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1.
Nature ; 574(7777): 200-205, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31582858

RESUMO

The responses of CD8+ T cells to hepatotropic viruses such as hepatitis B range from dysfunction to differentiation into effector cells, but the mechanisms that underlie these distinct outcomes remain poorly understood. Here we show that priming by Kupffer cells, which are not natural targets of hepatitis B, leads to differentiation of CD8+ T cells into effector cells that form dense, extravascular clusters of immotile cells scattered throughout the liver. By contrast, priming by hepatocytes, which are natural targets of hepatitis B, leads to local activation and proliferation of CD8+ T cells but not to differentiation into effector cells; these cells form loose, intravascular clusters of motile cells that coalesce around portal tracts. Transcriptomic and chromatin accessibility analyses reveal unique features of these dysfunctional CD8+ T cells, with limited overlap with those of exhausted or tolerant T cells; accordingly, CD8+ T cells primed by hepatocytes cannot be rescued by treatment with anti-PD-L1, but instead respond to IL-2. These findings suggest immunotherapeutic strategies against chronic hepatitis B infection.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada/imunologia , Vírus da Hepatite B/imunologia , Hepatócitos/imunologia , Hepatócitos/virologia , Animais , Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Cromatina/metabolismo , Feminino , Hepatite B/tratamento farmacológico , Hepatite B/imunologia , Hepatite B/virologia , Humanos , Tolerância Imunológica , Interleucina-2/imunologia , Interleucina-2/uso terapêutico , Macrófagos do Fígado/imunologia , Ativação Linfocitária , Masculino , Camundongos , Transcriptoma/genética
2.
Nature ; 572(7768): 199-204, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31292543

RESUMO

The human liver is an essential multifunctional organ. The incidence of liver diseases is rising and there are limited treatment options. However, the cellular composition of the liver remains poorly understood. Here we performed single-cell RNA sequencing of about 10,000 cells from normal liver tissue from nine human donors to construct a human liver cell atlas. Our analysis identified previously unknown subtypes of endothelial cells, Kupffer cells, and hepatocytes, with transcriptome-wide zonation of some of these populations. We show that the EPCAM+ population is heterogeneous, comprising hepatocyte-biased and cholangiocyte populations as well as a TROP2int progenitor population with strong potential to form bipotent liver organoids. As a proof-of-principle, we used our atlas to unravel the phenotypic changes that occur in hepatocellular carcinoma cells and in human hepatocytes and liver endothelial cells engrafted into a mouse liver. Our human liver cell atlas provides a powerful resource to enable the discovery of previously unknown cell types in normal and diseased livers.


Assuntos
Células Epiteliais/citologia , Hepatócitos/citologia , Fígado/citologia , Células-Tronco/citologia , Adulto , Animais , Antígenos de Neoplasias/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Moléculas de Adesão Celular/metabolismo , Quimera/imunologia , Quimera/metabolismo , Células Endoteliais/citologia , Células Endoteliais/imunologia , Células Epiteliais/imunologia , Feminino , Regulação da Expressão Gênica , Hepatócitos/imunologia , Hepatócitos/metabolismo , Humanos , Fígado/imunologia , Masculino , Camundongos , Organoides/metabolismo , RNA Citoplasmático Pequeno/genética , Reprodutibilidade dos Testes , Células-Tronco/imunologia
3.
J Agric Food Chem ; 67(31): 8510-8519, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31294559

RESUMO

Acrylamide, mainly formed in Maillard browning reaction during food processing, causes defects in liver circadian clock and mitochondrial function by inducing oxidative stress. Resveratrol is a polyphenol that has powerful antioxidant and anti-inflammatory activity. However, the preventive effects of resveratrol on acrylamide-triggered oxidative damage and circadian rhythm disorders are unclear at the current stage. The present research revealed that resveratrol pretreatment prevented acrylamide-induced cell death, mitochondrial dysfunction, and inflammatory responses in HepG2 liver cells. Acrylamide significantly triggered disorders of circadian genes transcription and protein expressions including Bmal1 and Cry 1 in primary hepatocytes, which were prevented by resveratrol pretreatment. Moreover, we found that the beneficial effects of resveratrol on stimulating Nrf2/NQO-1 pathway and mitochondrial respiration complex expressions in acrylamide-treated cells were Bmal1-dependent. Similarly, the inhibitory effects of resveratrol on inflammation signaling NF-κB were Cry1-dependent. In conclusion, these results demonstrated resveratrol could be a promising compound in suppressing acrylamide-induced hepatotoxicity and balancing the circadian clock.


