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[This corrects the article DOI: 10.3389/fonc.2019.01253.].
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Chemerin is a multifunctional protein acting mainly through the G protein-coupled receptor ChemR23/CMKLR1/Chemerin1. Its expression is frequently downregulated in human tumors, including in melanoma and squamous cell carcinoma of the skin and anti-tumoral properties of chemerin were reported in mouse tumor graft models. In the present study, we report the development of spontaneous skin tumors in aged ChemR23-deficient mice. In order to test the potential therapeutic benefit of chemerin analogs, a transgenic model in which bioactive chemerin is over-expressed by basal keratinocytes was generated. These animals are characterized by increased levels of chemerin immunoreactivity and bioactivity in the skin and the circulation. In a chemical carcinogenesis model, papillomas developed later, were less numerous, and their progression to carcinomas was delayed. Temporal control of chemerin expression by doxycycline allowed to attribute its effects to late stages of carcinogenesis. The protective effects of chemerin were partly abrogated by ChemR23 invalidation. These results demonstrate that chemerin is able to delay very significantly tumor progression in a model that recapitulates closely the evolution of solid cancer types in human and suggest that the chemerin-ChemR23 system might constitute an interesting target for therapeutic intervention in the cancer field.
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Chronic hepatitis B virus (HBV) infection is a serious public health burden, and current therapies cannot achieve satisfactory cure rate. There are high unmet medical needs of novel therapeutic agents with differentiated mechanism of action (MOA) from the current standard of care. RG7834, a compound from the dihydroquinolizinone (DHQ) chemical series, is a first-in-class highly selective and orally bioavailable HBV inhibitor which can reduce both viral antigens and viral DNA with a novel mechanism of action. Here we report the discovery of RG7834 from a phenotypic screening and the structure-activity relationship (SAR) of the DHQ chemical series. RG7834 can selectively inhibit HBV but not other DNA or RNA viruses in a virus panel screening. Both in vitro and in vivo profiles of RG7834 are described herein, and the data support further development of this compound as a chronic HBV therapy.
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Regulación Viral de la Expresión Génica/efectos de los fármacos , Virus de la Hepatitis B/efectos de los fármacos , Quinolinas/farmacología , Quinolinas/farmacocinética , Administración Oral , Animales , Disponibilidad Biológica , Células CACO-2 , Células Hep G2 , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/metabolismo , Humanos , Masculino , Ratones , Fenotipo , Quinolinas/administración & dosificación , Quinolinas/química , Relación Estructura-ActividadRESUMEN
BACKGROUND & AIMS: The hallmarks of chronic HBV infection are a high viral load (HBV DNA) and even higher levels (>100-fold in excess of virions) of non-infectious membranous particles containing the tolerogenic viral S antigen (HBsAg). Currently, standard treatment effectively reduces viremia but only rarely results in a functional cure (defined as sustained HBsAg loss). There is an urgent need to identify novel therapies that reduce HBsAg levels and restore virus-specific immune responsiveness in patients. We report the discovery of a novel, potent and orally bioavailable small molecule inhibitor of HBV gene expression (RG7834). METHODS: RG7834 antiviral characteristics and selectivity against HBV were evaluated in HBV natural infection assays and in a urokinase-type plasminogen activator/severe combined immunodeficiency humanized mouse model of HBV infection, either alone or in combination with entecavir. RESULTS: Unlike nucleos(t)ide therapies, which reduce viremia but do not lead to an effective reduction in HBV antigen expression, RG7834 significantly reduced the levels of viral proteins (including HBsAg), as well as lowering viremia. Consistent with its proposed mechanism of action, time course RNA-seq analysis revealed a fast and selective reduction in HBV mRNAs in response to RG7834 treatment. Furthermore, oral treatment of HBV-infected humanized mice with RG7834 led to a mean HBsAg reduction of 1.09â¯log10 compared to entecavir, which had no significant effect on HBsAg levels. Combination of RG7834, entecavir and pegylated interferon α-2a led to significant reductions of both HBV DNA and HBsAg levels in humanized mice. CONCLUSION: We have identified a novel oral HBV viral gene expression inhibitor that blocks viral antigen and virion production, that is highly selective for HBV, and has a unique antiviral profile that is clearly differentiated from nucleos(t)ide analogues. LAY SUMMARY: We discovered a novel small molecule viral expression inhibitor that is highly selective for HBV and unlike current therapy inhibits the expression of viral proteins by specifically reducing HBV mRNAs. RG7834 can therefore potentially provide anti-HBV benefits and increase HBV cure rates, by direct reduction of viral agents needed to complete the viral life cycle, as well as a reduction of viral agents involved in evasion of the host immune responses.
