Therapeutic Inhaled Sphingosine for Treating Lung Infection in a Mouse Model of Critical Illness
Nadine Beckmanna Amanda M. Pugha Nicholas J. Auteria Michael J. Edwardsa
Erich Gulbinsa,b Charles C. Caldwella,c
aDepartment of Surgery, University of Cincinnati, Cincinnati, OH, USA, bDepartment of Molecular Biology, University of Duisburg-Essen, Essen, Germany, cDivision of Research, Shriners Hospital for Children, Cincinnati, OH, USA
Key Words
Pneumonia Pseudomonas aeruginosa Sphingosine Antibiotic resistance Sepsis
Abstract
Background/Aims: Sphingosine, a sphingoid long chain base, is a natural lipid with antimicrobial properties. Recent animal studies have shown that preventive sphingosine inhalation can rescue susceptible mice, such as cystic fibrosis-, burn injured- or aged mice from bacterial pulmonary infection. While preventing lung infections in susceptible patients has obvious clinical merit, treatment strategies for an established infection are also direly needed, particularly in the times of rising antibiotic resistance. Here, we tested the potential of sphingosine in treating an established pulmonary infection. Methods: We used a cecal ligation and puncture (CLP) model in male CF-1 mice and a Pseudomonas aeruginosa strain that was isolated from a septic patient (P. aeruginosa 762). We determined susceptibility to intranasal infection and ascertained when the pulmonary infection was established by continuous core body temperature monitoring. We quantified sphingosine levels in the tracheal epithelium by immunohistochemistry and studied the effects on sphingosine on bacterial membrane permeabilization and intracellular acidification using fluorescent probes. Results: We first determined that septic mice are highly susceptible to P. aeruginosa infection 2 days after inducing sepsis. Additionally, at this time, sphingosine levels in the tracheal epithelium are significantly reduced as compared to levels in healthy mice. Secondly, upon intranasal Pseudomonas inoculation, we ascertained that pulmonary infection was established as early as 2.5 h after inoculation as evidenced by a significant drop in core body temperature. Using these times of infection susceptibility and detection (2 days post CLP, 2.5h after inoculation) we treated with inhaled sphingosine and observed pulmonary bacterial loads reduced to levels found in infected healthy mice after inoculation and decreased infection-associated mortality. Further, our data demonstrate that sphingosine induces outer membrane permeabilization, disrupting the membrane potential and leading to intracellular acidification of the bacteria. Conclusion: Sphingosine shows efficacy in treating P. aeruginosa lung infections not only prophylactically, but also therapeutically.
Pneumonia is the fourth most common cause of death worldwide and the leading cause of death in low-income countries [1]. Both community-acquired and hospital-acquired bacterial pneumonia are associated with increased morbidity and mortality. The typical treatment for these lung infections is supportive care and antibiotics. Despite this care, a recent study from France described treatment failure rates as high as 31 % [2]. This failure rate is likely due to growing appearance of antibiotic resistant bacteria [3]. Cystic fibrosis and Chronic obstructive pulmonary disease patients, the elderly and critically ill patients are at a particularly high risk for pneumonia. Among the hospitalized group, septic patients, patients who suffered a major trauma (i.e. burn victims) and any patients requiring mechanical ventilation are especially susceptible [4-7].
Sphingosine is a natural lipid and part of the innate defense barrier of the skin and airways [8, 9]. The antimicrobial properties of sphingosine against both gram-positive and gram-negative bacteria were first reported in 1992 [10]. Bacterial killing occurs at low micromolar concentrations [10-13], but the bactericidal mechanism is still unknown. Studies with artificial liposomes and ghost membranes have shown that sphingosine can permeabilize membranes [14, 15]. Additionally, changes in the cell surface of sphingosine-treated bacteria were observed using electron microscopy [12]. The cytoplasmic membrane of the tested Gram-negative bacteria (Escherichia coli) appeared intact, whereas the tested Gram-positive bacteria (Staphylococcus aureus) showed signs of membrane disruption [12]. Taken together, this indicates that sphingosine may kill Gram-positive bacteria through membrane permeabilization, but how death of Gram-negative bacteria is induced if the inner membrane stays intact remains unclear.
Recently, animal studies have shown that sphingosine inhalation can prevent pulmonary infections with common pathogens such as Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) [13, 16-19]. No toxic effects of sphingosine inhalation were observed in mice [20] and mini pigs [7]. In these studies, inhaled sphingosine was administered prior to inoculation with bacteria or at very early time-points after inoculation, successfully preventing pneumonia [13, 16-19]. While preventing a lung infection in high-risk patients altogether is certainly an elegant approach, there is still a high demand for effective therapies for established infections. In contrast to these studies, we tested the therapeutic - and not the prophylactic - potential of inhaled sphingosine. We chose a murine model of polymicrobial sepsis - cecal ligation and puncture (CLP) - since septic patients are among the most vulnerable to pneumonia. We first established when after sepsis mice are susceptible to infection. We used a multi-drug resistant clinical P. aeruginosa isolate (P. aeruginosa 762), as pneumonia caused by P. aeruginosa is a major cause of morbidity and mortality in hospitalized patients and antibiotic resistance is a leading cause for antibiotic therapy failure. We confirmed that sphingosine kills this multi-drug resistant strain. We next determined how long it takes for the mice to develop pneumonia after inoculation with Pseudomonas and then treated the mice with aerosolized sphingosine at this time to determine therapeutic efficacy. To gain a better understanding of the mechanism underlying sphingosine-mediated bacterial killing, we determined bacterial outer and inner membrane permeabilization, inner membrane potential and intracellular acidification upon in vitro sphingosine treatment.
