RESUMO
Plasmacytoid DCs (pDCs) are crucial mediators in the establishment of immunity against most viruses, given their extraordinary capacity to produce a massive quantity of type I IFN. In this study we investigate the response of pDCs to infection with EBV, a γ-herpes virus that persists with an asymptomatic infection in immunocompetent hosts, although in certain conditions it can promote development of cancers or autoimmune diseases. We show that high amounts of type I IFNs were released from isolated pDCs after exposure to EBV by a mechanism requiring TLRs and a functional autophagic machinery. We next demonstrate that EBV can infect pDCs via viral binding to MHC class II molecule HLA-DR and that pDCs express EBV-induced latency genes. Furthermore, we observe that EBV is able to induce activation but not maturation of pDCs, which correlates with an impaired TNF-α release. Accordingly, EBV-infected pDCs are unable to mount a full T-cell response, suggesting that impaired pDC maturation, combined with a concomitant EBV-mediated upregulation of the T-cell inhibitory molecules B7-H1 and ICOS-L, could represent an immune-evasion strategy promoted by the virus. These mechanisms might lead to persistence in immunocompetent hosts or to dysregulated immune responses linked to EBV-associated diseases.
Assuntos
Células Dendríticas/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/fisiologia , Linfócitos T/imunologia , Receptor Toll-Like 9/imunologia , Autofagia/imunologia , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Células Cultivadas , Células Dendríticas/virologia , Antígenos HLA-DR/metabolismo , Herpesvirus Humano 4/patogenicidade , Humanos , Evasão da Resposta Imune , Ligante Coestimulador de Linfócitos T Induzíveis/genética , Ligante Coestimulador de Linfócitos T Induzíveis/metabolismo , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Ativação Linfocitária , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Internalização do Vírus , Latência Viral/genética , Replicação ViralRESUMO
RATIONALE: Neutrophils play an important role in the inflammatory process associated with chronic obstructive pulmonary disease (COPD). Lung-infiltrating neutrophils secrete elastinolytic proteases that participate in elastin breakdown and the formation of elastin peptides (EPs). OBJECTIVES: We hypothesized that circulating neutrophil-associated immune response may be modulated by EPs during COPD. METHODS: Neutrophils obtained from patients with either stable or exacerbated COPD and controls were cultured with or without EPs. Cell chemotaxis was analysed by the Boyden method and cytokine expression was analysed by ELISA and real-time reverse transcriptase PCR. Bacterial phagocytosis and killing of ingested bacteria were evaluated after incubation with Pseudomonas aeruginosa. Reactive oxygen species (ROS) measurement and elastin receptor expression were determined by flow cytometry. RESULTS: Chemotactic activity of neutrophils from patients with COPD towards the VGVAPG EP was reduced compared with controls. VGVAPG increased proinflammatory cytokine synthesis and bacterial load, but reduced ROS production in neutrophils from controls and from patients with stable COPD. Patients with exacerbated COPD were unresponsive to VGVAPG treatment. These findings were associated with a decreased or almost complete loss of S-Gal elastin receptor expression in neutrophils from patients with stable or exacerbated COPD, respectively. CONCLUSIONS: The study demonstrates that the response of neutrophils from patients with COPD to VGVAPG varied according to COPD phase and critical level of S-Gal expression. S-Gal downregulation could result from a feedback mechanism induced by high levels of EPs.
Assuntos
Citocinas/biossíntese , Ativação de Neutrófilo , Neutrófilos/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Receptores de Superfície Celular/biossíntese , Idoso , Células Cultivadas , Quimiotaxia , Elastina , Feminino , Citometria de Fluxo , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Neutrófilos/patologia , Doença Pulmonar Obstrutiva Crônica/patologia , Espécies Reativas de Oxigênio/metabolismo , Fumar/efeitos adversos , Escarro/citologia , Escarro/metabolismoRESUMO
Mycobacterium tuberculosis (Mtb) evades the immune response by impairing the functions of different antigen-presenting cells. We have recently shown that Mtb hijacks differentiation of monocytes into dendritic cells (DCs). To further characterize the mechanisms underlying this process, we investigated the consequences of inducing dendritic cell differentiation using interferon-α and granulocyte-macrophage colony-stimulating factor in the presence of supernatants (SNs) obtained from monocyte cultures treated with or without heat-inactivated Mtb. Although the SNs from control cultures do not interfere with the generation of fully differentiated DCs, monocytes stimulated with SNs from Mtb-stimulated cells (SN Mtb) remained CD14(+) and poorly differentiated into CD1a(+) cells. Among cytokines known to affect dendritic cell differentiation, we observed a robust production of interleukin-1ß, interleukin-6, interleukin-10 and tumor necrosis factor-α upon Mtb stimulation. However, only interleukin-10 neutralization through the addition of soluble interleukin-10 receptor reversed the inhibitory activity of SN Mtb. Accordingly, the addition of recombinant interleukin-10 was able to significantly reduce CD1a expression. The interaction of Mtb with differentiating monocytes rapidly activates p38 mitogen-activated protein kinase, signal transducer and activator of transcription pathways, which are likely involved in interleukin-10 gene expression. Taken together, our results suggest that Mtb may inhibit the differentiation of bystander non-infected monocytes into DCs through the release of interleukin-10. These results shed light on new aspects of the host-pathogen interaction, which might help to identify innovative immunological strategies to limit Mtb virulence.
