Fig 1.
Simplified overview of key metabolic pathways involved in BMDC activation-associated metabolic reprogramming.
Schematic representations of important metabolic pathways used in (A) resting DCs, (B) during acute metabolic reprogramming events, and (C) during sustained metabolic reprogramming. Assays assessing metabolic changes described in this manuscript are noted where most relevant in the time course of activation.
Fig 2.
NO produced via iNOS in BMDCs suppresses mitochondrial respiration in a dose dependent manner.
(A) Nos2 gene transcription assessed by RT-qPCR from BMDCs stimulated for 5 hours with U or 100 ng/mL LPS. Signals normalized to β-actin as the housekeeping gene via the 2(ΔCt) method. Analyzed by t test, adjusted p values are reported (p value > 0.05 ns), n = 4 biological replicates, representative of at least three independent experiments. (B) Western blot for iNOS from protein lysates isolated from BMDCs stimulated for 24 hours with U or 100 ng/mL LPS. β-actin serves as a loading control, n = 4 biological replicates, representative of at least three independent experiments. (C) Griess nitrite assay on media supernatant from BMDCs stimulated for 24 hours with U or 100 ng/mL LPS. Analyzed by t test, adjusted p values are reported (p value > 0.05 ns), n = 4 biological replicates, representative of at least three independent experiments. (D) Mitochondrial stress test OCR kinetic trace of extracellular flux analysis on BMDCs stimulated for 24 hours with U or 100 ng/mL LPS. Oligo, FCCP, and Rot/Ant are short for injections of oligomycin; ATP synthase inhibitor, fluoro-carbonyl cyanide phenylhydrazone; mitochondrial membrane uncoupler, and rotenone/antimycin A; electron transport chain complex I and III inhibitors, respectively. Injections indicated by dashed vertical lines, n = 1 biological replicate, representative of at least three independent experiments. (E) Mitochondrial-linked OCR calculated from kinetic traces in (D) by subtracting the average OCR post inhibition of the electron transport chain by rotenone/antimycin A from average baseline OCR before any injections. Analyzed by t test, adjusted p values are reported (p value > 0.05 ns), n = 4 biological replicates, representative of at least three independent experiments. (F) Mitochondrial-linked OCR calculated from kinetic traces during a standard mitochondrial stress test on BMDCs treated with a titration of LPS from 0 ng/mL up to 100 ng/mL, n = 2 biological replicates, representative of three or more independent experiments.
Fig 3.
NO-mediated suppression of mitochondrial respiration occurs at a finite threshold and is directly linked to TLR stimulus concentration.
(A) Nos2 gene transcription assessed by RT-qPCR from BMDCs stimulated for 5 hours with U, 5 ng/mL, 10 ng/mL, or 25 ng/mL LPS. Signals normalized to β-actin as the housekeeping gene via the 2(ΔCt) method. Analyzed by one-way ANOVA, adjusted p values are reported (p value > 0.05 ns), n = 4 biological replicates, representative of at least three independent experiments. (B) Western blot for iNOS from protein lysates isolated from BMDCs stimulated for 24 hours with U, 5 ng/mL, 10 ng/mL, or 25 ng/mL LPS. β-actin serves as a loading control, n = 4 biological replicates, representative of at least three independent experiments. (C) Griess nitrite assay on media supernatant from BMDCs stimulated for 24 hours with U, 12–14 ng/mL, or 25 ng/mL LPS. Analyzed by one-way ANOVA, adjusted p values are reported (p value > 0.05 ns), n = 4 biological replicates, representative of at least three independent experiments. (D) Mitochondrial stress test OCR kinetic trace of extracellular flux analysis on