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1.
Sci Rep ; 9(1): 10878, 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31350436

RESUMO

As a first host barrier, the skin is constantly exposed to environmental insults that perturb its integrity. Tight regulation of skin homeostasis is largely controlled by the aryl hydrocarbon receptor (AhR). Here, we demonstrate that Henna and its major pigment, the naphthoquinone Lawsone activate AhR, both in vitro and in vivo. In human keratinocytes and epidermis equivalents, Lawsone exposure enhances the production of late epidermal proteins, impacts keratinocyte differentiation and proliferation, and regulates skin inflammation. To determine the potential use of Lawsone for therapeutic application, we harnessed human, murine and zebrafish models. In skin regeneration models, Lawsone interferes with physiological tissue regeneration and inhibits wound healing. Conversely, in a human acute dermatitis model, topical application of a Lawsone-containing cream ameliorates skin irritation. Altogether, our study reveals how a widely used natural plant pigment is sensed by the host receptor AhR, and how the physiopathological context determines beneficial and detrimental outcomes.

2.
EMBO Rep ; 20(4)2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30872316

RESUMO

Cyclic dinucleotides (CDNs) are important second messenger molecules in prokaryotes and eukaryotes. Within host cells, cytosolic CDNs are detected by STING and alert the host by activating innate immunity characterized by type I interferon (IFN) responses. Extracellular bacteria and dying cells can release CDNs, but sensing of extracellular CDNs (eCDNs) by mammalian cells remains elusive. Here, we report that endocytosis facilitates internalization of eCDNs. The DNA sensor cGAS facilitates sensing of endocytosed CDNs, their perinuclear accumulation, and subsequent STING-dependent release of type I IFN Internalized CDNs bind cGAS directly, leading to its dimerization, and the formation of a cGAS/STING complex, which may activate downstream signaling. Thus, eCDNs comprise microbe- and danger-associated molecular patterns that contribute to host-microbe crosstalk during health and disease.

3.
Front Immunol ; 10: 89, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30766535

RESUMO

Human immune system mice are highly valuable for in vivo dissection of human immune responses. Although they were employed for analyzing tuberculosis (TB) disease, there is little data on the spatial organization and cellular composition of human immune cells in TB granuloma pathology in this model. We demonstrate that human immune system mice, generated by transplanted human fetal liver derived hematopoietic stem cells develop a continuum of pulmonary lesions upon Mycobacterium tuberculosis aerosol infection. In particular, caseous necrotic granulomas, which contribute to prolonged TB treatment time, developed, and had cellular phenotypic spatial-organization similar to TB patients. By comparing two recommended drug regimens, we confirmed observations made in clinical settings: Adding Moxifloxacin to a classical chemotherapy regimen had no beneficial effects on bacterial eradication. We consider this model instrumental for deeper understanding of human specific features of TB pathogenesis and of particular value for the pre-clinical drug development pipeline.

4.
Sci Rep ; 8(1): 1520, 2018 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-29367626

RESUMO

Mycobacterium tuberculosis (Mtb) is a life-threatening pathogen in humans. Bacterial infection of macrophages usually triggers strong innate immune mechanisms, including IL-1 cytokine secretion. The newer member of the IL-1 family, IL-36, was recently shown to be involved in cellular defense against Mtb. To unveil the underlying mechanism of IL-36 induced antibacterial activity, we analyzed its role in the regulation of cholesterol metabolism, together with the involvement of Liver X Receptor (LXR) in this process. We report that, in Mtb-infected macrophages, IL-36 signaling modulates cholesterol biosynthesis and efflux via LXR. Moreover, IL-36 induces the expression of cholesterol-converting enzymes and the accumulation of LXR ligands, such as oxysterols. Ultimately, both IL-36 and LXR signaling play a role in the regulation of antimicrobial peptides expression and in Mtb growth restriction. These data provide novel evidence for the importance of IL-36 and cholesterol metabolism mediated by LXR in cellular host defense against Mtb.

