Human samples

All experiments on human samples were performed with the permission of the Ethics Committee of the University Hospital Essen, permission number 17–7326-BO.

Freshly removed bronchial pieces and lung parenchyma from human lung transplant tissue were immediately cut into similarly sized pieces (10 × 10 × 10 mm), washed three times in Dulbecco's Modified Eagle Medium (DMEM), and placed in a 24-well plate for infection with 3.5 × 105 M. smegmatis, prepared as described below, in 500 µL DMEM.

Lung parenchyma tissues were transferred into a petri dish and cut into small pieces with scissors. The tissues were then transferred into a 50-mL Falcon tube with ice-cold phosphate-buffered saline (PBS) to wash away remaining blood on the tissues, and the PBS was removed by passing the tissue through a 40-µm cell strainer. The tissues on the top of the strainer were transferred to a new Falcon tube containing 0.6 mg/mL collagenase (Merck, Darmstadt, Germany; # C2-22-BC), 0.02 mg/mL Elastase (Serva, Heidelberg, Germany; #20,930.01), and 17 units/mL DNase (QIAGEN, Hilden, Germany; # 79254) for 30 min at 37 °C. Samples were then washed in 10% fetal calf serum (FCS) in PBS, and the cell suspension was passed through a 70-µm strainer to a 15-mL Falcon tube for centrifugation at 300 × g for 5 min at 4 °C. The cell pellets were resuspended in 2 mL of red blood cell lysis buffer (Biolegend, San Diego, CA, USA) for 2 min at room temperature, followed by the addition of 10 mL RPMI-1640/10% FCS and pelleted again by centrifugation. Cells were resuspended in RPMI-1640 medium (with HEPES buffer), counted by trypan blue staining, and seeded at 1 × 104 cells per mL in 96-well plates for infection.

For both bronchial and parenchymal samples, M. smegmatis was prepared as below and used for infection. After 4 h of infection, the non-internalized bacteria were extensively washed off, and fresh medium containing 1 µM sphingosine or medium containing 0.025% OGP was added for 1 h. Samples were washed, and cells were lysed by adding saponin to a final concentration of 0.5% for 15 min to release intracellular bacteria. The lysates were diluted and plated on LB agar plates. Colonies were counted after 3 days of incubation at 37 °C.

Mouse experiments

Procedures performed on the animals were in accordance with the North Rhine–Westphalia (NRW) State Office for Nature, Environment and Consumer Protection (LANUV), Recklinghausen, Germany, permission number: AZ 81–02.04.2019.A148.

We used wild-type mice on a C57BL/6 background. Mice were housed and bred in the mouse facilities of the University Hospital, University of Duisburg-Essen, Germany, and the University of Cincinnati (Cincinnati, OH, USA). They were tested for common murine pathogens according to the 2002 recommendations of the Federation of European Laboratory Animal Science Associations (FELASA), and they were kept free of pathogens. All experiments were performed according to the FELASA regulations and the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

Mycobacteria preparation

A green fluorescent protein (GFP)-expressing BCG (GFP-BCG) strain was used for both in vivo and in vitro infections. The GFP-BCG strain was constructed by transforming BCG with the dual reporter plasmid pSMT3L × EGFP. For the infection experiments, bacteria were cultured and shaken at 120 rpm at 37 °C in Erlenmeyer flasks containing 10 mL Middlebrook 7H9 Broth supplemented with glycerol (BD Biosciences, Heidelberg, Germany) supplemented with 50 µg/mL hygromycin B for maintaining GFP plasmids. After 5 to 7 days of incubation, the bacteria were used for experiments. M. smegmatis (ATCC19420) was prepared as above and used for experiments after 16 to 24 h growth. M. tuberculosis (ATCC 27294, H37Rv) was cultured in Mycobacteria Growth Indicator Tubes with PANTA supplement (Becton–Dickinson Microbiology System, Sparks, MD, USA) at 37 °C for 5 to 7 days.

