Endothelial ENaC-α Restrains Oxidative Stress in Lung Capillaries in Murine Pneumococcal Pneumonia-associated Acute Lung Injury

Abstract

Infection of lung endothelial cells with pneumococci activates the superoxide-generating enzyme NOX2 (nicotinamide adenine dinucleotide phosphate hydrogen [NADPH] oxidase 2), involving the pneumococcal virulence factor PLY (pneumolysin). Excessive NOX2 activity disturbs capillary barriers, but its global inhibition can impair bactericidal phagocyte activity during pneumococcal pneumonia. Depletion of the α subunit of ENaC (epithelial sodium channel) in pulmonary endothelial cells increases expression and PMA-induced activity of NOX2. Direct ENaC activation by TIP peptide improves capillary barrier function-measured by electrical cell substrate impedance sensing in endothelial monolayers and by Evans blue dye incorporation in mouse lungs-after infection with pneumococci. PLY-induced hyperpermeability in human lung microvascular endothelial cell monolayers is abrogated by both NOX2 inhibitor gp91dstat and TIP peptide. Endothelial NOX2 expression is assessed by increased surface membrane presence of phosphorylated p47^phox subunit (Western blotting) in vitro and by colocalization of CD31 and gp91^phox in mouse lung slices using DuoLink, whereas NOX2-generated superoxide is measured by chemiluminescence. TIP peptide blunts PMA-induced NOX2 activity in cells expressing ENaC-α, but not in neutrophils, which lack ENaC. Conditional endothelial ENaC-α knockout (enENaC-α knockout) mice develop increased capillary leak upon intratracheal instillation with PLY or pneumococci, compared with wild-type animals. TIP peptide diminishes capillary leak in Streptococcus pneumoniae-infected wild-type mice, without significantly increasing lung bacterial load. Lung slices from S. pneumoniae-infected enENaC-α knockout mice have significantly increased endothelial NOX2 expression, compared with infected cyclization recombination mice. In conclusion, enENaC may represent a novel therapeutic target to reduce NOX2-mediated oxidative stress and capillary leak in acute respiratory distress syndrome, without impairing host defense.

Keywords: NOX2; acute respiratory distress syndrome; capillary leak; endothelial ENaC; pneumococcal pneumonia.

Figure 1.

Human lung microvascular endothelial cell (HL-MVEC) ENaC (epithelial sodium channel) expression and open probability (Po) is activated by TIP peptide and inhibited by PLY (pneumolysin). (A) Typical record showing many low conductance (5 pS) channels (ENaC, a.k.a. highly selective channel) in HL-MVECs. (B) Current voltage (I-V) plot of ENaC/highly selective channel in HL-MVECs fitted to the Goldman equation from SigmaPlot 14. (C) ENaC response in HL-MVECs to two consecutive applications of TIP peptide (5 pS currents). (D) TIP peptide increases ENaC Po in HL-MVECs (P < 0.001 vs. untreated; n = 30). (E) ENaC current trace in HL-MVECs demonstrating that the application of PLY (30 ng/ml) potently inhibits ENaC activity within minutes. (F) PLY inhibits ENaC Po within 10 minutes in HL-MVECs (n = 5; P < 0.01 vs. untreated). (G) TIP peptide partially preserves ENaC-α acetylation in PLY-treated HL-MVECs. Confluent monolayers of HL-MVECs (one 100-mm dish per condition) were treated for 30 minutes with vehicle (CTRL), PLY (30 ng/ml), or PLY in the presence of TIP peptide (50 μg/ml). After 30 minutes, acetylated lysine residues within ENaC-α were assessed upon co-IP with anti–ENaC-α antibody (Ab) (Novus) and anti-acetylated lysine Ab (Cell Signaling). (H) Relative expression of acetylated ENaC-α in vehicle-, PLY-, or (TIP + PLY)-treated HL-MVECs. Ctrl = control; co-IP = co-immunoprecipitation (co-IP).

Figure 2.

NOX2 (nicotinamide adenine dinucleotide phosphate hydrogen [NADPH] oxidase 2) inhibition blunts PLY-induced hyperpermeability in HL-MVEC monolayers. HL-MVEC grown to confluence on ECIS arrays (ECIS 1600R, Applied Biophysics; initial resistance between 1,800 and 2,000 Ohms) were treated with (A) 15 ng/ml PLY (low concentration), or (B) 60 ng/ml PLY (high concentration) for 8 hours, in the presence or absence of vehicle or the NOX2 inhibitor peptide gp91dstat (10 μM, applied 30 min before, or, in the case of the higher concentration, 30 min after PLY) (n = 4 per group; mean ± SD; *P < 0.05 between PLY +  gp91dstat vs. PLY for the time period indicated by the bold line).

Figure 3.

