RESUMO
Klebsiella pneumoniae (Kp) is one of the most common pathogens in nosocomial infections and is becoming increasingly multidrug resistant. However, the underlying molecular pathogenesis of this bacterium remains elusive, limiting the therapeutic options. Understanding the mechanism of its pathogenesis may facilitate the development of anti-bacterial therapeutics. Here, we show that Lyn, a pleiotropic Src tyrosine kinase, is involved in host defense against Kp by regulating phagocytosis process and simultaneously downregulating inflammatory responses. Using acute infection mouse models, we observed that lyn(-/-) mice were more susceptible to Kp with increased mortality and severe lung injury compared with WT mice. Kp infected-lyn(-/-) mice exhibited elevated inflammatory cytokines (IL-6 and TNF-α), and increased superoxide in the lung and other organs. In addition, the phosphorylation of p38 and NF-κB p65 subunit increased markedly in response to Kp infection in lyn(-/-) mice. We also demonstrated that the translocation of p65 from cytoplasm to nuclei increased in cultured murine lung epithelial cells by Lyn siRNA knockdown. Furthermore, lipid rafts clustered with activated Lyn and accumulated in the site of Kp invasion. Taken together, these findings revealed that Lyn may participate in host defense against Kp infection through the negative modulation of inflammatory cytokines.
Assuntos
Infecções por Klebsiella/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases da Família src/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Infecções por Klebsiella/genética , Infecções por Klebsiella/imunologia , Infecções por Klebsiella/mortalidade , Klebsiella pneumoniae , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Knockout , Oxirredução , Fagocitose/imunologia , Pneumonia/genética , Pneumonia/imunologia , Pneumonia/metabolismo , Pneumonia/mortalidade , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Quinases da Família src/deficiência , Quinases da Família src/genéticaRESUMO
SHIP-1 is an inositol phosphatase that hydrolyzes phosphatidylinositol 3-kinase (PI3K) products and negatively regulates protein kinase B (Akt) activity, thereby modulating a variety of cellular processes in mammals. However, the role of SHIP-1 in bacterial-induced sepsis is largely unknown. Here, we show that SHIP-1 regulates inflammatory responses during Gram-negative bacterium Pseudomonas aeruginosa infection. We found that infected-SHIP-1-/- mice exhibited decreased survival rates, increased inflammatory responses, and susceptibility owing to elevated expression of PI3K than wild-type (WT) mice. Inhibiting SHIP-1 via siRNA silencing resulted in lipid raft aggregates, aggravated oxidative damage, and bacterial burden in macrophages after PAO1 infection. Mechanistically, SHIP-1 deficiency augmented phosphorylation of PI3K and nuclear transcription of signal transducer and activator of transcription 5 (STAT5) to induce the expression of Trib1, which is critical for differentiation of M2 but not M1 macrophages. These findings reveal a previously unrecognized role of SHIP-1 in inflammatory responses and macrophage homeostasis during P. aeruginosa infection through a PI3K/Akt-STAT5-Trib1 axis.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Macrófagos/fisiologia , Fagocitose , Fosfatidilinositol 3-Quinases/fisiologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/fisiologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/fisiologia , Infecções por Pseudomonas/imunologia , Fator de Transcrição STAT5/fisiologia , Animais , Polaridade Celular , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Serina-Treonina Quinases/fisiologiaRESUMO
Transient receptor potential channel 1 (TRPC1) is a nonselective cation channel that is required for Ca(2+) homeostasis necessary for cellular functions. However, whether TRPC1 is involved in infectious disease remains unknown. Here, we report a novel function for TRPC1 in host defense against Gram-negative bacteria. TRPC1(-/-) mice exhibited decreased survival, severe lung injury, and systemic bacterial dissemination upon infection. Furthermore, silencing of TRPC1 showed decreased Ca(2+) entry, reduced proinflammatory cytokines, and lowered bacterial clearance. Importantly, TRPC1 functioned as an endogenous Ca(2+) entry channel critical for proinflammatory cytokine production in both alveolar macrophages and epithelial cells. We further identified that bacterium-mediated activation of TRPC1 was dependent on Toll-like receptor 4 (TLR4), which induced endoplasmic reticulum (ER) store depletion. After activation of phospholipase Cγ (PLC-γ), TRPC1 mediated Ca(2+) entry and triggered protein kinase Cα (PKCα) activity to facilitate nuclear translocation of NF-κB/Jun N-terminal protein kinase (JNK) and augment the proinflammatory response, leading to tissue damage and eventually mortality. These findings reveal that TRPC1 is required for host defense against bacterial infections through the TLR4-TRPC1-PKCα signaling circuit.