5.
Autophagy ; 13(12): 2041-2055, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29251248

RESUMO

The E3 ubiquitin ligase NEDD4 has been intensively studied in processes involved in viral infections, such as virus budding. However, little is known about its functions in bacterial infections. Our investigations into the role of NEDD4 in intracellular bacterial infections demonstrate that Mycobacterium tuberculosis and Listeria monocytogenes, but not Mycobacterium bovis BCG, replicate more efficiently in NEDD4 knockdown macrophages. In parallel, NEDD4 knockdown or knockout impaired basal macroautophagy/autophagy, as well as infection-induced autophagy. Conversely, NEDD4 expression promoted autophagy in an E3 catalytic activity-dependent manner, thereby restricting intracellular Listeria replication. Mechanistic studies uncovered that endogenous NEDD4 interacted with BECN1/Beclin 1 and this interaction increased during Listeria infection. Deficiency of NEDD4 resulted in elevated K48-linkage ubiquitination of endogenous BECN1. Further, NEDD4 mediated K6- and K27- linkage ubiquitination of BECN1, leading to elevated stability of BECN1 and increased autophagy. Thus, NEDD4 participates in killing of intracellular bacterial pathogens via autophagy by sustaining the stability of BECN1.

6.
J Infect Dis ; 214(3): 464-74, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27389350

RESUMO

Tuberculosis remains a major killer worldwide, not the least because of our incomplete knowledge of protective and pathogenic immune mechanism. The roles of the interleukin 1 (IL-1) and interleukin 18 pathways in host defense are well established, as are their regulation through the inflammasome complex. In contrast, the regulation of interleukin 36γ (IL-36γ), a recently described member of the IL-1 family, and its immunological relevance in host defense remain largely unknown. Here we show that Mycobacterium tuberculosis infection of macrophages induces IL-36γ production in a 2-stage-regulated fashion. In the first stage, microbial ligands trigger host Toll-like receptor and MyD88-dependent pathways, leading to IL-36γ secretion. In the second stage, endogenous IL-1ß and interleukin 18 further amplify IL-36γ synthesis. The relevance of this cytokine in the control of M. tuberculosis is demonstrated by IL-36γ-induced antimicrobial peptides and IL-36 receptor-dependent restriction of M. tuberculosis growth. Thus, we provide first insight into the induction and regulation of the proinflammatory cytokine IL-36γ during tuberculosis.


Assuntos
Interleucina-1/metabolismo , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Animais , Linhagem Celular , Humanos , Interleucina-1/deficiência , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout
7.
Cell Microbiol ; 18(12): 1846-1856, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27279134

RESUMO

Pulmonary tuberculosis (TB) is an airborne disease caused by the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb). Alveolar epithelial cells and macrophages are the first point of contact for Mtb in the respiratory tract. However, the mechanisms of mycobacterial attachment to, and internalization by, nonprofessional phagocytes, such as epithelial cells, remain incompletely understood. We identified syndecan 4 (Sdc4) as mycobacterial attachment receptor on alveolar epithelial cells. Sdc4 mRNA expression was increased in human and mouse alveolar epithelial cells after mycobacterial infection. Sdc4 knockdown in alveolar epithelial cells or blocking with anti-Sdc4 antibody reduced mycobacterial attachment and internalization. At the molecular level, interactions between epithelial cells and mycobacteria involved host Sdc and the mycobacterial heparin-binding hemagglutinin adhesin. In vivo, Sdc1/Sdc4 double-knockout mice were more resistant to Mtb colonization of the lung. Our work reveals a role for distinct Sdcs in promoting mycobacterial entry into alveolar epithelial cells with impact on outcome of TB disease.


Assuntos
Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , Pulmão/microbiologia , Sindecana-4/imunologia , Tuberculose Pulmonar/imunologia , Células A549 , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Células Epiteliais/imunologia , Regulação da Expressão Gênica , Humanos , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/microbiologia , Camundongos , Camundongos Knockout , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Sindecana-1/deficiência , Sindecana-1/genética , Sindecana-1/imunologia , Sindecana-4/antagonistas & inibidores , Sindecana-4/deficiência , Sindecana-4/genética , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia
8.
J Infect Dis ; 211(11): 1831-41, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25505299

