LAMP3 deficiency affects surfactant homeostasis in mice

Lysosome-associated membrane glycoprotein 3 (LAMP3) is a type I transmembrane protein of the LAMP protein family with a cell-type-specific expression in alveolar type II cells in mice and hitherto unknown function. In type II pneumocytes, LAMP3 is localized in lamellar bodies, secretory organelles releasing pulmonary surfactant into the extracellular space to lower surface tension at the air/liquid interface. The physiological function of LAMP3, however, remains enigmatic. We generated Lamp3 knockout mice by CRISPR/Cas9. LAMP3 deficient mice are viable with an average life span and display regular lung function under basal conditions. The levels of a major hydrophobic protein component of pulmonary surfactant, SP-C, are strongly increased in the lung of Lamp3 knockout mice, and the lipid composition of the bronchoalveolar lavage shows mild but significant changes, resulting in alterations in surfactant functionality. In ovalbumin-induced experimental allergic asthma, the changes in lipid composition are aggravated, and LAMP3-deficient mice exert an increased airway resistance. Our data suggest a critical role of LAMP3 in the regulation of pulmonary surfactant homeostasis and normal lung function.


Introduction
The lysosome-associated membrane glycoprotein (LAMP) family consists of five members (LAMP1, LAMP2, LAMP3 / DC-LAMP, CD68 / macrosialin, and LAMP5 (BAD-LAMP)). All family members are heavily N-and O-glycosylated type I transmembrane proteins localized to the limiting membrane of lysosomes and lysosome-related organelles [1]. LAMP1 and LAMP2 are ubiquitously expressed [1], while LAMP3 (synonymously called DC-LAMP), CD68, and LAMP5 show cell-type-specific or tissue-specific expression. CD68 expression is restricted to macrophages, monocytes, and microglia [2], whereas LAMP5 is expressed exclusively in the brain [3]. The LAMP3-expression pattern between humans and mice differs: LAMP3 is expressed in humans in alveolar type II (AT2) cells and dendritic cells (DC) (eponymously for its alternative name "DC-LAMP" or CD208). In mice,LAMP3 is found exclusively in AT2 cells but not in DCs [4][5][6][7]. These findings suggest a conserved and critical function of LAMP3 in AT2 cells. On the subcellular levels, LAMP3 is localized in lamellar bodies (LBs) [4]. However, the function of LAMP3 in these cells remains enigmatic until now; if and how LAMP3 affects surfactant homeostasis, e.g., mediating surfactant release via LBs, is also poorly understood.
The primary function of AT2 cells is mainly attributed to pulmonary surfactant release to lower surface tension at the lung's air/liquid interface. Pulmonary surfactant is a mixture of lipids and proteins [8]: The major hydrophobic protein components, surfactant proteins B (SP-B) and C (SP-C), are small proteins deeply embedded into the phospholipids of the surfactant. Notably, deficiency in SP-B or SP-C due to genetic mutations in SFTPB or SFTPC cause hereditary forms of childhood interstitial lung disease [9,10]. Sftpb gene-targeted mice die of respiratory failure after birth, associated with irregular and dysfunctional LBs and tubular myelin [11]. SP-C deficient mice have a regular life span and only show subtle deficits in lung mechanics and surfactant stability [12].
The composition of surfactant lipids has been analyzed extensively [13][14][15][16][17]. The most abundant and characteristic lipid class is phosphatidylcholine (PC). PC makes up approximately 70% of the total surfactant mass with dipalmitoylphosphatidylcholine (DPPC; PC 16:0/16:0) as the primary lipid species [18]. An overall decrease of PC lipids is coupled to higher surface tension and lower surfactant protein inclusion [8]. Other highly abundant and functionally essential phospholipid classes in surfactant include phosphatidylglycerol (PG) and phosphatidylinositol (PI), while phosphatidylethanolamine (PE), and sphingomyelin (SM) are reported as minor components of surfactant and most likely derived from other cell membranes. In addition, lysophosphatidylcholine (LPC) can be found in low abundance in pulmonary surfactant [18,19]. Preassembled surfactant is stored in secretory organelles known as LB that fuse with the cell membranes and release pulmonary surfactant. LBs are multivesicular body / late endosome-derived specialized organelles filled with surfactant characterized by a typical "myelin"-like appearance by electron microscopy. Phospholipids are directly transferred from the endoplasmic reticulum to LB through a vesicle-independent mechanism by the phospholipid transporter "ATP binding cassette subfamily A member 3" (ABCA3) [20,21]. Like SFTPB or SFTPC, loss-of-function mutations in ABCA3 were identified in full-term infants who died from unexplained fatal respiratory distress syndrome [22]. ABCA3 mutations represent the most frequent form of congenital surfactant deficiencies [23].
A presumably loss-of-function mutation in LAMP3 (p.(E387K)) was recently identified in an Airedale Terrier dog breed, leading to severe symptoms and pathology similar to clinical symptoms of the most severe neonatal forms of human surfactant deficiency [24]. The affected puppies' symptoms included lethal hypoxic respiratory distress and occurred within the first days or weeks of life [24]. LBs were smaller, contained fewer lamellae, and occasionally revealed a disrupted common limiting membrane [24].
To conclusively address the physiological role of LAMP3 in surfactant homeostasis using a genetically defined animal model, we generated Lamp3 knockout mice (KO, Lamp3 -/-) by CRISPR/Cas9. In contrast to dogs bearing a natural mutation in LAMP3, Lamp3 -/mice are vital, display a regular lung function at the basal level, and show no increased prenatal lethality. The lungs of Lamp3 -/animals did neither macroscopically nor microscopically differ from those of wild type littermates. However, biochemical analysis of broncho-alveolar lavage (BAL) fluid clearly revealed increased pro-SP-C levels and altered lipid composition in the Lamp3 -/mice. These differences are further pronounced under diseased conditions as evoked by allergen-induced experimental asthma, and most notably, diseased Lamp3 -/mice revealed significantly increased airway resistance, when stressed during methacholine provocation testing. In conclusion, our data suggest a critical but not essential role of LAMP3 in pulmonary surfactant homeostasis associated with normal lung function.

