CL was quantitated in tracheal aspirates from critically ill subjects with non-pulmonary illnesses (n=five), clinically diagnosed pneumonia (n=17), or congestive heart failure (CHF) (n=six, Supplementary Table I). Control patients with non-pulmonary illnesses included those requiring mechanical ventilation for liver failure, hemolysis elevated liver enzymes low platelets (HELLP) syndrome, Guillain-Barre syndrome, renal failure, and gastrointestinal tract bleeding. Subjects with pneumonia were identified as having new or changing radiographic pulmonary infiltrates, increasing sputum quantity or change in sputum character, and clinical features such as fever, elevated white blood cell count and hypoxemia without dependence upon sputum culture results or distinction between community or hospital acquisition. No bacterial growth was detected in 39% (7/17) of subjects with pneumonia while tracheal aspirates grew Streptococcus pneumoniae in four subjects, Pseudomonas aeruginosa and Staphylococcus aureus in three subjects each, and Haemophilus influenza in one subject. 90% (26/28) of all subjects were on broad-spectrum antibiotics on the day of tracheal aspirate collection (Supplementary Table. 1). Subjects with pneumonia had significantly higher levels of CL (median=12.9 Mol%) in tracheal aspirates compared to subjects with non-pulmonary diagnoses (~9.7 fold) or CHF (~6 fold, Kruskal-Wallis P=0.0007, Fig. 1a). Importantly, CL levels did not correlate with culture status, bacterial pathogen, duration of mechanical ventilation, gender, or age (Supplementary Fig. 1).

Mice were also infected with strains of H. influenzae or E. coli that cause pneumonia ^9,10. Bronchoalveolar lavage (BAL) fluid from infected mice had greater levels of CL than BAL isolated from uninfected mice (Fig. 1b). To assess if elevated CL levels were due to reduced cellular uptake, primary mouse type II lung epithelia were cultured with [³H] CL. Infection with H. influenzae or E. coli resulted in significantly reduced cellular uptake of [³H] CL (Fig. 1c). Thus, CL is increased in lung fluid of both patients and animal models with pneumonitis and this may be due to decreased epithelial uptake of the phospholipid.

Phospholipids (CL, LPC [lysophosphatidylcholine], PS [phosphatidylserine]) were incorporated into Infasurf (a commercial apoprotein containing surfactant) to generate lipid vesicles that were used to assay surface-tension (Fig. 2a). Lipids were also extracted from Infasurf to generate protein-deficient preparations that were reconstituted with phospholipids for testing (Fig. 2b). Unlike PS, increasing CL concentrations (> 3 Mol%) resulted in increased surface-tension. CL impaired surface-activity to a greater extent than LPC, a positive control ¹¹. These adverse effects of CL were more pronounced when testing lipid extracts devoid of surfactant-associated apoproteins (Fig. 2b). Mice given intratracheal (i.t.) CL had significantly decreased lung compliance and increased elastance and resistance compared to controls (Fig. 2c-f). CL also increased BAL protein concentration (Supplementary Fig. 2a), differentially altered surfactant proteins (Supplementary Fig. 2b), reduced γ-interferon and IL-2 and increased IL-10 (Supplementary Fig. 2c,d), but did not affect the distribution of various inflammatory cells (Supplementary Fig. 2e) in vivo. Thus, CL in lung fluid adversely affects lung mechanics by greatly impairing surfactant activity. The lipid also modulates expression of cytokine networks that could impact lung stability.

Mice were subjected to live imaging of lungs by micro CT scanning (Fig. 3a). Compared to diluent, mice given CL (50 nmol) had more prominent markings in parenchyma with scattered patchy areas of alveolar consolidation (arrows). These abnormalities were more severe after high doses of CL. Histological analysis identified areas of alveolar infiltration and appearance of foamy cells within alveoli (Fig. 3a, lowest, mid panel, arrows). High dose CL produced edema and disruption of alveolar lining cells (Fig. 3a, lower right panels, Fig. 3b,c) that contributed to fatality of mice within 2-3 h. CL activated the apoptotic program in cells (Fig. 3d, left) and in tissue (Fig. 3d, right panel) and it decreased cell viability (Fig. 3e) and increased cell toxicity (Fig. 3f).

