RESUMO
Gastrointestinal helminths are a major health threat worldwide. Alternatively activated macrophages (AAMs) have been shown to contribute to host protection during secondary helminth infections. AAMs express effector molecules that depend on activation of the IL-4- or IL-13-induced transcription factor signal transducer and activator of transcription 6 (STAT6). However, the specific role of STAT6-regulated genes like Arginase-1 (Arg1) from AAMs or STAT6-regulated genes in other cell types for host protection remains unclear. To address this point, we generated mice expressing STAT6 only in macrophages (Mac-STAT6 mouse). In the model of Heligmosomoides polygyrus bakeri (Hpb) infection, Mac-STAT6 mice could not trap larvae in the submucosa of the small intestine after secondary infection. Further, mice lacking Arg1 in hematopoietic and endothelial cells were still protected from secondary Hpb infection. On the other hand, specific deletion of IL-4/IL-13 in T cells blunted AAM polarization, activation of intestinal epithelial cells (IECs) and protective immunity. Deletion of IL-4Rα on IEC also caused loss of larval trapping while AAM polarization remained intact. These results show that Th2-dependent and STAT6-regulated genes in IECs are required and AAMs are not sufficient for protection against secondary Hpb infection by mechanisms that remain to be investigated.
Assuntos
Coinfecção , Nematospiroides dubius , Infecções por Strongylida , Camundongos , Animais , Nematospiroides dubius/metabolismo , Camundongos Knockout , Interleucina-4/genética , Interleucina-4/metabolismo , Interleucina-13/metabolismo , Larva/metabolismo , Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismo , Infecções por Strongylida/genéticaRESUMO
Eosinophilia is associated with various persisting inflammatory diseases and often coincides with chronic fungal infections or fungal allergy as in the case of allergic bronchopulmonary aspergillosis (ABPA). Here, we show that intranasal administration of live Aspergillus fumigatus conidia causes fatal lung damage in eosinophilic interleukin-5 (IL-5)-transgenic mice. To further investigate the activation of eosinophils by A. fumigatus, we established a coculture system of mouse bone marrow-derived eosinophils (BMDE) with different A. fumigatus morphotypes and analyzed the secretion of cytokines, chemokines, and eicosanoids. A. fumigatus-stimulated BMDE upregulated expression of CD11b and downregulated CD62L and CCR3. They further secreted several proinflammatory mediators, including IL-4, IL-13, IL-18, macrophage inflammatory protein-1α (MIP-1α)/CC chemokine ligand 3 (CCL3), MIP-1ß/CCL4, and thromboxane. This effect required direct interaction and adherence between eosinophils and A. fumigatus, as A. fumigatus culture supernatants or A. fumigatus mutant strains with impaired adhesion elicited a rather poor eosinophil response. Unexpectedly, canonical Toll-like receptor (TLR) or C-type-lectin receptor (CLR) signaling was largely dispensable, as the absence of MYD88, TRIF, or caspase recruitment domain-containing protein 9 (CARD9) resulted in only minor alterations. However, transcriptome analysis indicated a role for the PI3K-AKT-mTOR pathway in A. fumigatus-induced eosinophil activation. Correspondingly, we could show that phosphatidylinositol 3-kinase (PI3K) inhibitors successfully prevent A. fumigatus-induced eosinophil activation. The PI3K pathway in eosinophils may therefore serve as a potential drug target to interfere with undesired eosinophil activation in fungus-elicited eosinophilic disorders. IMPORTANCE Allergic bronchopulmonary aspergillosis (ABPA) is caused by the fungus Aspergillus fumigatus, afflicts about five million patients globally, and is still a noncurable disease. ABPA is associated with pronounced lung eosinophilia. Activated eosinophils enhance the inflammatory response not only by degranulation of toxic proteins but also by secretion of small effector molecules. Receptors and signaling pathways involved in activation of eosinophils by A. fumigatus are currently unknown. Here, we show that A. fumigatus-elicited activation of eosinophils requires direct cell-cell contact and results in modulation of cell surface markers and rapid secretion of cytokines, chemokines, and lipid mediators. Unexpectedly, this activation occurred independently of canonical Toll-like receptor or C-type lectin receptor signaling. However, transcriptome analysis indicated a role for the PI3K-AKT-mTOR pathway, and PI3K inhibitors successfully prevented A. fumigatus-induced eosinophil activation. The PI3K pathway may therefore serve as a potential drug target to interfere with undesired eosinophil activation in fungus-elicited eosinophilic disorders.