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Antivirales , Regulación Viral de la Expresión Génica/efectos de los fármacos , Virus de la Hepatitis B , Hepatitis B Crónica , Bibliotecas de Moléculas Pequeñas , Administración Oral , Animales , Antivirales/administración & dosificación , Antivirales/efectos adversos , Antivirales/farmacocinética , Disponibilidad Biológica , ADN Viral/aislamiento & purificación , Modelos Animales de Enfermedad , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/virología , Ratones , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/efectos adversos , Bibliotecas de Moléculas Pequeñas/farmacocinética , Resultado del Tratamiento , Carga Viral/efectos de los fármacosRESUMEN
The liver is the largest gland in the human body and functions as an innate immune organ. Liver macrophages called Kupffer cells (KC) constitute the largest group of macrophages in the human body. Innate immune responses involving KC represent the first line of defense against pathogens in the liver. Human monocyte-derived macrophages have been used to characterize inflammasome responses that lead to the release of the proinflammatory cytokines IL-1ß and IL-18, but it has not yet been determined whether human KC contain functional inflammasomes. We show, to our knowledge for the first time, that KC express genes and proteins that make up several different inflammasome complexes. Moreover, activation of KC in response to the absent in melanoma 2 (AIM2) inflammasome led to the production of IL-1ß and IL-18, which activated IL-8 transcription and hepatic NK cell activity, respectively. Other inflammasome responses were also activated in response to selected bacteria and viruses. However, hepatitis B virus inhibited the AIM2 inflammasome by reducing the mRNA stability of IFN regulatory factor 7, which regulated AIM2 transcription. These data demonstrate the production of IL-1ß and IL-18 in KC, suggesting that KC contain functional inflammasomes that could be important players in the innate immune response following certain infections of the liver. We think our findings could potentially aid therapeutic approaches against chronic liver diseases that activate the inflammasome.
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Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Inflamasomas/metabolismo , Células Asesinas Naturales/inmunología , Macrófagos del Hígado/fisiología , Hígado/inmunología , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Humanos , Inmunidad Innata , Factor 7 Regulador del Interferón/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Activación de LinfocitosRESUMEN
In HBV-infected patients, therapies with nucleoside analogues or IFNα remain ineffective in eradicating the infection. Our aim was to re-analyze the anti-HBV activity of a large panel of IFNs and cytokines in vitro using non-transformed cultured hepatocytes infected with HBV, to identify new immune-therapeutic options. HepaRG cells and primary human hepatocytes were infected with HBV and, when infection was established, treated with various concentrations of different IFNs or inflammatory cytokines. Viral parameters were evaluated by quantifying HBV nucleic acids by qPCR and Southern Blot, and secreted HBV antigens were evaluated using ELISA. The cytokines tested were type-I IFNs, IFNγ, type-III IFNs, TNFα, IL-6, IL-1ß, IL-18 as well as nucleos(t)ide analogues tenofovir and ribavirin. Cytokines and drugs, with the exception of IL-18 and ribavirin, exhibited a suppressive effect on HBV replication at least as strong as, but often stronger than, IFNα. The cytokine presenting the highest effect on HBV DNA was IL-1ß, which exerted its inhibition within picomolar range. Importantly, we noticed differential effects on other parameters (HBV RNA, HBeAg, HBsAg) between both IFNs and inflammatory cytokines, thus suggesting different mechanisms of action. The combination of IL-1ß and already used therapies, i.e. IFNα or tenofovir, demonstrated a stronger or similar anti-HBV activity. IL-1ß was found to have a very potent antiviral effect against HBV in vitro. HBV was previously shown to promptly inhibit IL-1ß production in Kupffer cells. Strategies aiming at unlocking this inhibition and restoring local production of IL-1ß may help to further inhibit HBV replication in vivo.