Assuntos
Fatores de Transcrição ARNTL/imunologia , Acrilamida/toxicidade , Transtornos Cronobiológicos/imunologia , Criptocromos/imunologia , Hepatócitos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Resveratrol/farmacologia , Fatores de Transcrição ARNTL/genética , Animais , Transtornos Cronobiológicos/tratamento farmacológico , Transtornos Cronobiológicos/genética , Transtornos Cronobiológicos/fisiopatologia , Ritmo Circadiano/efeitos dos fármacos , Criptocromos/genética , Células Hep G2 , Hepatócitos/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Mitocôndrias/imunologia
4.
Artigo em Inglês | MEDLINE | ID: mdl-31119110

RESUMO

Toxoplasma gondii is an important human and animal pathogen that causes life-threatening toxoplasmosis. The host immune system produces interferon-γ (IFN-γ) to inhibit T. gondii proliferation. IFN-γ-inducible indole-2,3-dioxygenase 1 (IDO1), which mediates tryptophan degradation, has a major role in anti-T. gondii immune responses in various human cells. In response to the host's immune system, T. gondii secretes many virulence molecules into the host cells to suppress IFN-γ-dependent antiparasitic immune responses. The GRA15-induced proparasitic mechanism for suppressing IDO1-dependent immune responses has previously been tested only in human hepatocyte and monocyte co-cultures. Thus, whether human cells other than hepatocytes contain this virulence mechanism remains unclear. Here, we show that the GRA15-dependent virulence mechanism for suppressing the IDO1-dependent anti-T. gondii response operates in human neuronal cell lines and primary human neurons. Analysis of various human cell lines revealed that IL-1ß-induced iNOS-dependent reduction of IDO1 mRNA expression occurred in brain cell lines (A172; glioblastoma, IMR-32; neuroblastoma, and T98G; glioblastoma) and liver cell lines (Huh7 and HepG2), but not in other cell lines. Moreover, co-culturing type II T. gondii-infected THP-1 human monocytes with the brain cell lines inhibited the IDO1-mediated anti-T. gondii response in a GRA15-dependent manner. These data suggest that a GRA15-dependent virulence mechanism antagonizes the IDO1-dependent host immune response in human brain cells.


Assuntos
Antígenos de Protozoários/metabolismo , Antiparasitários/metabolismo , Interferon gama/metabolismo , Neurônios/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Toxoplasmose/imunologia , Antígenos de Protozoários/imunologia , Antiparasitários/farmacologia , Linhagem Celular , Hepatócitos/imunologia , Interações Hospedeiro-Parasita/imunologia , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/farmacologia , Interferon gama/imunologia , Interleucina-1beta/metabolismo , Monócitos/imunologia , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas de Protozoários/imunologia , RNA Mensageiro/metabolismo , Toxoplasma/efeitos dos fármacos , Toxoplasma/imunologia , Virulência
5.
Nat Microbiol ; 4(7): 1096-1104, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30988429

RESUMO

Current models of cell-intrinsic immunity to RNA viruses centre on virus-triggered inducible antiviral responses initiated by RIG-I-like receptors or Toll-like receptors that sense pathogen-associated molecular patterns, and signal downstream through interferon regulatory factors (IRFs), transcription factors that induce synthesis of type I and type III interferons1. RNA viruses have evolved sophisticated strategies to disrupt these signalling pathways and evade elimination by cells, attesting to their importance2. Less attention has been paid to how IRFs maintain basal levels of protection against viruses. Here, we depleted antiviral factors linked to RIG-I-like receptor and Toll-like receptor signalling to map critical host pathways restricting positive-strand RNA virus replication in immortalized hepatocytes and identified an unexpected role for IRF1. We show that constitutively expressed IRF1 acts independently of mitochondrial antiviral signalling (MAVS) protein, IRF3 and signal transducer and activator of transcription 1 (STAT1)-dependent signalling to provide intrinsic antiviral protection in actinomycin D-treated cells. IRF1 localizes to the nucleus, where it maintains the basal transcription of a suite of antiviral genes that protect against multiple pathogenic RNA viruses, including hepatitis A and C viruses, dengue virus and Zika virus. Our findings reveal an unappreciated layer of hepatocyte-intrinsic immunity to these positive-strand RNA viruses and identify previously unrecognized antiviral effector genes.


