Clearance Of Hepatocytes Research Articles

An unexpected role of neutrophils in clearing apoptotic hepatocytes in vivo.

Billions of apoptotic cells are removed daily in a human adult by professional phagocytes (e.g. macrophages) and neighboring nonprofessional phagocytes (e.g. stromal cells). Despite being a type of professional phagocyte, neutrophils are thought to be excluded from apoptotic sites to avoid tissue inflammation. Here, we report a fundamental and unexpected role of neutrophils as the predominant phagocyte responsible for the clearance of apoptotic hepatic cells in the steady state. In contrast to the engulfment of dead cells by macrophages, neutrophils burrowed directly into apoptotic hepatocytes, a process we term perforocytosis, and ingested the effete cells from the inside. The depletion of neutrophils caused defective removal of apoptotic bodies, induced tissue injury in the mouse liver, and led to the generation of autoantibodies. Human autoimmune liver disease showed similar defects in the neutrophil-mediated clearance of apoptotic hepatic cells. Hence, neutrophils possess a specialized immunologically silent mechanism for the clearance of apoptotic hepatocytes through perforocytosis, and defects in this key housekeeping function of neutrophils contribute to the genesis of autoimmune liver disease.

An unexpected role of neutrophils in clearing apoptotic hepatocytes in vivo

Billions of apoptotic cells are removed daily in a human adult by professional phagocytes (e.g. macrophages) and neighboring nonprofessional phagocytes (e.g. stromal cells). Despite being a type of professional phagocyte, neutrophils are thought to be excluded from apoptotic sites to avoid tissue inflammation. Here, we report a fundamental and unexpected role of neutrophils as the predominant phagocyte responsible for the clearance of apoptotic hepatic cells in the steady state. In contrast to the engulfment of dead cells by macrophages, neutrophils burrowed directly into apoptotic hepatocytes, a process we term perforocytosis, and ingested the effete cells from the inside. The depletion of neutrophils caused defective removal of apoptotic bodies, induced tissue injury in the mouse liver, and led to the generation of autoantibodies. Human autoimmune liver disease showed similar defects in the neutrophil-mediated clearance of apoptotic hepatic cells. Hence, neutrophils possess a specialized immunologically silent mechanism for the clearance of apoptotic hepatocytes through perforocytosis, and defects in this key housekeeping function of neutrophils contribute to the genesis of autoimmune liver disease.

Alternating Arenavirus Vector Immunization Generates Robust Polyfunctional Genotype Cross-Reactive Hepatitis B Virus-Specific CD8 T-Cell Responses and High Anti-Hepatitis B Surface Antigen Titers.

Hepatitis B Virus (HBV) is a major driver of infectious disease mortality. Curative therapies are needed and ideally should induce CD8 T cell-mediated clearance of infected hepatocytes plus anti-hepatitis B surface antigen (HBsAg) antibodies (anti-HBs) to neutralize residual virus. We developed a novel therapeutic vaccine using non-replicating arenavirus vectors. Antigens were screened for genotype conservation and magnitude and genotype reactivity of T cell response, then cloned into Pichinde virus (PICV) vectors (recombinant PICV, GS-2829) and lymphocytic choriomeningitis virus (LCMV) vectors (replication-incompetent, GS-6779). Alternating immunizations with GS-2829 and GS-6779 induced high-magnitude HBV T cell responses, and high anti-HBs titers. Dose schedule optimization in macaques achieved strong polyfunctional CD8 T cell responses against core, HBsAg, and polymerase and high titer anti-HBs. In AAV-HBV mice, GS-2829 and GS-6779 were efficacious in animals with low pre-treatment serum HBsAg. Based on these results, GS-2829 and GS-6779 could become a central component of cure regimens.

Ethanol attenuates presentation of cytotoxic T-lymphocyte epitopes on hepatocytes of HBV-infected humanized mice.

