Evaluation of the influence of immunoglobulin G4 (IgG4) on the innate immune response and tight junction-mediated barrier function of biliary epithelial cells in chronic inflammatory cholangiopathies

越来越多的证据表明，对肝内和肝外胆管衬里的胆管上皮细胞（BEC）中胆汁或门静脉血来源的肠道病原体相关分子模式（PAMP）的不适当先天免疫应答以及连续的胆管屏障功能障碍有助于慢性炎性胆管病的发病机制，如原发性硬化性胆管炎（PSC）和免疫球蛋白G4（IgG 4）相关性胆管炎（IAC）。含有肠PAMPs如脂多糖（LPS）的胆汁的后续胆周渗漏可能会加速胆道损伤。与PSC相反，IAC是一种激素敏感的慢性炎症性胆道疾病，涉及胰胆系统的不同部分，但其发病机制知之甚少。我们能够证明，在晚期PSC中存在主要的T辅助细胞（Th）1型细胞因子环境的情况下，激活的BEC表现出不适当的模式识别受体（PRR）介导的对肠内毒素的先天性免疫应答，以及随后的内毒素不耐受，因为增强的PRR信号传导，这加速了慢性胆道炎症。由于TNF-α抑制部分恢复了保护性先天免疫耐受，内源性TNF-α分泌可能导致活化的BEC中不适当的内毒素反应。与PSC肝脏相反，来自IAC患者的胆汁样品表现出Th 2细胞因子如白细胞介素（IL）-4和IL-5水平的显著增加。在胆汁样本中未检测到IL-13，但与对照组相比，这些患者肝外胆管的刷检细胞学样本显示IL-13 mRNA水平升高。在体外，IL-4和IL-13通过激活claudin-2介导的细胞旁孔通路显著降低紧密连接（TJ）相关的BEC屏障功能。这些结果表明，在IAC中的特定的Th 2细胞因子环境可能有助于在这些患者中的慢性胆道炎症的发病机制，由于增加的衬里胆管上皮细胞旁通透性的诱导。Th 2细胞因子也损害BEC单层的伤口闭合。IgG 4在IAC发病机制中的作用仍有待确定。我们的初步工作表明，IgG 4选择性结合肝外胆管的BEC。值得注意的是，在PSC患者中也描述了针对BEC的自身反应性抗体。我们假设自身反应性IgG 4通过诱导促炎性和促纤维化介质（如TNF-α或TGF-β）的分泌增强，促进BEC中异常的先天免疫应答和TJ介导的屏障功能障碍，从而促进这些患者慢性胆道炎症的发病机制。这项资助计划旨在阐明这一假说的潜在病理机制。"

There is growing evidence that inappropriate innate immune responses to bile- or portal venous blood-derived intestinal pathogen-associated molecular patterns (PAMPs) in biliary epithelial cells (BECs) lining the intra- and extra-hepatic bile ducts and consecutive biliary barrier dysfunction contribute to the pathogenesis of chronic inflammatory cholangiopathies, such as primary sclerosing cholangitis (PSC) and immunoglobulin G4 (IgG4)-associated cholangitis (IAC). Subsequent peribiliary leakage of bile containing intestinal PAMPs such as lipopolysaccharide (LPS) likely accelerates biliary damage. In contrast to PSC, IAC is a steroid-sensitive chronic inflammatory biliary disease that involves different parts of the pancreatobiliary system, but little is known about its mechanisms of pathogenesis.We isolated primary human BECs from explanted livers from patients with PSC. We were able to show that in the presence of the predominant T helper (Th) cell type 1 cytokine milieu in advanced PSC, activated BECs exhibit inappropriate pattern recognition receptor (PRR)-mediated innate immune responses to intestinal endotoxins and subsequent endotoxin intolerance because of enhanced PRR signaling, which accelerated chronic biliary inflammation. As TNF-alpha inhibition partly restored protective innate immune tolerance, endogenous TNF-alpha secretion likely contributed to inappropriate endotoxin responses in activated BECs. In contrast to PSC livers, bile samples from patients with IAC exhibited significant increases in levels of Th2 cytokines such as interleukin (IL)-4 and IL-5. IL-13 was not detected in bile samples, but brush cytology samples from the extra-hepatic bile ducts of these patients revealed enhanced levels of IL-13 mRNA, compared with controls. In vitro, IL-4 and IL-13 significantly reduced tight junction (TJ)-associated BEC barrier function by activating claudin-2-mediated paracellular pore pathways. These findings suggested that the specific Th2 cytokine milieu in IAC likely contributed to the pathogenesis of chonic biliary inflammation in these patients due to the induction of increased paracellular permeability of the lining biliary epithelium. Th2 cytokines also impaired wound closure in BEC monolayers. The role of IgG4 in the pathogenesis of IAC remains to be determined. Our preliminary work suggests that IgG4 selectively bind to BECs of the extra-hepatic bile ducts. Of note, autoreactive antibodies to BECs have also been described in patients with PSC. We hypothesize that autoreactive IgG4 promote aberrant innate immune responses and TJ-mediated barrier dysfunction in BECs by the induction of an enhanced secretion of pro-inflammatory respectively pro-fibriotic mediators such as TNF-alpha or TGF-ß and thus contribute to the pathogenesis of chronic biliary inflammation in these patients. This grant proposal aims to elucidate potential pathomechanisms underlying this hypothesis."