GFP-BCG and M. smegmatis were finally collected by centrifugation at 3000 × g for 10 min. The bacterial pellet was resuspended in PBS buffer (pH 7.4) and vortexed for 5 min. Additionally, GFP-BCG was passed through a syringe with a 25-gauge needle for 10 times to avoid clumps. The bacteria were then bath-sonicated for 5 min at 4 °C to suspend any remaining clumps. Unseparated bacterial clumps were finally removed by centrifugation at 220 × g for 2 min. The supernatant containing single GFP-BCG or M. smegmatis was carefully collected. We calculated the bacterial counts on the basis of the optical density (OD) by using an Eppendorf (Hamburg, Germany) BioPhotometer 6131 (OD600 1 = 108 bacteria).

M. tuberculosis (ATCC 27294, H37Rv) was collected when the bacteria reached logarithm growth, transferred into a Falcon tube, and sonicated for 5 min. Remaining clumps were allowed to sediment for 10 min, the supernatant containing single bacteria was carefully collected, and the bacterial number was calculated on the basis of the OD as measured with an Eppendorf Biophotometer 6131.

Macrophage preparation

For preparation of BMDMs, mice were put to death, and femurs and tibias were rinsed with minimal essential medium (MEM; Gibco, Paisley, UK) supplemented with 10% FCS (Gibco), 10 mM HEPES (Roth GmbH, Karlsruhe, Germany; pH 7.4), 2 mM l-glutamine, 1 mM sodium pyruvate, 100 μM nonessential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). The isolated cells were flushed through a 23G needle for obtaining single cells. The isolated cells were washed, and 3 to 6 × 106 cells were cultured in Petri dishes in MEM containing 20% L-cell supernatant as a source of macrophage colony-stimulating factor (M-CSF). Fresh MEM/L-cell supernatant medium was applied to the culture every 3 days. Macrophages matured within the next 6 days and were used on day 8 of culture. Cells were grown at 37 °C in 5% CO2.

For alveolar macrophages, mice were put to death, the trachea was exposed and catheterized with a polyethylene tube, and bronchoalveolar lavage was performed with a total of 15 mL ice-cold PBS. The fluid was collected and centrifuged for 5 min at 300 × g at 4 °C. The pellets containing alveolar macrophages were resuspended in MEM/HEPES, counted, and then seeded for further experiments.

Infection and treatment

For in vitro assays, BMDMs or alveolar macrophages were seeded in 24- or 96-well plates, left uninfected, or infected with GFP-BCG in MEM/10 mM HEPES (pH 7.4) at a bacteria-to-host cell ratio (multiplicity of infection, MOI) of 1:1 to 10:1 for a specified indicated time. Synchronous infection conditions and enhanced interactions between bacteria and host cells were achieved by centrifuging (55 × g) the bacteria onto the cells for 5 min. The end of centrifugation was defined as the starting point of infection.

For in vivo infections, bacteria were prepared as described above and then pelleted at 3,000 × g for 10 min. BCG bacteria were resuspended in 0.9% NaCl, and 5 × 104 CFU bacteria in 50 µL were intranasally infected into mice. After 3 weeks of infection, the mice were left untreated or were treated with 0.9% NaCl or 125 µM sphingosine (dissolved in 0.9% NaCl) twice a day for one week by inhalation in a Buxco Inhalation Tower (Data Science International, New Brighton, MN, USA). At the end of the infection and treatment, the mice were put to death by cervical dislocation, blood was collected, lungs were removed, and the right upper lobe was separated for counting CFUs. The remaining lung tissue was fixed in 4% paraformaldehyde (PFA) and embedded in paraffin after serial dehydration with an ethanol-to-xylol gradient, or embedded in Tissue-Tec (Sakura Finetek USA, Torrance, CA, USA) and shock-frozen in liquid nitrogen.

