RESUMO
Myeloid dendritic cells, including BDCA3hi DCs and BDCA1+ DCs (hereafter dubbed DC1 and DC2 for clarity), play a pivotal role in the induction and regulation of immune responses. Interestingly, a fraction of DC2 also express low to intermediate levels of BDCA3. It is unknown whether BDCA3+ DC2 also share other traits with DC1 that are absent in BDCA3- DC2 and/or whether BDCA3 expression renders DC2 functionally distinct from their BDCA3-lacking counterparts. Here, we used expression analysis on a predefined set of immunology-related genes to determine divergence between BDCA3-positive and BDCA3-negative DC2 and their relation to bona fide BDCA3hi DC1. Results showed that mRNA fingerprints of BDCA3+ DC2 and BDCA3- DC2 are very similar, and clearly distinct from that of DC1. Differences in mRNA expression, however, were observed between BDCA3+ DC2 and BDCA3- DC2 that pointed toward a more activated status of BDCA3+ DC2. In line with this, higher steady state maturation marker expression and TLR-induced maturation marker expression and inflammatory cytokine production by BDCA3+ DC2 were observed. This dataset provides insight into the relationship between myeloid DC populations and contributes to further understanding of DC immunobiology.
Assuntos
Antígenos de Superfície/metabolismo , Biomarcadores/metabolismo , Células Dendríticas/metabolismo , Células Mieloides/metabolismo , Transcrição Gênica , Citocinas/biossíntese , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , TrombomodulinaRESUMO
UNLABELLED: Due to a scarcity of immunocompetent animal models for viral hepatitis, little is known about the early innate immune responses in the liver. In various hepatotoxic models, both pro- and anti-inflammatory activities of recruited monocytes have been described. In this study, we compared the effect of liver inflammation induced by the Toll-like receptor 4 ligand lipopolysaccharide (LPS) with that of a persistent virus, lymphocytic choriomeningitis virus (LCMV) clone 13, on early innate intrahepatic immune responses in mice. LCMV infection induces a remarkable influx of inflammatory monocytes in the liver within 24 h, accompanied by increased transcript levels of several proinflammatory cytokines and chemokines in whole liver. Importantly, while a single LPS injection results in similar recruitment of inflammatory monocytes to the liver, the functional properties of the infiltrating cells are dramatically different in response to LPS versus LCMV infection. In fact, intrahepatic inflammatory monocytes are skewed toward a secretory phenotype with impaired phagocytosis in LCMV-induced liver inflammation but exhibit increased endocytic capacity after LPS challenge. In contrast, F4/80(high)-Kupffer cells retain their steady-state endocytic functions upon LCMV infection. Strikingly, the gene expression levels of inflammatory monocytes dramatically change upon LCMV exposure and resemble those of Kupffer cells. Since inflammatory monocytes outnumber Kupffer cells 24 h after LCMV infection, it is highly likely that inflammatory monocytes contribute to the intrahepatic inflammatory response during the early phase of infection. Our findings are instrumental in understanding the early immunological events during virus-induced liver disease and point toward inflammatory monocytes as potential target cells for future treatment options in viral hepatitis. IMPORTANCE: Insights into how the immune system deals with hepatitis B virus (HBV) and HCV are scarce due to the lack of adequate animal model systems. This knowledge is, however, crucial to developing new antiviral strategies aimed at eradicating these chronic infections. We model virus-host interactions during the initial phase of liver inflammation 24 h after inoculating mice with LCMV. We show that infected Kupffer cells are rapidly outnumbered by infiltrating inflammatory monocytes, which secrete proinflammatory cytokines but are less phagocytic. Nevertheless, these recruited inflammatory monocytes start to resemble Kupffer cells on a transcript level. The specificity of these cellular changes for virus-induced liver inflammation is corroborated by demonstrating opposite functions of monocytes after LPS challenge. Overall, this demonstrates the enormous functional and genetic plasticity of infiltrating monocytes and identifies them as an important target cell for future treatment regimens.