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Antivirales/farmacología , Citocinas/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/fisiología , Hepatocitos/virología , Mediadores de Inflamación/farmacología , Interferones/farmacología , Antígenos Virales/inmunología , Antígenos Virales/metabolismo , Línea Celular , Células Cultivadas , ADN Viral , Hepatocitos/efectos de los fármacos , Humanos , Inmunidad Innata , Factores Inmunológicos/farmacología , ARN Viral , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND & AIMS: The outcome of hepatitis B virus (HBV) infection may be influenced by early interactions between the virus and hepatocyte innate immune responses. To date, the study of such interactions during the very early step of infection has not been adequately investigated. METHODS: We used the HepaRG cell line, as well as primary human hepatocytes to analyze, within 24h of exposure to HBV, either delivered by a physiologic route or baculovirus vector (Bac-HBV), the early modulation of the expression of selected antiviral/pro-inflammatory cytokines and interferon stimulated genes. Experiments were also performed in the presence or absence of innate receptor agonists to investigate early HBV-induced blockade of innate responses. RESULTS: We show that hepatocytes themselves could detect HBV, and express innate genes when exposed to either HBV virions or Bac-HBV. Whereas Bac-HBV triggered a strong antiviral cytokine secretion followed by the clearance of replicative intermediates, a physiologic HBV exposure led to an abortive response. The early inhibition of innate response by HBV was mainly evidenced on Toll-like receptor 3 and RIG-I/MDA5 signaling pathways upon engagement with exogenous agonist, leading to a decreased expression of several pro-inflammatory and antiviral cytokine genes. Finally, we demonstrate that this early inhibition of dsRNA-mediated response is due to factor(s) present in the HBV inoculum, but not being HBsAg or HBeAg themselves, and does not require de novo viral protein synthesis and replication. CONCLUSIONS: Our data provide strong evidence that HBV viral particles themselves can readily inhibit host innate immune responses upon virion/cell interactions, and may explain, at least partially, the "stealthy" character of HBV.
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Virus de la Hepatitis B/inmunología , Hepatocitos/inmunología , Hepatocitos/virología , Inmunidad Innata , Línea Celular , Células Cultivadas , Expresión Génica , Antígenos de Superficie de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/inmunología , Antígenos e de la Hepatitis B/genética , Antígenos e de la Hepatitis B/inmunología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/patogenicidad , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata/genética , Interleucina-6/biosíntesis , ARN Bicatenario/genética , ARN Bicatenario/inmunologíaRESUMEN
BACKGROUND & AIMS: HepaRG cells are considered as the best surrogate model to primary human hepatocyte (PHH) culture to investigate host-pathogen interactions. Yet their innate immune functions remain unknown. In this study, we explored the expression and functionality of Toll-like (TLR) and retinoic acid-inducible gene-1 (RIG-I)-like receptors (RLR) in these cells. METHODS: Gene and protein expression levels of TLR-1 to 9 and RLR in HepaRG were mainly compared to PHH, by RT-qPCR, FACS, and Western blotting. Their functionality was assessed, by measuring the induction of toll/rig-like themselves and several target innate gene expressions, as well as the secretion of IL-6, IP-10, and type I interferon (IFN), upon agonist stimulation. Their functionality was also shown by measuring the antiviral activity of some TLR/RLR agonists against hepatitis B virus (HBV) infection. RESULTS: The basal gene and protein expression profile of TLR/RLR in HepaRG cells was similar to PHH. Most receptors, except for TLR-7 and 9, were expressed as proteins and functionally active as shown by the induction of some innate genes, as well as by the secretion of IL-6 and IP-10, upon agonist stimulation. The highest levels of IL-6 and IP-10 secretion were obtained by TLR-2 and TLR-3 agonist stimulation respectively. The highest preventive anti-HBV activity was obtained following TLR-2, TLR-4 or RIG-I/MDA-5 stimulations, which correlated with their high capacity to produce both cytokines. CONCLUSIONS: Our results indicate that HepaRG cells express a similar pattern of functional TLR/RLR as compared to PHH, thus qualifying HepaRG cells as a surrogate model to study pathogen interactions within a hepatocyte innate system.
