Metabolic Activation of Intrahepatic CD8+ T Cells and NKT Cells Causes Nonalcoholic Steatohepatitis and Liver Cancer via Cross-Talk with Hepatocytes

SummaryHepatocellular carcinoma (HCC), the fastest rising cancer in the United States and increasing in Europe, often occurs with nonalcoholic steatohepatitis (NASH). Mechanisms underlying NASH and NASH-induced HCC are largely unknown. We developed a mouse model recapitulating key features of human metabolic syndrome, NASH, and HCC by long-term feeding of a choline-deficient high-fat diet. This induced activated intrahepatic CD8+ T cells, NKT cells, and inflammatory cytokines, similar to NASH patients. CD8+ T cells and NKT cells but not myeloid cells promote NASH and HCC through interactions with hepatocytes. NKT cells primarily cause steatosis via secreted LIGHT, while CD8+ and NKT cells cooperatively induce liver damage. Hepatocellular LTβR and canonical NF-κB signaling facilitate NASH-to-HCC transition, demonstrating that distinct molecular mechanisms determine NASH and HCC development

Multiplexed pancreatic genome engineering and cancer induction by transfection-based CRISPR/Cas9 delivery in mice

Mouse transgenesis has provided fundamental insights into pancreatic cancer, but is limited by the long duration of allele/model generation. Here we show transfection-based multiplexed delivery of CRISPR/Cas9 to the pancreas of adult mice, allowing simultaneous editing of multiple gene sets in individual cells. We use the method to induce pancreatic cancer and exploit CRISPR/Cas9 mutational signatures for phylogenetic tracking of metastatic disease. Our results demonstrate that CRISPR/Cas9-multiplexing enables key applications, such as combinatorial gene-network analysis, in vivo synthetic lethality screening and chromosome engineering. Negative-selection screening in the pancreas using multiplexed-CRISPR/Cas9 confirms the vulnerability of pancreatic cells to Brca2-inactivation in a Kras-mutant context. We also demonstrate modelling of chromosomal deletions and targeted somatic engineering of inter-chromosomal translocations, offering multifaceted opportunities to study complex structural variation, a hallmark of pancreatic cancer. The low-frequency mosaic pattern of transfection-based CRISPR/Cas9 delivery faithfully recapitulates the stochastic nature of human tumorigenesis, supporting wide applicability for biological/preclinical research

Multiplexed pancreatic genome engineering and cancer induction by transfection-based CRISPR/Cas9 delivery in mice.

Mouse transgenesis has provided fundamental insights into pancreatic cancer, but is limited by the long duration of allele/model generation. Here we show transfection-based multiplexed delivery of CRISPR/Cas9 to the pancreas of adult mice, allowing simultaneous editing of multiple gene sets in individual cells. We use the method to induce pancreatic cancer and exploit CRISPR/Cas9 mutational signatures for phylogenetic tracking of metastatic disease. Our results demonstrate that CRISPR/Cas9-multiplexing enables key applications, such as combinatorial gene-network analysis, in vivo synthetic lethality screening and chromosome engineering. Negative-selection screening in the pancreas using multiplexed-CRISPR/Cas9 confirms the vulnerability of pancreatic cells to Brca2-inactivation in a Kras-mutant context. We also demonstrate modelling of chromosomal deletions and targeted somatic engineering of inter-chromosomal translocations, offering multifaceted opportunities to study complex structural variation, a hallmark of pancreatic cancer. The low-frequency mosaic pattern of transfection-based CRISPR/Cas9 delivery faithfully recapitulates the stochastic nature of human tumorigenesis, supporting wide applicability for biological/preclinical research

Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques

Hepatitis B virus (HBV) is a major global health concern, and the development of curative therapeutics is urgently needed. Such efforts are impeded by the lack of a physiologically relevant, pre-clinical animal model of HBV infection. Here, we report that expression of the HBV entry receptor, human sodium-taurocholate cotransporting polypeptide (hNTCP), on macaque primary hepatocytes facilitates HBV infection in vitro, where all replicative intermediates including covalently closed circular DNA (cccDNA) are present. Furthermore, viral vector-mediated expression of hNTCP on hepatocytes in vivo renders rhesus macaques permissive to HBV infection. These in vivo macaque HBV infections are characterized by longitudinal HBV DNA in serum, and detection of HBV DNA, RNA, and HBV core antigen (HBcAg) in hepatocytes. Together, these results show that expressing hNTCP on macaque hepatocytes renders them susceptible to HBV infection, thereby establishing a physiologically relevant model of HBV infection to study immune clearance and test therapeutic and curative approaches

NASH limits anti-tumour surveillance in immunotherapy-treated HCC.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1-5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment

The NF-κB Subunit RelA/p65 Is Dispensable for Successful Liver Regeneration after Partial Hepatectomy in Mice

