Dexamethasone inhibits Mycobacterium tuberculosis-induced glycolysis but preserves antimicrobial function in primary human macrophages.
Data files
Sep 08, 2025 version files 90.93 KB
-
README.md
10.66 KB
-
Thong_2025_Sci_Reports_Dryad_File.xlsx
80.27 KB
Abstract
This data belongs to the figures from Thong et al 2025 Scientific Reports (Dexamethasone inhibits Mycobacterium tuberculosis-induced glycolysis but preserves antimicrobial function in primary human macrophages). The data is presented in a single excel file. Each tab in the excel file represents a different figure from the above paper.
It is highly recommended to read the excel file in conjunction with each figure within the associated paper. The data has been organised based on the layout of figures in the associated Scientific Reports paper.
This data consists of human monocyte derived macrophages and human alveolar macrophages. Human macrophages were left untreated or treated with vehicle control (ethanol, 0.02%) or dexamethasone (0.05-5 μM, as indicated). To model pathways of cellular death, MDM were treated with staurosporine (1 μM) for 24 h. The effects of dexamethasone on macrophage metabolism was examined by Seahorse metabolic flux analysis using the glycolytic rate assays and Mitostress tests (Excel sheet 1 – labelled Figure 1 hMDM, Excel sheet 2 – labelled Figure 2 hAM, Excel sheet 3 – labelled Figure 3 hMDM). The expression of mRNA transcripts of metabolic enzymes were assessed to support the functional metabolic data (Excel sheet 1 – labelled Figure 1 hMDM, Excel sheet 3 – labelled Figure 3 hMDM). The ability of dexamethasone to alter the secretion of cytokines IL-1β, TNF, IL-6, IL-8 and IL-10 was assessed in supernatants from uninfected and Mtb infected (H37Ra) macrophages (Excel Sheet 4 – labelled Figure 4 hMDM). The effect of dexamethasone cell death and caspase activation in macrophages was examined (Excel Sheet 5 – labelled Figure 5 hMDM). Finally bacterial control of macrophages was examined in dexamethasone treated macrophages in the presence of bafilomycin or rapamycin. Bafilomycin A1 (50 nM) or rapamycin (250 ng/ml) were used to examine the role of autophagy in dexamethasone induced bacterial control (Excel Sheet 6 – labelled Figure 6 hMDM). These data were calculated by assessing colony forming units assays on Middlebrook agar. Supplemental Figures 1 and 2 are supporting data for Seahorse Assays (Excel Sheet 7- labelled Figure S1 hMDM, Excel Sheet 8- labelled Figure S2 hMDM). Supplemental Figure 3 is supporting data for bacterial growth assays demonstrating phagocytosis on day 0 (Excel Sheet 9- labelled Figure S3 hMDM).
Dataset DOI: 10.5061/dryad.rxwdbrvp0
Description of the data and file structure
Description: Data for figures presented in Dexamethasone inhibits Mycobacterium tuberculosis-induced glycolysis but preserves antimicrobial function in primary human macrophages. Thong et al., 2025 Scientific Reports.
The data is presented in a single excel file.
Files and variables
File: Thong_2025_Sci_Reports_Dryad_File.xlsx
Variables
- Cell type (either human monocyte derived macrophage (hMDM) or human airway macrophage (hAM)) is listed in box 1A of every data set within each excel sheet.
Excel Sheet 1 (Figure 1-hMDM): Data is derived from human monocyte derived macrophages (MDM) that were treated with vehicle control (VC; ethanol 0.02%) or dexamethasone for 24 h prior to analysis in the Seahorse XFe24 Analyzer using a glycolytic rate assay (a-e). Alternatively, human MDM were treated with VC or dexamethasone for 4 hours, lysed and RNA extracted (f-i). Outputs are listed in columns E, AA, AG,AM and AS. These outputs correspond to subparts of figure 1, i.e. Fig 1a, 1b etc. in the accompanying paper in Scientific Reports.
Outputs in Excel Sheet 1:
Glyco Proton Extrusion Rate (GlycoPER; pmol/min), a marker of glycolysis. This output relates to Figure 1a, 1b, and 1c of the associated paper.
Oxygen Consumption Rate (OCR, pmol/min), a surrogate marker of oxidative phosphorylation. This output relates to Figure 1d, and 1e of the associated paper.
Gene expression relative to 18S (δδCT) for PFKFB3. This is a gene encoding a glycolytic enzyme. This output relates to Figure 1f of the associated paper.
Gene expression relative to 18S (δδCT) for GAPDH. This is a gene encoding a glycolytic enzyme. This output relates to Figure 1g of the associated paper.
Gene expression relative to 18S (δδCT) for PKM2. This is a gene encoding a glycolytic enzyme. This output relates to Figure 1h of the associated paper.