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Proteína 58 DEAD Box/genética , ADN Viral/genética , Regulación de la Expresión Génica , Virus de la Hepatitis B/genética , Hepatitis B/genética , Hepatocitos/patología , Receptores Toll-Like/genética , Western Blotting , Células Cultivadas , Proteína 58 DEAD Box/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Hepatitis B/metabolismo , Hepatitis B/virología , Hepatocitos/metabolismo , Hepatocitos/virología , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Citoplasmáticos y Nucleares , Receptores Inmunológicos , Receptores Toll-Like/biosíntesis , Replicación ViralRESUMEN
Chemerin was initially described as a chemoattractant factor for leukocyte populations. More recently, the protein has also been reported to be an adipokine, regulating adipocyte differentiation in vitro via its receptor ChemR23, and to be correlated with BMI and other parameters of the metabolic syndrome in humans. The aim of this study was to investigate the role of the chemerin/ChemR23 axis in the regulation of metabolism in vivo, using a mouse knockout (KO) model for ChemR23 (Cmklr1) in a C57BL/6 genetic background. Body weight and adipose tissue mass did not differ significantly in young animals, but were significantly higher in ChemR23 KO mice aged above 12 months. Glucose tolerance was unaffected. No significant modifications in the levels of blood lipids were observed and no increase in the levels of inflammatory markers was observed in the adipose tissue of KO mice. A high-fat diet did not exacerbate the obese phenotype in ChemR23 KO mice. No obvious defect in adipocyte differentiation was detected, while a marker of lipogenic activity (GPD1 expression) was found to be elevated. In conclusion, the chemerin/ChemR23 system does not appear to play a major role in adipocyte differentiation in vivo, but it may be involved in adipose tissue homeostasis.
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Adipogénesis , Envejecimiento , Factores Quimiotácticos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Obesidad/metabolismo , Sobrepeso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Tejido Adiposo Blanco/enzimología , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/patología , Adiposidad , Animales , Biomarcadores/metabolismo , Quimiocinas , Factores Quimiotácticos/genética , Dieta Alta en Grasa/efectos adversos , Femenino , Glicerolfosfato Deshidrogenasa/genética , Glicerolfosfato Deshidrogenasa/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Isoenzimas/metabolismo , Lipogénesis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/etiología , Obesidad/patología , Sobrepeso/etiología , Sobrepeso/patología , Receptores de Quimiocina , Receptores Acoplados a Proteínas G/genética , Caracteres Sexuales , Aumento de PesoRESUMEN
Macrophages constitute a major component of innate immunity and play an essential role in defense mechanisms against external aggressions and in inflammatory responses. Chemerin, a chemoattractant protein, is generated in inflammatory conditions, and recruits cells expressing the G protein-coupled receptor ChemR23, including macrophages. Chemerin was initially expected to behave as a pro-inflammatory agent. However, recent data described more complex activities that are either pro- or anti-inflammatory, according to the disease model investigated. In the present study, peritoneal macrophages were generated from WT or ChemR23(-/-) mice, stimulated with lipopolyssaccharide in combination or not with IFN-γ and the production of pro- (TNF-α, IL-1ß and IL-6) and anti-inflammatory (IL-10) cytokines was evaluated using qRT-PCR and ELISA. Human macrophages generated from peripheral blood monocytes were also tested in parallel. Peritoneal macrophages from WT mice, recruited by thioglycolate or polyacrylamide beads, functionally expressed ChemR23, as assessed by flow cytometry, binding and chemotaxis assays. However, chemerin had no effect on the strong upregulation of cytokine release by these cells upon stimulation by LPS or LPS/IFN-γ, whatever the concentration tested. Similar data were obtained with human macrophages. In conclusion, our results rule out the direct anti-inflammatory effect of chemerin on macrophages ex vivo, described previously in the literature, despite the expression of a functional ChemR23 receptor in these cells.
