146 The Impact of Cirrhosis or Prior Liver Transplant On Obstetrical Outcomes: A National Study

Abstract

BACKGROUND: TLR signaling in biliary epithelial cells (BECs) is tightly controlled to prevent excessive innate immune responses in the healthy liver. We examined the effect of chronic inflammation on TLR-mediated endotoxin tolerance in human BECs in patients with end-stage chronic inflammatory liver disease (ESLD), which protects the host from permanent BEC activation in the face of ubiquitous intestinal TLR ligands in the bile and portal venous blood. METHODS: We examined patients undergoing liver transplantation for immune-mediated (PBC, PSC), alcoholic and viral (chronic hepatitis B and C) ESLD. Northern blots, real-time RT-PCR, western blots and immunocytochemistry were used for mRNA and protein expression studies of innate immune proteins in whole ESLD tissue and isolated primary BECs. Flow cytometry for incorporated endotoxin, ELISAs for secreted proinflammatory mediators such as IL-6, IL-8, and MCP-1, NF-κB reporter assays and TLR over-expression studies were used for functional studies. RESULTS: Freshly isolated human BECs in ESLD showed increased TLR activation profiles and markedly enhanced ICAM-1 expression, which have recently been linked to decreased hepatic endotoxin tolerance. Whole ESLD tissue exhibited increased expression of IFN-γ and TNF-α. Affected BECs depicted activated IFN-γ-dependent genes such as CXCL9, CXCL10 and CXCL11. We also found enhanced activation of IRF-1 and STAT1 in activated BECs, which have been shown to promote TLR signaling. These findings suggested that chronic inflammation led to enhanced TLR-mediated innate immune responses and decreased endotoxin tolerance in affected BECs in ESLD. In line with this hypothesis, primary human BECs isolated from patients with early disease stages depicted normal TLR and IL-6, IL-8, MCP-1 and ICAM-1 expression. These data suggested that increased PRR signaling and diminished endotoxin tolerance in BECs did not play a role in the primary pathogenesis of these diseases and argued for a secondary phenomenon due to chronic inflammation. Further In Vitro studies confirmed that pro-inflammatory cytokines and repetitive endotoxin challenges disrupted homoand hetero-tolerance to endotoxins in BECs. Enhanced TLR surface expression and subsequently increased endotoxin incorporation in cytokine-primed BECs contributed to increased TLR sensitivity. CONCLUSIONS: Chronic inflammation promotes a state of hyper-responsiveness to TLR ligands in ESLD and breaks the protective endotoxin tolerance in BECs. Loss of TLR tolerance appear to be especially deleterious in the face of increased intestinal endotoxin levels, which have been described in patients with ESLD of different etiologies.