Assuntos
Efeito Espectador , Diferenciação Celular , Células Dendríticas/citologia , Células Dendríticas/imunologia , Interleucina-10/imunologia , Mycobacterium tuberculosis/fisiologia , Tuberculose/imunologia , Efeito Espectador/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Citocinas/biossíntese , Humanos , Interferon-alfa/imunologia , Interferon-alfa/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Mycobacterium tuberculosis/imunologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Knowledge of the molecular events regulating the innate response to Mycobacterium tuberculosis (Mtb) is critical for understanding immunological pathogenesis and protection from tuberculosis. To this aim, the regulation and the expression of regulatory and proinflammatory cytokines were investigated in human primary monocytes upon Mtb infection. We found that Mtb-infected monocytes preferentially express a proinflammatory cytokine profile, including IL-6, TNF-α, and IL-1ß. Conversely, among the regulatory cytokines, Mtb elicited IL-10 and IL-23 release while no expression of IL-12p70, IL-27, and IFN-ß was observed. The analysis of the signalling pathways leading to this selective cytokine expression showed that in monocytes Mtb activates MAPK and NF-κB but is unable to stimulate IRF-3 phosphorylation, a transcription factor required for IL-12p35 and IFN-ß gene expression. Thus, by inducing a specific cytokine profile, Mtb can influence the immunoregulatory properties of monocytes, which represent important target of novel vaccinal strategies against Mtb infection.
Assuntos
Citocinas , Expressão Gênica , Monócitos , Técnicas de Cultura de Células , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Expressão Gênica/imunologia , Humanos , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Sistema de Sinalização das MAP Quinases/imunologia , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/microbiologia , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/metabolismo , NF-kappa B/imunologia , NF-kappa B/metabolismo , Fosforilação , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Equilíbrio Th1-Th2RESUMO
Although data show the importance of type I interferons (IFNs) in the regulation of the innate and adaptive immunity elicited in response to viral, bacterial and parasitic infections, the functional activities of these cytokines during fungal infections are poorly understood. We examined here the impact of IFN-ß on the response of human monocyte-derived dendritic cells (DCs) infected in vitro with Aspergillus fumigatus. Having found that A. fumigatus-infected DCs do not express IFN-ß, we evaluated the effect of the exogenous addition of IFN-ß on the maturation of human DCs induced by the infection with A. fumigatus conidia. Although the phagocytosis of the fungus was not affected by IFN-ß treatment, the expression of CD86 and CD83 induced upon A. fumigatus challenge was enhanced in IFN-ß-conditioned DCs, which also showed an increased expression of IL-27 and IL-12p70, members of IL-12 family. Through these modifications, IFN-ß improved the capacity of DCs to promote an anti-Aspergillus T helper type 1 response, as evaluated by mixed leucocyte reaction, which plays a crucial role in the control of invasive aspergillosis. Our results identified a novel effect of IFN-ß on anti-Aspergillus immune responses which, in turn, might open new perspectives on the use of IFN-ß in immunotherapy for fungal infections aimed at enhancing the immunological functions of DCs.