Materials and Methods
Mouse housing
CF-1 outbred mice were obtained from Charles River Laboratories (Wilmington, MA). Male mice aged 6-8 weeks were used for experiments and randomized into experimental groups. Mice were housed in standard environmental conditions with corn-cob bedding in groups of four. A standard pellet diet and water was provided ab libitum. Mice were acclimated for at least one week prior to experimentation. All murine experiments were performed between 8 AM and 1 PM.
Cecal ligation and puncture
A non-lethal CLP (mortality rate less than 10 %) was used to induce polymicrobial sepsis as previously described [21, 22]: Briefly, mice were anesthetized with 2% isoflurane in oxygen, shaved over the abdominal area, disinfected with povidone-iodine, and placed supine on a heating pad. Following a 1 to 2 cm midline laparotomy incision, 33 % of the cecum was ligated with a 4-0 silk tie (Syneture, Norwalk, CT). The cecum was punctured on the anti-mesenteric side with a 25-gauge needle and a small amount of feces was extruded through the puncture hole to ensure perforation. The cecum was replaced in its original location, and the midline incision was closed in two layers with 4-0 silk. Following CLP, mice were resuscitated with 1 mL sterile normal saline (NS), (Hospira, Lake Forest, IL, USA) injected subdermally and monitored to ensure that they wake up from the anesthesia. They were subsequently allowed to recover on a 42.0°C heating pad for one hour. Mice were then returned to their home cage and monitored for any complications twice a day until the experiment was completed. Sham mice underwent the above process except for ligation and puncture.
Pseudomonas aeruginosa infection
We used P. aeruginosa strain 762, which was isolated from a septic patient (strain fist described in [23]). The strain is non-mucoid and multidrug-resistant. Bacteria from frozen stocks were plated on fresh tryptic soy agar (TSA) plates (BD Biosciences, Franklin Lakes, NJ) and cultured overnight at 37°C. Bacteria were then resuspended in 40 mL Trypticase soy broth medium. The inoculated medium was incubated for 1 h at 37°C with shaking at 125 rpm. Bacteria were washed twice and resuspended in PBS to a concentration of 50 x 106 Colony Forming Units (CFU)/mL based on a standard absorption curve. Mice were then infected with 106 CFU P. aeruginosa 762 (20 µl) intranasally under short anesthesia with 3 % isoflurane using a 31-gauge blunted needle. Mice were returned to their home cage and monitored for any complications twice a day until the experiment was completed. Non-infected mice underwent the same process, but were administered PBS.
Sphingosine immunofluorescence analysis
Sphingosine staining was performed as previously described [13, 19]. Mice were euthanized with CO2 at a 30 % fill rate 4 h after infection and lungs were inflated with 1 mL 10 % neutral buffered formalin (Thermo Fisher Scientific, Waltham, MA), harvested and fixed in 10 % neutral buffered formalin for 48 h. The tissue was then dehydrated with ethanol, embedded in paraffin and sectioned at 5-10 µm. Sections were dewaxed and rehydrated. Antigen-retrieval was conducted with Pepsin Digest All (Invitrogen, Thermo Fisher Scientific) at 37°C for 30 min followed by blocking with 5 % Fetal Calf Serum (FCS). Sections were stained with monoclonal mouse anti-sphingosine antibody (clone NHSPH, Alfresa Pharma Corporation, Osaka, Japan) followed by Cy3-coupled donkey anti-mouse IgM F(ab)2 fragments (polyclonal, RRID AB_2340815, Jackson ImmunoResearch, West Grove, PA). Sections were washed, embedded with Mowiol and analyzed on a Leica confocal microscope (Leica, Wetzlar, Germany). 3-4 sections per group were analyzed and 15-20 vision fields of tracheal epithelial cells per section were imaged at 400x magnification. The sphingosine signal in these images was quantified using ImageJ software (NIH, Bethesda, MD). The specificity of the anti-sphingosine antibody has been previously described. Briefly, binding to ceramide, sphingosine-1-phosphate, sphinganine, sphingomyelin, cerebroside, gangliosides, phosphatidylserine, lyso-phosphatidylserine and lyso-phosphatidylcholine has been excluded [13].
Determination of bacterial load
Mice were euthanized with CO2 at a 30 % fill rate 4 h after infection with P. aeruginosa 762 and bronchoalveolar lavage (BAL) fluid was harvested: 1 mL of sterile normal saline was flushed through the trachea into the lungs with a 20-gauge catheter as previously described [19]. Approximately 0.7 mL of fluid was then recovered through the same syringe. Samples were serially diluted in sterile normal saline and cultured on TSA plates at 37°C. Colonies were counted the next morning.
In vitro antimicrobial activity
Bacteria were prepared as described above for both P. aeruginosa 762 and ATCC19660 strains. Sphingosine was diluted in normal saline and sonicated 10 min prior to use. 106 CFUs were treated with 100 µM sphingosine or normal saline for 4 h at 37°C. Samples were then diluted in normal saline, plated on TSA plates and incubated at 37°C. Colonies were counted the next day.
Core body temperature monitoring
An Anipill temperature-monitoring implant (Data Science International, New Brighton, MN) was inserted into the abdomen during the CLP surgery. Core temperature was recorded electronically every 15 min until the mice were sacrificed.
