Work Description

Description

Targeted metabolomics analysis of primary WT murine bone marrow-derived macrophages (BMDM) mock-treated or infected with methicillin-resistant Staphylococcus aureus (MRSA) (File S1). Targeted metabolomics analysis of mock-treated or MRSA-infected wildtype (WT) or Interferon alpha/beta receptor 1 knockout (Ifnar1-/-) immortalized BMDM (iBMDM) (File S2).

Methodology

Cell culture
Murine primary bone marrow-derived macrophages (BMDM) and immortalized BMDM (iBMDM) were prepared as previously reported (Reynolds et al. 2023, Sci Adv). Femurs and tibiae from the C57BL/6J lineage mice of various genotypes were flushed and grown for 6 days in macrophage differentiation media [50% DMEM, 1 mM sodium pyruvate, 2 mM L-glutamine, 20% heat-inactivated fetal bovine serum (FBS) (Biowest), 30% L929 cell-conditioned medium, penicillin (50 U/ml), and streptomycin (50 μg/ml)]. For immortalization, recombinant Cre-J2 virus containing v-Raf and v-Myc oncogenes was generated in 3T3 fibroblasts grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated FBS and penicillin (50 U/ml) and streptomycin (50 μg/ml). Sterile-filtered culture supernatants containing Cre-J2 virus were stored at −80°C. Bone marrow isolates were transduced with Cre-J2 virus in macrophage differentiation media to generate iBMDMs, and iBMDMs were grown for at least 1 month to ensure successful immortalization. L-929 cells were cultured in minimum essential Eagle’s medium supplemented with 1 mM nonessential amino acid, 1 mM sodium pyruvate, 2 mM l-glutamine, 10 mM Hepes, and 10% FBS. L929-conditioned medium was sterile filtered prior to use. All experiments were performed in DMEM supplemented with 2 mM l-glutamine, 1 mM sodium pyruvate, and 10% FBS, without antibiotics unless otherwise indicated. Ifnar1 -/- (strain #010830) and wild-type (WT) age and sex-matched mouse pairs were obtained from the Jackson Laboratory. BMDMs were derived according to the protocol described above. Ifnar1 -/- deletion was validated using PCR with primer sequences provided by the Jackson laboratory (5’-CGA GGC GAA GTG GTT AAA AG-3’, 5’-ACG GAT CAA CCT CAT TCC AC-3’, and 5’-AAT TCG CCA ATG ACA AGA CG-3’) Cell lines were confirmed to be mycoplasma-free with the Lookout Mycoplasma PCR Detection Kit (Sigma-Aldrich). All cells were cultured at 37°C in 5% CO2.

MRSA infection of macrophage cultures in vitro
Methicillin-resistant Staphylococcus aureus (MRSA) USA300 isolate JE2 glycerol stocks were struck out on tryptic soy broth (TSB) agar plates within 3 days of planned experiments. For each experiment, a single MRSA colony was picked and grown overnight at 37°C, slanted, with shaking. Overnight cultures were washed three times with PBS and diluted to an optical density (OD) of 1 as measured by a spectrophotometer, which was determined to equate to a bacterial density of 1e9 CFU/mL. Macrophages were infected at a multiplicity of infection (MOI) of 20 CFU/macrophage and incubated at 37°C in a tissue culture incubator for 1 h. After 1 h, cultures were supplemented with 10 U/mL of lysostaphin (Sigma) to kill extracellular bacteria. Infections were then allowed to proceed to the endpoint according to individual experimental protocols.