Assuntos
Aspergilose Broncopulmonar Alérgica , Eosinofilia , Fosfatidilinositol 3-Quinase , Animais , Aspergilose Broncopulmonar Alérgica/genética , Aspergilose Broncopulmonar Alérgica/metabolismo , Aspergillus fumigatus , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Eosinofilia/genética , Eosinofilia/metabolismo , Eosinófilos/metabolismo , Lectinas Tipo C/metabolismo , Camundongos , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Mitogênicos/metabolismo , Serina-Treonina Quinases TOR , Receptores Toll-Like/metabolismoRESUMO
Eosinophils are potent innate effector cells associated mainly with type 2 immune responses elicited by helminths and allergens. Their activity needs to be tightly controlled to prevent severe inflammation and tissue damage. Eosinophil degranulation and secretion of inflammatory effector molecules, including cytokines, chemokines, and lipid mediators, can be regulated by activating and inhibitory receptors on the cell surface. In this study, we investigated the modulation of proliferation, apoptosis, gene expression, and cytokine/chemokine secretion from IL-33-activated Mus musculus eosinophils on cross-linking of the transmembrane receptor Sialic acid-binding Ig-like lectin F (Siglec-F). Siglec-F contains an ITIM plus an ITIM-like motif in its intracellular tail and is mainly regarded as an inhibitory and apoptosis-inducing receptor. In vitro costimulation of bone marrow-derived eosinophils with anti-Siglec-F and IL-33 compared with treatment with either alone led to enhanced STAT6 phosphorylation, stronger induction of hypoxia/glycolysis-related proinflammatory genes, and elevated secretion of type 2 cytokines (IL-4, IL-13) and chemokines (CCL3, CCL4) with only minor effects on proliferation and apoptosis. Using a competitive mixed bone marrow chimera approach with wild-type and Siglec-F-deficient eosinophils, we observed no evidence for Siglec-F-regulated inhibition of Aspergillus fumigatus-elicited lung eosinophilia. Truncation of the Siglec-F cytoplasmic tail, but not mutation of the ITIM and ITIM-like motifs, ablated the effect of enhanced cytokine/chemokine secretion. This provides evidence for an ITIM phosphorylation-independent signaling pathway from the cytoplasmic tail of the Siglec-F receptor that enhances effector molecule release from activated eosinophils.
Assuntos
Aspergilose/imunologia , Eosinofilia/imunologia , Eosinófilos/imunologia , Interleucina-33/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Animais , Apoptose/imunologia , Aspergilose/patologia , Aspergillus fumigatus/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Quimiocina CCL3/metabolismo , Quimiocina CCL4/metabolismo , Humanos , Interleucina-13/metabolismo , Interleucina-33/imunologia , Interleucina-4/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Fator de Transcrição STAT6/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genéticaRESUMO
Cure of infections with Leishmania major is critically dependent on the ability of macrophages to induce the type 2 nitic oxide (NO) synthase (NOS2) that produces high levels of NO in the presence of ample oxygen. Therefore, we analyzed the oxygen levels found in leishmanial skin lesions and their effect on the NOS2-dependent leishmanicidal activity of macrophages (MΦ). When L. major skin lesions of self-healing C57BL/6 mice reached their maximum size, the infected tissue displayed low oxygen levels (pO2â¼21 Torr). MΦ activated under these oxygen tensions failed to produce sufficient amounts of NO to clear L. major. Nos2-deficient and hypoxic wild-type macrophages displayed a similar phenotype. Killing was restored when MΦ were reoxygenated or exposed to a NO donor. The resolution of the lesion in C57BL/6 mice was paralleled by an increase of lesional pO2. When mice were kept under normobaric hypoxia, this caused a persistent suppression of the lesional pO2 and a concurrent increase of the parasite load. In Nos2-deficient mice, there was no effect of atmospheric hypoxia. Low oxygen levels found at leishmanial skin lesions impaired the NOS2-dependent leishmanicidal activity of MΦ. Hence, tissue oxygenation represents an underestimated local milieu factor that participates in the persistence of Leishmania.
Assuntos
Hipóxia/metabolismo , Leishmania major/metabolismo , Leishmaniose Cutânea/metabolismo , Macrófagos/parasitologia , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Animais , Arginase/genética , Arginase/metabolismo , Câmaras de Exposição Atmosférica , Hipóxia/parasitologia , Hipóxia/patologia , Leishmaniose Cutânea/patologia , Macrófagos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/genética , Oxigênio/metabolismo , Fenótipo , Pele/metabolismo , Pele/parasitologia , Pele/patologiaRESUMO
In infected tissues oxygen tensions are low. As innate immune cells have to operate under these conditions, we analyzed the ability of macrophages (Mφ) to kill Escherichia coli or Staphylococcus aureus in a hypoxic microenvironment. Oxygen restriction did not promote intracellular bacterial growth but did impair the bactericidal activity of the host cells against both pathogens. This correlated with a decreased production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates. Experiments with phagocyte NADPH oxidase (PHOX) and inducible NO synthase (NOS2) double-deficient Mφ revealed that in E. coli- or S. aureus-infected cells the reduced antibacterial activity during hypoxia was either entirely or partially independent of the diminished PHOX and NOS2 activity. Hypoxia impaired the mitochondrial activity of infected Mφ. Inhibition of the mitochondrial respiratory chain activity during normoxia (using rotenone or antimycin A) completely or partially mimicked the defective antibacterial activity observed in hypoxic E. coli- or S. aureus-infected wild-type Mφ, respectively. Accordingly, inhibition of the respiratory chain of S. aureus-infected, normoxic PHOX(-/-) NOS2(-/-) Mφ further raised the bacterial burden of the cells, which reached the level measured in hypoxic PHOX(-/-) NOS2(-/-) Mφ cultures. Our data demonstrate that the reduced killing of S. aureus or E. coli during hypoxia is not simply due to a lack of PHOX and NOS2 activity but partially or completely results from an impaired mitochondrial antibacterial effector function. Since pharmacological inhibition of the respiratory chain raised the generation of ROI but nevertheless phenocopied the effect of hypoxia, ROI can be excluded as the mechanism underlying the antimicrobial activity of mitochondria.