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Quimiocinas/metabolismo , Factores Quimiotácticos/metabolismo , Mediadores de Inflamación/metabolismo , Inflamación/patología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Macrófagos Peritoneales/metabolismo , Receptores de Quimiocina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Resinas Acrílicas , Animales , Citocinas/biosíntesis , Humanos , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Microesferas , TioglicolatosRESUMEN
Viral diseases of the respiratory tract, which include influenza pandemic, children acute bronchiolitis, and viral pneumonia of the elderly, represent major health problems. Plasmacytoid dendritic cells play an important role in anti-viral immunity, and these cells were recently shown to express ChemR23, the receptor for the chemoattractant protein chemerin, which is expressed by epithelial cells in the lung. Our aim was to determine the role played by the chemerin/ChemR23 system in the physiopathology of viral pneumonia, using the pneumonia virus of mice (PVM) as a model. Wild-type and ChemR23 knock-out mice were infected by PVM and followed for functional and inflammatory parameters. ChemR23(-/-) mice displayed higher mortality/morbidity, alteration of lung function, delayed viral clearance and increased neutrophilic infiltration. We demonstrated in these mice a lower recruitment of plasmacytoid dendritic cells and a reduction in type I interferon production. The role of plasmacytoid dendritic cells was further addressed by performing depletion and adoptive transfer experiments as well as by the generation of chimeric mice, demonstrating two opposite effects of the chemerin/ChemR23 system. First, the ChemR23-dependent recruitment of plasmacytoid dendritic cells contributes to adaptive immune responses and viral clearance, but also enhances the inflammatory response. Second, increased morbidity/mortality in ChemR23(-/-) mice is not due to defective plasmacytoid dendritic cells recruitment, but rather to the loss of an anti-inflammatory pathway involving ChemR23 expressed by non-leukocytic cells. The chemerin/ChemR23 system plays important roles in the physiopathology of viral pneumonia, and might therefore be considered as a therapeutic target for anti-viral and anti-inflammatory therapies.
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Factores Quimiotácticos/metabolismo , Células Dendríticas/inmunología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Virus de la Neumonía Murina/inmunología , Neumonía Viral/inmunología , Infecciones por Pneumovirus/inmunología , Receptores Acoplados a Proteínas G/inmunología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Quimiocinas , Factores Quimiotácticos/biosíntesis , Células Dendríticas/metabolismo , Mediadores de Inflamación , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Interferón Tipo I/biosíntesis , Interferón Tipo I/deficiencia , Pulmón/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Virus de la Neumonía Murina/metabolismo , Virus de la Neumonía Murina/patogenicidad , Neumonía Viral/metabolismo , Infecciones por Pneumovirus/metabolismo , Receptores de Quimiocina , Receptores Acoplados a Proteínas G/biosíntesis , Receptores Acoplados a Proteínas G/genética , Carga ViralRESUMEN
Chemerin is the ligand of the ChemR23 receptor and a chemoattractant factor for human immature dendritic cells (DCs), macrophages, and NK cells. In this study, we characterized the mouse chemerin/ChemR23 system in terms of pharmacology, structure-function, distribution, and in vivo biological properties. Mouse chemerin is synthesized as an inactive precursor (prochemerin) requiring, as in human, the precise processing of its C terminus for generating an agonist of ChemR23. Mouse ChemR23 is highly expressed in immature plasmacytoid DCs and at lower levels in myeloid DCs, macrophages, and NK cells. Mouse prochemerin is expressed in most epithelial cells acting as barriers for pathogens but not in leukocytes. Chemerin promotes calcium mobilization and chemotaxis on DCs and macrophages and these functional responses were abrogated in ChemR23 knockout mice. In a mouse model of acute lung inflammation induced by LPS, chemerin displayed potent anti-inflammatory properties, reducing neutrophil infiltration and inflammatory cytokine release in a ChemR23-dependent manner. ChemR23 knockout mice were unresponsive to chemerin and displayed an increased neutrophil infiltrate following LPS challenge. Altogether, the mouse chemerin/ChemR23 system is structurally and functionally conserved between human and mouse, and mouse can therefore be considered as a good model for studying the anti-inflammatory role of this system in the regulation of immune responses and inflammatory diseases.
