RESUMO
The central pathogen-immune interface in tuberculosis is the granuloma, a complex host immune structure that dictates infection trajectory and physiology. Granuloma macrophages undergo a dramatic transition in which entire epithelial modules are induced and define granuloma architecture. In tuberculosis, relatively little is known about the host signals that trigger this transition. Using the zebrafish-Mycobacterium marinum model, we identify the basis of granuloma macrophage transformation. Single-cell RNA-sequencing analysis of zebrafish granulomas and analysis of Mycobacterium tuberculosis-infected macaques reveal that, even in the presence of robust type 1 immune responses, countervailing type 2 signals associate with macrophage epithelialization. We find that type 2 immune signaling, mediated via stat6, is absolutely required for epithelialization and granuloma formation. In mixed chimeras, stat6 acts cell autonomously within macrophages, where it is required for epithelioid transformation and incorporation into necrotic granulomas. These findings establish the signaling pathway that produces the hallmark structure of mycobacterial infection.
Assuntos
Granuloma/patologia , Imunidade/fisiologia , Infecções por Mycobacterium não Tuberculosas/patologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular , Modelos Animais de Doenças , Células Epitelioides/citologia , Células Epitelioides/imunologia , Células Epitelioides/metabolismo , Granuloma/imunologia , Granuloma/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Interferon gama/metabolismo , Interleucina-12/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium marinum/isolamento & purificação , Mycobacterium marinum/fisiologia , Necrose , RNA Guia de Cinetoplastídeos/metabolismo , Receptores de Interleucina-4/antagonistas & inibidores , Receptores de Interleucina-4/genética , Receptores de Interleucina-4/metabolismo , Fator de Transcrição STAT6/antagonistas & inibidores , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismoRESUMO
Mycobacterium tuberculosis infection in humans triggers formation of granulomas, which are tightly organized immune cell aggregates that are the central structure of tuberculosis. Infected and uninfected macrophages interdigitate, assuming an altered, flattened appearance. Although pathologists have described these changes for over a century, the molecular and cellular programs underlying this transition are unclear. Here, using the zebrafish-Mycobacterium marinum model, we found that mycobacterial granuloma formation is accompanied by macrophage induction of canonical epithelial molecules and structures. We identified fundamental macrophage reprogramming events that parallel E-cadherin-dependent mesenchymal-epithelial transitions. Macrophage-specific disruption of E-cadherin function resulted in disordered granuloma formation, enhanced immune cell access, decreased bacterial burden, and increased host survival, suggesting that the granuloma can also serve a bacteria-protective role. Granuloma macrophages in humans with tuberculosis were similarly transformed. Thus, during mycobacterial infection, granuloma macrophages are broadly reprogrammed by epithelial modules, and this reprogramming alters the trajectory of infection and the associated immune response.
Assuntos
Epitélio/imunologia , Macrófagos/imunologia , Mycobacterium marinum/imunologia , Animais , Caderinas/imunologia , Epitélio/microbiologia , Granuloma/imunologia , Granuloma/microbiologia , Macrófagos/microbiologia , Mycobacterium tuberculosis/imunologia , Peixe-ZebraRESUMO
BACKGROUND: Neutrophil migration is critical to the initiation and resolution of inflammation. Macrophage-1 antigen (Mac-1; CD11b/CD18, αMß2) is a leukocyte integrin essential for firm adhesion to endothelial ICAM-1 (intercellular adhesion molecule 1) and migration of neutrophils in the shear forces of the circulation. PDI (protein disulfide isomerase) has been reported to influence neutrophil adhesion and migration. We aimed to elucidate the molecular mechanism of PDI control of Mac-1 affinity for ICAM-1 during neutrophil migration under fluid shear. METHODS: Neutrophils isolated from whole blood were perfused over microfluidic chips coated with ICAM-1. Colocalization of Mac-1 and PDI on neutrophils was visualized by fluorescently labeled antibodies and confocal microscopy. The redox state of Mac-1 disulfide bonds was mapped by differential cysteine alkylation and mass spectrometry. Wild-type or disulfide mutant Mac-1 was expressed recombinantly in Baby Hamster Kidney cells to measure ligand affinity. Mac-1 conformations were measured by conformation-specific antibodies and molecular dynamics simulations. Neutrophils crawling on immobilized ICAM-1 were measured in