RESUMO
Liver disease is one of the leading comorbidities in HIV infection. The risk of liver fibrosis development is potentiated by alcohol abuse. In our previous studies, we reported that hepatocytes exposed to HIV and acetaldehyde undergo significant apoptosis, and the engulfment of apoptotic bodies (ABs) by hepatic stellate cells (HSC) potentiates their pro-fibrotic activation. However, in addition to hepatocytes, under the same conditions, ABs can be generated from liver-infiltrating immune cells. The goal of this study is to explore whether lymphocyte-derived ABs trigger HSC profibrotic activation as strongly as hepatocyte-derived ABs. ABs were generated from Huh7.5-CYP2E1 (RLW) cells and Jurkat cells treated with HIV+acetaldehyde and co-culture with HSC to induce their pro-fibrotic activation. ABs cargo was analyzed by proteomics. ABs generated from RLW, but not from Jurkat cells activated fibrogenic genes in HSC. This was driven by the expression of hepatocyte-specific proteins in ABs cargo. One of these proteins is Hepatocyte-Derived Growth Factor, for which suppression attenuates pro-fibrotic activation of HSC. In mice humanized with only immune cells but not human hepatocytes, infected with HIV and fed ethanol, liver fibrosis was not observed. We conclude that HIV+ABs of hepatocyte origin promote HSC activation, which potentially may lead to liver fibrosis progression.
Assuntos
Vesículas Extracelulares , Infecções por HIV , Camundongos , Animais , Células Estreladas do Fígado/metabolismo , Etanol/metabolismo , Infecções por HIV/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Cirrose Hepática/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Acetaldeído/metabolismo , Vesículas Extracelulares/metabolismoRESUMO
BACKGROUND AND AIMS: Approximately 3.5% of the global population is chronically infected with Hepatitis B Virus (HBV), which puts them at high risk of end-stage liver disease, with the risk of persistent infection potentiated by alcohol consumption. However, the mechanisms underlying the effects of alcohol on HBV persistence remain unclear. Here, we aimed to establish in vivo/ex vivo evidence that alcohol suppresses HBV peptides-major histocompatibility complex (MHC) class I antigen display on primary human hepatocytes (PHH), which diminishes the recognition and clearance of HBV-infected hepatocytes by cytotoxic T-lymphocytes (CTLs). METHODS: We used fumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chain knock-out (FRG-KO) humanized mice transplanted with human leukocyte antigen-A2 (HLA-A2)-positive hepatocytes. The mice were HBV-infected and fed control and alcohol diets. Isolated hepatocytes were exposed ex vivo to HBV 18-27-HLA-A2-restricted CTLs to quantify cytotoxicity. For mechanistic studies, we measured proteasome activities, unfolded protein response (UPR), and endoplasmic reticulum (ER) stress in hepatocytes from HBV-infected humanized mouse livers. RESULTS AND CONCLUSIONS: We found that alcohol feeding attenuated HBV core 18-27-HLA-A2 complex presentation on infected hepatocytes due to the suppression of proteasome function and ER stress induction, which diminished both the processing of HBV peptides and trafficking of HBV-MHC class I complexes to the hepatocyte surface. This alcohol-mediated decrease in MHC class I-restricted antigen presentation of the CTL epitope on target hepatocytes reduced the CTL-specific elimination of infected cells, potentially leading to HBV-infection persistence, which promotes end-stage liver disease outcomes.
Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Etanol/farmacologia , Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Hepatócitos/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Doença Hepática Terminal/virologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Antígeno HLA-A2/análise , Hepatócitos/transplante , Hepatócitos/virologia , Xenoenxertos , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Camundongos , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/fisiologia , Resposta a Proteínas não Dobradas/genéticaRESUMO
Alcohol consumption worsens hepatitis B virus (HBV) infection pathogenesis. We have recently reported that acetaldehyde suppressed HBV peptide-major histocompatibility complex I (MHC class I) complex display on hepatocytes, limiting recognition and subsequent removal of the infected hepatocytes by HBV-specific cytotoxic T lymphocytes (CTLs). This suppression was attributed to impaired processing of antigenic peptides by the proteasome. However, in addition to proteasome dysfunction, alcohol may induce endoplasmic reticulum (ER) stress and Golgi fragmentation in HBV-infected liver cells to reduce uploading of viral peptides to MHC class I and/or trafficking of this complex to the hepatocyte surface. Hence, the aim of this study was to elucidate whether alcohol-induced ER stress and Golgi fragmentation affect HBV peptide-MHC class I complex presentation on HBV+ hepatocytes. Here, we demonstrate that, while both acetaldehyde and HBV independently cause ER stress and Golgi fragmentation, the combined exposure provided an additive effect. Thus we observed an activation of the inositol-requiring enzyme 1α-X-box binding protein 1 and activation transcription factor (ATF)6α, but not the phospho PKR-like ER kinase-phospho eukaryotic initiation factor 2α-ATF4-C/EBP homologous protein arms of ER stress in HBV-transfected cells treated with acetaldehyde-generating system (AGS). In addition, Golgi proteins trans-Golgi network 46, GM130, and Giantin revealed punctate distribution, indicating Golgi fragmentation upon AGS exposure. Furthermore, the effects of acetaldehyde were reproduced by treatment with ER stress inducers, thapsigargin and tunicamycin, which also decreased the display of this complex and MHC class I turnover in HepG2.2.15 cells and HBV-infected primary human hepatocytes. Taken together, alcohol-induced ER stress and Golgi fragmentation contribute to the suppression of HBV peptide-MHC class I complex presentation on HBV+ hepatocytes, which may diminish their recognition by CTLs and promote persistence of HBV infection in hepatocytes.NEW & NOTEWORTHY Our current findings show that acetaldehyde accelerates endoplasmic reticulum (ER) stress by activating the unfolded protein response arms inositol-requiring enzyme 1α-X-box binding protein 1 and activation transcription factor (ATF)6α but not phospho PKR-like ER kinase-p eukaryotic initiation factor 2α-ATF4-C/EBP homologous protein in hepatitis B virus (HBV)-transfected HepG2.2.15 cells. It also potentiates Golgi fragmentation, as evident by punctate distribution of Golgi proteins, GM130, trans-Golgi network 46, and Giantin. While concomitantly increasing HBV DNA and HBV surface antigen titers, acetaldehyde-induced ER stress suppresses the presentation of HBV peptide-major histocompatibility complex I complexes on hepatocyte surfaces, thereby promoting the persistence of HBV infection in the liver.
Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Vírus da Hepatite B/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Fígado/virologia , Acetaldeído , Estresse do Retículo Endoplasmático/genética , Expressão Gênica/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Antígeno HLA-A2/análise , Células Hep G2 , Vírus da Hepatite B/genética , Antígenos de Histocompatibilidade Classe I/efeitos dos fármacos , Humanos , Fígado/imunologia , RNA Mensageiro/análise , Transfecção , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genéticaRESUMO
Nowadays, there is a strong request for the treatment of chronic HBV-infection with direct acting antivirals. Furthermore, prevalent human immunodeficiency virus (HIV-1) and hepatitis B (HBV) co-infections highlight an immediate need for dual long-acting and easily administered antivirals. To this end, we modified lamivudine (3TC), a nucleoside analog inhibitor of both viruses, into a lipophilic monophosphorylated prodrug (M23TC). Prior work demonstrated that nanoformulation of M23TC (NM23TC) enhanced drug stability, controlled dissolution and improved access to sites of viral replication. The present study evaluated the efficacy of a NM23TC in HBV-infected chimeric liver humanized mice. Levels of HBV DNA and HBsAg in plasma were monitored up to 8 weeks posttreatment. A single intramuscular dose of 75 mg/kg 3TC equivalents of nanoformulated NM23TC provided sustained drug levels and suppressed HBV replication in humanized mice for 4 weeks. The results support further development of this long-acting 3TC nanoformulation for HBV treatment and prevention.