Assuntos
Expressão Gênica , Hepatócitos/imunologia , Imunidade Inata/genética , Fator Regulador 1 de Interferon/genética , Vírus de RNA/fisiologia , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Fezes/virologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Fator Regulador 1 de Interferon/metabolismo , Cinética , Fígado/virologia , Camundongos , RNA Interferente Pequeno , Transdução de Sinais/genética , Replicação Viral
6.
Int J Mol Sci ; 20(6)2019 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-30909560

RESUMO

Apolipoprotein E (apoE) is mainly secreted by hepatocytes and incorporated into most plasma lipoproteins. Macrophages, which accumulate cholesterol and are critical for the development of the atherosclerotic plaque, are also an important, albeit smaller, apoE source. Distal regulatory elements control cell-specific activity of the apoE promoter: multienhancers (ME.1/2) in macrophages and hepatic control regions (HCR-1/2) in hepatocytes. A member of AP-1 cell growth regulator, c-Jun regulates the transcription of various apolipoproteins and proinflammatory molecules implicated in atherosclerosis. We aimed to investigate the effect of c-Jun on apoE expression in macrophages versus hepatocytes and to reveal the underlying molecular mechanisms. Herein we show that c-Jun had an opposite, cell-specific effect on apoE expression: downregulation in macrophages but upregulation in hepatocytes. Transient transfections using ME.2 deletion mutants and DNA pull-down (DNAP) assays showed that the inhibitory effect of c-Jun on the apoE promoter in macrophages was mediated by a functional c-Jun binding site located at 301/311 on ME.2. In hepatocytes, c-Jun overexpression strongly increased apoE expression, and this effect was due to c-Jun binding at the canonical site located at -94/-84 on the apoE proximal promoter, identified by transient transfections using apoE deletion mutants, DNAP, and chromatin immunoprecipitation assays. Overall, the dual effect of c-Jun on apoE gene expression led to decreased cholesterol efflux in macrophages resident in the atherosclerotic plaque synergized with an increased level of systemic apoE secreted by the liver to exacerbate atherogenesis.


Assuntos
Apolipoproteínas E/genética , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Macrófagos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Animais , Elementos Facilitadores Genéticos , Hepatócitos/imunologia , Macrófagos/imunologia , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas , Células RAW 264.7 , Fator de Transcrição AP-1/metabolismo
7.
Viral Immunol ; 32(3): 112-120, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30817236

RESUMO

The present report describes current concepts about the mechanism of liver cell injury caused by host immune response against hepatitis C virus (HCV) infection in human beings. This report is based on the observations from experimental studies and follow-up actions on human liver diseases. The results from different investigations suggest that liver injury depends on the presentation of viral antigen and the level of host immune response raised against HCV-related peptides. Both innate and adaptive immunity are triggered to counter the viral onset. During development of host immunity, the cell-mediated immune response involving CD4+ Th1 cells and CD8+ cytotoxic T-lymphocyte (CTL) cells were found to play a major role in causing liver damage. The hepatic Innate lymphoid cells (ILCs) subsets are involved in the immune regulation of different liver diseases: viral hepatitis, mechanical liver injury, and fibrosis. Humoral immunity and natural killer (NK) cell action also contributed in liver cell injury by antibody-dependent cellular cytotoxicity (ADCC). In fact, immunopathogenesis of HCV infection is a complex phenomenon where regulation of immune response at several steps decides the possibility of viral elimination or persistence. Regulation of immune response was noted starting from viral-host interaction to immune reaction cascade engaged in cell damage. The activation or suppression of interferon-stimulated genes, NK cell action, CTL inducement by regulatory T cells (Treg), B cell proliferation, and so on was demonstrated during HCV infection. Involvement of HLA in antigen presentation, as well as types of viral genotypes, also influenced host immune response against HCV peptides. The combined effect of all these effector mechanisms ultimately decides the progression of viral onset to acute or chronic infection. In conclusion, immunopathogenesis of liver injury after HCV infection may be ascribed mainly to host immune response. Second, it is cell-mediated immunity that plays a predominant role in liver cell damage.


Assuntos
Imunidade Adaptativa , Hepatite C/imunologia , Hepatite C/patologia , Imunidade Celular , Fígado/imunologia , Fígado/patologia , Citocinas/imunologia , Hepacivirus/imunologia , Hepatócitos/imunologia , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Linfócitos/virologia , Linfócitos T Citotóxicos/imunologia
8.
J Med Microbiol ; 68(4): 642-648, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30747617

RESUMO

The liver-expressed pattern recognition receptors mannose-binding lectin (MBL), ficolin-2 and ficolin-3 contribute to the innate immune response by activating complement. Binding of soluble ficolin-2 to viral pathogens can directly neutralize virus entry. We observed that the human hepatoma cell line HuH7.5, which is routinely used for the study of hepatotropic viruses, is deficient in expression of MBL, ficolin-2 and ficolin-3. We generated a cell line that expressed and secreted ficolin-2. This cell line (HuH7.5 [FCN2]) was more resistant to infection with hepatitis C virus (HCV), ebolavirus and vesicular stomatitis virus, but surprisingly was more susceptible to infection with rabies virus. Cell-to-cell spread of HCV was also inhibited in ficolin-2 expressing cells. This illustrates that ficolin-2 expression in hepatocytes contributes to innate resistance to virus infection, but some viruses might utilize ficolin-2 to facilitate entry.