Assuntos
Hepatite Viral Animal/patologia , Células de Kupffer/imunologia , Fígado/patologia , Vírus da Coriomeningite Linfocítica/imunologia , Monócitos/imunologia , Animais , Citocinas/metabolismo , Perfilação da Expressão Gênica , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Based on their localization, Kupffer cells (KCs) likely interact with hepatitis B virus (HBV). However, the role of KCs in inducing immunity toward HBV is poorly understood. Therefore, the interaction of hepatitis B surface antigen (HBsAg) and KCs, and possible functional consequences, were assessed. METHODS: KCs in liver tissue from patients with chronic HBV were analyzed for presence of HBsAg and their phenotype, and compared with KCs in control liver tissue. Liver graft perfusate-derived KCs and in vitro-generated monocyte-derived macrophages were investigated for functional interaction with patient-derived HBsAg. RESULTS: Intrahepatic KCs were HBsAg positive and more activated than those from control livers. KCs internalized HBsAg in vitro, which did not change their phenotype, but strongly induced proinflammatory cytokine production. Additionally, monocyte-derived macrophages also interacted with HBsAg, leading to activation and cytokine production. Furthermore, HBsAg-exposed macrophages and KC activated natural killer (NK) cells, resulting in increased CD69 expression and interferon-γ production. CONCLUSIONS: KCs directly interact with HBsAg in vivo and in vitro. HBsAg-induced cytokine production by KCs and monocyte-derived macrophages and subsequent NK cell activation may be an early event in viral containment and may support induction of HBV-specific immunity upon HBV infection, but may also contribute to liver pathology.
Assuntos
Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Inflamação/imunologia , Interferon gama/imunologia , Células Matadoras Naturais/imunologia , Células de Kupffer/imunologia , Adulto , Células Dendríticas/imunologia , Humanos , Técnicas In Vitro/métodos , Fígado/imunologia , Ativação Linfocitária/imunologia , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/imunologia , Adulto JovemRESUMO
BACKGROUND & AIMS: Natural killer (NK) cells play an important role in the immune response to viruses. As the hepatitis B virus (HBV) replicates in hepatocytes, examination of the liver of chronic hepatitis B (CHB) patients is crucial to better understand the role of NK cells in HBV. HBeAg-negative CHB differs in many aspects from HBeAg-positive CHB, and until now little is known about the intrahepatic NK cell response in HBeAg-negative patients. Intrahepatic immune control might be different in HBeAg-negative as compared with HBeAg-positive patients. METHODS: Liver NK cells were investigated in 21 HBeAg-positive and 35 HBeAg-negative CHB patients. Biopsy specimens were processed for routine histopathology and staging according to Ishak scores. Intrahepatic and blood NK cell frequencies, activation status and function of NK cells were analysed by flow cytometry. RESULTS: In HBeAg-negative CHB patients, compared to blood, liver NK cells displayed a more activated phenotype and stimulation further increased the activation status, but production of IFN-γ was markedly less. There was no difference with HBeAg-positive CHB. Only in HBeAg-negative CHB, but not in HBeAg-positive CHB, NK cell activation was inversely correlated with HBsAg levels. CONCLUSIONS: The present study indicates that liver NK cells of CHB have a higher activation status compared to blood. However, they are not capable to increase cytokine production above levels reached by activated blood NK cells. In HBeAg-negative CHB, the levels of HBsAg may contribute to the incapacity of activated liver NK cells to increase cytokine production.
Assuntos
DNA Viral/sangue , Antígenos de Superfície da Hepatite B/sangue , Antígenos E da Hepatite B/sangue , Hepatite B Crônica/imunologia , Células Matadoras Naturais/imunologia , Fígado/patologia , Adulto , Células Cultivadas , Feminino , Vírus da Hepatite B/fisiologia , Humanos , Interferon gama/imunologia , Fígado/imunologia , Ativação Linfocitária/imunologia , Masculino , Replicação ViralRESUMO
There is increasing evidence that the function of NK cells in patients with chronic hepatitis B (CHB) infection is impaired. The underlying mechanism for the impaired NK cell function is still unknown. Since myeloid dendritic cells (mDC) are potent inducers of NK cells, we investigated the functional interaction of mDC and NK cells in CHB and the influence of antiviral therapy. Blood BDCA1(+) mDC and NK cells were isolated from 16 healthy controls or 39 CHB patients at baseline and during 6 months of antiviral therapy. After activation of mDC with poly(I · C) and gamma interferon (IFN-γ), mDC were cocultured with NK cells. Phenotype and function were analyzed in detail by flow cytometry and enzyme-linked immunosorbent assay. Our findings demonstrate that on poly(I · C)/IFN-γ-stimulated mDC from CHB patients, the expression of costimulatory molecules was enhanced, while cytokine production was reduced. In cocultures of poly(I · C)/IFN-γ-stimulated mDC and NK cells obtained from CHB patients, reduced mDC-induced NK cell activation (i.e., CD69 expression) and IFN-γ production compared to those in healthy individuals was observed. Antiviral therapy normalized mDC activity, since decreased expression of CD80 and CD86 on DC and of HLA-E on NK cells was observed, while poly(I · C)/IFN-γ-induced cytokine production by mDC was enhanced. In parallel, successful antiviral therapy resulted in improved mDC-induced NK cell activation and IFN-γ production. These data demonstrate that CHB patients display a diminished functional interaction between poly(I · C)/IFN-γ activated mDC and NK cells due to impaired mDC function, which can be partially restored by antiviral therapy. Enhancing this reciprocal interaction could reinforce the innate and thus the adaptive T cell response, and this may be an important step in achieving effective antiviral immunity.