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Factores Quimiotácticos/metabolismo , Células Dendríticas/inmunología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Macrófagos/inmunología , Neumonía/inmunología , Receptores Acoplados a Proteínas G/metabolismo , Enfermedad Aguda , Aequorina/inmunología , Aequorina/metabolismo , Animales , Apoproteínas/inmunología , Apoproteínas/metabolismo , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Calcio/inmunología , Calcio/metabolismo , Quimiocinas , Factores Quimiotácticos/inmunología , Factores Quimiotácticos/farmacología , Quimiotaxis/efectos de los fármacos , Quimiotaxis/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Péptidos y Proteínas de Señalización Intercelular/inmunología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Lipopolisacáridos/farmacología , Pulmón/inmunología , Pulmón/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Péptidos/inmunología , Péptidos/metabolismo , Neumonía/inducido químicamente , Neumonía/metabolismo , Receptores de Quimiocina , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
Posttranslational proteolytic processing of chemokines is a natural mechanism to regulate inflammation. In this study, we describe modification of the CXC chemokine stromal cell-derived factor 1alpha/CXCL12 by peptidylarginine deiminase (PAD) that converts arginine residues into citrulline (Cit), thereby reducing the number of positive charges. The three NH(2)-terminal arginines of CXCL12, Arg(8), Arg(12), and Arg(20), were citrullinated upon incubation with PAD. The physiologic relevance of citrullination was demonstrated by showing coexpression of CXCL12 and PAD in Crohn's disease. Three CXCL12 isoforms were synthesized for biologic characterization: CXCL12-1Cit, CXCL12-3Cit, and CXCL12-5Cit, in which Arg(8), Arg(8)/Arg(12)/Arg(20), or all five arginines were citrullinated, respectively. Replacement of only Arg(8) caused already impaired (30-fold reduction) CXCR4 binding and signaling (calcium mobilization, phosphorylation of ERK and protein kinase B) properties. Interaction with CXCR4 was completely abolished for CXCL12-3Cit and CXCL12-5Cit. However, the CXCR7-binding capacities of CXCL12-1Cit and CXCL12-3Cit were, respectively, intact and reduced, whereas CXCL12-5Cit failed to bind CXCR7. In chemotaxis assays with lymphocytes and monocytes, CXCL12-3Cit and CXCL12-5Cit were completely devoid of activity, whereas CXCL12-1Cit, albeit at higher concentrations than CXCL12, induced migration. The antiviral potency of CXCL12-1Cit was reduced compared with CXCL12 and CXCL12-3Cit and CXCL12-5Cit (maximal dose 200 nM) could not inhibit infection of lymphocytic MT-4 cells with the HIV-1 strains NL4.3 and HE. In conclusion, modification of CXCL12 by one Cit severely impaired the CXCR4-mediated biologic effects of this chemokine and maximally citrullinated CXCL12 was inactive. Therefore, PAD is a potent physiologic down-regulator of CXCL12 function.