Sphingosine quantification by liquid chromatography–tandem mass spectrometry (LC–MS/MS)

Samples of cell culture media were filled with water to 1 mL (if necessary) followed by the addition of 110 μL 10 × Baker buffer (300 mM citric acid, 400 mM disodium hydrogen phosphate, pH 3.0). For lipid extraction, 2 mL 1-butanol and 1 mL water-saturated 1-butanol were added. The extraction solvent contained sphingosine-d7 (Sph-d7) (Avanti Polar Lipids, Alabaster, AL, USA) as an internal standard. Extraction was facilitated by intensive vortexing (1500 rpm) for 10 min at room temperature. Afterwards, samples were centrifuged for 5 min at 2200 g (4 °C). The upper organic phase was dried under reduced pressure with a Savant SpeedVac concentrator (Thermo Fisher Scientific, Dreieich, Germany). Dried residues were reconstituted in 200 μL acetonitrile/methanol/water (47.5:47.5:5 v:v:v; 0.1% formic acid) and subjected to LC–MS/MS sphingosine quantification as described41. The instrumentation used consisted of a 1260 Infinity liquid chromatography (LC) system equipped with a Poroshell 120 EC-C8 column (3.0 × 150 mm, 2.7 μm) coupled to a 6490 triple-quadrupole mass spectrometer (all from Agilent Technologies, Waldbronn, Germany) operating in positive electrospray ionization (ESI +) mode. The mass transitions m/z 300.3 → 282.3 (252.3) for sphingosine (Sph) and m/z 307.3 → 289.3 (259.3) for Sph-d7 were recorded (qualifier product ions in parentheses). Sph was directly quantified via its deuterated internal standard Sph-d7 (0.25 pmol on column). Data evaluation was performed with MassHunter Quantitative Analysis software (Agilent Technologies).

Colony forming assay

To study bacterial killing by macrophages, we seeded cells in 96-well plates (1 × 104 per well) and infected them with a bacteria-to-host cell ratio (MOI) of 1:1 for 4 h. The cells were washed and left untreated or treated with sphingosine for a specified time. At the end of infection, the cells were washed and lysed by incubation in 0.5% saponin at 37 °C for 15 min. The lysates were diluted and plated out on 7H10 agar plates, and colonies were counted after 21 days of incubation at 37 °C.

To count CFUs in the lungs of infected mice, the right upper lobe was collected and transferred to a 6-well plate, cut into small pieces, and homogenized with a scalpel and the plunger of a 5 mL syringe in 2 mL of 0.5% saponin for 15 min of incubation at 37 °C. The lysates were diluted with PBS and plated on 7H10 agar plates. The bacteria were grown for 21 days at 37 °C, and colonies were counted.

Immunofluorescence and hematoxylin & eosin staining

Macrophages were grown on coverslips and were left uninfected or were infected with GFP-BCG. The infected cells were left untreated or were treated with sphingosine as described above. At the end of infection, cells were washed, fixed in 1% PFA (Sigma) in PBS (pH 7.4) for 10 min, and washed three times with PBS. For immunofluorescence staining, macrophages were permeabilized with 0.1% Triton X-100/PBS for 5 min at room temperature, washed with PBS, and incubated for 1 h with 5% FCS (Thermo Fisher Scientific, Waltham, MA, USA) to block nonspecific binding. The samples were then washed three times with PBS and were mounted with Mowiol (Kuraray Specialities Europe GmbH, Okayama, Japan) or DAPI (Abcam, Cambridge, UK).

For hematoxylin and eosin (H&E) staining, paraffin-embedded lung samples were sectioned at 6 µm, dewaxed, rehydrated, and stained with Mayer’s hemalaun solution (Roth, Karlsruhe, Germany; #T865.1) for 5 min. Samples were rinsed with water for 15 min, stained with 1% eosin solution for 2 min, and washed again with water. Stained sections were dehydrated with ethanol to xylene and embedded in Eukitt mounting medium (Sigma-Aldrich). Specimens were examined with a Leica DMi8 transmitted light microscope with a 40 × lens using identical settings. All images were analyzed with ImageJ software (Fiji, Wisconsin, USA).