Assuntos
Lamivudina/química , Animais , Antivirais/química , Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Imuno-Histoquímica , Lamivudina/farmacologia , Masculino , Camundongos , Camundongos Knockout , Ratos , Ratos Sprague-Dawley , Replicação Viral/efeitos dos fármacosRESUMO
Hepatitis B virus (HBV) infection and alcoholism are major public health problems worldwide, contributing to the development of end-stage liver disease. Alcohol intake affects HBV infection pathogenesis and treatment outcomes. HBV-specific cytotoxic T lymphocytes (CTLs) play an important role in HBV clearance. Many previous studies have focused on alcohol-induced impairments of the immune response. However, it is not clear whether alcohol alters the presentation of HBV peptide-major histocompatibility complex (MHC) class I complexes on infected hepatocytes resulting in escape of its recognition by CTLs. Hence, the focus of this study was to investigate the mechanisms by which ethanol metabolism affects the presentation of CTL epitope on HBV-infected hepatocytes. As demonstrated here, although continuous cell exposure to acetaldehyde-generating system (AGS) increased HBV load in HepG2.2.15 cells, it decreased the expression of HBV core peptide 18-27-human leukocyte antigen-A2complex (CTL epitope) on the cell surface. Moreover, we observed AGS-induced suppression of chymotrypsin- and trypsin-like proteasome activities necessary for peptide processing by proteasome as well as a decline in IFNγ-stimulated immunoproteasome (IPR) function and expression of PA28 activator and immunoproteasome subunits LMP7 and LMP2. Furthermore, IFNγ-induced activation of peptide-loading complex (PLC) components, such as transporter associated with antigen processing (TAP1) and tapasin, were suppressed by AGS. The attenuation of IPR and PLC activation was attributed to AGS-triggered impairment of IFNγ signaling in HepG2.2.15 cells. Collectively, all these downstream events reduced the display of HBV peptide-MHC class I complexes on the hepatocyte surface, which may suppress CTL activation and the recognition of CTL epitopes on HBV-expressing hepatocytes by immune cells, thereby leading to persistence of liver inflammation.NEW & NOTEWORTHY Our study shows that in HBV-expressing HepG2.2.15 cells, acetaldehyde alters HBV peptide processing by suppressing chymotrypsin- and trypsin-like proteasome activities and decreases IFNγ-stimulated immunoproteasome function and expression of PA28 activator and immunoproteasome subunits. It also suppresses IFNγ-induced activation of peptide-loading complex (PLC) components due to impairment of IFNγ signaling via the JAK-STAT1 pathway. These acetaldehyde-induced dysfunctions reduced the display of HBV peptide-MHC class I complexes on the hepatocyte surface, thereby leading to persistence of HBV infection.
Assuntos
Acetaldeído/metabolismo , Quimases/metabolismo , Etanol/metabolismo , Hepatite B , Complexo Principal de Histocompatibilidade/imunologia , Serina Endopeptidases/metabolismo , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Apresentação de Antígeno , Antígenos HLA-D/imunologia , Células Hep G2 , Hepatite B/imunologia , Hepatite B/metabolismo , Vírus da Hepatite B/imunologia , Humanos , Interferon gama/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Linfócitos T Citotóxicos/imunologiaRESUMO
HIV-HCV co-infection causes rapid progression of liver fibrosis. These outcomes to liver cirrhosis can be improved, but not stopped by specific antiviral therapies. Due to high significance of HIV-HCV interactions for morbidity and mortality in co-infected patients, our attention was attracted to the multi-component pathogenesis of fibrosis progression as the transition to end-stage liver disease development. In this study, we hypothesize that increased matrix stiffness enhances apoptosis in HCV-HIV-co-infected hepatocytes and that capturing of apoptotic bodies (AB) derived from these infected hepatocytes by hepatic stellate cells (HSC) drives the fibrosis progression. As the source of viruses, JFH1 (HCV genotype 2a) and HIV-1ADA (either purified or containing in infected macrophage supernatants) were chosen. Using Huh7.5-CYP (RLW) cells and primary human hepatocytes mono-infected with HCV and HIV or co-infected, we have shown that both HCV and HIV RNA levels were increased in co-infected cells, which was accompanied by hepatocyte apoptosis. This apoptosis was attenuated by azidothymidine treatment. The levels of both infections and apoptosis were more prominent in primary hepatocytes cultured on substrates mimicking fibrotic stiffness (24â¯kPa-stiff) compared to substrates mimicking healthy liver (2.4â¯kPa-soft). The engulfment of AB from pathogen-exposed hepatocytes activated pro-fibrotic mRNAs in HSC. Overall, the increased matrix stiffness is not only a consequence of liver inflammation/fibrosis, but the condition that further accelerates liver fibrosis development. This is attributed to the switching of HSC to pro-fibrotic phenotype by capturing of excessive amounts of apoptotic HCV- and HIV-infected hepatocytes.