Assuntos
Células Dendríticas/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Células Matadoras Naturais/imunologia , Receptor 3 Toll-Like/metabolismo , Adulto , Antivirais/farmacologia , Antivirais/uso terapêutico , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/imunologia , Técnicas de Cocultura , Citocinas/biossíntese , Células Dendríticas/efeitos dos fármacos , Feminino , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunofenotipagem , Células Matadoras Naturais/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Antígenos HLA-ERESUMO
Background & Aims: Antigen-specific immunotherapy is a promising strategy to treat HBV infection and hepatocellular carcinoma (HCC). To facilitate killing of malignant and/or infected hepatocytes, it is vital to know which T cell targets are presented by human leucocyte antigen (HLA)-I complexes on patient-derived hepatocytes. Here, we aimed to reveal the hepatocyte-specific HLA-I peptidome with emphasis on peptides derived from HBV proteins and tumour-associated antigens (TAA) to guide development of antigen-specific immunotherapy. Methods: Primary human hepatocytes were isolated with high purity from (HBV-infected) non-tumour and HCC tissues using a newly designed perfusion-free procedure. Hepatocyte-derived HLA-bound peptides were identified by unbiased mass spectrometry (MS), after which source proteins were subjected to Gene Ontology and pathway analysis. HBV antigen and TAA-derived HLA peptides were searched for using targeted MS, and a selection of peptides was tested for immunogenicity. Results: Using unbiased data-dependent acquisition (DDA), we acquired a high-quality HLA-I peptidome of 2 × 105 peptides that contained 8 HBV-derived peptides and 14 peptides from 8 known HCC-associated TAA that were exclusive to tumours. Of these, 3 HBV- and 12 TAA-derived HLA peptides were detected by targeted MS in the sample they were originally identified in by DDA. Moreover, 2 HBV- and 2 TAA-derived HLA peptides were detected in samples in which no identification was made using unbiased MS. Finally, immunogenicity was demonstrated for 5 HBV-derived and 3 TAA-derived peptides. Conclusions: We present a first HLA-I immunopeptidome of isolated primary human hepatocytes, devoid of immune cells. Identified HBV-derived and TAA-derived peptides directly aid development of antigen-specific immunotherapy for chronic HBV infection and HCC. The described methodology can also be applied to personalise immunotherapeutic treatment of liver diseases in general. Lay summary: Immunotherapy that aims to induce immune responses against a virus or tumour is a promising novel treatment option to treat chronic HBV infection and liver cancer. For the design of successful therapy, it is essential to know which fragments (i.e. peptides) of virus-derived and tumour-specific proteins are presented to the T cells of the immune system by diseased liver cells and are thus good targets for immunotherapy. Here, we have isolated liver cells from patients who have chronic HBV infection and/or liver cancer, analysed what peptides are presented by these cells, and assessed which peptides are able to drive immune responses.