presence of oxidized or reduced PDI, and the effect of PDI inhibition using isoquercetin on neutrophil crawling on inflamed endothelial cells was examined. Migration indices in the X- and Y-direction were determined and the crawling speed was calculated. RESULTS: PDI colocalized with high-affinity Mac-1 at the trailing edge of stimulated neutrophils when crawling on ICAM-1 under fluid shear. PDI cleaved 2 allosteric disulfide bonds, C169-C176 and C224-C264, in the ßI domain of the ß2 subunit, and cleavage of the C224-C264 disulfide bond selectively controls Mac-1 disengagement from ICAM-1 under fluid shear. Molecular dynamics simulations and conformation-specific antibodies reveal that cleavage of the C224-C264 bond induces conformational change and mechanical stress in the ßI domain. This allosterically alters the exposure of an αI domain epitope associated with a shift of Mac-1 to a lower-affinity state. These molecular events promote neutrophil motility in the direction of flow at high shear stress. Inhibition of PDI by isoquercetin reduces neutrophil migration in the direction of flow on endothelial cells during inflammation. CONCLUSIONS: Shear-dependent PDI cleavage of the neutrophil Mac-1 C224-C264 disulfide bond triggers Mac-1 de-adherence from ICAM-1 at the trailing edge of the cell and enables directional movement of neutrophils during inflammation.
Assuntos
Molécula 1 de Adesão Intercelular , Antígeno de Macrófago 1 , Humanos , Antígeno de Macrófago 1/fisiologia , Adesão Celular/fisiologia , Células Endoteliais , Inflamação , Movimento Celular/fisiologia , NeutrófilosRESUMO
Pathogenic mycobacteria actively dysregulate protective host immune signalling pathways during infection to drive the formation of permissive granuloma microenvironments. Dynamic regulation of host microRNA (miRNA) expression is a conserved feature of mycobacterial infections across host-pathogen pairings. Here we examine the role of miR-206 in the zebrafish model of Mycobacterium marinum infection, which allows investigation of the early stages of granuloma formation. We find miR-206 is upregulated following infection by pathogenic M. marinum and that antagomir-mediated knockdown of miR-206 is protective against infection. We observed striking upregulation of cxcl12a and cxcr4b in infected miR-206 knockdown zebrafish embryos and live imaging revealed enhanced recruitment of neutrophils to sites of infection. We used CRISPR/Cas9-mediated knockdown of cxcl12a and cxcr4b expression and AMD3100 inhibition of Cxcr4 to show that the enhanced neutrophil response and reduced bacterial burden caused by miR-206 knockdown was dependent on the Cxcl12/Cxcr4 signalling axis. Together, our data illustrate a pathway through which pathogenic mycobacteria induce host miR-206 expression to suppress Cxcl12/Cxcr4 signalling and prevent protective neutrophil recruitment to granulomas.
Assuntos
Quimiocina CXCL12/metabolismo , MicroRNAs/genética , Infiltração de Neutrófilos/imunologia , Receptores CXCR4/metabolismo , Animais , Quimiocina CXCL12/imunologia , Técnicas de Silenciamento de Genes/métodos , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium marinum/metabolismo , Receptores CXCR4/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Peixe-Zebra/imunologiaRESUMO
BACKGROUND: Accumulation of lipid in the liver is the first hallmark of both alcohol-related liver disease (ALD) and non-alcohol-related fatty liver disease (NAFLD). Recent studies indicate that specific mutations in lipid genes confer risk and might influence disease progression to irreversible liver cirrhosis. This study aimed to understand the function/s of lipid risk genes driving disease development in zebrafish genetic models of alcohol-related and non-alcohol-related fatty liver. METHODS: We used zebrafish larvae to investigate the effect of alcohol and high fat to model fatty liver and tested the utility of this model to study lipid risk gene functions. CRISPR/Cas9 gene editing was used to create knockdowns in 5 days post-fertilisation zebrafish larvae for the available orthologs of human cirrhosis risk genes (pnpla3, faf2, tm6sf2). To establish fatty liver models, larvae were exposed to ethanol and a high-fat diet (HFD) consisting of chicken egg yolk. Changes in morphology (imaging), survival, liver injury (biochemical tests, histopathology), gene expression (qPCR) and lipid accumulation (dye-specific live imaging) were analysed across treatment groups to test the functions of these genes. RESULTS: Exposure of 5-day post-fertilisation (dpf) WT larvae to 2% ethanol or HFD for 48 h developed measurable hepatic steatosis. CRISPR-Cas9 genome editing depleted pnpla3, faf2 and tm6sf2 gene expression in these CRISPR knockdown larvae (crispants). Depletion significantly