Targeted metabolomics
WT BMDM derived from five different C57BL/6J mice, 2 male and 3 females, (1e6 cells) were infected with MRSA (MOI 20) for 24 h or left uninfected. Alternatively, WT and Ifnar1 -/- iBMDM derived from three different mice per genotype, 2 males and 1 female, were infected with MRSA (MOI 20) for 24 h or left uninfected. All experiments were performed in DMEM supplemented with 2 mM l-glutamine, 1 mM sodium pyruvate, and 10% FBS. At the experimental endpoint, cells were washed with ice cold PBS and metabolites were extracted with 80% methanol for 10 min on dry ice. Metabolite extracts were centrifuged at 16,000 g for 10 min at 4°C to remove insoluble material, and the supernatant was collected. Extracts were protein normalized by adjusting volumes according to a Bradford assay from paired samples for each experiment. Equivalent volumes of normalized extracts were dried using a SpeedVac at room temperature (RT) for 2.5h. After drying, the metabolite pellet was dissolved in 50% methanol-water and analyzed by liquid chromatography-mass spectrometry (LC-MS) with an Agilent 1290 Infinity II LC-6470 Triple Quadrupole (QqQ) tandem mass spectrometer system in the negative mode. Agilent ZORBAX RRHD Extend-C18, 2.1 × 150 mm, 1.8 µm and ZORBAX Extend Fast Guards for UHPLC were used in separation. The LC gradient utilized 3 solutions: Solution A was 97% water and 3% methanol with 15 mM acetic acid and 10 mM Tributylamine (TBA) at pH 5. Solution B was 15 mM acetic acid and 10 mM TBA in methanol. Washing Solvent C was acetonitrile. The following LC gradient profile was used: from 0-2.5 min, 0.25 ml/min of 100% A; from 2.5-7.5 min, 0.25 ml/min of 80% A and 20% B; from 7.5-13 min, 0.25 ml/min of 55% A and 45% B; from 13-20 min, 0.25 ml/min of 1% A and 99% B; from 20-24 min, 0.25 ml/min of 1% A and 99% B; from 24.05-27 min, 0.25 ml/min of 1% A and 99% C; from 27.5-31.35 min, 0.8 ml/min of 1% A and 99% C; from 31.5-32.25, 0.6 ml/min of 1% A and 99% C; from 32.25-39.9 min, 0.4 ml/min of 1% A and 99% C; from 39.9-40 min, 0.25 ml/min of 100% A. Targeted metabolomics analysis of measured MS/MS spectra with a reference library of 230 standard metabolites was performed using Skyline open-source software, and major findings were validated by an independent assessment using the Agilent Masshunter Workstation Software LC/MS Data Acquisition for 6400 Series Triple Quadrupole MS with Version B.08.02. The LC-MS/MS dynamic multiple reaction monitoring (dMRM) method for detection of 220 metabolites based on MS/MS spectra, retention time (RT) windows, and explicit RTs was developed by Agilent using 220 individual metabolite standards. An additional 10 metabolites were added to the acquisition method using an identical chromatographic method with compound standards. 0.07-min peak width was used in dMRM scans with an acquisition time of 24 min. Isomers and isobaric peaks from the standard database were integrated separately and annotated appropriately in subsequent analysis.

Files contained here:

File S1 - Targeted metabolomics analysis of primary WT murine bone marrow-derived macrophages (BMDM) mock-treated or infected with methicillin-resistant Staphylococcus aureus (MRSA)
Column headers:
- Metabolite name: name of metabolite reference standard used for LC-MS method
- HMDB ID: Human metabolome database identifier for standard metabolite (https://hmdb.ca/)
- Mock 1-5: 5 mock (uninfected) BMDM biological replicates
- MRSA 1-5: 5 MRSA-infected BMDM biological replicates
- Method notes: Additional method details, including noted limitations such as isobaric peaks or isomers.

File S2 - Targeted metabolomics analysis of mock-treated or MRSA-infected WT or Ifnar1-/- iBMDM
Column headers:
- Metabolite name: name of metabolite reference standard used for LC-MS method
- HMDB ID: Human metabolome database identifier for standard metabolite (https://hmdb.ca/)
- WT Mock 1-3: 3 wildtype (WT) mock (uninfected) immortalized bone marrow-derived macrophage (iBMDM) biological replicates
- WT MRSA 1-3: 3 wildtype (WT) MRSA-infected iBMDM biological replicates
- IFNAR1 KO Mock 1-3: 3 Interferon alpha/beta receptor 1 knockout (IFNAR1 KO) mock (uninfected) iBMDM biological replicates
- IFNAR1 KO MRSA 1-3: 3 Interferon alpha/beta receptor 1 knockout (IFNAR1 KO) MRSA-infected iBMDM biological replicates
- Method notes: Additional method details, including noted limitations such as isobaric peaks or isomers.

Related publication(s):
Type I interferon governs immunometabolic checkpoints that coordinate inflammation during Staphylococcal infection Mack B. Reynolds, Benjamin Klein, Michael J. McFadden, Norah K. Judge, Hannah E. Navarrete, Britton C Michmerhuizen, Dominik Awad, Tracey L. Schultz, Paul W. Harms, Li Zhang, Teresa R. O’Meara, Jonathan Z. Sexton, Costas A. Lyssiotis, J. Michelle Kahlenberg, Mary X. O’Riordan bioRxiv 2024.01.10.575104; doi: https://doi.org/10.1101/2024.01.10.575104Use and Access:

Use and Access:
This data set is made available under a Creative Commons Public Domain license ( http://creativecommons.org/licenses/by/4.0/)

To Cite this Work:
Reynolds, M. B., Navarrete, H. E., Judge, N. K., McFadden, M. J., Klein, B., Michmerhuizen, B. C., Awad, D., Schultz, T. L., Harms, P. W., Zhang, L., O'Meara, T. R., Sexton, J. Z., Lyssiotis, C. A., Kahlenberg, J. M., O'Riordan, M. X. Targeted metabolomics analysis of wildtype and interferon alpha/beta receptor 1-deficient murine bone marrow-derived macrophages following methicillin-resistant Staphylococcus aureus infection [Data set], University of Michigan - Deep Blue Data. https://doi.org/10.7302/h15v-j558