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Fármacos Anti-VIH/antagonistas & inhibidores , Quimiocina CXCL12/metabolismo , Citrulina/metabolismo , Mediadores de Inflamación/antagonistas & inhibidores , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/metabolismo , Receptores CXCR/antagonistas & inhibidores , Receptores CXCR/metabolismo , Animales , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacología , Unión Competitiva , Células CHO , Línea Celular Tumoral , Células Cultivadas , Quimiocina CXCL12/biosíntesis , Quimiocina CXCL12/genética , Quimiocina CXCL12/fisiología , Cricetinae , Cricetulus , Enfermedad de Crohn/enzimología , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/metabolismo , Humanos , Hidrolasas/biosíntesis , Hidrolasas/genética , Hidrolasas/metabolismo , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/fisiología , Unión Proteica/inmunología , Desiminasas de la Arginina Proteica , Receptores CXCR/fisiología , Receptores CXCR4/fisiologíaRESUMEN
The role of specific microbial Ags in the induction of experimental inflammatory bowel disease is poorly understood. Oral infection of susceptible C57BL/6 mice with Toxoplasma gondii results in a lethal ileitis within 7-9 days postinfection. An immunodominant Ag of T. gondii (surface Ag 1 (SAG1)) that induces a robust B and T cell-specific response has been identified and a SAG1-deficient parasite (Deltasag1) engineered. We investigated the ability of Deltasag1 parasite to induce a lethal intestinal inflammatory response in susceptible mice. C57BL/6 mice orally infected with Deltasag1 parasites failed to develop ileitis. In vitro, the mutant parasites replicate in both enterocytes and dendritic cells. In vivo, infection with the mutant parasites was associated with a decrease in the chemokine and cytokine production within several compartments of the gut-associated cell population. RAG-deficient (RAG1(-/-)) mice are resistant to the development of the ileitis after T. gondii infection. Adoptive transfer of Ag-specific CD4(+) effector T lymphocytes isolated from C57BL/6-infected mice into RAG(-/-) mice conferred susceptibility to the development of the intestinal disease. In contrast, CD4(+) effector T lymphocytes from mice infected with the mutant Deltasag1 strain failed to transfer the pathology. In addition, resistant mice (BALB/c) that fail to develop ileitis following oral infection with T. gondii were rendered susceptible following intranasal presensitization with the SAG1 protein. This process was associated with a shift toward a Th1 response. These findings demonstrate that a single Ag (SAG1) of T. gondii can elicit a lethal inflammatory process in this experimental model of pathogen-driven ileitis.
Asunto(s)
Antígenos de Protozoos/inmunología , Ileítis/parasitología , Intestinos/patología , Proteínas Protozoarias/inmunología , Toxoplasmosis Animal/inmunología , Toxoplasmosis Animal/patología , Enfermedad Aguda , Traslado Adoptivo , Animales , Antígenos de Protozoos/genética , Citocinas/inmunología , Femenino , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Ileítis/inmunología , Intestinos/parasitología , Ratones , Mutación , Proteínas Protozoarias/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Toxoplasma/inmunologíaRESUMEN
BACKGROUND & AIMS: The loss of homeostasis is a hallmark of inflammatory bowel disease. Oral infection of susceptible mice with Toxoplasma gondii results in an acute lethal ileitis characterized by increased interferon gamma, tumor necrosis factor alpha, and inducible nitric oxide synthase; homeostasis results from transforming growth factor beta production by intraepithelial lymphocytes. The synthetic oleanane triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) is a potent anti-inflammatory molecule previously shown in vitro to suppress the de novo synthesis of inducible nitric oxide synthase and to induce the transcription and activation of genes from the transforming growth factor beta signaling pathway. METHODS: We evaluated the immune response in the small intestine and by intraepithelial lymphocytes after a single intraperitoneal dose of CDDO at the time of T. gondii oral infection. We abrogated the homeostatic effects of CDDO by blocking transforming growth factor beta in vivo. RESULTS: CDDO acid prevented ileitis development through the global down-regulation of inflammatory cytokines and chemokines. Total transforming growth factor beta(1) production by the intraepithelial lymphocytes increased, as did Smad2 expression. Blocking transforming growth factor beta reversed CDDO-induced protection and prevented the up-regulation of Smad2 in the small intestine. CONCLUSIONS: CDDO acid is a novel anti-inflammatory molecule capable of preventing ileitis by activating the transforming growth factor beta signaling pathway in a pathogen-driven ileitis model. This could represent a new treatment of inflammatory bowel disease.
Asunto(s)
Antiinflamatorios no Esteroideos/administración & dosificación , Ileítis/parasitología , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/administración & dosificación , Toxoplasmosis/complicaciones , Factor de Crecimiento Transformador beta/efectos de los fármacos , Animales , Citocinas/efectos de los fármacos , Citocinas/inmunología , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Femenino , Ileítis/prevención & control , Inyecciones Intraperitoneales , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Ratones , Modelos Animales , Transducción de Señal/inmunología , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Toxoplasma gondii, an intracellular parasite infects the host through the oral route. Infection induces a cascade of immunological events that involve both the components of the innate and adaptative immune responses. Alteration of the homeostatic balance of infected intestine results in an acute inflammatory ileitis in certain strains of inbred mice. Both the infected enterocytes as well as the CD4 T cells from the lamina propria produce chemokines and cytokines that are necessary to clear the parasite whereas CD8 intraepithelial lymphocytes secrete transforming growth factor beta that reduces the inflammation. In this review, we describe the salient features of this complex network of interactions among the different components of the gut-associated lymphoid tissue cell population that are induced after oral infection with T. gondii.