Assuntos
Hepacivirus , Hepatócitos/virologia , Imunidade Inata , Lectinas/metabolismo , Carcinoma Hepatocelular , Linhagem Celular , Linhagem Celular Tumoral , Ativação do Complemento , Células HEK293 , Hepatócitos/imunologia , Humanos , Lectinas/genética , Ligação Proteica , Internalização do Vírus
9.
Immunity ; 50(2): 403-417.e4, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30709740

RESUMO

The tolerogenic microenvironment of the liver is associated with impaired hepatic T cell function. Here, we examined the contribution of liver-resident natural killer (LrNK) cells, a prominent hepatic NK cell compartment, to T cell antiviral responses in the liver. The number of virus-specific T cells increased in LrNK-cell-deficient mice during both acute and chronic lymphocytic choriomeningitis virus infection. Upon infection with adenovirus, hepatic T cells from these mice produced more cytokines, which was accompanied by reduced viral loads. Transfer of LrNK cells into LrNK-cell-deficient or wild-type mice inhibited hepatic T cell function, resulting in impaired viral clearance, whereas transfer of conventional NK cells promoted T cell antiviral responses. LrNK-cell-mediated inhibition of T cell function was dependent on the PD-1-PD-L1 axis. Our findings reveal a role for LrNK cells in the regulation of T cell immunity and provide insight into the mechanisms of immune tolerance in the liver.


Assuntos
Antígeno B7-H1/imunologia , Células Matadoras Naturais/imunologia , Fígado/imunologia , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T/imunologia , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Hepatócitos/imunologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Células Matadoras Naturais/metabolismo , Fígado/metabolismo , Fígado/virologia , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/metabolismo , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Linfócitos T/virologia , Transcriptoma/genética , Transcriptoma/imunologia
10.
Mol Biol Rep ; 46(1): 1127-1138, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30603953

RESUMO

Hepatitis is the principal cause of hepatocellular carcinoma (HCC) and decompensated cirrhosis. HCC is amongst the leading causes of deaths worldwide. Current therapeutic options have proven to be unsuccessful in treating this disease due to multifactorial nature of the disease. The present study was designed to investigate the role of IL-22 mediated survival of hepatocytes during cirrhosis and HCC. Resected/explanted liver tissue samples of patients with End Stage Liver Disease were obtained from Hepato-Pancreato-Biliary Liver Transplant Unit of Sheikh Zayed Hospital, Lahore, Pakistan. Qualitative expression of IL-22, SOCS3, and IL-22 induced anti-apoptotic protein, B-cell lymphoma extra-large (Bcl-xL), were evaluated by Immunohistochemical analysis (IHC). The IHC analysis revealed significantly high expression of IL-22, SOCS3, and Bcl-xL within explanted livers of HCC patients. Overall, the expression of SOCS3 was higher than any other protein, and the expression of all proteins showed significant variation in different group of patients based on clincopathological features. The results of the current study indicated that IL-22 mediated JAK-STAT pathway i.e. liver regeneration and healing is dependent on the disease progression and type of agent responsible for causing the infection in the first place. However, quantitative analysis of these factors in future can provide further evidence of the role of this pathway in HCC for development of anti-HCC therapies.


Assuntos
Doença Hepática Terminal/imunologia , Interleucinas/fisiologia , Regeneração Hepática/imunologia , Adulto , Idoso , Carcinoma Hepatocelular/patologia , Doença Hepática Terminal/fisiopatologia , Feminino , Hepatócitos/imunologia , Hepatócitos/fisiologia , Humanos , Interleucinas/metabolismo , Fígado/metabolismo , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , Regeneração Hepática/fisiologia , Masculino , Pessoa de Meia-Idade , Paquistão , Proteína 3 Supressora da Sinalização de Citocinas/análise , Proteína bcl-X/análise
11.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(4): 500-511, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30639734

