Project description:BACKGROUND & AIMS: Recent studies revealed that hemoglobin is expressed in some non-erythrocytes and it suppresses oxidative stress when overexpressed. Oxidative stress plays a critical role in the pathogenesis of non-alcoholic steatohepatitis (NASH). This study was to investigate whether hemoglobin is expressed in hepatocytes and how it is related to oxidative stress in NASH patients. METHODS: Microarray was performed to identify differentially expressed genes in NASH. Quantitative real time PCR (qRT-PCR) was used to examine gene expression levels. Western blotting and immunofluorescence staining were employed to examine hemoglobin proteins. Flow cytometry was used to measure intracellular oxidative stress. RESULTS: Analysis of microarray gene expression data has revealed a significant increase in the expression of hemoglobin alpha (HBA1) and beta (HBB) in liver biopspies from NASH patients. Increased hemoglobin expression in NASH was validated by qRT-PCR. However, the expression of erythrocyte specific marker genes such as SPTA, SPTB, GYPA, GATA1, and ALAS2 did not change, indicating that increased hemoglobin expression in NASH was not from erythropoiesis, but could result from increased expression in hepatocytes. Immunofluorescence staining demonstrated positive HBA1 and HBB expression in the hepatocytes of NASH livers. Hemoglobin expression was also observed in human hepatocellular carcinoma HepG2 cell line. Furthermore, treatment with hydrogen peroxide, a known oxidative stress inducer, induced a dose dependent increase in HBA1 expression in HepG2 cells. Intriguingly, forced hemoglobin expression suppressed oxidative stress. CONCLUSIONS: Oxidative stress upregulates hemoglobin expression in hepatocytes. Suppression of oxidative stress by hemoglobin could be a mechanism to protect hepatocytes from oxidative damage. These findings suggest that hemoglobin is an inducible antioxidant in hepatocytes in response to increased oxidative stress as found in NASH livers. Twelve biopsy diagnosed NASH patients were included in this study. For control groups, total RNA from 5 different subjects were purchased from ADMET. These subjects are free from liver disease.

Project description:Hepatocellular carcinoma (HCC) has dismal treatment responses to systemic therapies and is caused by both, viral and non-viral etiologies, including non-alcoholic steatohepatitis (NASH) 1–5. NASH - triggered by high caloric intake and sedentary lifestyle - has become an important driver for HCC development. Immunotherapy has been recently approved for HCC but stratification of responders versus non-responders has remained an unmet need 5–8. Here, we found a progressive accumulation of non-classical activated CD8+PD-1+ T-cells in livers of NASH-affected patients and mice. PD-L1/PD-1-targeted immunotherapy of NASH-induced HCC administered either at cancer-initiation or after cancer-establishment lacked beneficial effects in mice. On the contrary, PD-1-targeted immunotherapy drove hepatic necro-inflammation and increased liver cancer incidence, tumor number, and nodule size. Anti-CD8 or anti-CD8/anti-NK1.1 treatment suppressed liver cancer development, implicating CD8+ T-cells as liver cancer drivers in the context of NASH. In line, PD-1-/- mice challenged with a NASH-inducing diet displayed early-onset of liver cancer. Mechanistically, PD-1-targeted immunotherapy triggered necro-inflammation and a pro-tumorigenic environment in the context of NASH, increasing the abundance of hepatic TOX+CXCR6+ expressing CD8+PD-1+ TNF+ T-cells. Anti-CD8/anti-PD-1 or anti-TNF/anti-PD-1, but not anti-CD4/anti-PD-1 treatment reverted anti-PD1 treatment-related increase of liver inflammation, NASH severity and tumorigenesis. Gene expression profile and increased abundance of murine hepatic CD8+PD-1+ T-cells were corroborated in human CD8+PD-1+ T cells derived from NASH patients. In a meta-analysis of clinical trials testing PD-1-targeting immune checkpoint inhibitors alone (i.e. pembrolizumab) or in combination in 1656 patients with advanced HCC, immunotherapy was not favorable over to control treatment in non-viral- when compared to viral-related HCC. Similarly, in two clinical cohorts tested, patients with NASH-driven HCC had a significantly worse overall survival with PD-1-targeted immunotherapy than HCC patients with other etiologies. Our data identify non-viral etiologies, particularly NASH, as potentially non-responsive in the context of HCC immunotherapy, providing a strong rational basis for patient stratification.

