Trypanosoma cruzi Infection Is a Potent Risk Factor for Non-alcoholic Steatohepatitis Enhancing Local and Systemic Inflammation Associated with Strong Oxidative Stress and Metabolic Disorders

BackgroundThe immune mechanisms underlying experimental non-alcoholic steatohepatitis (NASH), and more interestingly, the effect of T. cruzi chronic infection on the pathogenesis of this metabolic disorder are not completely understood.Methodology/Principal FindingsWe evaluated immunological parameters in male C57BL/6 wild type and TLR4 deficient mice fed with a standard, low fat diet, LFD (3% fat) as control group, or a medium fat diet, MFD (14% fat) in order to induce NASH, or mice infected intraperitoneally with 100 blood-derived trypomastigotes of Tulahuen strain and also fed with LFD (I+LFD) or MFD (I+MFD) for 24 weeks. We demonstrated that MFD by itself was able to induce NASH in WT mice and that parasitic infection induced marked metabolic changes with reduction of body weight and steatosis revealed by histological studies. The I+MFD group also improved insulin resistance, demonstrated by homeostasis model assessment of insulin resistance (HOMA-IR) analysis; although parasitic infection increased the triglycerides and cholesterol plasma levels. In addition, hepatic M1 inflammatory macrophages and cytotoxic T cells showed intracellular inflammatory cytokines which were associated with high levels of IL6, IFNγ and IL17 plasmatic cytokines and CCL2 chemokine. These findings correlated with an increase in hepatic parasite load in I+MFD group demonstrated by qPCR assays. The recruitment of hepatic B lymphocytes, NK and dendritic cells was enhanced by MFD, and it was intensified by parasitic infection. These results were TLR4 signaling dependent. Flow cytometry and confocal microscopy analysis demonstrated that the reactive oxygen species and peroxinitrites produced by liver inflammatory leukocytes of MFD group were also exacerbated by parasitic infection in our NASH model.ConclusionsWe highlight that a medium fat diet by itself is able to induce steatohepatitis. Our results also suggest a synergic effect between damage associated with molecular patterns generated during NASH and parasitic infection, revealing an intense cross-talk between metabolically active tissues, such as the liver, and the immune system. Thus, T. cruzi infection must be considered as an additional risk factor since exacerbates the inflammation and accelerates the development of hepatic injury.背景 实验性非酒精性脂肪性肝炎（non-alcoholic steatohepatitis, NASH）的潜在免疫机制，以及更值得关注的克氏锥虫（T. cruzi）慢性感染对该代谢紊乱发病机制的影响，目前尚未完全阐明。 研究方法与主要结果 我们对雄性C57BL/6野生型及Toll样受体4（TLR4）缺陷型小鼠开展免疫参数检测：小鼠分别喂食标准低脂饲料（low fat diet, LFD，脂肪含量3%，作为对照组）、中等脂肪饲料（medium fat diet, MFD，脂肪含量14%，用于诱导NASH模型）；另有部分小鼠经腹腔接种100株图拉亨株（Tulahuen strain）血液来源的锥鞭毛体，并分别喂食LFD（记为I+LFD组）或MFD（记为I+MFD组），饲养周期均为24周。我们的实验证实，单独使用MFD即可在野生型小鼠中诱导NASH；而寄生虫感染可引发显著的代谢改变，表现为体重下降，且经组织学研究证实可降低肝脂肪变性程度。I+MFD组还可改善胰岛素抵抗，这一点通过胰岛素抵抗稳态模型评估（homeostasis model assessment of insulin resistance, HOMA-IR）得到证实；不过寄生虫感染会升高血浆甘油三酯与胆固醇水平。此外，肝脏内的M1型炎性巨噬细胞与细胞毒性T细胞可表达胞内炎性细胞因子，这与血浆中高水平的IL6、IFNγ、IL17细胞因子及CCL2趋化因子水平相关。上述结果与实时定量聚合酶链反应（quantitative polymerase chain reaction, qPCR）检测证实的I+MFD组肝脏寄生虫负荷升高相一致。MFD可促进肝脏B淋巴细胞、自然杀伤细胞（NK细胞）及树突状细胞的招募，而寄生虫感染可进一步增强这一效应，且该过程依赖于TLR4信号通路。流式细胞术与共聚焦显微镜分析显示，在本研究的NASH模型中，MFD组肝脏炎性白细胞产生的活性氧与过氧亚硝基阴离子水平，可因寄生虫感染而进一步升高。 结论 我们证实，单独使用中等脂肪饲料即可诱导脂肪性肝炎。本研究结果还提示，NASH过程中产生的损伤相关分子模式与寄生虫感染之间存在协同效应，揭示了肝脏等代谢活跃组织与免疫系统之间存在强烈的交叉对话。因此，克氏锥虫（T. cruzi）感染可作为额外的危险因素，因其会加剧炎症反应并加速肝损伤的进展。