Clinical Development of Sphingosine as Anti-Bacterial Drug: Inhalation of Sphingosine in Mini Pigs has no Adverse Side Effects
Henning Carstensa Fabian Schumacherb,c Simone Keitschc Melanie Kramerc
Claudine Kühnc Carolin Sehlc Matthias Soddemannc Barbara Wilkerc
Daniel Herrmannb Ashraf Swaidanc Burkhard Kleuserb Rabea Verhaeghc
Gero Hilkend Michael J. Edwardse Marko Dubicanacd Alexander Carpinteiroc,f
Andreas Wissmannd Katrin Anne Beckerc Markus Kamlera Erich Gulbinsc,e
aThoracic Transplantation, Thoracic and Cardiovascular Surgery, University Clinic, University of Duisburg-Essen, Essen, Germany, bFaculty of Mathematics and Natural Science, Institute of Nutritional Science, Department of Toxicology, University of Potsdam, Nuthetal, Potsdam, Germany, cDepartment of Molecular Biology, University Clinic, University of Duisburg-Essen, Essen, Germany, dCentral Animal Facility, University Clinic, University of Duisburg-Essen, Essen, Germany, eDepartment of Surgery, College of Medicine, University of Cincinnati, Cincinnati, Ohio, USA, fDepartment of Hematology, University Clinic, University of Duisburg-Essen, Essen, Germany
Key Words
Sphingosine • Inhalation • Lung • Side effects • Inflammation • Cell death
Abstract
Background/Aims: Pulmonary infections with Pseudomonas aeruginosa (P. aeruginosa) or Staphylococcus aureus (S. aureus) are of utmost clinical relevance in patients with cystic fibrosis, chronic obstructive pulmonary disease, after trauma and burn, upon ventilation or in immuno-compromised patients. Many P. aeruginosa and S. aureus strains are resistant to many known antibiotics and it is very difficult or often impossible to eradicate the pathogens in patient´s lungs. We have recently shown that the sphingoid base sphingosine very efficiently kills many pathogens, including for instance P. aeruginosa, S. aureus or Acinetobacter baumannii, in vitro. In vivo experiments of our group on cystic fibrosis mice indicated that inhalation of sphingosine prevents or eliminates existing acute or chronic pneumonia with P. aeruginosa or S. aureus in these mice. We also demonstrated that sphingosine is safe to use for inhalation up to high doses, at least in mice. To facilitate development of sphingosine to an anti-bactericidal drug that can be used in humans for inhalation, safety data on non-rodents, larger animals are absolutely required. Methods: Here, we inhaled mini pigs with increasing doses of sphingosine for 10 days and analyzed the uptake of sphingosine into epithelial cells of bronchi as well as into the trachea and lung and the systemic circulation. Moreover, we measured the generation of ceramide and sphingosine 1-phosphate that potentially mediate inflammation, the influx of leukocytes, epithelial cell death and disruption of the epithelial cell barrier. Results: We demonstrate that inhalation of sphingosine results in increased levels of sphingosine in the luminal membrane of bronchi and the trachea, but not in systemic accumulation. Inhaled sphingosine had no side effects up to very high doses. Conclusion: In summary, we demonstrate that inhalation of sphingosine results in an increase of sphingosine concentrations in the luminal plasma membrane of tracheal and bronchial epithelial cells. The inhalation has no systemic or local side effects.
Introduction
Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) are ubiquitous and opportunistic pathogens that cause severe respiratory tract and systemic infections, especially among patients with cystic fibrosis (CF), previous viral infections, burn wounds, trauma, or sepsis [1-6]. Worldwide, P. aeruginosa has become the most common gram-negative pathogen associated with community-acquired pneumonia (17% of all cases), nosocomial pneumonia (25% of all cases) [7], and ventilator-associated pneumonia in intensive care units (28% of all cases) [8]. Reported mortality rates associated with ventilator-associated pneumonia range from 33% to 72% [8]. Chronic airway infections with P. aeruginosa are also common among patients with bronchiectasis and advanced-stage chronic obstructive pulmonary disease (COPD) [9], which affects approximately 15% of the populations of North America and Europe. Intermittent colonization with P. aeruginosa is observed in approximately 30% of patients with COPD. Chronic P. aeruginosa airway infections that lead to substantial morbidity and mortality rates occur among 5% of COPD patients [10].
