RESUMO
Research on liver-related conditions requires a robust and efficient method to purify viable hepatocytes, lymphocytes and all other liver resident cells, such as Kupffer or liver sinusoidal endothelial cells. Here we describe a novel purification method using liver enzymatic digestion, followed by a downstream optimized purification. Using this enzymatic digestion protocol, the resident liver cells as well as viable hepatocytes could be captured, compared to the classical mechanical liver disruption method. Moreover, single-cell RNA-sequencing demonstrated higher quality lymphocyte data in downstream analyses after the liver enzymatic digestion, allowing for studying of immunological responses or changes. In order to also understand the peripheral immune landscape, a protocol for lymphocyte purification from mouse systemic whole blood was optimized, allowing for efficient removal of red blood cells. The combination of microbeads and mRNA blockers allowed for a clean blood sample, enabling robust single-cell RNA-sequencing data. These two protocols for blood and liver provide important new methodologies for liver-related studies such as NASH, hepatitis virus infections or cancer research but also for immunology where high-quality cells are indispensable for further downstream assays.
Assuntos
Leucócitos , Fígado , Animais , Camundongos , Fígado/imunologia , Leucócitos/imunologia , Hepatócitos/imunologia , Separação Celular/métodos , Análise de Célula Única/métodos , Camundongos Endogâmicos C57BL , Masculino , Linfócitos/imunologia , Linfócitos/citologiaRESUMO
Apical membrane antigen-1 (AMA1) is a conserved malarial vaccine candidate essential for the formation of tight junctions with the rhoptry neck protein (RON) complex, enabling Plasmodium parasites to invade human erythrocytes, hepatocytes, and mosquito salivary glands. Despite its critical role, extensive surface polymorphisms in AMA1 have led to strain-specific protection, limiting the success of AMA1-based interventions beyond initial clinical trials. Here, we identify an i-body, a humanised single-domain antibody-like molecule that recognises a conserved pan-species conformational epitope in AMA1 with low nanomolar affinity and inhibits the binding of the RON2 ligand to AMA1. Structural characterisation indicates that the WD34 i-body epitope spans the centre of the conserved hydrophobic cleft in AMA1, where interacting residues are highly conserved among all Plasmodium species. Furthermore, we show that WD34 inhibits merozoite invasion of erythrocytes by multiple Plasmodium species and hepatocyte invasion by P. falciparum sporozoites. Despite a short half-life in mouse serum, we demonstrate that WD34 transiently suppressed P. berghei infections in female BALB/c mice. Our work describes the first pan-species AMA1 biologic with inhibitory activity against multiple life-cycle stages of Plasmodium. With improved pharmacokinetic characteristics, WD34 could be a potential immunotherapy against multiple species of Plasmodium.
Assuntos
Antígenos de Protozoários , Eritrócitos , Fígado , Proteínas de Membrana , Camundongos Endogâmicos BALB C , Proteínas de Protozoários , Animais , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Feminino , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Humanos , Eritrócitos/parasitologia , Eritrócitos/imunologia , Fígado/parasitologia , Fígado/imunologia , Fígado/metabolismo , Vacinas Antimaláricas/imunologia , Malária/imunologia , Malária/parasitologia , Malária/prevenção & controle , Reações Cruzadas/imunologia , Plasmodium falciparum/imunologia , Plasmodium berghei/imunologia , Epitopos/imunologia , Hepatócitos/parasitologia , Hepatócitos/imunologia , Hepatócitos/metabolismo , Plasmodium/imunologia , Merozoítos/imunologia , Merozoítos/metabolismoRESUMO
Therapeutics for eradicating hepatitis B virus (HBV) infection are still limited and current nucleos(t)ide analogs (NAs) and interferon are effective in controlling viral replication and improving liver health, but they cannot completely eradicate the hepatitis B virus and only a very small number of patients are cured of it. The TCR-like antibodies recognizing viral peptides presented on human leukocyte antigens (HLA) provide possible tools for targeting and eliminating HBV-infected hepatocytes. Here, we generated three TCR-like antibodies targeting three different HLA-A2.1-presented peptides derived from HBV core and surface proteins. Bispecific antibodies (BsAbs) were developed by fuzing variable fragments of these TCR-like mAbs with an anti-CD3ϵ antibody. Our data demonstrate that the BsAbs could act as T cell engagers, effectively redirecting and activating T cells to target HBV-infected hepatocytes in vitro and in vivo. In HBV-persistent mice expressing human HLA-A2.1, two infusions of BsAbs induced marked and sustained suppression in serum HBsAg levels and also reduced the numbers of HBV-positive hepatocytes. These findings highlighted the therapeutic potential of TCR-like BsAbs as a new strategy to cure hepatitis B.
