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.

Therapeutic siRNAs Targeting the JAK/STAT Signalling Pathway in Inflammatory Bowel Diseases.

BACKGROUND AND AIMS: Inflammatory bowel diseases are highly debilitating conditions that require constant monitoring and life-long medication. Current treatments are focused on systemic administration of immunomodulatory drugs, but they have a broad range of undesirable side-effects. RNA interference is a highly specific endogenous mechanism that regulates the expression of the gene at the transcript level, which can be repurposed using exogenous short interfering RNA [siRNA] to repress expression of the target gene. While siRNA therapeutics can offer an alternative to existing therapies, with a high specificity critical for chronically administrated drugs, evidence of their potency compared to chemical kinase inhibitors used in clinics is still lacking in alleviating an adverse inflammatory response. METHODS: We provide a framework to select highly specific siRNA, with a focus on two kinases strongly involved in pro-inflammatory diseases, namely JAK1 and JAK3. Using western-blot, real-time quantitative PCR and large-scale analysis, we assessed the specificity profile of these siRNA drugs and compared their efficacy to the most recent and promising kinase inhibitors for Janus kinases [Jakinibs], tofacitinib and filgotinib. RESULTS: siRNA drugs can reach higher efficiency and selectivity at lower doses [5 pM vs 1 µM] than Jakinibs. Moreover, JAK silencing lasted up to 11 days, even with 6 h pulse transfection. CONCLUSIONS: The siRNA-based drugs developed hold the potential to develop more potent therapeutics for chronic inflammatory diseases.

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.

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.

Tuning the Immunostimulation Properties of Cationic Lipid Nanocarriers for Nucleic Acid Delivery.

Nonviral systems, such as lipid nanoparticles, have emerged as reliable methods to enable nucleic acid intracellular delivery. The use of cationic lipids in various formulations of lipid nanoparticles enables the formation of complexes with nucleic acid cargo and facilitates their uptake by target cells. However, due to their small size and highly charged nature, these nanocarrier systems can interact in vivo with antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages. As this might prove to be a safety concern for developing therapies based on lipid nanocarriers, we sought to understand how they could affect the physiology of APCs. In the present study, we investigate the cellular and metabolic response of primary macrophages or DCs exposed to the neutral or cationic variant of the same lipid nanoparticle formulation. We demonstrate that macrophages are the cells affected most significantly and that the cationic nanocarrier has a substantial impact on their physiology, depending on the positive surface charge. Our study provides a first model explaining the impact of charged lipid materials on immune cells and demonstrates that the primary adverse effects observed can be prevented by fine-tuning the load of nucleic acid cargo. Finally, we bring rationale to calibrate the nucleic acid load of cationic lipid nanocarriers depending on whether immunostimulation is desirable with the intended therapeutic application, for instance, gene delivery or messenger RNA vaccines.

Multimodal Imaging with NanoGd Reveals Spatiotemporal Features of Neuroinflammation after Experimental Stroke.

The purpose of this study is to propose and validate a preclinical in vivo magnetic resonance imaging (MRI) tool to monitor neuroinflammation following ischemic stroke, based on injection of a novel multimodal nanoprobe, NanoGd, specifically designed for internalization by phagocytic cells. First, it is verified that NanoGd is efficiently internalized by microglia in vitro. In vivo MRI coupled with intravenous injection of NanoGd in a permanent middle cerebral artery occlusion mouse model results in hypointense signals in the ischemic lesion. In these mice, longitudinal two-photon intravital microscopy shows NanoGd internalization by activated CX3CR1-GFP/+ cells. Ex vivo analysis, including phase contrast imaging with synchrotron X-ray, histochemistry, and transmission electron microscopy corroborate NanoGd accumulation within the ischemic lesion and uptake by immune phagocytic cells. Taken together, these results confirm the potential of NanoGd-enhanced MRI as an imaging biomarker of neuroinflammation at the subacute stage of ischemic stroke. As far as it is known, this work is the first to decipher the working mechanism of MR signals induced by a nanoparticle passively targeted at phagocytic cells by performing intravital microscopy back-to-back with MRI. Furthermore, using a gadolinium-based rather than an iron-based contrast agent raises future perspectives for the development of molecular imaging with emerging computed tomography technologies.

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.

Hybrid multimodal contrast agent for multiscale in vivo investigation of neuroinflammation.

