RESUMEN
BACKGROUND: Previous researches have clarified that miR-155 is increased in methicillin-resistant Staphylococcus aureus (MRSA) pneumonia, and modulates Th9 differentiation. Like Th9 cells, Th17 cells were also a subset of CD4+ T cells and involved in MRSA pneumonia progression. This work aimed to investigate the role and mechanism of miR-155 in Th17 differentiation. METHODS: Bronchoalveolar lavage fluid (BALF) was collected from children with MRSA pneumonia and bronchial foreign bodies. MRSA-infected murine model was established followed by collecting BALF and lung tissues. qRT-PCR, ELISA and flow cytometry were performed to examine the mRNA expression and concentration of IL-17 and the number of Th17 cells in above samples. HE and ELISA were used to evaluate inflammatory responses in lung. Furthermore, CD4+ T cells were isolated from BALF of children for in vitro experiments. After treatments with miR-155 mimic/inhibitor, the roles of miR-155 in Th17/IL-17 regulation were determined. The downstream of miR-155 was explored by qRT-PCR, western blotting, dual luciferase reporter analysis and RIP assay. RESULTS: The levels of IL-17 and the proportion of Th17 cells were increased in children with MRSA pneumonia. A similar pattern was observed in MRSA-infected mice. On the contrary, IL-17 neutralization abolished the activation of Th17/IL-17 induced by MRSA infection. Furthermore, IL-17 blockade diminished the inflammation caused by MRSA. In vitro experiments demonstrated miR-155 positively regulated IL-17 expression and Th17 differentiation. Mechanistically, FOXP3 was a direct target of miR-155. miR-155 inhibited FOXP3 level via binding between FOXP3 and Argonaute 2 (AGO2), the key component of RNA-induced silencing complex (RISC). FOXP3 overexpression reversed elevated IL-17 levels and Th17 differentiation induced by miR-155. CONCLUSIONS: miR-155 facilitates Th17 differentiation by reducing FOXP3 through interaction of AGO2 and FOXP3 to promote the pathogenesis of MRSA pneumonia. IL-17 blockade weakens the inflammation due to MRSA, which provides a nonantibiotic treatment strategy for MRSA pneumonia.
Asunto(s)
Diferenciación Celular , Factores de Transcripción Forkhead , Inflamación , Interleucina-17 , Staphylococcus aureus Resistente a Meticilina , MicroARNs , Células Th17 , MicroARNs/genética , MicroARNs/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Animales , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Humanos , Ratones , Interleucina-17/metabolismo , Inflamación/metabolismo , Masculino , Líquido del Lavado Bronquioalveolar , Femenino , Niño , Neumonía Estafilocócica/inmunología , Neumonía Estafilocócica/metabolismo , Neumonía Estafilocócica/microbiología , PreescolarRESUMEN
In this study, we examined the effect of Il9 deletion on macrophages in methicillin-resistant Staphylococcus aureus (MRSA) infection. MRSA-infected mice were employed for the in vivo experiments, and RAW264.7 cells were stimulated with MRSA for the in vitro experiments. Macrophage polarization was determined by flow cytometry and quantitative real-time PCR; macrophage phagocytosis was assessed by flow cytometry and laser scanning confocal microscopy; cell apoptosis was assessed by flow cytometry and western blotting. Il9 deletion markedly elevated macrophage phagocytosis and M2 macrophages in MRSA infection, which was accompanied by elevated expression of Il10 and Arg1 and reduced expression of Inos, tumor necrosis factor-α (Tnfα), and Il6. Il9 deletion also inhibited macrophage apoptosis in MRSA infection, which was manifested by elevated B-cell lymphoma 2 (BCL-2) protein level and reduced protein levels of cleaved cysteine protease 3 (CASPASE-3) and BCL2-Associated X (BAX). Both the in vivo and in vitro experiments further showed the activation of phosphoinositide 3-kinase (PI3K)/AKT (also known as protein kinase B, PKB) signaling pathway in MRSA infection and that the regulation of Il9 expression may be dependent on Toll-like receptor (TLR) 2/PI3K pathway. The above results showed that Il9 deletion exhibited a protective role against MRSA infection by promoting M2 polarization and phagocytosis of macrophages and the regulation of Il9 partly owing to the activation of TLR2/PI3K pathway, proposing a novel therapeutic strategy for MRSA-infected pneumonia.
