Flagella from five Cronobacter species induce pro-inflammatory cytokines in macrophage derivatives from human monocytes

• Published in: PloS one Vol. 7; no. 12; p. e52091
• Main Authors: Cruz-Córdova, Ariadnna, Rocha-Ramírez, Luz M, Ochoa, Sara A, González-Pedrajo, Bertha, Gónzalez-Pedrajo, Bertha, Espinosa, Norma, Eslava, Carlos, Hernández-Chiñas, Ulises, Mendoza-Hernández, Guillermo, Rodríguez-Leviz, Alejandra, Valencia-Mayoral, Pedro, Sadowinski-Pine, Stanislaw, Hernández-Castro, Rigoberto, Estrada-García, Iris, Muñoz-Hernández, Onofre, Rosas, Irma, Xicohtencatl-Cortes, Juan
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 21.12.2012; Public Library of Science (PLoS)
• Subjects: Amino Acid Sequence; Antibodies; Antibodies - immunology; Antibodies - pharmacology; Bacteremia; Bacteria; Biofilms; Biology; Cell activation; Cronobacter; Cronobacter - immunology; Cronobacter - ultrastructure; Cytokines; Cytokines - metabolism; Derivatives; Dilution; E coli; Electron microscopy; Enterocolitis; Epidemiology; Epithelial cells; Escherichia coli; Flagella; Flagella - chemistry; Flagella - drug effects; Flagella - immunology; Flagella - ultrastructure; Flagellin; Flagellin - chemistry; Gel electrophoresis; Gels; Gene expression; Health aspects; HEK293 Cells; Humans; Immunoglobulin A; Immunoglobulin A - immunology; Immunoglobulin A - pharmacology; Infections; Inflammation; Inflammation Mediators - metabolism; Inflammatory response; Interleukin 10; Interleukin 8; Macrophages; Macrophages - cytology; Macrophages - immunology; Macrophages - metabolism; Mass spectrometry; Mass spectroscopy; Medicine; Meningitis; Molecular Sequence Data; Monocytes; Monocytes - cytology; Monomers; Necrotizing enterocolitis; Neonates; Opportunist infection; Pathogenesis; Pathogens; Peptides; Rodents; Salmonella; Sequence Alignment; Sodium lauryl sulfate; TLR5 protein; Toll-Like Receptor 5 - antagonists & inhibitors; Toll-Like Receptor 5 - immunology; Toll-like receptors; Transfection; Transmission electron microscopy; Tumor necrosis factor
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0052091

Cronobacter spp. are opportunistic pathogens linked to lie-threatening infections in neonates and contaminated powdered infant formula that has been epidemiologically associated with these cases. Clinical symptoms of Cronobacter include necrotizing enterocolitis, bacteremia, and meningitis. Flagella from C. sakazakii are involved in biofilm formation and its adhesion to epithelial cells. We investigated the role of flagella from C. sakazakii ST1 and ST4, C. malonaticus, C. muytjensii, C. turicensis and C. dublinensis during the activation of cytokines (IL-8, TNF-α, and IL-10) in macrophage derivatives from human monocytes, which has not been extensively studied. The production and identity of flagella from the five Cronobacter species were visualized and recognized with anti-flagella antibodies by immunogold labeling through transmission electron microscopy. Purified flagella were dissociated into monomers in 12% SDS-PAGE Coomassie blue-stained gels showing a band of ∼28 kDa and, in addition, mass spectrometry revealed the presence of several peptides that correspond to flagellin. Flagella (100 ng) induced the release of IL-8 (3314-6025 pg/ml), TNF-α (39-359 pg/ml), and IL-10 (2-96 pg/ml), in macrophage isolates from human monocytes and similar results were obtained when flagella were dissociated into monomers. Inhibition assays using three dilutions of anti-flagella antibodies (1∶10, 1∶100, and 1∶200) suppressed the secretion of IL-8, TNF-α, and IL-10 between 95-100% using 100 ng of protein. A transfection assay using 293-hTLR5 cells showed IL-8 release of 197 pg/ml and suppression in the secretion of IL-8 when anti-hTLR5-IgA antibodies were used at different concentrations. These observations suggest that flagella and flagellin are involved in an inflammatory response dependent on TLR5 recognition, which could contribute to the pathogenesis of the bacteria.