Assuntos
Aspergillus fumigatus/imunologia , Células Dendríticas/imunologia , Interferon beta/farmacologia , Células Th1/imunologia , Antígenos CD/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Proliferação de Células , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Expressão Gênica/genética , Expressão Gênica/imunologia , Humanos , Interferon beta/genética , Interferon beta/metabolismo , Interferon gama/metabolismo , Interleucina-4/metabolismo , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/imunologia , Teste de Cultura Mista de Linfócitos , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Células Th1/citologiaRESUMO
The beneficial effects of Bifidobacterium are partly due to its immunostimulatory properties. These immunostimulatory properties may be linked to the presence of unmethylated CpG motifs specific to bacterial DNA, which may induce a TH1 response by activating Toll-like receptors (TLR). Using in silico analyses, PCR amplification, and dot blotting, we characterized the CpG content of various bifidobacterial strains and evaluated the immunostimulatory properties and genomic heterogeneity of these motifs in the genus. Our in silico study, based on entire genome sequences from five bifidobacterial strains, showed that Bifidobacterium genomes contain numerous CpG motifs, including 5'-purine-purine-CG-pyrimidine-pyrimidine-3' and 5'-purine-TCG-pyrimidine-pyrimidine-3' motifs, and biologically active sequences previously identified in lactic acid bacteria. We identified four CpG-rich sequences with Bifidobacterium longum NCC2705. Two sequences with a percent G+C of about 68% included 14 and 16 CpG motifs. Two sequences with a percent G+C of about 60% included 16 and 6 CpG motifs. These sequences induce the production of monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor alpha (TNF-alpha) through a pattern of TLR9 stimulation on RAW 264.7 macrophages. No link could be established between their immunostimulatory properties, the number of CpG motifs, and percent G+C. We investigated inter- and intraspecies heterogeneity in 71 strains of various origins. These sequences were highly conserved in the genus. No link was found between the presence of the CpG-rich sequence and the origin of the strains (healthy, allergic, or preterm infants). The high frequency of CpG motifs in the DNA of Bifidobacterium may play an important role in the immunostimulatory properties of commensal or probiotic bifidobacterial strains.
Assuntos
Adjuvantes Imunológicos/farmacologia , Bifidobacterium/genética , Ilhas de CpG , DNA Bacteriano/farmacologia , Fosfatos de Dinucleosídeos/farmacologia , Adjuvantes Imunológicos/genética , Sequência de Bases , DNA Bacteriano/química , Humanos , Lactente , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Cryptosporidium parvum is a protozoan parasite that causes intestinal malabsorptive syndrome and malnutrition. Considering the importance of di-tripeptide absorption for nutritional status, we previously investigated the regulation of PepT1 transporter in the suckling rat model of acute cryptosporidiosis and showed that PepT1 protein expression and activity were not modified in the parasitized intestine. Here we used confocal microscopy performed on intestinal villi to determine the subcellular localization of PepT1 together with f-actin and parasites. For this purpose, confocal microscopy using vibratome thick sections was developed on the distal small intestine, the preferential site of parasite implantation. Results showed major heterogeneity of apical PepT1 expression among enterocytes, which did not correlate with actin staining or parasite implantation. These results underscore the importance of considering the effect of C. parvum at the cellular scale and not only in the entire epithelium.
Assuntos
Criptosporidiose/parasitologia , Cryptosporidium parvum/fisiologia , Proteínas de Membrana Transportadoras/biossíntese , Actinas/análise , Animais , Animais Recém-Nascidos , Cryptosporidium parvum/química , Feminino , Mucosa Intestinal/parasitologia , Microscopia Confocal , Microscopia Eletrônica de Varredura , RatosRESUMO
The roles of plasmacytoid dendritic cells (pDCs) and their response to interferon (IFN)-beta therapy in multiple sclerosis (MS) patients are poorly understood. We identified pDC accumulation in white matter lesions and leptomeninges of MS brains and abundant expression of the Type I IFN-induced protein MxA, mainly in perivascular CD3+ lymphocytes in lesions, indicating Type I IFN production by activated pDCs. The pDC chemoattractant chemerin was detected in intralesional cerebrovascular endothelial cells, and the chemerin receptor was expressed on infiltrating leukocytes, including pDCs. The effect of IFN-beta on pDC phenotype and function was evaluated in MS patients before and during IFN-beta treatment. Although IFN-beta did not modify the frequency and immature phenotype of circulating pDC, they showed lower expression of major histocompatibility complex Class II