RESUMO
Treatment of HIV-1ADA-infected CD34+ NSG-humanized mice with long-acting ester prodrugs of cabotegravir, lamivudine, and abacavir in combination with native rilpivirine was followed by dual CRISPR-Cas9 C-C chemokine receptor type five (CCR5) and HIV-1 proviral DNA gene editing. This led to sequential viral suppression, restoration of absolute human CD4+ T cell numbers, then elimination of replication-competent virus in 58% of infected mice. Dual CRISPR therapies enabled the excision of integrated proviral DNA in infected human cells contained within live infected animals. Highly sensitive nucleic acid nested and droplet digital PCR, RNAscope, and viral outgrowth assays affirmed viral elimination. HIV-1 was not detected in the blood, spleen, lung, kidney, liver, gut, bone marrow, and brain of virus-free animals. Progeny virus from adoptively transferred and CRISPR-treated virus-free mice was neither detected nor recovered. Residual HIV-1 DNA fragments were easily seen in untreated and viral-rebounded animals. No evidence of off-target toxicities was recorded in any of the treated animals. Importantly, the dual CRISPR therapy demonstrated statistically significant improvements in HIV-1 cure percentages compared to single treatments. Taken together, these observations underscore a pivotal role of combinatorial CRISPR gene editing in achieving the elimination of HIV-1 infection.
Assuntos
Infecções por HIV , Soropositividade para HIV , Camundongos , Animais , Humanos , Antirretrovirais/uso terapêutico , Edição de Genes , Provírus/genética , Receptores CCR5RESUMO
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
BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by pathological deposition of misfolded self-protein amyloid beta (Aß) which in kind facilitates tau aggregation and neurodegeneration. Neuroinflammation is accepted as a key disease driver caused by innate microglia activation. Recently, adaptive immune alterations have been uncovered that begin early and persist throughout the disease. How these occur and whether they can be harnessed to halt disease progress is unclear. We propose that self-antigens would induct autoreactive effector T cells (Teffs) that drive pro-inflammatory and neurodestructive immunity leading to cognitive impairments. Here, we investigated the role of effector immunity and how it could affect cellular-level disease pathobiology in an AD animal model. METHODS: In this report, we developed and characterized cloned lines of amyloid beta (Aß) reactive type 1 T helper (Th1) and type 17 Th (Th17) cells to study their role in AD pathogenesis. The cellular phenotype and antigen-specificity of Aß-specific Th1 and Th17 clones were confirmed using flow cytometry, immunoblot staining and Aß T cell epitope loaded haplotype-matched major histocompatibility complex II IAb (MHCII-IAb-KLVFFAEDVGSNKGA) tetramer binding. Aß-Th1 and Aß-Th17 clones were adoptively transferred into APP/PS1 double-transgenic mice expressing chimeric mouse/human amyloid precursor protein and mutant human presenilin 1, and the mice were assessed for memory impairments. Finally, blood, spleen, lymph nodes and brain were harvested for immunological, biochemical, and histological analyses. RESULTS: The propagated Aß-Th1 and Aß-Th17 clones were confirmed stable and long-lived. Treatment of APP/PS1 mice with Aß reactive Teffs accelerated memory impairment and systemic inflammation, increased amyloid burden, elevated microglia activation, and exacerbated neuroinflammation. Both Th1 and Th17 Aß-reactive Teffs progressed AD pathology by downregulating anti-inflammatory and immunosuppressive regulatory T cells (Tregs) as recorded in the periphery and within the central nervous system. CONCLUSIONS: These results underscore an important pathological role for CD4+ Teffs in AD progression. We posit that aberrant disease-associated effector T cell immune responses can be controlled. One solution is by Aß reactive Tregs.