Asunto(s)
Mucosa Intestinal/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Animales , Citocinas/inmunología , Homeostasis/inmunología , Humanos , Inmunidad Mucosa , RatonesRESUMEN
Intraepithelial lymphocytes (IEL) play a key role in gut homeostasis and are critical effector cells preventing the inflammatory intestinal lesions induced in mice following oral infection with Toxoplasma gondii. In this intestinal inflammatory model, CD4(+) T lymphocytes from the lamina propria (LP) synergize with the infected enterocytes to secrete pro-inflammatory chemokines and cytokines. In this study, we assessed the mechanisms accounting for the ability of IEL to modulate the inflammatory activity of these cells. Adoptive transfer of IEL purified from wild-type mice, or CD154-,CD95L- or IL-10-deficient mice infected with T. gondii completely impairs the development of the lethal ileitis in recipient mice orally infected with T. gondii. Compared with unprimed IEL isolated from naive mice, the CD8 alpha beta TCR alpha beta subset of primed IEL, isolated from T. gondii-infected mice, secretes increased amount of TGF-beta. IEL interact with the LP CD4(+) T lymphocytes, down-regulate their production of inflammatory cytokines such as IFN-gamma and reduce their proliferative activity. These effects are linked to the secretion of TGF-beta and are correlated with a shift in the balance between Smad7/T-bet down-regulation and Smad2/Smad3 up-regulation in LP CD4(+) T lymphocytes.
Asunto(s)
Proteínas de Unión al ADN/inmunología , Ileítis/inmunología , Inflamación/inmunología , Linfocitos T/inmunología , Transactivadores/inmunología , Traslado Adoptivo , Animales , Femenino , Ileítis/parasitología , Immunoblotting , Inflamación/parasitología , Mucosa Intestinal/inmunología , Mucosa Intestinal/parasitología , Intestinos/inmunología , Intestinos/parasitología , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Proteínas Smad , Linfocitos T/parasitología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
BACKGROUND & AIMS: Toxoplasma gondii, an obligate intracellular parasite, can invade intestinal epithelial cells and elicit a robust Th1 immune response. In this model of intestinal inflammation, CD8(+) intraepithelial lymphocytes (IELs) secrete transforming growth factor (TGF)-beta, which appears necessary for the maintenance of homeostasis in the intestine. However, the mechanism responsible for the IEL migration to the inflamed intestine is still unclear. METHODS: An in vitro coculture cell system was used to quantify the IEL attraction by an infected intestinal epithelial cell line (m-IC(cl2)). We used CCR5-deficient mice to determine which chemokine receptor-chemokine interaction could be responsible for the recruitment of antigen-specific CD8(+) IELs to the small intestine for the promotion of parasite clearance and host recovery. RESULTS: We observed increased expression of several chemokine receptors (CCR1, CCR2, CCR5, CXCR3) in the infected ileum. In particular, CCR5 expression was markedly increased in antigen-primed CD8(+) IELs. Experiments using recombinant chemokines as well as blocking antibodies showed that macrophage inflammatory protein (MIP)-1alpha and MIP-1beta were critical for their homing. CD8(+) IELs isolated from CCR5-deficient mice (CCR5-/-), despite their high production of TGF-beta and overexpression of activation markers, were impaired in their ability to migrate in vitro to the m-IC(cl2) monolayer or in vivo to the inflamed intestine after adoptive transfer. CONCLUSIONS: Our data emphasize the biologic role of CCR5 as an important component in the migration of intraepithelial CD8(+) T cells and the regulation of the inflammatory response following parasite infection.