RESUMO

BACKGROUND: The recombinant BCG ΔureC::hly (rBCG) vaccine candidate induces improved protection against tuberculosis over parental BCG (pBCG) in preclinical studies and has successfully completed a phase 2a clinical trial. However, the mechanisms responsible for the superior vaccine efficacy of rBCG are still incompletely understood. Here, we investigated the underlying biological mechanisms elicited by the rBCG vaccine candidate relevant to its protective efficacy. METHODS: THP-1 macrophages were infected with pBCG or rBCG, and inflammasome activation and autophagy were evaluated. In addition, mice were vaccinated with pBCG or rBCG, and gene expression in the draining lymph nodes was analyzed by microarray at day 1 after vaccination. RESULTS: BCG-derived DNA was detected in the cytosol of rBCG-infected macrophages. rBCG infection was associated with enhanced absent in melanoma 2 (AIM2) inflammasome activation, increased activation of caspases and production of interleukin (IL)-1ß and IL-18, as well as induction of AIM2-dependent and stimulator of interferon genes (STING)-dependent autophagy. Similarly, mice vaccinated with rBCG showed early increased expression of Il-1ß, Il-18, and Tmem173 (transmembrane protein 173; also known as STING). CONCLUSIONS: rBCG stimulates AIM2 inflammasome activation and autophagy, suggesting that these cell-autonomous functions should be exploited for improved vaccine design.


Assuntos
Autofagia/imunologia , Vacina BCG/imunologia , Inflamassomos/imunologia , Tuberculose/imunologia , Vacinas Sintéticas/imunologia , Animais , Linhagem Celular , Feminino , Humanos , Inflamação , Interleucina-18/imunologia , Interleucina-1beta/imunologia , Linfonodos/química , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos C57BL
9.
Nature ; 512(7515): 387-92, 2014 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-25119038

RESUMO

The aryl hydrocarbon receptor (AhR) is a highly conserved ligand-dependent transcription factor that senses environmental toxins and endogenous ligands, thereby inducing detoxifying enzymes and modulating immune cell differentiation and responses. We hypothesized that AhR evolved to sense not only environmental pollutants but also microbial insults. We characterized bacterial pigmented virulence factors, namely the phenazines from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, as ligands of AhR. Upon ligand binding, AhR activation leads to virulence factor degradation and regulated cytokine and chemokine production. The relevance of AhR to host defence is underlined by heightened susceptibility of AhR-deficient mice to both P. aeruginosa and M. tuberculosis. Thus, we demonstrate that AhR senses distinct bacterial virulence factors and controls antibacterial responses, supporting a previously unidentified role for AhR as an intracellular pattern recognition receptor, and identify bacterial pigments as a new class of pathogen-associated molecular patterns.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Mycobacterium tuberculosis/imunologia , Pigmentos Biológicos/metabolismo , Pseudomonas aeruginosa/imunologia , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Animais , Antibacterianos/metabolismo , Células da Medula Óssea/citologia , Citocinas/imunologia , Citocinas/metabolismo , Retroalimentação Fisiológica , Humanos , Ligantes , Ativação de Macrófagos , Camundongos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Fenazinas/metabolismo , Pigmentos Biológicos/química , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Piocianina/metabolismo , Fatores de Virulência/química , Fatores de Virulência/metabolismo
10.
J Clin Invest ; 123(11): 4836-48, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24084739

RESUMO

The molecular mechanisms that control innate immune cell trafficking during chronic infection and inflammation, such as in tuberculosis (TB), are incompletely understood. During active TB, myeloid cells infiltrate the lung and sustain local inflammation. While the chemoattractants that orchestrate these processes are increasingly recognized, the posttranscriptional events that dictate their availability are unclear. We identified microRNA-223 (miR-223) as an upregulated small noncoding RNA in blood and lung parenchyma of TB patients and during murine TB. Deletion of miR-223 rendered TB-resistant mice highly susceptible to acute lung infection. The lethality of miR-223(­/­) mice was apparently not due to defects in antimycobacterial T cell responses. Exacerbated TB in miR-223(­/­) animals could be partially reversed by neutralization of CXCL2, CCL3, and IL-6, by mAb depletion of neutrophils, and by genetic deletion of Cxcr2. We found that miR-223 controlled lung recruitment of myeloid cells, and consequently, neutrophil-driven lethal inflammation. We conclude that miR-223 directly targets the chemoattractants CXCL2, CCL3, and IL-6 in myeloid cells. Our study not only reveals an essential role for a single miRNA in TB, it also identifies new targets for, and assigns biological functions to, miR-223. By regulating leukocyte chemotaxis via chemoattractants, miR-223 is critical for the control of TB and potentially other chronic inflammatory diseases.