Reagents and antibodies
Analytical grade chemicals were purchased, if not stated otherwise, from Sigma-Aldrich (MO., USA). Antibodies: The following antibodies were used in the study: Rat monoclonal LAMP3, clone 1006F7.05 (Dendritics), SP-C, rabbit polyclonal (Abcam), rabbit polyclonal antibodies against mature SP-B and Pro-SP-B (Seven Hills Bioreagents) were a generous gift from Jeffrey A. Whitsett, polyclonal β-Actin (Santa Cruz Biotechnology, Inc), polyclonal rabbit antiserum against ABCA3 was a kind gift from Michael L. Fitzgerald [20]. Fluorophore-conjugated secondary antibodies against the corresponding primary antibody species (AlexaFluor 488) were purchased from Invitrogen / Molecular Probes and were diluted 1:500.

Generation and housing of Lamp3 -/mice
Mice with a null mutation in the Lamp3 gene were generated in a C57BL/6N background using a CRISPR genome-editing system. For this purpose, Cas9 was combined with two single guide RNAs (sgRNAs) targeting the second exon in the Lamp3 gene with the following spacer sequences: sgRNA_Lamp3 F1: TCATCTACTGACGATACCAT and sgRNA_Lamp3 R1: GCTAGACTAGCTCTGGTTGT microinjected into fertilized zygotes. Genome editing was confirmed by PCR amplification in the resulting founder mice with the primers Lamp3F 5´-GATGGGGGAGGGATCTTTTA-3' and Lamp3R 5´-GTTGGCCTCTGATTGGTTGT-3'. A founder harboring a 40 bp deletion within the second exon leading to a frameshift mutation was selected for further breeding. Mice were housed under specific pathogen-free conditions (12 hours' light/dark cycle, constant room temperature, and humidity). Food and water were available ad libitum.

Ovalbumin model of allergic asthma
Female, 6-to 8-week-old Lamp3 -/and wildtype littermates mice (CAU, Kiel, Germany) were used for the allergic asthma model. They received ovalbumin (OVA)-free diet and water ad libitum.
Mice were sensitized to OVA by three intraperitoneal (i.p.) injections of 10 μg of OVA (OVA grade VI, Sigma-Aldrich, St. Louis, MO, USA) adsorbed to 150 μg of aluminum hydroxide (imject alum, Thermo Fisher Scientific, Waltham, MA, USA) on days 1, 14 and 21. To induce acute allergic airway inflammation, mice were exposed three times to an OVA (OVA grade V, Sigma-Aldrich) aerosol (1% wt/vol in PBS) on days 26, 27, and 28. Control animals were sham sensitized to PBS and subsequently challenged with OVA aerosol.

Lung function analysis
Steady-state lung parameters midexpiratory flow (EF50), frequency (f), functional residual capacity (Frc), minute volume (MV), peak expiratory flow (PEF), peak inspiratory flow (PIF), expiration time (Te), inspiration time (Ti), and tidal volume (TV) were measured in conscious, spontaneously breathing mice using FinePointe non-invasive airway mechanics double-chamber plethysmography (NAM, Data Science International, St. Paul, MN, USA). Steady-state airway resistance (RI) and dynamic compliance (Cdyn) were measured in anesthetized and ventilated mice using FinePointe RC Units (Data Science International). Airway responsiveness to methacholine (MCh, acetyl-β-methylcholine chloride; Sigma-Aldrich) challenge was invasively assessed on day 29 using FinePointe RC Units (Data Science International, St. Paul, MN, USA) by continuous measurement of RI. Animals were anesthetized with ketamine (90 mg/kg body weight; cp-pharma) and xylazine (10 mg/kg BW; cp-pharma) and tracheotomized with a cannula. Mechanical ventilation was previously described [25]. Measurements were taken at baseline (PBS) and in response to inhalation of increased concentrations of aerosolized methacholine (3.125; 6.25; 12.5; 25; 50; and 100 mg/mL). After assessment of lung function, all animals were sacrificed by cervical dislocation under deep anesthesia.

Bronchoalveolar lavage
For bronchoalveolar lavage, lungs were rinsed with 1 ml of fresh, ice-cold PBS containing protease inhibitor (Complete, Roche, Basel, Switzerland) via a tracheal cannula. Cells in BAL fluid were counted in the Neubauer chamber. Fifty microliter aliquots of lavage fluids were subjected to centrifugation (5 minutes at 320 g) (Zytozentrifuge Shandon Cytospin 4, Thermo Fisher Scientific), and cells were microscopically differentiated according to morphologic criteria.