However, most important are P. aeruginosa and S. aureus infections among patients with CF. CF is the most common recessively inherited disorder in North America and Europe [11, 12]: more than 80, 000 persons with CF live in the United States and Europe alone [https://www.cff.org]. The most frequent cause of morbidity and mortality among CF patients is chronic pulmonary infection with bacterial pathogens, in particular P. aeruginosa [13, 14]. Approximately 80% of all CF patients are chronically infected with P. aeruginosa by the age of 25. Many strains of P. aeruginosa and S. aureus are highly resistant to existing antibiotics, and attempts to eradicate pulmonary P. aeruginosa or S. aureus among CF or COPD patients usually fail [15, 16]. Thus, it is important to develop novel strategies for treating pulmonary infections caused by P. aeruginosa and S. aureus.
We and others have recently reported that the lipid sphingosine, a sphingoid long-chain base that is generated from ceramide via the enzyme (acid) ceramidase, efficiently kills many bacterial species in vitro and in vivo, including P. aeruginosa, S. aureus (even MRSA), Acinetobacter baumannii, Escherichia coli, and Neisseria meningitides [17-23]. Our studies demonstrated that sphingosine is abundantly expressed on the luminal surface of human nasal epithelial cells obtained from healthy subjects and on epithelial cells of trachea and conducting bronchi in healthy humans and wild-type mice, whereas it is almost undetectable on the surface of nasal epithelial cells from CF patients and on tracheal and bronchial cells from CF mice [17, 18, 22]. Thus, CF mice and patients exhibit a severe reduction of sphingosine concentrations in airway epithelial cells. Mechanistically, we have shown that the initial accumulation of ceramide in bronchial and tracheal epithelial cells seen in CF results in trapping and ectopic surface expression of β1 integrins in these epithelial cells, and that these integrin molecules, via still-unknown mechanisms, mediate a downregulation of acid ceramidase expression [18]. This downregulation of the expression of acid ceramidase, which converts ceramide into sphingosine, results in a feed-forward cycle of additional ceramide accumulation and β1-integrin clustering and in a further marked reduction in sphingosine levels [18].
Most importantly, treating CF mice with inhaled sphingosine eliminated existing chronic pulmonary P. aeruginosa infections and prevented new P. aeruginosa or S. aureus infections in these mice [17, 18], a finding demonstrating that sphingosine plays a key role in the innate and immediate defense of the upper respiratory tract. Likewise, the inhalation of recombinant human acid ceramidase by CF mice restored epithelial airway sphingosine levels and reversed acute and chronic infection with P. aeruginosa [18].
These studies indicate that sphingosine efficiently kills pathogens, but they did not investigate toxic effects of sphingosine. To develop sphingosine to a drug against bacterial pneumonia, it is essential to test for any side effects of inhaled sphingosine in different animal models. Here, we inhaled mini pigs with increasing doses of sphingosine and determined sphingosine uptake into bronchial epithelial cells, a possible generation of ceramide and sphingosine 1-phosphate, death of bronchial epithelial cells, influx of inflammatory cells into the lung and blood parameters. Sphingosine was incorporated into bronchial epithelial cells after inhalation. We did not observe any toxic or pro-inflammatory side effects of inhaled sphingosine.