BMDCs stimulated for 24 hours with U, 12–14 ng/mL, or 25 ng/mL LPS. Oligo, FCCP, and Rot/Ant are short for injections of oligomycin; ATP synthase inhibitor, fluoro-carbonyl cyanide phenylhydrazone; mitochondrial membrane uncoupler, and rotenone/antimycin A; electron transport chain complex I and III inhibitors, respectively. Injections indicated by dashed vertical lines, n = 1 biological replicate, representative of at least three independent experiments. (E) Mitochondrial-linked OCR calculated from kinetic traces in (D) by subtracting the average OCR post inhibition of the electron transport chain by rotenone/antimycin A from average baseline OCR before any injections. Analyzed by one-way ANOVA, adjusted p values are reported (p value > 0.05 ns), n = 4 biological replicates, representative of at least three independent experiments. (F) Griess nitrite assay on media supernatant from BMDCs stimulated for 24 hours with U, 12–14 ng/mL, or 25 ng/mL LPS. Analyzed by one-way ANOVA, adjusted p values are reported (p value > 0.05 ns), n = 4 biological replicates, representative of at least three independent experiments. (G) Percentage of live cells assessed by flow cytometry using a fixable Live/Dead stain on BMDCs treated with U, 12–14 ng/mL, or 25 ng/mL LPS for 96 hours, n = 3 biological replicates, representative of at least three independent experiments. (H) Live cell percentage of BMDCs treated with 10 ng/mL and 25 ng/mL LPS for 96 hours from (G). Analyzed by t test, adjusted p values are reported (p value > 0.05 ns), n = 3 biological replicates, representative of at least three independent experiments. If no p value is provided, the data is non-significant (p value > 0.05) and therefore, not included on the figure itself. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.
Fig 4.
Detection of non-mitochondrial oxygen consumption in the iNOS reaction using Real Time Extracellular Flux Analysis.
(A) Modified mitochondrial stress test OCR kinetic trace of extracellular flux analysis on BMDCs stimulated for 24 hours with U or 100 ng/mL LPS. Rot/Ant and SEITU are short for injections of Rotenone/Antimycin A; electron transport chain complex I and III inhibitors, and S-ethyl-iso-thiourea; iNOS inhibitor. Injections are indicated by dashed vertical lines, n = 1 biological replicate, representative of at least three independent experiments. (B) iNOS-linked OCR calculated from kinetic traces in (A) by subtracting the average OCR post inhibition of iNOS by SEITU from average baseline OCR before any injections. Analyzed by t test, adjusted p values are reported (p value > 0.05 ns), n = 4 biological replicates, representative of at least three independent experiments. (C) Modified mitochondrial stress test OCR kinetic trace of extracellular flux analysis on BMDCs stimulated for 24 hours with U, 12–14 ng/mL, or 25 ng/mL LPS. Injections are indicated by dashed vertical lines, n = 1 biological replicate, representative of at least three independent experiments. (D) iNOS-linked OCR calculated from kinetic traces as in (C) by subtracting the average OCR post inhibition of iNOS by SEITU from average baseline OCR before any injections. Analyzed by one-way ANOVA, adjusted p values are reported (p value > 0.05 ns), n = 4 biological replicates, representative of at least three independent experiments. If no p value is provided, the data is non-significant (p value > 0.05) and therefore, not included on the figure itself. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.
Table 1.
Binding affinity of common iNOS inhibitors to nitric oxide synthase (NOS) human isoforms.
Fig 5.
Differential kinetics of key iNOS inhibitors SEITU and 1400W.