Electron microscopy
Animals were anesthetized and lungs were fixed by perfusion through the right ventricle with HEPES buffer (pH 7.35) containing 1.5% glutaraldehyde and 1.5% formaldehyde, at a fixed positive inflation pressure of 25 cm H 2 O. After storage of the lungs in the same solution overnight, 3 mm sized pieces of lung tissue were postfixed first in 1% osmium tetroxide for 2 hours and then in 4% uranyl acetate overnight (both aqueous solutions). Samples were dehydrated in acetone and embedded in Epon. Ultrathin sections of 60 nm were poststained with aqueous uranyl acetate and lead citrate [26] and imaged in a Morgagni TEM (FEI, Eindhoven, NL).

Histology
Lungs were dissected from mice, and the left lung lobe was snap-frozen in liquid nitrogen. The other lobes were fixed ex-situ with 4% (wt/vol) formaldehyde via the trachea under constant pressure, removed and stored in 4% formaldehyde. Volume of these lobes was determined based on Archimedes' principle. In brief, the remaining lung lobes were placed in a water-filled container set on electronic scales. The weight of water displaced by the submerged lung equals the volume of the lung. Then lung tissues were embedded in paraffin. For analysis of lung inflammation, 2 μm sections were stained with periodic acid-Schiff (PAS). Photomicrographs were recorded by a digital camera (DP-25, Olympus, Tokyo, Japan) attached to a microscope (BX-51, Olympus) with a 20-fold magnification objective using Olympus cell^A software. Mucus quantification was performed as previously described [27]. Apoptotic cells were stained using ApopTag Peroxidase In Situ Apoptosis Detection Kit (Merck Millipore, MA, USA) according to manufacturer's instructions.

Immunofluorescence staining of lung tissue
4% Formaldehyde-fixed lungs were incubated in cryoprotectant solution (30% w/v sucrose in 0.1 M phosphate buffer, pH 7.4) over night. 35 μm thin sections were cut with a Leica 9000s sliding microtome (Leica, Wetzlar, Germany) and collected in cold 0.1 M phosphate buffer (PB). Free floating sections were stained by blocking in blocking solution (0.5% Triton-X 100, 4% normal goat serum in 0.1 M PB pH 7.4) for 1 hour at room temperature. Subsequently, sections were incubated in blocking solution containing the primary antibody at 4˚C over night. After washing three times with wash solution (0.1 M PB pH 7.4 containing 0.25% Triton-X 100), sections were incubated for 2 hours in secondary antibody in solution, washed again three times in wash solution containing 4 0 ,6-Diamidin-2-phenylindol (DAPI) and finally brought on glass slides and embedded in Mowiol/DABCO. Cells were imaged using a Zeiss confocal microscope equipoped with a 63x objective (Zeiss LSM980 AiryScan 2).

Homogenization of lung tissue
Deep frozen lungs were homogenized with mortar and pestle. An aliquot of 30 mg of lung powder was transferred into RLT buffer (Qiagen), and RNA was isolated with RNeasy Mini Kit (Qiagen) according to the manufacturer's guidelines. 1 μg of RNA was used for cDNA synthesis (First Strand cDNA Synthesis Kit, Thermo Fisher Scientific, Waltham, MA, USA). Another 30 mg aliquot of lung powder was transferred into radioimmunoprecipitation assay buffer (RIPA) buffer, and proteins were isolated. Protein concentration was determined by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Waltham, MA, USA).

Quantitative PCR
Quantitative RT-PCR was performed on the Roche LightCycler 480 Instrument II system using the LightCycler 480 SYBR Green I Master (Roche Applied Science, Mannheim, Germany). Template cDNA was diluted 1:10. RT-PCR was performed in triplicate in a total volume of 10 μl according to the manufacturer's instruction with a final primer concentration of 0.5 μM. Cycling conditions were as follows: 45 cycles at 95˚C for 10 s, touch-down annealing temperature (63 to 58˚C, temperature reduction 0.5˚C per cycle) for 10 s and 72˚C for 8 s. For each primer pair, a standard curve was established by serial cDNA dilutions. The following primer sequences for reference gene RPL32 were used: forward 5'-AAAATTAAGCGAAACT GGCG-3', reverse 5'-ATTGTGGACCAGGAACTTGC-3' (NM_172086.2,156 bp). QuantiTect Primer Assays from Qiagen (Venlo, The Netherlands) were used for Lamp3 (QT00157031, NM_177356, 105 bp), Sftpb (QT00124908, NM_011359, 125 bp) and Sftpc (QT00109424, NM_011359, 125 bp). Negative controls (reverse transcriptase/RNA template) were included to detect possible contaminations. Amplification specificity was checked using the melting curve and agarose gel (2%) electrophoreses with a Gene Ruler 50bp DNA ladder (Thermo Scientific). Specific mRNA levels were normalized to the level of the reference gene Rpl32 in the same sample. Data were analyzed employing the 'advanced relative quantification' and 'standard curve method'. A calibrator cDNA was included in each run to correct for run-to-run differences.

Chemicals and lipid standards for lipidomics
All solvents, reagents, and lipid standards were used in the highest available purity. Internal standards were acquired from Avanti Polar Lipids (Avanti Polar Lipids, Alabama, USA).

Lipid extraction
A customized methyl-tert-butyl ether (MTBE)-based lipid extraction method [28] was applied for BAL and lung tissue homogenates. Details can be found in the S1 Text. Lipid extracts were stored in a storage solution at -20˚C until further usage. Cholesterol determination was performed as described earlier [29] (S1 Text).