RESUMO
The chemokine receptor CCR5 is implicated in the pathogenesis of various inflammatory diseases, such as multiple sclerosis (MS), atherosclerosis, transplant rejection, and autoimmunity. In previous studies, we have shown that MS lesions are characterized by enhanced expression of transcription factors associated with stress responses, ie, IRF-1, NF-kappaB, and CREB-1, which modulate expression of both classes of major histocompatibility complex (MHC) molecules. The expression of MHC-I and MHC-II molecules greatly overlaps with the expression of CCR5 in MS lesions. Therefore, we investigated whether these factors are also involved in the transcriptional regulation of CCR5. Using in vitro assays, we determined that neither IRF-1 nor NF-kappaB is involved in the activation of the CCR5 promoter. This is corroborated by the finding that these factors are not involved in the induction of endogenous CCR5 transcription in various cell types. In contrast, we show that CCR5 expression is regulated by the cAMP/CREB pathway and that interference in this pathway affects endogenous CCR5 transcription. From this, we conclude that the cAMP/CREB pathway is involved in the regulation of CCR5 transcription and that, given the ubiquitous nature of CREB-1 protein expression, additional regulatory mechanisms must contribute to cell type-specific expression of CCR5.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regiões Promotoras Genéticas , Receptores CCR5/genética , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Células Cultivadas , Colforsina/farmacologia , Primers do DNA/genética , Células Dendríticas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fator Regulador 1 de Interferon/metabolismo , Microglia/metabolismo , Monócitos/metabolismo , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , NF-kappa B/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Linfócitos T/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ativação TranscricionalRESUMO
TLR7 agonists are of high interest for the treatment of cancer, auto-immunity and chronic viral infections. They are known to activate plasmacytoid dendritic cells (pDCs) to produce high amounts of Type I Interferon (IFN) and to facilitate T and B cell responses, the latter with the help of maturation markers such as CD40, CD80 and CD86. The TLR7 single nucleotide polymorphism (SNP) rs179008 (GLn11Leu), sex and chronic viral infection have all been reported to influence pDC IFN production. It is unknown, however, whether these factors also influence pDC phenotypic maturation and thereby IFN-independent pDC functions. Furthermore, it is unclear whether SNP rs179008 influences HBV susceptibility and/or clearance. Here we investigated whether the SNP rs179008, sex and HBV infection affected phenotypic maturation of pDCs from 38 healthy individuals and 28 chronic HBV patients. In addition, we assessed SNP prevalence in a large cohort of healthy individuals (nâ¯=â¯231) and chronic HBV patients (nâ¯=â¯1054). Consistent with previous reports, the rs179008 variant allele was largely absent in Asians and more prevalent in Caucasians. Among Caucasians, the SNP was equally prevalent in healthy and chronically infected males. The SNP was, however, significantly more prevalent in healthy females than in those with chronic HBV infection (42 versus 28%), suggesting that in females it may offer protection from chronic infection. Ex vivo experiments demonstrated that induction of the co-stimulatory molecules CD40 and CD86 by TLR7 ligands, but not TLR9 ligands, was augmented in pDCs from healthy SNP-carrying females. Furthermore, CD80 and CD86 upregulation was more pronounced in females independent of the SNP. Lastly, our data suggested that chronic HBV infection impairs pDC maturation. These findings provide insight into factors determining TLR7 responses, which is important for further clinical development of TLR7-based therapies.
Assuntos
Diferenciação Celular , Células Dendríticas/fisiologia , Hepatite B Crônica/imunologia , Interferon Tipo I/metabolismo , Polimorfismo de Nucleotídeo Único , Fatores Sexuais , Receptor 7 Toll-Like/genética , Resistência à Doença , Etnicidade , Hepatite B Crônica/genética , HumanosRESUMO
Chronic hepatitis B virus (HBV) infection results from inadequate HBV-specific immunity. BDCA3+ dendritic cells (DCs) are professional antigen presenting cells considered to be important for antiviral responses because of specific characteristics, including high interferon-λ production. BDCA3+ DCs may thus also have a role in the immune response against HBV, and immunotherapeutic strategies aiming to activate DCs, including BDCA3+ DCs, in patient livers may represent an interesting treatment option for chronic HBV. However, neither the effect of chronic hepatitis B (CHB) infection on the frequency and function of BDCA3+ DCs in liver and blood, nor the effect of the viral surface protein (HBsAg) that is abundantly present in blood of infected individuals are known. Here, we provide an overview of BDCA3+ DC frequency and functional capacity in CHB patients. We find that intrahepatic BDCA3+ DC numbers are increased in CHB patients. BDCA3+ DCs from patient blood are not more mature at steady state, but display an impaired capacity to mature and to produce interferon-λ upon polyI:C stimulation. Furthermore, in vitro experiments exposing blood and intrahepatic BDCA3+ DCs to the viral envelope protein HBsAg demonstrate that HBsAg does not directly induce phenotypical maturation of BDCA3+ DCs, but may reduce IFN-λ production via an indirect unknown mechanism. These results suggest that BDCA3+ DCs are available in the blood and on site in HBV infected livers, but measures may need to be taken to revive their function for DC-targeted therapy.
