Harnessing Immune Cell Metabolism to Modulate Alloresponse in Transplantation.

Immune cell metabolism plays a pivotal role in shaping and modulating immune responses. The metabolic state of immune cells influences their development, activation, differentiation, and overall function, impacting both innate and adaptive immunity. While glycolysis is crucial for activation and effector function of CD8 T cells, regulatory T cells mainly use oxidative phosphorylation and fatty acid oxidation, highlighting how different metabolic programs shape immune cells. Modification of cell metabolism may provide new therapeutic approaches to prevent rejection and avoid immunosuppressive toxicities. In particular, the distinct metabolic patterns of effector and suppressive cell subsets offer promising opportunities to target metabolic pathways that influence immune responses and graft outcomes. Herein, we review the main metabolic pathways used by immune cells, the techniques available to assay immune metabolism, and evidence supporting the possibility of shifting the immune response towards a tolerogenic profile by modifying energetic metabolism.

PD1 and TIM3 Expression is Associated with Very Early Hepatocellular Carcinoma Recurrence After Percutaneous Thermal Ablation.

Purpose: Percutaneous thermal ablation (PTA) is a cornerstone in the management of early-stage hepatocellular carcinoma (HCC). However, intrahepatic distant recurrence (IDR) occurs in the majority of patients after PTA. The aim of this study was to evaluate the immune signature associated with very early IDR. Patients and Methods: Thirty-one patients (26 men, 5 women; mean age:72.4 ± 8.6; age range:57-86 years) who underwent PTA for HCC were included in this study. After PTA for HCC, patients were followed and later divided into two groups: a "very early recurrence" group in case of IDR within 12 months after PTA, and a "prolonged recurrence-free" group in case of no recurrence before 12 months of follow-up. Freshly harvested intratumoral and nontumoral liver tissues and peripheral blood were obtained before PTA and explored by multiparametric flow cytometry. Results: The frequency of PD1+CD4+ T cells was higher in the early recurrence group than in the prolonged recurrence-free group in the peripheral blood (24.3%, IQR: 22.3-36.5 vs 14.0%, IQR: 11.5-16.4, p<0.0001), in the nontumoral liver (37.9%, IQR: 36.0-50.0 vs 22.5%, IQR: 18.0-29.9, p=0.0004), and in the tumor (37.6%, IQR: 32.3-39.3 vs 24.0%, IQR: 20.0-30.3, p=0.0137). Similarly, the frequency of TIM+CD8+ T cells was higher in the very early recurrence group in the peripheral blood (p=0.0021), non-tumoral liver (p=0.0012), and tumor (p=0.0239). Conclusion: The expression of immune checkpoint molecules, such as PD1 and TIM3 on T cells identified HCC patients at risk of very early IDR after PTA who would likely benefit from adjuvant immunotherapy. Thus, our study contributes to a better understanding of the potential association of PTA with adjuvant immunotherapies.

Nucleoside diphosphate kinases 1 and 2 regulate a protective liver response to a high-fat diet.

The synthesis of fatty acids from acetyl-coenzyme A (AcCoA) is deregulated in diverse pathologies, including cancer. Here, we report that fatty acid accumulation is negatively regulated by nucleoside diphosphate kinases 1 and 2 (NME1/2), housekeeping enzymes involved in nucleotide homeostasis that were recently found to bind CoA. We show that NME1 additionally binds AcCoA and that ligand recognition involves a unique binding mode dependent on the CoA/AcCoA 3' phosphate. We report that Nme2 knockout mice fed a high-fat diet (HFD) exhibit excessive triglyceride synthesis and liver steatosis. In liver cells, NME2 mediates a gene transcriptional response to HFD leading to the repression of fatty acid accumulation and activation of a protective gene expression program via targeted histone acetylation. Our findings implicate NME1/2 in the epigenetic regulation of a protective liver response to HFD and suggest a potential role in controlling AcCoA usage between the competing paths of histone acetylation and fatty acid synthesis.

Immune checkpoints on T and NK cells in the context of HBV infection: Landscape, pathophysiology and therapeutic exploitation.

In hepatitis B virus (HBV) infection, the interplay between the virus and the host immune system is crucial in determining the pathogenesis of the disease. Patients who fail to mount a sufficient and sustained anti-viral immune response develop chronic hepatitis B (CHB). T cells and natural killer (NK) cells play decisive role in viral clearance, but they are defective in chronic HBV infection. The activation of immune cells is tightly controlled by a combination of activating and inhibitory receptors, called immune checkpoints (ICs), allowing the maintenance of immune homeostasis. Chronic exposure to viral antigens and the subsequent dysregulation of ICs actively contribute to the exhaustion of effector cells and viral persistence. The present review aims to summarize the function of various ICs and their expression in T lymphocytes and NK cells in the course of HBV infection as well as the use of immunotherapeutic strategies targeting ICs in chronic HBV infection.

