LPS-TLR4 Pathway Mediates Ductular Cell Expansion in Alcoholic Hepatitis

Abstract

Alcoholic hepatitis (AH) is the most severe form of alcoholic liver disease for which there are no effective therapies. Patients with AH show impaired hepatocyte proliferation, expansion of inefficient ductular cells and high lipopolysaccharide (LPS) levels. It is unknown whether LPS mediates ductular cell expansion. We performed transcriptome studies and identified keratin 23 (KRT23) as a new ductular cell marker. KRT23 expression correlated with mortality and LPS serum levels. LPS-TLR4 pathway role in ductular cell expansion was assessed in human and mouse progenitor cells, liver slices and liver injured TLR4 KO mice. In AH patients, ductular cell expansion correlated with portal hypertension and collagen expression. Functional studies in ductular cells showed that KRT23 regulates collagen expression. These results support a role for LPS-TLR4 pathway in promoting ductular reaction in AH. Maneuvers aimed at decreasing LPS serum levels in AH patients could have beneficial effects by preventing ductular reaction development.

Figure 1. Comparative Functional Analysis of the Transcriptome in AH and NASH.

(a) Heatmap display of the genes with the most significantly different expression between patients with AH, patients with NASH and healthy controls. Rows represent genes, and columns represent samples. The intensity of each color denotes the standardized ratio between each value and the average expression of each gene across all samples. Red pixels correspond to an increased abundance of mRNA in the indicated liver biopsy sample, whereas green pixels indicate decreased mRNA levels. (b) Multidimensional scaling analysis representing the 30 samples that underwent array profiling. The different samples are placed in the three-dimensional space according to their mRNA expression. AH samples are represented in magenta, NASH in light blue and normal livers in red. (c) Venn diagram showing the overlapping genes that were significantly upregulated (red) or downregulated (green). Venn diagram shows genes specifically upregulated (1200 genes) or downregulated (854 genes) in AH as well as genes specifically upregulated (148 genes) or downregulated (102 genes) in NASH. (d) Heatmap display of keratins with the most significantly different expression between AH and NASH and control livers. The intensity of each color denotes the standardized ratio between each value and the average expression of each gene across all samples. Red pixels correspond to an increased abundance of mRNA in the indicated liver biopsy sample, whereas green pixels indicate decreased mRNA levels.

Figure 2. Identification of KRT23 as a Marker of Ductular Cells in AH.

(a) Representative micrographs of KRT23 protein expression in paraffin sections of liver biopsies from normal livers and from patients with AH, stained with anti-KRT23 antibody (x100 magnification). (b) Immunofluorescence staining shows co-localization of KRT23 and LPCs markers (KRT7 and EPCAM) in paraffin sections of liver biopsies from patients with AH (x100 magnification). (c) Krt23 hepatic gene expression measured in a mouse model of progenitor cell expansion (DDC diet for 4 weeks, n = 6 compared to uninjured mouse liver (control, n = 6). (d) Representative images of liver KRT23 staining in mice treated for 4 weeks with a DDC diet and in uninjured mice (x200 magnification). (e) Krt23 and LPCs markers (Epcam, Krt7, Krt19) gene expression was significantly enriched in LCP cells (n = 3) vs. a non-LPC population (n = 3).

Figure 3. KRT23 expression correlates with disease severity and key features of AH.

(a) KRT23 hepatic gene expression measured by qPCR in patients with AH (n = 51), HCV (n = 10), compensated cirrhosis (n = 10) and NASH (n = 14), compared to normal livers (n = 7). (b) KRT23 peripheral serum levels are elevated in patients with AH (n = 34) when compared to patients with cirrhosis (n = 14). (c) Correlation between KRT23 hepatic gene expression (fold change vs. normal livers) and clinical features in patients with AH (n = 51). Both MELD score as well as ABIC score correlated with KRT23 hepatic gene expression. (d) Kaplan-Meier’s curve analysis illustrates the relationship between KRT23 hepatic gene expression with 90-day mortality in patients with AH. A cut-off value of 250-fold expression (fold change vs. normal livers) defined patients with low and high KRT23 gene expression with the best sensitivity and specificity. Portal hypertension (hepatic venous pressure gradient – HVPG – mmHg) was higher in patients with higher KRT23 gene expression levels (>250-fold).

Figure 4. KRT23 hepatic expression in animal models of liver injury and in LPCs.

(a) Relationship between KRT23 hepatic gene expression and LPS serum levels in patients with AH (n = 39). Correlation between LPS serum levels and ABIC score in patients with AH. (b) Krt23 hepatic gene expression in a mouse model of acute liver injury. Krt23 gene expression in mice treated with LPS (n = 6) compared to control mice (n = 6). (c) Krt23 hepatic gene expression in a mouse model of advanced fibrosis. Krt23 in mice treated CCl₄ plus LPS (n = 8) compared to mice treated with CCl₄ alone (n = 6). (d) Gene expression of KRT23 and LPCs markers (KRT7 and EPCAM) in a model of liver progenitor cell differentiation from 3 independent experiments.

Figure 5. LPS-TLR4 induced liver damage is mediated by ductular cells in animal models of liver injury.

(a) Gene expression of Krt23 and LPCs markers (Krt7 and Epcam) in WT and Tlr4-KO mice subjected to sham operation (n = 3) or BDL. BDL mice were sacrificed 5 (n = 4) or 21 days (n = 8) after operation. (b) Representative micrographs of KRT23 protein expression in WT and Tlr4-KO mice subjected to sham operation or BDL. (c) Gene expression of Krt23 and LPCs markers in WT and Tlr4-KO mice subjected to Tsukamoto-French model of ethanol damage (4 weeks, n = 6) compared to uninjured control mice (n = 4). (d) Representative micrographs of Krt23 protein expression in WT and Tlr4-KO mice subjected to Tsukamoto-French model and uninjured control mice. Bars, 100 μm.

Figure 6. KRT23 regulation by HDACs in mice liver progenitor cells and KRT23 effects on collagen synthesis.

(a) Effect of HDAC inhibition by NaB administration on Krt23 gene expression in BAML cells (3 independent experiments). (b) Gene expression of Sirt1, an HDAC, in a BAML cells treated with NaB (3 independent experiments). (c) Correlation between KRT23 hepatic gene expression and COL1A1 hepatic gene expression in patients with AH (n = 39). (d) Effect of HDACs inhibition by NaB administration on Col1a1 gene expression in BAML cells. (e) Silencing of Krt23 by means of siRNA inhibited NaB induced Col1a1 gene expression in BAML cells (3 independent experiments).