Assuntos
Apoptose , Coinfecção/patologia , Progressão da Doença , Matriz Extracelular/metabolismo , Infecções por HIV/patologia , Hepatite C/patologia , Hepatócitos/virologia , Cirrose Hepática/virologia , Fenômenos Biomecânicos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Coinfecção/virologia , Módulo de Elasticidade , Infecções por HIV/virologia , Hepatite C/virologia , Hepatócitos/patologia , Humanos , Cirrose Hepática/patologia , RNA Viral/metabolismoRESUMO
Antiretroviral drug (ARV) metabolism is linked largely to hepatic cytochrome P450 activity. One ARV drug class known to be metabolized by intestinal and hepatic CYP3A are the protease inhibitors (PIs). Plasma drug concentrations are boosted by CYP3A inhibitors such as cobisistat and ritonavir (RTV). Studies of such drug-drug interactions are limited since the enzyme pathways are human specific. While immune-deficient mice reconstituted with human cells are an excellent model to study ARVs during human immunodeficiency virus type 1 (HIV-1) infection, they cannot reflect human drug metabolism. Thus, we created a mouse strain with the human pregnane X receptor, constitutive androstane receptor, and CYP3A4/7 genes on a NOD.Cg-Prkdcscid Il2rgtm1Sug /JicTac background (hCYP3A-NOG) and used them to evaluate the impact of human CYP3A metabolism on ARV pharmacokinetics. In proof-of-concept studies we used nanoformulated atazanavir (nanoATV) with or without RTV. NOG and hCYP3A-NOG mice were treated weekly with 50 mg/kg nanoATV alone or boosted with nanoformulated ritonavir (nanoATV/r). Plasma was collected weekly and liver was collected at 28 days post-treatment. Plasma and liver atazanavir (ATV) concentrations in nanoATV/r-treated hCYP3A-NOG mice were 2- to 4-fold higher than in replicate NOG mice. RTV enhanced plasma and liver ATV concentrations 3-fold in hCYP3A-NOG mice and 1.7-fold in NOG mice. The results indicate that human CYP3A-mediated drug metabolism is reduced compared with mouse and that RTV differentially affects human gene activity. These differences can affect responses to PIs in humanized mouse models of HIV-1 infection. Importantly, hCYP3A-NOG mice reconstituted with human immune cells can be used for bench-to-bedside translation.
Assuntos
Fármacos Anti-HIV/farmacologia , Citocromo P-450 CYP3A/genética , Receptor de Pregnano X/genética , Receptores Citoplasmáticos e Nucleares/genética , Animais , Fármacos Anti-HIV/farmacocinética , Receptor Constitutivo de Androstano , Interações Medicamentosas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Distribuição Tecidual , Pesquisa Translacional BiomédicaRESUMO
Human-specific HIV-1 and hepatitis co-infections significantly affect patient management and call for new therapeutic options. Small xenotransplantation models with human hepatocytes and hematolymphoid tissue should facilitate antiviral/antiretroviral drug trials. However, experience with mouse strains tested for dual reconstitution is limited, with technical difficulties such as risky manipulations with newborns and high mortality rates due to metabolic abnormalities. The best animal strains for hepatocyte transplantation are not optimal for human hematopoietic stem cell (HSC) engraftment, and vice versa. We evaluated a new strain of highly immunodeficient nonobese diabetic/Shi-scid (severe combined immunodeficiency)/IL-2Rγc(null) (NOG) mice that carry two copies of the mouse albumin promoter-driven urokinase-type plasminogen activator transgene for dual reconstitution with human liver and immune cells. Three approaches for dual reconstitution were evaluated: i) freshly isolated fetal hepatoblasts were injected intrasplenically, followed by transplantation of cryopreserved HSCs obtained from the same tissue samples 1 month later after treosulfan conditioning; ii) treosulfan conditioning is followed by intrasplenic simultaneous transplantation of fetal hepatoblasts and HSCs; and iii) transplantation of mature hepatocytes is followed by mismatched HSCs. The long-term dual reconstitution was achieved on urokinase-type plasminogen activator-NOG mice with mature hepatocytes (not fetal hepatoblasts) and HSCs. Even major histocompatibility complex mismatched transplantation was sustained without any evidence of hepatocyte rejection by the human immune system.
Assuntos
Coinfecção , Modelos Animais de Doenças , Transplante de Células-Tronco Hematopoéticas/métodos , Hepatócitos/transplante , Animais , Antineoplásicos Alquilantes/farmacologia , Bussulfano/análogos & derivados , Bussulfano/farmacologia , Infecções por HIV , Hepatite C , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Transgenes , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Antiviral therapy using nucleoside reverse transcriptase inhibitors (NRTIs) is neurotoxic and has low efficiency in eradication of HIV-1 harbored in central nervous system (CNS). Previously, we reported that active 5'-triphosphates of NRTIs encapsulated in cationic nanogels (nano-NRTIs) suppress HIV-1 activity more efficiently than NRTIs and exhibit reduced mitochondrial toxicity [Vinogradov SV, Poluektova LY, Makarov E, Gerson T, Senanayake MT. Nano-NRTIs: efficient inhibitors of HIV type-1 in macrophages with a reduced mitochondrial toxicity. Antivir Chem Chemother. 2010; 21:1-14. Makarov E, Gerson T, Senanayake T, Poluektova LY, Vinogradov. Efficient suppression of Human Immunodeficiency Virus in Macrophages by Nano-NRTIs. Antiviral Res. 2010; 86(1):A38-9]. Here, we demonstrated low neurotoxicity and excellent antiviral activity of nano-NRTIs decorated with the peptide (AP) binding brain-specific apolipoprotein E receptor. Nano-NRTIs induced lower levels of apoptosis and formation of reactive oxygen species, a major cause of neuron death, than free NRTIs. Optimization of size, surface decoration with AP significantly increased brain accumulation of nano-NRTIs. The efficient CNS delivery of nano-NRTIs resulted in up to 10-fold suppression of retroviral activity and reduced virus-associated inflammation in humanized mouse model of HIV-1 infection in the brain. Our data provide proof of the advanced efficacy of nano-NRTIs as safer alternative of current antiviral drugs. FROM THE CLINICAL EDITOR: This team of investigators demonstrated low neurotoxicity and excellent anti-HIV activity of nano-nucleoside reverse transcriptase inhibitors decorated with the peptide (AP) binding brain-specific apolipoprotein E receptor, providing proof of enhanced efficacy and a safer alternative compared with current antiviral drugs.