Intracellular accumulation and immunological response of NIR-II polymeric nanoparticles.

Polymeric nanoparticles (NPs) are extremely promising for theranostic applications. However, their interest depends largely on their interactions with immune system, including the capacity to activate inflammation after their capture by macrophages. In the present study, we generated monodisperse poly(ethyl methacrylate) (PEMA) NPs loaded with hydrophobic photoluminescent gold nanoclusters (Au NCs) emitting in the NIR-II optical windows and studied their interaction in vitro with J774.1A macrophages. PEMA NPs showed an efficient time and dose dependent cellular uptake with up to 70 % of macrophages labelled in 24 h without detectable cell death. Interestingly, PEMA and Au-PEMA NPs induced an anti-inflammatory response and a strong down-regulation of nitric oxide level on lipopolysacharides (LPS) activated macrophages, but without influence on the levels of reactive oxygen species (ROS). These polymeric NPs may thus present a potential interest for the treatment of inflammatory diseases.

Effect of Novel AKT Inhibitor Vevorisertib as Single Agent and in Combination with Sorafenib on Hepatocellular Carcinoma in a Cirrhotic Rat Model.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide. The AKT pathway is often activated in HCC cases, and a longer exposure to tyrosine kinase inhibitors such as sorafenib may lead to over-activation of the AKT pathway, leading to HCC resistance. Here, we studied the efficacy of a new generation of allosteric AKT inhibitor, vevorisertib, alone or in combination with sorafenib. To identify specific adverse effects related to the background of cirrhosis, we used a diethylnitrosamine (DEN)-induced cirrhotic rat model. Vevorisertib was tested in vitro on Hep3B, HepG2, HuH7 and PLC/PRF cell lines. Rats were treated weekly with intra-peritoneal injections of DEN for 14 weeks to obtain cirrhosis with fully developed HCC. After that, rats were randomized into four groups (n = 7/group): control, sorafenib, vevorisertib and the combination of vevorisertib + sorafenib, and treated for 6 weeks. Tumor progression was followed by MRI. We demonstrated that the vevorisertib is a highly potent treatment, blocking the phosphorylation of AKT. The tumor progression in the rat liver was significantly reduced by treatment with vevorisertib + sorafenib (49.4%) compared to the control group (158.8%, p < 0.0001). Tumor size, tumor number and tumor cell proliferation were significantly reduced in both the vevorisertib group and vevorisertib + sorafenib groups compared to the control group. Sirius red staining showed an improvement in liver fibrosis by vevorisertib and the combination treatment. Moreover, vevorisertib + sorafenib treatment was associated with a normalization in the liver vasculature. Altogether, vevorisertib as a single agent and its combination with sorafenib exerted a strong suppression of tumor progression and improved liver fibrosis. Thus, results provide a rationale for testing vevorisertib in clinical settings and confirm the importance of targeting AKT in HCC.

Enantioselective Approach for Expanding the Three-Dimensional Space of Tetrahydroquinoline to Develop BET Bromodomain Inhibitors.

The pharmaceutical industry has a pervasive need for chiral specific molecules with optimal affinity for their biological targets. However, the mass production of such compounds is currently limited by conventional chemical routes, that are costly and have an environmental impact. Here, we propose an easy access to obtain new tetrahydroquinolines, a motif found in many bioactive compounds, that is rapid and cost effective. Starting from simple raw materials, the procedure uses a proline-catalyzed Mannich reaction followed by the addition of BF3 ⋅ OEt2 , which generates a highly electrophilic aza-ortho-quinone methide intermediate capable of reacting with different nucleophiles to form the diversely functionalized tetrahydroquinoline. Moreover, this enantioselective one-pot process provides access for the first time to tetrahydroquinolines with a cis-2,3 and trans-3,4 configuration. As proof of concept, we demonstrate that a three-step reaction sequence, from simple and inexpensive starting compounds and catalysts, can generate a BD2-selective BET bromodomain inhibitor with anti-inflammatory effect.

Chronic Intermittent Hypoxia Increases Cell Proliferation in Hepatocellular Carcinoma.