Materials and Methods
Mini pigs
Goettingen mini pigs were obtained at an age between 15 and 25 weeks from Ellegaard Minipigs A/S, Soroe Landevej 302, DK-4261 Dalmose, Denmark. The pigs were allowed to adapt onto the new environment for at least 7 days. Animals were investigated according to FELASA criteria and were free of pig-specific pathogens (SPF-status). The health status was again tested upon arrival. Pigs were further tested for nasal methicillin-resistant S. aureus as well as fecal Campylobacter, Salmonella, Shigella, Aeromonas and Yersinia species. All tests were negative except a colonization of two pigs with Aeromonas caviae. The two pigs did not show any symptoms of a gastrointestinal infection and no further treatment was initiated. We also investigated for gastrointestinal parasites. These tests were all negative. Animals were prophylactically treated with Baycox (Toltrazuril, 20 mg/kg) against Coccidiosis. In one pig we detected Coccidia oocytes in the feces, which rapidly disappeared after treatment.
Pigs were housed within the pig facility of the University Hospital Essen, University of Duisburg-Essen, Germany. All procedures performed on the animals were approved by the LANUV, Recklinghausen, Germany, under the permission number # 84-02.04.2017.A266.
Inhalation experiments
Sphingosine (SPH) (Avanti Polar Lipids, Alabaster, USA) was resuspended as a 20 mM stock solution in distilled water containing 10% octylglucopyranoside (OGP) (Sigma). Prior to use, the sphingosine stock was sonicated in a bath sonicator for 10 min to promote the formation of micelles. The sphingosine stock was diluted in 0.9% saline to a 10 mM, 125 mM, 250 mM or 1 mM solution. Pigs inhaled 3 mL of these 10 mM, 125 mM, 250 mM or 1 mM sphingosine solution in 0.9% NaCl/0.005%, 0.0625%, 0.125% or 0.5% OGP, respectively. Control pigs were inhaled with 0.9% NaCl only or with 0.125% OGP in 0.9% NaCl or left untreated. Pigs were inhaled using a NEBUTEC device (# DM-2000/SaDoMa®-II, Germany), which produced particles of 3.54 mm size, which corresponds to the size often used in inhalation devices for treatment of cystic fibrosis patients. Pigs were inhaled twice daily for 14 days, except on weekends. This regimen is similar to planned clinical phase I and II studies on volunteers and patients, in which we would inhale 1-2-times per day over a period of 14 days with a break on the weekends for safety reasons. Pigs were placed in a hammock for inhalation and manually held in the hammock. The entire inhalation procedure was trained with the pigs using 0.9% NaCl for inhalation for at least 21 days prior to inhalation of sphingosine. This period of training allowed us to perform the subsequent experimental inhalation without stressing the animals and without any sedation, which may impact the inhalation.
Biopsies
Biopsies from the bronchi were obtained 17 hrs after the last inhalation of sphingosine. To this end, the pigs were sedated using intramuscular injection of Ketamine (30 mg/kg body weight) and Xylazine (2 mg/kg body weight) and anesthetized by intravenous injection of Ketamine and Midazolam until disappearance of relevant reflexes has been achieved. Pigs were then subjected to a bronchoscopy employing a fiberoptic videoscope from Ambo A/S, Baltorpbakken 13, DK-2750 Ballerup, Denmark. An illuminated spattle was used to push tongue and larynx caudally to allow intubation of the trachea. The bronchial system was examined for pathological changes especially signs of inflammation, epithelial disintegration and tumors. The videoscope was then positioned in target areas. Biopsies from the larger bronchi for histological and biochemical studies were taken using toothed (alligator) forceps and biopsies. The biopsy specimens were immediately fixed in 4% PFA for 40 hrs or shock-frozen in liquid nitrogen.
Uptake studies
To determine the uptake of sphingosine into the lung, circulation and other organs, we inhaled one group of pigs (n=4) once with 3.8 mL of 125 mM deuterated sphingosine (D-erythro-sphingosine-d7, Avanti Polar Lipids, # 860657P). The pigs were sacrificed 60 mins after this inhalation and tissue samples were obtained from the trachea, lungs (bronchi, alveoli in the periphery), liver, kidney, spleen and blood. The samples were immediately shock-frozen and analyzed by mass spectrometry as described below.