<div><h3>Background</h3><p>The transcription factor NF-κB consisting of the subunits RelA/p65 and p50 is known to be quickly activated after partial hepatectomy (PH), the functional relevance of which is still a matter of debate. Current concepts suggest that activation of NF-κB is especially critical in non-parenchymal cells to produce cytokines (TNF, IL-6) to adequately prime hepatocytes to proliferate after PH, while NF-κB within hepatocytes mainly bears cytoprotective functions.</p> <h3>Methods</h3><p>To study the role of the NF-κB pathway in different liver cell compartments, we generated conditional knockout mice in which the transactivating NF-κB subunit RelA/p65 can be inactivated specifically in hepatocytes (<em>Rela^F/FAlbCre</em>) or both in hepatocytes plus non-parenchymal cells including Kupffer cells (<em>Rela^F/FMxCre</em>). 2/3 and 80% PH were performed in controls (<em>Rela^F/F</em>) and conditional knockout mice (<em>Rela^F/FAlbCre</em> and <em>Rela^F/FMxCre</em>) and analyzed for regeneration.</p> <h3>Results</h3><p>Hepatocyte-specific deletion of RelA/p65 in <em>Rela^F/FAlbCre</em> mice resulted in an accelerated cell cycle progression without altering liver mass regeneration after 2/3 PH. Surprisingly, hepatocyte apoptosis or liver damage were not enhanced in <em>Rela^F/FAlbCre</em> mice, even when performing 80% PH. The additional inactivation of RelA/p65 in non-parenchymal cells in <em>Rela^F/FMxCre</em> mice reversed the small proliferative advantage observed after hepatocyte-specific deletion of RelA/p65 so that <em>Rela^F/FMxCre</em> mice displayed normal cell cycle progression, DNA-synthesis and liver mass regeneration.</p> <h3>Conclusion</h3><p>The NF-κB subunit RelA/p65 fulfills opposite functions in different liver cell compartments in liver regeneration after PH. However, the effects observed after conditional deletion of RelA/p65 are small and do not alter liver mass regeneration after PH. We therefore do not consider RelA/p65-containing canonical NF-κB signalling to be essential for successful liver regeneration after PH.</p> </div

Hepatocyte-specific inactivation of RelA/p65 in <i>Rela^F/FAlbCre</i> animals does not lead to enhanced liver injury after 2/3 PH.

<p>(<b>A</b>) <i>Rela^F/F</i> and <i>Rela^F/FAlbCre</i> animals were subjected to 2/3 PH and ALT serum levels were determined at the indicated time points. Data are expressed as mean ± SEM (n = 3−6 animals per time point and group). (<b>B</b>) No significant apoptosis was detected at any time point after PH in control or mutant mice as assessed by TUNEL-staining. Representative TUNEL stainings from control and mutant livers harvested 24 h post PH are shown in the upper row. Liver sections from TNF-injected (i.v. 10 ng/g BW) control and <i>Rela^F/FAlbCre</i> animals which are highly sensitive to TNF-induced apoptosis served as control (lower panel, magnification ×200).</p

Genetic deletion of WT RelA/p65 in all liver cells in <i>Rela^F/FMxCre</i> animals does not alter DNA-synthesis or liver mass regeneration after 2/3 PH.

<p>(<b>A</b>) DNA-synthesis as determined by BrdU-uptake and (<b>B</b>) liver mass regeneration was not different in control and mutant animals. (<b>C</b>) Representative TUNEL staining at 24 h and ALT levels did not reveal significant apoptosis or altered liver damage in <i>Rela^F/F</i> and <i>Rela^F/FMxCre</i> animals. Data are presented as the average ± SEM for 3–6 animals per time point per group.</p

Additional genetic deletion of RelA/p65 in all liver cells in <i>Rela^F/FMxCre</i> alters the cytokine response without significantly altering cell cycle progression after 2/3 PH.

<p>(<b>A–C</b>) 2/3 PH was performed on <i>Rela^F/F</i> and <i>Rela^F/FMxCre</i> animals and analyzed as indicated. (<b>A</b>) Levels of phospho-STAT3 in control and <i>Rela^F/FMxCre</i> animals were not different while phosphorylation of JNK was barely detected in either group as assessed be immunoblot analysis. (<b>B</b>) Induction of liver IL-6 mRNA was inhibited in <i>Rela^F/FMxCre</i> animals as determined by RT-PCR (upper image), however, IL-6 serum levels were significantly elevated in livers of <i>Rela^F/FMxCre</i> mice at 4 h post PH (lower image). (<b>C</b>) Immunoblot analysis of cell cycle associated proteins was performed as described in Fig. 2. Data from cytokine analysis and RT-PCR are presented as the average ± SEM for 3–6 animals per time point per group. *, p≤0.05 for mutant vs. control mice.</p