increased the effects of ethanol and HFD toxicity by increasing hepatic steatosis and hepatic neutrophil recruitment ≥2-fold in all three crispants. Furthermore, ethanol or HFD exposure significantly altered the expression of genes associated with ethanol metabolism (cyp2y3) and lipid metabolism-related gene expression, including atgl (triglyceride hydrolysis), axox1, echs1 (fatty acid ß-oxidation), fabp10a (transport), hmgcra (metabolism), notch1 (signalling) and srebp1 (lipid synthesis), in all three pnpla3, faf2 and tm6sf2 crispants. Nile Red staining in all three crispants revealed significantly increased lipid droplet size and triglyceride accumulation in the livers following exposure to ethanol or HFD. CONCLUSIONS: We identified roles for pnpla3, faf2 and tm6sf2 genes in triglyceride accumulation and fatty acid oxidation pathways in a zebrafish larvae model of fatty liver.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Peixe-Zebra , Humanos , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Etanol/toxicidade , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/patologia , Metabolismo dos Lipídeos/genética , Triglicerídeos/metabolismo , Ácidos Graxos/metabolismoRESUMO
Mycobacterium abscessus infections are of increasing global prevalence and are often difficult to treat due to complex antibiotic resistance profiles. While there are similarities between the pathogenesis of M. abscessus and tuberculous mycobacteria, including granuloma formation and stromal remodelling, there are distinct molecular differences at the host-pathogen interface. Here we have used a zebrafish-M. abscessus model and host-directed therapies that were previously identified in the zebrafish-M. marinum model to identify potential host-directed therapies against M. abscessus infection. We find efficacy of anti-angiogenic and vascular normalizing therapies against rough M. abscessus infection, but no effect of anti-platelet drugs.
Assuntos
Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Mycobacterium , Animais , Infecções por Mycobacterium não Tuberculosas/microbiologia , Peixe-ZebraRESUMO
Pidotimod (PDT) is a synthetic dipeptide molecule which can improve immune responses in mice and humans, protecting hosts from infection. However, the exact mechanism of protection remains ill-defined. The effect of pidotimod has not yet been investigated in the inflammatory response of zebrafish. In this study, we used tail wound and infection models of zebrafish to study the effect of PDT on inflammation. We found that zebrafish larvae were sensitive to PDT immersion causing toxicity at doses above 50 µg/mL. The tail wound assay showed that PDT increased the recruitment of neutrophils and macrophages to the wound site and promoted the transcription of the pro-inflammatory cytokine il1b. However, we did not observe protection of uropathogenic Escherichia coli or Mycobacterium marinum infected zebrafish larvae following PDT treatment. This study provides a new platform for PDT research, which is worthy of further research to identify further effects of PDT therapy.
Assuntos
Inflamação , Ácido Pirrolidonocarboxílico/análogos & derivados , Tiazolidinas/efeitos adversos , Peixe-Zebra , Animais , Inflamação/induzido quimicamente , Larva , Ácido Pirrolidonocarboxílico/efeitos adversosRESUMO
Pathogenic mycobacteria induce the formation of complex cellular aggregates called granulomas that are the hallmark of tuberculosis. Here we examine the development and consequences of vascularization of the tuberculous granuloma in the zebrafish-Mycobacterium marinum infection model, which is characterized by organized granulomas with necrotic cores that bear striking resemblance to those of human tuberculosis. Using intravital microscopy in the transparent larval zebrafish, we show that granuloma formation is intimately associated with angiogenesis. The initiation of angiogenesis in turn coincides with the generation of local hypoxia and transcriptional induction of the canonical pro-angiogenic molecule Vegfaa. Pharmacological inhibition of the Vegf pathway suppresses granuloma-associated angiogenesis, reduces infection burden and limits dissemination. Moreover, anti-angiogenic therapies synergize with the first-line anti-tubercular antibiotic rifampicin, as well as with the antibiotic metronidazole, which targets hypoxic bacterial populations. Our data indicate that mycobacteria induce granuloma-associated angiogenesis, which promotes mycobacterial growth and increases spread of infection to new tissue sites. We propose the use of anti-angiogenic agents, now being used in cancer regimens, as a host-targeting tuberculosis therapy, particularly in extensively drug-resistant disease for which current antibiotic regimens are largely ineffective.