Assuntos
Antivirais/administração & dosagem , Infecções por HIV/tratamento farmacológico , Polietilenoglicóis/administração & dosagem , Polietilenoimina/administração & dosagem , Inibidores da Transcriptase Reversa/administração & dosagem , Animais , Antivirais/efeitos adversos , Antivirais/química , Apoptose/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/patologia , Infecções por HIV/virologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , HIV-1/patogenicidade , Humanos , Camundongos , Camundongos Transgênicos , Nanogéis , Polietilenoglicóis/química , Polietilenoimina/química , Espécies Reativas de Oxigênio/metabolismo , Inibidores da Transcriptase Reversa/efeitos adversos , Inibidores da Transcriptase Reversa/químicaRESUMO
Long-acting injectable (LAI) formulations promise to deliver patient benefits by overcoming issues associated with non-adherence. A preclinical assessment of semi-solid prodrug nanoparticle (SSPN) LAI formulations of emtricitabine (FTC) is reported here. Pharmacokinetics over 28 days were assessed in Wistar rats, New Zealand white rabbits, and Balb/C mice following intramuscular injection. Two lead formulations were assessed for the prevention of an HIV infection in NSG-cmah-/- humanised mice to ensure antiviral activities were as anticipated according to the pharmacokinetics. Cmax was reached by 12, 48, and 24 h in rats, rabbits, and mice, respectively. Plasma concentrations were below the limit of detection (2 ng/mL) by 21 days in rats and rabbits, and 28 days in mice. Mice treated with SSPN formulations demonstrated undetectable viral loads (700 copies/mL detection limit), and HIV RNA remained undetectable 28 days post-infection in plasma, spleen, lung, and liver. The in vivo data presented here demonstrate that the combined prodrug/SSPN approach can provide a dramatically extended pharmacokinetic half-life across multiple preclinical species. Species differences in renal clearance of FTC mean that longer exposures are likely to be achievable in humans than in preclinical models.
RESUMO
Neuronal damage induced by ongoing human immunodeficiency virus type 1 (HIV-1) infection was investigated in humanized NOD/scid-IL-2Rγ(c)(null) mice transplanted at birth with human CD34-positive hematopoietic stem cells. Mice infected at 5 months of age and followed for up to 15 weeks maintained significant plasma viral loads and showed reduced numbers of CD4(+) T-cells. Prospective serial proton magnetic resonance spectroscopy tests showed selective reductions in cortical N-acetyl aspartate in infected animals. Diffusion tensor imaging revealed structural changes in cortical gray matter. Postmortem immunofluorescence brain tissue examinations for neuronal and glial markers, captured by multispectral imaging microscopy and quantified by morphometric and fluorescence emission, showed regional reduction of neuronal soma and synaptic architectures. This was evidenced by loss of microtubule-associated protein 2, synaptophysin, and neurofilament antigens. This study is the first, to our knowledge, demonstrating lost neuronal integrity after HIV-1 infection in humanized mice. As such, the model permits studies of the relationships between ongoing viral replication and virus-associated neurodegeneration.