Project description:Immunotherapy has opened hitherto unknown possibilities to treat cancer. Whereas some cancer types (e.g. melanoma) can be efficiently treated, others lack measurable positive effects (e.g. PDAC). Moreover, stratification of responders/non-responders is only possible in some cancer types (e.g. melanoma). Hepatocellular carcinoma (HCC) has a dismal prognosis, limited treatment options and survival benefit, and represents a potential cancer entity for successful immunotherapy. Here, we investigated NASH-triggered HCC in the context PD-1-targeted immunotherapy. Using flow cytometry, single cell RNA sequencing, immunohistochemistry and mass spectrometric analyses, we found a progressive increase of CD8+PD-1+ effector T-cells with a unique profile of exhaustion and activation markers rising with murine and human NASH severity. Notably, late-stage HCC treatment with PD-1-targeted immunotherapy enhanced hepatic carcinogenesis in mice. Dissecting potential mechanisms of action during tumor-initiation and -progression we analyzed the effects of PD-1-targeted immunotherapy at HCC initiation. PD-1-targeted immunotherapy induced a pro-tumorigenic environment, enhanced necro-inflammation and increased NAFLD-activation score (NAS), leading to increased liver cancer incidence, tumor number and nodule size. In contrast, anti-CD8 or anti-CD8/anti-NK1.1 treatment reduced NAS and abrogated the development of liver cancer, thus identifying CD8+PD-1+ T-cells as drivers of liver cancer in NASH-triggered HCC. Increased apoptotic signaling, STAT3 phosphorylation and hepatic proliferation were detected in intra-tumoral liver tissue upon PD-1-targeted immunotherapy. In line, PD-1-/- mice challenged with a NASH diet displayed early onset of hepatocarcinogenesis, corroborating the pro-tumorigenic role of absent or reduced PD-1. Mechanistically PD-1-targeted immunotherapy mainly affected hepatic abundance of CD8+PD-1+ T-cells, instead of altering the quality of Tox+CXCR6+ expressing CD8+PD-1+TNF+CD39+Gzmb+ T-cells found in NASH livers, leading to an aggressive, pro-tumorigenic liver environment. Single-cell mapping of human NASH-, borderline NASH- or unaffected livers corroborated our preclinical NASH results. Moreover, in human NASH livers a correlation of hepatic CD8+, PD-1+, TNF+ T-cells with fibrosis and NASH severity was observed. Accordingly, HCC patients with NASH etiology display a sharp increase in intra- and peri-tumoral CD8+ PD-1+ T-cells. In a cohort of 65 patients recruited across 6 centers in Germany and Austria, patients with NAFLD/NASH-driven HCC responded worse to PD-1-targeted immunotherapy by Nivolumab or Pembrolizumab compared to non-NAFLD patients. This resulted in significant reduced overall survival, in trends of faster disease progression and reduced progression free survival. Histological analysis of livers derived from HCC patients treated with PD-1-targeted immunotherapy displayed high levels of intra and peri-tumoral CD8+ PD-1+ T-cells and Ki67+ hepatocytes. Taken together, these data indicate that PD-1-targeted immunotherapy induces immune-related adverse effects in NAFLD/NASH-driven HCC through CD8+PD-1+ T-cells. Our data call for stratification of HCC patients subjected to PD-1-targeted immunotherapy, with NAFLD being a negative predictor.

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RNA Sequencing Facilitates Quantitative Analysis of Humanized Normal Livers, NASH Livers, and HCC Transcriptomes

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