RESUMEN
Since bacterial invasion into host cells is a critical step in the infection process and the predominance of multiple-antibiotic-resistant Klebsiella (K.) pneumoniae strains, using molecular agents to interfere with K. pneumoniae invasion is an attractive approach for the prevention of infection and suppress the immune inflammatory response. In previous studies by our group, high-mobility group nucleosome-binding domain 2 (HMGN2) protein was shown to exhibit anti-bacterial activity in vitro. The objective of the present study was to investigate the effects of HMGN2 protein on the invasion of K. pneumoniae 03183 in vivo. The results showed that pre-treatment with 128 µg/ml HMGN2 significantly reduced K. pneumoniae 03183 invasion into mouse lungs and increased the mRNA expression of CXCL1 and LCN2 within 2 h. Immunohistochemical staining showed that F-actin expression was significantly decreased, and fluorescence microscopy and western blot analysis further demonstrated that HMGN2 significantly blocked K. pneumoniae 03183-induced actin polymerization. These changes implied that HMGN2 may provide protection against K. pneumoniae 03183 infection in vivo.
Asunto(s)
Antibacterianos/farmacología , Proteína HMGN2/farmacología , Infecciones por Klebsiella/tratamiento farmacológico , Klebsiella pneumoniae/efectos de los fármacos , Pulmón/efectos de los fármacos , Neumonía Bacteriana/tratamiento farmacológico , Actinas/genética , Actinas/inmunología , Proteínas de Fase Aguda/agonistas , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/inmunología , Animales , Antibacterianos/biosíntesis , Quimiocina CXCL1/agonistas , Quimiocina CXCL1/genética , Quimiocina CXCL1/inmunología , Femenino , Expresión Génica , Proteína HMGN2/biosíntesis , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Infecciones por Klebsiella/inmunología , Infecciones por Klebsiella/microbiología , Infecciones por Klebsiella/patología , Klebsiella pneumoniae/fisiología , Lipocalina 2 , Lipocalinas/agonistas , Lipocalinas/genética , Lipocalinas/inmunología , Pulmón/microbiología , Ratones , Ratones Endogámicos C57BL , Proteínas Oncogénicas/agonistas , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/inmunología , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/microbiología , Neumonía Bacteriana/patología , ARN Mensajero/genética , ARN Mensajero/inmunología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacologíaAsunto(s)
Proteínas de Fase Aguda/genética , Antibióticos Antineoplásicos/farmacología , Doxorrubicina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Lipocalinas/genética , Metaloproteinasa 9 de la Matriz/genética , Paclitaxel/farmacología , Proteínas Proto-Oncogénicas/genética , Proteínas de Fase Aguda/agonistas , Proteínas de Fase Aguda/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Concentración 50 Inhibidora , Lipocalina 2 , Lipocalinas/agonistas , Lipocalinas/metabolismo , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas Proto-Oncogénicas/agonistas , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The generation of reactive oxygen species is a central feature of inflammation that results in the oxidation of host phospholipids. Oxidized phospholipids, such as 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC), have been shown to inhibit signaling induced by bacterial lipopeptide or lipopolysaccharide (LPS), yet the mechanisms responsible for the inhibition of Toll-like receptor (TLR) signaling by OxPAPC remain incompletely understood. Here, we examined the mechanisms by which OxPAPC inhibits TLR signaling induced by diverse ligands in macrophages, smooth muscle cells, and epithelial cells. OxPAPC inhibited tumor necrosis factor-alpha production, IkappaBalpha degradation, p38 MAPK phosphorylation, and NF-kappaB-dependent reporter activation induced by stimulants of TLR2 and TLR4 (Pam3CSK4 and LPS) but not by stimulants of other TLRs (poly(I.C), flagellin, loxoribine, single-stranded RNA, or CpG DNA) in macrophages and HEK-293 cells transfected with respective TLRs and significantly reduced inflammatory responses in mice injected subcutaneously or intraperitoneally with Pam3CSK4. Serum proteins, including CD14 and LPS-binding protein, were identified as key targets for the specificity of TLR inhibition as supplementation with excess serum or recombinant CD14 or LBP reversed TLR2 inhibition by OxPAPC, whereas serum accessory proteins or expression of membrane CD14 potentiated signaling via TLR2 and TLR4 but not other TLRs. Binding experiments and functional assays identified MD2 as a novel additional target of OxPAPC inhibition of LPS signaling. Synthetic phospholipid oxidation products 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine inhibited TLR2 signaling from approximately 30 microm. Taken together, these results suggest that oxidized phospholipid-mediated inhibition of TLR signaling occurs mainly by competitive interaction with accessory proteins that interact directly with bacterial lipids to promote signaling via TLR2 or TLR4.