and blood-dendritic cell antigen 2 molecules and upregulation of CD38 and B7H1 costimulatory molecules. On exposure to CpG (a site where cytosine [C] lies next to guanine [G] in the DNA sequence [the p indicates that C and G are connected by a phosphodiester bond]) oligodeoxynucleotides in vitro, pDCs from IFN-beta-treated MS patients showed reduced expression of the pDC maturation markers CD83 and CD86 molecules; in vitro IFN-beta treatment of pDCs from healthy donors resulted in lower secretion of proinflammatory cytokines, including IFN-alpha, and a decreased ability to stimulate allogeneic T cells in response to maturative stimuli. These data indicate that IFN-beta modulates the immunologic functions of pDC, thus identifying pDCs as a novel target of IFN-beta therapy in MS patients.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/imunologia , Células Dendríticas/efeitos dos fármacos , Interferon beta/farmacologia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Adulto , Antígenos CD/análise , Antígenos CD/metabolismo , Antígeno B7-H1 , Biomarcadores/análise , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Córtex Cerebral/fisiopatologia , Quimiocinas/efeitos dos fármacos , Quimiocinas/imunologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/imunologia , Células Dendríticas/imunologia , Feminino , Proteínas de Ligação ao GTP/efeitos dos fármacos , Proteínas de Ligação ao GTP/imunologia , Humanos , Fatores Imunológicos/farmacologia , Fatores Imunológicos/uso terapêutico , Peptídeos e Proteínas de Sinalização Intercelular , Interferon beta/uso terapêutico , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/fisiopatologia , Proteínas de Resistência a Myxovirus , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Fibras Nervosas Mielinizadas/imunologia , Fibras Nervosas Mielinizadas/ultraestrutura , Fenótipo , Plasmócitos/efeitos dos fármacos , Plasmócitos/imunologiaRESUMO
The transcription factor STAT-4 plays a pivotal role in the IL-12-mediated development of naive CD4+ T cells into the Th1 phenotype. Initially thought to be restricted to the lymphoid lineage, STAT-4 was subsequently shown to be expressed in the myeloid compartment, mainly in activated monocytes, macrophages, and dendritic cells (DC). Here, we have studied STAT-4 in human monocyte-derived DC, and we demonstrated that its expression can be induced by multiple stimuli, such as the ligands for TLR-4, TLR-2, and TLR-3, different pathogens, CD40 ligand, and the proinflammatory cytokines TNF-alpha and IL-1beta. It is interesting that we found that STAT-4 is tyrosine-phosphorylated in response to type I IFN but not IL-12 in human mature DC. Cloning and functional analysis of the STAT-4 promoter showed that a NF-kappaB binding site, localized at -969/-959 bp upstream of the transcriptional start site, is involved in the regulation of this gene in primary human DC. EMSAs using a probe containing this NF-kappaB binding sequence and chromatin immunoprecipitation indicated that p65/p50 and p50/p50 dimers were the main NF-kappaB/Rel proteins involved in STAT-4 gene expression in maturing DC. The mutation of this kappaB site or the overexpression of the repressor IkappaBalpha exerted an inhibitory effect on a STAT-4 promoter-driven reporter as well as on STAT-4 expression. Altogether, these results indicate that STAT-4 can be finely tuned along with DC maturation through NF-kappaB activation and that its induction may be involved in preparing the DC to be receptive to the cytokine environment present in lymphoid organs.
Assuntos
Células Dendríticas/metabolismo , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição STAT4/metabolismo , Sequência de Bases , Sítios de Ligação , Diferenciação Celular , Células Cultivadas , Células Dendríticas/fisiologia , Humanos , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Fator de Transcrição STAT4/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
Given the role played by chemokines in the selective homing of immune cells, we sought to characterize the profile of chemokines produced by human dendritic cells (DC) following in vitro Aspergillus fumigatus infection and their ability to recruit cells involved in the antifungal defense. At the onset of A. fumigatus infection, DC released elevated amounts of CXCL8 that promote the migration of polymorphonuclear cells (PMN). Moreover, soluble factors released from A. fumigatus-infected DC increased also the surface expression of two activation markers, CD11b and CD18, on PMN. A. fumigatus infection resulted also in CCL3, CCL4, CXCL10 and CCL20 productions that induce the migration of effector memory Th1 cells. Moreover, the late expression of CCL19 suggests that A. fumigatus-infected DC could be implicated in the migration of CCR7+ naïve T cells and mature DC in lymph nodes. Together these results suggested the involvement of human DC in the regulation of innate and adaptive immunity against A. fumigatus through the recruitment of cells active in the fungal destruction.