Assuntos
Anticorpos Biespecíficos , Modelos Animais de Doenças , Vírus da Hepatite B , Hepatite B , Hepatócitos , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Hepatócitos/virologia , Hepatócitos/imunologia , Camundongos , Humanos , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/genética , Hepatite B/imunologia , Hepatite B/virologia , Antígeno HLA-A2/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologiaRESUMO
Hepatitis B Virus (HBV) is a stealthy and insidious pathogen capable of inducing chronic necro-inflammatory liver disease and hepatocellular carcinoma (HCC), resulting in over one million deaths worldwide per year. The traditional understanding of Chronic Hepatitis B (CHB) progression has focused on the complex interplay among ongoing virus replication, aberrant immune responses, and liver pathogenesis. However, the dynamic progression and crucial factors involved in the transition from HBV infection to immune activation and intrahepatic inflammation remain elusive. Recent insights have illuminated HBV's exploitation of the sodium taurocholate co-transporting polypeptide (NTCP) and manipulation of the cholesterol transport system shared between macrophages and hepatocytes for viral entry. These discoveries deepen our understanding of HBV as a virus that hijacks hepatocyte metabolism. Moreover, hepatic niche macrophages exhibit significant phenotypic and functional diversity, zonal characteristics, and play essential roles, either in maintaining liver homeostasis or contributing to the pathogenesis of chronic liver diseases. Therefore, we underscore recent revelations concerning the importance of hepatic niche macrophages in the context of viral hepatitis. This review particularly emphasizes the significant role of HBV-induced metabolic changes in hepatic macrophages as a key factor in the transition from viral infection to immune activation, ultimately culminating in liver inflammation. These metabolic alterations in hepatic macrophages offer promising targets for therapeutic interventions and serve as valuable early warning indicators, shedding light on the disease progression.
Assuntos
Vírus da Hepatite B , Hepatite B Crônica , Fígado , Macrófagos , Humanos , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/fisiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Animais , Fígado/imunologia , Fígado/virologia , Fígado/metabolismo , Fígado/patologia , Hepatite B Crônica/imunologia , Hepatite B Crônica/metabolismo , Hepatite B Crônica/virologia , Inflamação/imunologia , Inflamação/metabolismo , Hepatócitos/metabolismo , Hepatócitos/imunologia , Hepatócitos/virologiaRESUMO
Liver is the largest internal organ of the body with vital functions. In addition to its endocrine and exocrine activities, liver also plays a pivotal role in the immune system, including haematopoietic functions. Liver parenchymal cells, which are epithelial cells, have been found to possess innate immune functions by expressing pattern-recognition receptors (PRRs), producing complement components, and secreting cytokines. Intriguingly, in recent years, it has been discovered that liver epithelial cells also produce immunoglobulins (Igs), which have long been thought to be produced exclusively by B cells. Notably, even liver epithelial cells from B lymphocyte-deficient mice, including SCID mice and µMT mice, could also produce Igs. Compelling evidence has revealed both the physiological and pathological functions of liver-derived Igs. For instance, liver epithelial cells-derived IgM can serve as a source of natural and specific antibodies that contribute to innate immune responses, while liver-produced IgG can act as a growth factor to promote cell proliferation and survival in normal hepatocytes and hepatocarcinoma. Similar to that in B cells, the toll-like receptor 9 (TLR9)-MyD88 signaling pathway is also actively involved in promoting liver epithelial cells to secrete IgM. Liver-derived Igs could potentially serve as biomarkers, prognostic indicators, and therapeutic targets in the clinical setting, particularly for liver cancers and liver injury. Nevertheless, despite significant advances, much remains unknown about the mechanisms governing Ig transcription in liver cells, as well as the detailed functions of liver-derived Igs and their involvement in diseases and adaptive immunity. Further studies are still needed to reveal these underlying, undefined issues related to the role of liver-derived Igs in both immunity and diseases.
