Journal: eLife

Article Title: Elevated glycolytic metabolism of monocytes limits the generation of HIF1A-driven migratory dendritic cells in tuberculosis

doi: 10.7554/eLife.89319

Figure Lengend Snippet: Monocyte-derived DCs (Mo-DCs) were treated (or not) with HIF1A inhibitor PX-478 (PX) or LDH inhibitor oxamate (OX) and stimulated with iMtb for 24 hr. ( A ) Lactate release as measured in supernatants in DCs stimulated or not with iMtb in the presence of OX (N = 5). ( B ) Percentage of migrated cells toward CCL21 relative to the number of initial cells per condition (N = 6). ( C, D ) Three-dimensional amoeboid migration of DCs through a collagen matrix after 24 hr. Cells within the matrix were fixed and stained with DAPI. Images of the membrane of each insert were taken and the percentage of cells per field were counted. ( C ) Mo-DCs stimulated with iMtb for 24 hr (N = 5). ( D ) Mo-DCs infected with Mtb for 24 hr (N = 4). The data are represented as scatter plots, where each circle represents a microphotograph sourced from either five ( C ) or four ( D ) independent donors, with each experiment typically including between 5 to 10 microphotographs. ( E ) Representative schematic of the experimental setup for in vivo migration assays. ( F ) Percentages of migrating bone marrow-derived DCs (BMDCs) (CFSE-labeled among CD11c + ) recovered from inguinal lymph nodes (N = 3). Statistical significance assessed by ( A, B ) ANOVA followed by Dunnett’s multiple-comparisons test (*p<0.05; **p<0.01); ( C, D ) Nested ANOVA followed by Dunnett’s multiple-comparisons test (*p<0.05; **p<0.01); ( F ) ANOVA followed by Holm–Sidak’s multiple-comparisons test (*p<0.05).

Article Snippet: Each DC population (4 × 10 5 cells in 75 μl) was placed on the upper chamber of a transwell insert (5 μm pore size, 96-well plate; Corning), and 230 μl of media (RPMI with 0.5% FCS) with human recombinant CCL21 (200 ng/ml) (Peprotech) was placed in the lower chamber.

Techniques: Derivative Assay, Migration, Staining, Membrane, Infection, In Vivo, Labeling

Journal: eLife

Article Title: Elevated glycolytic metabolism of monocytes limits the generation of HIF1A-driven migratory dendritic cells in tuberculosis

doi: 10.7554/eLife.89319

Figure Lengend Snippet: ( A, B ) Monocyte-derived DCs (Mo-DCs) were treated or not with the glycolysis inhibitor GSK2837808A and stimulated with iMtb. ( A ) Lactate release (N = 5). ( B ) Chemotactic activity toward CCL21 (N = 5). ( C, D ) Murine bone marrow-derived DCs (BMDCs) were treated or not with PX-478 or oxamate and stimulated with iMtb for 24 hr. ( C ) Lactate release (N = 3). ( D ) Representative dot blots showing the percentages of migrating BMDCs (CD11c + , CFSE-labeled) determined from inguinal lymph nodes. ( E ) Mo-DCs were stimulated with iMtb in the presence of either PX-478 or oxamate and CCR7 expression was measured by FACS (N = 6). Two-way ANOVA followed by Tukey’s multiple-comparisons test (*p<0.05; **p<0.01; ***p<0.001), as depicted by lines. The data are represented as scatter plots, with each circle representing a single individual, means ± SEM are shown.

Article Snippet: Each DC population (4 × 10 5 cells in 75 μl) was placed on the upper chamber of a transwell insert (5 μm pore size, 96-well plate; Corning), and 230 μl of media (RPMI with 0.5% FCS) with human recombinant CCL21 (200 ng/ml) (Peprotech) was placed in the lower chamber.