Because CL is internalized by alveolar cells, we hypothesized that cells would express an import protein that regulates CL levels in lung fluid. ATP8b1 was a candidate protein as it internalizes phospholipids and patients with ATP8b1 defects are prone to pneumonia ⁸. Type II cells had high levels of ATP8b1 compared to macrophages or fibroblasts (Supplementary Fig. 3a, [inset]), ATP8b1 expression was regulated (Supplementary Fig. 3b), and it exhibited surface expression (Supplementary Fig. 3c). Lentiviral ATP8b1 expressing cells had higher levels of mRNA and protein expression coupled with a robust increase in the uptake of fluorescent (nitrobenzoxadiazole [NBD]) labeled NBD-CL or NBD-PS (positive control) compared to non-transduced cells (Supplementary Fig. 3d, Fig. 4a, b). ATP8b1 overexpressing cells had greater fluorescence on both the cell surface and inside the cells, indicating that the NBD-CL was located on the plasma membrane and within the cytoplasm. Although ATP8b1 internalized CL, it did not enhance GFP-labeled E. coli uptake (Supplementary Fig. 3e).

Mice were also administered adenovirus (Ad5) expressing ATP8b1 or empty adenovirus prior to E. coli infection. Mice infected with E. coli at 1 × 10⁶ CFU/mouse cleared the pathogens by 48 h of analysis. These mice exhibited decreased BAL macrophages with a neutrophilic infiltrate typical of pneumonia, a profile not affected by Ad5-ATP8b1 gene transfer (Supplementary Fig. 4a). Ad5 alone or Ad5 encoding ATP8b1 tended to have high BAL protein concentrations associated with release of pro-inflammatory cytokines after bacterial infection (Supplementary Fig. 4b,c). ATP8b1 gene delivery before E. coli infection also reduced collectins, surfactant protein A (SP-A) and surfactant protein D (SP-D), but did not affect surfactant protein B (SP-B), the latter essential for surfactant activity (Supplementary Fig. 4d). Importantly, ATP8b1 gene delivery effectively increased pump levels, reduced CL levels, and reversed E. coli induced impairment of lung mechanics (Fig. 4c,d, Supplementary Fig. 4e) compared to infected mice given Ad5. ATP8b1 gene transfer did not reduce apoptosis after bacterial infection (Fig. 4e). Thus, the primary mechanism for beneficial effects of increased ATP8b1 expression is via reduced CL availability, thereby preserving surfactant function and improving lung mechanics. These effects were sufficient to result in increased survival of mice (Fig. 4f).

Additional loss-of-function studies were performed using siRNA and ATP8b1 mutant mice that harbor a single amino acid substitution (Gly³⁰⁸→Val) resulting in an apparent defect in PS importability ^12,13. ATP8b1 siRNA produced a significant decrease in CL uptake and selectively reduced immunoreactive ATP8b1 compared to control RNA (Fig. 5a). HaeIII restriction digest fragment patterns were used to genotype mutant, wild-type, and heterozygous mice revealing that mutants lacked 500 bp and 300 bp digest fragments (Fig. 5b). We detected low level ATP8b1 expression in mutant liver and lung compared to wild-type tissues (Fig. 5c). ATP8b1 mutant mice had significantly higher BAL CL versus wild-type littermates (Fig. 5d). Consistent with ATP8b1’s inability to alter bacterial uptake, bacterial loads did not differ between ATP8b1 mutant and wild-type mice (Supplementary Fig. 5a). Both ATP8b1 defective and wild-type littermates exhibited no differences in BAL protein content (Supplementary Fig. 5b), wet/dry lung weight ratios (Supplementary Fig. 5c), or cellular inflammation (Supplementary Fig. 5d) after infection. Interestingly, ATP8b1 defective mice exhibited a blunted cytokine response to Th1 cytokines (interferon-γ, TNF-α, IL-β) compared to wild-type littermates after E. coli infection (Supplementary Fig. 6a,b) which may be secondary to increased expression of SP-A and SP-D (Supplementary Fig. 6c)^14,15. Primary type II cells isolated from ATP8b1 mutant mice exhibited a blunted response with regard to NBD-CL uptake compared to cells isolated from wild-type littermates (Fig. 5e). Mutant mice also had impaired biophysical properties compared to wild-type mice particularly following E. coli infection (Figs. 5f,g, Supplementary Fig. 6d). Although mutant mice also were more prone to apoptosis (Fig. 5h), there was no significant difference in mortality between ATP8b1 mutant and wild-type mice with infection (Fig. 5i). Collectively, these studies strongly suggest that ATP8b1 is a bona fide alveolar epithelial CL import pump.