Obstructive sleep apnea (OSA) syndrome is characterized by chronic intermittent hypoxia and is associated with an increased risk of all-cause mortality, including cancer mortality. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, characterized by increasing incidence and high mortality. However, the link between HCC and OSA-related chronic intermittent hypoxia remains unclear. Herein, we used a diethylnitrosamine (DEN)-induced HCC model to investigate whether OSA-related chronic intermittent hypoxia has an impact on HCC progression. To elucidate the associated mechanisms, we first evaluated the hypoxia status in the DEN-induced HCC model. Next, to simulate OSA-related intermittent hypoxia, we exposed cirrhotic rats with HCC to intermittent hypoxia during six weeks. We performed histopathological, immunohistochemical, RT-qPCR, and RNA-seq analysis. Chronic DEN injections strongly promoted cell proliferation, fibrosis, disorganized vasculature, and hypoxia in liver tissue, which mimics the usual events observed during human HCC development. Intermittent hypoxia further increased cell proliferation in DEN-induced HCC, which may contribute to an increased risk of HCC progression. In conclusion, our observations suggest that chronic intermittent hypoxia may be a factor worsening the prognosis of HCC.

GNS561, a clinical-stage PPT1 inhibitor, is efficient against hepatocellular carcinoma via modulation of lysosomal functions.

Hepatocellular carcinoma is the most frequent primary liver cancer. Macroautophagy/autophagy inhibitors have been extensively studied in cancer but, to date, none has reached efficacy in clinical trials. In this study, we demonstrated that GNS561, a new autophagy inhibitor, whose anticancer activity was previously linked to lysosomal cell death, displayed high liver tropism and potent antitumor activity against a panel of human cancer cell lines and in two hepatocellular carcinoma in vivo models. We showed that due to its lysosomotropic properties, GNS561 could reach and specifically inhibited its enzyme target, PPT1 (palmitoyl-protein thioesterase 1), resulting in lysosomal unbound Zn2+ accumulation, impairment of cathepsin activity, blockage of autophagic flux, altered location of MTOR (mechanistic target of rapamycin kinase), lysosomal membrane permeabilization, caspase activation and cell death. Accordingly, GNS561, for which a global phase 1b clinical trial in liver cancers was just successfully achieved, represents a promising new drug candidate and a hopeful therapeutic strategy in cancer treatment.Abbreviations: ANXA5:annexin A5; ATCC: American type culture collection; BafA1: bafilomycin A1; BSA: bovine serum albumin; CASP3: caspase 3; CASP7: caspase 7; CASP8: caspase 8; CCND1: cyclin D1; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; CQ: chloroquine; iCCA: intrahepatic cholangiocarcinoma; DEN: diethylnitrosamine; DMEM: Dulbelcco's modified Eagle medium; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HCC: hepatocellular carcinoma; HCQ: hydroxychloroquine; HDSF: hexadecylsulfonylfluoride; IC50: mean half-maximal inhibitory concentration; LAMP: lysosomal associated membrane protein; LC3-II: phosphatidylethanolamine-conjugated form of MAP1LC3; LMP: lysosomal membrane permeabilization; MALDI: matrix assisted laser desorption ionization; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MKI67: marker of proliferation Ki-67; MTOR: mechanistic target of rapamycin kinase; MRI: magnetic resonance imaging; NH4Cl: ammonium chloride; NtBuHA: N-tert-butylhydroxylamine; PARP: poly(ADP-ribose) polymerase; PBS: phosphate-buffered saline; PPT1: palmitoyl-protein thioesterase 1; SD: standard deviation; SEM: standard error mean; vs, versus; Zn2+: zinc ion; Z-Phe: Z-Phe-Tyt(tBu)-diazomethylketone; Z-VAD-FMK: carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone.

Immunomodulation for hepatocellular carcinoma therapy: current challenges.

PURPOSE OF REVIEW: The emergence of novel immunotherapies, such as immune-checkpoint inhibitors has changed the landscape of systemic cancer treatment. In hepatocellular carcinoma (HCC) patients, despite initial enthusiasm, the proportion of responders to immune-checkpoint inhibitors remains low. We provide a brief update of this rapidly evolving field, with specific focus on the development in the field of predictive factors and the immunomodulation induced by locoregional therapies. RECENT FINDINGS: Even if the immune contexture of HCC before the treatment remains the most promising predictive marker for response to immunotherapies, recent findings show that the cause of HCC may have also a key role. Specific inflammatory mechanisms induced by NASH may result in limited efficacy of immunotherapy compared with viral HCC. Other recent findings showed that percutaneous ablations are responsible for intratumoral immune changes and systemic immune system activation that may help to prevent recurrence when combined with immunotherapies. In case of multifocal HCC, transarterial therapies (TACE and SIRT) may help to turn a cold tumor type to a hot tumor type and could be associated with immune-checkpoint inhibitors to improve outcomes. SUMMARY: The future HCC management will focus on patient stratification for specific immunotherapies depending on the signature and cause of HCC and the best combined approaches in which locoregional therapies may play a pivotal role.

