Hepatocyte IKK2 Protects Mdr2−/− Mice from Chronic Liver Failure

Mice lacking the Abc4 protein encoded by the multidrug resistance-2 gene (Mdr2−/−) develop chronic periductular inflammation and cholestatic liver disease resulting in the development of hepatocellular carcinoma (HCC). Inhibition of NF-κB by expression of an IκBα super-repressor (IκBαSR) transgene in hepatocytes was shown to prevent HCC development in Mdr2−/− mice, suggesting that NF-κB acts as a tumour promoter in this model of inflammation-associated carcinogenesis. On the other hand, inhibition of NF-κB by hepatocyte specific ablation of IKK2 resulted in increased liver tumour development induced by the chemical carcinogen DEN. To address the role of IKK2-mediated NF-κB activation in hepatocytes in the pathogenesis of liver disease and HCC in Mdr2−/− mice, we generated Mdr2-deficient animals lacking IKK2 specifically in hepatocytes using the Cre-loxP system. Mdr2−/− mice lacking IKK2 in hepatocytes developed spontaneously a severe liver disease characterized by cholestasis, major hyperbilirubinemia and severe to end-stage fibrosis, which caused muscle wasting, loss of body weight, lethargy and early spontaneous death. Cell culture experiments showed that primary hepatocytes lacking IKK2 were more sensitive to bile acid induced death, suggesting that hepatocyte-specific IKK2 deficiency sensitized hepatocytes to the toxicity of bile acids under conditions of cholestasis resulting in greatly exacerbated liver damage. Mdr2−/−IKK2Hep-KO mice remarkably recapitulate chronic liver failure in humans and might be of special importance for the study of the mechanisms contributing to the pathogenesis of end-stage chronic liver disease or its implications on other organs. Conclusion: IKK2-mediated signaling in hepatocytes protects the liver from damage under conditions of chronic inflammatory cholestasis and prevents the development of severe fibrosis and liver failure

Characterization of distinct subpopulations of hepatic macrophages in HFD/obese mice.

The current dogma is that obesity-associated hepatic inflammation is due to increased Kupffer cell (KC) activation. However, recruited hepatic macrophages (RHMs) were recently shown to represent a sizable liver macrophage population in the context of obesity. Therefore, we assessed whether KCs and RHMs, or both, represent the major liver inflammatory cell type in obesity. We used a combination of in vivo macrophage tracking methodologies and adoptive transfer techniques in which KCs and RHMs are differentially labeled with fluorescent markers. With these approaches, the inflammatory phenotype of these distinct macrophage populations was determined under lean and obese conditions. In vivo macrophage tracking revealed an approximately sixfold higher number of RHMs in obese mice than in lean mice, whereas the number of KCs was comparable. In addition, RHMs comprised smaller size and immature, monocyte-derived cells compared with KCs. Furthermore, RHMs from obese mice were more inflamed and expressed higher levels of tumor necrosis factor-α and interleukin-6 than RHMs from lean mice. A comparison of the MCP-1/C-C chemokine receptor type 2 (CCR2) chemokine system between the two cell types showed that the ligand (MCP-1) is more highly expressed in KCs than in RHMs, whereas CCR2 expression is approximately fivefold greater in RHMs. We conclude that KCs can participate in obesity-induced inflammation by causing the recruitment of RHMs, which are distinct from KCs and are not precursors to KCs. These RHMs then enhance the severity of obesity-induced inflammation and hepatic insulin resistance

Mdr2^−/−IKK2^Hep-KO mice show impaired growth and signs of jaundice.

