Dynamic regulation of cardiolipin by the lipid pump Atp8b1 determines the severity of lung injury in experimental pneumonia

Abstract

Pneumonia remains the leading cause of death from infection in the US, yet fundamentally new conceptual models underlying its pathogenesis have not emerged. We show that humans and mice with bacterial pneumonia have markedly elevated amounts of cardiolipin, a rare, mitochondrial-specific phospholipid, in lung fluid and find that it potently disrupts surfactant function. Intratracheal cardiolipin administration in mice recapitulates the clinical phenotype of pneumonia, including impaired lung mechanics, modulation of cell survival and cytokine networks and lung consolidation. We have identified and characterized the activity of a unique cardiolipin transporter, the P-type ATPase transmembrane lipid pump Atp8b1, a mutant version of which is associated with severe pneumonia in humans and mice. Atp8b1 bound and internalized cardiolipin from extracellular fluid via a basic residue-enriched motif. Administration of a peptide encompassing the cardiolipin binding motif or Atp8b1 gene transfer in mice lessened bacteria-induced lung injury and improved survival. The results unveil a new paradigm whereby Atp8b1 is a cardiolipin importer whose capacity to remove cardiolipin from lung fluid is exceeded during inflammation or when Atp8b1 is defective. This discovery opens the door for new therapeutic strategies directed at modulating the abundance or molecular interactions of cardiolipin in pneumonia.

Figure 1. Quantification of cardiolipin (CL) in pneumonia

a. Panel a depicts the median (gray line) and distribution (black circles) of CL levels in tracheal aspirates from subjects with non-pulmonary critical illness (NPCI, n=five), pneumonia (PNA, n=17), and congestive heart failure (CHF, n=six). Groups were compared by Kruskal Wallis test (P=0.0007) followed by post hoc Wilcoxon Rank Sum analysis with Bonferroni correction for multiple testing b. C57BL/6 mice (three control, five H. influenzae, four E.coli) were infected intratracheally (i.t.) with E. coli (1 ×10⁶ CFU/mouse) or H. influenzae (2 × 10⁸ CFU/mouse). Mice were euthanized 48 h (E. coli) or 72 h (H. influenzae) later, lungs lavaged, and processed for CL assay. Inset: Mice (six/group) were given HCL (pH 1.5, 2 ml/kg i.t.) prior to being euthanized 30 min later for analysis of CL. c. Primary type II lung epithelia (from n=ten mice) were cultured with [³H]-CL containing the commercial surfactant, Infasurf, for 2 h at 37 °C in the presence or absence of E. coli (MOI=100) or H. influenzae (MOI=10) and cellular CL uptake was determined. In (b-c), ^** represents means ± S.D. with P<0.01, and ^***represents P<0.001 vs. control as determined by one-way ANOVA analysis.

Figure 2. Biophysical effects of CL. a-b. Surface-tension

Cardiolipin (CL), lysophosphatidylcholine (LPC), and phosphatidylserine (PS) were reconstituted with Infasurf (liposomes in CaCl₂ (5 mM)) with apoprotein (a) or without apoprotein (b) and dynamic surface tension (γ_min) was measured using a pulsating bubble surfactometer. Increasing amounts of CL in Infasurf resulted in significantly increased surface tension compared to LPC or PS (magnified in inset). c-f. Mice (three-five/group) were anesthetized, paralyzed, and mechanically ventilated with a PEEP=3 and compliance (c), quasi-static pressure-volume curves (d), elastance (e), and resistance (f) was determined using a FlexiVent system after i.t. application of phosphatidylglycerol (PG) or CL. Significance was determined by a one-way ANOVA analysis where ^*P<0.05, ^*P<0.01, ^***P<0.001 vs. control.

Figure 3. CL disrupts lung structure and alters epithelial cell viability

a. MicroCT scan images were obtained on live mice (in vivo) 1 h after i.t. administration of CL (50 nmole, [low], 100 nmole [high]) vs. control mice (above panels). In separate studies, mice were given CL as above and lungs were fixed and processed for microCT scanning (ex vivo). Data represents two mice/group. Below, third row: fixed tissue was processed for H & E staining (20× magnification). Lower row (middle) shows a high magnification (100×) image of foamy cells. b-c. CL induces lung edema. Mice were deeply anesthetized and administered CL (15 mM in 50 μl saline, i.t.; n = three) or vehicle (50 μl saline; n = 3). 30 min after CL administration Evan’s Blue dye was injected intravenously. Wet/dry weights of lungs were also determined (c). ^***P<0.01 vs. control. d. Primary mouse type II cells were serum starved for 24 h and then exposed to Infasurf (120 nmol/ml) or CL (5-15 Mol%) for various times prior to harvest for detection of poly(ADP-ribose) polymerase (PARP) cleavage (d, arrows, left panel); mice were also given i.t. CL (50 nmol) for analysis of lung DNA fragmentation using a DNA ladder extraction kit ([BioVision], d, right panel) or assayed for cell viability (e) and LDH release (f, primary type II cells). NP-40 was used as a positive control for LDH release. Data represents three separate experiments where ^*P<0.05, ^**P<0.01, and ^***P<0.001 vs. control as determined by one-way ANOVA analysis.