The Chicken Embryo Model: A Novel and Relevant Model for Immune-Based Studies.

Dysregulation of the immune system is associated with many pathologies, including cardiovascular diseases, diabetes, and cancer. To date, the most commonly used models in biomedical research are rodents, and despite the various advantages they offer, their use also raises numerous drawbacks. Recently, another in vivo model, the chicken embryo and its chorioallantoic membrane, has re-emerged for various applications. This model has many benefits compared to other classical models, as it is cost-effective, time-efficient, and easier to use. In this review, we explain how the chicken embryo can be used as a model for immune-based studies, as it gradually develops an embryonic immune system, yet which is functionally similar to humans'. We mainly aim to describe the avian immune system, highlighting the differences and similarities with the human immune system, including the repertoire of lymphoid tissues, immune cells, and other key features. We also describe the general in ovo immune ontogeny. In conclusion, we expect that this review will help future studies better tailor their use of the chicken embryo model for testing specific experimental hypotheses or performing preclinical testing.

DEN-Induced Rat Model Reproduces Key Features of Human Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. The majority of HCC cases are associated with liver fibrosis or cirrhosis developing from chronic liver injuries. The immune system of the liver contributes to the severity of tissue damage, the establishment of fibrosis and the disease's progression towards HCC. Herein, we provide a detailed characterization of the DEN-induced HCC rat model during fibrosis progression and HCC development with a special focus on the liver's inflammatory microenvironment. Fischer 344 male rats were treated weekly for 14 weeks with intra-peritoneal injections of 50 mg/kg DEN. The rats were sacrificed before starting DEN-injections at 0 weeks, after 8 weeks, 14 weeks and 20 weeks after the start of DEN-injections. We performed histopathological, immunohistochemical, RT-qPCR, RNA-seq and flow cytometry analysis. Data were compared between tumor and non-tumor samples from the DEN-treated versus untreated rats, as well as versus human HCCs. Chronic DEN injections lead to liver damage, hepatocytes proliferation, liver fibrosis and cirrhosis, disorganized vasculature, and a modulated immune microenvironment that mimics the usual events observed during human HCC development. The RNA-seq results showed that DEN-induced liver tumors in the rat model shared remarkable molecular characteristics with human HCC, especially with HCC associated with high proliferation. In conclusion, our study provides detailed insight into hepatocarcinogenesis in a commonly used model of HCC, facilitating the future use of this model for preclinical testing.

Increased Intrahepatic Expression of Immune Checkpoint Molecules in Autoimmune Liver Disease.

Immune checkpoint molecules (ICM) are critical in maintaining immunologic homeostasis and participate in preventing or promoting autoimmune disease development. Exploring a large panel of intrahepatic inhibitory and stimulatory ICM is necessary for drawing a general picture of the immune alterations in autoimmune hepatitis (AIH). Here, we performed a multiparametric analysis of ICM, including PD-1, TIM3, LAG3, CTLA-4, OX40 and 4-1BB, and we determined their expression on intrahepatic lymphocyte subsets in untreated and in treated patients with AIH in comparison to normal liver tissue. AIH patient-derived liver tissue revealed the overexpression of ICM, mainly PD-1 and 4-1BB, as well as the strong correlation between PD-1+ CD8+ T-cell abundance and severity of AIH (alanine transaminase and aspartate transaminase levels). Our results show that the ICM play an important role in the loss of immune homeostasis in the liver, providing an attractive approach to investigate their role as targets for effective therapeutic interventions.

GNS561, a New Autophagy Inhibitor Active against Cancer Stem Cells in Hepatocellular Carcinoma and Hepatic Metastasis from Colorectal Cancer.

