Purchase this article with an account.
M. Ni, A. Verma, D.J. Evans, R. Ramphal, S. Hawgood, S.M. J. Fleiszig; Pseudomonas aeruginosa Induced Surfactant Protein D Upregulation by Human Corneal Epithelial Cells (HCEs) Is MyD88 Independent and Does Not Require the TLR5 Binding Site of Flagellin . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1887.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We have shown that SP–D can inhibit bacterial invasion of HCEs, that bacterial exposure upregulates SP–D expression by HCEs, and that both bacterial flagellin and LPS participate in that upregulation. Since flagellin and LPS are ligands for Toll like receptors (TLR), we tested the hypothesis that factors shown to be involved in TLR mediated responses participated in upregulation of SP–D by bacteria.
HCEs were exposed to heat killed P. aeruginosa (strain PAK) with or without inhibiting the activation of NF–kB (CAPE), p38 (SB203580), ERK (UO126), or JNK (SP600125). Transcripts for MyD88 were knocked down using siRNA. The effect on SP–D levels in cell lysates and secretions versus controls was examined using Western Blot. Bacteria expressing mutant flagellin lacking the ability to bind TLR5 due to a single amino acid mutation were compared to bacteria expressing normal flagellin. Recombinant wild type flagellin and the mutant flagellin were also compared for their effect on SP–D upregulation.
MyD88 knockdown reduced MyD88 protein expression without having significant impact on SP–D upregulation in response to bacterial challenge. Inhibition of JNK or ERK (but not of NF–kB or p38) significantly reduced P. aeruginosa induced SP–D upregulation. Bacteria expressing mutant flagellin defective in TLR5 binding induced an even stronger SP–D response than bacteria with normal flagellin. Experiments with the recombinant flagellin proteins confirmed that mutation of the TLR5 binding site did not reduce the capacity of flagellin to upregulate SP–D.
Upregulation of SP–D by P. aeruginosa may involve different mechanisms other than TLR5–flagellin–mediated upregulation of innate defenses that depend on NF–kB activation and MyD88. Since mutation of the TLR5 binding site of flagellin did not reduce capacity to upregulate SP–D, this data suggest that corneal epithelial cells may possess multiple receptors that recognize different domains on bacterial flagellins. Whether TLR5 mediated signaling actively inhibits SP–D production is yet to be determined.
This PDF is available to Subscribers Only