Sphingosine inhalation
Mice were stratified into two groups based on their weight loss after CLP surgery. One group received aerosolized normal saline and the other aerosolized sphingosine. In preparation, sphingosine (D-erythro-sphingosine [d18:1], Tocris, Minneapolis, MN) was diluted to 125 µM in normal saline and sonicated for 10 min. Mice were restrained in a Buxco Inhalation Tower (Data Science International) and inhaled 333 µl/mouse 125 µM sphingosine for 15 min at -0.25 cm H20 pressure and with an In Flow of 0.25 l/min/mouse 2.5 h after infection with P. aeruginosa 762. The time for inhalation was chosen based on the results of core body temperature monitoring in response to pulmonary infection with P. aeruginosa 762. Fig. 1 summarizes the experimental timeline.
Assessment of outer membrane permeabilization
Outer membrane permeabilization was assessed using N-phenyl-1-naphtylamine (NPN) as previously described [24]. Briefly, when the integrity of the outer membrane of Gram-negative bacteria is disturbed, its barrier properties are compromised and NPN accumulates in its hydrophobic core, resulting in increased fluorescence [25-27]. Bacteria (P. aeruginosa 762) were prepared as described for pulmonary infection. 3 x 106 CFUs were plated in 96 well plates. Sphingosine was sonicated for 10 min. NPN was added to a final concentration of 10 µM. Sphingosine was added to a final concentration of 100 µM sphingosine. Positive controls were treated with ethanol at a final concentration of 67 % to determine maximum NPN uptake and blanks without bacteria were assessed to subtract background fluorescence. Measurements were made using a Cytation 5 microplate reader (BioTek, Winooski, VT) and initiated immediately after compound addition. The signal was read every minute for five minutes for four wells per group and results were averaged. Background fluorescence was subtracted and the signal obtained for ethanol-treated samples defined as 100 %. NPN uptake was then calculated for sphingosine and vehicle-treated samples.
Assessment of inner membrane permeabilization
Inner membrane permeabilization was assessed using propidium iodide (PI). PI fluorescence increases upon binding to nucleic acids, but PI cannot cross intact membranes. Bacteria (P. aeruginosa 762) were prepared as described for pulmonary infection. 2 x 105 CFUs were plated in 96 well plates. Sphingosine was sonicated for 10 min. PI (ThermoFisher Scientific) was added to a final concentration of 60 µM. Sphingosine was added to a final concentration of 100 µM sphingosine. Positive controls were treated with ethanol at a final concentration of 67 % to determine maximum PI uptake. Measurements were made using a Cytation 5 microplate reader (BioTek) every 15 minutes over 4 h. The signal obtained for ethanol-treated samples was defined as 100 %. PI uptake was then calculated for sphingosine and vehicle-treated samples.
Assessment of membrane potential
Gram-negative bacteria normally maintain an electrical potential gradient across the inner membrane, which is crucial for a number of physiological processes, including ATP synthesis [28]. DiOC2(3) and JC-1 are positively charged lipophilic probes with green fluorescence that bind to the inner side of the inner membrane in actively growing cells [28]. At higher concentrations, aggregates form in the cytoplasm that result in a shift towards red fluorescence [28]. The ratio of red to green fluorescence can be used to determine bacterial membrane potential and a flow cytometry-based assay has been previously described [29]. Briefly, P. aeruginosa 762 were prepared as described above and stained with 30 µM DiOC2(3) (ThermoFisher Scientific) or 2 µM JC-1. Sphingosine was sonicated for 10 min and then added to the samples for 30 min at a concentration of 100 µM. JC-1 was added to the samples for the final 10 min of incubation with sphingosine. Controls were treated with normal saline. Fluorescence was measured on an Attune NxT flow cytometer (ThermoFisher Scientific) upon excitation with a blue 488 nm laser and detected through 530/30 nm (green) and 590/40 nm (red) emission filters.
Assessment of intracellular acidification
DND-189, also known as LysoSensor Green, is an acidotropic, membrane-permeable dye that can be used to track pH changes in living cells, as its fluorescence increases in acidic environments [30]. Bacteria (P. aeruginosa 762) were prepared as described above and stained with 1 µM DND-189 (ThermoFisher Scientific). Sphingosine was sonicated for 10 min and then added to the samples for 30 min at a concentration of 100 µM. Controls were treated with normal saline. Fluorescence was measured on an Attune NxT flow cytometer (ThermoFisher Scientific) upon excitation with a blue 488 nm laser and detected through 530/30 nm (green) emission filter.
Statistical analysis
Prism software (GraphPad, La Jolla, CA) was used for all statistical comparisons. Survival differences were assessed using log-rank test. ROUT method was used to test for statistically significant outliers and data points were removed when the criteria were met (false discovery rate Q = 1 %). For comparisons between two groups, Student t-test was used, and for multiple comparisons 1-way analysis of variance (ANOVA) with the indicated post-test. Data are graphed as mean + standard error of the mean. A p-value of p ≤ 0.05 was considered statistically significant.
Results
Septic mice are susceptible to pulmonary P. aeruginosa infection up to three days after CLP and their airway sphingosine levels are reduced
Pulmonary infections with P. aeruginosa are a major cause of morbidity and mortality in hospitalized patients [31]. Septic patients, in particular, are vulnerable to P. aeruginosa pneumonia [32]. Antibiotic resistance is a leading cause for antibiotic therapy failure, so novel therapeutics are direly needed. Sphingosine inhalation has been shown to protect mice from pneumonia [13, 16-19]. However, it is currently still unclear if it can mitigate an established pulmonary infection. We first tested when after sepsis mice are susceptible to lung infection. For this, we infected mice 1 day, 2 days, 3 days and 4 days after CLP surgery. Sham operated mice were infected on respective days and assessed as controls. We did not observe mortality in any of the sham-operated cohorts or in the septic mice that were infected on day 4 (Fig. 2A). For the other septic cohorts, the mortality rates were approximately 60 % for infection on day 1, 75 % for infection on day 2 and 40 % for infection on day 3 (Fig. 2A).