Blood samples
In addition to obtaining tissue biopsies, we collected venous blood samples from the ear veins of the pigs and analyzed blood cell counts and a panel of blood chemistry values.
Mass spectrometry
Biopsies were shock-frozen for mass spectrometry.
Sphingolipids. Porcine tissue was homogenized in aqueous buffered solution with a Bead Ruptor 12 (Omni International, Kennesaw, USA). Aliquots of the homogenates were subjected to lipid extraction with 1.5 mL methanol/chloroform (2:1, v:v) as described [24]. The extraction solvent contained C17-ceramide (Cer17), C16-d31-sphingomyelin (d31-SM16), d7-sphingosine (d7-SPH) and d7-sphingosine 1-phosphate (d7-S1P) (all Avanti Polar Lipids) as internal standards. To study the uptake of inhaled deuterated sphingosine, the latter two internal reference compounds were replaced by C17-sphinganine (C17 dhSPH) (Avanti Polar Lipids). Lipid extracts were subjected to LC-MS/MS analysis using a 1260 Infinity HPLC coupled to a 6490 triple-quadrupole mass spectrometer (both Agilent Technologies, Waldbronn, Germany) operating in the positive electrospray ionization mode (ESI+). Sphingolipids and their deuterated analogues (m/z ratios given in parentheses) were analyzed by selected reaction monitoring (SRM) using the following mass transitions: m/z 288.5 → 270.5 for C17 dhSPH, m/z 300.3 (307.3) → 282.3 (289.3) for SPH, m/z 380.3 (387.3) → 264.3 (271.3) for S1P, m/z 520.5 (527.6) → 264.3 (271.3) for Cer16, m/z 534.5 → 264.3 for Cer17, m/z 548.5 (555.6) → 264.3 (271.3) for Cer18, m/z 576.6 (583.6) → 264.3 (271.3) for Cer20, m/z 604.6 (611.6) → 264.3 (271.3) for Cer22, m/z 630.6 (637.7) → 264.3 (271.3) for Cer24:1, m/z 632.6 (639.7) → 264.3 (271.3) for Cer24, m/z 703.6 (710.6) → 184.1 for SM16, m/z 731.6 (738.7) → 184.1 for SM18, m/z 734.8 → 184.1 for d31-SM16, m/z 759.6 (766.7) → 184.1 for SM20, m/z 787.7 (794.7) → 184.1 for SM22, m/z 813.7 (820.7) → 184.1 for SM24:1 and m/z 815.7 (822.7) → 184.1 for SM24. Quantification was performed with MassHunter Software (Agilent Technologies). Determined sphingolipid amounts were normalized to the actual protein content of the tissue homogenate used for lipid extraction.
Fatty aldehydes and fatty acids (d7-SPH uptake study only). Aliquots of tissue homogenates prepared as described above were subjected to fatty acid and aldehyde extraction according to our optimized protocol [25]. Pentadecanal and heptadecanoic acid (both Sigma-Aldrich, Taufkirchen, Germany) served as internal standards. Dried organic extracts were derivatized using 2-diphenylacetyl- 1, 3-indandione-1-hydrazone (DAIH) and N-(3-dimethylaminopropyl)-N′-ethyl- carbodiimide hydrochloride (EDC hydrochloride) (both from Sigma-Aldrich) to form fatty aldehyde and acid derivatives ionizable by negative mode electrospray ionization (ESI-) [26]. To investigate whether inhaled d7-SPH exited the sphingolipid metabolism pathway via S1P lyase-catalyzed S1P break-down, we analyzed the derivatized samples for (2E)-hexadecenal-d7 (2EHD-d7), (2E)-hexadecenoic acid-d7 (2EHD-COOH-d7) and hexadecanoic acid-d7 (palmitate-d7) using the above mentioned LC-MS/MS instrumentation. The following multiple reaction monitoring (MRM) transitions were recorded: m/z 580.4 → 256.2 / 323.1 for 2EHD-d7, m/z 596.4 → 159.1 / 402.4 for 2EHD-COOH-d7 and m/z 598.4 → 159.1 / 404.4 for palmitate-d7. Methodological details and MS/MS parameter of unlabeled fatty aldehydes and acids (including internal standards) that were measured simultaneously can be taken from ref. [26].