Shotgun lipidomics measurements
Aliquots of 10 μL of the lipid extract and cholesterol derivatization were diluted in 190 μL ESI spray mixture for each measurement, vigorously mixed, and centrifuged prior to loading into a 96 well plate (Eppendorf, Hamburg, Germany). The well plate was sealed with aluminum sealing foil and kept at 15˚C during the measurement process. All measurements were performed in duplicate. All mass spectrometric acquisitions were performed using a Triversa Nanomate (Advion, Ithaca, USA) as an autosampler and nano-ESI source, applying a spray voltage of 1.1 kV and backpressure of 1.1 psi in ionization modes. For the acquisition of tandem mass spectrometric data, a Q Exactive Plus was used (Thermo Fisher Scientific, Bremen, Germany; methodological details are found in the S1 Text).

Lipidomics data processing
For data interpretation, the software pipeline described earlier was utilized (see details S1 Text) [30]. Briefly, mass spectra raw files were converted to mzML format with the software module "msconvert" from ProteoWizard version 3.0.18212-6dd99b0f6 [31]. Converted files were then imported into LipidXplorer version 1.2.8.1 [32]. For each ESI mode, a different set of MFQL [33] files is used. The files generated in LipidXplorer were used in lxPostman for further post-processing, including quality control and quantitation. All details on how the lipidome data (S1 Data) was generated for visualization and statistical analysis is available at LIFS webportal https://lifs-tools.org/publications/99-shotgun-lipidomics-data-set-lamp3-is-criticalfor-surfactant-homeostasis-in-mice.html

Statistical analysis for lipidomic data
Two-way ANOVA was performed with the parameters matching time points for each row, mixed-effects with full fitting, and within each row comparing columns with correcting for multiple comparisons via Tukey. Lipid values are expressed as mean ± standard deviation and significance is designated as � , P < 0.05; �� , P < 0.01; ��� , P < 0.001.
For easier visualization of significant lipid results, lipids from two-way ANOVA were autoscaled. In this procedure, data are centered on the means of groups and then divided by the standard deviation, removing the impact of differences between lipid abundance.

Bubble surfactometer
Cell free BAL was weighed (= 400-800 μl) and ultra-centrifuged (J2-MC, Beckman Coulter, Krefeld, Germany) for 1h at 4˚C at 38,730 g. The supernatant was decanted. The pellet was weighed and resuspended using saline containing 1.5 mmol/L CaCL 2 equivalent to a concentration of 1:50 (w/w) compared to the basic BAL. Two μl of the concentrated BAL were analyzed for content of choline-containing phospholipids (DPPC, lysolecithin, sphingomyelin; LabAssay Phospholipids, FUJIFILM WAKO). Then, samples were normalized to final phospholipids-, i.e. choline containing phospholipids-, concentration of 1.5 mg/ml and analyzed in the Pulsating Bubble Surfactometer [34]. A Pulsating Bubble Surfactometer -sample chamber was prefilled using degassed 10% sucrose in saline plus 1.5 mmol/l CaCL 2 . Then, 5 μl of each normalized BAL sample were pipetted on the top of the sucrose prefill close to the chimney, where it remained by buoyancy. Subsequently, to mounting of the chamber in the Pulsating Bubble Surfactometer, an air bubble was created by aspiration of ambient air through the chimney and phospholipids were allowed to adsorb for 30s to the air/liquid interface. Then, the bubble was cyclically expanded and compressed at a frequency of 20/min, a compression rate of 50% surface area and at 37˚C. Surface tension at maximum (γmax) and minimum (γmin) bubble size was calculated by the Pulsating Bubble Surfactometer and recorded for a 300s period.
Statistical analyses. If not stated otherwise, a two-tailed unpaired t-test was performed using GraphPad Prism Software Version 5.03. Significant values were considered at P < 0.05. Values are expressed as mean ± standard error of the mean (SEM) and significance is designated as � , P < 0.05; �� , P < 0.01; ��� , P < 0.001. Single datapoints for plotting data are available as S2 Data.

