or an HSP60-peptide activates T cell regulationand reduces atherosclerosis. Arterioscler Thromb Vasc Biol27:2677-2683.
[45] Faria AM, Weiner HL (2005) Oral tolerance. Immunol Rev206:232-259.
[46] Kang Y, Xu L, Wang B, Chen A, Zheng G (2008) Cutting edge: Immunosuppressant asadjuvant for tolerogenic immunization. J Immunol180:5172-5176.
[47] Nilsson J, Bjorkbacka H, Fredrikson GN (2012) Apolipoprotein B100autoimmunity andatherosclerosis-disease mechanisms and therapeutic potential. Curr Opin Lipidol23:422-428.
[48] Yang H, Mohamed AS, Zhou SH (2012) Oxidized low density lipoprotein, stem cells,and atherosclerosis. Lipids Health Dis11:85.
[49] Yan M, Mehta JL, Zhang W, Hu C (2011) LOX-1, oxidative stress and inflammation: anovel mechanism for diabetic cardiovascular complications. Cardiovasc Drugs Ther25:451-459.
[50] Mitra S, Deshmukh A, Sachdeva R, Lu J, Mehta JL (2011) Oxidized low-densitylipoprotein and atherosclerosis implications in antioxidant therapy. Am J Med Sci342:135-142.
[51] Tamura Y, Torigoe T, Kukita K, Saito K, Okuya K, et al.(2012) Heat-shock proteins asendogenous ligands building a bridge between innate and adaptive immunity.Immunotherapy4:841-852.
[52] van Noort JM, Bsibsi M, Nacken P, Gerritsen WH, Amor S (2012) The link betweensmall heat shock proteins and the immune system. Int J Biochem Cell Biol44:1670-1679.
[53] Vidal Magalhaes W, Gouveia Nogueira MF, Kaneko TM (2012) Heat shock proteins(HSP): dermatological implications and perspectives. Eur J Dermatol22:8-13.
[54] Pisetsky D (2011) Cell death in the pathogenesis of immune-mediated diseases: the roleof HMGB1and DAMP-PAMP complexes. Swiss Med Wkly141: w13256.
[55] Henderson B, Pockley AG (2012) Proteotoxic stress and circulating cell stress proteinsin the cardiovascular diseases. Cell Stress Chaperones17:303-311.
[56] Quintana FJ, Cohen IR (2011) The HSP60immune system network. Trends Immunol32:89-95.
[57] Grundtman C, Wick G (2011) The autoimmune concept of atherosclerosis. Curr OpinLipidol22:327-334.
[58] Hassan GA, Sliem HA, Ellethy AT, Salama Mel S (2012) Role of immune systemmodulation in prevention of type1diabetes mellitus. Indian J Endocrinol Metab16:904-909.
[59] Coelho V, Faria AM (2011) HSP60: issues and insights on its therapeutic use as animmunoregulatory agent. Front Immunol2:97.
[60] Marker T, Sell H, Zillessen P, Glode A, Kriebel J, et al.(2012) Heat shock protein60asa mediator of adipose tissue inflammation and insulin resistance. Diabetes61:615-625.
[61] Bower AN, Oyen LJ (2005) Interaction between dexamethasone treatment and thecorticotropin stimulation test in septic shock. Ann Pharmacother39:335-338.
[62] Jeklova E, Leva L, Jaglic Z, Faldyna M (2008) Dexamethasone-induced immunosu-ppression: a rabbit model. Vet Immunol Immunopathol122:231-240.
[63] Kerzerho J, Wunsch D, Szely N, Meyer HA, Lurz L, et al.(2012) Effects of systemicversus local administration of corticosteroids on mucosal tolerance. J Immunol188:470-476.
[64] Weichhart T, Brandt O, Lassnig C, Muller M, Horl WH, et al.(2010) The anti-inflammatory potency of dexamethasone is determined by the route of application invivo. Immunol Lett129:50-52.
[65] Piemonti L, Monti P, Allavena P, Sironi M, Soldini L, et al.(1999) Glucocorticoidsaffect human dendritic cell differentiation and maturation. J Immunol162:6473-6481.
[66] Matyszak MK, Citterio S, Rescigno M, Ricciardi-Castagnoli P (2000) Differentialeffects of corticosteroids during different stages of dendritic cell maturation. Eur JImmunol30:1233-1242.
[67] Banchereau J, Steinman RM (1998) Dendritic cells and the control of immunity. Nature392:245-252.
[68] Jonuleit H, Schmitt E, Schuler G, Knop J, Enk AH (2000) Induction of interleukin10-producing, nonproliferating CD4(+) T cells with regulatory properties by repetitivestimulation with allogeneic immature human dendritic cells. J Exp Med192:1213-1222.