Gene expression relative to 18S (δδCT) for ATP5B. This is a gene encoding a enzyme required for oxidative phosphorylation. This output relates to Figure 1i of the associated paper.
Excel Sheet 2 (Figure 2-hAM): Data is derived from human airway macrophages (MDM) that were treated with vehicle control (VC; ethanol 0.02%) or dexamethasone for 24 h prior to analysis in the Seahorse XFe24 Analyzer using a glycolytic rate assay (a-f). Outputs are listed in columns E, AD, AJ and AP. These outputs correspond to subparts of figure 2, i.e. Fig 2a, 2b etc. in the accompanying paper in Scientific Reports.
Outputs in Excel Sheet 2:
Glyco Proton Extrusion Rate (GlycoPER; pmol/min), a marker of glycolysis. This output relates to Figure 2a, 2b, 2c and 2f of the associated paper.
Data in blue with an asterisk were excluded for generating figure 2a in the associated paper due to a technical fault with the Seahorse analyzer in AM4 at timepoint 9.199336 minutes. GlycoPER cannot be a negative value. For transparency these data are included in the data table, however to denote the exclusion they are highlighted in blue with an asterisk, rather than being omitted.
Oxygen Consumption Rate (OCR, pmol/min), a surrogate marker of oxidative phosphorylation. This output relates to Figure 2d, 2e and 2f of the associated paper.
Excel Sheet 3 (Figure 3-hMDM): Data is derived from human monocyte derived macrophages (MDM) that were treated with vehicle control (VC; ethanol 0.02%) or dexamethasone for immediately prior to transferring into the Seahorse XFe24 Analyzer. MDM were then stimulated with media or irradiated Mtb (iH37Rv) and assessed using a glycolytic rate assay (a-f). Alternatively, MDM were treated with VC or dexamethasone for 1 hour prior to infection with Mtb for 3h, MDM were then lysed and RNA extracted (g-j). Outputs are listed in columns E, AE, AL, AT and BA. These outputs correspond to subparts of figure 3, i.e. Fig 3a, 3b etc. in the accompanying paper in Scientific Reports.
Outputs in Excel Sheet 3:
Glyco Proton Extrusion Rate (GlycoPER; pmol/min), a marker of glycolysis. This output relates to Figure 3a, 3b, 3c, 3d, 3e and 3f of the associated paper.
Oxygen Consumption Rate (OCR, pmol/min), a surrogate marker of oxidative phosphorylation. This output relates to Figure 3e and 3f of the associated paper.
Gene expression relative to 18S (δδCT) for PFKFB3. This is a gene encoding a glycolytic enzyme. This output relates to Figure 3g of the associated paper.
Gene expression relative to 18S (δδCT) for GAPDH. This is a gene encoding a glycolytic enzyme. This output relates to Figure 3h of the associated paper.
Gene expression relative to 18S (δδCT) for PKM2. This is a gene encoding a glycolytic enzyme. This output relates to Figure 3i of the associated paper.
Gene expression relative to 18S (δδCT) for ATP5B. This is a gene encoding a enzyme required for oxidative phosphorylation. This output relates to Figure 3j of the associated paper.
Excel Sheet 4 (Figure 4-hMDM): Data is derived from the measurement of IL-1β, TNF, IL-6, IL-8 and IL-10 secretion from supernatants of uninfected and Mtb (strain H37Ra) infected MDM. MDM were treated with VC (ethanol 0.02%) or dexamethasone (0.05, 0.5 or 5 μM) for 1 hour prior to infection with Mtb (MOI 1-10). Outputs are listed in column E. These outputs correspond to subparts of figure 4, i.e. Fig 4a, 4b etc. in the accompanying paper in Scientific Reports.
Outputs in Excel Sheet 4:
Secreted levels of IL-1β (pg/mL). This output relates to Figure 4a of the associated paper.
Secreted levels of TNF (pg/mL). This output relates to Figure 4b of the associated paper.
Secreted levels of IL-6 (pg/mL). This output relates to Figure 4c of the associated paper.
Secreted levels of IL-8 (pg/mL). This output relates to Figure 4d of the associated paper.
Secreted levels of IL-10 (pg/mL). This output relates to Figure 4e of the associated paper.
NA indicates a sample was not assayed for an entire sample.
Excel Sheet 5 (Figure 5-hMDM): Data is derived from the measurement of cell death by propidium iodide or caspase activation in human MDM treated with VC (ethanol 0.02%) or dexamethasone (0.05, 0.5 or 5 μM). MDM were then infected with Mtb (MOI 1-10) or stimulated with staurosporine (1μM). Outputs are listed in column E. These outputs correspond to subparts of figure 5, i.e. Fig 5a, 5b etc. in the accompanying paper in Scientific Reports.
Outputs in Excel Sheet 5:
Cell Death calculated as a percentage (%) of total cells. This output relates to Figures 5a and 5C of the associated paper.
