Lipoteichoic Acid from <em>Staphylococcus aureus</em> Induces Lung Endothelial Cell Barrier Dysfunction: Role of Reactive Oxygen and Nitrogen Species

<div><p>Tunneled central venous catheters (TCVCs) are used for dialysis access in 82% of new hemodialysis patients and are rapidly colonized with Gram-positive organism (e.g. <em>Staphylococcus aureus</em>) biofilm, a source of recurrent infections and chronic inflammation. Lipoteichoic acid (LTA), a cell wall ribitol polymer from Gram-positive organisms, mediates inflammation through the Toll-like receptor 2 (TLR2). The effect of LTA on lung endothelial permeability is not known. We tested the hypothesis that LTA from <em>Staphylococcus aureus</em> induces alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM) that result from activation of TLR2 and are mediated by reactive oxygen/nitrogen species (RONS). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin, the activation of the TLR2 pathway was assessed by Western blot, and the generation of RONS was measured by the fluorescence of oxidized dihydroethidium and a dichlorofluorescein derivative. Treatment with LTA or the TLR2 agonist Pam₍₃₎CSK₍₄₎ induced significant increases in albumin permeability, IκBα phosphorylation, IRAK1 degradation, RONS generation, and endothelial nitric oxide synthase (eNOS) activation (as measured by the p-eNOS^ser1177:p-eNOS^thr495 ratio). The effects on permeability and RONS were effectively prevented by co-administration of the superoxide scavenger Tiron, the peroxynitrite scavenger Urate, or the eNOS inhibitor L-NAME and these effects as well as eNOS activation were reduced or prevented by pretreatment with an IRAK1/4 inhibitor. The results indicate that the activation of TLR2 and the generation of ROS/RNS mediates LTA-induced barrier dysfunction in PMEM.</p> </div

TLR2 agonist-induced IRAK activity increases endothelial nitric oxide synthase (eNOS) activity.

<p>PMEM pre-treated for 2 hours in the absence or presence of IRAK1/4 inhibitor (IRI: 10 µM) were then co-treated with LTA or PAM (both 10 µg/mL) for 0.5, 4, and 24 hours. (<i>a</i>) Representative Western blots for each treatment period of the eNOS activation site phosphorylation (p-eNOS^S1177, upper band) and the eNOS inhibitory site phosphorylation (p-eNOS^T495, lower band). The labels are: Control (C), LTA (L), PAM (P), IRI alone (I), IRI+LTA (IL), and IRI+PAM (IP). (<i>b</i>) Western blot band density ratios of p-eNOS^S1177/p-eNOS^T495 for all blots of all treatment groups at each time point represented in panel (a). Values represent means ± SEM (N ≥4). <b>*</b> p<0.03 vs. media alone, <b>#</b> p<0.03 vs. TLR2 agonist alone.</p

IRAK mediates eNOS activation and inhibitory site phosphorylation in LTA-treated PMEM.

<p>Western blots were generated from PMEM pre-treated for 2 hours in the absence or presence of increasing concentrations of IRAK1/4 inhibitor (IRI) and then co-treated with LTA (10 µg/mL) for 4 hours. (<i>a</i>) The Western blot band densities of phosphorylated serine 1177 of eNOS, its major activation site. (<i>b</i>) The Western blot band densities of phosphorylated threonine 495 of eNOS, a major inhibitory site. (<i>c</i>) The ratios of the band densities in panel (a) over those in panel (b). The units for panels a – c are relative density units (RDU). (<i>d</i>) Representative Western blots. Values represent means ± SEM (N ≥4). <b>*</b> p<0.03 vs. media alone, <b>#</b> p<0.03 vs. LTA alone.</p