Assuntos
Imunidade Inata , Fígado , Animais , Fígado/metabolismo , Fígado/imunologia , Humanos , Imunoglobulinas/metabolismo , Imunoglobulinas/imunologia , Imunoglobulinas/genética , Transdução de Sinais , Imunoglobulina M/imunologia , Imunoglobulina M/metabolismo , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Camundongos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Hepatócitos/metabolismo , Hepatócitos/imunologia , Relevância ClínicaRESUMO
Chronic hepatitis B virus (HBV) infection affects 300 million patients worldwide1,2, in whom virus-specific CD8 T cells by still ill-defined mechanisms lose their function and cannot eliminate HBV-infected hepatocytes3-7. Here we demonstrate that a liver immune rheostat renders virus-specific CD8 T cells refractory to activation and leads to their loss of effector functions. In preclinical models of persistent infection with hepatotropic viruses such as HBV, dysfunctional virus-specific CXCR6+ CD8 T cells accumulated in the liver and, as a characteristic hallmark, showed enhanced transcriptional activity of cAMP-responsive element modulator (CREM) distinct from T cell exhaustion. In patients with chronic hepatitis B, circulating and intrahepatic HBV-specific CXCR6+ CD8 T cells with enhanced CREM expression and transcriptional activity were detected at a frequency of 12-22% of HBV-specific CD8 T cells. Knocking out the inhibitory CREM/ICER isoform in T cells, however, failed to rescue T cell immunity. This indicates that CREM activity was a consequence, rather than the cause, of loss in T cell function, further supported by the observation of enhanced phosphorylation of protein kinase A (PKA) which is upstream of CREM. Indeed, we found that enhanced cAMP-PKA-signalling from increased T cell adenylyl cyclase activity augmented CREM activity and curbed T cell activation and effector function in persistent hepatic infection. Mechanistically, CD8 T cells recognizing their antigen on hepatocytes established close and extensive contact with liver sinusoidal endothelial cells, thereby enhancing adenylyl cyclase-cAMP-PKA signalling in T cells. In these hepatic CD8 T cells, which recognize their antigen on hepatocytes, phosphorylation of key signalling kinases of the T cell receptor signalling pathway was impaired, which rendered them refractory to activation. Thus, close contact with liver sinusoidal endothelial cells curbs the activation and effector function of HBV-specific CD8 T cells that target hepatocytes expressing viral antigens by means of the adenylyl cyclase-cAMP-PKA axis in an immune rheostat-like fashion.
Assuntos
Linfócitos T CD8-Positivos , Hepatite B Crônica , Fígado , Animais , Humanos , Masculino , Camundongos , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Hepatócitos/imunologia , Hepatócitos/virologia , Fígado/imunologia , Fígado/virologia , Fosforilação , Transdução de Sinais , Ativação LinfocitáriaRESUMO
Trained immunity is classically characterized by long-term functional reprogramming of innate immune cells to combat infectious diseases. Infection-induced organ injury is a common clinical severity phenotype of sepsis. However, whether the induction of trained immunity plays a role in protecting septic organ injury remains largely unknown. Here, through establishing an in vivo ß-glucan training and lipopolysaccharide (LPS) challenge model in zebrafish larvae, we observe that induction of trained immunity could inhibit pyroptosis of hepatocytes to alleviate septic liver injury, with an elevated trimethyl-histone H3 lysine 4 (H3K4me3) modification that targets mitophagy-related genes. Moreover, we identify a C-type lectin domain receptor in zebrafish, named DrDectin-1, which is revealed as the orchestrator in gating H3K4me3 rewiring-mediated mitophagy activation and alleviating pyroptosis-engaged septic liver injury in vivo. Taken together, our results uncover tissue-resident trained immunity in maintaining liver homeostasis at the whole-animal level and offer an in vivo model to efficiently integrate trained immunity for immunotherapies.
Assuntos
Hepatócitos , Piroptose , Sepse , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Hepatócitos/metabolismo , Hepatócitos/imunologia , Sepse/imunologia , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Lipopolissacarídeos , Fígado/patologia , Fígado/metabolismo , Fígado/imunologia , Mitofagia , Lectinas Tipo C/metabolismo , Imunidade Inata , Histonas/metabolismo , beta-Glucanas/farmacologia , Imunidade TreinadaRESUMO
Chronic hepatitis B poses a significant global burden, modulating immune cells, leading to chronic inflammation and long-term damage. Due to its hepatotropism, the hepatitis B virus (HBV) cannot infect other cells. The mechanisms underlying the intercellular communication among different liver cells in HBV-infected individuals and the immune microenvironment imbalance remain elusive. Exosomes, as important intercellular communication and cargo transportation tools between HBV-infected hepatocytes and immune cells, have been shown to assist in HBV cargo transportation and regulate the immune microenvironment. However, the role of exosomes in hepatitis B has only gradually received attention in recent years. Minimal literature has systematically elaborated on the role of exosomes in reshaping the immune microenvironment of the liver. This review unfolds sequentially based on the biological processes of exosomes: exosomes' biogenesis, release, transport, uptake by recipient cells, and their impact on recipient cells. We delineate how HBV influences the biogenesis of exosomes, utilizing exosomal covert transmission, and reshapes the hepatic immune microenvironment. And based on the characteristics and functions of exosomes, potential applications of exosomes in hepatitis B are summarized and predicted.