Caspase3+/7+ Cells calculated as a percentage (%) of total cells. This output relates to Figure 5b of the associated paper.
Excel Sheet 6 (Figure 6-hMDM): Data is derived from the counts of colony forming units (CFU) of Mycobacterium tuberculosis (Strain H37Ra) grown on Middlebrook agar. MDM were treated with vehicle control (VC; ethanol 0.02%) or dexamethasone for 1 hour prior to infection with Mtb (MOI 1-10). Alternatively, MDM were treated with bafilomycin A1 (50 nM) for 1 hour or rapamycin (250 ng/ml) for 24 h prior to dexamethasone. MDM were lysed at 3, 48 or 120 hours. Outputs are listed in column E. These outputs correspond to subparts of figure 6, i.e. Fig 6a, 6b etc. in the accompanying paper in Scientific Reports.
Outputs in Excel Sheet 6:
Colony Forming Units (CFU) per mL (CFU/mL). This output relates to Figures 6a, 6b and 6c of the associated paper.
Excel Sheet 7 (Figure S1-hMDM): Data is derived from human monocyte derived macrophages (MDM) that were left untreated or treated with vehicle control (VC; ethanol 0.02%) for 24 h prior to analysis in the Seahorse XFe24 Analyzer using a glycolytic rate assay. Outputs are listed in columns E, U and AA. These outputs correspond to subparts of supplemental figure S1, i.e. Fig S1a, S1b etc. in the accompanying paper in Scientific Reports.
Outputs in Excel Sheet 7:
Glyco Proton Extrusion Rate (GlycoPER; pmol/min), a marker of glycolysis. This output relates to Figure S1a, S1b, and S1c of the associated paper.
Oxygen Consumption Rate (OCR, pmol/min), a surrogate marker of oxidative phosphorylation. This output relates to Figure S1d, and S1e of the associated paper.
Excel Sheet 8 (Figure S2-hMDM): Data is derived from human monocyte derived macrophages (MDM) that were treated with vehicle control (VC; ethanol 0.02%) or dexamethasone for 24 h prior to analysis in the Seahorse XFe24 Analyzer using a mitostress test. Outputs are listed in columns D, AE, AK and AQ. These outputs correspond to subparts of supplemental figure S2, i.e. Fig S2a, S2b etc. in the accompanying paper in Scientific Reports.
Outputs in Excel Sheet 7:
Oxygen Consumption Rate (OCR, pmol/min), a surrogate marker of oxidative phosphorylation. This output relates to Figure S2a, S2b, S2e and S2f of the associated paper. OCR is also used to measure maximal respiration in Figure S2c and S2g and Spare Respiratory Capacity in Figure S2d and S2h.
Excel Sheet 9 (Figure S3-hMDM): Data is derived from the counts of colony forming units (CFU) of Mycobacterium tuberculosis (Strain H37Ra) grown on Middlebrook agar. MDM were treated with vehicle control (VC; ethanol 0.02%) or dexamethasone for 1 hour prior to infection with Mtb (MOI 1-10). Alternatively, MDM were treated with bafilomycin A1 (50 nM) for 1 hour or rapamycin (250 ng/ml) for 24 h prior to dexamethasone. MDM were lysed at 3 to examine phagocytosis inputs to support experiments in Figure 6. Outputs are listed in column E. These outputs correspond to subparts of supplemental figure S3, i.e. Fig S3a, S3b etc. in the accompanying paper in Scientific Reports.
A detailed methods section can be found in "Dexamethasone inhibits Mycobacterium tuberculosis-induced glycolysis but preserves antimicrobial function in primary human macrophages." Thong et al., 2025 Scientific Reports.
Blank values within a data sheet were not assayed for a given sample.
Code/software
NA
Human subjects data
All individuals provided written informed consent and were made aware that collective results arising from samples given would be published. Each donor was assigned an anonymized, sequential identification number (starting at 1) after data analysis and graphing. These identifiers are arbitrary and cannot be linked to any personal information, ensuring complete anonymisation.
Human Macrophage Culture
Buffy coats were obtained with consent from healthy donors (aged between 18–69; ethical approval, School of Medicine Research Ethics Committee, Trinity College Dublin). Peripheral blood mononuclear cells (PBMC) were isolated by density-gradient centrifugation over Lymphoprep (StemCell Technologies). Cells were resuspended in RPMI (Gibco) supplemented with 10% AB human serum (Merck) and plated onto non-treated tissue culture plates (Costar) for 7 days. Non-adherent cells were removed by washing every 2-3 days. Cultures were >90% pure based on co-expression of CD14 and CD68.
