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

Histologic and Clinical Effects of Different Topical Corticosteroids for Eosinophilic Esophagitis: Lessons from an Updated Meta-Analysis of Placebo-Controlled Randomized Trials

Background: Topical corticosteroids (TS) have become standard therapy for eosinophilic esophagitis (EoE). However, a variety of drug formulations have been used for which results of histological and clinical responses may be different. We aimed at determining the short-term histologic efficacy of TS for EoE based on randomized placebo-controlled trials and to review clinical response. Methods: We searched MEDLINE, ISI Web of Science, and clinicaltrials.gov for randomized controlled trials (RCTs) on TS versus placebo for active EoE published until June 2019. Treatment effects were calculated as risk ratios (RRs) comparing histologic remission between groups. Results: Nine RCTs (6 budesonide and 3 fluticasone) involving a total of 483 participants were included. A substantial overall effect of TS on acute histologic remission (RR 12.5, 95% confidence interval 6.0-25.9) was found despite varying definitions of histologic response. Indirect comparisons between drug and formulation types showed a trend for a better histologic efficacy of budesonide (RR 13.5 vs. 10.4 fluticasone) and for the orodispersible tablet (RR 46.2 vs. 11.5 suspension, and 10.4 nebulized formula/spray), but only based on small patient numbers. Scores used for clinical response assessment were different between studies, and short-term clinical results were less impressive: significant differences favoring TS were found in 4/9 RCTs (4/6 budesonide, 0/3 fluticasone). Conclusions: TS are effective for short-term induction of histological remission in EoE with less impressive clinical response rates. The mode of drug delivery to the esophagus may be a relevant factor for the degree of histologic remission. Further trials should use uniform assessment criteria and long-term patient-centered outcomes

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

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

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

Opposing Role of Tumor Necrosis Factor Receptor 1 Signaling in T Cell-Mediated Hepatitis and Bacterial Infection in Mice

Death receptor (DR) ligands such as tumor necrosis factor (TNF) have been identified as fundamental mediators of liver damage both in mouse models and in humans. While the essential site of function of DR signaling is conceivably the hepatocyte, a systematic analysis is missing. Using mice with conditional gene ablation, we analyzed the tissue-specific function of DR signaling in T cell-dependent (concanavalin A) and independent (lipopolysaccharide/galactosamine) hepatitis and in models of bacterial infection (Listeria monocytogenes, lipopolysaccharide). We report that lipopolysaccharide/galactosamine-induced liver injury depends on hepatocyte-intrinsic TNF receptor 1 (p55, TNFR1). In contrast, we show that T cell-induced hepatitis was independent of TNFR1 signaling in hepatocytes, T cells, or endothelial cells. Moreover, T cell-induced hepatitis was independent of hepatocyte-intrinsic Fas-associated protein with death domain, TNF-related apoptosis-inducing ligand receptor, or Fas signaling. Instead, concanavalin A-induced hepatitis was completely prevented in mice with myeloid-derived cell (MDC)-specific deletion of TNFR1. Significantly, however, mice lacking TNFR1 in MDCs succumbed to listeria infection, although they displayed similar sensitivity toward endotoxin-induced septic shock when compared to control mice. These results suggest that TNFR1 signaling in MDCs is a critical mediator of both the detrimental and the protective functions of TNF in T cell-induced hepatitis and bacterial infection, respectively. Conclusion: The critical site of action of DRs is completely dependent on the nature of hepatitis; the data specify MDCs as the essential cell type of TNFR1 function in T cell-mediated hepatitis and in the response to listeria, thereby identifying the opposing role of MDC TNFR1 in autoimmunity and bacterial infection

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