Approximately 3.5% of the global population is chronically infected with Hepatitis B Virus (HBV), which puts them at high risk of end-stage liver disease, with the risk of persistent infection potentiated by alcohol consumption. However, the mechanisms underlying the effects of alcohol on HBV persistence remain unclear. Here, we aimed to establish in vivo/ex vivo evidence that alcohol suppresses HBV peptides-major histocompatibility complex (MHC) class I antigen display on primary human hepatocytes (PHH), which diminishes the recognition and clearance of HBV-infected hepatocytes by cytotoxic T-lymphocytes (CTLs). We used fumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chain knock-out (FRG-KO) humanized mice transplanted with human leukocyte antigen-A2 (HLA-A2)-positive hepatocytes. The mice were HBV-infected and fed control and alcohol diets. Isolated hepatocytes were exposed ex vivo to HBV 18-27-HLA-A2-restricted CTLs to quantify cytotoxicity. For mechanistic studies, we measured proteasome activities, unfolded protein response (UPR), and endoplasmic reticulum (ER) stress in hepatocytes from HBV-infected humanized mouse livers. We found that alcohol feeding attenuated HBV core 18-27-HLA-A2 complex presentation on infected hepatocytes due to the suppression of proteasome function and ER stress induction, which diminished both the processing of HBV peptides and trafficking of HBV-MHC class I complexes to the hepatocyte surface. This alcohol-mediated decrease in MHC class I-restricted antigen presentation of the CTL epitope on target hepatocytes reduced the CTL-specific elimination of infected cells, potentially leading to HBV-infection persistence, which promotes end-stage liver disease outcomes.

Cellular senescence and hepatitis B-related hepatocellular carcinoma: An intriguing link.

Chronic hepatitis B is mainly responsible for the morbidity and mortality from hepatitis B virus (HBV)-related complications, including hepatocellular carcinoma (HCC) and decompensated cirrhosis. Hepatocellular carcinoma remains the main challenge in the management of not only undiagnosed and/or untreated but also diagnosed and treated patients with chronic HBV infection, as its incidence decreases but is not eliminated even after many years of effective anti-HBV therapy. The exact mechanisms used by HBV to cause malignant transformation remain uncertain, although much of the available data are in favour of a pathogenetic role of HBx protein. Senescence is a cellular state, in which cells lose their ability to proliferate. This biological mechanism may function in a dual mode, namely being both cancer-protective as a result of reduced cellular proliferation, but also cancer-enhancing as a result of modulation of the tissular microenvironment by immune cells during persistent accumulation of senescent cells. Protein X of HBV protein exhibits many similarities in terms of the implemented mechanisms of action and pathways related to the biological process of cellular senescence. Concurrently, insufficient clearance of both senescent and precancerous hepatocytes combined with inadequate immune surveillance as a result of immunosenescence caused by chronic HBV infection may lead to hepatocarcinogenesis. Thus, the effect of HBV seems to be critical as a connecting link between cellular senescence and development of HCC. An ongoing research is underway towards identifying and validating markers of hepatocyte senescence, which could improve the landscape for evaluation of chronic liver disease, thereby providing valuable information in terms of HBV-related carcinogenesis.

Enhanced T Cell Responses Induced by a Necrotic Dendritic Cell Vaccine, Expressing HCV NS3.

A vaccine that induces potent, broad and sustained cell-mediated immunity, resulting in effective memory has the potential to restrict hepatitis C (HCV) virus infection. Early, multi-functional CD4+ and CD8+ T cell responses against non-structural protein 3 (NS3) have been associated with HCV clearance. Necrotic cells generate strong immune responses and represent a major antigenic source used by dendritic cells (DC) for processing and presentation, but there is conflicting evidence as to their immunogenicity in vaccination. Immunization with DC loaded with viral antigens has been done in the past, but to date the immunogenicity of live vs. necrotic DC vaccines has not been investigated. We developed a DC2.4 cell line stably expressing HCV NS3, and compared the NS3-specific responses of live vs. necrotic NS3 DC. Vaccination of mice with necrotic NS3 DC increased the breadth of T-cell responses and enhanced the production of IL-2, TNF-α, and IFN-γ by effector memory CD4+ and CD8+T cells, compared to mice vaccinated with live NS3 DC. A single dose of necrotic NS3 DC vaccine induced a greater influx and activation of cross-presenting CD11c+ CD8α+ DC and necrosis-sensing Clec9A+ DC in the draining lymph nodes. Furthermore, using a hydrodynamic challenge model necrotic NS3 DC vaccination resulted in enhanced clearance of NS3-positive hepatocytes from the livers of vaccinated mice. Taken together, the data demonstrate that necrotic DC represent a novel and exciting vaccination strategy capable of inducing broad and multifunctional T cell memory.

