RESUMO
Tuberculosis is a chronic bacterial disease with a complex pathogenesis. An effective immunity against Mycobacterium tuberculosis requires both the innate and adaptive immune responses, including proper T helper (Th) type 1 cell function. FURIN is a proprotein convertase subtilisin/kexin (PCSK) enzyme, which is highly expressed in Th1 type cells. FURIN expression in T cells is essential for maintaining peripheral immune tolerance, but its role in the innate immunity and infections has remained elusive. Here, we utilized Mycobacterium marinum infection models in zebrafish (Danio rerio) to investigate how furin regulates host responses against mycobacteria. In steady-state furinAtd204e/+ fish reduced furinA mRNA levels associated with low granulocyte counts and elevated Th cell transcription factor expressions. Silencing furin genes reduced the survival of M. marinum-infected zebrafish embryos. A mycobacterial infection upregulated furinA in adult zebrafish, and infected furinAtd204e/+ mutants exhibited a proinflammatory phenotype characterized by elevated tumor necrosis factor a (tnfa), lymphotoxin alpha (lta) and interleukin 17a/f3 (il17a/f3) expression levels. The enhanced innate immune response in the furinAtd204e/+ mutants correlated with a significantly decreased bacterial burden in a chronic M. marinum infection model. Our data show that upregulated furinA expression can serve as a marker for mycobacterial disease, since it inhibits early host responses and consequently promotes bacterial growth in a chronic infection.
Assuntos
Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium marinum/imunologia , Pró-Proteína Convertases/imunologia , Subtilisina/imunologia , Proteínas de Peixe-Zebra/imunologia , Peixe-Zebra/imunologia , Animais , Complexo CD3/biossíntese , Diferenciação Celular/imunologia , Modelos Animais de Doenças , Inativação Gênica , Predisposição Genética para Doença , Granulócitos/citologia , Granulócitos/imunologia , Imunidade Inata/genética , Interleucina-17/metabolismo , Linfotoxina-alfa/metabolismo , Morfolinos/genética , Pró-Proteína Convertases/genética , RNA Mensageiro/biossíntese , Células Th1/imunologia , Tuberculose/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genéticaRESUMO
Tuberculosis remains a major global health challenge despite extensive vaccination schemes with the current live vaccine, Bacillus Calmette-Guérin. Tuberculosis vaccine research has been hampered by a scarcity of animal models which replicate human disease and are suitable for large-scale studies. We have shown recently that Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, causes an infection resembling human tuberculosis in adult zebrafish (Danio rerio). In the present study we use this model to show that BCG vaccination as well as DNA vaccination with selected mycobacterial antigens (Ag85B, CFP-10 and ESAT-6) protects adult zebrafish from mycobacterial infection. Using a low-dose (â¼20-30 bacteria) intraperitoneal M. marinum infection, both the number of granulomas and the amount of infected organs were reduced in the DNA vaccinated fish. Likewise, when infecting with a lethal infection dose (â¼20,000-27,000 bacteria), vaccination significantly reduced both mortality and bacterial counts in a manner dependent on the adaptive immune response. Protective effects of vaccination were associated with enhanced expression of interferon gamma. Our results indicate that the zebrafish is a promising new model for preclinical tuberculosis vaccine research.
Assuntos
Modelos Animais de Doenças , Descoberta de Drogas/métodos , Mycobacterium marinum/imunologia , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/imunologia , Vacinas contra a Tuberculose/isolamento & purificação , Tuberculose/prevenção & controle , Animais , Humanos , Interferon gama/metabolismo , Mycobacterium marinum/patogenicidade , Análise de Sobrevida , Tuberculose/imunologia , Tuberculose/patologia , Vacinas contra a Tuberculose/administração & dosagem , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Peixe-ZebraRESUMO
The mechanisms leading to latency and reactivation of human tuberculosis are still unclear, mainly due to the lack of standardized animal models for latent mycobacterial infection. In this longitudinal study of the progression of a mycobacterial disease in adult zebrafish, we show that an experimental intraperitoneal infection with a low dose (≈ 35 bacteria) of Mycobacterium marinum, results in the development of a latent disease in most individuals. The infection is characterized by limited mortality (25%), stable bacterial loads 4 weeks following infection and constant numbers of highly organized granulomas in few target organs. The majority of bacteria are dormant during a latent mycobacterial infection in zebrafish, and can be activated by resuscitation promoting factor ex vivo. In 5-10% of tuberculosis cases in humans, the disease is reactivated usually as a consequence of immune suppression. In our model, we are able to show that reactivation can be efficiently induced in infected zebrafish by γ-irradiation that transiently depletes granulo/monocyte and lymphocyte pools, as determined by flow cytometry. This immunosuppression causes reactivation of the dormant mycobacterial population and a rapid outgrowth of bacteria, leading to 88% mortality in four weeks. In this study, the adult zebrafish presents itself as a unique non-mammalian vertebrate model for studying the development of latency, regulation of mycobacterial dormancy, as well as reactivation of latent or subclinical tuberculosis. The possibilities for screening for host and pathogen factors affecting the disease progression, and identifying novel therapeutic agents and vaccine targets make this established model especially attractive.