Techniques: Derivative Assay, Activity Assay, Labeling, Expressing

Journal: eLife

Article Title: Elevated glycolytic metabolism of monocytes limits the generation of HIF1A-driven migratory dendritic cells in tuberculosis

doi: 10.7554/eLife.89319

Figure Lengend Snippet: Tolerogenic Mo-DCs were generated by dexamethasone (Dx) treatment and were stimulated (or not) with irradiated Mycobacterium tuberculosis (iMtb) in the presence or absence of HIF1A activator dimethyloxalylglycine (DMOG). ( A ) Lactate release and glucose uptake as measured in supernatant (N = 8). ( B ) Mean fluorescence intensity (MFI) of HIF1A. Representative histograms and quantification are shown (N = 6). ( C ) Three-dimensional amoeboid migration of DCs through a collagen matrix. After 24 hr of migration, images of stacks within the matrix were taken every 30 µm. Percentage of migrating cells was defined as cells in the stacks within the matrix relative to total number of cells (N = 6). ( D ) Chemotactic activity toward CCL21 in vitro (N = 6). ( E–H ) Mo-DCs were generated from healthy subjects (HS) or TB patients, and DCs were stimulated (or not) with iMtb. ( E ) Chemotaxis index toward CCL21 (relative to unstimulated DCs) (N = 6). ( F ) Lactate production ratio relative to unstimulated DCs (N = 6). ( G ) Glycolytic capacity assessed by SCENITH (N = 4). ( H ) Chemotactic activity toward CCL21 of Mo-DCs from TB patients stimulated with iMtb and treated or not with DMOG (N = 6). Statistical significance assessed by ( A–D ) two-way ANOVA followed by Tukey’s multiple-comparisons test (*p<0.05; **p<0.01); ( E–G ) unpaired t -test (*p<0.05); ( H ) paired t -test (*p<0.05). The data are represented as scatter plots, with each circle representing a single individual, means ± SEM are shown.

Article Snippet: Each DC population (4 × 10 5 cells in 75 μl) was placed on the upper chamber of a transwell insert (5 μm pore size, 96-well plate; Corning), and 230 μl of media (RPMI with 0.5% FCS) with human recombinant CCL21 (200 ng/ml) (Peprotech) was placed in the lower chamber.

Techniques: Generated, Irradiation, Fluorescence, Migration, Activity Assay, In Vitro, Chemotaxis Assay

Journal: eLife

Article Title: Elevated glycolytic metabolism of monocytes limits the generation of HIF1A-driven migratory dendritic cells in tuberculosis

doi: 10.7554/eLife.89319

Figure Lengend Snippet: ( A ) Ex vivo determination of HIF1A expression by monocytes from healthy subjects (HS) or TB patients (TB) for each monocyte subset (CD14 + CD16 - , CD14 + CD16 + , and CD14 dim CD16 + ) (N = 6). ( B, C ) Monocytes from HS were treated with dimethyloxalylglycine (DMOG) during the first 24 hr of differentiation with IL-4/GM-CSF (earlyDMOG) and removed afterward. On day 6 of differentiation, cells were stimulated (or not) with irradiated Mycobacterium tuberculosis (iMtb). ( B ) Monocyte lactate release after 24 hr of DMOG addition (N = 4). ( C ) Chemotactic activity toward CCL21 of DCs (N = 5). Statistical significance was assessed by ( B ) paired t -test (p<0.01); ( C ) two-way ANOVA followed by Tukey’s multiple-comparisons test (*p<0.05). The data are represented as scatter plots, with each circle representing a single individual, means ± SEM are shown.

Article Snippet: Each DC population (4 × 10 5 cells in 75 μl) was placed on the upper chamber of a transwell insert (5 μm pore size, 96-well plate; Corning), and 230 μl of media (RPMI with 0.5% FCS) with human recombinant CCL21 (200 ng/ml) (Peprotech) was placed in the lower chamber.

Techniques: Ex Vivo, Expressing, Irradiation, Activity Assay

Journal: eLife

Article Title: Elevated glycolytic metabolism of monocytes limits the generation of HIF1A-driven migratory dendritic cells in tuberculosis

doi: 10.7554/eLife.89319

Figure Lengend Snippet:

Article Snippet: Each DC population (4 × 10 5 cells in 75 μl) was placed on the upper chamber of a transwell insert (5 μm pore size, 96-well plate; Corning), and 230 μl of media (RPMI with 0.5% FCS) with human recombinant CCL21 (200 ng/ml) (Peprotech) was placed in the lower chamber.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Sequencing, Software