Assuntos
Antígenos de Superfície/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Hepatite B Crônica/imunologia , Contagem de Células , Células Dendríticas/imunologia , Antígenos de Superfície da Hepatite B/metabolismo , Hepatite B Crônica/metabolismo , Humanos , Interferons/biossíntese , Fígado/imunologia , Fígado/virologia , TrombomodulinaRESUMO
Statins, the main therapy for hypercholesterolemia, are currently considered as possible immunomodulatory agents. Statins inhibit the production of proinflammatory cytokines and reduce the expression of several immunoregulatory molecules, including major histocompatibility complex class II (MHC-II) molecules. In this study, we investigated the mechanism by which simvastatin reduces the membrane expression of MHC-II molecules on several human cell types. We demonstrate that the reduction of MHC-II membrane expression by simvastatin correlates with disruption of cholesterol-containing microdomains, which transport and concentrate MHC-II molecules to the cell surface. In addition, we demonstrate that statins reduce cell-surface expression of other immunoregulatory molecules, which include MHC-I, CD3, CD4, CD8, CD28, CD40, CD80, CD86, and CD54. Our observations indicate that the downregulation of MHC-II at the cell surface contributes to the immunomodulatory properties of statins and is achieved through disruption of cholesterol-containing microdomains, which are involved in their intracellular transport.
Assuntos
Colesterol/metabolismo , Antígenos de Histocompatibilidade Classe II/biossíntese , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/imunologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Membrana Celular/metabolismo , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/imunologia , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestrutura , Expressão Gênica/efeitos dos fármacos , Células HeLa , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Microdomínios da Membrana/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/imunologia , Biossíntese de Proteínas/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/imunologia , Sinvastatina/farmacologia , Células U937RESUMO
Macrophages play an important role in immunity and homeostasis. Upon pathogen recognition via specific receptors, they rapidly induce inflammatory responses. This process is tightly controlled at the transcriptional level. The DNA binding zinc-finger protein CCCTC-binding factor (Ctcf) is a crucial regulator of long-range chromatin interactions and coordinates specific communication between transcription factors and gene expression processes. In this study, the Ctcf gene was specifically deleted in myeloid cells by making use of the transgenic Cre-LoxP system. Conditional deletion of the Ctcf gene in myeloid cells induced a mild phenotype in vivo. Ctcf-deficient mice exhibited significantly reduced expression of major histocompatibility complex (MHC) class II in the liver. Ctcf-deficient macrophages demonstrated a normal surface phenotype and phagocytosis capacity. Upon Toll-like receptor (TLR) stimulation, they produced normal levels of the pro-inflammatory cytokines IL-12 and IL-6, but manifested a strongly impaired capacity to produce tumor-necrosis factor (TNF) and IL-10, as well as to express the IL-10 family members IL-19, IL-20 and IL-24. Taken together, our data demonstrate a role of Ctcf that involves fine-tuning of macrophage function.
Assuntos
Citocinas/metabolismo , Regulação da Expressão Gênica , Fígado/metabolismo , Macrófagos/metabolismo , Complexo Principal de Histocompatibilidade/fisiologia , Células Mieloides/metabolismo , Proteínas Repressoras/fisiologia , Animais , Western Blotting , Fator de Ligação a CCCTC , Células Cultivadas , Citocinas/genética , Citometria de Fluxo , Técnicas Imunoenzimáticas , Integrases/metabolismo , Lipopolissacarídeos/farmacologia , Fígado/citologia , Macrófagos/citologia , Camundongos , Camundongos Knockout , Células Mieloides/citologia , Fagocitose/fisiologia , Fenótipo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
The combination of ribavirin and peginterferon is the current standard of anti-viral treatment for chronic HCV patients. However, little is known on the mode of action of ribavirin in the anti-viral treatment of HCV patients. To investigate the immunomodulatory mechanism of ribavirin, we studied peginterferon alone versus peginterferon and ribavirin in chronic HBV patients. The addition of ribavirin did not affect the number of myeloid dendritic cells (mDC) or plasmacytoid dendritic cells (pDC), nor did it enhance T-helper-1 cell activity or T-cell proliferation. In contrast, it increased upregulation of activation markers on mDC and pDC, which was sustained throughout treatment. However, the addition of ribavirin had no effect on IFNα production by pDC. Our findings demonstrate that, although ribavirin does not lead to a viral load decline, in vivo treatment with ribavirin affects the activation of pDC and mDC in chronic HBV patients.