[69] Dhodapkar MV, Steinman RM, Krasovsky J, Munz C, Bhardwaj N (2001) Antigen-specific inhibition of effector T cell function in humans after injection of immaturedendritic cells. J Exp Med193:233-238.
[70] Rutella S, Lemoli RM (2004) Regulatory T cells and tolerogenic dendritic cells: frombasic biology to clinical applications. Immunol Lett94:11-26.
[71] Kummrow A, Frankowski M, Bock N, Werner C, Dziekan T, et al.(2013) Quantitativeassessment of cell viability based on flow cytometry and microscopy. Cytometry A83:197-204.
[72] North ML, Khanna N, Marsden PA, Grasemann H, Scott JA (2009) Functionallyimportant role for arginase1in the airway hyperresponsiveness of asthma. Am J PhysiolLung Cell Mol Physiol296: L911-920.
[73] Lehner B, Sandner B, Marschallinger J, Lehner C, Furtner T, et al.(2011) The dark sideof BrdU in neural stem cell biology: detrimental effects on cell cycle, differentiation andsurvival. Cell Tissue Res345:313-328.
[74] Schlichting CL, Schareck WD, Nickel T, Weis M (2005) Dendritic cells aspharmacological targets for the generation of regulatory immunosuppressive effectors.New implications for allo-transplantation. Curr Med Chem12:1921-1930.
[75] Rozkova D, Horvath R, Bartunkova J, Spisek R (2006) Glucocorticoids severely impairdifferentiation and antigen presenting function of dendritic cells despite upregulation ofToll-like receptors. Clin Immunol120:260-271.
[76] Huwyler J, Drewe J, Krahenbuhl S (2008) Tumor targeting using liposomalantineoplastic drugs. Int J Nanomedicine3:21-29.
[77] Schnyder A, Huwyler J (2005) Drug transport to brain with targeted liposomes.NeuroRx2:99-107.
[78] van Rooijen N, Sanders A, van den Berg TK (1996) Apoptosis of macrophages inducedby liposome-mediated intracellular delivery of clodronate and propamidine. J ImmunolMethods193:93-99.
[79] Immordino ML, Dosio F, Cattel L (2006) Stealth liposomes: review of the basic science,rationale, and clinical applications, existing and potential. Int J Nanomedicine1:297-315.
[80] Schaaf MJ, Cidlowski JA (2002) AUUUA motifs in the3'UTR of human glucocort-icoid receptor alpha and beta mRNA destabilize mRNA and decrease receptor proteinexpression. Steroids67:627-636.
[81] Bamberger CM, Bamberger AM, de Castro M, Chrousos GP (1995) Glucocorticoidreceptor beta, a potential endogenous inhibitor of glucocorticoid action in humans. JClin Invest95:2435-2441.
[82] Zhang GL, Khan AM, Srinivasan KN, Heiny A, Lee K, et al.(2008) Hotspot Hunter: acomputational system for large-scale screening and selection of candidateimmunological hotspots in pathogen proteomes. BMC Bioinformatics9Suppl1: S19.
[83] Salomon J, Flower DR (2006) Predicting Class II MHC-Peptide binding: a kernel basedapproach using similarity scores. BMC Bioinformatics7:501.
[84] Valatas V, Xidakis C, Roumpaki H, Kolios G, Kouroumalis EA (2003) Isolation of ratKupffer cells: a combined methodology for highly purified primary cultures. Cell BiolInt27:67-73.
[85] Baecher-Allan C, Brown JA, Freeman GJ, Hafler DA (2001) CD4+CD25high regulatorycells in human peripheral blood. J Immunol167:1245-1253.
[86] Sakaguchi S (2005) Naturally arising Foxp3-expressing CD25+CD4+regulatory T cellsin immunological tolerance to self and non-self. Nat Immunol6:345-352.
[87] Shevach EM (2004) Regulatory/suppressor T cells in health and disease. ArthritisRheum50:2721-2724.
[88] Baeck C, Wehr A, Karlmark KR, Heymann F, Vucur M, et al.(2012) Pharmacologicalinhibition of the chemokine CCL2(MCP-1) diminishes liver macrophage infiltrationand steatohepatitis in chronic hepatic injury. Gut61:416-426.
[89] Koteish A, Diehl AM (2001) Animal models of steatosis. Semin Liver Dis21:89-104.
[90] Koteish A, Mae Diehl A (2002) Animal models of steatohepatitis. Best Pract Res ClinGastroenterol16:679-690.
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