RESUMO

Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters (CE) and triglycerides (TG) to generate fatty acids (FA) and cholesterol. LAL deficiency (LAL-D) in both humans and mice leads to hepatomegaly, hypercholesterolemia, and shortened life span. Despite its essential role in lysosomal neutral lipid catabolism, the cell type-specific contribution of LAL to disease progression is still elusive. To investigate the role of LAL in the liver in more detail and to exclude the contribution of LAL in macrophages, we generated hepatocyte-specific LAL-deficient mice (Liv-Lipa-/-) and fed them either chow or high fat/high cholesterol diets (HF/HCD). Comparable to systemic LAL-D, Liv-Lipa-/- mice were resistant to diet-induced obesity independent of food intake, movement, and energy expenditure. Reduced body weight gain was mainly due to reduced white adipose tissue depots. Furthermore, Liv-Lipa-/- mice exhibited improved glucose clearance during glucose and insulin tolerance tests compared to control mice. Analysis of hepatic lipid content revealed a massive reduction of TG, whereas CE concentrations were markedly increased, leading to CE crystal formation in the livers of Liv-Lipa-/- mice. Elevated plasma transaminase activities, increased pro-inflammatory cytokines and chemokines as well as hepatic macrophage infiltration indicated liver inflammation. Our data provide evidence that hepatocyte-specific LAL deficiency is sufficient to alter whole-body lipid and energy homeostasis in mice. We conclude that hepatic LAL plays a pivotal role by preventing liver damage and maintaining lipid and energy homeostasis, especially during high lipid availability.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Hepatite/genética , Hepatócitos/enzimologia , Obesidade/prevenção & controle , Esterol Esterase/deficiência , Animais , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Hepatócitos/imunologia , Homeostase , Metabolismo dos Lipídeos , Masculino , Camundongos , Obesidade/induzido quimicamente , Obesidade/genética , Esterol Esterase/genética , Esterol Esterase/metabolismo
12.
Nat Commun ; 10(1): 377, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670689

RESUMO

The circadian clock regulates immune responses to microbes and affects pathogen replication, but the underlying molecular mechanisms are not well understood. Here we demonstrate that the circadian components BMAL1 and REV-ERBα influence several steps in the hepatitis C virus (HCV) life cycle, including particle entry into hepatocytes and RNA genome replication. Genetic knock out of Bmal1 and over-expression or activation of REV-ERB with synthetic agonists inhibits the replication of HCV and the related flaviruses dengue and Zika via perturbation of lipid signaling pathways. This study highlights a role for the circadian clock component REV-ERBα in regulating flavivirus replication.


Assuntos
Fatores de Transcrição ARNTL/genética , Relógios Circadianos/genética , Flavivirus/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Replicação Viral/efeitos dos fármacos , Fatores de Transcrição ARNTL/imunologia , Fatores de Transcrição ARNTL/farmacologia , Linhagem Celular , Relógios Circadianos/imunologia , Replicação do DNA , Dengue , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/genética , Flavivirus/efeitos dos fármacos , Flavivirus/metabolismo , Flavivirus/patogenicidade , Regulação da Expressão Gênica/genética , Genes Essenciais/genética , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/imunologia , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/farmacologia , Proteômica , RNA Mensageiro/metabolismo , Internalização do Vírus/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Zika virus/genética , Infecção por Zika virus
13.
Virchows Arch ; 474(3): 365-374, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30539318

RESUMO

Reconstitution of hepatocytes by hematopoietic stem cells-a phenomenon which occurs in rodents under highly selective conditions-results from infrequent fusion between incoming myelomonocytes and host hepatocytes, with subsequent proliferation. Human hematopoietic stem cell transplant recipients have been little studied, with some support for transdifferentiation (direct differentiation). We studied routinely obtained autopsy liver tissue of four female hematopoietic cell transplant recipients with male donors, using a highly specific conjoint immunohistochemistry in situ hybridization light microscopic technique. Hepatocyte nuclei were identified by cytokeratin (Cam5.2) staining and evaluated for X and Y chromosome content. Over 1.6 million hepatocytes were assessed for rare instances of donor origin, revealing a Y chromosome in 67. Mixed tetraploids (XXXY) and their nuclear truncation products (XXY, XY, Y) were directly demonstrated, with no detection of the male tetraploids (XXYY) that may result from transdifferentiation with subsequent tetraploidization, nor their unique truncation products (XYY, YY), implicating fusion as the mechanism. To determine whether it is the sole mechanism, we modeled the chromosome distribution based on the same probability of detection of each X chromosome, deriving parameters of sensitivity and female tetraploidy by best fit. We then hypothesized that the distribution of Y chromosome-containing cells could be predicted by a similar model. After modification to account for "clumpy" Y chromosomes, the observed results were in accord with the predicted results (p = 0.6). These results suggest that all the Y-containing cells, including apparent XY cells, derive from mixed tetraploids, consistent with fusion as the sole mechanism.