Assuntos
Progressão da Doença , Infecções por HIV/patologia , HIV-1/imunologia , Rede Nervosa/patologia , Neurônios/patologia , Animais , Antígenos CD34/administração & dosagem , Antígenos CD34/biossíntese , Transtornos Cognitivos/imunologia , Transtornos Cognitivos/patologia , Infecções por HIV/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Rede Nervosa/imunologia , Neuroglia/imunologia , Neuroglia/patologia , Neuroglia/virologia , Neurônios/imunologia , Neurônios/virologia , Estudos Prospectivos , Carga Viral/métodos , Replicação Viral/imunologiaRESUMO
Stable engraftment of human lymphoid tissue in NOD/scid-IL-2Rgammacnull mice after CD34+ hematopoietic stem cell reconstitution permits the evaluation of ongoing HIV-1 infection for weeks to months. We demonstrate that HIV-1-infected rodents develop virus-specific cellular immune responses. CD8+ cell depletion, 2 or 5-7 wk after viral infection, resulted in a significant increase of HIV-1 load, robust immune cell activation, and cytopathology in lymphoid tissues but preserved CD4/CD8 double-positive thymic T cell pools. Human CD8+ cells reappeared in circulation as early as 2-3 wk. These data support a role of CD8+ cells in viral surveillance and the relevance of this humanized mouse model for the studies of HIV-1 pathobiology and virus-specific immunity.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Animais , Separação Celular , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Receptores de Interleucina-2/deficiência , Receptores de Interleucina-2/genéticaRESUMO
Recently, we found that both HIV and acetaldehyde, an alcohol metabolite, induce hepatocyte apoptosis, resulting in the release of large extracellular vesicles called apoptotic bodies (ABs). The engulfment of these hepatocyte ABs by hepatic stellate cells (HSC) leads to their profibrotic activation. This study aims to establish the mechanisms of HSC activation after engulfment of ABs from acetaldehyde and HIV-exposed hepatocytes (ABAGS+HIV). In vitro experiments were performed on Huh7.5-CYP (RLW) cells to generate hepatocyte ABs and LX2 cells were used as HSC. To generate ABs, RLW cells were pretreated for 24 h with acetaldehyde, then exposed overnight to HIV1ADA and to acetaldehyde for 96 h. Thereafter, ABs were isolated from cell suspension by a differential centrifugation method and incubated with LX2 cells (3:1 ratio) for profibrotic genes and protein analyses. We found that HSC internalized ABs via the tyrosine kinase receptor, Axl. While the HIV gag RNA/HIV proteins accumulated in ABs elicited no productive infection in LX2 and immune cells, they triggered ROS and IL6 generation, which, in turn, activated profibrotic genes via the JNK-ERK1/2 and JAK-STAT3 pathways. Similarly, ongoing profibrotic activation was observed in immunodeficient NSG mice fed ethanol and injected with HIV-derived RLW ABs. We conclude that HSC activation by hepatocyte ABAGS+HIV engulfment is mediated by ROS-dependent JNK-ERK1/2 and IL6 triggering of JAK-STAT3 pathways. This can partially explain the mechanisms of liver fibrosis development frequently observed among alcohol abusing PLWH.
RESUMO
Few rodent models of human immunodeficiency virus type one (HIV-1) infection can reflect the course of viral infection in humans. To this end, we investigated the relationships between progressive HIV-1 infection, immune compromise, and neuroinflammatory responses in NOD/scid-IL-2Rγ(c)(null) mice reconstituted with human hematopoietic CD34(+) stem cells. Human blood-borne macrophages repopulated the meninges and perivascular spaces of chimeric animals. Viral infection in lymphoid tissue led to the accelerated entry of human cells into the brain, marked neuroinflammation, and HIV-1 replication in human mononuclear phagocytes. A meningitis and less commonly an encephalitis followed cM-T807 antibody-mediated CD8(+) cell depletion. We conclude that HIV-1-infected NOD/scid-IL-2Rγ(c)(null) humanized mice can, at least in part, recapitulate lentiviral neuropathobiology. This model of neuroAIDS reflects the virological, immunological, and early disease-associated neuropathological components of human disease.
Assuntos
Complexo AIDS Demência/etiologia , Modelos Animais de Doenças , Infecções por HIV/complicações , HIV-1/fisiologia , Camundongos , Complexo AIDS Demência/patologia , Complexo AIDS Demência/virologia , Animais , Encéfalo/imunologia , Encéfalo/virologia , Células Cultivadas , Progressão da Doença , Infecções por HIV/imunologia , Infecções por HIV/patologia , Infecções por HIV/virologia , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Meningite Viral/complicações , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos TransgênicosRESUMO
Defining the latent human immunodeficiency virus type 1 (HIV-1) burden in the human brain during progressive infection is limited by sample access. Human hematopoietic stem cells (hu-HSCs)-reconstituted humanized mice provide an opportunity for this study. The model mimics, in measure, HIV-1 pathophysiology, transmission, treatment, and elimination in an infected human host. However, to date, brain HIV-1 latency in hu-HSC mice during suppressive antiretroviral therapy (ART) was not studied. To address this need, hu-HSC mice were administered long acting (LA) ART 14 days after HIV-1 infection was established. Animals were maintained under suppressive ART for 3 months, at which time HIV-1 infection was detected at low levels in brain tissue by droplet digital polymerase chain reaction (ddPCR) test on DNA. Notably, adoptive transfer of cells acquired from the hu-HSC mouse brains and placed into naive hu-HSC mice demonstrated viral recovery. These proof-of-concept results demonstrate replication-competent HIV-1 reservoir can be established in hu-HSC mouse brains that persists during long-term ART treatment. Hu-HSC mice-based mouse viral outgrowth assay (hu-MVOA) serves as a sensitive tool to interrogate latent HIV-1 brain reservoirs.