We mapped the ATP8b1-CL binding domain (CBD). Synthesized deletion mutants (Supplementary Fig. 7a,b) were reacted with fifteen lipids pre-spotted onto hydrophobic lipid strips (Supplementary Fig. 7c). Using this system, FL ATP8b1 and specific mutants were observed to bind CL and also sulfatide (Supplementary Fig. 7c). C–terminal truncation mutants containing only the first 810 or 771 residues did not bind CL suggesting that a CBD resides between residues 810 to 850 within ATP8b1. This was confirmed by testing a fragment (residues 771-850) that was sufficient for CL binding indicating that a novel CBD resides within the ATP8b1 carboxyl-terminus (Supplementary Fig. 7c).

A cDNA encoding the putative CBD was fused to GST, for expression of purified protein from cells. Cells cultured with GST-CBD peptide exhibited an 83% decrease in [³H] CL uptake versus GST peptide alone (Fig. 6a). Mice infected with or without E. coli and given CBD peptide compared to vehicle exhibited a significantly increased proportion of macrophages and modestly reduced protein content and BAL neutrophils (Supplementary Fig. 8a,b). Importantly, CBD peptide profoundly reduced TNF-α, and IL-β levels and significantly reduced GM-CSF levels after bacterial infection (Fig 6, Supplementary Fig. 8c). CBD peptide did not alter surfactant apoproteins nor the degree of apoptosis (Supplementary Fig. 9a,c). Infected mice given vehicle exhibited impaired lung mechanics, effects that were reversed after CBD peptide administration (Fig. 6b,c,d, Supplementary Fig. 9b). These beneficial effects of CBD peptide on pulmonary homeostasis led to significantly improved survival in mice (Fig. 6f). Thus, the CBD within ATP8b1 is functional with regard to substrate binding and this peptide exerts biological effects in concert with its activity in antagonizing actions of CL in vivo.

The study was approved by respective institutional review boards. After obtaining informed consent, tracheal aspirates were collected using an inline suction catheter without saline dilution. Patients were diagnosed with pneumonia (clinical diagnosis and confirmed with infiltrates on chest x-ray), CHF (clinical diagnosis and confirmed with pulmonary edema on chest x-ray) and included control subjects who were intubated for non-pulmonary illnesses (normal chest x-ray). Aliquots were sent for routine cultures.

Mouse lung epithelial (MLE) cells were cultured as described ³². Cells were serum starved for 24 h then exposed to 120 nmol/ml Infasurf, PC liposomes, or CL (5-15 Mol %). Mouse alveolar type II cells, macrophages, and fibroblasts were isolated as described ³². Cell viability was determined using the CellTiter-Glo Luminescent Cell Viability assay (Promega). LDH release was assayed by monitoring the NAD-NADH reaction at a 340 nm wavelength.

[³H]- CL (Moravek Biochemicals, Inc.) was reconstituted in liposomes using Infasurf (Forest Pharmaceuticals Inc.) and added to medium for 2 h at 37 °C. Cellular uptake was terminated by washing with serum-free cold media twice and 2% fatty acid free BSA in PBS. Lipids were extracted (1 ml hexane:isopropanol (3:2, v/v)), solvents dried, and radioactivity (dpm) in lipids measured by scintillation counting.