Patients with advanced hepatocellular carcinoma (HCC) or metastatic colorectal cancer (mCRC) have a very poor prognosis due to the lack of efficient treatments. As observed in several other tumors, the effectiveness of treatments is mainly hampered by the presence of a highly tumorigenic sub-population of cancer cells called cancer stem cells (CSCs). Indeed, CSCs are resistant to chemotherapy and radiotherapy and can regenerate the tumor bulk. Hence, innovative drugs that are efficient against both bulk tumor cells and CSCs would likely improve cancer treatment. In this study, we demonstrated that GNS561, a new autophagy inhibitor that induces lysosomal cell death, showed significant activity against not only the whole tumor population but also a sub-population displaying CSC features (high ALDH activity and tumorsphere formation ability) in HCC and in liver mCRC cell lines. These results were confirmed in vivo in HCC from a DEN-induced cirrhotic rat model in which GNS561 decreased tumor growth and reduced the frequency of CSCs (CD90+CD45-). Thus, GNS561 offers great promise for cancer therapy by exterminating both the tumor bulk and the CSC sub-population. Accordingly, a global phase 1b clinical trial in liver cancers was recently completed.

Modeling Diet-Induced NAFLD and NASH in Rats: A Comprehensive Review.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, characterized by hepatic steatosis without any alcohol abuse. As the prevalence of NAFLD is rapidly increasing worldwide, important research activity is being dedicated to deciphering the underlying molecular mechanisms in order to define new therapeutic targets. To investigate these pathways and validate preclinical study, reliable, simple and reproducible tools are needed. For that purpose, animal models, more precisely, diet-induced NAFLD and nonalcoholic steatohepatitis (NASH) models, were developed to mimic the human disease. In this review, we focus on rat models, especially in the current investigation of the establishment of the dietary model of NAFLD and NASH in this species, compiling the different dietary compositions and their impact on histological outcomes and metabolic injuries, as well as external factors influencing the course of liver pathogenesis.

Targeting Akt in Hepatocellular Carcinoma and Its Tumor Microenvironment.

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related deaths worldwide, and its incidence is rising. HCC develops almost exclusively on the background of chronic liver inflammation, which can be caused by chronic alcohol consumption, viral hepatitis, or an unhealthy diet. The key role of chronic inflammation in the process of hepatocarcinogenesis, including in the deregulation of innate and adaptive immune responses, has been demonstrated. The inhibition of Akt (also known as Protein Kinase B) directly affects cancer cells, but this therapeutic strategy also exhibits indirect anti-tumor activity mediated by the modulation of the tumor microenvironment, as demonstrated by using Akt inhibitors AZD5363, MK-2206, or ARQ 092. Moreover, the isoforms of Akt converge and diverge in their designated roles, but the currently available Akt inhibitors fail to display an isoform specificity. Thus, selective Akt inhibition needs to be better explored in the context of HCC and its possible combination with immunotherapy. This review presents a compact overview of the current knowledge concerning the role of Akt in HCC and the effect of Akt inhibition on the HCC and liver tumor microenvironment.

Circulating IL-13 Is Associated with De Novo Development of HCC in HCV-Infected Patients Responding to Direct-Acting Antivirals.

Direct-acting antivirals (DAAs) are highly effective in targeting hepatitis C virus (HCV) infections, but the incidence of HCV-related hepatocellular carcinoma (HCC) remains still high. In this study, we investigated a cohort of HCV-infected patients treated with DAAs who were followed up for 4 years after sustained virological response (SVR) achievement. Patients who developed de novo HCC following DAA treatment were compared to matched controls who did not develop HCC. These control patients were selected based on DAA treatment, sex, age, fibrosis status, and platelet counts. We evaluated serum levels of 30 immune mediators before, during, at the end of, and three months after DAA treatment using Luminex technology. We identified the immune factors associated with de novo HCC occurrence following DAA treatment. Specifically, interleukin (IL)-4 and IL-13 levels were significantly higher before start of the DAA treatment in the serum of patients who later developed HCC than in controls and stayed higher at each subsequent time point. Least absolute shrinkage and selection operator (LASSO) regression revealed IL-13 as the only strong factor associated with HCC development in this cohort of HCV patients. The difference was observed already at baseline of DAA treatment, which confirms the existence of a specific immune profile in these patients who later develop HCC.

Reply to Comment on "Jilkova, Z.M.; et al. Predictive Factors for Response to PD-1/PD-L1 Checkpoint Inhibition in the Field of Hepatocellular Carcinoma: Current Status and Challenges" Cancers 2019, 11, 1554.

We appreciate the comments made on our review paper focused on possible predictive factors for responses to PD-1/PD-L1 checkpoint inhibitors in the field of hepatocellular carcinoma [...].