Lipoteichoic acid from Staphylococcus aureus induces lung endothelial cell barrier dysfunction: role of reactive oxygen and nitrogen species

Abstract

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. Staphylococcus aureus) 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 Staphylococcus aureus 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.

Figure 1. LTA- or PAM-induced activation of the TLR2 pathway in pulmonary microvessel endothelial monolayers (PMEM).

(a) Representative Western blot of TLR2 in PMEM after treatment with vehicle, TNFα 100 ng/mL, LTA from S. aureus and PAM (both 30 µg/mL) for 1 hour, confirming presence of receptors. (b) Representative Western blots of IRAK1, IRAK4, IκBα, and p-IκBα^Ser32/36 from Control (C) or 0.5, 4 and 24 hour LTA or PAM-treated (both 10 µg/mL) PMEM and (c–f) the blot band densities in Relative Density Units (RDU) for these proteins from all blots of similar treatments. Values represent means ± SEM (N ≥4). * p<0.05 vs. Control.

Figure 2. IRAK1/4 inhibition of the TLR2 pathway.

(a) Western blots of p-IκBα^Ser32/36 from 0.5, 4 and 24 hour Control (C), LTA (L) or PAM (P) -treated (both 10 µg/mL) PMEM in the absence (upper bands) or presence (lower bands) of IRAK1/4 inhibitor (IRI: 10 µM). (c–d) Western blot band densities in Relative Density Units (RDU) for p-IκBα^Ser32/36 from all blots represented by (a). Values represent means ± SEM (N ≥4). * p<0.003 vs. Control, # p<0.02 vs. TLR2 agonist alone.

Figure 3. TLR2 agonist-induced PMEM barrier dysfunction.

The PMEM clearance rate over 1 hour of Evans Blue-labeled albumin was measured in a transwell system. Cell monolayers were treated for 24 hours with LTA (30 µg/mL) or PAM (10 µg/mL) alone or co-treated with RNS scavenger, Urate (5 µM), ROS scavenger, Tiron (5 mM), or NOS inhibitor, L-NAME (100 µM) or co-treated for 24 hours following a 2 hour pretreatment with IRAK1/4 inhibitor, (IRI: 10 µM). Units are % change in µL/min from Control values (media alone). Values represent means ± SEM (N ≥4). * p<0.02 vs. media alone, # p<0.02 vs. TLR2 agonist alone.

Figure 4. TLR2 agonist-induced production of reactive oxygen/nitrogen species (RONS).

(a) Fluorescence of oxidized dihydroethidium (DHE) in cell sonicate after a 30 minute treatment of PMEM with LTA (30 µg/mL) or PAM (10 µg/mL) alone or co-treated with RNS scavenger, Urate (5 µM), ROS scavenger, Tiron (5 mM), or NOS inhibitor, L-NAME (100 µM). (b) Fluorescence of oxidized DCFDA in intact cell monolayers in 96-well plate following a 2 hour treatment with LTA or PAM alone (both 10 µg/mL) or co-treatment following a 2 hour pretreatment with IRAK1/4 inhibitor, (IRI: 10 µM). Units are % change in relative fluorescence units from Control values (media alone). Values represent means ± SEM (N ≥4). * p<0.01 vs. media alone, # p<0.04 vs. TLR2 agonist alone.

Figure 5. TLR2 agonist-induced increase in endothelial nitric oxide synthase (eNOS) activity.

An index of eNOS activity was calculated from the ratio of Western blot band densities of eNOS activation site phosphorylation over eNOS inhibitory site phosphorylation (phospho-eNOS (Ser1177)/phospho-eNOS (Thr495)). PMEM were treated for 0.5, 4 and 24 hours with LTA or PAM (both 10 µg/mL). Values represent means ± SEM (N ≥4). * p<0.05 vs. Control (media alone).

Figure 6. TLR2 agonist-induced IRAK activity increases endothelial nitric oxide synthase (eNOS) activity.

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. (a) 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). (b) 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). * p<0.03 vs. media alone, # p<0.03 vs. TLR2 agonist alone.

Figure 7. IRAK mediates eNOS activation and inhibitory site phosphorylation in LTA-treated PMEM.

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. (a) The Western blot band densities of phosphorylated serine 1177 of eNOS, its major activation site. (b) The Western blot band densities of phosphorylated threonine 495 of eNOS, a major inhibitory site. (c) 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). (d) Representative Western blots. Values represent means ± SEM (N ≥4). * p<0.03 vs. media alone, # p<0.03 vs. LTA alone.