Cell competition in liver carcinogenesis.

Cell competition is now a well-established quality control strategy to optimize cell and tissue fitness in multicellular organisms. While pursuing this goal, it is also effective in selecting against altered/defective cells with putative (pre)-neoplastic potential, thereby edging the risk of cancer development. The flip side of the coin is that the molecular machinery driving cell competition can also be co-opted by neoplastic cell populations to expand unchecked, outside the boundaries of tissue homeostatic control. This review will focus on information that begins to emerge regarding the role of cell competition in liver physiology and pathology. Liver repopulation by normal transplanted hepatocytes is an interesting field of investigation in this regard. The biological coordinates of this process share many features suggesting that cell competition is a driving force for the clearance of endogenous damaged hepatocytes by normal donor-derived cells, as previously proposed. Intriguing analogies between liver repopulation and carcinogenesis will be briefly discussed and the potential dual role of cell competition, as a barrier or a spur to neoplastic development, will be considered. Cell competition is in essence a cooperative strategy organized at tissue level. One facet of such cooperative attitude is expressed in the elimination of altered cells which may represent a threat to the organismal community. On the other hand, the society of cells can be disrupted by the emergence of selfish clones, exploiting the molecular bar codes of cell competition, thereby paving their way to uncontrolled growth.

From the Editor’s Desk…: October 2019

From the Editor’s Desk…: October 2019

Outcome of Antiviral Immunity in the Liver Is Shaped by the Level of Antigen Expressed in Infected Hepatocytes.

The liver bears unique immune properties that support both immune tolerance and immunity, but the mechanisms responsible for clearance versus persistence of virus‐infected hepatocytes remain unclear. Here, we dissect the factors determining the outcome of antiviral immunity using recombinant adenoviruses that reflect the hepatropism and hepatrophism of hepatitis viruses. We generated replication‐deficient adenoviruses with equimolar expression of ovalbumin, luciferase, and green fluorescent protein driven by a strong ubiquitous cytomegalovirus (CMV) promoter (Ad‐CMV‐GOL) or by 100‐fold weaker, yet hepatocyte‐specific, transthyretin (TTR) promoter (Ad‐TTR‐GOL). Using in vivo bioluminescence to quantitatively and dynamically image luciferase activity, we demonstrated that Ad‐TTR‐GOL infection always persists, whereas Ad‐CMV‐GOL infection is always cleared, independent of the number of infected hepatocytes. Failure to clear Ad‐TTR‐GOL infection involved mechanisms acting during initiation as well as execution of antigen‐specific immunity. First, hepatocyte‐restricted antigen expression led to delayed and curtailed T‐cell expansion—10,000‐fold after Ad‐CMV‐GOL versus 150‐fold after Ad‐TTR‐GOL‐infection. Second, CD8 T‐cells primed toward antigens selectively expressed by hepatocytes showed high PD‐1/Tim‐3/LAG‐3/CTLA‐4/CD160 expression levels similar to that seen in chronic hepatitis B. Third, Ad‐TTR‐GOL but not Ad‐CMV‐GOL‐infected hepatocytes escaped being killed by effector T‐cells while still inducing high PD‐1/Tim‐3/LAG‐3/CTLA‐4/CD160 expression, indicating different thresholds of T‐cell receptor signaling relevant for triggering effector functions compared with exhaustion. Conclusion: Our study identifies deficits in the generation of CD8 T‐cell immunity toward hepatocyte‐expressed antigens and escape of infected hepatocytes expressing low viral antigen levels from effector T‐cell killing as independent factors promoting viral persistence. This highlights the importance of addressing both the restauration of CD8 T‐cell dysfunction and overcoming local hurdles of effector T‐cell function to eliminate virus‐infected hepatocytes.