Asunto(s)
Interleucina-9 , Staphylococcus aureus Resistente a Meticilina , Fagocitosis , Neumonía Estafilocócica , Animales , Ratones , Interleucina-9/genética , Interleucina-9/metabolismo , Macrófagos/metabolismo , Staphylococcus aureus Resistente a Meticilina/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Neumonía Estafilocócica/tratamiento farmacológico , Neumonía Estafilocócica/inmunologíaRESUMEN
Methicillin-resistant Staphylococcus aureus (MRSA) is an important etiology of pneumonia. Interleukin (IL)-9 is a T helper 9 (Th9) cytokine and participates in the pathogenesis of infectious diseases. Here, we investigated the role of IL-9 by using an MRSA pneumonia animal model. The BALB/c mice underwent nasal inhalation with an ST239 MRSA strain to establish the mouse model of MRSA pneumonia, and a subset of mice were intravenously injected with IL-9 neutralizing antibody or immunoglobulin (Ig) G. At 3 and 8 days postinfection, the peripheral blood, bronchioalveolar lavage fluid (BALF), and lung tissues were collected. The frequencies of Th9 cells and the levels of cytokines in peripheral blood, BALF, and lung tissues were determined by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. The colony counts of MRSA in BALF and lung tissue were detected. The lung pathological changes were examined using hematoxylin and eosin staining. Data from flow cytometry, qRT-PCR, and ELISA showed that MRSA-infected mice exhibited higher frequency of Th9 cells and higher IL-9 mRNA and protein levels in the peripheral blood, BALF, and lung tissues of mice. In contrast, the neutralization of IL-9 abrogated MRSA inoculation-induced Th9 cell generation and IL-9 production in BALF and lung tissues. Furthermore, bacterial counting and histological examination showed that the numbers of bacteria in BALF and lungs and the lung pathological scores induced by MRSA inoculation were attenuated by the neutralization of IL-9. Moreover, cell counting and ELISA results demonstrated that IL-9 neutralization diminished the MRSA inoculation-induced count of neutrophils and macrophages and levels of pro-inflammatory cytokines in BALF. Collectively, IL-9 neutralization attenuated inflammation of MRSA pneumonia by regulating Th9/IL-9 expression.
Asunto(s)
Inflamación/prevención & control , Interleucina-9/metabolismo , Staphylococcus aureus Resistente a Meticilina , Neumonía/microbiología , Animales , Anticuerpos/farmacología , Líquido del Lavado Bronquioalveolar/microbiología , Recuento de Colonia Microbiana , Citocinas/metabolismo , Modelos Animales de Enfermedad , Interleucina-9/antagonistas & inhibidores , Interleucina-9/genética , Interleucina-9/inmunología , Pulmón/microbiología , Ratones , Ratones Endogámicos BALB C , Neumonía/patología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/patologíaRESUMEN
Th9 is a subset of CD4+ T cells that mainly secrete IL-9. Th9/IL-9 participates in immune response during Staphylococcus aureus and methicillin-resistant Staphylococcus aureus pneumonia (MRSA) infection. Here, we collected bronchoalveolar lavage fluid (BALF) from 30 children with MRSA pneumonia (MRSA group) and 10 children with bronchial foreign bodies (Control group). RT-PCR, ELISA and flow cytometry were used to detect the expression of miR-155 and IL-9 in BALF and the number of Th9 cells. CD4+ T cells isolated from BALF of MRSA and Control group were transfected with miR-155 mimic or inhibitor, and then induced Th9 cell differentiation. The results showed that the expression of miR-155 and IL-9 were significantly increased in BALF and Th9 cell of MRSA group, as well as the number of Th9 cells. miR-155 mimic upregulated IL-9 mRNA expression, IL-9 secretion and increased number of Th9 cells. On the contrary, miR-155 inhibitor inhibited IL-9 mRNA expression, IL-9 secretion and decreased number of Th9 cells. The dual luciferase assays demonstrated miR-155 can target binding to SIRT1 3'UTR. Moreover, overexpression of SIRT1 could reverse the effect of miR-155 mimic on IL-9 expression level, Th9 cell number and transcription factors PU.1 and IRF4 expression. In conclusion, miR-155 regulates Th9 differentiation in children with MRSA by targeting SIRT1.
Asunto(s)
Diferenciación Celular/genética , Staphylococcus aureus Resistente a Meticilina/inmunología , MicroARNs/genética , Sirtuina 1/genética , Infecciones Estafilocócicas/etiología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Regiones no Traducidas 3' , Biomarcadores , Diferenciación Celular/inmunología , Niño , Preescolar , Susceptibilidad a Enfermedades/inmunología , Femenino , Expresión Génica , Regulación de la Expresión Génica , Genes Reporteros , Predisposición Genética a la Enfermedad , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunofenotipificación , Masculino , Interferencia de ARN , Infecciones Estafilocócicas/metabolismoRESUMEN
A series of octahedral Ru(ii) complexes with an x-y planar tetradentate and two trans-z-axis ammonia ligands were synthesized. High stabilization of G-quadruplexes was achieved upon π-stacking with aromatic planar ligands. Unique interaction from z-axis ligands bestowed both excellent binding resistance to duplex DNA and selectivity towards the antiparallel topology.