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Over the last decades, organoids have been established from most of the tissue-resident stem and iPS cells. They hold great promise for our understanding of mammalian organ development, but also for the study of disease or even personalised medicine. In recent years, several reports hinted at intraculture organoid variability, but a systematic analysis of such heterogeneity has not been performed before. Here, we used RNA-seq of individual intrahepatic cholangiocyte organoids to address this question. We find that batch-to-batch variation is very low, whereas passage number has a profound impact on gene expression profiles. On the other hand, there is organoid-toorganoid variability within a culture. Using differential gene expression, we did not identify specific pathways that drive this variability, pointing towards possible effects of the microenvironment within the culture condition. Taken together, our study provides a framework for organoid researchers to properly consider experimental design.

SQSTM1/p62 is a multitasking protein that functions as an autophagy receptor, but also as a signaling hub regulating diverse cellular pathways. p62 accumulation in mice with autophagydeficient hepatocytes mediates liver damage and hepatocarcinogenesis through Nrf2 overactivation, yet the role of the p62-Keapl-Nrf2 axis in cell death and hepatocarcinogenesis in the absence of underlying autophagy defects is less clear. Here, we addressed the role of p62 and Nrf2 activation in a chronic liver disease model, namely mice with liver parenchymal cell-specific knockout of NEMO (NEMOLPC-KO), in which we demonstrate that they show no inherent autophagy impairment. Unexpectedly, systemic p62 ablation aggravated the phenotype and caused early postnatal lethality in NEMO(LPC-KO )mice. Expression of a p62 mutant (p624Ex2-5), which retains the ability to form aggregates and activate Nrf2 signaling, did not cause early lethality, but exacerbated hepatocarcinogenesis in these mice. Our immunohistological and molecular analyses showed that the increased tumor burden was only consistent with increased expression/stability of p624Ex2-5 driving Nrf2 hyperactivation, but not with other protumorigenic functions of p62, such as mTOR activation, cMYC upregulation or increased fibrosis. Surprisingly, forced activation of Nrf2 per se did not increase liver injury or tumor burden in NEMOLPC-KO mice, suggesting that autophagy impairment is a necessary prerequisite to unleash the Nrf2 oncogenic potential in mice with autophagy-competent hepatocytes.

There is a medical need of biomarkers for disease stratification in cholestatic liver diseases that come along with changes in hepatocyte polarity. Neighbor of Punc E11 (Nope) is an oncofetal marker that is lost after final differentiation and polarization of hepatocytes. We analyzed the expression pattern of Nope and connexin (Cx) 26 as markers of hepatocyte polarization during murine liver development as well as in adult liver with or without bile duct ligation (BDL) by quantitative real-time reverse transcription polymerise chain reaction (qRT-PCR), western blotting (WB), and immunohistochemistry. Nope is highly expressed in fetal and postnatal liver but barely detectable thereafter. Cx26, however, is much higher expressed in adult than in fetal liver. Postnatally, Nope is directed to the sinusoidal membrane of early hepatocytes while Cx26 remains distributed over the whole membrane indicating limited polarization. In the adult liver, only Cx26 is detectable and restricted to the bile canalicular domain indicating fully polarized hepatocytes. After BDL, Nope is again >300-fold upregulated while Cx26 is reduced rapidly. By immunohistochemistry, Nope identifies a subset of hepatocytes with randomly distributed Cx26. In summary, Nope identifies depolarized adult hepatocytes after cholestatic liver injury resembling early postnatal hepatocytes. Therefore, Nope might be a valuable histochemical biomarker allowing stage-specific stratifications in cholestatic liver diseases.

Mitochondria are essential organelles that host crucial metabolic pathways and produce adenosine triphosphate. The mitochondrial proteome is heterogeneous among tissues and can dynamically change in response to different metabolic conditions. Although the transcriptional programs that govern mitochondrial biogenesis and respiratory function are well known, posttranscriptional regulatory mechanisms remain unclear. In this study, we show that the cytosolic RNA-binding protein clustered mitochondria homologue (CLUH) regulates the expression of a mitochondrial protein network supporting key metabolic programs required under nutrient deprivation. CLUH exerts its function by controlling the stability and translation of target messenger RNAs. In the absence of Cluh, mitochondria are severely depleted of crucial enzymes involved in catabolic energy-converting pathways. CLUH preserves oxidative mitochondrial function and glucose homeostasis, thus preventing death at the fetal-neonatal transition. In the adult liver, CLUH ensures maximal respiration capacity and the metabolic response to starvation. Our results shed new light on the posttranscriptional mechanisms controlling the expression of mitochondrial proteins and suggest novel strategies to tailor mitochondrial function to physiological and pathological conditions.

Regulated cell death pathways have important functions in host defense and tissue homeostasis. Studies in genetic mouse models provided evidence that cell death could cause inflammation in different tissues. Inhibition of RIPK3-MLKL-dependent necroptosis by FADD and caspase-8 was identified as a key mechanism preventing inflammation in epithelial barriers. Moreover, the interplay between IKK/NF-kappa B and RIPK1 signaling was recognized as a critical determinant of tissue homeostasis and inflammation. NEMO was shown to regulate RIPK1 kinase activity-mediated apoptosis by NF-kappa B-dependent and -independent functions, which are critical for averting chronic tissue injury and inflammation in the intestine and the liver. In addition, RIPK1 was shown to exhibit kinase activity-independent functions that are essential for preventing cell death, maintaining tissue architecture and inhibiting inflammation. In the intestine, RIPK1 acts as a scaffold to prevent epithelial cell apoptosis and preserve tissue integrity. In the skin, RIPK1 functions via its RHIM to counteract ZBP1/DAI-dependent activation of RIPK3-MLKL-dependent necroptosis and inflammation. Collectively, these studies provided evidence that the regulation of cell death signaling plays an important role in the maintenance of tissue homeostasis, and suggested that cell death could be causally involved in the pathogenesis of inflammatory diseases.

Intestinal epithelial cells (IECs) regulate gut immune homeostasis, and impaired epithelial responses are implicated in the pathogenesis of inflammatory bowel diseases (IBD). IEC-specific ablation of nuclear factor kappa B (NF-kappa B) essential modulator (NEMO) caused Paneth cell apoptosis and impaired antimicrobial factor expression in the ileum, as well as colonocyte apoptosis and microbiota-driven chronic inflammation in the colon. Combined RelA, c-Rel, and RelB deficiency in IECs caused Paneth cell apoptosis but not colitis, suggesting that NEMO prevents colon inflammation by NF-kappa B-independent functions. Inhibition of receptor-interacting protein kinase 1 (RIPK1) kinase activity or combined deficiency of Fas-associated via death domain protein (FADD) and RIPK3 prevented epithelial cell death, Paneth cell loss, and colitis development in mice with epithelial NEMO deficiency. Therefore, NEMO prevents intestinal inflammation by inhibiting RIPK1 kinase activity-mediated IEC death, suggesting that RIPK1 inhibitors could be effective in the treatment of colitis in patients with NEMO mutations and possibly in IBD.

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.

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-kappa B by expression of an I kappa B alpha super-repressor (I kappa B alpha SR) transgene in hepatocytes was shown to prevent HCC development in Mdr2(-/-) mice, suggesting that NF-kappa B acts as a tumour promoter in this model of inflammation-associated carcinogenesis. On the other hand, inhibition of NF-kappa B 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-kappa B 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(-/-) IKK2(Hep-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.