Assuntos
Exossomos , Vírus da Hepatite B , Hepatite B Crônica , Hepatócitos , Fígado , Exossomos/imunologia , Exossomos/metabolismo , Humanos , Vírus da Hepatite B/imunologia , Fígado/imunologia , Fígado/virologia , Animais , Hepatite B Crônica/imunologia , Hepatócitos/virologia , Hepatócitos/imunologia , Comunicação Celular , Microambiente Celular/imunologia , Hepatite B/imunologia , Hepatite B/virologiaRESUMO
In the fight against antimicrobial resistance, host defense peptides (HDPs) are increasingly referred to as promising molecules for the design of new antimicrobial agents. In terms of their future clinical use, particularly small, synthetic HDPs offer several advantages, based on which their application as feed additives has aroused great interest in the poultry sector. However, given their complex mechanism of action and the limited data about the cellular effects in production animals, their investigation is of great importance in these species. The present study aimed to examine the immunomodulatory activity of the synthetic HDP Pap12-6 (PAP) solely and in inflammatory environments evoked by lipoteichoic acid (LTA) and polyinosinic-polycytidylic acid (Poly I:C), in a primary chicken hepatocyte-non-parenchymal cell co-culture. Based on the investigation of the extracellular lactate dehydrogenase (LDH) activity, PAP seemed to exert no cytotoxicity on hepatic cells, suggesting its safe application. Moreover, PAP was able to influence the immune response, reflected by the decreased production of interleukin (IL)-6, IL-8, and "regulated on activation, normal T cell expressed and secreted"(RANTES), as well as the reduced IL-6/IL-10 ratio in Poly I:C-induced inflammation. PAP also diminished the levels of extracellular H2O2 and nuclear factor erythroid 2-related factor 2 (Nrf2) when applied together with Poly I:C and in both inflammatory conditions, respectively. Consequently, PAP appeared to display potent immunomodulatory activity, preferring to act towards the cellular anti-inflammatory and antioxidant processes. These findings confirm that PAP might be a promising alternative for designing novel antimicrobial immunomodulatory agents for chickens, thereby contributing to the reduction of the use of conventional antibiotics.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Hepatócitos , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Células Cultivadas , Galinhas , Técnicas de Cocultura , Citocinas/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/imunologia , Hepatócitos/metabolismo , Agentes de Imunomodulação/farmacologia , Agentes de Imunomodulação/química , Lipopolissacarídeos/farmacologia , Poli I-C/farmacologia , Ácidos Teicoicos/farmacologiaRESUMO
Infections of hepatotropic viruses cause a wide array of liver diseases including acute hepatitis, chronic hepatitis and the consequently developed cirrhosis and hepatocellular carcinoma (HCC). Among the five classical hepatotropic viruses, hepatitis B virus (HBV) and hepatitis C virus (HCV) usually infect human persistently and cause chronic hepatitis, leading to major troubles to humanity. Previous studies have revealed that several types of inflammasomes are involved in the infections of HBV and HCV. Here, we summarize the current knowledge about their roles in hepatitis B and C. NLRP3 inflammasome can be activated and regulated by HBV and HCV. It is found to exert antiviral function or mediates inflammatory response in viral infections depending on different experimental models. Besides NLRP3 inflammasome, IFI16 and AIM2 inflammasomes participate in the pathological process of hepatitis B, and NALP3 inflammasome may sense HCV infection in hepatocytes. The inflammasomes affect the pathological process of viral hepatitis through its downstream secretion of inflammatory cytokines interleukin-1ß (IL-1ß) and IL-18 or induction of pyroptosis resulting from cleaved gasdermin D (GSDMD). However, the roles of inflammasomes in different stages of viral infection remains mainly unclear. More proper experimental models of viral hepatitis should be developed for specific studies in future, so that we can understand more about the complexity of inflammasome regulation and multifunction of inflammasomes and their downstream effectors during HBV and HCV infections.