Chimeric liver transplantation reveals interspecific graft remodelling

A major limitation in the field of liver transplantation is the shortage of transplantable organs. Chimeric animals carrying human tissue have the potential to solve this problem. However, currently available chimeric organs retain a high level of xenogeneic cells, and the transplantation of impure organs needs to be tested. We created chimeric livers by injecting Lewis rat hepatocytes into C57Bl/6Fah-/-Rag2-/-Il2rg-/- mice, and further transplanted them into newly weaned Lewis rats (45 ± 3 g) with or without suboptimal immunosuppression (tacrolimus 0.6 mg/kg/day for 56 or 112 days). Control donors included wild-type C57Bl/6 mice (xenogeneic) and Lewis rats (syngeneic). Without immunosuppression, recipients of chimeric livers experienced acute rejection, and died within 8 to 11 days. With immunosuppression, they all survived for >112 days with normal weight gain compared to syngeneic controls, while all xenogeneic controls died within 98 days due to rejection with Banff scores >6 (p = 0.0014). The chimeric grafts underwent post-transplant remodelling, growing by 670% on average. Rat hepatocytes fully replaced mouse hepatocytes starting from day 56 (absence of detectable mouse serum albumin, histological clearance of mouse hepatocytes). In addition, rat albumin levels reached those of syngeneic recipients. Four months after transplantation of chimeric livers, we observed the development of diffuse mature rat bile ducts through transdifferentiation of hepatocytes (up to 72% of cholangiocytes), and patchy areas of portal endothelium originating from the host (seen in one out of five recipients). Taken together, these data demonstrate the efficacy of transplanting rat-to-mouse chimeric livers into rats, with a high potential for post-transplant recipient-oriented graft remodelling. Validation in a large animal model is still needed. Chimeric animals are composed of cells from different species. Chimeric animals carrying human tissue have the potential to increase the availability of transplantable organs. We transplanted rat-to-mouse liver grafts into newly weaned rats. The chimeric grafts underwent post-transplant remodelling with rat hepatocytes replacing all mouse hepatocytes within 56 days. In addition, we observed the post-transplant development of diffuse mature rat bile ducts through the transformation of hepatocytes, and patchy areas of portal endothelium originating from the host. These data demonstrate the efficacy of transplanting rat-to-mouse chimeric livers into rats, with a high potential for post-transplant graft remodelling.

Diet induced obesity has an influence on intrahepatic T cell responses

AimObesity is accompanied with systemic inflammation and pre-conditions to severe alterations in liver environment and functions. So far, it remains elusive to which extent obesity modulates immune responses during hepatotropic virus infections as well as in autoimmune hepatitis. In this study we investigated the influence of obesity on the intrahepatic immune response, in particular on the function of CD8 T cells as the crucial players in clearance of virus infected hepatocytes. MethodsWe established high fat induced obesity in transgenic mouse models with hepatocyte specific expression of a model antigen (Ova). We investigated the immune response upon adoptive transfer of antigen specific T cells and in mice with continuous thymic output of antigen specific T cells, mimicking the situations upon acute infection and autoimmunity, respectively. ResultsIrrespective of the metabolic condition, adoptive T cell transfer resulted in a transient hepatitis with no obvious differences concerning the acute T cell response. In the situation of autoimmunity, we observed a transient hepatitis in lean mice, whereas an extended hepatitis with a reduced antigen clearance capacity was found in obese mice. ConclusionOur results demonstrate that obesity affects T cell function and increases the severity of autoimmune hepatitis while it has no impact on the acute T cell response.

GS-014 - CXCR6-dependent hepatic iNKT cells limit hepatocarcinogenesis in vivo by promoting clearance of senescent hepatocytes

GS-014 - CXCR6-dependent hepatic iNKT cells limit hepatocarcinogenesis in vivo by promoting clearance of senescent hepatocytes

Depletion of myeloid cells exacerbates hepatitis and induces an aberrant increase in histone H3 in mouse serum.

These findings indicate an unexpected role of myeloid cells in decreasing serum IL-6, TNFα, and histone H3 levels via the suppression of TNFα-induced hepatocyte apoptosis. (Hepatology 2017;65:237-252).