Assuntos
Aspergilose/imunologia , Aspergillus fumigatus/patogenicidade , Quimiocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Infiltração de Neutrófilos , Células Th1/imunologia , Aspergilose/microbiologia , Aspergillus fumigatus/imunologia , Quimiocina CCL19 , Quimiocina CCL20 , Quimiocinas/imunologia , Quimiocinas CC/metabolismo , Quimiotaxia de Leucócito/imunologia , Células Dendríticas/metabolismo , Humanos , Interleucina-8/metabolismo , Ativação Linfocitária , Proteínas Inflamatórias de Macrófagos/metabolismoRESUMO
Plasmacytoid dendritic cells (pDCs) display altered immune-phenotype in multiple sclerosis (MS) patients and are found actively recruited in postmortem MS brain lesions, implying that their immune regulation may represent an important aspect of MS pathogenesis. Because of the reported Toll-like receptor 7 (TLR7) implication in autoimmunity, in this study we characterized how IFN-ß therapy impacts on pDC activation to TLR7 triggering in MS patients, aspect only poorly investigated so far. In vivo IFN-ß administration regulates pDC functions in TLR7-treated peripheral blood mononuclear cell (PBMC) cultures differently from what is observed in isolated cells, suggesting that IFN-ß may activate inhibitory mechanisms in MS peripheral blood involved in turning off pDC response to dampen the ongoing inflammation. Indeed, IL-10, a key regulatory cytokine found increased upon TLR7 stimulation in in vivo IFN-ß-exposed PBMCs, directly reduced pDC-mediated IFN-α production. IFN-ß therapy also shaped T-cell responses by decreasing TLR7-induced pDC maturation and inducing T-cell inhibitory molecules. Accordingly, raised pDC-induced IL-27 and decreased IL-23 expression, together with high IL-10 level, contribute to inhibit Th17 cell differentiation. Our study uncovered a role for IFN-ß in the regulation of TLR7-mediated pDC responses in MS toward an anti-inflammatory phenotype opening new opportunities to better understand mechanisms of action of this drug in controlling MS immunopathogenesis.
Assuntos
Anti-Inflamatórios/uso terapêutico , Células Dendríticas/imunologia , Interferon beta/imunologia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Receptor 7 Toll-Like/imunologia , Adulto , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Células Dendríticas/efeitos dos fármacos , Feminino , Humanos , Fatores Imunológicos/imunologia , Interferon-alfa/imunologia , Interleucina-10/imunologia , Interleucina-23/imunologia , Interleucina-27/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
Etoposide (VP-16) is a hydrophobic anticancer agent inhibiting Topoisomerase II, commonly used in pediatric brain chemotherapeutic schemes as mildly toxic. Unfortunately, despite its appropriate solubilization in vehicle solvents, its poor bioavailability and limited passage of the blood-brain barrier concur to disappointing results requiring the development of new delivery system forms. In this study, etoposide formulated as a parenteral injectable solution (Teva®) was loaded into all-biocompatible poly(lactide-co-glycolide) (PLGA) or PLGA/P188-blended nanoparticles (size 110-130 nm) using a fully biocompatible nanoprecipitation technique. The presence of coprecipitated P188 on encapsulation efficacies and in vitro drug release was investigated. Drug encapsulation was determined using HPLC. Inflammatory response was checked by FACS analysis on human monocytes. Cytotoxic activity of the various simple (Teva®) or double (Teva®-loaded NPs) formulations was studied on the murine C6 and F98 cell lines. Obtained results suggest that, although noninflammatory neither nontoxic by themselves, the use of PLGA and PLGA/P188 nanoencapsulations over pre-existing etoposide formulation could induce a greatly improved cytotoxic activity. This approach demonstrated a promising perspective for parenteral delivery of VP16 and potential development of a therapeutic entity.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Portadores de Fármacos/química , Etoposídeo/administração & dosagem , Ácido Láctico/química , Nanopartículas/química , Poloxâmero/química , Ácido Poliglicólico/química , Animais , Antineoplásicos Fitogênicos/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Portadores de Fármacos/metabolismo , Etoposídeo/farmacologia , Glioma/tratamento farmacológico , Glioma/patologia , Humanos , Ácido Láctico/metabolismo , Camundongos , Monócitos/efeitos dos fármacos , Nanopartículas/ultraestrutura , Tamanho da Partícula , Poloxâmero/metabolismo , Ácido Poliglicólico/metabolismo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Propriedades de SuperfícieRESUMO
Emerging evidence points to an important role of autophagy in the immune response mediated by dendritic cells (DC) against Mycobacterium tuberculosis (Mtb). Since current vaccination based on Bacillus Calmette-Guerin (BCG) is unable to stop the tuberculosis epidemic, a deeper comprehension of the alterations induced by Mtb in DC is essential for setting new vaccine strategies. Here, we compared the capacity of virulent (H37Rv) and avirulent (H37Ra) Mtb strains as well as BCG to modulate autophagy in human primary DC. We found that Mtb H37Rv impairs autophagy at the step of autophagosome-lysosome fusion. In contrast, neither Mtb H37Ra nor BCG strains were able to hamper autophagosome maturation. Both these attenuated strains have a functional inhibition of the 6kD early secreted antigenic target ESAT-6, an effector protein of the ESAT-6 Secretion System-1(ESX-1)/type VII secretion system. Notably, the ability to inhibit autophagy was fully restored in recombinant BCG and Mtb H37Ra strains in which ESAT-6 secretion was re-established by genetic complementation using either the ESX-1 region from Mtb (BCG::ESX-1) or the PhoP gene (Mtb H37Ra::PhoP), a regulator of ESAT-6 secretion. Importantly, the autophagic block induced by Mtb was overcome by rapamycin treatment leading to an increased interleukin-12 expression and, in turn, to an enhanced capacity to expand a Th1-oriented response. Collectively, our study demonstrated that Mtb alters the autophagic machinery through the ESX-1 system, and thereby opens new exciting perspectives to better understand the relationship between Mtb virulence and its ability to escape the DC-mediated immune response.