Assuntos
Hepacivirus , Vírus da Hepatite B , Hepatite B Crônica , Hepatite C Crônica , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Humanos , Inflamassomos/metabolismo , Inflamassomos/imunologia , Hepatite C Crônica/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Hepacivirus/imunologia , Hepatite B Crônica/imunologia , Hepatite B Crônica/metabolismo , Vírus da Hepatite B/imunologia , Proteínas de Ligação a DNA/metabolismo , Interleucina-1beta/metabolismo , Piroptose , Animais , Fosfoproteínas/metabolismo , Proteínas Nucleares/metabolismo , Hepatócitos/virologia , Hepatócitos/imunologia , Interleucina-18/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , GasderminasRESUMO
BACKGROUND AND AIMS: Activation of the cGAS-STING pathway induces the production of type I interferons, initiating the antiviral immune response, which contributes to the clearance of pathogens. Previous studies have shown that STING agonists promote hepatitis B virus (HBV) clearance; however, few studies have investigated the effect of activating the cGAS-STING pathway in macrophages on HBV. METHODS: The polarization status of HBV particle-stimulated RAW264.7 macrophages was analyzed. After stimulation with HBV particles, the analysis focused on determining whether the DNA sensors in RAW264.7 macrophages recognized the viral double-stranded DNA (dsDNA) and evaluating the activation of the cGAS-STING pathway. Coculture of mouse macrophages and hepatocytes harboring HBV was used to study the antiviral activity of HBV-stimulated RAW264.7 macrophages. RESULTS: After stimulation with HBV particles, HBV relaxed circular DNA (rcDNA) was detected in RAW264.7 macrophages, and the protein expression of phospho-STING, phospho-TBK1, and phospho-IRF3 in the STING pathway was increased, as shown by Western blot analysis, which revealed that M1 polarization of macrophages was caused by increased expression of CD86. RT-PCR analyses revealed elevated expression of M1 macrophage polarization-associated cytokines such as TNFα, IL-1ß, iNOS, and IFNα/ß. In the coculture experiment, both HBsAg and HBeAg expression levels were significantly decreased in AML12-HBV1.3 cells cocultured with the supernatants of HBV-stimulated RAW264.7 macrophages. CONCLUSION: The results suggest that macrophages can endocytose HBV particles. Additionally, viral dsDNA can be recognized by DNA pattern recognition receptors, which in turn activate the cGAS-STING pathway, promoting the M1 polarization of macrophages, while no significant M2 polarization is observed. Macrophages stimulated with HBV particles exhibit enhanced antiviral activity against HBV.
Assuntos
DNA Viral , Vírus da Hepatite B , Macrófagos , Proteínas de Membrana , Nucleotidiltransferases , Transdução de Sinais , Vírus da Hepatite B/fisiologia , Vírus da Hepatite B/imunologia , Animais , Nucleotidiltransferases/metabolismo , Camundongos , Macrófagos/imunologia , Macrófagos/virologia , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Células RAW 264.7 , Hepatite B/imunologia , Hepatite B/virologia , Humanos , Ativação de Macrófagos/imunologia , Hepatócitos/virologia , Hepatócitos/imunologia , Hepatócitos/metabolismo , Fator Regulador 3 de Interferon/metabolismoRESUMO
The Plasmodium parasites that cause malaria undergo asymptomatic development in the parenchymal cells of the liver, the hepatocytes, prior to infecting erythrocytes and causing clinical disease. Traditionally, hepatocytes have been perceived as passive bystanders that allow hepatotropic pathogens such as Plasmodium to develop relatively unchallenged. However, now there is emerging evidence suggesting that hepatocytes can mount robust cell-autonomous immune responses that target Plasmodium, limiting its progression to the blood and reducing the incidence and severity of clinical malaria. Here we discuss our current understanding of hepatocyte cell-intrinsic immune responses that target Plasmodium and how these pathways impact malaria.