Human AM were retrieved at bronchoscopy, (ethical approval, the SJH/TUH Joint Research Ethics Committee). AM were plated in RPMI (Gibco) supplemented with 10% FBS (Gibco), fungizone (2.5 μg/ml; Gibco) and cefotaxime (50 μg/ml; Melford Biolaboratories). Cells were incubated for 24 h before washing to remove non-adherent cells. Adherent cells (predominantly AM) were used for experiments.
Macrophage Stimulation
Human macrophages were left untreated or treated with vehicle (ethanol, 0.02%) or dexamethasone (0.05-5 μM, as indicated). To model pathways of cellular death, MDM were treated with staurosporine (1 μM; Tocris) for 24h.
Macrophage Infection
Mtb H37Ra was obtained from the American Type Culture Collection (ATCC 25177™; Manassas, VA) and propagated in Middlebrook 7H9 medium supplemented with ADC (Beckton Dickinson), to log phase. Irradiated H37Rv (iH37Rv) was gifted by BEI Resources. The multiplicity of infection (MOI) and donor variation in phagocytosis of Mtb was adjusted for by Auramine-O staining. MDM or AM were plated on 8-well Lab-Tek chamber slides (Nunc) and infected with a range of bacterial concentrations for 3 h before extracellular bacteria were thoroughly washed off. Cells were fixed with 2% PFA, stained with Hoechst 33342 (10 μg/ml; Merck), and rapid Auramine O staining set (Scientific Device Laboratory Inc). The numbers of bacilli per cell were counted in at least 30 fields of vision per well on a fluorescent microscope (Olympus IX51).
Metabolic Flux Analysis
MDM were placed in ice-cold PBS and incubated at 4°C on ice for 30 min, then gently scraped and counted using trypan blue. MDM (1x105 cells/well) were re-plated onto 24 well Seahorse plates (Agilent). AM (1x105 cells/well) were directly plated onto Seahorse plates and washed after 24 h. Glycolytic rate assays and Mitostress tests were conducted as per manufacturer’s protocol.
Cytokine Assays
IL-1β, IL-6, IL-8, IL-10 (BioLegend), and TNF (Invitrogen) concentrations in supernatants were quantified by ELISA, according to manufacturer’s protocol.
Gene Expression Analysis
RNA extractions from monocytes were performed using an RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA content and quality were quantified and assayed, respectively, using a Nanodrop (Thermo Fisher Scientific) and RNA reverse transcribed using the SensiFast Reverse-Transcription Kit (Meridian Biosciences). Catalogued TaqMan (Thermo Fisher Scientific) predesigned gene primer probes attached to the FAM dye were used: PFKFB3 (Hs00998698_m1), GAPDH (Hs02786624_g1), PKM2 (Hs00761782_s1), and ATP5B (Hs00969569_m1). 18S (Hs03003631_g1) was used as the reference gene primer, attached to the VIC dye. RT-qPCR was performed using SensiFast Probe Mix (Meridian Biosciences, catalogue BIO-82005) on a QuantStudio 5 RT-qPCR System (Applied Biosystems). Relative quantitative data were obtained and analysed utilizing the 2–ΔΔCt method.
PI inclusion Assay
Human MDM were stained with propidium iodide (PI; 5 μg/ml; Merck), and Hoechst 33258 and 33342 (both 50 μg/ml; Merck) at the indicated time-points post-infection with Mtb or treatment with staurosporine or TNF. Cells were incubated for 30 min at room temperature in the dark. Imaging was performed with a LionHeart FX® Automated Microscope (Agilent) at 10X. The ratio of PI–positive cells to the total number of Hoechst 33258/Hoechst 33242-stained nuclei in 4 fields was determined by the Gen5 software (Biotek) and used to calculate the percentage of cell death.
Caspase Assay
MDM were stained with CellEvent™ Caspase 3/7 Green Detection Reagent (Invitrogen) as per manufacturer’s instructions as well as Hoechst 33258 and 33342 (both 50 μg/ml; Merck). Imaging was performed using a LionHeart FX® Automated Microscope (Agilent) at 10X. The ratio of caspase 3/7–positive cells to the total number of Hoechst 33258/Hoechst 33342-stained nuclei in 4 fields was determined by the Gen5 software (Biotek) and used to calculate percentage Caspase 3^+/7+^ MDM.
Bacterial Growth Assays
Colony forming units (CFU) were determined at 3, 48 or 120 h post-infection, as indicated. MDM were also treated with bafilomycin A1 (50 nM) or rapamycin (250 ng/ml) to examine the role of autophagy in dexamethasone induced bacterial control. Cells were lysed with triton-X 100 (0.1%) and pooled with bacterial pellets (except 3 h) from the centrifugation of supernatants. Bacteria were diluted in Middlebrook 7H9 broth and plated onto Middlebrook 7H10 agar supplemented with OADC (both Becton Dickinson) and cycloheximide (Merck). Agar plates were incubated at 37°C for 21 days and CFU were then enumerated.