Lipidomic Profiling of Bronchoalveolar Lavage Fluid Extracellular Vesicles Indicates Their Involvement in Lipopolysaccharide-Induced Acute Lung Injury

Abstract

Emerging data support the pivotal role of extracellular vesicles (EVs) in normal cellular physiology and disease conditions. However, despite their abundance, there is much less information about the lipid mediators carried in EVs, especially in the context of acute lung injury (ALI). Our data demonstrate that C57BL/6 mice subjected to intranasal Escherichia coli lipopolysaccharide (LPS)-induced ALI release, a higher number of EVs into the alveolar space, compared to saline-treated controls. EVs released during ALI originated from alveolar epithelial cells, macrophages, and neutrophils and carry a diverse array of lipid mediators derived from ω-3 and ω-6 polyunsaturated fatty acids (PUFA). The eicosanoids in EVs correlated with cellular levels of arachidonic acid, expression of cytosolic phospholipase A2, cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome epoxygenase p450 proteins in pulmonary macrophages. Furthermore, EVs from LPS-toll-like receptor 4 knockout (TLR4-/-) mice contained significantly lower amounts of COX and LOX catalyzed eicosanoids and ω-3 PUFA metabolites. More importantly, EVs from LPS-treated wild-type mice increased TNF-α release by macrophages and reduced alveolar epithelial monolayer barrier integrity compared to EVs from LPS-treated TLR4-/- mice. In summary, our study demonstrates for the first time that the EV carried PUFA metabolite profile in part depends on the inflammatory status of the lung macrophages and modulates pulmonary macrophage and alveolar epithelial cell function during LPS-induced ALI.

Keywords: Acute lung injury; Extracellular vesicles; Inflammation; Macrophage; Polyunsaturated fatty acids.

Fig. 1

Characterization of EVs from mouse BALF. BALF was subjected to sequential centrifugation to separate cells, apoptotic bodies, MVs, and EVs. aSize distribution of vesicles in total BALF, MVs, EVs, and EV-depleted supernatant was assayed by NTA. bDifference in release of EVs into BALF from control (saline) and LPS-treated mice. cDetermination of EV size by transmission electron microscopy. dExpression of EV marker proteins CD9, CD63 in EVs. eTNF-α levels in EV-depleted BALF in saline and LPS treatment after 6 h, 3, and 15 days. fComparison of phospholipid content of MVs, EVs, and EV-depleted BALF. The error bars represent the mean ± SEM from five mice in each group. Statistical difference between different treatments calculated by one-way ANOVA with Bonferroni correction. b*p ≤ 0.05 LPS versus saline control (e), ***/•••p ≤ 0.001 LPS versus saline control. f***p ≤ 0.001 EV or MV versus EV-depleted BALF. NTA, nanoparticle tracking analysis.

Fig. 2

BALF EVs from LPS-treated mice carry a diverse array of ω-6 and ω-3 PUFA metabolites. WT mice were treated with LPS or saline (i.n.), and BALF was collected after 6 h, 3, and 15 days later. EVs from BALF of individual animals (n = 4) were isolated by ultracentrifugation and confirmed by size determination. a, bThe relative amount of AA, EPA, DHA, and LA-derived metabolites from enzymatic action of COX, LOX, CYP450, and nonenzymatic lipid peroxidation were determined by LC-MS. Heat map constructed using Graphpad Prism v9.0. Scale bar represents picogram of metabolite per milligram of EV protein. Samples with no detectable metabolites are in white.

Fig. 3

TLR4^−/− mice are resistant to LPS-induced ALI, release lower number of macrophage and neutrophil-derived EVs into BALF. WT and TLR4^−/− mice were treated with LPS or saline (i.n.). After 3 days of LPS challenge mice were euthanized, and lung injury severity was determined by measuring BALF levels of IL6 (a), TNF-α (b), extravasated protein (c), and lung wet to dry ratios (d). BALF EVs were attached to CD9-Exo-Flow capture beads, stained with F4/80-APC, CD31-FITC, CD326-PE, Ly6G-BV421, and analyzed on BD LSR Fortessa Flow Cytometer (e). Data were analyzed using FlowJo v10.8.1. The total number of BALF EVs compared between WT and TLR4^−/− mice, subjected to control or LPS-induced ALI (f). Lung sections of WT and TLR4^−/− mice subjected to control or LPS-induced ALI were stained with H & E. Representative sections showing lung injury and neutrophilic infiltration (g). a–dError bars represent mean ± SEM from 5 mice in each group. Statistical difference calculated by one-way ANOVA with Bonferroni correction. a, b, d***p ≤ 0.001 WT LPS versus WT-saline control; **p ≤ 0.01 WT-LPS versus TLR4^−/−LPS. c**p ≤ 0.01 WT-LPS versus WT-saline control, *p ≤ 0.05 WT-LPS versus TLR4^−/− LPS.