Neuroinflammation is a process common to several brain pathologies. Despites its medical relevance, it still remains poorly understood; there is therefore a need to develop new in vivo preclinical imaging strategies to monitor inflammatory processes longitudinally. We here present the development of a hybrid imaging nanoprobe named NP3, that was specifically designed to get internalized by phagocytic cells and imaged in vivo with MRI and bi-photon microscopy. NP3 is composed of a 16 nm core of gadolinium fluoride (GdF3), coated with bisphosphonate polyethylene glycol (PEG) and functionalized with a Lemke-type fluorophore. It has a hydrodynamic diameter of 28 ± 8 nm and a zeta potential of -42 ± 6 mV. The MR relaxivity ratio at 7 T is r1/r2 = 20; therefore, NP3 is well suited as a T2/T2* contrast agent. In vitro cytotoxicity assessments performed on four human cell lines revealed no toxic effects of NP3. In addition, NP3 is internalized by macrophages in vitro without inducing inflammation or cytotoxicity. In vivo, uptake of NP3 has been observed in the spleen and the liver. NP3 has a prolonged vascular remanence, which is an advantage for macrophage uptake in vivo. The proof-of-concept that NP3 may be used as a contrast agent targeting phagocytic cells is provided in an animal model of ischemic stroke in transgenic CX3CR1-GFP/+ mice using three complementary imaging modalities: MRI, intravital two-photon microscopy and phase contrast imaging with synchrotron X-rays. In summary, NP3 is a promising preclinical tool for the multiscale and multimodal investigation of neuroinflammation.

Impact of Gold Nanoparticles on the Functions of Macrophages and Dendritic Cells.

Gold nanoparticles (AuNPs) have demonstrated outstanding performance in many biomedical applications. Their safety is recognised; however, their effects on the immune system remain ill defined. Antigen-presenting cells (APCs) are immune cells specialised in sensing external stimulus and in capturing exogenous materials then delivering signals for the immune responses. We used primary macrophages (Ms) and dendritic cells (DCs) of mice as an APC model. Whereas AuNPs did not alter significantly Ms and DCs functions, the exposure to AuNPs affected differently Ms and DCs in their responses to subsequent stimulations. The secretion of inflammatory molecules like cytokines (IL-6, TNF-α), chemokine (MCP-1), and reactive oxygen species (ROS) were altered differently in Ms and DCs. Furthermore, the metabolic activity of Ms was affected with the increase of mitochondrial respiration and glycolysis, while only a minor effect was seen on DCs. Antigen presentation to T cells increased when DCs were exposed to AuNPs leading to stronger Th1, Th2, and Th17 responses. In conclusion, our data provide new insights into the complexity of the effects of AuNPs on the immune system. Although AuNPs may be considered as devoid of significant effect, they may induce discrete modifications on some functions that can differ among the immune cells.

Synthesis and use of an amphiphilic dendrimer for siRNA delivery into primary immune cells.

Using siRNAs to genetically manipulate immune cells is important to both basic immunological studies and therapeutic applications. However, siRNA delivery is challenging because primary immune cells are often sensitive to the delivery materials and generate immune responses. We have recently developed an amphiphilic dendrimer that is able to deliver siRNA to a variety of cells, including primary immune cells. We provide here a protocol for the synthesis of this dendrimer, as well as siRNA delivery to immune cells such as primary T and B cells, natural killer cells, macrophages, and primary microglia. The dendrimer synthesis entails straightforward click coupling followed by an amidation reaction, and the siRNA delivery protocol requires simple mixing of the siRNA and dendrimer in buffer, with subsequent application to the primary immune cells to achieve effective and functional siRNA delivery. This dendrimer-mediated siRNA delivery largely outperforms the standard electroporation technique, opening a new avenue for functional and therapeutic studies of the immune system. The whole protocol encompasses the dendrimer synthesis, which takes 10 days; the primary immune cell preparation, which takes 3-10 d, depending on the tissue source and cell type; the dendrimer-mediated siRNA delivery; and subsequent functional assays, which take an additional 3-6 d.

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.

Modulating the Crosstalk between the Tumor and Its Microenvironment Using RNA Interference: A Treatment Strategy for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy with one of the highest mortality rates among solid cancers. It develops almost exclusively in the background of chronic liver inflammation, which can be caused by viral hepatitis, chronic alcohol consumption or an unhealthy diet. Chronic inflammation deregulates the innate and adaptive immune responses that contribute to the proliferation, survival and migration of tumor cells. The continuous communication between the tumor and its microenvironment components serves as the overriding force of the tumor against the body's defenses. The importance of this crosstalk between the tumor microenvironment and immune cells in the process of hepatocarcinogenesis has been shown, and therapeutic strategies modulating this communication have improved the outcomes of patients with liver cancer. To target this communication, an RNA interference (RNAi)-based approach can be used, an innovative and promising strategy that can disrupt the crosstalk at the transcriptomic level. Moreover, RNAi offers the advantage of specificity in comparison to the treatments currently used for HCC in clinics. In this review, we will provide the recent data pertaining to the modulation of a tumor and its microenvironment by using RNAi and its potential for therapeutic intervention in HCC.

