RESUMO
Due to poor diagnostics and increased co-infections, HIV-associated Legionella infections are underreported. We aimed to retrospectively determine the frequency of Legionella infections in bronchoalveolar lavage (BAL) from HIV-associated pneumonia patients hospitalized in Medellin, Colombia, between February 2007 and April 2014. Although culture was negative, 17 BAL (36%) were positive for Legionella by quantitative polymerase chain reaction, most of which were in the Mycobacterium tuberculosis or Pneumocystis jirovecii co-infected patients, and included L. anisa (nâ¯=â¯6), L. bozemanae (nâ¯=â¯4), L. pneumophila (nâ¯=â¯3), and L. micdadei (nâ¯=â¯2). All L. bozemanae and L. micdadei associated with Pneumocystis, while all L. pneumophila associated with M. tuberculosis. Legionella probable cases had more complications and higher mortality rates (Pâ¯=â¯0.02) and were rarely administered empirical anti-Legionella therapy while in hospital. Clinicians should be aware of the possible presence of Legionella in HIV and M. tuberculosis or P. jirovecii co-infected patients.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Coinfecção/microbiologia , Legionella/fisiologia , Legionelose/microbiologia , Pneumonia/microbiologia , Infecções Oportunistas Relacionadas com a AIDS/epidemiologia , Adulto , Líquido da Lavagem Broncoalveolar/microbiologia , Coinfecção/epidemiologia , Colômbia/epidemiologia , Feminino , Humanos , Legionella/genética , Legionelose/epidemiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/fisiologia , Pneumocystis carinii/isolamento & purificação , Pneumonia/epidemiologia , Reação em Cadeia da Polimerase , Estudos Retrospectivos , RiscoRESUMO
Legionella pneumophila is a gram-negative bacterium that infects many species of unicellular protozoa in freshwater environments. The human infection is accidental, and the bacteria may not have evolved strategies to bypass innate immune signaling in mammalian macrophages. Thus, L. pneumophila triggers many innate immune pathways including inflammasome activation. The inflammasomes are multimolecular platforms assembled in the host cell cytoplasm and lead to activation of inflammatory caspases. Inflammasome activation leads to secretion of inflammatory cytokines, such as IL-1ß and IL-18, and an inflammatory form of cell death called pyroptosis, which initiates with the induction of a pore in the macrophage membranes. In this chapter we provide detailed protocols to evaluate Legionella-induced inflammasome activation in macrophages, including real-time pore formation assay, western blotting to detect activation of inflammatory caspases (cleavage and pulldown), and the measurement of inflammatory cytokines.
Assuntos
Interações Hospedeiro-Patógeno , Inflamassomos/metabolismo , Legionella/fisiologia , Legionelose/metabolismo , Legionelose/microbiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Animais , Caspases/genética , Caspases/metabolismo , Citocinas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Mediadores da Inflamação/metabolismo , Legionelose/imunologia , Macrófagos/imunologia , Camundongos , Camundongos KnockoutRESUMO
Gram-negative bacteria from the Legionella genus are intracellular pathogens that cause a severe form of pneumonia called Legionnaires' disease. The bacteria replicate intracellularly in macrophages, and the restriction of bacterial replication by these cells is critical for host resistance. The activation of the NAIP5/NLRC4 inflammasome, which is readily triggered in response to bacterial flagellin, is essential for the restriction of bacterial replication in murine macrophages. Once activated, this inflammasome induces pore formation and pyroptosis and facilitates the restriction of bacterial replication in macrophages. Because investigations related to the NLRC4-mediated restriction of Legionella replication were performed using mice double deficient for caspase-1 and caspase-11, we assessed the participation of caspase-1 and caspase-11 in the functions of the NLRC4 inflammasome and the restriction of Legionella replication in macrophages and in vivo. By using several species of Legionella and mice singly deficient for caspase-1 or caspase-11, we demonstrated that caspase-1 but not caspase-11 was required for pore formation, pyroptosis, and restriction of Legionella replication in macrophages and in vivo. By generating F1 mice in a mixed 129 × C57BL/6 background deficient (129 × Casp-11(-/-) ) or sufficient (129 × C57BL/6) for caspase-11 expression, we found that caspase-11 was dispensable for the restriction of Legionella pneumophila replication in macrophages and in vivo. Thus, although caspase-11 participates in flagellin-independent noncanonical activation of the NLRP3 inflammasome, it is dispensable for the activities of the NLRC4 inflammasome. In contrast, functional caspase-1 is necessary and sufficient to trigger flagellin/NLRC4-mediated restriction of Legionella spp. infection in macrophages and in vivo.