Assuntos
Diferenciação Celular , Cromossomos Humanos X , Cromossomos Humanos Y , Transplante de Células-Tronco Hematopoéticas , Hepatócitos/fisiologia , Adulto , Autopsia , Biomarcadores/análise , Fusão Celular , Feminino , Marcadores Genéticos , Genótipo , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/mortalidade , Hepatócitos/imunologia , Humanos , Células Híbridas , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Queratinas/análise , Masculino , Microscopia/métodos , Pessoa de Meia-Idade , Fenótipo , Ploidias , Resultado do Tratamento
14.
Gut ; 68(8): 1493-1503, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30487267

RESUMO

OBJECTIVE: In order to refine new therapeutic strategies in the pipeline for HBV cure, evaluation of virological and immunological changes compartmentalised at the site of infection will be required. We therefore investigated if liver fine needle aspirates (FNAs) could comprehensively sample the local immune landscape in parallel with viable hepatocytes. DESIGN: Matched blood, liver biopsy and FNAs from 28 patients with HBV and 15 without viral infection were analysed using 16-colour multiparameter flow cytometry. RESULTS: The proportion of CD4 T, CD8 T, Mucosal Associated Invariant T cell (MAIT), Natural Killer (NK) and B cells identified by FNA correlated with that in liver biopsies from the same donors. Populations of Programmed Death-1 (PD-1)hiCD39hi tissue-resident memory CD8 T cells (CD69+CD103+) and liver-resident NK cells (CXCR6+T-betloEomeshi), were identified by both FNA and liver biopsy, and not seen in the blood. Crucially, HBV-specific T cells could be identified by FNAs at similar frequencies to biopsies and enriched compared with blood. FNAs could simultaneously identify populations of myeloid cells and live hepatocytes expressing albumin, Scavenger Receptor class B type 1 (SR-B1), Programmed Death-Ligand 1 (PD-L1), whereas hepatocytes were poorly viable after the processing required for liver biopsies. CONCLUSION: We demonstrate for the first time that FNAs identify a range of intrahepatic immune cells including locally resident sentinel HBV-specific T cells and NK cells, together with PD-L1-expressing hepatocytes. In addition, we provide a scoring tool to estimate the extent to which an individual FNA has reliably sampled intrahepatic populations rather than contaminating blood. The broad profiling achieved by this less invasive, rapid technique makes it suitable for longitudinal monitoring of the liver to optimise new therapies for HBV.


Assuntos
Biópsia por Agulha Fina/métodos , Hepatite B Crônica , Hepatócitos , Adulto , Algoritmos , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , DNA Viral/sangue , Feminino , Citometria de Fluxo/métodos , Vírus da Hepatite B/genética , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Hepatócitos/imunologia , Hepatócitos/patologia , Humanos , Células Matadoras Naturais/imunologia , Masculino , Receptor de Morte Celular Programada 1/imunologia , Reprodutibilidade dos Testes , Receptores Depuradores Classe B/imunologia
15.
J Immunol ; 202(1): 20-28, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30587570

RESUMO

A highly efficacious malaria vaccine that prevents disease and breaks the cycle of infection remains an aspirational goal of medicine. Whole parasite vaccines based on the sporozoite forms of the parasite that target the clinically silent pre-erythrocytic stages of infection have emerged as one of the leading candidates. In animal models of malaria, these vaccines elicit potent neutralizing Ab responses against the sporozoite stage and cytotoxic T cells that eliminate parasite-infected hepatocytes. Among whole-sporozoite vaccines, immunization with live, replication-competent whole parasites engenders superior immunity and protection when compared with live replication-deficient sporozoites. As such, the genetic design of replication-competent vaccine strains holds the promise for a potent, broadly protective malaria vaccine. In this report, we will review the advances in whole-sporozoite vaccine development with a particular focus on genetically attenuated parasites both as malaria vaccine candidates and also as valuable tools to interrogate protective immunity against Plasmodium infection.


Assuntos
Hepatócitos/imunologia , Vacinas Antimaláricas/imunologia , Malária/imunologia , Plasmodium/fisiologia , Esporozoítos/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Anticorpos Neutralizantes/metabolismo , Anticorpos Antiprotozoários/metabolismo , Antígenos de Protozoários/imunologia , Engenharia Genética , Hepatócitos/parasitologia , Humanos , Malária/prevenção & controle
16.
Methods Mol Biol ; 1911: 459-479, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30593646

RESUMO

The complete life cycle of the hepatitis C virus (HCV) can be recapitulated in vivo using immunodeficient mice that have had their livers extensively repopulated with human hepatocytes. These human liver chimeric mouse models have enabled the study of many aspects of the HCV life cycle, including antiviral interventions that have helped to shape the curative landscape that is available today. The first human liver chimeric mouse model capable of supporting the HCV life cycle was generated in SCID-uPA mice. Although other human liver chimeric mouse models have since been developed, the SCID-uPA mouse model remains one of the most robust in vivo systems available for HCV studies. This chapter reviews development, validation and application of the SCID-uPA mouse model, and discusses their potential application for studying other liver-centric diseases and pathogens and for the design and testing of vaccine candidates for the eradication of HCV.