Assuntos
Inibidores da Angiogênese/farmacologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium marinum/efeitos dos fármacos , Mycobacterium marinum/crescimento & desenvolvimento , Neovascularização Patológica/microbiologia , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra/microbiologia , Inibidores da Angiogênese/uso terapêutico , Animais , Antibióticos Antituberculose/farmacologia , Carga Bacteriana/efeitos dos fármacos , Modelos Animais de Doenças , Sinergismo Farmacológico , Granuloma/tratamento farmacológico , Granuloma/metabolismo , Granuloma/microbiologia , Granuloma/patologia , Hipóxia/metabolismo , Hipóxia/microbiologia , Hipóxia/patologia , Larva/efeitos dos fármacos , Larva/microbiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Macrófagos/patologia , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/metabolismo , Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium marinum/patogenicidade , Neovascularização Patológica/tratamento farmacológico , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , Tuberculose/patologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
T lymphocytes utilize amoeboid migration to navigate effectively within complex microenvironments. The precise rearrangement of the actin cytoskeleton required for cellular forward propulsion is mediated by actin regulators, including the actin-related protein 2/3 (Arp2/3) complex, a macromolecular machine that nucleates branched actin filaments at the leading edge. The consequences of modulating Arp2/3 activity on the biophysical properties of the actomyosin cortex and downstream T cell function are incompletely understood. We report that even a moderate decrease of Arp3 levels in T cells profoundly affects actin cortex integrity. Reduction in total F-actin content leads to reduced cortical tension and disrupted lamellipodia formation. Instead, in Arp3-knockdown cells, the motility mode is dominated by blebbing migration characterized by transient, balloon-like protrusions at the leading edge. Although this migration mode seems to be compatible with interstitial migration in three-dimensional environments, diminished locomotion kinetics and impaired cytotoxicity interfere with optimal T cell function. These findings define the importance of finely tuned, Arp2/3-dependent mechanophysical membrane integrity in cytotoxic effector T lymphocyte activities.
Assuntos
Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteína 3 Relacionada a Actina/metabolismo , Movimento Celular/genética , Linfócitos T Citotóxicos/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteína 3 Relacionada a Actina/genética , Actinas/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Regulação para Baixo , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Interferente Pequeno , Análise de Célula Única , Linfócitos T Citotóxicos/citologia , Peixe-ZebraRESUMO
Risk, severity, and outcome of infection depend on the interplay of pathogen virulence and host susceptibility. Systematic identification of genetic susceptibility to infection is being undertaken through genome-wide association studies, but how to expeditiously move from genetic differences to functional mechanisms is unclear. Here, we use genetic association of molecular, cellular, and human disease traits and experimental validation to demonstrate that genetic variation affects expression of VAC14, a phosphoinositide-regulating protein, to influence susceptibility to Salmonella enterica serovar Typhi (S Typhi) infection. Decreased VAC14 expression increased plasma membrane cholesterol, facilitating Salmonella docking and invasion. This increased susceptibility at the cellular level manifests as increased susceptibility to typhoid fever in a Vietnamese population. Furthermore, treating zebrafish with a cholesterol-lowering agent, ezetimibe, reduced susceptibility to S Typhi. Thus, coupling multiple genetic association studies with mechanistic dissection revealed how VAC14 regulates Salmonella invasion and typhoid fever susceptibility and may open doors to new prophylactic/therapeutic approaches.
Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Salmonella typhi/genética , Linhagem Celular Tumoral , Colesterol/genética , Colesterol/metabolismo , Ezetimiba , Variação Genética/genética , Estudo de Associação Genômica Ampla , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Polimorfismo de Nucleotídeo Único , Salmonella/genética , Salmonella/patogenicidade , Salmonella typhi/metabolismo , Salmonella typhi/patogenicidade , Febre Tifoide/metabolismo , Febre Tifoide/fisiopatologia , Virulência/genéticaRESUMO
BACKGROUND: Infection-induced thrombocytosis is a clinically important complication of tuberculosis infection. Recent studies have highlighted the utility of aspirin as a host-directed therapy modulating the inflammatory response to infection but have not investigated the possibility that the effect of aspirin is related to an antiplatelet mode of action. METHODS: In this study, we utilize the zebrafish-Mycobacterium marinum model to show mycobacteria drive host hemostasis through the formation of granulomas. Treatment of infected zebrafish with aspirin markedly reduced mycobacterial burden. This effect is reproduced by treatment with platelet-specific glycoprotein IIb/IIIa inhibitors demonstrating a detrimental role for infection-induced thrombocyte activation. RESULTS: We find that the reduction in mycobacterial burden is dependent on macrophages and granuloma formation, providing the first in vivo experimental evidence that infection-induced platelet activation compromises protective host immunity to mycobacterial infection. CONCLUSIONS: Our study illuminates platelet activation as an efficacious target of aspirin, a widely available and affordable host-directed therapy candidate for tuberculosis.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/imunologia , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium marinum/imunologia , Peixe-Zebra/imunologia , Peixe-Zebra/microbiologia , Animais , Proteínas de Bactérias/imunologia , Modelos Animais de Doenças , Granuloma/tratamento farmacológico , Granuloma/imunologia , Granuloma/microbiologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/microbiologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium marinum/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Tuberculose/tratamento farmacológico , Tuberculose/imunologia , Tuberculose/microbiologiaRESUMO
The spectre of the coming post-antibiotic age demands novel therapies for infectious diseases. Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the single deadliest infection throughout human history. M. tuberculosis has acquired antibiotic resistance at an alarming rate with some strains reported as being totally drug resistant. Host-directed therapies (HDTs) attempt to overcome the evolution of antibiotic resistance by targeting relatively immutable host processes. Here, I hypothesise the induction of hypoxia via anti-angiogenic therapy will be an efficacious HDT against TB. I argue that anti-angiogenic therapy is a modernisation of industrial revolution era sanatoria treatment for TB, and present a view of the TB granuloma as a 'bacterial tumour' that can be treated with anti-angiogenic therapies to reduce bacterial burden and spare host immunopathology. I suggest two complementary modes of action, induction of bacterial dormancy and activation of host hypoxia-induced factor (HIF)-mediated immunity, and define the experimental tools necessary to test this hypothesis.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Antituberculosos/uso terapêutico , Granuloma/tratamento farmacológico , Mycobacterium tuberculosis/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Inibidores da Angiogênese/efeitos adversos , Animais , Antibióticos Antituberculose/uso terapêutico , Antituberculosos/efeitos adversos , Hipóxia Celular , Farmacorresistência Bacteriana , Granuloma/imunologia , Granuloma/microbiologia , Granuloma/fisiopatologia , Interações Hospedeiro-Patógeno , Humanos , Viabilidade Microbiana , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/imunologia , Tuberculose Resistente a Múltiplos Medicamentos/imunologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/fisiopatologiaRESUMO
Changes to lipid metabolism are well-characterised consequences of human tuberculosis infection but their functional relevance are not clearly elucidated in these or other host-mycobacterial systems. The zebrafish-Mycobacterium marinum infection model is used extensively to model many aspects of human-M. tuberculosis pathogenesis but has not been widely used to study the role of infection-induced lipid metabolism. We find mammalian mycobacterial infection-induced alterations in host Low Density Lipoprotein metabolism are conserved in the zebrafish model of mycobacterial pathogenesis. Depletion of LDLR, a key lipid metabolism node, decreased M. marinum burden, and corrected infection-induced altered lipid metabolism resulting in decreased LDL and reduced the rate of macrophage transformation into foam cells. Our results demonstrate a conserved role for infection-induced alterations to host lipid metabolism, and specifically the LDL-LDLR axis, across host-mycobacterial species pairings.