Results
Lamp3 knockout mice are viable with normal lung anatomy and pulmonary function LAMP3 is expressed highest in humans and mice in the lung's AT2 cells, where it localizes to LBs [4,5]. Notably, a naturally occurring recessive missense LAMP3 variant has recently been associated with a fatal neonatal interstitial lung disease in Airedale Terrier dogs [24]. Therefore, we aimed to analyze the function of LAMP3 in the lung in a genetically more defined animal model and generated Lamp3 knockout mice (Lamp3 -/-). CRISPR/Cas9 editing of the Lamp3 allele in mice with guide RNAs targeting exon 2 (Fig 1A and 1B) yielded a 40 bp deletion resulting in an early Stop-codon in one founder, and the mutation was transmitted in the germline (Fig 1C). PCR with primers spanning this deletion was used for genotyping ( Fig  1D). Reverse transcriptase PCR and quantitative realtime PCR with total lung RNA as a template with primers specific for LAMP3 revealed a complete absence of LAMP3 transcript, indicating nonsense-mediated decay (Fig 1E and 1F). LAMP3 was readily detectable by immunohistochemistry on lung sections with a monoclonal antibody in AT2 cells of wildtype mice but was completely absent in Lamp3 -/mice, validating the knockout at the protein level (Fig 1G). We hypothesized that knockout of Lamp3 in mice causes abnormal surfactant homeostasis and postnatal death. However, in contrast to dogs with a LAMP3 mutation, homozygous Lamp3 -/mice were surprisingly born according to the expected Mendelian distribution and survived the weaning phase without increased mortality (Fig 1H). No premature death was observed until 15 months-of-age, indicating that LAMP3 is not essential for survival in mice. Macroscopically, Lamp3 -/mice were indistinguishable from wildtype littermates with no obvious phenotype. Histology and analysis of semi-thin sections revealed normal microanatomy of the lung with a typical distribution of AT2 cells, type I pneumocytes, alveolar macrophages, and regular capillaries (Fig 1I). Macroscopic signs of multifocal emphysema or edema (as previously described in dogs with a mutation in LAMP3, [24]) were absent in Lamp3 -/mice (Fig 1J). The lung volume, normalized to the body weight in 3-month-old animals, was comparable between wildtype and Lamp3 -/mice (Fig 1K).
Furthermore, lung function analysis revealed no abnormalities in the breathing pattern of Lamp3 -/mice regarding the frequency, flow, volume, and time parameters as non-invasively assessed in spontaneously breathing animals as well as for airway resistance and compliance as recorded invasively in relaxed, ventilated animals ( Table 1). Analysis of the BAL cellular composition revealed a tendency towards a higher number of lymphocytes and neutrophils in Lamp3 -/mice, which, however, did not reach statistical significance. The number of macrophages was moderately increased in Lamp3 -/mice (Fig 1L).

LAMP3 deficiency in mice causes changes in surfactant protein levels
We reasoned that a lack of LAMP3 might affect the levels of the surfactant proteins SP-B and SP-C, major components of the LB-stored-and secreted surfactant. Indeed, quantitative PCR analysis revealed reduced levels of Sftpb transcript and a similar trend (though not statistically significant) for Sftpc in total lung RNA (Fig 2A) of Lamp3 knockout mice. In contrast, immunoblot analysis of lung tissue lysates from wildtype and Lamp3 -/mice for SP-B and SP-C showed normal levels of pro-SP-B and a striking increase in the levels of pro-SP-C, the immature pro-form of SP-C, indicating a critical role of LAMP3 in the biosynthesis or turnover of SP-C (Fig 2B and 2C). The levels of mature SP-B were also significantly increased, though only to a moderate level compared to the increase in SP-C.
Immunofluorescence analysis on lung sections from wildtype and Lamp3 -/mice revealed no major differences in the staining pattern of the essential LB proteins ABCA3 or mature SP-B. Both proteins localized in wildtype and Lamp3 -/mice to ring-like structures of AT2 cells, representing LBs as determined by high-resolution fluorescence microscopy (Fig 2D).

The lipid composition of the bronchoalveolar lavage is altered in Lamp3 -/mice
Given the apparent differences in SP-C levels and mature SP-B in protein extracts of lung tissue lysates, we next analyzed the lipid composition of the BAL and total lung homogenates by a semi-targeted shotgun lipidomics approach. After shotgun lipidomics, a total of 208 different lipid species in the BAL were quantified. We first determined if the two groups (wildtype and Lamp3 -/-BAL lipids) could be separated by analyzing the lipidomics dataset by unsupervised principal component analysis (PCA) (Fig 3A). The apparent separation was characterized by increased PGs and LPG levels in the wildtype while SMs are increased in the Lamp3 -/mice. To gain better insight into the perturbations on the lipid species level, we further applied hierarchical clustering (Fig 3B). In conjunction with the PCA, wildtype and Lamp3 -/mouse BAL lipid profiles were clearly distinguished by the underlying Spearman Rank correlation. According to the genotype, the abundance of lipid species correlated for PG, LPG, and DAG, showing a relative increase in wildtype mice, which was also confirmed by statistical analysis for the selected indicated lipids species (Fig 3C). In contrast, PI and LPI were decreased in Lamp3 -/mice. Notably, PG is an essential component of the pulmonary surfactant, and as multiple lipids of the same class were affected, a disruption of the normal BAL physical properties is likely. In contrast to BAL's lipid profiles, no statistical differences were identified between wild type and Lamp3 -/mice by lipidomic analysis of the perfused total lung tissue (S1A-S1C Fig). These findings, in summary, indicate that LAMP3 deficiency leads to subtle but statistically significant changes in the lipidome and particularly surfactant-relevant lipids of the BAL.