The susceptibility to pneumonia has previously been linked to reduced levels of sphingosine in the airways [13, 16-19]. To test for a possible reduction of sphingosine levels in our septic mice, we harvested lungs on those days after surgery when mice had shown susceptibility to P. aeruginosa infection (pCLPd1-3) and quantified sphingosine in the respiratory epithelium by immunofluorescence staining (Fig. 2B). Septic mice had significantly reduced sphingosine levels in their respiratory epithelium on day 2 and day 3 after CLP surgery, with pCLPd2 showing the most pronounced reduction by approximately 35 % (Fig. 2C).
Septic mice have higher pulmonary bacterial loads upon P. aeruginosa infection two days after CLP
Given that the highest mortality rate was observed in the post-CLP day 2 (pCLPd2) cohort, we determined lung bacterial loads in these mice by harvesting BAL fluid 4 h after infection. The bacterial burden in septic mice was approximately 10-fold higher than in sham-operated controls (Fig. 3). In light of the high mortality rate and significant bacterial load in pCLPd2 mice upon infection, we chose this time point for subsequent experiments.
Sphingosine has antimicrobial activity against clinical P. aeruginosa isolates
Sphingosine has been reported to exhibit antimicrobial properties against a wide range of Gram-positive and Gram-negative bacteria. Reports on its effectiveness against Pseudomonas strains have varied [11, 13]. To confirm that sphingosine can kill the multi-drug resistant Pseudomonas isolate used in our study, we treated bacteria with 100 µM sphingosine in vitro for 4 h. This time-point was chosen as it corresponds to the time at which we determined bacterial loads in the airways after infection of septic mice (Fig. 2B). The samples were then diluted and plated to determine CFUs. Even when significantly lower dilutions for the sphingosine samples were plated (1:10 rather than 1:100,000) no colonies were observed for P. aeruginosa 762, indicating more than 99.999 % of bacteria were killed (Fig. 4A). The antimicrobial activity against another strain, ATCC19660, was not quite as pronounced, averaging 72 % (Fig. 4B).
Sphingosine inhalation rescues septic mice from an established lung infection
In order to determine at what time after inoculation septic mice develop a lung infection, we continuously monitored core body temperature. The CLP surgery led to an initial drop of core body temperature, which had normalized in the two days prior to the P. aeruginosa inoculation (Fig. 5A). Core body temperature started to drop again approximately 90 min after inoculation and remained significantly reduced between 2.5 h and 7.75 h after infection (Fig. 5A).
Based on the observed temperature drop, mice were inhaled with either normal saline or sphingosine 2.5 h after inoculation and subsequently monitored for survival. Bacterial loads were again determined in BAL fluid 4 h after the inoculation (90 minutes after sphingosine inhalation, please refer to Fig. 1 for experimental timeline). As before, sham mice showed no mortality from P. aeruginosa infection irrespective of treatment (Fig. 5B). Septic mice that received aerosolized normal saline had a 43.8 % mortality rate, whereas only one septic mouse that was treated with aerosolized sphingosine died, corresponding to mortality rate of only 5.6 % (Fig. 5B). Regarding bacterial loads, sphingosine inhalation reduced the bacterial loads approximately 10-fold compared to inhalation with normal saline, and actually lowered them to the levels found in healthy mice infected with P. aeruginosa (Fig. 5C).
Sphingosine permeabilizes the outer membrane and disrupts inner membrane potential
Membrane permeabilization has been suggested to underlie the antimicrobial activity of sphingosine [14, 15]. To determine if sphingosine induces outer membrane permeabilization in Pseudomonas, we incubated bacteria with sphingosine in the presence of NPN. When the integrity of the outer membrane of Gram-negative bacteria is compromised, its barrier properties are disturbed and NPN can accumulate in its hydrophobic core, which increases NPN fluorescence [25-27]. Within 5 minutes of sphingosine addition, NPN fluorescence was significantly increased to 35 % of its signal upon complete membrane disruption (Fig. 6A).
To test for the kinetics of inner membrane permeabilization, we utilized PI. Intact membranes are impermeable for PI, whose fluorescence increases upon binding to nucleic acids. Sphingosine caused a significant increase in PI fluorescence 75 minutes after its addition (Fig. 6B).
To determine if the function of the inner membrane is compromised, we assessed membrane potential. Gram-negative bacteria normally maintain an electrical potential across the inner membrane, which is crucial for a number of physiological processes, including ATP synthesis [28]. DiOC2(3) and JC-1 are positively charged lipophilic probes with green fluorescence that bind to the inner side of the inner membrane in actively growing cells [28]. At higher concentrations, aggregates form in the cytoplasm that result in a shift towards red fluorescence [28]. The ratio of red to green fluorescence can be used to determine bacterial membrane potential. We noted a decrease in the fluorescence ratio of both DIOC2(3) (Fig. 6C) and JC-1 (Fig. 6D) 30 minutes after sphingosine addition, indicating reduced dye uptake and thus a reduction of the membrane potential.