PTU-085 A NOTCH1-Driven Secretome Switch Modulates Immune Surveillance In Ras-Induced Senescence

Introduction Oncogene-induced senescence (OIS) is an intrinsic tumour suppressor mechanism, but its impact on tumorigenesis is largely dependent on the nature of the senescence-associated secretory phenotype (SASP). Major components of the SASP include TGFβ1 and pro-inflammatory cytokines, such as IL1, IL6, and IL8, that have pleiotropic context-dependent effects. Methods We utilised the well validated ER:Ras G12V IMR90 HDF in vitro model which undergo Ras-induced senescence (RIS) with 4 OHT. Genetic manipulation was achieved through retroviral gene transfer; transcriptional profiling by mRNA-seq; validation through qPCR and immunoblotting. In vivo hepatocyte senescence was achieved through hydrodynamic tail-vein delivery of NRAS G12V -containing transposons. Results We have demonstrated that NOTCH1, a highly conserved receptor is up-regulated in RIS. In contrast to the up-regulation of NOTCH1, downstream signalling is dynamically regulated: the cleaved, active intracellular domain of NOTCH1 (N1ICD) and NOTCH-target genes were transiently up-regulated at an early phase of RIS, but down-regulated at full senescence. The dynamic expression patterns of N1ICD and TGF-β1 expression were nearly identical, and inversely correlated with the cytokines, IL1 and IL8. Inhibition of NOTCH1 signalling, through expression of a dominant-negative form of the NOTCH1 binding partner MAML1, led to a reduction in TGF-β1, but increased IL1 and IL8 expression during RIS. In addition, ectopic restoration of N1ICD in established RIS cells drove reciprocal secretome changes with reduced IL1 and IL8 and increased TGF-β1, suggesting NOTCH1 signalling plays a critical role in secretome switching. Utilising an NRAS-drive hepatocyte senescence model, NOTCH1 was upregulated during RIS in vivo. Previously it has been shown that the SASP in RIS is critical for the immune-mediated clearance of senescent hepatocytes. Consistent with the enhancement of the pro-inflammatory secretome, co-delivery of the NOTCH inhibitor dnMAML1 with RAS, accelerated the infiltration of CD3+ T-cells into the liver and promoted the clearance of senescent hepatocytes. Restoration of N1ICD in hepatocyte RIS led to senescence bypass and tumour formation. Conclusion The transition to RIS is correlated with a NOTCH1-mediated switch from a TGFβ-rich secretome to a proinflammatory secretome. Promotion of this proinflammatory secretome, through NOTCH inhibition enhances immune surveillance of senescent hepatocytes. This raises the possibility of therapeutically promoting the clearance of senescent cells in vivo. Disclosure of Interest None Declared

Diminished Innate Antiviral Response to Adenovirus Vectors in cGAS/STING-Deficient Mice Minimally Impacts Adaptive Immunity.

Infection by adenovirus, a nonenveloped DNA virus, induces antiviral innate and adaptive immune responses. Studies of transformed human and murine cell lines using short hairpin RNA (shRNA) knockdown strategies identified cyclic guanine adenine synthase (cGAS) as a pattern recognition receptor (PRR) that contributes to the antiadenovirus response. Here we demonstrate how the cGAS/STING cascade influences the antiviral innate and adaptive immune responses in a murine knockout model. Using knockout bone marrow-derived dendritic cells (BMDCs) and bone marrow-derived macrophages (BMMOs), we determined that cGAS and STING are essential to the induction of the antiadenovirus response in these antigen-presenting cells (APCs) in vitro We next determined how the cGAS/STING cascade impacts the antiviral response following systemic administration of a recombinant adenovirus type 5 vector (rAd5V). Infection of cGAS(-/-) and STING(-/-) mice results in a compromised early antiviral innate response compared to that in wild-type (WT) controls: significantly lower levels of beta interferon (IFN-β) secretion, low levels of proinflammatory chemokine induction, and reduced levels of antiviral transcript induction in hepatic tissue. At 24 h postinfection, levels of viral DNA and reporter gene expression in the liver were similar in all strains. At 28 days postinfection, clearance of infected hepatocytes in cGAS or STING knockout mice was comparable to that in WT C57BL/6 mice. Levels of neutralizing anti-Ad5V antibody were modestly reduced in infected cGAS mice. These data support a dominant role for the cGAS/STING cascade in the early innate antiviral inflammatory response to adenovirus vectors. However, loss of the cGAS/STING pathway did not affect viral clearance, and cGAS deficiency had a modest influence on the magnitude of the antiviral humoral immune response to adenovirus infections. The detection of viral infection by host sentinel immune cells contributes to the activation of a complex and varied antiviral innate and adaptive immune response, which limits virus replication, spread, and susceptibility to infection. In this study, we have characterized how the cGAS/STING DNA-sensing cascade contributes to early detection of adenovirus infections. cGAS influences APC activation and early innate antiviral inflammatory immune responses, but adaptive immune pathways associated with virus clearance and anti-Ad antibody production were minimally influenced by the loss of the cGAS PRR signaling cascade.