Assuntos
Antivirais/administração & dosagem , Células Dendríticas/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Interferon-alfa/administração & dosagem , Polietilenoglicóis/administração & dosagem , Ribavirina/administração & dosagem , Adulto , Idoso , Células Dendríticas/imunologia , Quimioterapia Combinada , Feminino , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/administração & dosagem , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Carga Viral/efeitos dos fármacosRESUMO
The immunostimulatory role of Kupffer cells in various inflammatory liver diseases is still not fully understood. In this study, phenotypic and functional aspects of Kupffer cells from healthy C57BL/6 mice were analyzed and compared with those of splenic and peritoneal macrophages to generate a blueprint of the cells under steady-state conditions. In the mouse liver, only one population of Kupffer cells was identified as F4/80(high)CD11b(low) cells. We observed that freshy isolated Kupffer cells are endocytic and show a relatively high basal ROS content. Interestingly, despite expression of TLR mRNA on Kupffer cells, ligation of TLR4, TLR7/8, and TLR9 resulted in a weak induction of IL-10, low or undetectable levels of IL-12p40 and TNF, and up-regulation of CD40 on the surface. Kupffer cells and splenic macrophages show functional similarities, in comparison with peritoneal macrophages, as reflected by comparable levels of TLR4, TLR7/8, and TLR9 mRNA and low or undetectable levels of TNF and IL-12p40 produced upon TLR ligation. The unique, functional characteristics of Kupffer cells, demonstrated in this study, suggest that Kupffer cells under steady-state conditions are specialized as phagocytes to clear and degrade particulates and only play a limited immunoregulatory role via the release of soluble mediators.
Assuntos
Células de Kupffer/fisiologia , Macrófagos Peritoneais/fisiologia , Macrófagos/fisiologia , Baço/imunologia , Animais , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Antígeno CD11b/análise , Células Cultivadas , Quinases Associadas a Receptores de Interleucina-1/genética , Interleucina-10/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/fisiologiaRESUMO
Chronic hepatitis B virus (HBV) infection is caused by inadequate anti-viral immunity. Activation of plasmacytoid dendritic cells (pDC) leading to IFNα production is important for effective anti-viral immunity. Hepatitis B virus (HBV) infection lacks IFNα induction in animal models and patients and chronic HBV patients display impaired IFNα production by pDC. Therefore, HBV and HBV-derived proteins were examined for their effect on human pDC in vitro. In addition, the in vitro findings were compared to the function of pDC derived from chronic HBV patients ex vivo. In contrast to other viruses, HBV did not activate pDC. Moreover, HBV and HBsAg abrogated CpG-A/TLR9-induced, but not Loxoribine/TLR7-induced, mTOR-mediated S6 phosphorylation, subsequent IRF7 phosphorylation and IFNα gene transcription. HBV/HBsAg also diminished upregulation of co-stimulatory molecules, production of TNFα, IP-10 and IL-6 and pDC-induced NK cell function, whereas TLR7-induced pDC function was hardly affected. In line, HBsAg preferentially bound to TLR9-triggered pDC demonstrating that once pDC are able to bind HBV/HBsAg, the virus exerts its immune regulatory effect. HBV not only directly interfered with pDC function, but also indirectly by interfering with monocyte-pDC interaction. Also HBeAg diminished pDC function to a certain extent, but via another unknown mechanism. Interestingly, patients with HBeAg-positive chronic hepatitis B displayed impaired CpG-induced IFNα production by pDC without significant alterations in Loxoribine-induced pDC function compared to HBeAg-negative patients and healthy controls. The lack of activation and the active inhibition of pDC by HBV may both contribute to HBV persistence. The finding that the interaction between pDC and HBV may change upon activation may aid in the identification of a scavenging receptor supporting immunosuppressive effects of HBV and also in the design of novel treatment strategies for chronic HBV.
