RESUMO
The innate recognition of fungi by leukocytes is mediated by pattern recognition receptors (PRR), such as Dectin-1, and is thought to occur at the cell surface triggering intracellular signalling cascades which lead to the induction of protective host responses. In the lung, this recognition is aided by surfactant which also serves to maintain the balance between inflammation and pulmonary function, although the underlying mechanisms are unknown. Here we have explored pulmonary innate recognition of a variety of fungal particles, including zymosan, Candida albicans and Aspergillus fumigatus, and demonstrate that opsonisation with surfactant components can limit inflammation by reducing host-cell fungal interactions. However, we found that this opsonisation does not contribute directly to innate fungal recognition and that this process is mediated through non-opsonic PRRs, including Dectin-1. Moreover, we found that pulmonary inflammatory responses to resting Aspergillus conidia were initiated by these PRRs in acidified phagolysosomes, following the uptake of fungal particles by leukocytes. Our data therefore provides crucial new insights into the mechanisms by which surfactant can maintain pulmonary function in the face of microbial challenge, and defines the phagolysosome as a novel intracellular compartment involved in the innate sensing of extracellular pathogens in the lung.
Assuntos
Aspergillus fumigatus/imunologia , Candida albicans/imunologia , Candidíase/imunologia , Imunidade Inata , Aspergilose Pulmonar/imunologia , Animais , Antígenos de Fungos/imunologia , Aspergillus fumigatus/fisiologia , Líquido da Lavagem Broncoalveolar , Feminino , Interações Hospedeiro-Patógeno , Lectinas Tipo C/metabolismo , Pneumopatias Fúngicas/imunologia , Lisossomos/imunologia , Lisossomos/microbiologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Fagocitose/imunologia , Fagossomos/imunologia , Fagossomos/microbiologia , Pneumonia/imunologia , Pneumonia/metabolismo , Pneumonia/microbiologia , Ligação Proteica , Surfactantes Pulmonares/metabolismo , Receptores Imunológicos/metabolismo , Esporos Fúngicos/imunologia , Zimosan/imunologiaRESUMO
Immune suppression increases the incidence of invasive fungal infections, particularly those caused by the opportunistic mold Aspergillus fumigatus. Previous investigations revealed that members of the TLR family are not absolutely required for host defense against A. fumigatus in nonimmunosuppressed hosts, suggesting that other pattern recognition receptors are involved. We show in this study that naive mice (i.e., not pharmacologically immunosuppressed) lacking the beta-glucan receptor Dectin-1 (Dectin-1(-/-)) are more sensitive to intratracheal challenge with A. fumigatus than control mice, exhibiting >80% mortality within 5 days, ultimately attributed to a compromise in respiratory mechanics. In response to A. fumigatus challenge, Dectin-1(-/-) mice demonstrated impaired IL-1alpha, IL-1beta, TNF-alpha, CCL3/MIP-1alpha, CCL4/MIP-1beta, and CXCL1/KC production, which resulted in insufficient lung neutrophil recruitment and uncontrolled A. fumigatus lung growth. Alveolar macrophages from Dectin-1(-/-) mice failed to produce proinflammatory mediators in response to A. fumigatus, whereas neutrophils from Dectin-1(-/-) mice had impaired reactive oxygen species production and impaired killing of A. fumigatus. We further show that IL-17 production in the lung after A. fumigatus challenge was Dectin-1 dependent, and that neutralization of IL-17 significantly impaired A. fumigatus clearance. Collectively, these results support a requisite role for Dectin-1 in in vivo defense against A. fumigatus.
Assuntos
Aspergilose/imunologia , Aspergillus fumigatus/imunologia , Pneumopatias Fúngicas/imunologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Animais , Aspergilose/genética , Aspergilose/metabolismo , Aspergilose/patologia , Suscetibilidade a Doenças , Interleucina-17/biossíntese , Lectinas Tipo C , Pneumopatias Fúngicas/genética , Pneumopatias Fúngicas/metabolismo , Pneumopatias Fúngicas/patologia , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Neutrófilos/imunologia , Taxa de Sobrevida , Fatores de TempoRESUMO
CLEC-2 is a member of the "dectin-1 cluster" of C-type lectin-like receptors and was originally thought to be restricted to platelets. In this study, we demonstrate that murine CLEC-2 is also expressed by peripheral blood neutrophils, but only weakly by bone marrow or elicited inflammatory neutrophils. On circulating neutrophils, CLEC-2 can mediate phagocytosis of Ab-coated beads and the production of proinflammatory cytokines, including TNF-alpha, in response to the CLEC-2 ligand, rhodocytin. CLEC-2 possesses a tyrosine-based cytoplasmic motif similar to that of dectin-1, and we show using chimeric analyses that the activities of this receptor are dependent on this tyrosine. Like dectin-1, CLEC-2 can recruit the signaling kinase Syk in myeloid cells, however, stimulation of this pathway does not induce the respiratory burst. These data therefore demonstrate that CLEC-2 expression is not restricted to platelets and that it functions as an activation receptor on neutrophils.