(A) Griess nitrite assay on media supernatant from BMDCs stimulated for 24 hours with U, 100 ng/mL LPS +/- SEITU or 1400W. Analyzed by one-way ANOVA, adjusted p values are reported (p value > 0.05 ns), n = 6 biological replicates for U & LPS-100 ng/mL, and 3 for LPS-100 ng/mL + SEITU or + 1400W groups, representative of at least three independent experiments. (B) Modified mitochondrial stress test OCR kinetic trace of extracellular flux analysis on BMDCs stimulated for 24 hours with U, 100 ng/mL LPS +/- SEITU or 1400W and the iNOS inhibitors in the run media. Oligo is short for injection of oligomycin, ATP synthase inhibitor. Injection is indicated by dashed vertical lines, n = 1 biological replicate, representative of at least three independent experiments. (C) ATP-linked OCR calculated from kinetic traces in (B) by subtracting the average OCR post injection of oligomycin from average baseline OCR before any injections. Analyzed by one-way ANOVA, adjusted p values are reported (p value > 0.05 ns), n = 4 for U, L100 group, n = 3 for SEITU and 1400W group, representative of three independent experiments. (D) Modified mitochondrial stress test OCR kinetic trace of extracellular flux analysis on BMDCs stimulated for 24 hours with U, 100 ng/mL LPS. Rot/Ant and iNOSib are short for injections of Rotenone/Antimycin A; electron transport chain complex I and III inhibitors, and iNOS inhibitor SEITU or 1400W were indicated in the key. Injections are indicated by dashed vertical lines, n = 1 biological replicate, representative of at least three independent experiments. (E) iNOS-linked OCR calculated from kinetic traces in (D) by subtracting the average OCR post inhibition of iNOS by SEITU or 1400W from average baseline OCR before any injections. n = 4 for SEITU group, n = 2 for 1400W group, representative of three independent experiments. No statistical testing performed due to n = 2 for 1400W group. (F) Percentage of live cells assessed by flow cytometry using a fixable Live/Dead stain on BMDCs treated with 100 ng/mL LPS +/- SEITU or 1400W for 24 hours. Analyzed by one-way ANOVA, adjusted p values are reported (p value > 0.05 ns), n = 4 biological replicates, representative of at least three independent experiments. (G) iNOS inhibitor kinetic assay OCR kinetic trace of extracellular flux analysis on BMDCs stimulated for 24 hours with 100 ng/mL LPS. Injection is indicated by a vertical dashed line, iNOSib is either SEITU or 1400W were indicated in the figure key, n = 1 biological replicate, representative of at least three independent experiments. (H) Maximal iNOS-linked OCR calculated from kinetic traces in (D) by subtracting the average OCR post inhibition of iNOS by SEITU or 1400W at 150 minutes from average baseline OCR before any injections. Analyzed by one-way ANOVA, adjusted p values are reported (p value > 0.05 ns), n = 4 biological replicates, representative of at least three independent experiments. If no p value is provided, the data is non-significant (p value > 0.05) and therefore, not included on the figure itself. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.
Fig 6.
Visualization of NO modulation of DC metabolic reprogramming in real time.
(A) Glycolytic burst assay ECAR kinetic trace of extracellular flux analysis on freshly harvested BMDCs. Cells were stimulated by injection with 100 ng/mL LPS +/- SEITU. Injection is indicated by dashed a vertical dashed line, n = 1 biological replicate, representative of at least three independent experiments. (B) NO-independent glycolytic burst calculated from kinetic traces in (A) by subtracting the average ECAR post stimulation with LPS at the highest point ~100 minutes from average baseline OCR before any injections. Analyzed by one-way ANOVA using pairwise comparisons by biological replicate, adjusted p values are reported (p value > 0.05 ns), n = 6 independent BMDC cultures from 3 mice, representative of at least three independent experiments. (C) NO-dependent glycolytic reprogramming calculated from kinetic traces in (A) by subtracting the average ECAR post stimulation with LPS at 600 minutes from average baseline OCR before any injections. Analyzed by one-way ANOVA, adjusted p values are reported (p value > 0.05 ns), n = 6 independent BMDC cultures from 3 mice, representative of at least three independent experiments. (D) Glycolytic burst assay OCR kinetic trace of extracellular flux analysis on freshly harvested BMDCs. Cells were stimulated by injection with 100 ng/mL LPS +/- SEITU. Injection is indicated by dashed a vertical dashed line, n = 1 biological replicate, representative of at least three independent experiments. (E) NO-dependent respiration inhibition calculated from kinetic traces in (D) by subtracting the average OCR post stimulation with LPS at 600 minutes from average baseline OCR before any injections. Analyzed by one-way ANOVA using pairwise comparisons by biological replicate, adjusted p values are reported (p value > 0.05 ns), n = 6 independent BMDC cultures from 3 mice, representative of at least three independent experiments. If no p value is provided, the data is non-significant (p value > 0.05) and therefore, not included on the figure itself.
Fig 7.
Overview of different assays to assess NO-mediated metabolic effects in DCs.
Schematic representations of experimental and timing considerations for mitochondrial stress tests (A), iNOS inhibitor kinetic assays (B), and glycolytic burst assays (C).