Assuntos
Doença de Alzheimer/patologia , Linfócitos T CD4-Positivos/patologia , Presenilina-1/genética , Precursor de Proteína beta-Amiloide/genética , Amiloidose/patologia , Animais , Transtornos Cognitivos/patologia , Transtornos Cognitivos/psicologia , Inflamação/genética , Camundongos , Camundongos Transgênicos , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th1/patologia , Células Th17/imunologia , Células Th17/patologiaRESUMO
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
BACKGROUND: The use of immunodeficient mice transplanted with human hematopoietic stem cells is an accepted approach to study human-specific infectious diseases such as HIV-1 and to investigate multiple aspects of human immune system development. However, mouse and human are different in sialylation patterns of proteins due to evolutionary mutations of the CMP-N-acetylneuraminic acid hydroxylase (CMAH) gene that prevent formation of N-glycolylneuraminic acid from N-acetylneuraminic acid. How changes in the mouse glycoproteins' chemistry affect phenotype and function of transplanted human hematopoietic stem cells and mature human immune cells in the course of HIV-1 infection are not known. RESULTS: We mutated mouse CMAH in the NOD/scid-IL2Rγc-/- (NSG) mouse strain, which is widely used for the transplantation of human cells, using the CRISPR/Cas9 system. The new strain provides a better environment for human immune cells. Transplantation of human hematopoietic stem cells leads to broad B cells repertoire, higher sensitivity to HIV-1 infection, and enhanced proliferation of transplanted peripheral blood lymphocytes. The mice showed no effect on the clearance of human immunoglobulins and enhanced transduction efficiency of recombinant adeno-associated viral vector rAAV2/DJ8. CONCLUSION: NSG-cmah-/- mice expand the mouse models suitable for human cells transplantation, and this new model has advantages in generating a human B cell repertoire. This strain is suitable to study different aspects of the human immune system development, provide advantages in patient-derived tissue and cell transplantation, and could allow studies of viral vectors and infectious agents that are sensitive to human-like sialylation of mouse glycoproteins.
Assuntos
Glicoproteínas/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1 , Linfócitos/imunologia , Linfócitos/metabolismo , Linfócitos/virologia , Animais , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Loci Gênicos , Infecções por HIV/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/virologia , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Imunofenotipagem , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Knockout , FenótipoRESUMO
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
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
The widespread use of antiretroviral therapy for treatment of human immunodeficiency virus (HIV) infections has dramatically improved the quality and duration of life for HIV-positive individuals. Despite this success, HIV persists for the life of an infected person in tissue reservoirs including the nervous system. Thus, whether HIV exacerbates age-related brain disorders such as Parkinson's disease (PD) is of concern. In support of this idea, HIV infection can be associated with motor and gait abnormalities that parallel late-stage manifestations of PD including dopaminergic neuronal loss. With these findings in hand, we investigated whether viral infection could affect nigrostriatal degeneration or exacerbate chemically induced nigral degeneration. We now demonstrate an additive effect of EcoHIV on dopaminergic neuronal loss and neuroinflammation induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intoxication. HIV-1-infected humanized mice failed to recapitulate these EcoHIV results suggesting species-specific neural signaling. The results demonstrate a previously undefined EcoHIV-associated neurodegenerative response that may be used to model pathobiological aspects of PD.
Assuntos
Infecções por HIV/complicações , Intoxicação por MPTP/complicações , Substância Negra/patologia , Substância Negra/virologia , Animais , Infecções por HIV/patologia , HIV-1 , Humanos , Intoxicação por MPTP/patologia , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/induzido quimicamente , Degeneração Neural/virologiaRESUMO
BACKGROUND: Despite improved clinical outcomes seen following antiretroviral therapy (ART), resting CD4+ T cells continue to harbor latent human immunodeficiency virus type one (HIV-1). However, such cells are not likely the solitary viral reservoir and as such defining where and how others harbor virus is imperative for eradication measures. To such ends, we used HIV-1ADA-infected NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ mice reconstituted with a human immune system to explore two long-acting ART regimens investigating their abilities to affect viral cell infection and latency. At 6 weeks of infection animals were divided into four groups. One received long-acting (LA) cabotegravir (CAB) and rilpivirine (RVP) (2ART), a second received LA CAB, lamivudine, abacavir and RVP (4ART), a third were left untreated and a fourth served as an uninfected control. After 4 weeks of LA ART treatment, blood, spleen and bone marrow (BM) cells were collected then phenotypically characterized. CD4+ T cell subsets, macrophages and hematopoietic progenitor cells were analyzed for HIV-1 nucleic acids by droplet digital PCR. RESULTS: Plasma viral loads were reduced by two log10 or to undetectable levels in the 2 and 4ART regimens, respectively. Numbers and distributions of CD4+ memory and regulatory T cells, macrophages and hematopoietic progenitor cells were significantly altered by HIV-1 infection and by both ART regimens. ART reduced viral DNA and RNA in all cell and tissue compartments. While memory cells were the dominant T cell reservoir, integrated HIV-1 DNA was also detected in the BM and spleen macrophages in both regimen-treated mice. CONCLUSION: Despite vigorous ART regimens, HIV-1 DNA and RNA were easily detected in mature macrophages supporting their potential role as an infectious viral reservoir.