Direct treatment of mycobacteria with sphingosine

Sphingosine (Avanti Polar Lipids, Alabaster, AL, USA; #860490P) was resuspended in 7.5% OGP at 20 mM or 100 mM concentration as a stock. Sphingosine was sonicated (Bandelin Sonorex, Berlin, Germany) for 10 min before each use and diluted in 0.0075% OGP or in 0.9% NaCl to 1 to 100 µM.

Bacteria were diluted in PBS to a concentration of 1 × 106 CFU/mL for GFP-BCG and M. smegmatis, or of 1 × 105 CFU/mL for M. tuberculosis. Bacteria were incubated with sphingosine for up to 24 h for BCG and M. smegmatis or up to 3 weeks for M. tuberculosis at 37 °C.

For determination of CFU, BCG and M. smegmatis were diluted and plated in 7H10 agar plates, and colonies were counted after incubation for 14 days (for BCG) or 3 days (for M. smegmatis) at 37 °C. For M. tuberculosis, growth was observed by BacTec MGIT automated mycobacterial detection system (BD) every 1 to 3 days for up to 21 days.

For membrane permeability, control or sphingosine-treated BCG were incubated with 100 nM TO-PRO-3 iodide (Life Technologies, Inc., Arcadia, CA, USA) for 10 min, pelleted, and analyzed by flow cytometry with Attune NxT (Life Technologies, Inc.). M. tuberculosis was incubated with OGP or sphingosine for 10 min, followed by centrifugation at 3,000 × g for 10 min. The pelleted bacteria were resuspended with 100 µL zombie violet (1:100 dilution; Biolegend) and incubated for 15 min. Bacteria were washed with PBS, pelleted, fixed with 4% PFA for 15 min, and washed with PBS for flow cytometry analysis.

For measuring the remaining ATP, control or sphingosine-treated BCG were pelleted and resuspended with BacTiter-Glo (Promega, Madison, WI, USA) according to the manufacturer’s instructions and were measured with a luminescence reader.

Transmission electron microscopy

Samples were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) overnight at 4 °C. The samples were postfixed with 1% osmium tetroxide plus potassium ferrocyanide 1% in 0.1 M sodium cacodylate buffer for 1 h at 4°. After three water washes, samples were dehydrated in a graded ethanol series and embedded in an epoxy resin (Sigma-Aldrich). Ultrathin Sects. (60–70 nm) were obtained with an Ultratome Leica Ultracut EM UC7 ultramicrotome, counterstained with uranyl acetate and lead citrate, and viewed with a Tecnai G2 (FEI) transmission electron microscope operating at 100 kV. Images were captured with a Veleta (Olympus Soft Imaging System, Muenster, Germany) digital camera.

Cell viability assay

To study the viability of the macrophages upon sphingosine treatment, we seeded 1 × 104 cells per well in a 96-well plate. The cells were left untreated or were treated with various concentrations of sphingosine for specified times. The viability of the cells was measured by CellTiter 96 assay (Promega). Briefly, cells were washed after incubation and resuspended in tetrazolium containing medium for 4 h. The plate was analyzed by a plate reader at 570 nm.

Statistical analysis and quantification

All data were obtained from independent experiments and presented as arithmetic mean ± standard deviation (SD); n represents the number of independent experiments or mice. Student's t-test for single comparisons and ANOVA for multiple comparisons were used to detect statistically significant differences; statistical significance was set at the level of p ≤ 0.05. If appropriate, data were quantified with ImageJ or Flowjo. GraphPad Prism 9 statistical software (GraphPad Software, La Jolla, CA, USA) was used for statistical analysis and graphical presentations.