Assuntos
Modelos Animais de Doenças , Hepacivirus/fisiologia , Hepatite C/imunologia , Fígado/imunologia , Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Hepatite C/virologia , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Fígado/virologia , Transplante de Fígado , Camundongos , Camundongos SCID , Quimeras de Transplante , Ativador de Plasminogênio Tipo Uroquinase/imunologia
17.
Neonatal Netw ; 37(5): 271-280, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30567809

RESUMO

Gestational alloimmune liver disease (GALD) is initiated by maternal antibodies that attack fetal hepatocytes. The fetal immune response to the antibodies causes liver damage. The incidence of GALD is four per 100,000 live births in the United States. Frequently, liver injury leads to fetal loss or neonatal demise; nonetheless, the presentation of GALD has a wide range of severity. Survival rates have increased from 20 to 80 percent changes in treatment and understanding of GALD. Current treatment is focused on supportive care with intravenous immunoglobulin (IVIG) and exchange transfusions. Mortality risk is positively associated with the timing of diagnosis. Although there has been an increase in understanding this disease, the discovery of the specific alloantigen is still needed. Relevant embryology, pathophysiology, clinical manifestations, diagnosis, medical treatment, and prognosis are discussed to aid health care professionals in the early identification and treatment for the neonate and family unit.


Assuntos
Feto/imunologia , Feto/fisiopatologia , Imunoglobulinas Intravenosas/uso terapêutico , Doenças do Recém-Nascido/tratamento farmacológico , Hepatopatias/tratamento farmacológico , Hepatopatias/imunologia , Complicações na Gravidez/tratamento farmacológico , Adulto , Feminino , Hepatócitos/imunologia , Hepatócitos/patologia , Humanos , Recém-Nascido , Doenças do Recém-Nascido/imunologia , Masculino , Gravidez , Complicações na Gravidez/imunologia , Resultado do Tratamento , Estados Unidos
18.
J Autoimmun ; 95: 34-46, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30401504

RESUMO

Autoimmune hepatitis (AIH) is an orphan disease characterized by an autoimmune attack against hepatocytes. The exact sequence of events that leads to a breach of tolerance is incompletely understood. Current hypotheses suggest that environmental agents such as toxins or infectious agents like viruses cause a tissue damage that initiates autoimmunity in genetically susceptible individuals. The growing knowledge of the multi-facetted immune dysregulation, which involves Th1/Th17 polarization and the suspected inability of regulatory T cells to revert autoimmunity in the otherwise tolerogenic milieu of the liver, offers multiple new therapeutic approaches and targets. Standard of care (SOC) is treatment with corticosteroids with or without azathioprine, which is sufficient to induce remission in the majority of patients. However, it rarely cures AIH or restores intrahepatic immune tolerance. Hence, life-long therapy is required in the majority of patients. In addition, several studies suggest a weakening of immune regulation mediated by intrahepatic T cells under current therapies. Furthermore, second line therapies for non-responders, intolerant or otherwise difficult to treat patients are urgently needed as this is relevant for at least one fifth of all patients with inefficacy or intolerance to SOC. Current second line therapies are mainly based on single center retrospective experiences and none of them have been approved by regulatory authorities for AIH, yet. This article highlights new therapeutic approaches based on our growing knowledge on the pathophysiology of AIH. It focuses on cell-based therapies that strengthen or restore immune tolerance. An additional focus lies on new therapeutic agents showing promising results in non-hepatic autoimmune diseases that have a potential for treating AIH. The dynamics in the whole field of innovative therapies for non-hepatic autoimmune diseases will hopefully improve the therapeutic armamentarium for AIH patients in the future.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Dieta/métodos , Hepatite Autoimune/terapia , Fígado/efeitos dos fármacos , Terapia de Alvo Molecular/métodos , Linfócitos T Reguladores/efeitos dos fármacos , Corticosteroides/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Autoanticorpos/biossíntese , Azatioprina/uso terapêutico , Hepatite Autoimune/imunologia , Hepatite Autoimune/patologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/imunologia , Hepatócitos/patologia , Humanos , Tolerância Imunológica , Fígado/imunologia , Fígado/patologia , Proteínas da Gravidez/uso terapêutico , Padrão de Cuidado , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/patologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/patologia
19.
Nanotoxicology ; 12(10): 1198-1214, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30422028