Assuntos
Hepatócitos , Malária , Plasmodium , Plasmodium/imunologia , Plasmodium/fisiologia , Humanos , Malária/imunologia , Malária/parasitologia , Hepatócitos/parasitologia , Hepatócitos/imunologia , AnimaisRESUMO
The pathogenesis of viral infection is attributed to two folds: intrinsic cell death pathway activation due to the viral cytopathic effect, and immune-mediated extrinsic cellular injuries. The immune system, encompassing both innate and adaptive immunity, therefore acts as a double-edged sword in viral infection. Insufficient potency permits pathogens to establish lifelong persistent infection and its consequences, while excessive activation leads to organ damage beyond its mission to control viral pathogens. The innate immune response serves as the front line of defense against viral infection, which is triggered through the recognition of viral products, referred to as pathogen-associated molecular patterns (PAMPs), by host cell pattern recognition receptors (PRRs). The PRRs-PAMPs interaction results in the induction of interferon-stimulated genes (ISGs) in infected cells, as well as the secretion of interferons (IFNs), to establish a tissue-wide antiviral state in an autocrine and paracrine manner. Cumulative evidence suggests significant variability in the expression patterns of PRRs, the induction potency of ISGs and IFNs, and the IFN response across different cell types and species. Hence, in our understanding of viral hepatitis pathogenesis, insights gained through hepatoma cell lines or murine-based experimental systems are uncertain in precisely recapitulating the innate antiviral response of genuine human hepatocytes. Accordingly, this review article aims to extract and summarize evidence made possible with bona fide human hepatocytes-based study tools, along with their clinical relevance and implications, as well as to identify the remaining gaps in knowledge for future investigations.
Assuntos
Vírus Delta da Hepatite , Hepatócitos , Imunidade Inata , Interferons , Receptores de Reconhecimento de Padrão , Humanos , Hepatite D/imunologia , Hepatite D/virologia , Vírus Delta da Hepatite/imunologia , Vírus Delta da Hepatite/fisiologia , Hepatócitos/virologia , Hepatócitos/imunologia , Interações Hospedeiro-Patógeno/imunologia , Interferons/imunologia , Interferons/metabolismo , Moléculas com Motivos Associados a Patógenos/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Receptores de Reconhecimento de Padrão/imunologiaRESUMO
Hepatitis B virus (HBV) is a major driver of chronic hepatic inflammation, which regularly leads to liver cirrhosis or hepatocellular carcinoma. Immediate innate immune cell response is crucial for the rapid clearance of the infection. Here, natural killer (NK) cells play a pivotal role in direct cytotoxicity and the secretion of antiviral cytokines as well as regulatory function. The aim of this study was to further elucidate NK cell responses triggered by an HBV infection. Therefore, we optimized HBV in vitro models that reliably stimulate NK cells using hepatocyte-like HepG2 cells expressing the Na+-taurocholate co-transporting polypeptide (NTCP) and HepaRG cells. Immune cells were acquired from healthy platelet donors. Initially, HepG2-NTCP cells demonstrated higher viral replication compared to HepaRG cells. Co-cultures with immune cells revealed increased production of interferon-γ and tumor necrosis factor-α by NK cells, which was no longer evident in isolated NK cells. Likewise, the depletion of monocytes and spatial separation from target cells led to the absence of the antiviral cytokine production of NK cells. Eventually, the combined co-culture of isolated NK cells and monocytes led to a sufficient cytokine response of NK cells, which was also apparent when communication between the two immune cell subpopulations was restricted to soluble factors. In summary, our study demonstrates antiviral cytokine production by NK cells in response to HBV+ HepG2-NTCP cells, which is dependent on monocyte bystander activation.
Assuntos
Técnicas de Cocultura , Citocinas , Vírus da Hepatite B , Hepatite B , Células Matadoras Naturais , Monócitos , Humanos , Células Matadoras Naturais/imunologia , Monócitos/imunologia , Monócitos/virologia , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/fisiologia , Citocinas/metabolismo , Células Hep G2 , Hepatite B/imunologia , Hepatite B/virologia , Replicação Viral , Interferon gama/metabolismo , Interferon gama/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Hepatócitos/virologia , Hepatócitos/imunologiaRESUMO
BACKGROUND & AIMS: The immune tolerance induced by hepatitis B virus (HBV) is a major challenge for achieving effective viral clearance, and the mechanisms involved are not well-understood. One potential factor involved in modulating immune responses is mesencephalic astrocyte-derived neurotrophic factor (MANF), which has been reported to be increased in patients with chronic hepatitis B. In this study, our objective is to examine the role of MANF in regulating immune responses to HBV. METHODS: We utilized a commonly used HBV-harboring mouse model, where mice were hydrodynamically injected with the pAAV/HBV1.2 plasmid. We assessed the HBV load by measuring the levels of various markers including hepatitis B surface antigen, hepatitis B envelope antigen, hepatitis B core antigen, HBV DNA, and HBV RNA. RESULTS: Our study revealed that following HBV infection, both myeloid cells and hepatocytes exhibited increased expression of MANF. Moreover, we observed that mice with myeloid-specific MANF knockout (ManfMye-/-) displayed reduced HBV load and improved HBV-specific T cell responses. The decreased HBV-induced tolerance in ManfMye-/- mice was associated with reduced accumulation of myeloid-derived suppressor cells (MDSCs) in the liver. Restoring MDSC levels in ManfMye-/- mice through MDSC adoptive transfer reinstated HBV-induced tolerance. Mechanistically, we found that MANF promoted MDSC expansion by activating the IL-6/STAT3 pathway. Importantly, our study demonstrated the effectiveness of a combination therapy involving an hepatitis B surface antigen vaccine and nanoparticle-encapsulated MANF siRNA in effectively clearing HBV in HBV-carrier mice. CONCLUSION: The current study reveals that MANF plays a previously unrecognized regulatory role in liver tolerance by expanding MDSCs in the liver through IL-6/STAT3 signaling, leading to MDSC-mediated CD8+ T cell exhaustion.
