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，肝内外胆管内胆管上皮细胞对胆汁或门静脉血源性肠道病原体相关分子模式(PAMPs)的不适当天然免疫反应以及连续的胆道屏障功能障碍导致了慢性炎症性胆管病的发病，如原发性硬化性胆管炎(PSC)和免疫球蛋白G4(Ig G4)相关性胆管炎(IAC)。含有肠道PAMP(如脂多糖)的胆汁随后在胆管周围渗漏可能会加速胆道损伤。与PSC不同的是，IAC是一种激素敏感型慢性炎症性胆道疾病，累及胰胆管系统的不同部位，但其发病机制尚不清楚。我们能够证明，在晚期PSC中存在主要的T辅助(Th)细胞1型细胞因子环境下，激活的BEC表现出不适当的模式识别受体(PRR)介导的对肠道内毒素的先天免疫反应，以及随后的内毒素耐受，这是由于PRR信号的增强，从而加速了慢性胆道炎症。由于对肿瘤坏死因子-α的抑制部分恢复了保护性的先天免疫耐受，内源性肿瘤坏死因子-α的分泌可能导致激活的内皮细胞产生不适当的内毒素反应。与PSC肝脏相比，IAC患者的胆汁样本中IL-4和IL-5等Th2细胞因子水平显著升高。在胆汁样本中未检测到IL-13，但这些患者的肝外胆管刷状细胞学样本显示，与对照组相比，IL-13的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，IAC中特定的Th2细胞因子环境可能通过诱导衬里胆管上皮细胞旁通透性增加而导致慢性胆管炎的发病。Th2细胞因子也损害了BEC单层伤口的闭合。IgG4在IAC发病机制中的作用尚不清楚。我们的初步工作表明，IgG4选择性地与肝外胆管的BECs结合。值得注意的是，在PSC患者中也描述了针对BECs的自身反应抗体。我们推测，自身反应性IgG4通过诱导促炎介质如肿瘤坏死因子-α或转化生长因子-β等促炎介质的分泌增加，促进BECs异常的先天免疫反应和TJ介导的屏障功能障碍，从而在这些患者的慢性胆管炎的发病机制中起作用。这项拨款建议旨在阐明这一假说背后的潜在致病机制。

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."