Assuntos
Autofagia , Sistemas de Secreção Bacterianos , Células Dendríticas/microbiologia , Células Dendríticas/patologia , Mycobacterium tuberculosis/fisiologia , Autofagia/efeitos dos fármacos , Sistemas de Secreção Bacterianos/efeitos dos fármacos , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/ultraestrutura , Humanos , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/fisiologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/ultraestrutura , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Sirolimo/farmacologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Tuberculose/imunologia , Tuberculose/microbiologia , Tuberculose/patologiaRESUMO
The early steps of echovirus 6 (E6) infection remain poorly understood and the only described receptor for haemagglutinating E6 strains is the decay accelerating factor (DAF). There is, however, accumulating evidence suggesting that E6 interaction with DAF is necessary but not sufficient for infection. In this report, we investigated the role of the coxsackie-adenovirus-receptor (CAR) as a potential DAF co-receptor during E6 infection. Using stably transfected Chinese Hamster Ovary (CHO) cells expressing CAR and DAF receptors, we found that DAF expression allowed attachment of both haemagglutinating and non-haemagglutinating E6 strains but was not sufficient for promoting E6 cell entry. Interestingly, the co-expression of DAF and CAR rendered 0.1-0.2% of cells permissive to some E6 strains' infection. Although our results did not show a major role of the CAR/DAF cooperation for E6 infection, it nevertheless indicated the use of CAR in the cell entry step of some minor E6 quasispecies. Moreover, the present report validates the use of recombinant CHO cells as valuable cellular model for the further characterisation of E6 receptors.
Assuntos
Antígenos CD55/metabolismo , Echovirus 6 Humano/fisiologia , Receptores Virais/metabolismo , Internalização do Vírus , Animais , Células CHO , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Cricetinae , Cricetulus , HumanosRESUMO
Given the variable protective efficacy provided by Mycobacterium bovis bacillus Calmette-Guérin (BCG), there is an urgent need to develop new vaccines against tuberculosis. As dendritic cells (DC) play a critical role in initiating and regulating a protective T cell response against the pathogens, the comprehension of mycobacterium-induced modulation of DC functions is critical to pinpoint new, immunological strategies. To this end, a comparative analysis of the effect induced by BCG and Mycobacterium tuberculosis (Mtb) infection on the DC immunophenotype indicated that BCG is less efficient in inducing DC maturation than Mtb. In addition, BCG-infected DC poorly expressed IFN-beta and displayed a reduced production of IL-12 as compared with Mtb-stimulated cells. The impaired expression of IL-12p35 and IFN-beta is likely a result of the inability of BCG to induce the activation of the IFN regulatory factor-3. Taking into account these data, we sought to investigate whether the exogenous addition of IFN-beta, a cytokine that exerts important effects on the immune system, could enhance the Th1-polarizing capacity of BCG-infected DC. Interestingly, when DC infected by BCG were pretreated in vitro with IFN-beta, they displayed a fully mature phenotype and released a significant amount of bioactive IL-12p70, which resulted in an enhanced Th1 response. This study demonstrates that IFN-beta potentiates DC immunological functions following BCG infection, thus suggesting IFN-beta as a possible candidate as vaccine adjuvant.
