Legionella pneumophila causes community-acquired pneumonia with high mortality, but little is known about its interaction with the alveolar epithelium. The aim of this study was to investigate whether L. pneumophila infection of lung epithelial cells (A549) resulted in proinflammatory activation.L. pneumophila infection induced liberation of interleukin (IL)-2, -4, -6, -8 and -17, monocyte chemoattractant protein-1, tumour necrosis factor-a, IL-1b, interferon-c and granulocyte colonystimulating factor, but not of IL-5, -7, -10, -12 (p70) or -13 or granulocyte-macrophage colonystimulating factor. The present study focused on IL-8 and found induction by L. pneumophila strains 130b, Philadelphia 1, Corby and, to a lesser extent, JR32. Knockout of dotA, a central gene involved in type IVB secretion, did not alter IL-8 induction, whereas lack of flagellin significantly reduced IL-8 release by Legionella. Moreover, p38 mitogen-activated protein kinase (MAPK) was activated and kinase inhibition reduced secretion of induced cytokines, with the exception of IL-2 and granulocyte colony-stimulating factor. In contrast, inhibition of the MAPK kinase 1/ extracellular signal-regulated kinase pathway only reduced the expression of a few cytokines. L. pneumophila also induced binding of nuclear factor-kB subunit RelA/p65 and RNA polymerase II to the il8 promoter, and a specific inhibitor of the inhibitor of nuclear factor-kB complex dosedependently lowered IL-8 expression.Taken together, Legionella pneumophila activated p38 mitogen-activated protein kinase-and nuclear factor-kB/RelA pathway-dependent expression of a complex pattern of cytokines by human alveolar epithelial cells, presumably contributing to the immune response in legionellosis.

Legionella pneumophila causes severe pneumonia. Acetylation of histones is thought to be an important regulator of gene transcription, but its impact on L. pneumophila-induced expression of proinflammatory cytokines is unknown. L. pneumophila strain 130b induced the expression of the important chemoattractant IL-8 and genome-wide histone modifications in human lung epithelial A549 cells. We analyzed the IL-8-promoter and found that histone H4 was acetylated and H3 was phosphorylated at Ser10 and acetylated at Lys14, followed by transcription factor NF-κB. Recruitment of RNA polymerase II to the IL-8 promoter corresponded with increases in gene transcription. Histone modification and IL-8 release were dependent on p38 kinase and NF-κB pathways. Legionella-induced IL-8 expression was decreased by histone acetylase (HAT) inhibitor anacardic acid and enhanced by histone deacetylase (HDAC) inhibitor trichostatin A. After Legionella infection, HATs p300 and CREB-binding protein were time-dependently recruited to the IL-8 promoter, whereas HDAC1 and HDAC5 first decreased and later reappeared at the promoter. Legionella specifically induced expression of HDAC5 but not of other HDACs in lung epithelial cells, but knockdown of HDAC1 or 5 did not alter IL-8 release. Furthermore, Legionella-induced cytokine release, promoter-specific histone modifications, and RNA polymerase II recruitment were reduced in infection with flagellin-deletion mutants. Legionella-induced histone modification as well as HAT-/HDAC-dependent IL-8 release could also be shown in primary lung epithelial cells. In summary, histone acetylation seems to be important for the regulation of proinflammatory gene expression in L. pneumophila infected lung epithelial cells. These pathways may contribute to the host response in Legionnaires’ disease.

Severe community-and hospital-acquired pneumonia is caused by Legionella pneumophila. Lung airway and alveolar epithelial cells comprise an important sentinel system in airborne infections. Although interleukin (IL)-6 is known as a central regulator of the immune response in pneumonia, its regulation in the lung is widely unknown.Herein, we demonstrate that different L. pneumophila strains induce delayed expression of IL-6 in comparison with IL-8 by human lung epithelial cells. IL-6 expression depended, at early time points, on flagellin recognition by Toll-like receptor (TLR)5, activity of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)1 and p38 mitogen-activated protein (MAP) kinase, and, at later time points, on the type-IV secretion system. In the same manner, but more rapidly, the recently described transcription factor IkBf was induced by Legionella infection and, binding to the nuclear factor (NF)-kB subunit p50 -recruited to the il6 promoter together with CCAAT-enhancer-binding protein b and phosphorylated activator protein-1 subunit cJun. Similarly, histone modifications and NF-kB subunit p65/RelA appeared at the ikbf and subsequently at the il6 gene promoter, thereby initiating gene expression. Gene silencing of IkBf reduced Legionella-related IL-6 expression by 41%.Overall, these data indicate a sequence of flagellin/TLR5-and type IV-dependent IkBf expression, recruitment of IkBf/p50 to the il6 promoter, chromatin remodelling and subsequent IL-6 transcription in L. pneumophila-infected lung epithelial cells.

Maturation of nerve growth factor (NGF) in neuronal cells requires endoproteolytic processing of the precursor protein proNGF to β-NGF by the proprotein convertase furin. Pro- and β-NGF elicit opposite biological functions by differential neurotrophin-receptor binding, leading to apoptosis via sortilin or survival via neurotrophic tyrosine kinase receptor type-1 (TrkA), respectively. The present study was done to investigate the impact of furin-dependent proNGF processing on vascular smooth muscle cell (VSMC) function. We found that β-NGF mRNA and protein expression was upregulated in platelet-derived growth factor-BB/transforming growth factor-β1-stimulated, proliferating rat aortic VSMCs. Although β-NGF itself did not affect VSMC proliferation, it promoted VSMC motility in an autocrine fashion via TrkA/Akt-dependent integrin inside-out signalling. The β-NGF-induced migration of VSMCs required proNGF processing by furin, which was co-regulated with NGF. Furin-inhibition increased proNGF and reduced β-NGF secretion, leading to apoptosis rather than migration. In line with our in vitro demonstration, we found co- and upregulation of NGF, its convertase furin and its high-affinity receptor TrkA in the neointima of balloon-injured rodent arteries. These results indicate that furin determines the balance between proNGF and β-NGF in proliferating VSMCs, thus impacting on VSMC survival and migration and is also important in neointima formation.

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