IFN-γ increases energetic metabolism in the AM but enhances Warburg 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 primed with IFN-γ or IL-4 (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, irradiated Mtb H37Rv (iH37Rv; MOI 1-10) or LPS (100 ng/ml), 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). Each linked data point represents one individual donor (MDM; n=8-9, AM; n=9-10). Statistically significant differences were determined using two-way ANOVA (A-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 primed with IFN-γ or IL-4 (both 10 ng/ml) for 24 h. AM or MDM were left unstimulated or stimulated with iH37Rv (MOI 1-10) or LPS (100 ng/ml). 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 and MDM based on the average of their respective no cytokine controls. Each linked data point represents one individual donor (n=8-9). Statistically significant differences were determined using two-way ANOVA (A-F); *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 primed with IFN-γ or IL-4 (both 10 ng/ml) for 24 h. Cells were treated with 2DG (5mM) 1 h prior to stimulation with iH37Rv (MOI 1-10) or LPS (100 ng/ml). 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 one individual donor (n=8-9). Statistically significant differences were determined using two-way ANOVA (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, were differentiated and adherence purified for 7 days in 10% human serum. Cells were primed with IFN-γ or IL-4 (both 10 ng/ml) for 24 h. AM or MDM were left unstimulated or stimulated iH37Rv (MOI 1-10) or LPS (100 ng/ml). 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 (A) and LPS (B). Each linked data point represents one individual donor (AM; n=12-13, MDM; n=8-10). Statistically significant differences were determined using two-way ANOVA (A-D); *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 or #P≤0.05, ##P≤0.01, ####P≤0.0001 (IFN-γ excluded for analysis).

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 primed with IFN-γ or IL-4 (both 10 ng/ml) for 24 h. Cells were treated with 2DG (5mM) for 1 h prior to stimulation with iH37Rv (MOI 1-10) or LPS (100 ng/ml). 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 one individual donor (AM; n=12-13, MDM; n=8-10). Statistically significant differences were determined using two-way ANOVA (A-D); *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 or #P≤0.05, ##P≤0.01 (IFN-γ excluded for analysis).