RESUMO

Ultra-small superparamagnetic iron oxide nanoparticles (USPIO-NPs) are widely used as clinical magnetic resonance imaging contrast agents for hepatic diseases diagnosis. USPIO-NPs often damage the hepatocytes and affect the function of liver but its mechanism of action remains unclear. In the present study, USPIO-NPs caused higher cytotoxicity and lactate dehydrogenase (LDH) leakage in hepatic L02 cells than SPIO-NPs. Subsequently, USPIO-NPs affected more genes' expression than SPIO-NPs analyzed through microarray and bioinformatics analysis. The affected genes were involved in several biological processes, including calcium ion homeostasis, inflammatory response-related leukocyte chemotaxis, and migration. In addition, the level of endoplasmic reticulum (ER) calcium ion was increased by USPIO-NPs. USPIO-NPs also upregulated the genes related to acute-phase inflammation, including IL1B, IL6, IL18, TNFSF12, TNFRSF12, SAA1, SAA2, JAK1, STAT5B, and CXCL14. Furthermore, interleukin-6 (IL-6) secretion was elevated by USPIO-NPs as detected using ELISA. On the other hand, USPIO-NPs changed the morphology of ER and triggered the ER stress and unfolded protein response PERK/ATF4 pathway. Furthermore, blocking ER stress with inhibitor or ATF4 small interfering RNA counteracted IL-6-related acute-phase inflammation and cytotoxicity caused by USPIO-NPs. Taken together, we found that the USPIO-NPs could trigger stronger IL-6-related acute-phase inflammation than SPIO-NPs in hepatocytes. We demonstrated, for the first time, that IL-6-related acute-phase inflammation caused by NPs was regulated by PERK/ATF4 signaling. The PERK/ATF4 pathway explored in this study could be a candidate for diagnostic and therapeutic target against NPs-induced liver injury and cytotoxicity, which would be helpful for USPIO-NPs medical application.


Assuntos
Reação de Fase Aguda/induzido quimicamente , Meios de Contraste/toxicidade , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Nanopartículas de Magnetita/toxicidade , Reação de Fase Aguda/imunologia , Animais , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/imunologia , Hepatócitos/imunologia , Hepatócitos/metabolismo , Humanos , Interleucina-6/biossíntese , Tamanho da Partícula , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia
20.
J Immunol ; 201(12): 3731-3740, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30397035

RESUMO

Humoral alloimmunity negatively impacts both short- and long-term cell and solid organ transplant survival. We previously reported that alloantibody-mediated rejection of transplanted hepatocytes is critically dependent on host macrophages. However, the effector mechanism(s) of macrophage-mediated injury to allogeneic liver parenchymal cells is not known. We hypothesized that macrophage-mediated destruction of allogeneic hepatocytes occurs by cell-cell interactions requiring FcγRs. To examine this, alloantibody-dependent hepatocyte rejection in CD8-depleted wild-type (WT) and Fcγ-chain knockout (KO; lacking all functional FcγR) transplant recipients was evaluated. Alloantibody-mediated hepatocellular allograft rejection was abrogated in recipients lacking FcγR compared with WT recipients. We also investigated anti-FcγRI mAb, anti-FcγRIII mAb, and inhibitors of intracellular signaling (to block phagocytosis, cytokines, and reactive oxygen species [ROS]) in an in vitro alloantibody-dependent, macrophage-mediated hepatocytoxicity assay. Results showed that in vitro alloantibody-dependent, macrophage-mediated hepatocytotoxicity was critically dependent on FcγRs and ROS. The adoptive transfer of WT macrophages into CD8-depleted FcγR-deficient recipients was sufficient to induce alloantibody-mediated rejection, whereas adoptive transfer of macrophages from Fcγ-chain KO mice or ROS-deficient (p47 KO) macrophages was not. These results provide the first evidence, to our knowledge, that alloantibody-dependent hepatocellular allograft rejection is mediated by host macrophages through FcγR signaling and ROS cytotoxic effector mechanisms. These results support the investigation of novel immunotherapeutic strategies targeting macrophages, FcγRs, and/or downstream molecules, including ROS, to inhibit humoral immune damage of transplanted hepatocytes and perhaps other cell and solid organ transplants.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Rejeição de Enxerto/imunologia , Hepatócitos/imunologia , Macrófagos/imunologia , Receptores de IgG/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Células Cultivadas , Citotoxicidade Imunológica , DNA Helicases/genética , Humanos , Isoanticorpos/metabolismo , Transplante de Fígado , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo , Receptores de IgG/genética , Transdução de Sinais
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