Virus evolution during chronic hepatitis B virus infection as revealed by ultradeep sequencing data.

Despite chronic hepatitis B virus (HBV) infection (CHB) being a leading cause of liver cirrhosis and cancer, HBV evolution during CHB is not fully understood. Recent studies have indicated that virus diversity progressively increases along the course of CHB and that some virus mutations correlate with severe liver conditions such as chronic hepatitis, cirrhosis and hepatocellular carcinoma. Using ultradeep sequencing (UDS) data from an intrafamilial case, we detected such mutations at low frequencies among three immunotolerant patients and at high frequencies in an inactive carrier. Furthermore, our analyses indicated that the HBV population from the seroconverter patient underwent many genetic changes in response to virus clearance. Together, these data indicate a potential use of UDS for developing non-invasive biomarkers for monitoring disease changes over time or in response to specific therapies. In addition, our analyses revealed that virus clearance seemed not to require the virus effective population size to decline. A detailed genetic analysis of the viral lineages arising during and after the clearance suggested that mutations at or close to critical elements of the core promoter (enhancer II, epsilon encapsidation signal, TA2, TA3 and direct repeat 1-hormone response element) might be responsible for a sustained replication. This hypothesis requires the decline in virus load to be explained by constant clearance of virus-producing hepatocytes, consistent with the sustained progress towards serious liver conditions experienced by many CHB patients.

Abstract 4456: Prevention of MICA shedding from HBV infected hepatocytes to activate NK cells for better clearance of malignant foci and virus-infected cells

Abstract Introduction: Hepatocellular carcinoma (HCC) is an important issue to public health of the world. By a genome-wide association study, we previously identified that a single nucleotide polymorphism (SNP) in the promoter region of the MHC class I polypeptide-related sequence A (MICA) gene is significantly associated with the risk of hepatitis-virus-related HCC. MICA is a ligand for NKG2D, a cell receptor of NK cells, which activates NK cells to clear the pathogenic cells expressing MICA such as infected cells or malignant cells as a “danger” signal. However, unexpectedly, the Hepatitis B virus (HBV)-infected patients with risk allele of MICA gene showed higher levels of serum MICA. This suggested that the serum MICA, which is excreted from the infected hepatocytes by a shedding mechanism, may work as a decoy for NK cells and inhibit their activities. Therefore, increasing cellular MICA levels by preventing those shedding and release may contribute to the clearance of the pathogenic cells and the prevention of HCC. In this study, we constructed a convenient MICA shedding assay system and, by a comprehensive screening, we identified several compounds which may prevent MICA shedding from the cells. Methods: We constructed lentivirual vector expressing MICA protein tagged with nano-luciferase (nanoLuc) in its N terminal because N terminal of MICA is excreted from the cells by shedding. We expressed this construct in HBV replicating cells, HepG2.2.15, and after adding compounds, we determined the luciferase activities in the supernatant and cell lysates. Results: We picked up the samples which showed low activities in supernatant with high activities in the cell lysates, indicating low levels of shedding and high levels of remaining cellular MICA. Out of over 1,000 compounds screened, we determined four candidate compounds which efficiently prevented MICA shedding. We confirmed the effects of these compounds in other cell systems infected with HBV. Conclusion: Because these compounds enhanced the expression levels of cellular MICA and decreased its shedding in HBV-replicating human hepatocytes, using these compounds may be favorable during HBV chronic infection to enhance the NK cell-mediated clearance of the pathogenic hepatocytes and to prevent HCC. Citation Format: Motoko Ohno, Motoyuki Otsuka, Takahiro Kishikawa, Takeshi Yoshikawa, Akemi Takata, Kazuhiko Koike. Prevention of MICA shedding from HBV infected hepatocytes to activate NK cells for better clearance of malignant foci and virus-infected cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4456. doi:10.1158/1538-7445.AM2015-4456