Assuntos
Vírus da Hepatite B , Tolerância Imunológica , Camundongos Knockout , Fatores de Crescimento Neural , Fator de Transcrição STAT3 , Animais , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/genética , Vírus da Hepatite B/imunologia , Camundongos , Fator de Transcrição STAT3/metabolismo , Modelos Animais de Doenças , Humanos , Hepatócitos/metabolismo , Hepatócitos/virologia , Hepatócitos/imunologia , Hepatite B/imunologia , Hepatite B/virologia , Carga Viral , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos de Superfície da Hepatite B/imunologia , Fígado/imunologia , Fígado/metabolismo , Fígado/virologia , Camundongos Endogâmicos C57BL , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologiaRESUMO
BACKGROUND AND AIMS: The outcomes of HBV infections are related to complex immune imbalances; however, the precise mechanisms by which HBV induces immune dysfunction are not well understood. METHODS: HBV transgenic (HBs-Tg) mice were used to investigate intrahepatic NK cells in two distinct subsets: conventional NK (cNK) and liver-resident NK (LrNK) cells during a chronic HBV infection. RESULTS: The cNK cells, but not the LrNK cells, were primarily responsible for the increase in the number of bulk NK cells in the livers of ageing HBs-Tg mice. The hepatic cNK cells showed a stronger ability to produce IL-10, coupled with a higher expression of CD69, TIGIT and PD-L1, and lower NKG2D expression in ageing HBs-Tg mice. A lower mitochondrial mass and membrane potential, and less polarized localization were observed in the hepatic cNK cells compared with the splenic cNK cells in the HBs-Tg mice. The enhanced galectin-3 (Gal-3) secreted from HBsAg+ hepatocytes accounted for the IL-10 production of hepatic cNK cells via ITGB1 signaling. For humans, LGALS3 and ITGB1 expression is positively correlated with IL-10 expression, and negatively correlated with the poor clinical progression of HCC. CONCLUSIONS: Gal-3-ITGB1 signaling shapes hepatic cNK cells but not LrNK cells during a chronic HBV infection, which may correlate with HCC progression.
Assuntos
Carcinoma Hepatocelular , Vírus da Hepatite B , Interleucina-10 , Células Matadoras Naturais , Neoplasias Hepáticas , Transdução de Sinais , Animais , Feminino , Humanos , Masculino , Camundongos , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/virologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Progressão da Doença , Galectina 3/genética , Galectina 3/metabolismo , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatócitos/virologia , Hepatócitos/metabolismo , Hepatócitos/imunologia , Interleucina-10/genética , Interleucina-10/metabolismo , Células Matadoras Naturais/imunologia , Fígado/patologia , Fígado/imunologia , Fígado/virologia , Fígado/metabolismo , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/virologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Macrophages are functionally heterogeneous cells essential for apoptotic cell clearance. Apoptotic cells are defined by homogeneous characteristics, ignoring their original cell lineage identity. We found that in an interleukin-4 (IL-4)-enriched environment, the sensing of apoptotic neutrophils by macrophages triggered their tissue remodeling signature. Engulfment of apoptotic hepatocytes promoted a tolerogenic phenotype, whereas phagocytosis of T cells had little effect on IL-4-induced gene expression. In a mouse model of parasite-induced pathology, the transfer of macrophages conditioned with IL-4 and apoptotic neutrophils promoted parasitic egg clearance. Knockout of phagocytic receptors required for the uptake of apoptotic neutrophils and partially T cells, but not hepatocytes, exacerbated helminth infection. These findings suggest that the identity of apoptotic cells may contribute to the development of distinct IL-4-driven immune programs in macrophages.
