Figure 6
Ex vivo LPS-activated inflammatory moDCs display an iNOS-dependent block in mitochondrial respiration and depend on glycolysis for survival in vivo. Inflammatory splenic moDCs were isolated from mice infected for 2 days with L monocytogenes (strain ΔActA, 2 × 105 CFU, intravenous) by sorting by flow cytometry for CD11c+CD11bintMHCII+Ly6Chi cells. Two subsets of resident splenic DCs were sorted from naive spleens based on expression of CD11chiMHCIIhiCD11b+CD4+ and CD11chiMHCIIhiDEC205+CD8α+. (A) Splenic DC subsets were left unstimulated or stimulated ex vivo with LPS for 24 hours and subsequently analyzed for intracellular iNOS expression. iNOS−/− cells were used to draw gates on iNOS+ cells. Representative FACS plots of 1 of 3 experiments are shown. (B) Supernatants from cultures of 24 hours ex vivo–stimulated inflammatory DCs were analyzed for nitrite levels. Data represent means ± SD of 3 experiments. (C) Ex vivo inflammatory moDCs were seeded in a Seahorse XF-24 analyzer, stimulated with medium or LPS for 24 hours in the presence or absence of SEITU, and real-time OCR was measured in response to sequential treatments with oligomycin, FCCP, and antimycin A/rotenone. Data represent means ± SD of triplicates. One of 2 experiments is shown. (D) Splenic DC subsets were cultured and stimulated as in panel C and the ratio between basal ECAR and OCR was calculated and plotted relative to the ratio of unstimulated DCs, which was set to 1. Data represent means ± SD of triplicates. One of 2 experiments is shown. (E) Mice infected for 1 day with L monocytogenes (strain ΔActA, 2 × 105 CFU, intravenous) were injected intraperitoneally with PBS or 2-DG (4 g/kg), and 6 hours later DC frequencies were determined in spleens. Data are representative of 5 individual mice per group. One of 2 independent experiments is shown. (F) Percentage of inflammatory moDCs staining positive for iNOS after PBS or 2-DG treatment as described in panel E. (G) Frequency of iNOS+ inflammatory moDCs in spleens after PBS or 2-DG treatment as described in panel E. Data are representative of 5 individual mice per group. One of 2 independent experiments is shown. **P < .01; ***P < .001.

Ex vivo LPS-activated inflammatory moDCs display an iNOS-dependent block in mitochondrial respiration and depend on glycolysis for survival in vivo. Inflammatory splenic moDCs were isolated from mice infected for 2 days with L monocytogenes (strain ΔActA, 2 × 105 CFU, intravenous) by sorting by flow cytometry for CD11c+CD11bintMHCII+Ly6Chi cells. Two subsets of resident splenic DCs were sorted from naive spleens based on expression of CD11chiMHCIIhiCD11b+CD4+ and CD11chiMHCIIhiDEC205+CD8α+. (A) Splenic DC subsets were left unstimulated or stimulated ex vivo with LPS for 24 hours and subsequently analyzed for intracellular iNOS expression. iNOS−/− cells were used to draw gates on iNOS+ cells. Representative FACS plots of 1 of 3 experiments are shown. (B) Supernatants from cultures of 24 hours ex vivo–stimulated inflammatory DCs were analyzed for nitrite levels. Data represent means ± SD of 3 experiments. (C) Ex vivo inflammatory moDCs were seeded in a Seahorse XF-24 analyzer, stimulated with medium or LPS for 24 hours in the presence or absence of SEITU, and real-time OCR was measured in response to sequential treatments with oligomycin, FCCP, and antimycin A/rotenone. Data represent means ± SD of triplicates. One of 2 experiments is shown. (D) Splenic DC subsets were cultured and stimulated as in panel C and the ratio between basal ECAR and OCR was calculated and plotted relative to the ratio of unstimulated DCs, which was set to 1. Data represent means ± SD of triplicates. One of 2 experiments is shown. (E) Mice infected for 1 day with L monocytogenes (strain ΔActA, 2 × 105 CFU, intravenous) were injected intraperitoneally with PBS or 2-DG (4 g/kg), and 6 hours later DC frequencies were determined in spleens. Data are representative of 5 individual mice per group. One of 2 independent experiments is shown. (F) Percentage of inflammatory moDCs staining positive for iNOS after PBS or 2-DG treatment as described in panel E. (G) Frequency of iNOS+ inflammatory moDCs in spleens after PBS or 2-DG treatment as described in panel E. Data are representative of 5 individual mice per group. One of 2 independent experiments is shown. **P < .01; ***P < .001.

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