Disturbances in membrane potential indicate an altered permeability for ions. This, in turn, is crucial for maintaining pH homeostasis [33]. Failure to maintain an appropriate pH has deleterious effects on the activity of biological macromolecules, significantly reducing growth and viability [34]. To see if the disturbance of the membrane potential resulted in intracellular acidification, we used DND-189. This compound, more commonly known as LysoSensor Green, is a membrane-permeable dye with pH-sensitive fluorescence [30]. We noted that the fluorescence of DND-189 approximately doubled 30 minutes after sphingosine addition to the bacteria (Fig. 6E).
Discussion
Using a 2-hit model of sepsis and subsequent nosocomial infection (Fig. 1), our data demonstrate that septic mice are most susceptible to P. aeruginosa lung infection two days after sepsis induction (Fig. 2A). This coincided with a reduction of sphingosine in the tracheal epithelium (Fig. 2B+C). Sphingosine killed 99.999% of bacteria of the multi-drug resistant, clinical P. aeruginosa isolate used for the lung infection in vitro (Fig. 4A). To tests sphingosines efficacy in vivo, we determined at what time after inoculation mice have established pneumonia. Based on a significant decrease in core body temperature, this was 2.5 h after inoculation (Fig. 5A). When inhaled sphingosine was administered at this time, the airway bacterial burden in septic mice was reduced to the levels found in healthy mice after inoculation (Fig. 5C). This was associated with a significant survival benefit (Fig. 5B).
Previous studies have established that sphingosine can be inhaled preventively, to restore reduced sphingosine levels in susceptible mice. For instance, using a protocol with a net sphingosine inhalation comparable to our method, Pewzner-Jung et al. reported that sphingosine inhalation of cystic fibrosis mice increases sphingosine levels in respiratory epithelial cells from approx. 3 pmol/105 cells in control-inhaled mice to 11 pmol/105 cells in sphingosine-inhaled mice, which corresponds to the level found in healthy mice [13]. This and other studies have shown that this is sufficient to protect susceptible mice from a subsequent infection [13, 16-19].
Which step in sphingolipid metabolism is affected and results in the reduction of sphingosine in susceptible mice is currently unknown and may differ based on the context (i.e. cystic fibrosis, trauma/sepsis). PAR1-SIP3 signaling has been reported to amplify inflammation in septic mice, suggesting that sphingosine phosphorylation may be enhanced. However, the study focused on dendritic cells, not the airway and sphingosine-1-phosphate levels were not quantified [35]. On the other hand, sphingosine reductions in cystic fibrosis mice have been linked to downregulation of acid ceramidase (ASAH1) [36]. We have also observed an increase in ceramide levels in the airways of critically ill mice (data not shown). This is a further commonality between critically ill and cystic fibrosis mice and indicates that the defect is with sphingosine production, rather than with sphingosine conversion to sphingosine-1-phosphate. However, further studies are necessary to elucidate the exact molecular mechanisms underlying the decrease of sphingosine levels in septic mice.
So far, it has not been investigated how long the prophylactic effect of sphingosine inhalation lasts. Additionally, it remained unknown if sphingosine would also be beneficial when administered after an infection. Our study addressed the second question. We can now show that sphingosine is also effective when given therapeutically, rather than preventively. We tested the efficacy of sphingosine in treating a pulmonary infection with a multidrug-resistant P. aeruginosa strain, which is of particular clinical importance, since P. aeruginosa is frequently the cause of hospital-acquired pneumonia and bacterial resistance is a leading cause of treatment failure. Antimicrobial lipids, such as sphingosine, have garnered much interest in the last years, as resistance to classical antibiotics is an increasing problem in virtually all microbes (reviewed in [37]).
Sphingosine is also a promising new treatment, because it has been reported to kill many different Gram-positive and Gram-negative bacteria in vitro. In this current study, we did not assess whether therapeutic sphingosine is also effective against pathogens other than P. aeruginosa. However, the previous prophylactic in vivo studies have also shown effectiveness against different pathogens, including S. aureus, another common hospital-acquired pathogen [13, 16-19].
Preliminary results indicate that side effects from sphingosine inhalation are likely to be minimal: No significant signs of toxicity in the respiratory epithelium were found upon sphingosine inhalation in mice [20] and mini pigs [7]. Additionally, sphingosine is a natural part of the host defense of the airways and has been reported to be reduced in cohorts that are susceptible to pulmonary infection [13, 16-19]. Thus, when dosed correctly, inhalation with sphingosine or sphingosine donors restores the pathologic loss, but does not increase it above normal levels [18]. Further, there are some indications that sphingosine inhalation may even mitigate tissue injury by reducing inflammatory cytokine levels [13, 17].
Although the antimicrobial activity of sphingosine was first described in 1992 [10], little is still known regarding the underlying mechanism. Studies with artificial liposomes and ghost membranes have shown that sphingosine can permeabilize membranes [14, 15]. In line with this Fischer et al. observed changes in the cell surface of sphingosine-treated bacteria using electron microscopy [12]. While the tested Gram-positive bacteria (Staphylococcus aureus) showed signs of membrane disruption, the cytoplasmic membrane of the tested Gram-negative bacteria (Escherichia coli) appeared intact [12]. Taken together, this indicates that sphingosine may kill Gram-positive bacteria through membrane permeabilization, but how death of Gram-negative bacteria is induced if the inner membrane stays intact remains unclear. To gain further insight, we first assessed outer membrane permeabilization in Pseudomonas, which occurred within 5 min of sphingosine addition (Fig. 6A). Inner membrane permeabilization, however, was not evident until 65 min after sphingosine addition (Fig. 6B). The integrity of the outer membrane contributes to the maintenance of an electronic gradient across the inner membrane [38]. We noted a disruption of inner membrane potential 30 min after sphingosine treatment (Fig. 6C+D). Preserving the correct membrane potential is critical for many physiological processes, including ATP synthesis and ion transport. The latter, in turn, is crucial for maintaining pH homeostasis. Failure to prevent intracellular acidification severely effects the activity of biological macromolecules, significantly reducing growth and viability [34]. In line with a disturbance of membrane potential, we also noted increased intracellular acidification upon sphingosine treatment (Fig. 6E). Taken together, these data indicate that sphingosine kills Pseudomonas by rapidly permeabilizing the outer membrane, resulting in loss of membrane potential and increased intracellular acidification. Bacteria can have protective strategies for dealing with acidic stress, e.g. the acid tolerance response [34]. As these may vary between strains, this could explain the different efficacies of sphingosine-mediated bacterial killing observed in our study. Further, outer membrane composition may influence the susceptibility to permeabilization by sphingosine [15, 39]. Further studies will be necessary to elucidate the mechanism underlying sphingosines antimicrobial properties.