Assuntos
Células Dendríticas/virologia , Vírus da Hepatite B/imunologia , Estudos de Casos e Controles , Comunicação Celular/imunologia , Células Cultivadas , Células Dendríticas/imunologia , Humanos , Imunidade , Monócitos/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Proteínas Virais/farmacologiaRESUMO
UNLABELLED: Chronicity of hepatitis B virus (HBV) infection is characterized by a weak immune response to the virus. CD4+CD25+ regulatory T cells (Treg) are present in increased numbers in the peripheral blood of chronic HBV patients, and these Treg are capable of suppressing the HBV-specific immune response. The aim of this study was to abrogate Treg-mediated suppression of the HBV-specific immune response. Therefore, Treg and a Treg-depleted cell fraction were isolated from peripheral blood of chronic HBV patients. Subsequently, the suppressive effect of Treg on the response to HBV core antigen (HBcAg) and tetanus toxin was compared, and the effect of exogenous tumor necrosis factor alpha (TNF-alpha), interleukin-1-beta (IL-1beta), or neutralizing antibodies against interleukin-10 (IL-10) or transforming growth factor beta (TGF-beta) on Treg-mediated suppression was determined. The results show that Treg of chronic HBV patients had a more potent suppressive effect on the response to HBcAg compared with the response to tetanus toxin. Neutralization of IL-10 and TGF-beta or exogenous IL-1beta had no effect on Treg-mediated suppression of the anti-HBcAg response, whereas exogenous TNF-alpha partially abrogated Treg-mediated suppression. Preincubation of Treg with TNF-alpha demonstrated that TNF-alpha had a direct effect on the Treg. No difference was observed in the type II TNF receptor expression by Treg from chronic HBV patients and healthy controls. CONCLUSION: Treg-mediated suppression of the anti-HBV response can be reduced by exogenous TNF-alpha. Because chronic HBV patients are known to produce less TNF-alpha, these data implicate an important role for TNF-alpha in the impaired antiviral response in chronic HBV.
Assuntos
Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Tolerância Imunológica , Linfócitos T Reguladores/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Anticorpos/farmacologia , Feminino , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Humanos , Tolerância Imunológica/efeitos dos fármacos , Interleucina-10/antagonistas & inibidores , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , Masculino , Receptores Tipo II do Fator de Necrose Tumoral , Linfócitos T Reguladores/efeitos dos fármacos , Toxina Tetânica/imunologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
In patients with chronic hepatitis B virus (HBV), 2 predominant precursor dendritic cell (DC) subtypes, the myeloid dendritic cell (mDC) and the plasmacytoid dendritic cell (pDC), were recently found to be functionally impaired. HBV DNA was found to be present in the DC subtypes, but no viral replication could be detected. The question remains whether simply the presence of the virus and viral proteins causes this dysfunction of DCs. To address this issue, the effect of viral load reduction resulting from treatment with the nucleotide analogue adefovir dipivoxil on the number and functionality of circulating DCs was studied during 6 months of treatment. Treatment resulted in a mean 5 log(10) decrease in the viral load and normalization of alanine aminotransferase within 3 months. The number of mDCs, but not of pDCs, increased significantly over 6 months of treatment to a level comparable to that of uninfected healthy controls. The allostimulatory capacity of isolated and in vitro matured mDCs increased significantly after 3 months of treatment. Accordingly, mDCs exhibited an increased capacity to produce tumor necrosis factor alpha and interleukin-12 after 3-6 months of treatment. There was no change in interferon alpha production by pDCs during treatment. In conclusion, adefovir treatment results in an improvement in the number and functionality of mDCs, but not of pDCs. Our findings provide clues for the reasons why current antiviral therapy does not lead to consistently sustained viral eradication.
Assuntos
Adenina/análogos & derivados , Antivirais/farmacologia , Células Dendríticas/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Imunossupressores/farmacologia , Células Mieloides/efeitos dos fármacos , Organofosfonatos/farmacologia , Adenina/farmacologia , Adenina/uso terapêutico , Adulto , Idoso , Alanina Transaminase/sangue , Alanina Transaminase/efeitos dos fármacos , Antígenos CD/metabolismo , Antivirais/uso terapêutico , Citocinas/metabolismo , DNA Viral/sangue , DNA Viral/efeitos dos fármacos , Células Dendríticas/metabolismo , Feminino , Hepatite B Crônica/metabolismo , Hepatite B Crônica/virologia , Humanos , Imunossupressores/uso terapêutico , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Células Mieloides/metabolismo , Organofosfonatos/uso terapêutico , Carga ViralRESUMO
Downregulation of major histocompatibility complex (MHC) molecules by tumor cells impairs cellular immune recognition and contributes to inefficient cell-mediated tumor eradication. Low or lack of expression of MHC molecules is frequently observed in early developmental or embryonically derived tumor cells. Considering the central role of the class II transactivator (CIITA) in MHC class II- and class I-mediated antigen presentation, we compared the induction of CIITA by interferon-gamma (IFN-gamma) in a diverse panel of developmental and more differentiated tumor cell lines. In contrast to the more differentiated tumor cell lines, none of the developmental tumor cell lines were capable of expressing CIITA after treatment with IFN-gamma. Remarkably, in transient transfection assays, CIITA promoter IV (CIITA-PIV) was found to be induced by IFN-gamma. Southern blot analysis of genomic DNA obtained from the developmental tumor cell lines indicated that the absence of endogenous CIITA induction was due to methylation of the CIITA-PIV region. Exposure to 5-azacytidine restored induction of CIITA and congruent HLA-DRA expression in these cells. The observation that only developmental tumor cell lines, originating from various tissues, employ methylation to silence CIITA expression may reflect the natural status of CIITA expression during early development rather than oncogenic transformation.