Assuntos
Antirretrovirais/administração & dosagem , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/crescimento & desenvolvimento , Macrófagos/virologia , Animais , DNA Viral/análise , DNA Viral/genética , Reservatórios de Doenças , HIV-1/fisiologia , Humanos , Camundongos SCID , Plasma/virologia , Reação em Cadeia da Polimerase , Provírus/genética , RNA Viral/análise , Resultado do Tratamento , Carga Viral , Latência ViralRESUMO
BACKGROUND: Alcohol consumption exacerbates the pathogenesis of hepatitis C virus (HCV) infection and worsens disease outcomes. The exact reasons are not clear yet, but they might be partially attributed to the ability of alcohol to further suppress the innate immunity. Innate immunity is known to be already decreased by HCV in liver cells. METHODS: In this study, we aimed to explore the mechanisms of how alcohol metabolism dysregulates IFNα signaling (STAT1 phosphorylation) in HCV+ hepatoma cells. To this end, CYP2E1+ Huh7.5 cells were infected with HCV and exposed to the acetaldehyde (Ach) generating system (AGS). RESULTS: Continuously produced Ach suppressed IFNα-induced STAT1 phosphorylation, but increased the level of a protease, USP18 (both measured by Western blot), which interferes with IFNα signaling. Induction of USP18 by Ach was confirmed in primary human hepatocyte cultures and in livers of ethanol-fed HCV transgenic mice. Silencing of USP18 by specific siRNA attenuated the pSTAT1 suppression by Ach. The mechanism by which Ach down-regulates pSTAT1 is related to an enhanced interaction between IFNαR2 and USP18 that finally dysregulates the cross talk between the IFN receptor on the cell surface and STAT1. Furthermore, Ach decreases ISGylation of STAT1 (protein conjugation of a small ubiquitin-like modifier, ISG15, Western blot), which preserves STAT1 activation. Suppressed ISGylation leads to an increase in STAT1 K48 polyubiquitination which allows pSTAT1 degrading by proteasome. CONCLUSIONS: We conclude that Ach disrupts IFNα-induced STAT1 phosphorylation by the up-regulation of USP18 to block the innate immunity protection in HCV-infected liver cells, thereby contributing to HCV-alcohol pathogenesis. This, in part, may explain the mechanism of HCV-infection exacerbation/progression in alcohol-abusing patients.
Assuntos
Acetaldeído/farmacologia , Endopeptidases/metabolismo , Hepatite C/metabolismo , Interferon-alfa/metabolismo , Fígado/efeitos dos fármacos , Fator de Transcrição STAT1/metabolismo , Consumo de Bebidas Alcoólicas/efeitos adversos , Consumo de Bebidas Alcoólicas/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Ubiquitina TiolesteraseRESUMO
During studies to extend the half-life of crystalline nanoformulated antiretroviral therapy (nanoART) the mixed lineage kinase-3 inhibitor URMC-099, developed as an adjunctive neuroprotective agent was shown to facilitate antiviral responses. Long-acting ritonavir-boosted atazanavir (nanoATV/r) nanoformulations co-administered with URMC-099 reduced viral load and the numbers of HIV-1 infected CD4+ T-cells in lymphoid tissues more than either drug alone in infected humanized NOD/SCID/IL2Rγc-/- mice. The drug effects were associated with sustained ART depots. Proteomics analyses demonstrated that the antiretroviral responses were linked to affected phagolysosomal storage pathways leading to sequestration of nanoATV/r in Rab-associated recycling and late endosomes; sites associated with viral maturation. URMC-099 administered with nanoATV induced a dose-dependent reduction in HIV-1p24 and reverse transcriptase activity. This drug combination offers a unique chemical marriage for cell-based viral clearance. From the Clinical Editor: Although successful in combating HIV-1 infection, the next improvement in antiretroviral therapy (nanoART) would be to devise long acting therapy, such as intra-cellular depots. In this report, the authors described the use of nanoformulated antiretroviral therapy given together with the mixed lineage kinase-3 inhibitor URMC-099, and showed that this combination not only prolonged drug half-life, but also had better efficacy. The findings are hoped to be translated into the clinical setting in the future.