Assessment of PLY-induced mitochondrial reactive oxygen species (ROS) production. (A) Representative confocal images of MitoSox Red staining in HL-MVECs treated for 15 minutes with PLY (30 ng/ml), upon a 30-minute pretreatment or not with TIP peptide (50 μg/ml). Scale bars, 100 μm. (B) Data points in the graph indicate the intensity of red fluorescence staining with MitoSOX normalized to the number of DAPI-positive nuclei (n = 3 wells for each treatment). ImageJ was used for image analysis and data graphed using GraphPad Prism. Mean and error bars indicating the SD are included in the dot blot image. Statistical analysis was performed using t test. *P < 0.0004 versus Ctrl. **P < 0.02 versus PLY.

Figure 4.

Absence of ENaC-α increases NOX2 expression in mouse and human lung endothelial cells. (A) Left: Representative Western blot (10% agarose gel) of basal expression of ENaC-α in mouse lung endothelial cells isolated from control cyclization recombination (CRE) mice (CRE) or EC-specific ENaC-α knockout (EC ENaC-α KO) mice. Right: ENaC-α to β-actin expression ratio is blunted in endothelial cells from ENaC-α KO mouse, compared with endothelial cells from CRE mice (***P < 0.01, compared with CRE group; n = 4–5 in each group). (B) Left: Representative Western blot (10% agarose gel) of basal expression of NOX2 in isolated lung endothelial cells from control CRE mice (CRE) or EC-specific ENaC-α knockout (EC ENaC-α KO) mice. Right: Ratio of NOX2 over β -actin expression is markedly increased in endothelial cells from the ENaC-α KO group, compared with those from the CRE group (***P < 0.01, compared with the CRE control group; n = 4–5 in each group). (C) Left: Representative Western blot of basal expression of ENaC-α in HL-MVECs. Right: ENaC-α depletion using siRNA ENaC-α (72 h, StressMarq rabbit polyclonal antibody) (***P < 0.001, compared with CTL group; n = 4 in each group). (D) Left: Representative western blotting (10% agarose gel) of basal expression of NOX2 in HL-MVECs. Cells were transfected at 70–80% confluence with transfection reagent (Lipofectamine RNAiMAX, Invitrogen) with siRNA control (CTL) or siRNA ENaC-α. Right: Depletion of ENaC-α markedly increases NOX2 expression on HL-MVECs at 72 hours after transfection (**P < 0.01, compared with CTL group; n = 4 in each group).

Figure 5.

Co-IP of ENaC-α and NOX2 in lung lysate (3 h and overnight precipitation with Protein A beads). (A) Total lung tissue (8- to 10-week-old female C57Bl6 mice). ENaC-α pAb-treated lysate (10 μg; Stressmarq SPC-403) was precipitated with Invitrogen Ag Dynabeads. Lane 1: 30 μg lung lysate incubated with antibody overnight before precipitation with Ag beads; lane 2: 30 μg lung lysate incubated with antibody overnight before precipitation with Protein A beads; lane 3: supernatant from #1 after binding to beads (unbound target protein); lane 4: supernatant from #2 after binding to beads (unbound target protein); lane 5: whole lysate used for #1; lane 6: whole lysate used for #2. Upper panel: blot was detected with ENaC-α rabbit polyclonal Ab (Stressmarq) to determine the efficiency of the immunoprecipitation. Lower panel: blot with anti-NOX2 (gp91^phox Ab) to determine ENaC–α-NOX2 coprecipitation. (B) Efficacy of immunoprecipitation. The density of the ENaC-α and NOX2 bands in the immunoprecipitate (lanes 1 and 2) and the supernatant (lanes 3 and 4) was quantified for four blots like those shown in A, using the Fiji variant of ImageJ. The ratios of density of the immunoprecipitate to supernatant are shown in the graph (mean ± SD; n = 4). The efficacy of the ENaC-α antibody is good, but, as expected, the coimmunoprecipitation of NOX2 is less efficient but still recovers as much from the lysate as is left in the supernatant. This could also be interpreted to mean that not every ENaC-α has a NOX2 associated with it.

Figure 6.

