Mechanisms of Legionella pneumophila-induced interleukin-8 expression in human lung epithelial cells

<p>Abstract</p> <p>Background</p> <p><it>Legionella pneumophila </it>is a facultative intracellular bacterium, capable of replicating within the phagosomes of macrophages and monocytes, but little is known about its interaction with human lung epithelial cells. We investigated the effect of <it>L. pneumophila </it>on the expression of interleukin-8 (IL-8) in human A549 alveolar and NCI-H292 tracheal epithelial cell lines.</p> <p>Results</p> <p>Infection of <it>L. pneumophila </it>strain, but not heat-killed strain, resulted in upregulation of IL-8. IL-8 mRNA expression was induced immediately after the infection and its signal became gradually stronger until 24 h after infection. On the other hand, IL-8 expression in A549 cells infected with <it>L. pneumophila </it>lacking a functional type IV secretion system was transient. The IL-8 expression was slightly induced at 16 h and increased at 24 h after infection with flagellin-deficient <it>Legionella</it>. Activation of the IL-8 promoter by <it>L. pneumophila </it>infection occurred through the action of nuclear factor-κB (NF-κB). Transfection of dominant negative mutants of NF-κB-inducing kinase, IκB kinase and IκB inhibited <it>L. pneumophila</it>-mediated activation of IL-8 promoter. Treatment with hsp90 inhibitor suppressed <it>L. pneumophila</it>-induced IL-8 mRNA due to deactivation of NF-κB.</p> <p>Conclusion</p> <p>Collectively, these results suggest that <it>L. pneumophila </it>induces activation of NF-κB through an intracellular signaling pathway that involves NF-κB-inducing kinase and IκB kinase, leading to IL-8 gene transcription, and that hsp90 acts as a crucial regulator in <it>L. pneumophila</it>-induced IL-8 expression, presumably contributing to immune response in <it>L. pneumophila</it>. The presence of flagellin and a type IV secretion system are critical for <it>Legionella </it>to induce IL-8 expression in lung epithelial cells.</p

Mechanisms of -induced interleukin-8 expression in human lung epithelial cells-8

<p><b>Copyright information:</b></p><p>Taken from "Mechanisms of -induced interleukin-8 expression in human lung epithelial cells"</p><p>http://www.biomedcentral.com/1471-2180/7/102</p><p>BMC Microbiology 2007;7():102-102.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2213657.</p><p></p> varying concentrations of AA100jm strain for 6 h. RT-PCR was performed to check the changes of IL-8 mRNA expression after 17-AAG treatment in -infected A549 cells. (B) Attenuation of -induced NF-κB DNA binding by 17-AAG treatment. A549 cells were treated with (+) or without (-) 17-AAG for 16 h prior to infection with varying concentrations of for 3 h. The nuclear extracts were isolated from A549 cells infected with and incubated with P-labeled oligonucleotides corresponding to NF-κB. (C) hsp90 protects IKKα and IKKβ from proteasomal degradation. A549 cells either were pretreated with LLnL (20 μM) for 1 h, followed or not followed by addition of 17-AAG (1 μM) and incubation for 16 h, or were treated with 17-AAG for 16 h or left untreated as indicated. Whole cell extracts were immunoblotted with specific antibodies against each protein. Representative results of three similar experiments in each panel are shown

Mechanisms of -induced interleukin-8 expression in human lung epithelial cells-2

<p><b>Copyright information:</b></p><p>Taken from "Mechanisms of -induced interleukin-8 expression in human lung epithelial cells"</p><p>http://www.biomedcentral.com/1471-2180/7/102</p><p>BMC Microbiology 2007;7():102-102.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2213657.</p><p></p>manner. A549 cells were infected with varying concentrations of AA100jm, and the levels of IL-8 mRNA expression were examined by RT-PCR in cells harvested after 8 h. (B) Effect of heat-treatment of on the ability to induce IL-8 mRNA expression. Expression of IL-8 mRNA in A549 and NCI-H292 cells treated with heat-killed AA100jm was observed at 6 and 24 h after infection. A549 and NCI-H292 cells were infected with the untreated AA100jm at an MOI of 100. β-actin expression served as controls. Representative results of three similar experiments in each panel are shown

Mechanisms of -induced interleukin-8 expression in human lung epithelial cells-3

<p><b>Copyright information:</b></p><p>Taken from "Mechanisms of -induced interleukin-8 expression in human lung epithelial cells"</p><p>http://www.biomedcentral.com/1471-2180/7/102</p><p>BMC Microbiology 2007;7():102-102.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2213657.</p><p></p>D of three experiments. *, < 0.001 (compared to uninfected cells). #, < 0.001 (compared to cells infected with the wild-type strains)

Mechanisms of -induced interleukin-8 expression in human lung epithelial cells-9

<p><b>Copyright information:</b></p><p>Taken from "Mechanisms of -induced interleukin-8 expression in human lung epithelial cells"</p><p>http://www.biomedcentral.com/1471-2180/7/102</p><p>BMC Microbiology 2007;7():102-102.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2213657.</p><p></p> A549 cells were infected with AA100jm and mutant (MOI of 100). (C) A549 cells were infected with Corby and mutant (MOI of 100). The number of bacteria at 0 h point is set as 1 and that at the indicated points is presented as the relative logCFU in cultures. Each data point represents the mean ± SD of triplicate cell cultures. *, < 0.05; **, < 0.01 (compared to cells infected with the wild-type strains)

Mechanisms of -induced interleukin-8 expression in human lung epithelial cells-6

<p><b>Copyright information:</b></p><p>Taken from "Mechanisms of -induced interleukin-8 expression in human lung epithelial cells"</p><p>http://www.biomedcentral.com/1471-2180/7/102</p><p>BMC Microbiology 2007;7():102-102.</p><p>Published online 22 Nov 2007</p><p>PMCID:PMC2213657.</p><p></p> evaluated by EMSA. Nuclear extracts from A549 and NCI-H292 cells, infected with AA100jm strain (MOI of 100), for the indicated time periods, were mixed with either NF-κB (upper panels) or AP-1 P-labeled probes (lower panels). Probes NF-κB and AP-1 are -83 to -68 bp and -130 to -116 bp fragments containing NF-κB and AP-1 binding sites of the IL-8 promoter, respectively. (B) Sequence specificity of NF-κB binding activity and characterization of NF-κB proteins that bound to the NF-κB binding site of the IL-8 gene. Competition assays were performed with nuclear extracts from A549 and NCI-H292 cells infected with AA100jm strain (MOI of 100) for 12 h. The competitor used was the NF-κB site of the IL-8 gene (lane 2). An unrelated AP-1 binding site was also used as a competitor (lane 3). Where indicated, 100-fold excess amounts of each specific competitor oligonucleotide (lanes 2 and 3) were added to the reaction mixture with labeled NF-κB probe. Specific bands are indicated by arrows. Supershift assay of NF-κB DNA-binding complexes in the same nuclear extracts was also performed. Gel shift assay reaction mixtures containing the same nuclear extracts and indicated antibodies were incubated for 45 min, and then P-labeled probes were added (lanes 4–8). Supershifted bands with anti-NF-κB p50, anti-NF-κB p65 and anti-c-Rel antibodies are indicated by arrowheads (lanes 4–6). (C) infection leads to IκBα phosphorylation and degradation. A549 cells were infected with AA100jm strain (MOI of 100), for the indicated time periods. The cells were then lysed and analyzed by immunoblot with phospho-specific IκBα, IκBα and actin antibodies. Representative results of three similar experiments in each panel are shown