Fig. 4

BALF EVs from TLR4^−/− mice are devoid of AA-derived eicosanoids. EVs were isolated from saline or LPS-treated WT and TLR4^−/− mice and analyzed by LC-MS. COX metabolites in EVs from saline or LPS-treated mice (a). Similarly, LOX metabolites were determined in EVs isolated from saline or LPS-treated WT and TLR4^−/− mice, showing levels of 5-HETE (b), 12-HETE (c), 15-HETE (d), and LXA4 (e). n = 4 mice in each group, EVs from each mouse processed separately. Heat map constructed using Graphpad Prism v9.0 and statistical differences calculated by one-way ANOVA with Bonferroni correction. b–e***p ≤ 0.001 WT LPS versus WT-saline control; •••p ≤ 0.001 WT-LPS versus TLR4^−/−LPS. Scale bar represents picogram of metabolite per milligram of EV protein. Samples with no detectable metabolites are in white. 5-HETE, 5-hydroxyeicosatetraenoic acid; 12-HETE, 12-hydroxyeicosatetraenoic acid; 15-HETE, 15-hydroxyeicosatetraenoic acid; LXA4, lipoxin A4.

Fig. 5

BALF EVs from TLR4^−/− mice are devoid of DHA and EPA-derived lipid mediators. EVs were isolated from saline or LPS-treated WT and TLR4^−/− mice and analyzed for the presence of DHA auto-oxidation metabolites and DHA-derived resolvins. Levels of 4-HDoHE (a), 10-HDoHE (b), 20-HDoHE (c). Heat map showing relative levels of Resolvin D6, 8-oxoResolvin D1, and Maresin 1 (d). EPA-derived 12-HEPE (e). n = 4 mice in each group, EVs from each mouse processed separately. Statistical difference calculated by one-way ANOVA with Bonferroni correction. a–e***p ≤ 0.001 WT LPS versus WT-saline control; •••p ≤ 0.001 WT-LPS versus TLR4^-/−LPS. Scale bar represents picogram of metabolite per milligram of EV protein. Samples with no detectable metabolites are in white. 4-HdoHE, 4-Hydroxydocosahexaenoic acid; 10-HdoHE, 10-Hydroxydocosahexaenoic acid; 20-HdoHE, 20-Hydroxydocosahexaenoic acid; 12-HEPE, 12-hydroxyeicosapentaenoic acid.

Fig. 6

Eicosanoids in EVs correlate with expression levels FA metabolic proteins and ω-6 and ω-3 fatty acid composition of lung macrophages. Total lung macrophages were isolated from saline and LPS-treated WT and TLR4^−/− mice. aSpecific activity of cPLA2 measured by hydrolysis of μmol of Arachidonoyl Thio-PC hydrolyzed per min per mg cellular protein. bExpression levels of cPLA2, COX-1, COX-2, 5-LOX, 12-LOX, CYP450, and β-actin and arbitrary densitometric units of COX-1, COX-2, 5-LOX, 12/15-LOX, CYP450, and β-actin averaged from different bots, n = 8 mice in each group, each well corresponds to lung macrophages pooled from two mice. In addition, total lipids were extracted from saline and LPS-treated mouse lung macrophages and analyzed by HPLC, (c) % AA (d) % DHA (e) % EPA. Statistical difference calculated by one-way ANOVA with Bonferroni correction. a, c, e***p ≤ 0.001 WT LPS versus WT-saline control, •••p ≤ 0.001 WT-LPS versus TLR4^−/−LPS.

Fig. 7

BALF EVs alter alveolar epithelial and pulmonary macrophage function. aAlveolar epithelial cells andblung macrophages were grown in the presence of PKH26-labeled EVs from WT, TLR4^−/−, solvent control (Cont), Cytochalasin (CD −10 μg/mL) or Nystatin (50 μg/mL) for 16 h and washed with 1X HBSS as described in methods. EV uptake was measured as RFU. cEVs from WT and TLR4^−/− mice, and 10 kDa FITC-dextran were added to 100% confluent monolayer of alveolar epithelial cells grown on transwells. After 16 h, flux of FITC-dextran to the bottom wells of the cell culture plate was measured at 492/520 nm. dEVs were added to lung macrophages grown in 96-well plates, and after 16 h, the release of TNF-α into the cell culture medium was measured. eEndotoxin contamination in EV preparation was determined by the Pierce Endotoxin Detection kit. f13-HODE (100–1,000 nM) and 10 kDa FITC-dextran were added to 100% confluent monolayer of alveolar epithelial cells grown on transwells. After 16 h, flux of FITC-dextran to the bottom wells of the cell culture plate was measured at 492/520 nm. Statistical difference calculated by one-way ANOVA with Bonferroni correction. a, b*p ≤ 0.01 CD or Nystatin uptake versus Solvent control uptake of WT or TLR4^−/− EVs. c, d**p ≤ 0.01 WT LPS EVs versus WT-saline EVs, *p ≤ 0.05 WT-LPS versus TLR4^−/−LPS EVs, ^#p ≤ 0.05 WT-LPS EVs versus WT-LPS + Nystatin, and x p ≤ 0.05 WT-LPS EVs versus WT-LPS + CD pretreated alveolar epithelial cells. f****p ≤ 0.0001 13-HODE-treated alveolar epithelial cells versus vehicle-treated cells.