Assuntos
Células Dendríticas/imunologia , Interferon beta/farmacologia , Vacinas contra a Tuberculose/imunologia , Vacina BCG/imunologia , Células Dendríticas/microbiologia , Humanos , Interferon beta/uso terapêutico , Interleucina-12/biossíntese , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologiaRESUMO
Type I IFN are cytokines which play a central role in host resistance to viral or microbial infections and are important components linking innate and adaptive immunity. We and others have previously demonstrated that the production of IFN-beta by DC following bacterial infections or TLR triggering influences, in an autocrine manner, their maturation. In this study, we investigated whether IFN-beta release modulates the phenotype of the immature DC and their response to a subsequent TLR stimulation. The induction of CD86, HLA-DR, CD38 and B7H1 and the absence of CCR7 and CD83 expression upon IFN-beta treatment suggest that IFN-beta-primed DC remain at the site of infection acquiring an activated phenotype. These results prompted us to investigate the response of IFN-beta-primed DC to TLR stimulation. While IFN-beta pretreatment increases slightly the expression of maturation markers in TLR2- or TLR4-stimulated DC, it is able to modulate selectively the secretion of inflammatory and immuno-regulating cytokines. Interestingly, IL-27p28 subunit was induced by IFN-beta alone or during LPS-induced maturation of DC in a type I IFN-dependent manner through IFN regulatory factor-1 (IRF-1) activation. Taken together, our results shed light on the capacity of IFN-beta to finely tune DC response to invading pathogens.
Assuntos
Células Dendríticas/efeitos dos fármacos , Fator Regulador 1 de Interferon/fisiologia , Interferon beta/farmacologia , Interleucinas/biossíntese , Receptor 2 Toll-Like/fisiologia , Receptor 4 Toll-Like/fisiologia , Antígenos CD/biossíntese , Antígenos CD/genética , Antígeno B7-2/biossíntese , Antígeno B7-2/genética , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Células Dendríticas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoglobulinas/biossíntese , Imunoglobulinas/genética , Interleucina-10/metabolismo , Interleucina-12/biossíntese , Interleucina-12/genética , Interleucina-6/metabolismo , Interleucinas/genética , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Subunidades Proteicas , Receptores CCR7/biossíntese , Receptores CCR7/genética , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Antígeno CD83RESUMO
TLRs interact with a growing list of pathogen-derived products and these interactions drive the activation of innate and adaptive immune responses. Dendritic cells (DC) play a key role in these events expressing a heterogeneous repertoire of TLRs. We have previously demonstrated the production of type I IFNs in DC following bacterial infections and TLR triggering. In this study, we sought to characterize the transcriptome specifically induced in human DC by IFN-beta production stimulated upon LPS treatment. To this aim, by using cDNA microarrays, we compared the transcriptome of DC following LPS treatment in the absence or presence of neutralizing anti-type I IFN Abs. Interestingly, we found that the expression of TLR7 was induced during LPS-induced maturation of DC in a type I IFN-dependent manner. The induction of TLR7 in maturing DC was mainly a consequence of the transcriptional activity of IRF-1, whose binding site was located within TLR7 promoter. Moreover, we also demonstrated that "priming" of immature DC, that usually express TLR8 but not TLR7, with exogenous IFN-beta induced a functionally active TLR7. In fact, treatment with the TLR7-specific ligand 3M-001 up-regulated the expression of CD83, CD86, and CD38 in IFN-beta-primed DC but not in immature DC. Therefore, a robust enhancement in proinflammatory as well as regulatory cytokines was observed. These data suggest that TLR4-mediated type I IFN release activates specific transcription programs in DC amplifying the expression of pathogen sensors to correctly and combinatorially respond to a bacterial as well as viral infection.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Interferon beta/fisiologia , Lipopolissacarídeos/farmacologia , Receptor 7 Toll-Like/agonistas , Antígenos de Superfície/biossíntese , Antígenos de Superfície/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Citocinas/biossíntese , Citocinas/genética , Células Dendríticas/citologia , Perfilação da Expressão Gênica , Humanos , Imunidade Inata/genética , Mediadores da Inflamação/metabolismo , Fator Regulador 1 de Interferon/fisiologia , Ligantes , Receptor 7 Toll-Like/biossíntese , Receptor 7 Toll-Like/metabolismoRESUMO
Aspergillus fumigatus is one of the most prevalent airborne fungal pathogens, causing severe and often fatal infections. Its fungal virulence factors have not been clearly identified. Reactive oxygen species produced by phagocytic cells are potent fungicides for A. fumigatus. The aim of this study was to examine the influence of conidia pigmentation, fungal development stage and genotype strain on human leucocytes oxidative response. Various A. fumigatus strains were used and the oxidative response was analysed by flow cytometry. A significant difference was observed between live- and killed-conidia. A pigmentless strain gave an important intracellular oxidative response compared with pigmented strains. But no difference was observed between strains isolated from patients with invasive aspergillosis (IA) and bronchial colonisation. The modification of healthy phagocytes' oxidative response caused by A. fumigatus components is not sufficient to explain the virulence of fungus and to predict an evolution of patients with IA.