Assuntos
Doenças dos Peixes/metabolismo , Infecções por Mycobacterium não Tuberculosas/metabolismo , Receptores de LDL/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , LDL-Colesterol/metabolismo , Modelos Animais de Doenças , Embrião não Mamífero , Metabolismo dos Lipídeos , Infecções por Mycobacterium não Tuberculosas/veterinária , Receptores de LDL/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Pathogenic mycobacteria trigger formation of organized granulomas. As granulomas mature, they induce angiogenesis and vascular permeability. Here, in a striking parallel to tumor pro-angiogenic signaling, we identify angiopoietin-2 (ANG-2) induction as an important component of vascular dysfunction during mycobacterial infection. Mycobacterial infection in humans and zebrafish results in robust induction of ANG-2 expression from macrophages and stromal cells. Using a small-molecule inhibitor closely related to one currently in clinical trials, we link ANG-2/TIE2 signaling to vascular permeability during mycobacterial infection. Targeting granuloma-induced vascular permeability via vascular endothelial-protein tyrosine phosphatase inhibition limits mycobacterial growth, suggesting a new strategy for host-directed therapies against tuberculosis.
Assuntos
Angiopoietina-2/metabolismo , Permeabilidade Capilar , Infecções por Mycobacterium/patologia , Mycobacterium/crescimento & desenvolvimento , Angiopoietina-2/genética , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Regulação da Expressão Gênica , Granuloma/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Larva , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Mycobacterium/efeitos dos fármacos , Receptor TIE-2/metabolismo , Transdução de Sinais , Tuberculose/microbiologia , Peixe-ZebraRESUMO
The incidence of infections due to nontuberculous mycobacteria (NTM) has increased rapidly in recent years, surpassing tuberculosis in developed countries. Due to inherent antimicrobial resistance, NTM infections are particularly difficult to treat with low cure rates. There is an urgent need to understand NTM pathogenesis and to develop novel therapeutic approaches for the treatment of NTM diseases. Zebrafish have emerged as an excellent animal model due to genetic amenability and optical transparency during embryonic development, allowing spatiotemporal visualization of host-pathogen interactions. Furthermore, adult zebrafish possess fully functional innate and adaptive immunity and recapitulate important pathophysiological hallmarks of mycobacterial infection. Here, we report recent breakthroughs in understanding the hallmarks of NTM infections using the zebrafish model.
Assuntos
Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Infecções por Mycobacterium não Tuberculosas , Micobactérias não Tuberculosas , Peixe-Zebra , Peixe-Zebra/microbiologia , Animais , Infecções por Mycobacterium não Tuberculosas/microbiologia , Micobactérias não Tuberculosas/patogenicidade , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Imunidade AdaptativaRESUMO
Regulation of host miRNA expression is a contested node that controls the host immune response to mycobacterial infection. The host must counter subversive efforts of pathogenic mycobacteria to launch a protective immune response. Here, we examine the role of miR-126 in the zebrafish-Mycobacterium marinum infection model and identify a protective role for infection-induced miR-126 through multiple effector pathways. We identified a putative link between miR-126 and the tsc1a and cxcl12a/ccl2/ccr2 signalling axes resulting in the suppression of non-tnfa expressing macrophage accumulation at early M. marinum granulomas. Mechanistically, we found a detrimental effect of tsc1a expression that renders zebrafish embryos susceptible to higher bacterial burden and increased cell death via mTOR inhibition. We found that macrophage recruitment driven by the cxcl12a/ccl2/ccr2 signalling axis was at the expense of the recruitment of classically activated tnfa-expressing macrophages and increased cell death around granulomas. Together, our results delineate putative pathways by which infection-induced miR-126 may shape an effective immune response to M. marinum infection in zebrafish embryos.
Assuntos
Quimiocina CXCL12 , MicroRNAs , Infecções por Mycobacterium não Tuberculosas , Proteína 1 do Complexo Esclerose Tuberosa , Proteínas de Peixe-Zebra , Animais , Granuloma/genética , Macrófagos , MicroRNAs/genética , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/microbiologia , Peixe-Zebra , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Quimiocina CXCL12/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
The constant exposure of the fish branchial cavity to aquatic pathogens causes local mucosal immune responses to be extremely important for their survival. Here, we used a marker for T lymphocytes/natural killer (NK) cells (ZAP70) and advanced imaging techniques to investigate the lymphoid architecture of the zebrafish branchial cavity. We identified a sub-pharyngeal lymphoid organ, which we tentatively named "Nemausean lymphoid organ" (NELO). NELO is enriched in T/NK cells, plasma/B cells, and antigen-presenting cells embedded in a network of reticulated epithelial cells. The presence of activated T cells and lymphocyte proliferation, but not V(D)J recombination or hematopoiesis, suggests that NELO is a secondary lymphoid organ. In response to infection, NELO displays structural changes including the formation of T/NK cell clusters. NELO and gill lymphoid tissues form a cohesive unit within a large mucosal lymphoid network. Collectively, we reveal an unreported mucosal lymphoid organ reminiscent of mammalian tonsils that evolved in multiple teleost fish families.