Lamp3 knockout leads to rare changes in the ultrastructure of lamellar bodies and altered biophysical properties of pulmonary surfactant
Defects in surfactant homeostasis are typically accompanied by a change of the ultrastructure of LBs [11,12,24]. This finding prompted the analysis of the Lamp3 -/lungs by transmission Table 1. Steady state lung parameters in wildtype and Lamp3 -/mice. Airway resistance (RI) and dynamic compliance were measured in anesthetized and ventilated mice using FinePointe RC Units. Midexpiratory flow (EF50), frequency (f), functional residual capacity (Frc), minute volume (MV), peak expiratory flow (PEF), peak inspiratory flow (PIF), expiration time (Te), inspiration time (Ti), and tidal volume (TV) were measured in conscious, spontaneously breathing mice using FinePointe non-invasive airway mechanics (NAM) double chamber plethysmography. Values displayed as mean ± SEM, n = 7 per genotype. electron microscopy, focusing on LBs in the AT2 cells. Lamellar bodies from wildtype mice had the typical lamellar appearance (Fig 4A). In one of the three Lamp3 -/mice that were analyzed by transmission electron microscopy, conspicuous lamellar bodies could occasionally be observed within AT2 cells (Fig 4A). They consisted of several clews of lipid lamellae and seemingly homogenous areas that were surrounded by a common limiting membrane. At higher magnification, the homogenous areas also revealed lamellae that were not immediately obvious at lower magnification (Fig 4A). However, lamellar bodies in the other two animals had a regular appearance comparable to the wildtype. Accordingly, we tested the surfactant's biophysical properties and functionality obtained from BAL of wildtype and Lamp3 -/mice to address any functional relevance of such morphological alterations. Surfactant samples were investigated during dynamic compression of an air bubble in a pulsating bubble [34]. The surface tension (γ) of wildtype and Lamp3 -/mice is summarized at minimum (γ min ) (Fig 4B) and maximum (γ max ) bubble size (Fig 4C). Surfactant standardized at a phospholipid concentration of 1.5 mg/ml of both wildtype and Lamp3 -/mice reached a low physiological γ min close to 0 mN/m during the observational period of 300s, respectively (Fig 4B). During the compression period, γ min (0.95 vs 3.6 mN/m, median, p<0.01) and γ max (29.2 vs. 29.9 mN/m, median, p<0.0001) in Lamp3 -/mice were found to be moderatly lower compared to wild type surfactant. Thus initially, γ min of samples from Lamp3 -/mice decreased faster, reaching <10 mN/m after 40s compared to wildtype surfactant after 80s (mean) (Fig 4C), indicating a slightly increased adsorption property of surfactant from Lamp3 -/mice.

Lamp3 -/mice show increased airway resistance and aggravated BAL lipid changes under pathological conditions
The overall phenotype of Lamp3 -/mice was mild and did not affect lung function to the extent that functional lung tests could reveal alterations in healthy, spontaneously breathing animals  C)) and maximum (γmax; B) bubble size, derived from a pulsating bubble surfactometer [34] during a pulsating period of 300s. Eight pellets of BAL from Lamp3 -/and wild type mice were prepared by ultracentrifugation and resuspended in saline CaCl 2 to standardize phospholipids to 1.5 mg/ml each. Phospholipid concentration was determined using a commercial kit. Data are plotted as mean +/-SD. Statistical analysis was performed using an unpaired t-test.
https://doi.org/10.1371/journal.pgen.1009619.g004 (Table 1). We, therefore, wondered if LAMP3 is more critical under challenged, i.e., pathological conditions. We applied a well-described model of experimental allergic asthma [25,35] (Fig  5A) that is characterized by impaired lung physiology arising on the basis of allergic airway inflammation, mucus hyperproduction, and airway hyperresponsiveness (AHR), but provides the advantage that LAMP3-expressing AT2 cells remain largely untouched by the induced disease. Analysis of the different immune-cell populations (macrophages, lymphocytes, neutrophils, eosinophils) infiltrating the bronchoalveolar lumen in diseased animals did not unveil any differences between wildtype and Lamp3 -/mice (Fig 5B). The number of mucus-producing goblet cells and the amount of mucus stored in the airway mucosa also did not differ (S3 Fig). The degree of apoptotic cell death as revealed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining between wildtype and Lamp3 -/mice remained unchanged (S4 Fig). Remarkably, when animals were subjected to MCh-provocation testing to determine the degree of AHR, Lamp3 -/mice displayed significantly increased airway resistance in response to MCh-inhalation (Fig 5C). In contrast, baseline airway resistance values did not differ between OVA-sensitized and challenged wildtype and Lamp3 -/mice (S5 Fig).
To further investigate the impact of experimental asthma in mice, the lipid composition of the BAL and total lung tissue was determined (Fig 5D-5G). The response to experimentally induced asthma led to further divergence in the BAL lipid composition between Lamp3 -/mice and wildtype. Further increases in PG, LPG, and DAG abundance were observed in wildtype mice while levels of SM and PI increased in Lamp3 -/-. In the lipidomes of the perfused total lung tissue, no further changes were observed (S2 Fig). Hierarchical cluster analysis further revealed that the opposite response in the BAL lipidome of the wildtype and Lamp3 -/mice affected a wider range of lipid species, compared to the healthy animals (Figs 5E and 3B-3D). Spearman Rank correlation between the two distinct groups (wildtype and Lamp3 -/-) further confirmed this lipid phenotype by high correlation factor for the two major branches comprising lipid compositions of 8 animals in each group (Fig 5F). Lipid species of mainly PG, LPG, and DAG classes showed a relative increase in Lamp3 +/+ mice, which were statistically significant for indicated lipid species. In contrast, PI, LPE, Cer, and SM showed increased abundance in Lamp3 -/mice (Fig 5G). In summary, the lipid homeostasis for surfactant production in BAL of Lamp3 -/mice was more disturbed under the pathophysiologic conditions of experimental asthma when compared to the unchallenged animals.