Assuntos
MicroRNAs/genética , MicroRNAs/imunologia , Infiltração de Neutrófilos/genética , Tuberculose Pulmonar/genética , Animais , Quimiocina CCL3/metabolismo , Quimiocina CXCL2/metabolismo , Suscetibilidade a Doenças , Humanos , Imunidade Inata/genética , Interleucina-6/metabolismo , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/sangue , Infiltração de Neutrófilos/imunologia , Receptores de Interleucina-8B/deficiência , Receptores de Interleucina-8B/genética , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/patologia , Regulação para Cima
11.
PLoS One ; 6(5): e19829, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21611201

RESUMO

The generation of diversity and plasticity of transcriptional programs are key components of effective vertebrate immune responses. The role of Alternative Splicing has been recognized, but it is underappreciated and poorly understood as a critical mechanism for the regulation and fine-tuning of physiological immune responses. Here we report the generation of loss-of-function phenotypes for a large collection of genes known or predicted to be involved in the splicing reaction and the identification of 19 novel regulators of IL-1ß secretion in response to E. coli challenge of THP-1 cells. Twelve of these genes are required for IL-1ß secretion, while seven are negative regulators of this process. Silencing of SFRS3 increased IL-1ß secretion due to elevation of IL-1ß and caspase-1 mRNA in addition to active caspase-1 levels. This study points to the relevance of splicing in the regulation of auto-inflammatory diseases.


Assuntos
Interleucina-1beta/metabolismo , Processamento de RNA/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Caspase 1/metabolismo , Linhagem Celular , Ativação Enzimática , Escherichia coli , Regulação da Expressão Gênica , Inativação Gênica , Genes Reporter , Humanos , Interleucina-1beta/genética , Monócitos/metabolismo , NF-kappa B/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Reprodutibilidade dos Testes , Fatores de Processamento de Serina-Arginina , Transcrição Genética
12.
Cell Microbiol ; 12(8): 1046-63, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20148899

RESUMO

Interleukin-1beta (IL-1beta) represents one of the most important mediators of inflammation and host responses to infection. Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, induces IL-1beta secretion at the site of infection, but the underlying mechanism(s) are poorly understood. In this work we show that Mtb infection of macrophages stimulates caspase-1 activity and promotes the secretion of IL-1beta. This stimulation requires live intracellular bacteria expressing a functional ESX-1 secretion system. ESAT-6, an ESX-1 substrate implicated in membrane damage, is both necessary and sufficient for caspase-1 activation and IL-1beta secretion. ESAT-6 promotes the access of other immunostimulatory agents such as AG85 into the macrophage cytosol, indicating that this protein may contribute to caspase-1 activation largely by perturbing host cell membranes. Using a high-throughput shRNA-based screen we found that numerous NOD-like receptors (NLRs) and CARD domain-containing proteins (CARDs) were important for IL-1beta secretion upon Mtb infection. Most importantly, NLRP3, ASC and caspase-1 form an infection-inducible inflammasome complex that is essential for IL-1beta secretion. In summary, we show that recognition of Mtb infection by the NLRP3 inflammasome requires the activity of the bacterial virulence factor ESAT-6, and the subsequent IL-1beta response is regulated by a number of NLR/CARD proteins.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/biossíntese , Proteínas do Citoesqueleto/biossíntese , Interações Hospedeiro-Patógeno , Interleucina-1beta/metabolismo , Macrófagos/microbiologia , Mycobacterium tuberculosis/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/imunologia , Caspase 1/biossíntese , Proteínas do Citoesqueleto/imunologia , Humanos , Macrófagos/imunologia , Mycobacterium tuberculosis/patogenicidade , Proteína 3 que Contém Domínio de Pirina da Família NLR
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