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. 2015 Mar 15;308(6):L550-62.
doi: 10.1152/ajplung.00248.2014. Epub 2015 Jan 9.

Oxidized phospholipids protect against lung injury and endothelial barrier dysfunction caused by heat-inactivated Staphylococcus aureus

Angelo Y Meliton  1 , Fanyong Meng  1 , Yufeng Tian  1 , Nicolene Sarich  1 , Gokhan M Mutlu  1 , Anna A Birukova  2 , Konstantin G Birukov  1
Affiliations

Oxidized phospholipids protect against lung injury and endothelial barrier dysfunction caused by heat-inactivated Staphylococcus aureus

Angelo Y Meliton et al. Am J Physiol Lung Cell Mol Physiol. .

Abstract

Increased endothelial cell (EC) permeability and vascular inflammation along with alveolar epithelial damage are key features of acute lung injury (ALI). Products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine oxidation (OxPAPC) showed protective effects against inflammatory signaling and vascular EC barrier dysfunction induced by gram-negative bacterial wall lipopolysaccharide (LPS). We explored the more general protective effects of OxPAPC and investigated whether delayed posttreatment with OxPAPC boosts the recovery of lung inflammatory injury and EC barrier dysfunction triggered by intratracheal injection of heat-killed gram-positive Staphylococcus aureus (HKSA) bacteria. HKSA-induced pulmonary EC permeability, activation of p38 MAP kinase and NF-κB inflammatory cascades, secretion of IL-8 and soluble ICAM1, fibronectin deposition, and expression of adhesion molecules ICAM1 and VCAM1 by activated EC were significantly attenuated by cotreatment as well as posttreatment with OxPAPC up to 16 h after HKSA addition. Remarkably, posttreatment with OxPAPC up to 24 h post-HKSA challenge dramatically accelerated lung recovery by restoring lung barrier properties monitored by Evans blue extravasation and protein content in bronchoalveolar lavage (BAL) fluid and reducing inflammation reflected by decreased MIP-1, KC, TNF-α, IL-13 levels and neutrophil count in BAL samples. These studies demonstrate potent in vivo and in vitro protective effects of posttreatment with anti-inflammatory oxidized phospholipids in the model of ALI caused by HKSA. These results warrant further investigations into the potential use of OxPAPC compounds combined with antibiotic therapies as a treatment of sepsis and ALI induced by gram-positive bacterial pathogens.

Keywords: cytoskeleton; inflammation; oxidized phospholipids; pulmonary endothelium; vascular leak.