Figure 4. ATP8b1 overexpression

a-c. A lentiviral transduced V5-ATP8b1 stable cell line was labeled with NBD-CL or NBD-PS and cells were processed for fluorescence uptake by time-lapse microscopy. The lentiviral transduced ATP8b1 cell line exhibited increased uptake of NBD-labeled CL and NBD-labeled PS compared to untransduced control cells as quantitated by fluorescence spectrophotometry in (b). ^**P<0.05 and ^*** P<0.001 by an unpaired t test. c-d. Mice (eight/group) received an empty vector (Ad5) or Ad5-ATP8b1 (2.5 ×10⁸ PFU) i.t. and 24 h later given E. coli at 10⁶ CFU/mouse for 48 h. Animals were anesthetized, paralyzed and mechanically ventilated and lung mechanics determined as in Fig. 2. The data in (c) top panel is a representative immunoblot showing levels of V5-immunoreactive ATP8b1 in lung tissue from two mice receiving Ad5 or Ad5-ATP8b1. In (d) CL levels were assayed in BAL after Ad5-ATP8b1 or Ad5 infection. Significance was determined by a one-way ANOVA where in (c) and (d) ^*P<0.05 for empty + E. coli vs. other groups. e. Mice treated as in (c) were also analyzed for lung DNA fragmentation. f. Kaplan-Meier survival curve for mice infected with Ad5 empty or Ad5-ATP8b1 and infected with E. coli. (5 × 10⁶ cfu/mouse, n=14 mice/group, P<0.001, log rank test).

Figure 5. ATP8b1 defective mice are prone to bacterial-induced lung injury

a. ATP8b1 siRNA knockdown. Human A549 ATII cells were transfected with ATP8b1 siRNA or control RNA and incubated with NBD-labeled CL or PS for 30 min at 37 °C prior to harvest for uptake (left) and immunoblotting (right) using 25 μg of protein loaded/lane. Blots were probed with ATP8b1, LPCAT1, Erk1, importin-α, and β-actin antibodies. ^**P<0.01. b. ATP8b1^G308V/G308V mutants genomic DNA has a Hae III restriction site (GGCC) in which the second G is mutated to T (glycine to valine) and is not recognized in restriction digests. Mutant genomic DNA generates an 800 bp fragment upon HaeIII digestion compared to a 500 bp fragment with wild-type genomic DNA. The HaeIII digestion pattern was used for genotyping wild-type (Wt), heterozygous (Het), and mutant (Mut) mice. c. ATP8b1 immunoblotting in mouse tissues. Below: densitometric analysis from n=six mice/group. d. CL was assayed in lung lavage from ATP8b1 mutant and wild-type littermates (three/group). ^**P<0.01. e. Primary type II epithelia isolated from mutants or wild-type littermates (five mice/group) were incubated with NBD-CL and cellular uptake was assayed initially after labeling (t=0) and after 1 min. f-g. Mutants and wild-type littermates uninfected or infected (seven/group) with E. coli at 10⁶ CFU/mouse for 48 h were analyzed for lung mechanics. Mice were mechanically ventilated for determination of quasi-static volume-pressure curves (f, [uninfected]), and elastance (g). Statistical significance was determined by a one-way ANOVA where in (g) ^** WT vs. MUT + E.coli, P<0.01 and ^* WT+ E. coli vs. MUT+ E. coli, P<0.05. h. Wild type and mutant mice treated as in (g) were also analyzed for lung DNA fragmentation. i. Kaplan-Meier survival curve for wild-type and ATP8b1 defective mice infected with E. coli, (5 × 10⁶ CFU/mouse, seven mice/group, P=0.11, log rank test).

Figure 6. Cardiolipin binding domain (CBD) peptide blocks bacterial lung injury

a. ATP8b1 CBD-GST fusion peptide (CBD) or GST (control) were purified and incubated with MLE cells (10 μg of GST peptide or GST-CBD peptide/dish) in serum-free medium after labeling for 2 h with [³H]-CL. Cells were harvested and processed for cellular [³H]-CL uptake, ^**P<0.01. b-d. C57/BL6 mice (six mice/group) were uninfected (control) or given E. coli at 10⁶ CFU/mouse. After 48 h, mice were given vehicle, or CBD peptide into lungs using an aerosolizer. After 10 min, biophysical measurements were taken. Mice were mechanically ventilated for determination of quasi-static volume-pressure curves (b), compliance (c), and elastance (d). Significance was determined by a one-way ANOVA where in panel (c) ^*** P<0.001 vehicle vs. all other groups, (d) ^* P<0.05 for 25 μg vs. either control or vehicle and ^*** P<0.001 vehicle vs. control, 100 μg, or 250 μg. (e). Mice (n=four/group) were inoculated with 1 × 10⁶ CFU E. coli or uninfected for 48 h prior to i.t. administration of CBD. Mice were lavaged, and BAL supernatant assayed for cytokines. All P values are <0.05 for vehicle buffer + E. coli. (0+) vs. other groups. f. Kaplan-Meier survival curve for mice given i.t. CBD peptide (100 μg) and infected with E. coli, (5 × 10⁶ CFU/mouse, n=14 mice/group, P=0.001, log rank test).