TLR4信号通路调控HBx介导的肾小管上皮-免疫细胞转分化的机制研究

Inflammatory cell infiltration in tubular and renal interstitial companied with local immune microenvironment disorders were important factors for local kidney damage. Renal tubular epithelial cells (REC), as non-dedicated antigen-presenting cells, can occur in the epithelium-immune cells trans-differentiation and start host defense mechanisms against pathogens. Our preliminary studies found that REC transfected with HBx plasmids could up-regulate the expression of MHC-II and CD40, suggesting HBx could promote renal tubular epithelial-immune cell trans-differentiation. In vivo studies also found that REC in the renal tissues of Hepatitis B Virus-related glomerulonephritis (HBV-GN) highly expressed TLR4 (one of pattern recognition receptors) and Notch1 (a signal of immune cells trans-differentiation). Along with increasing of TLR4, local inflammation exacerbated, suggesting that TLR4 maybe a key factor to start renal tubular epithelium cell trans-differentiation and promote local immune microenvironment disorders. On this basis, from the view of epithelium-immune cell trans-differentiation and TLR4 regulation, we will choose HBx, a trans-activating factor, as an incentive to explore expressions of the markers of renal tubule-immune cells trans-differentiation and the effect on immune regulation function. Cross talks between TLR4 signaling pathway and Notch1 signaling pathway will also be explored. We aimed to improve the local immune microenvironment disorders, interfere with the REC differentiation, and provide a theoretical basis for the clinical treatment of HBV-GN.

肾小管及间质炎细胞浸润及局部免疫微环境紊乱是造成肾脏局部损伤的重要因素，肾小管上皮细胞（REC）作为非专职的抗原呈递细胞，可发生上皮-免疫细胞转分化，启动病原体-宿主间反应及防御机制。我们前期研究首先发现转染HBx质粒的REC可上调表达MHC-II、CD40分子，推测HBx可通过促进肾小管上皮-免疫细胞转分化导致局部免疫微环境紊乱。同时体内研究发现乙肝肾患者REC可高表达模式识别受体TLR4和影响免疫细胞分化的Notch1，且伴随TLR4上调，局部炎症加剧。提示TLR4可能是启动细胞转分化致免疫紊乱的关键因素。在此基础上，以上皮-免疫细胞转分化及TLR4调控机制为切入点，采用反式激活因子HBx为诱因，调控TLR4信号后观察肾小管上皮-免疫细胞转分化及免疫调节功能的变化，并深入探讨TLR4信号与Notch1信号的串话作用，为进一步阐释HBV损伤肾脏局部免疫的机制提供新的理论依据和干预策略。

乙型肝炎病毒相关性肾炎（Hepatitis B Virus-related glomerulonephritis, HBV-GN）已成为我国主要的继发性肾脏疾病之一。HBV可引起肾脏局部固有免疫乃至适应性免疫异常，且炎症及免疫反应是HBV感染致肾脏局部损伤的重要病理生理机制。我们前期研究发现HBx可上调肾小管上皮细胞MHC-II、CD40分子的表达，同时体内研究发现HBV-GN患者肾小管上皮细胞可高表达模式识别受体TLR4和影响免疫细胞分化的Notch1，且伴随TLR4上调，局部炎症加剧。故本项目想进一步系统的研究：1）HBx对肾小管上皮细胞TLR4信号通路的影响；2）TLR4对HBx介导的肾小管上皮-免疫细胞转分化、固有免疫以及抗原呈递功能的调控；3）TLR4信号通路与Notch1信号通路的串话作用。. 在本项目实施期间，我们首次发现了HBx可以在肾小管上皮细胞中表达，并促进肾小管上皮细胞TLR4及其下游信号分子TRIF、TRAM、NF-κB信号通路的激活。HBx亦可以促进肾小管上皮细胞表面免疫分子的表达，表现出抗原提呈细胞的活性，并进一步激活T细胞免疫反应，亦能诱导CD4+T细胞及巨噬细胞在肾小管间质的浸润，在这一过程中TLR4发挥了重要作用。阻断TLR4的表达，可以减少CD4+T细胞及巨噬细胞的浸润，降低肾小管上皮细胞免疫分子的表达，激活T细胞免疫反应减弱。由于前期研究发现Notch1受体在HBV-GN患者肾组织中异常表达，且与肾小管上皮细胞损伤密切相关，故我们进一步的探索TLR4和Notch1的串话。我们研究发现TLR4可呈剂量依赖性的促进Notch1受体及其下游NICD1的表达。本研究证实了HBx使TLR4信号通路的异常可促进肾小管上皮-免疫细胞转分化，干扰肾小管上皮细胞抗原呈递功能，加剧细胞的免疫损伤及局部免疫微环境紊乱的形成，最终逐渐导致肾小管及间质纤维化，使肾脏功能恶化。阻断TLR4信号通路可能将抑制肾小管上皮-免疫细胞转分化的作用，改善肾脏局部免疫微环境，延缓HBV-GN进展。本研究也证实TLR4信号通路与Notch1信号通路之间存在着串话作用。