Antibodies and reagents
Ceramide or sphingosine stainings were performed with the monoclonal mouse anti-ceramide antibody clone S58-9 (#MAB_0011, Glycobiotech), or the monoclonal mouse anti-sphingosine antibody clone NHSPH (#ALF-274042010, Alfresa Pharma Corporation), which were visualized with Cy3 donkey anti-mouse IgM F(ab)2 fragments (#715-166-020; Jackson ImmunoResearch).
Immunohistochemistry
Stainings were performed as previously reported [17, 18, 22, 27, 28]. The PFA-fixed tissue biopsies were serially dehydrated with an ethanol to xylol gradient and then embedded in paraffin. Samples were sectioned at 7 mm, dewaxed and rehydrated. To achieve retrieval of the antigens, sections were treated with pepsin (Digest All; #003009, Invitrogen) for 30 min at 37°C and then washed with distilled water. Unspecific binding sites were blocked by 10 min incubation with PBS, 0.05% Tween 20 (Sigma) and 5% fetal calf serum (FCS) at room temperature. The samples were then incubated with anti-ceramide- (1:100 dilution) or anti-sphingosine- (1:1000 dilution) antibodies in H/S (132 mM NaCl, 20 mM HEPES [pH 7.4], 5 mM KCl, 1 mM CaCl2, 0.7 mM MgCl2, 0.8 mM MgSO4) plus 1% FCS at room temperature for 45 min. Samples were then washed three times with PBS plus 0.05% Tween 20, 5 min per wash step, and once with PBS. The sections were secondarily labeled with Cy3-coupled anti-mouse IgM F(ab)2 fragments (Jackson Immunoresearch) in H/S plus 1% FCS for 30 min. The sections were again washed three times with PBS plus 0.05% Tween 20, each 5 min, and once with PBS. The samples were embedded in Mowiol and analyzed on a Leica TCS-SP5 confocal microscope employing a 40 × lens. Images were analyzed with Leica LCS software version 2.61 (Leica Microsystems, Mannheim, Germany). All comparative samples were measured at identical settings.
All immunostainings were controlled with control antibodies that showed no or very weak staining. These were mouse IgM for the antibodies against sphingosine and ceramide. We also included controls with secondary Cy3-coupled antibodies only. These controls showed again no or very weak staining.
TUNEL assays
PFA-fixed sections were dewaxed and rehydrated as above. The sections were then treated in 0.1 M sodium citrate (pH 6.0) in a microwave at 450 W for 5 min, washed twice in PBS and the TUNEL reaction was performed with 5 mL TUNEL enzyme, 20 ml TMR label and 25 mL TUNEL dilution buffer following the instructions of the vendor (Roche). Samples were incubated for 60 min at 37°C, washed 3-times in PBS. The samples were then incubated for 10 min at 70oC in PBS to reduce background staining, washed once in PBS, and were finally embedded in Mowiol.
Hemalaun stainings
Lung paraffin sections were dewaxed, rehydrated and washed as above. The specimens were stained for 5 min with hemalaun. Samples were embedded in Mowiol and analyzed on a Leica DMIRE2. In the hemalaun staining studies we used a score for analysis: Grade 0: no change of the epithelial cell layer, basal membrane intact, no evidence of leukocyte influx, less than 2% pyknotic, i.e. dead epithelial cells. Grade 1: small disruptions of the epithelial cell layer, basal membrane intact, very minor leukocyte influx with few singular cells in the epithelial cell layer, less than 5% pyknotic, i.e. dead epithelial cells. Grade 2: Larger disruptions of the epithelial cell layer, basal membrane still intact, scattered leukocyte influx, less than 10% pyknotic, i.e. epithelial dead cells. Grade 3: Larger disruptions of the epithelial cell layer, disrupted basal membrane, massive leukocyte influx, more than 10% pyknotic, i.e. dead epithelial cells.