Assuntos
Sulfato de Atazanavir/administração & dosagem , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Nanocápsulas/química , Piridinas/administração & dosagem , Pirróis/administração & dosagem , Animais , Antirretrovirais/administração & dosagem , Terapia Antirretroviral de Alta Atividade/métodos , Quimioterapia Combinada/métodos , Infecções por HIV/diagnóstico , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Camundongos , Camundongos SCID , Nanocápsulas/administração & dosagem , Nanocápsulas/ultraestrutura , Inibidores de Proteínas Quinases/administração & dosagem , Resultado do Tratamento , MAP Quinase Quinase Quinase 11 Ativada por MitógenoRESUMO
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.
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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
Disordered immunity, aging, human immunodeficiency virus type one (HIV-1) infection, and responses to antiretroviral therapy are linked. However, how each factor is linked with the other(s) remains incompletely understood. It has been reported that accelerated aging, advanced HIV-1 infection, inflammation, and host genetic factors are associated with host cellular, mitochondrial, and metabolic alterations. However, the underlying mechanism remains elusive. With these questions in mind, we used chronically HIV-1-infected CD34-NSG humanized mice (hu-mice) to model older people living with HIV and uncover associations between HIV-1 infection and aging. Adult humanized mice were infected with HIV-1 at the age of 20 weeks and maintained for another 40 weeks before sacrifice. Animal brains were collected and subjected to transcriptomics, qPCR, and immunofluorescence assays to uncover immune disease-based biomarkers. CD4+ T cell decline was associated with viral level and age. Upregulated C1QA, CD163, and CXCL16 and downregulated LMNA and CLU were identified as age-associated genes tied to HIV-1 infection. Ingenuity pathway analysis affirmed links to innate immune activation, pyroptosis signaling, neuroinflammation, mitochondrial dysfunction, cellular senescence, and neuronal dysfunction. In summary, CD34-NSG humanized mice are identified as a valuable model for studying HIV-1-associated aging. Biomarkers of immune senescence and neuronal signaling are both age- and virus-associated. By exploring the underlying biological mechanisms that are linked to these biomarkers, interventions for next generation HIV-1-infected patients can be realized.
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INTRODUCTION: Liver disease is known as one of the leading co-morbidities in HIV infection, with 18% of non-AIDS-related mortality. There is constant crosstalk between liver parenchymal (hepatocytes) and non-parenchymal cells (macrophages, hepatic stellate cells, endothelial cells), and extracellular vesicles (EVs) are one of the most important ways of cell-to-cell communication. AREAS COVERED: We briefly cover the role of EVs in liver disease as well as what is known about the role of small EVs, exosomes, in HIV-induced liver disease potentiated by alcohol as one of the second hits. We also touch large EVs, apoptotic bodies (ABs), in HIV-induced liver injury, the mechanisms of their formation and potentiation by second hits, and their role in the progression of liver disease. EXPERT OPINION/COMMENTARY: Liver cells are an important source of EVs, which may provide the connection between different organs via secretion into the circulating blood (exosomes) or serve for the communication between the cells within the organ (ABs). Understanding the role of liver EVs in HIV infection and the involvement of second hits in EV generation would provide a new angle for the analysis of HIV-related liver disease pathogenesis and progression to end-stage liver disease.
People living with HIV-1 (PLWH) are at risk of developing chronic liver disease. They are often coinfected with hepatitis viruses B and C and misuse alcohol. Combining these 'second hits' factors with HIV infection greatly accelerates liver damage. The exact mechanisms are not clear. The liver contains different types of cells. Most are hepatocytes, which cannot replicate HIV but can be stressed by HIV and alcohol metabolites. During this process, hepatocytes release small extracellular vesicles called exosomes.Liver exosomes can carry toxic viral proteins and nucleic acids. When hepatocyte exosomes are captured by other liver cells, such as stellate cells, Kupffer macrophages, and sinusoidal endothelial cells, they change the functions of these cells.If hepatocytes die due to high HIV and alcohol toxicity, they form large particles called apoptotic bodies.Like exosomes, these large apoptotic bodies also change the functions of neighboring cells they capture.Macrophages and stellate cells exposed to extracellular vesicles containing HIV proteins and nucleic acids promote liver inflammation and fibrosis.