Assuntos
Apoptose , Interleucina-4 , Macrófagos , Fagocitose , Esquistossomose mansoni , Animais , Camundongos , Apoptose/imunologia , Hepatócitos/imunologia , Interleucina-4/genética , Interleucina-4/metabolismo , Macrófagos/imunologia , Camundongos Knockout , Neutrófilos/imunologia , Fagocitose/imunologia , Esquistossomose mansoni/genética , Esquistossomose mansoni/imunologia , Modelos Animais de DoençasRESUMO
T cell infiltration into tumors is a favorable prognostic feature, but most solid tumors lack productive T cell responses. Mechanisms that coordinate T cell exclusion are incompletely understood. Here we identify hepatocyte activation via interleukin-6/STAT3 and secretion of serum amyloid A (SAA) proteins 1 and 2 as important regulators of T cell surveillance of extrahepatic tumors. Loss of STAT3 in hepatocytes or SAA remodeled the tumor microenvironment with infiltration by CD8+ T cells, while interleukin-6 overexpression in hepatocytes and SAA signaling via Toll-like receptor 2 reduced the number of intratumoral dendritic cells and, in doing so, inhibited T cell tumor infiltration. Genetic ablation of SAA enhanced survival after tumor resection in a T cell-dependent manner. Likewise, in individuals with pancreatic ductal adenocarcinoma, long-term survivors after surgery demonstrated lower serum SAA levels than short-term survivors. Taken together, these data define a fundamental link between liver and tumor immunobiology wherein hepatocytes govern productive T cell surveillance in cancer.
Assuntos
Linfócitos T CD8-Positivos , Hepatócitos , Interleucina-6 , Fator de Transcrição STAT3 , Proteína Amiloide A Sérica , Proteína Amiloide A Sérica/metabolismo , Proteína Amiloide A Sérica/genética , Hepatócitos/metabolismo , Hepatócitos/imunologia , Animais , Humanos , Camundongos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Microambiente Tumoral/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Evasão Tumoral , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Transdução de Sinais , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/patologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linhagem Celular TumoralRESUMO
Previously, we reported a neutralizing monoclonal antibody, A8A11, raised against a novel conserved epitope within the hepatitis C virus (HCV) E2 protein, that could significantly reduce HCV replication. Here, we report the nucleotide sequence of A8A11 and demonstrate the efficacy of a single-chain variable fragment (scFv) protein that mimics the antibody, inhibits the binding of an HCV virus-like particle to hepatocytes, and reduces viral RNA replication in a cell culture system. More importantly, scFv A8A11 was found to effectively restrict the increase of viral RNA levels in the serum of HCV-infected chimeric mice harbouring human hepatocytes. These results suggest a promising approach to neutralizing-antibody-based therapeutic interventions against HCV infection.
Assuntos
Epitopos , Hepacivirus , Hepatócitos , Anticorpos de Cadeia Única , Proteínas do Envelope Viral , Internalização do Vírus , Hepacivirus/imunologia , Hepacivirus/genética , Hepacivirus/fisiologia , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/genética , Hepatócitos/virologia , Hepatócitos/imunologia , Animais , Humanos , Epitopos/imunologia , Camundongos , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Hepatite C/virologia , Hepatite C/imunologia , Anticorpos Neutralizantes/imunologia , Replicação Viral , Anticorpos Monoclonais/imunologiaRESUMO
The aim of this paper is to develop and investigate a novel mathematical model of the dynamical behaviors of chronic hepatitis B virus infection. The model includes exposed infected hepatocytes, intracellular HBV DNA-containing capsids, uses a general incidence function for viral infection covering a variety of special cases available in the literature, and describes the interaction of cytotoxic T lymphocytes that kill the infected hepatocytes and the magnitude of B-cells that send antibody immune defense to neutralize free virions. Further, one time delay is incorporated to account for actual capsids production. The other time delays are used to account for maturation of capsids and free viruses. We start with the analysis of the proposed model by establishing the local and global existence, uniqueness, non-negativity and boundedness of solutions. After defined the threshold parameters, we discuss the stability properties of all possible steady state constants by using the crafty Lyapunov functionals, the LaSalle's invariance principle and linearization methods. The impacts of the three time delays on the HBV infection transmission are discussed through local and global sensitivity analysis of the basic reproduction number and of the classes of infected states. Finally, an application is provided and numerical simulations are performed to illustrate and interpret the theoretical results obtained. It is suggested that, a good strategy to eradicate or to control HBV infection within a host should concentrate on any drugs that may prolong the values of the three delays.