PTH-097 Notch1 is a master regulator of the senescence secretome through repression of c/ebpΒ

<h3>Introduction</h3> Oncogene-induced senescence (OIS) is an intrinsic tumour suppressor mechanism, but its impact on tumorigenesis is largely dependent on the nature of SASP, senescence-associated secretory phenotype. Major components of the SASP include TGFβ1 and pro-inflammatory cytokines, such as IL1A, IL6, and IL8 that have pleiotropic context-dependent effects. <h3>Method</h3> We utilised the well validated ER:Ras^G12VIMR90 HDF <i>in vitro</i>model which undergo Ras-induced senescence (RIS) with 4OHT. <i>In vivo</i>hepatocyte RIS was achieved with hydrodynamic tail-vein injection of NRas^G12V-containing transposons. <h3>Results</h3> Previously, we have shown that Notch1, a highly conserved receptor is up-regulated in RIS. In contrast to the up-regulation of Notch1, downstream signalling is dynamically regulated; Notch-target genes were transiently up-regulated at an early phase of RIS, but down-regulated at full senescence. The dynamic expression pattern of N1ICD and TGF-β1 expression were nearly identical, and inversely correlated with the cytokines, IL1A and IL8. Inhibition of Notch1 signalling, through expression of a dominant-negative form of the Notch1 binding partner MAML1, led to a reduction in TGF-β1, but increased IL1A and IL8 expression during RIS, suggesting Notch1 signalling plays a critical role in secretome switching. It has been shown that the SASP in RIS is regulated by two major transcription factors, NFkB and CEBPβ. Strikingly, over-expression of N1ICD strongly down-regulated CEBPβ, but not NFkB, in fully established RIS cells. Further, expression of ectopic CEBPβ in N1ICD-expressing cells partially restored levels of IL6/8. N1ICD was also able to suppress pro-inflammatory cytokine expression in TNF-α stimulated cells, through repression of CEBPβ. These data indicate that Notch1 represses pro-inflammatory cytokines by down-regulating CEBPβ. Finally, Notch1 up-regulation in OIS was confirmed in Nras-driven hepatocyte senescence; Notch1 inhibition through dnMAML1 promoted immune-mediated clearance of senescent hepatocytes. <h3>Conclusion</h3> We propose that the transition to OIS is correlated with a switch from Notch1-driven TGFβ-rich secretome to a CEBPβ-driven IL1/8 rich secretome, and that dynamic Notch1 signalling modulates senescence and its long-term fate strictly through a non-cell-autonomous fashion. <h3>Disclosure of interest</h3> None Declared.

Hepatology Highlights

Hepatology Highlights

Proteasome- and ethanol-dependent regulation of HCV-infection pathogenesis.

This paper reviews the role of the catabolism of HCV and signaling proteins in HCV protection and the involvement of ethanol in HCV-proteasome interactions. HCV specifically infects hepatocytes, and intracellularly expressed HCV proteins generate oxidative stress, which is further exacerbated by heavy drinking. The proteasome is the principal proteolytic system in cells, and its activity is sensitive to the level of cellular oxidative stress. Not only host proteins, but some HCV proteins are degraded by the proteasome, which, in turn, controls HCV propagation and is crucial for the elimination of the virus. Ubiquitylation of HCV proteins usually leads to the prevention of HCV propagation, while accumulation of undegraded viral proteins in the nuclear compartment exacerbates infection pathogenesis. Proteasome activity also regulates both innate and adaptive immunity in HCV-infected cells. In addition, the proteasome/immunoproteasome is activated by interferons, which also induce “early” and “late” interferon-sensitive genes (ISGs) with anti-viral properties. Cleaving viral proteins to peptides in professional immune antigen presenting cells and infected (“target”) hepatocytes that express the MHC class I-antigenic peptide complex, the proteasome regulates the clearance of infected hepatocytes by the immune system. Alcohol exposure prevents peptide cleavage by generating metabolites that impair proteasome activity, thereby providing escape mechanisms that interfere with efficient viral clearance to promote the persistence of HCV-infection.