Human airway macrophages are metabolically reprogrammed by IFN-γ resulting in glycolysis-dependent functional plasticity
Data files
Nov 19, 2024 version files 103.73 KB
Abstract
Airway macrophages (AM) are the predominant immune cells in the lung and play a crucial role in preventing infection, making them a target for host-directed therapy. Macrophage effector functions are associated with cellular metabolism. A knowledge gap remains in understanding metabolic reprogramming and functional plasticity of distinct human macrophage subpopulations, especially in lung resident AM. We examined tissue-resident AM and monocyte-derived macrophages (MDM; as a model of blood-derived macrophages) in their resting state and after priming with IFN-γ or IL-4 to model the Th1/Th2 axis in the lung. Human macrophages, regardless of origin, had a strong induction of glycolysis in response to IFN-γ or upon stimulation. IFN-γ significantly enhanced cellular energetics in both AM and MDM by upregulating both glycolysis and oxidative phosphorylation. Upon stimulation, AM does not decrease oxidative phosphorylation, unlike MDM which shifts to “Warburg”-like metabolism. IFN-γ priming promoted cytokine secretion in AM. Blocking glycolysis with 2-deoxyglucose significantly reduced IFN-γ driven cytokine production in AM, indicating that IFN-γ induces functional plasticity in human AM, which is mechanistically mediated by glycolysis. Directly comparing responses between macrophages, AM was more responsive to IFN-γ priming and dependent on glycolysis for cytokine secretion than MDM. Interestingly, TNF production was under the control of glycolysis in AM and not in MDM. MDM exhibited glycolysis-dependent upregulation of HLA-DR and CD40, whereas IFN-γ upregulated HLA-DR and CD40 on AM independently of glycolysis. These data indicate that human AM are functionally plastic and respond to IFN-γ in a manner distinct from MDM. These data provide evidence that human AM is a tractable target for inhalable immunomodulatory therapies for respiratory diseases.
https://doi.org/10.5061/dryad.98sf7m0t5
Description of the data and file structure
The data is presented in a single Excel file. All data in a column on each tab of the Excel file is matched to the same donor. Data is not matched across Excel tabs i.e., different donors may have been used for different experiments due to limitations in cellular yield from samples. 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 eLife paper.
This data consists of human alveolar macrophages and human monocyte-derived macrophages which were left unprimed or primed with IFN-γ or IL-4 (both 10 ng/ml) for 24h. Both cell types were then left unstimulated or stimulated with irradiated Mycobacterium tuberculosis (Strain H37Rv; iH37v) or lipopolysaccharide (LPS; 100 ng/ml). To assess the contribution of glycolysis to human macrophage function both alveolar macrophages and monocyte-derived macrophages were treated with 2-deoxyglucose (2DG; 5 mM), where indicated. Cellular metabolism was examined by Seahorse metabolic flux analysis (Excel sheet 1 – labelled Figure 1). Expression of macrophage activation markers (HLA-DR, CD40 and CD86) was assessed by flow cytometry (Excel Sheet 2 – labelled Figure 2). The dependency of unprimed or cytokine-primed macrophages on glycolysis for the expression activation markers was examined utilising the glycolytic inhibitor (Excel Sheet 2 – labelled Figure 2). The ability of primed macrophages to secrete the cytokines IL-1β, TNF and IL-10 were assessed in supernatants from unstimulated and stimulated macrophages (Excel Sheet 4 – labelled Figure 4). The dependency of human alveolar macrophages and monocyte-derived macrophages on glycolysis to produce the cytokines IL-1β, TNF and IL-10 was examined by ELISA (Excel Sheet 5 – labelled Figure 5).
Files and variables
File: Cox_et_al_2024_eLife_Human_airway_macrophages_are_metabolically_reprogrammed_by_IFN-γ_resulting_in_glycolysis_dependent_functional_plasticity.xlsx
Variables
Cell type (either human alveolar macrophage or human monocyte-derived macrophage) is listed above every data set within each Excel sheet.
Excel Sheet 1 (Figure 1): Data is derived from human alveolar macrophages and monocyte-derived macrophages that were left unprimed or primed with IFN-γ or IL-4 prior to stimulation as outlined above and assessed by Seahorse metabolic flux analysis. Outputs are listed in columns B, O and AB. These outputs correspond to subparts of Figure 1, i.e., Fig 1A, 1B etc. in the accompanying paper in eLife. Subparts of Figure 1 are also listed in columns B, O and AB immediately above the output. All data within a given column in this data sheet is matched to the same donor.
Outputs in Excel Sheet 1:
Extracellular Acidification Rate (ECAR, Fold Change to no cytokine unstimulated), a surrogate marker of glycolysis. This output relates to Figures 1A, 1E, and 1I of the associated paper.
Oxygen Consumption Rate (OCR, Fold Change to no cytokine unstimulated), a surrogate marker of oxidative phosphorylation. This output relates to Figure 1B, 1F, and 1J of the associated paper.
ECAR (% Change compared to respective baseline) percentage change compared to respective baselines was assessed to control for the effect of priming on glycolysis. This output relates to Figure 1C, 1G of the associated paper.
OCR (% Change compared to respective baseline) percentage change compared to respective baselines was assessed to control for the effect of priming on oxidative phosphorylation. This output relates to Figure 1D, 1H of the associated paper.