Discussion
The two best-characterized family members of the LAMP family, LAMP1 and LAMP2, are ubiquitously expressed and quantitatively account for a substantial fraction of the lysosomal membrane proteome [21,36]. Though the function of LAMP1 and LAMP2 are still not fully understood, they are supposed to play pivotal roles in the fusion of membranes, autophagy, microtubule-dependent positioning of lysosomes, and especially as structural components of the lysosomal membrane [37]. In contrast, the expression of the structurally related LAMP3 protein is limited to specific cell types, implicating a more defined and tissue/cell-type-specific function. Together with the fact that LAMP3 localizes to LBs rather than lysosomes, a critical function of LAMP3 in LB function and surfactant homeostasis is suggested. In fact, the recent finding that a natural mutation (p.(E387K) in LAMP3 leads to clinical symptoms and pathology similar to the most severe neonatal forms of surfactant deficiency in an Airedale Terrier dog breed suggested a critical role in surfactant biology and lung physiology for LAMP3. Therefore, we aimed to investigate the role of LAMP3 in lung physiology by using a genuine knockout model. To our surprise, our findings obtained from Lamp3 -/mice revealed striking differences to those found in dogs with the recessive missense LAMP3 variant: While lethal  [24], Lamp3 -/mice developed normally and did not display signs of lung pathology and dysfunction after birth. We can only speculate about the apparent difference between the different animal models: On the one hand, in contrast to laboratory mouse strains, dog breeds like the Airedale Terrier were generated by inbreeding that aimed to pronounce or produce distinct anatomical features. Thus, it could not be excluded that other genetic factors, possibly homozygous in the inbred dogs, impact this phenotype. On the other hand, the point mutation (p.(E387K)) found in the dogs resulted in the expression of a LAMP3 variant that could still be detected with a LAMP3-specific antibody in lung tissue [24], suggesting that the overall protein structure of the LAMP3 variant is at least in part untouched by the mutation. This finding leaves the possibility of an impaired function of the mutated variant, which is not the case when LAMP3 is completely knocked-out by CRISPR/Cas9. Another possibility potentially explaining the differences between the complete knockout and the point-mutation is genetic compensation, a process of transcriptional adaptation which acts independently of protein feedback loops [38]. While the knockout might lead to the upregulation of compensatory mechanisms, attenuating the phenotype in mice, the point mutation might not induce such compensatory pathways. These findings, however, should be considered when human patients suffering from surfactant deficiencies are analyzed: Our data suggest that LAMP3 mutations might also present with a mild clinical phenotype, perhaps not immediately presenting with the typical signs of a full surfactant deficiency in human patients suffering from childhood interstitial lung disease. Since neither snap-frozen tissue samples nor BAL from the affected Airedale Terrier puppies could be obtained, characterization of the phenotype caused by a mutation of the LAMP3 gene in dogs is quite limited in relation to the lipidome profile of BAL and the function of the surfactant.
The lipidomics analyses performed in this study are in good agreement with earlier reported quantities of main lipid species and classes by Prueitt et al. [16]. We find PC at 67.1 mol% in wildtype mice (16 mol% neutral lipids excluded as in study) compared to the previously reported 75.2 mol% [16]. The main contributing PC class lipid species is DPPC (PC 16:0/16:0) with 43.9% compared to the 30-60% reported in the literature [14,17,39,40]. PG was found at 9 mol% in agreement with the values reported by others [16]. Our analyses show a clear separation of the genotypes according to different lipid compositions and upon induction of experimental asthma. In the lipidomes of the perfused lung tissues, only minor Lamp3 genotype-dependent changes were observed, which underlines the importance of LAMP3 to experimental groups (wildtype, Lamp3 -/-, wildtype + OVA, Lamp3 -/-+OVA). N = 8 (per genotype), significances calculated for methacholine provocation test after 100 mg/ml methacholine exposure, ns = not significant; 0.05; ���� = p � 0.0001. (D) Twodimensional PCA loadings plot of the BAL lipidome of wild type (black), Lamp3 -/-(green), wild type +OVA (blue), and Lamp3 -/-+OVA (red) mice. The datasets for wild type (black), Lamp3 -/-(green) are identical to (Fig 3A). Each point represents one individual. Arrows show the 10 lipid species with the strongest effect on all principal components. Ellipses indicate the 95% confidence interval of the experimental groups, where PC1 and PC2 describe 48.9% of the variability in the data set. The separation of the groups is in PC1, and PC2 is not complete for all groups. A clear separation between the Lamp3 +/+ +PBS and Lamp3 +/+ +OVA can be seen. The separation between untreated Lamp3 -/and treated Lamp3 -/is not complete. (E) Hierarchical clustering of 140 lipid species (of 208 after application of a 90% occupation threshold) identified in 8 wild type +OVA and 8 Lamp3 -/-+OVA mice BAL fluid samples. Each row represents a lipid species and each column a sample. Red boxes indicate selections shown in (F). The two major branches of the tree represent the genotypes without false assignments. Selected segments of the hierarchical clustered tree where a relative decrease for 21 lipids for Lamp3 -/-, mainly DAG and PG species, compared to the Lamp3 +/+ mice are observed. The lower selection shows, in contrast, a cluster of 26 lipids with a relative increase in quantity in the mutant compared to Lamp3 +/+ . regulate the lipid secretion in the surfactant. It is further noteworthy that the BAL lipid composition was mostly affected by the reduction in the abundance of PGs, LPG, and DAG in the Lamp3 -/mice. In transmission electron microscopy, phospholipids typically appear as bilayer lamellae, whereas neutral lipids typically form homogenous lipid droplets. Thus, the occurrence of conspicuous lamellar bodies in one out of three transgenic mice may indicate altered metabolism and/or trafficking of lipids in AT2 cells; however, the finding that LBs in the other two animals had a regular shape comparable to the wildtype puts this interpretation into perspective.