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Figures

Fig. 1.
Fig. 1.
Effects of heat-killed gram-positive Staphylococcus aureus (HKSA) on pulmonary endothelial cell (EC) permeability and F-actin remodeling. A: EC grown on microelectrodes to confluence were treated with HKSA (1 × 108, 2 × 108, 3 × 108, 5 × 108, or 10 × 108 bacterial particles/ml) and used for measurements of transendothelial electrical resistance (TER). B: EC grown in 96-well plates with immobilized biotinylated gelatin were treated with vehicle of HKSA (5 × 108 particles/ml) for indicated time periods followed by addition of FITC-avidin tracer in the last 5 min of incubation. Unbound FITC-avidin was removed, and FITC fluorescence was measured. Data are expressed as means ± SD of 4 independent experiments; *P < 0.05. XPerT, express micromolecule permeability testing assay. C: EC monolayers were treated with HKSA (5 × 108 particles/ml), and cytoskeletal remodeling was assessed by immunofluorescence staining for F-actin with Texas red phalloidin at 2, 6, or 24 h of HKSA stimulation. Paracellular gaps are shown by arrows. Bar graphs depict quantitative analysis of stress fibers and gap area in EC monolayers after incubation with HKSA. Data are expressed as means ± SD of 3 independent experiments; *P < 0.05.
Fig. 2.
Fig. 2.
Effects of HKSA on inflammatory signaling. A: human pulmonary artery endothelial cells (HPAEC) were challenged with HKSA (5 × 108 bacterial particles/ml) for indicated periods of time. Phosphorylation of p38 MAP kinase and degradation of IκBα was detected by immunoblotting with corresponding antibodies. Equal protein loading was confirmed by determination of β-tubulin content in total cell lysates. B: after EC treatment with HKSA, the content of NF-κB p65-subunit in nuclear (Nucl) and cytosolic (Cyto) fractions was performed using immunoblotting. The level of phosphorylated NF-κB p65 in the whole cell lysates (WCL) was monitored by Western blot with phospho-specific NF-κB p65 antibody. C: HKSA-induced nuclear translocation of NF-κB was visualized by immunofluorescence staining of EC culture with NF-κB antibody. D: time-dependent expression of ICAM1, VCAM1, and fibronectin (FN) caused by HKSA stimulation of pulmonary was monitored by immunoblotting. Equal protein loading was confirmed by determination of β-tubulin content in total cell lysates. The results of Western blot quantitative densitometry are presented as means ± SD of 5 independent experiments.
Fig. 3.
Fig. 3.
Effects of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine oxidation (OxPAPC) cotreatment on HKSA-induced EC permeability, F-actin, and adherens junction remodeling. A: pulmonary EC grown in 96-well plates with immobilized biotinylated gelatin were treated with HKSA (5 × 108 particles/ml) with or without cotreatment with OxPAPC or DMPC (15 μg/ml), and permeability for FITC-avidin was evaluated by fluorimetric XPerT assay described in methods. Data are expressed as means ± SD of 6 independent experiments; *P < 0.05. B: endothelial monolayers were challenged with HKSA (5 × 108 particles/ml) with or without OxPAPC cotreatment and incubated for 6 h. Cytoskeletal remodeling was assessed by immunofluorescence staining for F-actin with Texas red phalloidin. VE-cadherin was detected by staining with VE-cadherin antibody. Shown are merged images of F-actin (red) and VE-cadherin (green) staining. Bar graphs depict quantitative analysis of the area of VE-cadherin-positive adherens junctions and intercellular gaps in control and treated EC monolayers. Data are expressed as means ± SD of 3 independent experiments; *P < 0.05. Veh, vehicle.
Fig. 4.
Fig. 4.
Effects of OxPAPC cotreatment on HKSA-induced inflammatory signaling. A: pulmonary EC were challenged with HKSA (5 × 108 particles/ml) with or without cotreatment with OxPAPC, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), or dimirystoyl-sn-glycero-3-phosphorylcholine (DMPC) (15 μg/ml) and incubated for 2 or 24 h. Phosphorylation of p38 MAP kinase, degradation of IκBα, and expression of ICAM1 and VCAM1 were detected by immunoblotting with corresponding antibodies. Equal protein loading was verified by membrane probing with β-tubulin antibody. The results of Western blot quantitative densitometry are presented as means ± SD; n = 4. B: ICAM1 expression in HKSA-stimulated EC with and without OxPAPC cotreatment after 48 h of culture was visualized by immunofluorescence staining with ICAM1 antibody (green). Cell nuclei were visualized by DAPI staining (blue). C: fibronectin deposition by control stimulated EC after 48 h of culture was visualized by immunofluorescence staining of coverslips after cell detachment. D and E: EC were cultured for 24 h with vehicle, HKSA, with or without OxPAPC, PAPC, or DMPC. Secretion of soluble ICAM1 (D) or IL-8 (E) was evaluated by ELISA assay. Data are expressed as means ± SD of 5 independent experiments; *P < 0.05.
Fig. 5.
Fig. 5.
Time-dependent effects of OxPAPC posttreatment on HKSA-induced EC permeability and inflammatory activation. A: pulmonary EC were challenged with HKSA (5 × 108 particles/ml) with or without posttreatment with OxPAPC (15 μg/ml) at different times after HKSA, and TER was measured over 20 h. B: EC grown in on glass coverslips with immobilized biotinylated gelatin were stimulated with HKSA (5 × 108 particles/ml) followed by posttreatment with OxPAPC (6 h after HKSA challenge) and incubation during 24 h. After addition of FITC-avidin for 5 min at the end of experiment, unbound FITC-avidin was removed, and sites of increased EC monolayer permeability were visualized by FITC fluorescence. C: EC monolayers were treated with HKSA (5 × 108 particles/ml) with or without OxPAPC posttreatment (6 h), and cytoskeletal remodeling was assessed by immunofluorescence staining for F-actin with Texas red phalloidin after 24 h of HKSA stimulation. Bar graphs depict quantitative analysis of gap area in control and HKSA-challenged EC monolayers with or without OxPAPC posttreatment (6 h). Data are expressed as means ± SD of 3 independent experiments; *P < 0.05. D: EC challenged with HKSA (5 × 108 particles/ml) were posttreated with OxPAPC at different times after HKSA addition, and expression of ICAM1 and VCAM1 was monitored by immunoblotting. Probing for β-tubulin was used as a normalization control. E: secretion of sICAM1 was measured by ELISA assay of EC conditioned media. Data are expressed as means ± SD of 6 independent experiments; *P < 0.05.
Fig. 6.
Fig. 6.
Effects of OxPAPC posttreatment on HKSA-induced changes in bronchoalveolar lavage fluid (BALF) and MPO activity. A: C57BL/6J mice were treated with HKSA (2 × 108 cells/mouse it), and analysis of BALF cell counts and protein content was performed at different time points after HKSA challenge. Intratracheal injection of LPS (0.7 mg/kg) used in previous studies served as a reference control. B–D: after intratracheal injection of HKSA, 1.5 mg/kg OxPAPC was injected into jugular vein (i/v) or into retro-orbital sinus (r/o) after 5 h or 24 h of HKSA treatment. BALF protein content (B), BALF protein count (C) and lung tissue MPO activity (D) were measured as described in methods. Data are expressed as means ± SD of 4 independent experiments; *P < 0.05.
Fig. 7.
Fig. 7.
Effects of OxPAPC posttreatment on HKSA-induced tissue injury Evans blue extravasation and inflammatory markers. C57BL/6J mice were challenged with vehicle or HKSA (2 × 108 cells/mouse it) with or without OxPAPC posttreatment (1.5 mg/kg, 5 h after HKSA). Analysis of lung injury was performed 48 h after HKSA challenge. A: histological analysis of lung tissue by hematoxylin and eosin staining (×40 magnification). B: Evans blue dye (30 ml/kg iv) was injected 2 h before termination of the experiment. Lung vascular permeability was assessed by Evans blue accumulation in the lung tissue. The quantitative analysis of Evans blue-labeled albumin extravasation was performed by spectrophotometric analysis of Evans blue extracted from the lung tissue samples; *P < 0.05 vs. HKSA alone; n = 3. C: ICAM1 and VCAM1 protein expression in lung tissue samples was evaluated by immunoblotting analysis. Membrane probing with β-tubulin antibody was used as a normalization control. The results of Western blot quantitative densitometry are presented as means ± SD; n = 4. D: levels of mouse cytokines KC, MIP-1, TNF-α, and IL-13 were measured in BALF samples by ELISA assay. Data are expressed as means ± SD of 4 independent experiments; *P < 0.05.
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