Excel Sheet 2: Data is derived from the measurement of the macrophage activation markers (HLA-DR, CD40, and CD86) by flow cytometry from human alveolar macrophages and monocyte-derived macrophages that were left unprimed or primed with IFN-γ or IL-4 prior to stimulation with iH37Rv or LPS. Outputs are listed in columns B, O, and AA. These outputs correspond to subparts of Figure 2, i.e., Fig 2A, 2B, etc. in the accompanying paper in eLife. Subparts of Figure 2 are also listed in columns B, O, and AA immediately above the output. All data within a given column in this data sheet is matched to the same donor.
Outputs in Excel Sheet 2:
HLA-DR, Median Fluorescence Intensity (x104): This output relates to Figures 2A and 2B of the associated paper.
CD40, Median Fluorescence Intensity (x104): This output relates to Figure 2C, 2D of the associated paper.
CD86, Median Fluorescence Intensity (x104) This output relates to Figure 2E, 2F of the associated paper.
HLA-DR, Median Fluorescence Intensity (fold change): This was used to normalise data for comparison of alveolar macrophages and monocyte-derived macrophages. This output relates to Figure 2G of the associated paper.
CD40, Median Fluorescence Intensity (fold change): This was used to normalise data for comparison of alveolar macrophages and monocyte-derived macrophages. This output relates to Figure 2H of the associated paper.
CD86, Median Fluorescence Intensity (fold change): This was used to normalise data for comparison of alveolar macrophages and monocyte-derived macrophages. This output relates to Figure 2H of the associated paper.
Excel Sheet 3: Data is derived from the measurement of the macrophage activation markers (HLA-DR, CD40, and CD86) by flow cytometry from human alveolar macrophages and monocyte-derived macrophages that were left unprimed or primed with IFN-γ or IL-4. Where indicated glycolysis was inhibited with 2DG prior to stimulation with iH37Rv or LPS. Outputs are listed in columns B and O. These outputs correspond to subparts of Figure 3, i.e., Fig 3A, 3B, etc. in the accompanying paper in eLife. Subparts of Figure 3 are also listed in columns B and O immediately above the output. All data within a given column in this data sheet is matched to the same donor.
Outputs in Excel Sheet 3:
HLA-DR, Median Fluorescence Intensity (x104): This output relates to Figures 3A and 3B of the associated paper.
CD40, Median Fluorescence Intensity (x104): This output relates to Figures 3C and 3D of the associated paper.
CD86, Median Fluorescence Intensity (x104) This output relates to Figures 3E and 3F of the associated paper.
Excel Sheet 4: Data is derived from the measurement of IL-1β, TNF, and IL-10 secretion from supernatants of unprimed and primed human alveolar macrophages and human monocyte-derived macrophages in response to stimulation with the bacterial ligands iH37Rv and LPS. Outputs are listed in columns C, U, and AJ. These outputs correspond to subparts of Figure 4, i.e., Fig 4A, 4B, etc. in the accompanying paper in eLife. Subparts of Figure 4 are also listed in columns C, U, and AJ immediately above the output. All data within a given column in this data sheet is matched to the same donor.
Outputs in Excel Sheet 4:
Secreted levels of IL-1β (pg/mL) This output relates to Figures 4A and 4B of the associated paper.
Secreted levels of TNF (ng/mL) This output relates to Figures 4C and 4D of the associated paper.
Secreted levels of IL-10 (pg/mL) This output relates to Figure 4E of the associated paper.
Secreted levels of IL-10 (ng/mL) This output relates to Figure 4F of the associated paper.
Fold change in IL-1β, TNF, and IL-10 secretion was calculated compared to the average of the respective iH37Rv stimulated unprimed control. This output relates to Figure 4G of the associated paper.
Fold change in IL-1β, TNF, and IL-10 secretion was calculated compared to the average of the respective LPS-stimulated unprimed control. This output relates to Figure 4H of the associated paper.
Excel Sheet 5: Data is derived from the measurement of IL-1β, TNF, and IL-10 secretion from supernatants of unprimed and primed human alveolar macrophages and human monocyte-derived macrophages in response to stimulation with the bacterial ligands iH37Rv and LPS. Outputs are listed in columns C and U. These outputs correspond to subparts of Figure 5, i.e. Fig 5A, 5B, etc. in the accompanying paper in eLife. Subparts of Figure 5 are also listed in columns C and U immediately above the output. All data within a given column in this data sheet is matched to the same donor.
Outputs in Excel Sheet 5:
Secreted levels of IL-1β (pg/mL) This output relates to Figures 5A and 5B of the associated paper.
Secreted levels of TNF (ng/mL) This output relates to Figures 5C and 5D of the associated paper.
Secreted levels of IL-10 (pg/mL) This output relates to Figure 5E of the associated paper.
Secreted levels of IL-10 (ng/mL) This output relates to Figure 5F of the associated paper.
A detailed methods section can be found in “Human airway macrophages are metabolically reprogrammed by IFN-γ resulting in glycolysis dependent functional plasticity” Cox et al. 2024, eLife.
Blank values within a data sheet were not assayed for a given sample.
Code/software
NA
Metabolic Flux Analysis (Seahorse XFe 24), Flow Cytometry and ELISA.