Assuntos
Infecções por HIV , HIV-1 , Transferência Adotiva , Animais , Encéfalo , Linfócitos T CD4-Positivos , Modelos Animais de Doenças , Infecções por HIV/tratamento farmacológico , Camundongos , Carga Viral , Latência Viral , Replicação ViralRESUMO
Early-onset heart failure (HF) continues to be a major cause of morbidity and mortality in people living with human immunodeficiency virus type one (HIV-1) infection (PLWH), yet the molecular causes for this remain poorly understood. Herein NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ humanized mice (Hu-mice), plasma from PLWH, and autopsied cardiac tissues from deceased HIV seropositive individuals were used to assess if there is a link between the glycolysis byproduct methylglyoxal (MG) and HF in the setting of HIV-1 infection. At five weeks post HIV infection, Hu-mice developed grade III-IV diastolic dysfunction (DD) with an associated two-fold increase in plasma MG. At sixteen-seventeen weeks post infection, cardiac ejection fraction and fractional shortening also declined by 26 and 35%, and plasma MG increased to four-fold higher than uninfected controls. Histopathological and biochemical analyses of cardiac tissues from Hu-mice 17 weeks post-infection affirmed MG increase with a concomitant decrease in expression of the MG-degrading enzyme glyoxalase-1 (Glo1). The endothelial cell marker CD31 was found to be lower, and coronary microvascular leakage and myocardial fibrosis were prominent. Increasing expression of Glo1 in Hu-mice five weeks post-infection using a single dose of an engineered AAV2/9 (1.7 × 1012 virion particles/kg), attenuated the increases in plasma and cardiac MG levels. Increasing Glo1 also blunted microvascular leakage, fibrosis, and HF seen at sixteen weeks post-infection, without changes in plasma viral loads. In plasma from virally suppressed PLWH, MG was also 3.7-fold higher. In autopsied cardiac tissues from seropositive, HIV individuals with low viral log, MG was 4.2-fold higher and Glo1 was 50% lower compared to uninfected controls. These data show for the first time a causal link between accumulation of MG and HF in the setting of HIV infection.
RESUMO
Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease's pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.
Assuntos
Infecções por HIV/complicações , Hepatócitos/transplante , Hepatopatias/terapia , Animais , Modelos Animais de Doenças , HIV-1 , Humanos , Fígado/imunologia , Hepatopatias/complicações , Camundongos , Camundongos SCID , Condicionamento Pré-Transplante , Transplante HeterólogoRESUMO
In an era of improved survival due to modern antiretroviral therapy, liver disease has become a major cause of morbidity and mortality, resulting in death in 15-17% of human immunodeficiency virus (HIV)-infected patients. Alcohol enhances HIV-mediated liver damage and promotes the progression to advanced fibrosis and cirrhosis. However, the mechanisms behind these events are uncertain. Here, we hypothesize that ethanol metabolism potentiates accumulation of HIV in hepatocytes, causing oxidative stress and intensive apoptotic cell death. Engulfment of HIV-containing apoptotic hepatocytes by non-parenchymal cells (NPCs) triggers their activation and liver injury progression. This study was performed on primary human hepatocytes and Huh7.5-CYP cells infected with HIV-1ADA, and major findings were confirmed by pilot data obtained on ethanol-fed HIV-injected chimeric mice with humanized livers. We demonstrated that ethanol exposure potentiates HIV accumulation in hepatocytes by suppressing HIV degradation by lysosomes and proteasomes. This leads to increased oxidative stress and hepatocyte apoptosis. Exposure of HIV-infected apoptotic hepatocytes to NPCs activates the inflammasome in macrophages and pro-fibrotic genes in hepatic stellate cells. We conclude that while HIV and ethanol metabolism-triggered apoptosis clears up HIV-infected hepatocytes, continued generation of HIV-expressing apoptotic bodies may be detrimental for progression of liver inflammation and fibrosis due to constant activation of NPCs.