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Vesículas Extracelulares , Infecções por HIV , Hepatopatias , Humanos , Infecções por HIV/complicações , Células Endoteliais , Hepatopatias/etiologiaRESUMO
A major roadblock to achieving a cure for human immunodeficiency virus type one (HIV-1) is the persistence of latent viral infections in the cells and tissue compartments of an infected human host. Latent HIV-1 proviral DNA persists in resting memory CD4+ T cells and mononuclear phagocytes (MPs; macrophages, microglia, and dendritic cells). Tissue viral reservoirs of both cell types reside in the gut, lymph nodes, bone marrow, spleen, liver, kidney, skin, adipose tissue, reproductive organs, and brain. However, despite the identification of virus-susceptible cells, several limitations persist in identifying broad latent reservoirs in infected persons. The major limitations include their relatively low abundance, the precise identification of latently infected cells, and the lack of biomarkers for identifying latent cells. While primary MP and CD4+ T cells and transformed cell lines are used to interrogate mechanisms of HIV-1 persistence, they often fail to accurately reflect the host cells and tissue environments that carry latent infections. Given the host specificity of HIV-1, there are few animal models that replicate the natural course of viral infection with any precision. These needs underlie the importance of humanized mouse models as both valuable and cost-effective tools for studying viral latency and subsequently identifying means of eliminating it. In this review, we discuss the advantages and limitations of humanized mice for studies of viral persistence and latency with an eye toward using these models to test antiretroviral and excision therapeutics. The goals of this research are to use the models to address how and under which circumstances HIV-1 latency can be detected and eliminated. Targeting latent reservoirs for an ultimate HIV-1 cure is the task at hand.
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Background: Hepatitis B virus (HBV) infection develops as an acute or chronic liver disease, which progresses from steatosis, hepatitis, and fibrosis to end-stage liver diseases such as cirrhosis and hepatocellular carcinoma (HCC). An increased stromal stiffness accompanies fibrosis in chronic liver diseases and is considered a strong predictor for disease progression. The goal of this study was to establish the mechanisms by which enhanced liver stiffness regulates HBV infectivity in the fibrotic liver tissue. Methods: For in vitro studies, HBV-transfected HepG2.2.15 cells were cultured on polydimethylsiloxane gels coated by polyelectrolyte multilayer films of 2 kPa (soft) or 24 kPa (stiff) rigidity mimicking the stiffness of the healthy or fibrotic liver. For in vivo studies, hepatic fibrosis was induced in C57Bl/6 parental and HBV+ transgenic (HBVTg) mice by injecting CCl4 twice a week for 6 weeks. Results: We found higher levels of HBV markers in stiff gel-attached hepatocytes accompanied by up-regulated OPN content in cell supernatants as well as suppression of anti-viral interferon-stimulated genes (ISGs). This indicates that pre-requisite "fibrotic" stiffness increases osteopontin (OPN) content and releases and suppresses anti-viral innate immunity, causing a subsequent rise in HBV markers expression in hepatocytes. In vitro results were corroborated by data from HBVTg mice administered CCl4 (HBVTg CCl4). These mice showed higher HBV RNA, DNA, HBV core antigen (HBcAg), and HBV surface antigen (HBsAg) levels after liver fibrosis induction as judged by a rise in Col1a1, SMA, MMPs, and TIMPs mRNAs and by increased liver stiffness. Importantly, CCl4-induced the pro-fibrotic activation of liver cells, and liver stiffness was higher in HBVTg mice compared with control mice. Elevation of HBV markers and OPN levels corresponded to decreased ISG activation in HBVTg CCl4 mice vs HBVTg control mice. Conclusion: Based on our data, we conclude that liver stiffness enhances OPN levels to limit anti-viral ISG activation in hepatocytes and promote an increase in HBV infectivity, thereby contributing to end-stage liver disease progression.
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Carcinoma Hepatocelular , Doença Hepática Terminal , Hepatite B , Neoplasias Hepáticas , Camundongos , Animais , Vírus da Hepatite B , Camundongos Transgênicos , Cirrose Hepática/induzido quimicamente , Imunidade Inata , Antígenos do Núcleo do Vírus da Hepatite B , Antígenos de Superfície da Hepatite B , AntiviraisRESUMO
Chronic hepatitis B virus (HBV) infection leads to the development of cirrhosis and hepatocellular carcinoma. Lifelong treatment with nucleotides/nucleoside antiviral agents is effective at suppressing HBV replication, however, adherence to daily therapy can be challenging. This review discusses recent advances in the development of long-acting formulations for HBV treatment and prevention, which could potentially improve adherence. Promising new compounds that target distinct steps of the virus life cycle are summarized. In addition to treatments that suppress viral replication, curative strategies are focused on the elimination of covalently closed circular DNA and the inactivation of the integrated viral DNA from infected hepatocytes. We highlight promising long-acting antivirals and genome editing strategies for the elimination or deactivation of persistent viral DNA products in development.