It should be noted that relatively high concentrations of sphingosine were used in the present studies, while previous studies showed bacterial death already at sphingosine concentrations between 1 and 10 mM (4) and a more rapid onset of bacterial death [40]. However, our study also used considerably higher bacterial cell counts. Thus, the principal effects of sphingosine are similar in all of these studies, but the concentrations required for induction of death and the antimicrobial kinetics seem to slightly differ between strains, growth and medium conditions.
The administered sphingosine is not inert and thus could be metabolized further. We cannot exclude that the observed beneficial effects of sphingosine inhalation are mediated by a metabolite rather than by sphingosine itself. However, sphingosine inhalation does not seem to increase ceramide and sphingosine-1-phosphate levels [7]. Further, sphingosine inhalation was shown to increase sphingosine levels in the respiratory surface, but not the epithelial cell layer [7] and it has been suggested that sphingosine mostly remains in the mucus on top of the epithelial cells [40]. This would favor direct bacterial killing by sphingosine.
The use of already clinically approved drugs could potentially accelerate the translation of these findings to clinical use. Fingolimod, an approved sphingosine-1-phosphate receptor modulator, could be one potential option. However, as long as the molecular mechanisms have not been elucidated, we can only speculate on the effect of Fingolimod. The observation that ceramide is increased in critically ill mice indicates that the defect is with sphingosine production, rather than consumption. Further, direct antimicrobial properties of Fingolimod have so far only been demonstrated for Clostridium perfringens [41]. Assuming a direct bactericidal effect of sphingosine, as discussed above, the reconstitution of these bacterial killing properties is likely paramount to mitigate pulmonary infection in the model. Rather than Fingolimod, the use of sphingomyelinase inhibitors like Amitriptyline, are another promising option when it comes to already approved drugs. These substances are of particular interest for those situations in which sphingosine reductions occur concurrently with ceramide elevations, as ceramide elevations were shown to be responsible for the reduced expression of acid ceramidase and the subsequent reduction of sphingosine [36]. Indeed, two phase II studies with pulmonal amitriptyline already reported with favorable results [42]. Nevertheless, clinical development of sphingosine inhalation itself is also underway at least [7] and could potentially garner a lot more attention soon, as sphingosine was very recently shown to prevent viral entry of SARS-CoV-2 [43].
Conclusion
Our data provide proof-of-principle that sphingosine inhalation can be used to treat an established pulmonary infection with P. aeruginosa. Further studies are needed to test whether sphingosine is also effective as a therapeutic against infections with other bacteria.
Acknowledgements
NB, CCC: Conception and study design, drafting of the article. NB, AMP, NJA: Data acquisition and analysis. NB, AMP, MJE, EG, CCC: Data interpretation, revision of the article, final approval of the version to be submitted.
The authors would like to acknowledge L. England and H. Goetzman for excellent veterinarian and experimental support.
Funding Sources
This work was supported by the Shriners Hospital for Children Grant 85000-CIN-17 to CCC and by U.S. National Institutes of Health (NIH), National Institute of General Medical Sciences (NIGMS) Grant T32 GM08478 to AMP. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or NIGMS.
Statement of Ethics
All murine experiments were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati (protocol number: 08-09-19-01).
Disclosure Statement
The authors have no conflicts of interest to declare.