Assuntos
Lectinas Tipo C/biossíntese , Neutrófilos/metabolismo , Fagocitose/fisiologia , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Expressão Gênica , Regulação da Expressão Gênica , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lectinas Tipo C/genética , Camundongos , Neutrófilos/imunologia , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Explosão Respiratória/imunologia , Quinase Syk , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Dectin-1 is the archetypal signaling, non-Toll-like pattern recognition receptor that plays a protective role in immune defense to Candida albicans as the major leukocyte receptor for beta-glucans. Dectin-1-deficiency is associated with impaired recruitment of inflammatory leukocytes and inflammatory mediator production at the site of infection. In this study, we have used mice to define the mechanisms that regulate the dectin-1-mediated inflammatory responses. Myeloid cell activation by dectin-1 is controlled by inherent cellular programming, with distinct macrophage and dendritic cell populations responding differentially to the engagement of this receptor. The inflammatory response is further modulated by the progression of the phagocytosis, with "frustrated phagocytosis" resulting in dramatically augmented inflammatory responses. These studies demonstrate that dectin-1 in isolation is sufficient to drive a potent inflammatory response in a context-dependent manner. This has implications for the mechanism by which myeloid cells are activated during fungal infections and the processes involved in the therapeutic manipulation of the immune system via exogenous dectin-1 stimulation or blockade.
Assuntos
Inflamação/etiologia , Proteínas de Membrana/fisiologia , Células Mieloides/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Fagocitose , Animais , Candida albicans/imunologia , Células Dendríticas , Lectinas Tipo C , Macrófagos , Proteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Micoses/imunologia , Proteínas do Tecido Nervoso/deficiência , beta-Glucanas/imunologiaRESUMO
Recognition of microbial components by germ-line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal beta-glucan receptor, Dectin-1, which collaborates through an undefined mechanism with Toll-like receptor 2 (TLR2) to induce optimal cytokine responses in macrophages. We show here that Dectin-1 signaling through the spleen tyrosine kinase (Syk) pathway is required for this collaboration, which can also occur with TLR4, 5, 7 and 9. Deficiency of either Syk or the TLR adaptor MyD88 abolished collaborative responses, which include TNF, MIP-1alpha and MIP-2 production, and which are comparable to the previously described synergy between TLR2 and TLR4. Collaboration of the Syk and TLR/MyD88 pathways results in sustained degradation of the inhibitor of kappaB (IkappaB), enhancing NFkappaB nuclear translocation. These findings establish the first example of Syk- and MyD88-coupled PRR collaboration, further supporting the concept that paired receptors collaborate to control infectious agents.
Assuntos
Citocinas/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas Tirosina Quinases/fisiologia , Receptor 2 Toll-Like/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Humanos , Proteínas I-kappa B/metabolismo , Mediadores da Inflamação/fisiologia , Lectinas Tipo C , Ligantes , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/patologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Quinase SykRESUMO
The formation of genomic loops by proteins bound at sites scattered along a chromosome has a central role in many cellular processes, such as transcription, recombination and replication. Until recently, few such loops had been analyzed in any detail, and there was little agreement about the nature of the molecular ties maintaining these loops. Recent evidence suggests that loops are found in both prokaryotes and eukaryotes, and that the transcription machinery is a molecular tie. In addition, results obtained using site-specific recombination in bacteria and chromosome conformation capture in eukaryotes support the idea that active transcription units are in close contact. These data are consistent with a model for genome organization in which active polymerases cluster into transcription 'factories', which, inevitably, loops the intervening DNA. They are also consistent with the ties functioning as barriers, silencers, enhancers or locus control regions, depending on their positions relative to other genes.
Assuntos
Genoma , Modelos Genéticos , Transcrição Gênica , Animais , DNA/química , Replicação do DNA , Humanos , Região de Controle de Locus Gênico , Conformação de Ácido Nucleico , Recombinação Genética , Fatores de Transcrição/metabolismoRESUMO
Although we have detailed information on the alterations occurring in steady-state levels of all cellular mRNAs during differentiation, we still know little about more global changes. Therefore, we investigated the numbers of molecules of RNA polymerase II that are active--and the way those molecules are organized--as two mouse cells (aneuploid F9 teratocarcinoma, and euploid and totipotent embryonic stem cells) differentiate into parietal endoderm. Quantitative immunoblotting shows the number of active molecules roughly halves. Transcription sites (detected by light and electron microscopy after allowing engaged polymerases to extend nascent transcripts in bromouridine-triphosphate) are uniformly distributed throughout the nucleoplasm. The numbers of such sites fall during differentiation as nuclei become smaller, but site density and diameter remain roughly constant. Similar site densities and diameters are found in salamander (amphibian) cells with 11-fold larger genomes, and in aneuploid HeLa cells. We conclude that active polymerases and their nascent transcripts are concentrated in a limited number of discrete nucleoplasmic sites or factories, and we speculate that the organization of transcription is conserved during both differentiation and evolution to a high C value.