IFN-γ increases energetic metabolism in the AM but enhances “Warburg”-like metabolism in MDM in response to inflammatory stimuli. Human AM (A-D) were isolated from bronchoalveolar lavage fluid. PBMC were isolated from buffy coats and MDM (E-H) were differentiated and adherence purified for 7 days in 10% human serum. Cells were left unprimed (black) or primed with IFN-γ (red) or IL-4 (blue) (both 10 ng/ml) for 24 h. Baseline measurements of the Extracellular Acidification Rate (ECAR) and the Oxygen Consumption Rate (OCR) were established before AM or MDM were stimulated with medium (circle), irradiated Mtb H37Rv (iH37Rv; MOI 1-10; square) or LPS (100 ng/ml; triangle), in the Seahorse XFe24 Analyzer, then monitored at 20-minute intervals. At 150 minutes, post stimulation fold change in ECAR (A, E, I) and OCR (B, F, J) was analysed, and percentage change (from baseline of the respective treatment group) was also calculated for ECAR (C, G) and OCR (D, H) at 150 minutes. A direct comparison of AM (white bar) and MDM (black bar) was also assessed at 150 minutes (I, J). Each linked data point represents the average of technical duplicates for one individual biological donor (MDM; n=8-9, AM; n=9-10). Statistically significant differences were determined using two-way ANOVA with a Tukey (A-H) or Bonferroni post-test (I-J); *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.001.

IFN-γ boosts activation marker expression on MDM to a greater extent than AM.

Human AM (A, C, E) isolated from bronchoalveolar lavage fluid. PBMC were isolated from buffy coats and MDM (B, D, F) were differentiated and adherence purified for 7 days in 10% human serum. Cells were left unprimed (black) or primed with IFN-γ (red) or IL-4 (blue) (both 10 ng/ml) for 24 h. AM or MDM were left unstimulated (circle) or stimulated with iH37Rv (MOI 1-10; square) or LPS (100 ng/ml; triangle). After 24 h cells were detached from the plates by cooling and gentle scraping and stained for HLAR-DR (A, B), CD40 (C, D), CD86 (E, F) and analysed by flow cytometry. Fold change of HLA-DR (G), CD40 (H) and CD86 (I) was calculated for AM (white bar) and MDM (black bar) based on the average of their respective no cytokine controls. Each linked data point represents the average of technical duplicates for one individual biological donor (n=8-9). Statistically significant differences were determined using two-way ANOVA with a Tukey (A-F) or Bonferroni post-test (G-I); *P≤0.05, **P≤0.01, P***≤0.001, ****P≤0.001.

Glycolysis is required for IFN-γ induced expression of activation markers by MDM and not AM.

Human AM (A, C, E) isolated from bronchoalveolar lavage fluid. PBMC were isolated from buffy coats and MDM (B, D, F) were differentiated and adherence purified for 7 days in 10% human serum. Cells were left unprimed (black) or primed with IFN-γ (red) or IL-4 (blue) (both 10 ng/ml) for 24 h. Cells were left untreated (solid) or treated with 2DG (5 mM; empty) 1 h prior to stimulation with iH37Rv (MOI 1-10; square) or LPS (100 ng/ml; triangle) or left unstimulated (circle). After 24 h cells were detached from the plates by cooling and gentle scraping and stained for HLAR-DR (A, B), CD40 (C, D), CD86 (E, F) and analysed by flow cytometry. Each linked data point represents the average of technical duplicates for one individual biological donor (n=8-9). Statistically significant differences were determined using two-way ANOVA with a Tukey post-test (A-F); *P≤0.05, **P≤0.01, P***≤0.001, ****P≤0.001.

IFN-γ enhances cytokine production more in AM compared with MDM. Human AM (A, C, E) isolated from bronchoalveolar lavage fluid. PBMC were isolated from buffy coats and MDM (B, D, F) were differentiated and adherence purified for 7 days in 10% human serum. Cells were left unprimed (black) or primed with IFN-γ (red) or IL-4 (blue) (both 10 ng/ml) for 24 h. AM or MDM were left unstimulated (circle) or stimulated iH37Rv (MOI 1-10; square) or LPS (100 ng/ml; triangle). Supernatants were harvested 24 h after stimulation and concentrations of IL-1β (A, B), TNF (C, D) and IL-10 (E, F) were quantified by ELISA. Fold change in IL-1β, TNF and IL-10 was calculated for AM and MDM based on the average of respective no cytokine controls for iH37Rv (G) and LPS (H). Each linked data point represents the average of technical duplicates for one individual biological donor (AM; n=12-13, MDM; n=8-10). Statistically significant differences were determined using two-way ANOVA with a Tukey (A-F) or Bonferroni post-test (G-H); *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 or #P≤0.05, ##P≤0.01, ####P≤0.0001 (where IFN-γ treated data sets were excluded for post-test analysis to analyse statistical differences between no cytokine and IL-4 treated data sets).

Cytokine secretion by AM is more reliant on glycolysis than MDM.

Human AM (A, C, E) isolated from bronchoalveolar lavage fluid. PBMC were isolated from buffy coats and MDM (B, D, F) were differentiated and adherence purified for 7 days in 10% human serum. Cells were left unprimed (black) or primed with IFN-γ (red) or IL-4 (blue) (both 10 ng/ml) for 24 h. Cells were left untreated (solid) treated with 2DG (5 mM; empty) for 1 h prior to stimulation with iH37Rv (MOI 1-10; square) or LPS (100 ng/ml; triangle) or left unstimulated (circle). Supernatants were harvested 24 h after stimulation and concentrations of IL-1β (A, B), TNF (C, D) and IL-10(E, F) were quantified by ELISA. Each linked data point represents the average of technical duplicates for one individual biological donor (AM; n=12-13, MDM; n=8-10). Statistically significant differences were determined using two-way ANOVA with a Tukey post-test (A-D); *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 or #P≤0.05, ##P≤0.01 (where IFN-γ primed data sets were excluded for post-test analysis to analyse statistical differences between no cytokine and IL-4 treated data sets).