<p>(A) Immunoblot of whole liver lysates from 3 representative mice for each depicted genotype. Tubulin serves as loading control. (B) Weight curve of male mice for the denoted genotypes. * p<0,05 for Mdr2^+/− IKK2^Hep-KO vs. Mdr2^−/−, ** p<0,001 for Mdr2^−/− IKK2^Hep-KO vs. Mdr2^−/−. (C) Macroscopic appearance of littermate male mice at 12 weeks of age. Genotypes as specified in the picture. (D) Necropsy of littermate mice from (C). White arrowheads: enlarged gall bladder in double knockout mouse. Black arrowhead: jaundiced peritoneal serosa in double knockout mouse vs. normal appearance of serosa in Mdr2^−/− and Mdr2^+/−IKK2^Hep-KO mice. The ruler indicates centimeters in C and D.</p

Mdr2^−/−IKK2^Hep-KO mice exhibit increased liver damage, hepatocyte death and cholestasis.

<p>(A) Alanine aminotransferase (ALT), (B) alkaline phosphatase (AP), and (C) bilirubin levels in serum of adult male mice (8–26 weeks) with the indicated genotypes. Error bars indicate SEM. Number of mice: Mdr2^−/−IKK2^Hep−KO: n = 10, Mdr2^−/−: n = 7, Mdr2^+/−IKK2^Hep-KO: n = 7 and Mdr2^+/+IKK2^FL: n = 2. (D–E) Representative pictures of TUNEL (D) and PCNA staining (E) on sections of paraffin-embedded livers from mice with the indicated genotypes. The green signal in E corresponds to background auto-fluorescence and was used to visualize the general morphology of the liver tissues. Scale bars: 20 µm.</p

IKK2-deficient hepatocytes are more sensitive to bile acid induced apoptosis compared to wild type cells.

<p>(A) Representative images of DAPI stained primary WT and IKK2-KO hepatocyte cultures. Cells were left untreated or were stimulated with taurolithocholic acid 3-sulfate disodium salt (TLCS, 100 µM) for the indicated time periods. Arrowheads show apoptotic hepatocytes as indicated by smaller highly fluorescent nuclei with condensed chromatin. Scale bars, 100 µm. (B) Graphs showing the average proportion of apoptotic hepatocytes from 3 independent experiments. Mean values ± SEM shown. * p<0,05; ** p<0,01; n.s.: not statistically significant. (C) Immunoblot detection of cleaved Caspase-3. Primary hepatocytes were stimulated with glycochenodeoxy-cholate (GCDC, 50 µM, top) or TLCS (100 µM, bottom) for the indicated time periods. Tubulin serves as loading control.</p

Mdr2^−/−IKK2^Hep-KO mice develop severe fibrosis.

<p>(A) Representative images of Masson trichrome stained livers from 12-week-old littermate male mice and from 27–30 week-old male mice. (B) Representative images of Sirius Red stained livers from 12-week-old littermate male mice and from old adult male mice (27–30 weeks). Insert: High magnification of corresponding liver sections. Arrowhead: Pericellular fibers. Double arrowhead: Pericellular space free of stained fibers. Genotypes as in (A). (C) Quantification of fibrosis in mice with the depicted genotypes measured as percentage of Sirius Red positive area as a fraction of the total area of at least 10 high power fields per mouse. Left: young adult mice (males, 8–19 weeks), right: old adult mice (males, 20–42 weeks). Error bars indicate SEM. Scale bars, 100 µm.</p

Histological analysis of the liver pathology in Mdr2^−/−IKK2^Hep-KO mice.

<p>(A) Representative images of Hematoxylin & Eosin (HE) stained livers from young (12-week-old) littermate male mice and old (27–30 weeks) mice with the specified genotypes. Insert: High magnification of corresponding liver sections. (B) Immunofluorescence staining of cytokeratin 19 (CK19) in old male mice. DNA was stained with DAPI. The dashed line marks the distance from one portal field to the next, showing extreme abundance of CK19 positive cells in the Mdr2^−/− IKK2^Hep-KO as compared to Mdr2^−/− or Mdr2^+/−IKK2^Hep-KO mice. PV portal vein. CV central vein. BD bile duct. Scale bars, 100 µm.</p