DOI: 10.5061/dryad.rxwdbrvp0
title: Dexamethasone inhibits Mycobacterium tuberculosis-induced glycolysis but preserves antimicrobial function in primary human macrophages.

description: 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). 
description: <strong>Human Macrophage Culture</strong>
 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 &gt;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. <strong>Macrophage
 Stimulation</strong> 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.
 <strong>Macrophage Infection</strong>
 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). 
 <strong>Metabolic Flux Analysis</strong>
 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
 (1x10<sup>5</sup> cells/well) were re-plated onto 24 well
 Seahorse plates (Agilent). AM (1x10<sup>5</sup> 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. <strong>Cytokine
 Assays</strong> IL-1β, IL-6, IL-8, IL-10
 (BioLegend), and TNF (Invitrogen) concentrations in supernatants were
 quantified by ELISA, according to manufacturer’s protocol.
 <strong>Gene Expression Analysis</strong>
 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. <strong>PI inclusion
 Assay</strong> 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. <strong>Caspase
 Assay</strong> 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^+<sup>/7</sup>+^ MDM.
 <strong>Bacterial Growth Assays</strong>
 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.
description: # Dexamethasone inhibits Mycobacterium tuberculosis-induced glycolysis but
 preserves antimicrobial function in primary human macrophages. Dataset
 DOI: [10.5061/dryad.rxwdbrvp0](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.