References
|
1 World Health Organization: The top 10 causes of death. The 10 leading causes of death in the world, 2000-2016. URL: http://www.who.int/en/news-room/fact-sheets/detail/the-top-10-causes-of-death. |
|
|
|
|
|
2 Ibn Saied W, Mourvillier B, Cohen Y, Ruckly S, Reignier J, Marcotte G, Siami S, Bouadma L, Darmon M, de Montmollin E, Argaud L, Kallel H, Garrouste-Orgeas M, Soufir L, Schwebel C, Souweine B, Glodgran-Toledano D, Papazian L, Timsit JF: A Comparison of the Mortality Risk Associated With Ventilator-Acquired Bacterial Pneumonia and Nonventilator ICU-Acquired Bacterial Pneumonia. Crit Care Med 2019;47:345-352. |
|
|
|
|
|
3 Baker JE, Boudreau RM, Seitz AP, Caldwell CC, Gulbins E, Edwards MJ: Sphingolipids and Innate Immunity: A New Approach to Infection in the Post-Antibiotic Era? Surg Infect (Larchmt) 2018;19:792-803. |
|
|
|
|
|
4 Poch DS, Ost DE: What are the important risk factors for healthcare-associated pneumonia? Semin Respir Crit Care Med 2009;30:26-35. |
|
|
|
|
|
5 Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia. Am J Respir Crit Care Med 2005;171:388-416. |
|
|
|
|
|
6 Lanks CW, Musani AI, Hsia DW:
Community-acquired Pneumonia and Hospital-acquired Pneumonia. Med Clin North
Am 2019;103:487-501. |
|
|
|
|
|
7 Carstens H, Schumacher F, Keitsch S, Kramer M, Kühn C, Sehl C, Soddemann M, Wilker B, Herrmann D, Swaidan A, Kleuser B, Verhaegh R, Hilken G, Edwards MJ, Dubicanac M, Carpinteiro A, Wissmann A, Becker KA, Kamler M, Gulbins E: Clinical Development of Sphingosine as Anti-Bacterial Drug: Inhalation of Sphingosine in Mini Pigs has no Adverse Side Effects. Cell Physiol Biochem 2019;53:1015-1028. |
|
|
|
|
|
8 Becker KA, Riethmüller J, Seitz AP, Gardner A, Boudreau R, Kamler M, Kleuser B, Schuchman E, Caldwell CC, Edwards MJ, Grassmé H, Brodlie M, Gulbins E: Sphingolipids as targets for inhalation treatment of cystic fibrosis. Adv Drug Deliv Res 2018;133:66-75. |
|
|
|
|
|
9 Wertz PW: Lipids and the Permeability and
Antimicrobial Barriers of the Skin. J Lipids 2018;2018:5954034. |
|
|
|
|
|
10 Bibel DJ, Aly R, Shinefield HR: Antimicrobial
activity of sphingosines. J Invest Dermatol 1992;98:269-273. |
|
|
|
|
|
11 Fischer CL, Drake DR, Dawson DV, Blanchette DR, Brogden KA, Wertz PW: Antibacterial activity of sphingoid bases and fatty acids against Gram-positive and Gram-negative bacteria. Antimicrob Agents Chemother 2012;56:1157-1161. |
|
|
|
|
|
12 Fischer CL, Walters KS, Drake DR, Blanchette
DR, Dawson DV, Brogden KA, Wertz PW: Sphingoid bases are taken up by Escherichia
coli and Staphylococcus aureus and induce ultrastructural damage. Skin
Pharmacol Physiol 2013;26:36-44. |
|
|
|
|
|
13 Pewzner-Jung Y, Tavakoli Tabazavareh S, Grassmé H, Becker KA, Japtok L, Steinmann J, Joseph T, Lang S, Tuemmler B, Schuchman EH, Lentsch AB, Kleuser B, Edwards MJ, Futerman AH, Gulbins E: Sphingoid long chain bases prevent lung infection by Pseudomonas aeruginosa. EMBO Mol Med 2014;6:1205-1214. |
|
|
|
|
|
14 Contreras FX, Sot J, Alonso A, Goni FM:
Sphingosine increases the permeability of model and cell membranes. Biophys J
2006;90:4085-4092. |
|
|
|
|
|
15 Jimenez-Rojo N, Sot J, Viguera AR, Collado
MI, Torrecillas A, Gomez-Fernandez JC, Goni FM, Alonso A: Membrane
permeabilization induced by sphingosine: effect of negatively charged lipids.
Biophys J 2014;106:2577-2584. |
|
|
|
|
|
16 Tavakoli Tabazavareh S, Seitz A, Jernigan P, Sehl C, Keitsch S, Lang S, Kahl BC, Edwards M, Grassme H, Gulbins E, Becker KA: Lack of Sphingosine Causes Susceptibility to Pulmonary Staphylococcus Aureus Infections in Cystic Fibrosis. Cell Physiol Biochem 2016;38:2094-2102. |
|
|
|
|
|
17 Rice TC, Pugh AM, Seitz AP, Gulbins E, Nomellini V, Caldwell CC: Sphingosine rescues aged mice from pulmonary pseudomonas infection. J Surg Res 2017;219:354-359. |
|
|
|
|
|
18 Rice TC, Pugh AM, Xia BT, Seitz AP, Whitacre BE, Gulbins E, Caldwell CC: Bronchoalveolar Lavage Microvesicles Protect Burn-Injured Mice from Pulmonary Infection. J Am Coll Surg 2017;225:538-547. |
|
|
|
|
|
19 Rice TC, Seitz AP, Edwards MJ, Gulbins E, Caldwell CC: Frontline Science: Sphingosine rescues burn-injured mice from pulmonary Pseudomonas aeruginosa infection. J Leukoc Biol 2016;100:1233-1237. |
|
|
|
|
|
20 Martin GE, Boudreau RM, Couch C, Becker KA, Edwards MJ, Caldwell CC, Gulbins E, Seitz A: Sphingosine's role in epithelial host defense: A natural antimicrobial and novel therapeutic. Biochimie 2017;141:91-96. |
|
|
|
|
|
21 Baker CC, Chaudry IH, Gaines HO, Baue AE: Evaluation of factors affecting mortality rate after sepsis in a murine cecal ligation and puncture model. Surgery 1983;94:331-335. |