Chronic Oral Exposure to Synthetic Amorphous Silica (NM-200) Results in Renal and Liver Lesions in Mice.

INTRODUCTION: Silicon dioxide, produced as synthetic amorphous silica (SAS), is made of nanoparticles (NPs), either present as such or as agglomerates and aggregates, and is widely used in many types of food processes and products as an additive. To assess whether repeated, long-term exposure to SAS NPs may result in adverse effects, mice were exposed for 18 months via drinking water to NM-200, one of the reference nanostructured silica used for applications related to food, at 4.8 mg NM-200/kg body weight per day, a dose relevant to the estimated dietary exposure to SAS in humans. METHODS: The experiment focused on the kidney and liver as target organs and was carried out in parallel using 3 mouse lines (wild type and transgenic) differing for the expression of α-synuclein, that is, murine and human mutated (A53T). Sensitive determination of silicon revealed higher contents in liver and kidneys of NM-200-exposed mice compared with unexposed aged-matched controls. RESULTS: Histological abnormalities, such as vacuolization of tubular epithelial cells, were detected in all kidneys, as well as inflammatory responses that were also detected in livers of exposed animals. Less frequent but more deleterious, amyloidosis lesions were observed in glomeruli, associated with perivascular amyloid accumulation in liver. CONCLUSION: These histological findings, in conjunction with the observation of detectable deposition of silica, highlight that chronic oral intake of SAS may pose a health risk to humans and need to be examined further.

Polarization Induced Electro-Functionalization of Pore Walls: A Contactless Technology.

This review summarizes recent advances in micro- and nanopore technologies with a focus on the functionalization of pores using a promising method named contactless electro-functionalization (CLEF). CLEF enables the localized grafting of electroactive entities onto the inner wall of a micro- or nano-sized pore in a solid-state silicon/silicon oxide membrane. A voltage or electrical current applied across the pore induces the surface functionalization by electroactive entities exclusively on the inside pore wall, which is a significant improvement over existing methods. CLEF's mechanism is based on the polarization of a sandwich-like silicon/silicon oxide membrane, creating electronic pathways between the core silicon and the electrolyte. Correlation between numerical simulations and experiments have validated this hypothesis. CLEF-induced micro- and nanopores functionalized with antibodies or oligonucleotides were successfully used for the detection and identification of cells and are promising sensitive biosensors. This technology could soon be successfully applied to planar configurations of pores, such as restrictions in microfluidic channels.

Immunologic Features of Patients With Advanced Hepatocellular Carcinoma Before and During Sorafenib or Anti-programmed Death-1/Programmed Death-L1 Treatment.

INTRODUCTION: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Today, a promising treatment strategy is focused on the enhancement of antitumor immune responses by immune checkpoint modification. However, as only 20% of patients with HCC are responders, identification of predictive factors is urgently required. Therefore, for the first time, the features of the intrahepatic and circulating immune system in patients with advanced-stage HCC, before and during the treatment, were analyzed. METHODS: We collected fresh HCC biopsies, along with adjacent tumor-free liver tissues and peripheral blood samples, from 21 patients with advanced HCC. Furthermore, we performed an extensive immunomonitoring of patients with HCC treated with sorafenib or programmed death (PD)-1/PD-L1 pathway blockade using multiparametric flow cytometry. RESULTS: We observed that regardless of the treatment, low baseline intratumoral CD4/CD8 T-cell ratio was associated with better overall survival (P = 0.0002). The baseline frequency of intratumoral PD-1 CD8 T cells was significantly lower in patients responding to sorafenib treatment than in the nonresponders (P = 0.0117), and the frequency of circulating PD-1 T cells increased with tumor progression (P = 0.0329). By contrast, responders to PD-1/PD-L1 pathway blockade showed a trend of high baseline frequency of intratumoral PD-1 CD8 T cells. Moreover, we observed a trend of LAG3 and TIM3 upregulation on circulating T cells in nonresponding patients to PD-1/PD-L1 pathway blockade. DISCUSSION: Immunosuppressive state, characterized by an enhanced intratumoral CD4/CD8 T-cell ratio, was associated with poor prognosis. Additionally, our results suggest that the frequency of intratumoral PD-1 CD8 T cells may serve as a biomarker to identify which individuals will benefit from which treatment and support the use of combination strategies.