Assuntos
Doença Hepática Terminal , Etanol , Hepatócitos/efeitos dos fármacos , Acetaldeído/toxicidade , Animais , Apoptose , Linhagem Celular , Progressão da Doença , Doença Hepática Terminal/patologia , Doença Hepática Terminal/virologia , Etanol/metabolismo , Etanol/toxicidade , HIV/patogenicidade , Infecções por HIV/complicações , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/virologia , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Fígado/patologia , Fígado/virologia , Cirrose Hepática/patologia , Cirrose Hepática/virologia , Camundongos , Estresse OxidativoRESUMO
BACKGROUND: Microglia are the principal innate immune defense cells of the centeral nervous system (CNS) and the target of the human immunodeficiency virus type one (HIV-1). A complete understanding of human microglial biology and function requires the cell's presence in a brain microenvironment. Lack of relevant animal models thus far has also precluded studies of HIV-1 infection. Productive viral infection in brain occurs only in human myeloid linage microglia and perivascular macrophages and requires cells present throughout the brain. Once infected, however, microglia become immune competent serving as sources of cellular neurotoxic factors leading to disrupted brain homeostasis and neurodegeneration. METHODS: Herein, we created a humanized bone-marrow chimera producing human "microglia like" cells in NOD.Cg-PrkdcscidIl2rgtm1SugTg(CMV-IL34)1/Jic mice. Newborn mice were engrafted intrahepatically with umbilical cord blood derived CD34+ hematopoietic stem progenitor cells (HSPC). After 3 months of stable engraftment, animals were infected with HIV-1ADA, a myeloid-specific tropic viral isolate. Virologic, immune and brain immunohistology were performed on blood, peripheral lymphoid tissues, and brain. RESULTS: Human interleukin-34 under the control of the cytomegalovirus promoter inserted in NSG mouse strain drove brain reconstitution of HSPC derived peripheral macrophages into microglial-like cells. These human cells expressed canonical human microglial cell markers that included CD14, CD68, CD163, CD11b, ITGB2, CX3CR1, CSFR1, TREM2 and P2RY12. Prior restriction to HIV-1 infection in the rodent brain rested on an inability to reconstitute human microglia. Thus, the natural emergence of these cells from ingressed peripheral macrophages to the brain could allow, for the first time, the study of a CNS viral reservoir. To this end we monitored HIV-1 infection in a rodent brain. Viral RNA and HIV-1p24 antigens were readily observed in infected brain tissues. Deep RNA sequencing of these infected mice and differential expression analysis revealed human-specific molecular signatures representative of antiviral and neuroinflammatory responses. CONCLUSIONS: This humanized microglia mouse reflected human HIV-1 infection in its known principal reservoir and showed the development of disease-specific innate immune inflammatory and neurotoxic responses mirroring what can occur in an infected human brain.
Assuntos
Modelos Animais de Doenças , Infecções por HIV/imunologia , Infecções por HIV/virologia , Interleucinas , Microglia/virologia , Animais , Diferenciação Celular , HIV-1 , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Camundongos Endogâmicos NODRESUMO
Human immunodeficiency virus type 1 (HIV-1) infection impairs liver function, and liver diseases have become a leading cause of morbidity in infected patients. The immunopathology of liver damage caused by HIV-1 remains unclear. We used chimeric mice dually reconstituted with a human immune system and hepatocytes to address the relevance of the model to pathobiology questions related to human hepatocyte survival in the presence of systemic infection. TK-NOG males were transplanted with mismatched human hematopoietic stem/progenitor cells and hepatocytes, human albumin concentration and the presence of human immune cells in blood were monitored for hepatocytes and immune reconstitution, and mice were infected with HIV-1. HIV-1-infected animals showed a decline in human albumin concentration with a significant reduction in percentage of human hepatocytes compared to uninfected mice. The decrease in human albumin levels correlated with a decline in CD4+ cells in the liver and with an increase in HIV-1 viral load. HIV-1 infection elicited proinflammatory response in the immunological milieu of the liver in HIV-infected mice compared to uninfected animals, as determined by upregulation of IL23, CXCL10 and multiple toll-like receptor expression. The inflammatory reaction associated with HIV-1 infection in vivo could contribute to the depletion and dysfunction of hepatocytes. The dual reconstituted TK-NOG mouse model is a feasible platform to investigate hepatocyte-related HIV-1 immunopathogenesis.This article has an associated First Person interview with the first author of the paper.