|
|
|
|
|
22 Johnson BL, 3rd, Rice TC, Xia BT, Boone KI, Green EA, Gulbins E, Caldwell CC: Amitriptyline Usage Exacerbates the Immune Suppression Following Burn Injury. Shock 2016;46:541-548. |
|
|
|
|
|
23 Grassme H, Jendrossek V, Riehle A, von Kurthy G, Berger J, Schwarz H, Weller M, Kolesnick R, Gulbins E: Host defense against Pseudomonas aeruginosa requires ceramide-rich membrane rafts. Nat Med 2003;9:322-330. |
|
|
|
|
|
24 Sautrey G, Zimmermann L, Deleu M, Delbar A, Souza Machado L, Jeannot K, Van Bambeke F, Buyck JM, Decout JL, Mingeot-Leclercq MP: New amphiphilic neamine derivatives active against resistant Pseudomonas aeruginosa and their interactions with lipopolysaccharides. Antimicrob Agents Chemother 2014;58:4420-4430. |
|
|
|
|
|
25 Loh B, Grant C, Hancock RE: Use of the fluorescent probe 1-N-phenylnaphthylamine to study the interactions of aminoglycoside antibiotics with the outer membrane of Pseudomonas aeruginosa. Antimicrob Agents Chemother 1984;26:546-551. |
|
|
|
|
|
26 Wu M, Hancock RE: Interaction of the cyclic antimicrobial cationic peptide bactenecin with the outer and cytoplasmic membrane. J Biol Chem1999;274:29-35. |
|
|
|
|
|
27 Helander IM, Mattila-Sandholm T: Fluorometric
assessment of gram-negative bacterial permeabilization. J Appl Microbiol
2000;88:213-219. |
|
|
|
|
|
28 Strauber H, Muller S: Viability states of bacteria--specific mechanisms of selected probes. Cytometry 2010;77:623-634. |
|
|
|
|
|
29 Novo D, Perlmutter NG, Hunt RH, Shapiro HM: Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique. Cytometry 1999;35:55-63. |
|
|
|
|
|
30 Lin HJ, Herman P, Kang JS, Lakowicz JR:
Fluorescence lifetime characterization of novel low-pH probes. Anal Biochem
2001;294:118-125. |
|
|
|
|
|
31 Koulenti D, Tsigou E, Rello J: Nosocomial pneumonia in 27 ICUs in Europe: perspectives from the EU-VAP/CAP study. Eur J Clin Microbiol Infect Dis 2017;36:1999-2006. |
|
|
|
|
|
32 Hotchkiss RS, Karl IE: The pathophysiology and treatment of sepsis. N Engl J Med 2003;348:138-150. |
|
|
|
|
|
33 Booth IR: The regulation of intracellular pH in bacteria. Novartis Found Symp 1999;221:19-28; discussions 28-37. |
|
|
|
|
|
34 Lund P, Tramonti A, De Biase D: Coping with low pH: molecular strategies in neutralophilic bacteria. FEMS Microbiol Rev 2014;38:1091-1125. |
|
|
|
|
|
35 Niessen F, Schaffner F, Furlan-Freguia C, Pawlinski R, Bhattacharjee G, Chun J, Derian CK, Andrade-Gordon P, Rosen H, Ruf W: Dendritic cell PAR1-S1P3 signalling couples coagulation and inflammation. Nature 2008;452:654-658. |
|
|
|
|
|
36 Grassmé H, Henry B, Ziobro R, Becker KA, Riethmüller J, Gardner A, Seitz AP, Steinmann J, Lang S, Ward C, Schuchman EH, Caldwell CC, Kamler M, Edwards MJ, Brodlie M, Gulbins E: β1-Integrin Accumulates in Cystic Fibrosis Luminal Airway Epithelial Membranes and Decreases Sphingosine, Promoting Bacterial Infections. Cell Host Microbe 2017;21:707-718.e708. |
|
|
|
|
|
37 Baker JE, Boudreau RM, Seitz AP, Caldwell CC,
Gulbins E, Edwards MJ: Sphingolipids and Innate Immunity: A New Approach to
Infection in the Post-Antibiotic Era? Surg Infect (Larchmt) 2018;19:792-803. |
|
|
|
|
|
38 Benarroch JM, Asally M: The Microbiologist's Guide to Membrane Potential Dynamics. Trends Microbiol 2020;28:304-314. |
|
|
|
|
|
39 Verhaegh R, Becker KA, Edwards MJ, Gulbins E:
Sphingosine kills bacteria by binding to cardiolipin. J Biol Chem
2020;295:7686-7696. |
|
|
|
|
|
40 Seitz AP, Schumacher F, Baker J, Soddemann M, Wilker B, Caldwell CC, Gobble RM, Kamler M, Becker KA, Beck S, Kleuser B, Edwards MJ, Gulbins E: Sphingosine-coating of plastic surfaces prevents ventilator-associated pneumonia. J Mol Med (Berl) 2019;97:1195-1211. |
|
|
|
|
|
41 Rumah KR, Vartanian TK, Fischetti VA: Oral Multiple Sclerosis Drugs Inhibit the In vitro Growth of Epsilon Toxin Producing Gut Bacterium, Clostridium perfringens. Front Cell Infect Microbiol 2017;7:11. |
|
|
|
|
|
42 Adams C, Icheva V, Deppisch C, Lauer J, Herrmann G, Graepler-Mainka U, Heyder S, Gulbins E, Riethmueller J: Long-Term Pulmonal Therapy of Cystic Fibrosis-Patients with Amitriptyline. Cell Physiol Biochem 2016;39:565-572. |
|
|
|
|
|
43 Edwards MJ, Becker KA, Gripp B, Hoffmann M,
Keitsch S, Wilker B, Soddemann M, Gulbins A, Carpinteiro E, Patel SH, Wilson
GC, Pöhlmann S, Walter S, Fassbender K, Ahmad SA, Carpinteiro A, Gulbins E:
Sphingosine prevents binding of SARS-CoV-2 spike to its cellular receptor ACE2.
J Biol Chem 2020; DOI:10.1074/jbc.RA120.015249. |
|