FL ATP8b1 was amplified from an expression clone in pcDNA3.1D/V5-His using a forward and reverse primer with engineered EcoRI or SpeI restriction sites, respectively. The amplified product was directionally cloned into the EcoRI and BamHI sites of the adenovirus shuttle plasmid, pacAd5 CMV K-NpA. An adenovirus expression vector including V5-FL ATP8b1 was produced by the University of Iowa Gene Transfer Vector Core ³³.

C57BL/6 mice (Jackson Laboratories) were used according to Institutional Animal Care and Use Committee approved protocols. E. coli (ATCC 25922) (1 ×10⁶ CFU (colony forming units)) was administered intratracheally (i.t). Mice were infected i.t. (2 ×10⁸ CFU, nontypable H. influenzae) or given agarose ²⁹ for 72 h prior to lung lavage. For adenoviral gene transfer, mice received 2.5 ×10⁸ PFU (plaque forming units) i.t. and 24 h later given E. coli for 48 h. Mice were mechanically ventilated with a FlexiVent system ³⁴. Mice were also given E. coli, ventilated, and 48 h later diluent or ATP8b1 CBD peptide was delivered into lungs using a microsprayer aerosolizer. After 10 min, biophysical measurements were taken. For assay of lung edema, mice received CL (15 mM in 50 μl saline i.t.) or vehicle (50 μl saline). Evan’s Blue dye (40 μg/g in 100 μl saline) was injected intravenously via the femoral vein 30 min later, BAL was obtained, Dye concentration was determined using a spectrophotometer at a wavelength of 620 nm. For wet/dry weights lungs were removed, blotted, and placed in tared weigh boats and weighed. The lungs were then dried (24 h at 60 °C) and weighed again.

Cells were plated (35 mm culture dishes) and incubated with NBD-CL and NBD-PS (25 °C), and then immediately incubated at 4 °C for 5 min prior to washing with medium. Fluorescence was detected using an epifluorescent microscope (Olympus).

Lentivirus expressing ATP8b1 was produced using pLenti6/V5-DEST by the University of Iowa Gene Transfer Vector Core. Cells were transduced with a final concentration of 4 μg/ml of polybrene (Sigma) in MEM-F12, at a multiplicity of transduction of 10:1. A final concentration of 2.5 μg/ml blasticidin (Invitrogen) was used to select for transduced cells.

NBD-labeled phosphatidylserine (PS) (Avanti Polar Lipids, Inc.) and NBD- CL (Invitrogen) were incubated with cells for 30 min at 37 °C. Lipids were extracted twice with 1 ml hexane:isopropanol (3:2, v/v). The solvents were evaporated under nitrogen and lipids dissolved in methanol. The fluorescence of NBD-labeled lipids was measured (excitation and emission at 460/530 nm) and quantitated using standard curves for known amounts of the NBD-labeled lipids.

Lipids were extracted ^35,36 and CL bound to fluorescent nonyl acridine orange (NAO) was quantitated using a spectrophotometer measuring NAO excitation and emission (494/530 nm) using standard curves ³⁷. Values were normalized for total phospholipid phosphorus ³⁸ and expressed as ratios (Mol%) of phosphorus (nmol) in CL to total phospholipid (nmol) within samples.

Lipid strips (Echelon Biosciences, Inc.) were incubated with translation products in TTBS buffer with 1% fatty acid-free BSA. Strips were washed extensively and radiolabeled protein was detected with V5 antibody using immunoblotting.

A549 alveolar type II (ATII) cells were transfected twice with 2 μg of ATP8b1 ON TARGET plus SMARTpool siRNA or ON-TARGET plus Non-targeting Pool control siRNA (Dharmacon) for 48 h using Fugene 6 prior to harvest.

We used a Prism program version 4.03 (GraphPad Software, Inc.) using an ANOVA or unpaired t test with P<0.05 indicative of significance. Kaplan Meier survival estimates were done using SAS version 9.2 (SAS institute). For human data, nonparametric testing using a Kruskal-Wallis and post hoc Wilcoxon rank sum test was performed.