Quantification and statistical analysis
Data are expressed as arithmetic means ± SD. For the comparison of continuous variables from independent groups we used Student’s t-test for two groups and one-way ANOVA for more than two groups followed by post-hoc Student´s t-tests for all pairwise comparisons applying Bonferroni correction for multiple testing. The p-values for the pairwise comparisons were calculated after Bonferroni correction. All values were normally distributed. The statistical details (n-numbers, mean ± SD and tests) are given in the figure legends. The sample size planning was based on two-sided Wilcoxon-Mann-Whitney tests (software: G*Power Version 3.1.7 of the University of Duesseldorf, Germany). Investigators were blinded for histology analyses and animal identity. Ceramide and sphingosine fluorescence data were quantified using Image J and are expressed as arbitrary units (a.u.). In each specimen 5 randomly chosen areas with 20 different cells were quantified, i.e. 100 cells per specimen were analyzed. We analyzed 3 sections per animal, i.e. 300 cells per animal, in total 1200 cells from the 4 pigs per group. The fluorescence values were averaged with the values obtained in the other photos of the fluorescence microscopy studies. In the TUNEL and hemalaun stainings we analyzed again 300 epithelial cells per animal in 3 sections, a total of 1200 cells in all 4 pigs per group. The results are expressed as percentage of cells. For studying epithelial cell integrity, we used the above-described score.
Results
Inhalation of mini pigs with sphingosine for 14 days, i.e. 10 days of inhalation and breaks on the weekends, did not result in an obvious change of the health status of the mini pigs. We did not observe any inflammation in the nose, mouth or eyes, nor any systemic changes such as lack of activity, reduced food intake, weight loss or any other change of the general health status. We also did not observe any adverse behavior of the pigs during the inhalation.
Analysis of the histological sections by immunofluorescence microscopy demonstrated an accumulation of sphingosine specifically in bronchial epithelial cells after inhalation. Interestingly, we observed a maximum of the local sphingosine concentration with inhalation of a 125 mM sphingosine suspension (Fig. 1A and B). The inhalation of 250 mM and 1 mM sphingosine suspension did not result in a further increase of sphingosine in the epithelial cell layer (Fig. 1A and B).
In contrast to the accumulation of sphingosine in bronchial epithelial cells we did not observe an increase of ceramide in bronchial epithelial cells by immunofluorescence microscopy (Fig. 2A and B).
Next, we investigated the distribution and metabolism of inhaled sphingosine applying deuterated (d7) sphingosine and performing measurements after 60 minutes. Mass spectrometry analysis of the biopsies from pigs that were inhaled with 3.8 mL of a 125 mM suspension of deuterated (d7) sphingosine revealed a rapid metabolism of sphingosine. We detected deuterated sphingomyelin and very low levels of deuterated ceramide in the trachea and the lung (Table 1), but did not detect deuterated sphingosine, sphingosine 1-phosphate, (2E)-hexadecenal or its fatty acid derivatives. We also detected some very low amounts of deuterated sphingomyelin in inner organs such as liver, heart, kidney and spleen (Table 2), although the levels compared to endogenous levels of sphingomyelin were very low (Table 2). Very low levels of d7-ceramide were also detected in the lung, trachea and liver, but not in other organs (Table 3). We only detected d7-C16-ceramide and no d7-C18-, C20-, C22-, C24- and C24:1-ceramide in these tissues. We did not detect deuterated sphingosine or sphingosine 1-phosphate in inner organs.