Assuntos
Aspergillus fumigatus/patogenicidade , Granulócitos/imunologia , Monócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Aspergilose , Aspergillus fumigatus/imunologia , Aspergillus fumigatus/fisiologia , Citometria de Fluxo , Granulócitos/metabolismo , Humanos , Monócitos/imunologia , Fagócitos/imunologia , Fagócitos/metabolismo , Pigmentação , Rodaminas/metabolismo , Esporos FúngicosRESUMO
Aspergillus fumigatus is the most prevalent airborne fungal pathogen and causes fatal invasive aspergillosis in immunocompromised patients. Given the essential role of dendritic cells (DC) in initiating and regulating immune responses, we investigated the impact of A. fumigatus conidial infection on human DC. A. fumigatus conidia were rapidly internalized and induced the release of tumor necrosis factor alpha within the first 8 h. After A. fumigatus infection, the majority of DC underwent full maturation, although CCR7 expression was observed only in DC that had internalized the conidia. Additionally, the analysis of regulatory cytokines showed that infected DC simultaneously produced interleukin-12p70 (IL-12p70) and significant amounts of IL-10. IL-10 neutralization was not able to further increase IL-12p70 production from infected DC. Whereas the central role of IL-12 in the generation of Th1 cells has long been appreciated, recently two other members of the IL-12 family, IL-23 and IL-27, were reported to play important roles in the regulation of gamma interferon (IFN-gamma) production from naïve and memory T cells. A. fumigatus-infected DC were also able to express high levels of IL-23p19 and low levels of IL-27p28 at later stages of infection. According to this expression pattern, A. fumigatus-infected DC were able to prime IFN-gamma production of naïve T cells. Thus, this study on the expression of the new IL-12 family members controlling the Th1 response sheds light on a novel aspect of the contribution of DC to anti-Aspergillus immunity.
Assuntos
Aspergilose/imunologia , Aspergillus fumigatus/fisiologia , Citocinas/análise , Células Dendríticas/metabolismo , Receptores de Quimiocinas/metabolismo , Células Th1/imunologia , Aspergillus fumigatus/imunologia , Diferenciação Celular , Células Cultivadas/imunologia , Células Cultivadas/metabolismo , Células Dendríticas/microbiologia , Humanos , Interleucina-12/análise , Interleucina-12/metabolismo , Interleucina-17/análise , Interleucina-17/metabolismo , Interleucina-23 , Subunidade p19 da Interleucina-23 , Interleucinas/análise , Interleucinas/metabolismo , Ativação Linfocitária , Receptores CCR7 , Células Th1/efeitos dos fármacosRESUMO
The Mycobacterium tuberculosis genome encodes 13 sigma factors. We have previously shown that mutations in some of these transcriptional activators render M. tuberculosis sensitive to various environmental stresses and can attenuate the virulence phenotype. In this work, we focused on extracytoplasmic factor sigmaE and studied the effects induced by the deletion of its structural gene (sigE) in the infection of human monocyte-derived dendritic cells (MDDC). We found that the wild-type M. tuberculosis strain (H37Rv), the sigE mutant (ST28), and the complemented strain (ST29) were able to infect dendritic cells (DC) to similar extents, although at 4 days postinfection a reduced ability to grow inside MDDC was observed for the sigE mutant ST28. After mycobacterium capture, the majority of MDDC underwent full maturation and expressed both inflammatory cytokines, such as tumor necrosis factor alpha, and the regulatory cytokines interleukin-12 (IL-12), IL-18, and beta interferon (IFN-beta). Conversely, a higher level of production of IL-10 was observed in ST28-infected MDDC compared to H37Rv- or ST29-infected cell results. However, in spite of the presence of IL-10, supernatants from ST28-infected DC induced IFN-gamma production by T cells similarly to those from H37Rv-infected DC culture. On the other hand, IL-10 impaired CXCL10 production in sigE mutant-infected DC and, indeed, its neutralization restored CXCL10 secretion. In line with these results, supernatants from ST28-infected cells showed a decreased capability to recruit CXCR3+ CD4+ T cells compared to those obtained from H37Rv-infected DC culture. Thus, our findings suggest that the sigE mutant-induced secretion of IL-10 inhibits CXCL10 expression and, in turn, the recruitment of activated-effector cells involved in the formation of granulomas.