Assuntos
Tonsila Palatina , Peixe-Zebra , Humanos , Animais , Tecido Linfoide , Faringe , Linfócitos T , MamíferosRESUMO
Inflammatory bowel disease (IBD) results from dysfunctional interactions between the intestinal immune system and microbiota, influenced by host genetic susceptibility. Because a key feature of the pathology is intestinal epithelial damage, potential disease factors have been traditionally analyzed within the background of chemical colitis models in mice. The zebrafish has greatly complemented the mouse for modeling aspects of disease processes, with an advantage for high content drug screens. Larval zebrafish exposed to the haptenizing agent trinitrobenzene sulfonic acid (TNBS) displayed impaired intestinal homeostasis and inflammation reminiscent of human IBD. There was a marked induction of pro-inflammatory cytokines, the degradative enzyme mmp9 and leukocytosis. Enterocolitis was dependent on microbiota and Toll-like receptor signaling, that can be ameliorated by antibiotic and anti-inflammatory drug treatments. This system will be useful to rapidly interrogate in vivo the biological significance of the IBD candidate genes so far identified and to carry out pharmacological modifier screens.
Assuntos
Anti-Inflamatórios/uso terapêutico , Modelos Animais de Doenças , Enterocolite/microbiologia , Enterocolite/prevenção & controle , Trato Gastrointestinal/microbiologia , Metagenoma/fisiologia , Peixe-Zebra , Animais , Anti-Inflamatórios/farmacologia , Embrião não Mamífero , Enterocolite/induzido quimicamente , Enterocolite/patologia , Trato Gastrointestinal/irrigação sanguínea , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/patologia , Haptenos/imunologia , Haptenos/metabolismo , Humanos , Larva , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Leucócitos/patologia , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 88 de Diferenciação Mieloide/fisiologia , Ácido Trinitrobenzenossulfônico , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Uropathogenic Escherichia coli (UPEC) causes urinary tract infections that can result in sepsis. The haemostatic system is protective in the pyelonephritis stage of ascending UPEC infection, but the role of the haemostatic system has not been investigated during sepsis. Here we utilize a zebrafish-UPEC systemic infection model to visualize infection-induced coagulation and examine the effects of commonly prescribed anti-haemostatic medications on the infection severity. Treatment of systemically infected zebrafish with warfarin, aspirin, or ticagrelor reduced host survival, while stabilization of clots with aminocaproic acid increased host survival. Anti-haemostatic drug treatment increased UPEC burden. Our findings provide evidence that commonly prescribed anti-haemostatic medications may worsen the outcome of severe UPEC infection.
Assuntos
Infecções por Escherichia coli , Hemostáticos , Sepse , Infecções Urinárias , Animais , Modelos Animais de Doenças , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/patologia , Hemostáticos/farmacologia , Sepse/tratamento farmacológico , Índice de Gravidade de Doença , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/patologia , Escherichia coli Uropatogênica , Peixe-ZebraRESUMO
Heme oxygenase function is highly conserved between vertebrates where it plays important roles in normal embryonic development and controls oxidative stress. Expression of the zebrafish heme oxygenase 1 genes is known to be responsive to oxidative stress suggesting a conserved physiological function. In this study, we generate a knockout allele of zebrafish hmox1a and characterize the effects of hmox1a and hmox1b loss on embryonic development. We find that loss of hmox1a or hmox1b causes developmental defects in only a minority of embryos, in contrast to Hmox1 gene deletions in mice that cause loss of most embryos. Using a tail wound inflammation assay we find a conserved role for hmox1a, but not hmox1b, in normal macrophage migration to the wound site. Together our results indicate that zebrafish hmox1a has clearly a partitioned role from hmox1b that is more consistent with conserved functions of mammalian Heme oxygenase 1.