Consistent with these studies, we did not detect a significant change of sphingosine, ceramide, sphingomyelin or sphingosine 1-phosphate in lung biopsies after inhalation of 10 mM, 125 mM, 250 mM or 1 mM sphingosine compared to pigs that inhaled only 0.9% NaCl solution (Table 4).
Next, we determined death of bronchial epithelial cells, integrity of the bronchial epithelial cell layer and influx of leukocytes into the epithelial cell layer by TUNEL and hemalaun stainings. The results revealed that inhaled sphingosine did not induce any cell death (Fig. 3) and did not disturb the epithelial cell integrity (Fig. 4A and B). Likewise, we did not observe an influx of leukocytes into the epithelial cell layer (Fig. 4A and C).
In addition, blood studies showed that inhalation of sphingosine had no effects on blood parameters (Table 5). We also did not observe any indications of local effects such as an increase of amylase in the blood (Table 5), for instance by affecting the salivary glands.
Discussion
In the present study, we demonstrate that inhalation of sphingosine in healthy mini pigs has no side effects in the trachea and the lung. We also did not detect any systemic side effects of sphingosine. These studies suggest that sphingosine is a safe drug to treat bacterial pneumonia. Future studies on infected mini pigs are required to confirm this notion in infected animals and to prove the efficacy of sphingosine to eliminate bacteria in mini pig lungs in vivo.
Our data are consistent with previous data obtained in mice [29]: In these studies healthy wild-type mice were inhaled with sphingosine in 0.9% NaCl at concentrations ranging from 10 mM to 1 mM twice daily for 14 days. Similar to the present results, we did not observe any side effects, i.e. induction of inflammation, influx of leukocytes, cell death or disruption of the epithelial cell layers [29].
The local immune defense requires sphingosine in the luminal layer of the tracheal and bronchial epithelial cells [17, 18]. Thus, it is very important to determine the local concentration of sphingosine in the epithelial cell layer, which we performed by immune stainings of bronchial biopsies. The confocal microscopy studies indicate that inhalation of 10 mM sphingosine results in an approximately 3-fold increase of the surface sphingosine concentration in the bronchial epithelial cell layer. The inhalation of 125 mM sphingosine resulted in an approximately 7-fold increase of the local sphingosine concentration. These concentrations were previously shown to be sufficient to eliminate P. aeruginosa and S. aureus in pulmonary infected cystic fibrosis mice [17, 18, 22].
Surprisingly, we did not observe a significant increase of sphingosine in bronchial epithelial cells when we inhaled higher doses of sphingosine, i.e. 250 mM or 1 mM sphingosine. It might be possible that sphingosine forms larger micelles at higher concentration that remain in the nose and larynx and/or the mucus and do not reach the epithelial cell layer. In addition, higher concentrations of sphingosine might also trigger the expression of sphingosine kinases and sphingosine 1-phosphate lyase resulting in rapid degradation of sphingosine. This means that the optimal dose for a treatment with inhaled sphingosine would be a 100 - 125 mM suspension.
The confocal microscopy and mass spectrometry data consistently showed that the concentrations of ceramide did not significantly change after inhalation. The mass spectrometry data also excluded an accumulation of sphingosine 1-phosphate in the trachea and lung after inhalation of sphingosine.
Previous studies from our laboratory indicated that a relatively large portion of sphingosine remains in the mucus on top of the epithelial cell layer [30]. In these experiments, sphingosine was locally applied onto the surface. It is technically very difficult to measure the exact concentration of sphingosine in the mucus after inhalation, because the mucus is washed off during fixation and dehydration of histology sections. Biopsies contain a small surface portion and most of the tissue in biopsies consists of submucosal tissue; thus, sphingosine in mucus on the surface is difficult to be determined in biopsies. Lavage fluid would also not reflect the local concentration of sphingosine in the luminal membrane of bronchial epithelial cells, which are not present in the lavage fluid. Further, the lavage fluid contains surface mucus from different areas of the bronchial tree and even alveoli. The local surface concentration of sphingosine could be determined in frozen sections that are subjected to immune fluorescence staining or, as best option, the local surface concentration of sphingosine could be determined by a surface kinase assay specifically measuring the concentration of sphingosine in the mucus and the luminal membrane of the epithelial cell layer in the trachea and the bronchi as previously described [18, 30].