Assuntos
Metilação de DNA , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Genes MHC da Classe II , Antígenos HLA-DR/biossíntese , Interferon gama/farmacologia , Proteínas de Neoplasias/biossíntese , Proteínas Nucleares , Regiões Promotoras Genéticas/efeitos dos fármacos , Transativadores/biossíntese , Azacitidina/farmacologia , Linfoma de Burkitt/patologia , Carcinoma/patologia , Diferenciação Celular , Ilhas de CpG , Metilação de DNA/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Melanoma/patologia , Proteínas de Neoplasias/genética , Transdução de Sinais/efeitos dos fármacos , Transativadores/genética , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
beta(2)-Microglobulin (beta(2)m) is a chaperone of major histocompatibility complex (MHC) class I (-like) molecules that play a central role in antigen presentation, immunoglobulin transport, and iron metabolism. It is therefore of importance that beta(2)m is adequately expressed in cells that perform these functions, such as hematopoietic cells. In this study, we investigated the transcriptional regulation of beta(2)m in lymphoid and myeloid cell lines through a promoter containing a putative E box, Ets/interferon-stimulated response element (ISRE), and kappa B site. Here we show that upstream stimulatory factor 1 (USF1) and USF2 bind to the E box and regulate beta(2)m transactivation. The nuclear factor kappa B (NF-kappa B) subunits p50 and p65 bind to the kappa B box and p65 transactivates beta(2)m. Interferon regulatory factor 1 (IRF1), IRF2, IRF4, and IRF8, but not PU.1, bind to the Ets/ISRE, and IRF1 and IRF3 are strong transactivators of beta(2)m. Together, all 3 boxes are important for the constitutive and cytokine-induced levels of beta(2)m expression in lymphoid and myeloid cell types. As such, beta(2)m transactivation is under the control of important transcriptional pathways, which are activated during injury, infection, and inflammation.
Assuntos
Linfócitos/metabolismo , Células Mieloides/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Repressoras , Ativação Transcricional , Microglobulina beta-2/genética , Proteínas Aviárias , Linhagem Celular Transformada/efeitos dos fármacos , Linhagem Celular Transformada/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Infecções/genética , Inflamação/genética , Fator Regulador 1 de Interferon , Fator Regulador 2 de Interferon , Fator Regulador 3 de Interferon , Fatores Reguladores de Interferon , Interferon beta-1a , Interferon beta/farmacologia , Interferon gama/farmacologia , Modelos Genéticos , NF-kappa B/metabolismo , Fosfoproteínas/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Sequências Reguladoras de Ácido Nucleico , Transdução de Sinais , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Fatores Estimuladores Upstream , Ferimentos e Lesões/genéticaRESUMO
The expression of HLA-G in extravillous cytotrophoblast cells coincides with a general lack of classical major histocompatibility complex (MHC) class I expression in this tissue. This differential expression of HLA-G and classical HLA class I molecules in trophoblasts suggests a tight transcriptional control of MHC class I genes. Transactivation of the classical MHC class I genes is mediated by two groups of juxtaposed cis-acting elements that can be viewed as regulatory modules. Both modules are divergent in HLA-G, rendering this gene unresponsive to NF-kappaB, IRF1, and class II transactivator (CIITA)-mediated induction pathways. In this study, we searched for alternative regulatory elements in the 1438-bp HLA-G promoter region. HLA-G was not responsive to interferon-alpha (IFNalpha), IFNbeta, or IFNgamma, despite the presence of an upstream ISRE binding IRF1 in vitro. However, the HLA-G promoter contains three CRE/TRE elements with binding affinity for CREB/ATF and Fos/Jun proteins both in vitro and in vivo. In transient transfection assays, it was shown that HLA-G transactivation is regulated by CREB, CREB-binding protein (CBP), and p300. Moreover, immunohistochemical analysis demonstrated that HLA-G is co-expressed with CREB and CBP in extravillous cytotrophoblasts, revealing the in vivo relevance of this transactivation pathway. This implies a unique regulation of HLA-G transcription among the MHC class I genes.