TIP peptide represses NOX2-mediated ROS generation in cells expressing ENaC-α. (A) Increased ROS generation in PMA-treated lung endothelial cells from endothelial ENaC-α KO mice (KO), compared with endothelial cells from wild-type (WT) control mice. ROS generation was blunted by addition of superoxide dismutase (SOD) (n = 8 per group; P < 0.01 for all time points between 20 and 70 min after start of experiment). (B) Upper panel: Representative western blotting of HL-MVEC membrane/organelle fractions demonstrating that PMA (1 μM) within 15 minutes induces p47^phox phosphorylation at Ser359 in HL-MVECs, and this leads to recruitment of the subunit from the cytosol to the surface membrane. Lower panel: Densitometric analysis calibrating protein levels to AIF (Apoptosis Inducing Factor), a protein marker expressed exclusively in membranes/organelle fractions, demonstrating that TIP peptide significantly blunts p47 phosphorylation in the presence of PMA (n = 3; ***P < 0.00001 vs. vehicle ctrl; **P < 0.0001 vs. PMA; PMA + TIP: not significant [NS] vs. vehicle). (C) PMA significantly stimulates ROS generation in Cos p22^phox cells, which express only NOX2 subunits, and the positive control DPI (an inhibitor of NADPH oxidase) blocks this completely. ENaC-α activation with TIP peptide (−30 min) significantly blunts PMA-induced ROS generation at 60 minutes in these cells (*P < 0.05 between PMA and PMA + TIP; n = 6 per group; PMA: 1 μM; DPI: 5 μM; TIP peptide 50 μg/ml). Cos p22^phox cells express ENaC-α (inset). (D) TIP peptide does not inhibit PMA-induced ROS generation in mouse PMNs, which do not express ENaC-α (inset). PMNs are pretreated for 20 minutes with TIP peptide (50 μg/ml) or with vehicle, before addition of PMA (50 nM). PMA significantly increases ROS generation, and TIP peptide does not inhibit this (n = 8 per group). Although TIP peptide did not decrease PMA-induced ROS generation in PMNs, it significantly increased it after PMA stimulation for the time points between 14 and 80 minutes (P < 0.05). (E) Neither TIP peptide (50 μg/ml) nor the indirect ENaC activator aldosterone (1 μM) is able to inhibit PMA (1 μM)-induced ROS generation in THP-1 macrophages at 180 minutes, whereas SOD (150 U/ml) does (n = 6 per group; ****P < 0.001 vs. vehicle or, in case of SOD, vs. PMA).

Figure 7.

Endothelial ENaC-α (enENaC-α) expression strengthens barrier function in pneumococcal pneumonia. (A) 8- to 10-week-old male enENaC-α KO mice develop significantly more capillary hyperpermeability 24 later upon intratracheal instillation of a moderate PLY dose (1.5 µg/kg) than age-matched control CRE mice (n = 3–5 per group; **P < 0.02 vs. vehicle Ctrl; ***P < 0.05 vs. CRE + PLY). (B) TIP peptide (2.5 mg/kg) significantly protects from capillary leak induced by intratracheal instillation of 10⁷ CFU of Streptococcus pneumoniae (Sp) in 8- to 10-week-old male WT C57BL6 mice (n = 4 per group; *P < 0.01 vs. vehicle and **P < 0.04 vs. Sp). (C) Infection with 2 × 10⁶ CFU Sp induces higher capillary leak in enENaC-α KO mice than in control CRE mice (n = 3–6 per group; *P < 0.03 vs. vehicle control and **P < 0.05 vs. CRE + Sp). (D) TIP peptide does not protect from Sp-induced capillary leak in enENaC-α KO mice. enENaC-α KO mice develop significant capillary leak 24 hours later intratracheal instillation of 2 × 10⁶ CFU Sp compared with enENaC-α KO control mice instilled with saline. Treatment with TIP peptide (2.5 mg/kg) did not significantly differ from vehicle-treated infected mice (n = 3–5 per group; *P < 0.003 vs. ctrl). (E) Measurement of cytokine/chemokine expression in lung homogenates (mean ± SD) (MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel–Immunology Multiplex Assay, EMD Millipore). Compared with vehicle control, 8- to 10-week-old male C57BL6 mice (n = 4 per group), mice instilled with 10⁷ CFU Sp have a significantly higher expression of TNF (**P < 0.004 vs. ctrl), keratinocyte-derived chemokine (KC) (**P < 0.006 vs. ctrl), and IL-6 (**P < 0.002 vs. ctrl) at 24 hours in lung homogenates, whereas levels of IL-10 were not significantly increased (n = 4 per group). Coinstillation of TIP peptide does not significantly reduce expression of TNF (**P < 0.003 vs. ctrl; ^#NS vs. Sp) or KC (*P < 0.02 vs. ctrl; ^#NS vs. Sp), but it does partially, but significantly, inhibit IL-6 expression (**P < 0.03 vs. ctrl; *P < 0.05 vs. ctrl; ^#P < 0.01 vs. Sp; n = 5 per group). CFU = colony-forming unit; NS = not significant.

Figure 8.

(A–F) Representative images of lung sections from enENaC-α KO and Cre-driver mice infected with Sp via intratracheal instillation. Vehicle saline was used instead of bacteria in Cre-driver control mice (Saline Ctrl, A and B). Red immunofluorescence staining demonstrates successful binding of both gp91^phox and CD31 antibodies in close proximity (⩽40 nm) in the lung sections. Nuclei are stained with DAPI (blue). Scale bars, 2 μm. (G) Number of areas of positive proximity ligation assay signal (red) per field in different regions of mouse lung tissue. Mean and error bars indicating the SD are included in the dot blot image. Statistical analysis was performed using t test. *P < 0.000001 vs. vehicle control. **P < 0.00000003 vs. saline control. ^#P < 0.0000001 vs. Sp CRE.