Assuntos
Hipóxia Celular , Escherichia coli/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Staphylococcus aureus/imunologia , Animais , Transporte de Elétrons , Escherichia coli/crescimento & desenvolvimento , Macrófagos/microbiologia , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/deficiência , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Espécies Reativas de Nitrogênio/biossíntese , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
HIF1A is a transcription factor that plays a central role for the adaptation to tissue hypoxia and for the inflammatory response of myeloid cells, including DCs. HIF1A is stabilized by hypoxia but also by TLR ligands under normoxic conditions. The underlying signaling events leading to the accumulation of HIF1A in the presence of oxygen are still poorly understood. Here, we show that in contrast to hypoxic stabilization of HIF1A, normoxic, TLR-mediated HIF1A accumulation in DCs follows a different pathway that predominantly requires MYD88-dependent NF-κB activity. The TLR-induced HIF1A controls a subset of proinflammatory genes that are insufficiently induced following hypoxia-mediated HIF1A induction. Thus, TLR activation and hypoxia stabilize HIF1A via distinct signaling pathways, resulting in differential HIF1A-dependent gene expression.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Perfilação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia , Fator 88 de Diferenciação Mieloide/fisiologia , Transdução de Sinais , Receptor 4 Toll-Like/fisiologia , Animais , Biomarcadores/metabolismo , Western Blotting , Diferenciação Celular , Proliferação de Células , Imunoprecipitação da Cromatina , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Selective gene silencing by RNA interference (RNAi) is a valuable tool for the targeted manipulation of the development and/or function of cells. Using a fluorescein-labeled non-silencing siRNA duplex, we established a protocol for the electroporation of primary mouse macrophages which routinely yielded >95% transfected cells. Electroporation of siRNAs directed against MAPK1 and CD86 led to an efficient knock-down of cellular protein in bone marrow-derived mouse macrophages (BM-Mphi). Importantly, the electroporation procedure did not impair the viability of BM-Mphi, their ability to ingest or degrade E. coli or their capacity to express iNOS mRNA, to produce NO or to upregulate TNF and IL-6 mRNA in response to inflammatory stimuli such as LPS. Therefore, we propose that electroporation of silencing siRNAs into murine BM-Mphi is a highly efficient method to manipulate gene expression of BM-Mphi that does not cause toxicity or a non-specific alteration of macrophage biology.
Assuntos
Eletroporação , Macrófagos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transfecção , Animais , Antígeno B7-2/genética , Sobrevivência Celular , Células Cultivadas , Feminino , Interleucina-6/genética , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/genética , Óxido Nítrico Sintase Tipo II/genética , Fagocitose , RNA Mensageiro/metabolismo , Fatores de Tempo , Fator de Necrose Tumoral alfa/genéticaRESUMO
The ability of macrophages to release cytokines is crucial to the host response to intracellular infection. In particular, macrophage-derived TNF plays an important role in the host response to infection with the intracellular pathogen Mycobacterium tuberculosis. In mice, TNF is indispensable for the formation of tuberculous granulomas, which serve to demarcate the virulent bacterium. TNF is also implicated in many of the immunopathological features of tuberculosis. To investigate the role of TNF in the local immune response, we infected human alveolar macrophages with virulent and attenuated mycobacteria. Infection with virulent strains induced the secretion of significantly higher levels of bioactive TNF than attenuated strains correlating with their ability to multiply intracellularly. Treatment of infected macrophages with neutralizing anti-TNF Abs reduced the growth rate of intracellular bacteria, whereas bacterial replication was augmented by addition of exogenous TNF. Infected and uninfected macrophages contributed to cytokine production as determined by double-staining of M. tuberculosis and intracellular TNF. The induction of TNF by human alveolar macrophages at the site of infection permits the multiplication of intracellular bacteria and may therefore present an evasion mechanism of human pathogens.