Remarkably, the alterations of the surfactant lipid and protein composition were not associated with pronounced changes of its adsorption properties, which even appeared to be slightly increased in Lamp3 -/mice. Indeed, this is quite in line with our findings on the lung structure and function of these mice under healthy conditions. Nevertheless, it does not exclude the possibility that an altered surfactant composition could have an impact on lung physiology under disease conditions, when the functional reserves of the respiratory system are exhausted. The altered lipid composition and particularly the increase in SP-C could cause enhanced adsorption of surfactant, and a more detailed functional analysis of surfactant is warranted.
Hence, experimentally induced asthma further reduced the levels of essential surfactant lipids and, in parallel, led to an increase in lipids like SM, PI, and PS. Interestingly lipids containing arachidonic acid (AA, FA 20:4) were elevated in Lamp3 -/mice. Further effects on signaling were not investigated in this study, although AA plays a crucial role as educt of numerous lipid mediators [41]. The increase in SM, PI and PS might indicate a higher degree of cell lysis in conjunction with increased number of immune cells in BAL after OVA treatment and an excessive immune reaction. However, it should be noted that we did not observe major differences in either the numbers, the numbers of apoptotic cells in lung tissues or infiltrating immune cells (e.g. eosinophils, lymphocytes, etc.) in BAL, which indicates that the sensitization and subsequent inflammation of the airways in reaction to allergen inhalation are not impacted by the knockout of Lamp3. In turn, these findings suggest that the changes in surfactant composition and its function give rise to an altered inflammatory response in the airways or eventually related apoptosis of cells.
While our data support a role of LAMP3 in surfactant homeostasis, we can only speculate about the mechanisms of how LAMP3 might regulate surfactant levels secreted by AT2 cells. Different scenarios are feasible: On the one hand, it has been speculated previously that LAMP3 plays a role in surfactant recycling by retrieving remnants of secreted LB via re-endocytosis [4]. On the other hand, other LAMP family members act as accessory subunits of multi-transmembrane spanning proteins like LAMP1/LAMP2 for the lysosomal polypeptide transporter TAPL [42]. Likewise, UNC-46, the C. elegans orthologue of LAMP5, acts as a trafficking chaperone, essential for the correct targeting of the nematode vesicular GABA-transporter UNC-47 [43]. LAMP3 could take over a similar function, e.g., by modulating or regulating the trafficking or stability of one of the integral LB proteins like SP-C (which is synthesized as a type II transmembrane protein).
Interestingly, the transcript-levels of Sftpb and Sftpc were significantly reduced or showed a trend towards a decrease, while protein levels were distinctively increased, suggesting posttranslational mechanisms governing the increased stability thereby leading to higher steadystate protein levels: a finding that could hint towards a stabilizing effect of LAMP3.However, such a mechanism might not be essential and become relevant only under conditions where surfactant levels need to be regulated more tightly. It is interesting to note that an aggravation of the lipid changes under the pathological conditions of experimental allergic asthma was associated with the lung function differences observed after MCh-provocation, when lung function was pushed to the limit. In sensitized animals, inhalation of allergen aerosols results in an acute inflammatory reaction with infiltration of T helper 2 (TH2) cells and eosinophils into the airways and the release of a plethora of cytokines, chemokines, and cytotoxic metabolites [35]. Consequently, this causes activation of submucosal glands and differentiation of goblet cells, ultimately resulting in increased mucus production. TH2-type cytokines, together with eosinophil products, trigger the development of AHR that leads to an increased airway resistance in response to non-allergic stimuli and the formation of the typical asthma attack [44]. Therefore, in mice with experimental allergic asthma, the degree of allergic airway inflammation is closely correlated with the degree of mucus production, and AHR. Remarkably, this was not the case in Lamp3 -/mice: Numbers of inflammatory cells such as eosinophils and lymphocytes as well as the number of goblet cells and amount of mucus stored in the airway mucosa are not different between wildtype and Lamp3 -/mice. The lack of any differences between Lamp3 -/and wildtype animals could be explained by the fact that the pathologic features of experimental allergic asthma are restricted to the airways, while the knock-out of Lamp3 affects AT2 cells, which are located in the alveoli, which remain largely untouched. Nevertheless Lamp3 -/mice displayed a markedly increased airway resistance in response to MCh inhalation. Since this response appeared to be independent of both the degree of airway inflammation and mucus production, respectively, it is possible that the increase in airway resistance was not compelling due to an increased AHR. Whether the alterations of the surfactant lipid and protein composition, which we found in healthy Lamp3 -/mice, could be causative for the observed impairment of lung function remains speculative. To our knowledge alteration of surfactant lipid or SP-B/C composition have not been connected to increased MCh-response of the airways yet, the surfactant film lining the alveoli and conducting airways has an impact on airway resistance. Hence, during expiration this film can develop high surface pressure and thus low surface tension, respectively, which in turn counteracts the tendency of liquid to accumulate in the airway's most narrow section [45]. At this point, alterations of the surfactant composition may result in an increased liquid accumulation and consequently blocking of terminal airways, which further leads to an increased airway resistance.
Taken together, whereas the lack of LAMP3 appears to be compensable under physiological conditions, it impacts basal surfactant homeostasis and lung lipid composition and airway resistance in experimental allergic asthma.