The mass spectrometry experiments using deuterated d7-sphingosine suggested that the half-life time of sphingosine in the respiratory tract is very short and that most of the inhaled sphingosine is rapidly consumed. We did not detect a significant difference of d7-sphingomyelin amounts between the center of the lung and the periphery of the lung, indicating that we achieve a relatively homogenous distribution of the inhaled d7-sphingosine. The trachea and the lung together contain approximately 20 pmol d7-sphingomyelin/mg protein. The pieces of tissue, which we investigated for mass spectrometry contained approximately 1 cm2 epithelial cell layer and certainly much more other cells from the submucosa, cartilage, etc. We inhaled 475 nmol deuterated sphingosine. If we assume that the lung of a pig has a surface of only 10 m2 (the surface of the human lung is 100 m2 and the pigs have a weight of 20 kg and, thus, the surface of the lung is more likely close to 20 m2) the local concentration of the d7-sphingosine would be approximately 5 pmol/cm2. This suggests that most of the d7-sphingosine was inhaled, but rapidly degraded. It should be indicated that the organ concentrations of endogenous sphingomyelin are 500 - 20 000-fold higher than the amount of d7-sphingomyelin and, thus, they are not significantly changed by the inhalation.
It should be noted that the studies on biopsies shown in Table 4 were done with very small biopsies obtained by bronchoscopy, while the studies measuring d7 sphingosine in Table 3 were performed with larger specimen obtained after sacrificing the pigs. This very likely also explains the high standard deviations in the samples reported in Table 4, i.e. the bronchoscopy biopsies. Further, the bronchoscopy biopsies might be more variable and contain variable amounts of epithelial cells.
The fluorescence microscopy studies that were performed on tissues from pigs that were inhaled 17 hrs prior to the biopsies show a higher concentration of sphingosine in luminal membranes of the bronchial epithelial cells. The discrepancy between the mass spectrometry and immunofluorescence studies suggests that surface sphingosine within the plasma membrane is relatively stable, while sphingosine that is taken up by cells and the tissue is rapidly consumed and degraded. However, the luminal surface concentration of sphingosine has been shown to be approximately 1 pmol per total trachea surface in mice [17, 18]. This is beyond the detection limit of the employed mass spectrometry. We have previously shown that the anti-sphingosine antibody specifically detects sphingosine and does not detect sphingomyelin [17, 18]. The exact metabolism of sphingosine after inhalation and in particular the local surface concentration need to be determined in future studies employing surface kinase assays measuring the local sphingosine concentration using the activity of a sphingosine kinase in the presence of [32P]gATP as previously described [17, 18]. These studies should also include detailed kinetics of the sphingosine concentration in the epithelial cell layer and the mucus on top of the epithelial cell layer.
Conclusion
In summary, we demonstrate that inhalation of sphingosine results in an increase of sphingosine concentrations in the luminal plasma membrane of tracheal and bronchial epithelial cells. The inhalation has no systemic or local side effects.
Acknowledgements
HC and AC performed the bronchoscopies, FS and BK the mass spectrometry. SK, MK, CK, CS, RV and AS inhaled the pigs, MK and CK performed blood studies. AW and MD supervised housing of the pigs and performed the anesthesia. GH, MJE, MK, KAB and EG designed the studies. EG initiated the studies, supervised research and analyzed the histology studies.
Ethical Statement
All animal experiments conform to internationally accepted standards and have been approved by the appropriate institutional review body, i.e. LANUV, Recklinghausen, Germany, under the permission number # 84-02.04.2017.A266.
Funding Sources
The study was supported by DFG grant GU 335/35-1.
Disclosure Statement
The authors have no conflicts of interest to declare.
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