Salt Transiently Inhibits Mitochondrial Energetics in Mononuclear Phagocytes

Figures

• 

  Figure 1.. High salt inhibits mitochondrial respiration in murine and human mononuclear phagocytes. A, Pictogram of the generation and activation of murine bone marrow–derived macrophages (BMDMs). B through D, BMDMs were treated for 3 hours with lipopolysaccharide (LPS) or interleukin (IL) 4+IL13, under normal salt (NS) or high salt (HS) conditions. (B) Basal oxygen consumption rate (OCR) (pooled data n=10 from 3 independent experiments). Data are depicted as box and whisker with minimum to maximum. Significance was analyzed by unpaired, 2-tailed t test. C, Relative ATP content, with Oligomycin A (Oligo, 10 µM) treatment as positive control for ATP decrease (pooled data n=12 from 2 independent experiments). Data are depicted as box and whisker with minimum to maximum. Significance was analyzed by 1-way ANOVA with Tukey’s post hoc test. D, Relative TMRE mean fluorescence intensity (MFI), with mitochondrial uncoupler BAM15 (10 µM) as positive control for mitochondrial depolarization (pooled data n=10 from 2 independent experiments). Data are depicted as box and whisker with minimum to maximum. Significance was analyzed by Kruskal-Wallis test with Dunn’s post hoc test (LPS groups) or by…

• 

  Figure 2.. High salt inhibits mitochondrial electron transport chain at complex II. A, Pictogram of the central carbon metabolism and ETC. Glucose-derived 13C-label is represented as red dots. Glutamine-derived 13C-label is represented as blue dots. Mitochondrial membrane potential is ψ. B through D, Bone marrow–derived macrophages (BMDMs) treated for 3 h (B and D) or 24 h (C) with lipopolysaccharide (LPS) or interleukin (IL) 4+IL13 under normal salt (NS) or high salt (HS) conditions. B and C, 13C-glutamine-derived 13C-fumarate to 13C-succinate ratio as surrogate for succinate dehydrogenase/complex II activity (n=5). Data are depicted as box and whisker with minimum to maximum. Significance was analyzed by unpaired 2-tailed t test. D, Relative (deproteinized) FAD content (n=4). Data are depicted as box and whisker with minimum to maximum. Significance was analyzed by unpaired 2-tailed t test. E, Relative (deproteinized) FAD content in freshly isolated monocytes (n=6 donors), cultivated for 3 hours under NS or HS conditions. Data are depicted in relation to the donor-paired NS control. Significance was analyzed by Wilcoxon matched-pairs signed-rank 2-tailed test. F, Relative complex II…

• 

  Figure 3.. Mitochondrial morphology is not affected by HS. A through D, Representative electron micrographs of M(lipopolysaccharide [LPS]) and M(interleukin [IL] 4+IL13) bone marrow–derived macrophages (BMDMs), activated for 3 hours under normal salt (NS) or high salt (HS) conditions, depicting an entire cell, a representative mitochondrion, and (in yellow) a 3-dimensional reconstruction of the mitochondrial network in a representative section of the cell.

• 

  Figure 4.. Coupled electron transport chain is crucial for early macrophage activation and function. Relative expression of Nos2 and Retnla1 in untreated, lipopolysaccharide (LPS)- or interleukin (IL) 4+IL13-treated bone marrow–derived macrophages (BMDMs) activated for 3 hours under normal salt (NS) or high salt (HS) or NS+dimethyl malonate (DMM) (A) (pooled data n=16 from 2 independent experiments), NS+antimycin A (AA) (B) (pooled data n=8 from 2 independent experiments), or NS+BAM15 (C) (pooled data n=6 from 2 independent experiments). D and E, Relative Escherichia coli (E. coli) colony-forming units (CFUs) in BMDMs (D) (pooled data n=18 from 3 independent experiments) and freshly isolated human monocytes (E) (pooled data n=10 from 3 independent experiments), 3 hours after infection under NS, HS, or NS+BAM15 (10 µM) treatment. Relative gene expression of Nos2 (F) and Il6 (H), as well as nitrite in the supernatant (G) in untreated or LPS-treated BMDMs activated for 24 hours under NS or HS. F through H, Data (n=6 each) are depicted as box and whisker with minimum to maximum. Significance was analyzed by 1-way ANOVA with Tukey’s post hoc test (A through C, E through H) and Kruskal-Wallis…

• 

  Figure 5.. Minimal sodium (Na+) changes inhibit monocytic mitochondrial function. A, Plasma Na+ (n=8) as well as (B) fold-change in monocytic basal oxygen consumption rate (OCR) in relation to difference in plasma Na+ compared with baseline (n=5 for day 3, n=4 for day 14, n=6 for day 28) from a chronic salt intervention study in male volunteers. C, Basal OCR, (D) ATP level, and (E) tetramethylrhodamine ethyl ester (TMRE) mean fluorescence intensity (MFI) in freshly isolated monocytes from 10 male and 10 female volunteers, treated in vitro for 3 hours with +2 mM and +4 mM NaCl. A, Significance of plasma Na+ was analyzed by Friedman test, and false discovery rate (FDR) correction was performed via Benjamini-Hochberg procedure. B, Relationship between the change in plasma Na+ and the change in basal OCR (of days 3, 14, and 28 compared with baseline) was determined by linear mixed-effects model applied to rank-transformed between–time point difference data, accounting for donor. C through E, Data were analyzed by Friedman test with Benjamini-Hochberg FDR correction. NS indicates normal salt; and TMRE, tetramethylrhodamine ethyl ester.

• 

  Figure 6.. Single meal inhibits monocytic mitochondrial function. A, Scheme of the study protocol with a picture of a representative pizza containing 9.8±0.3 g NaCl. Before, 3 hours and 8 hours after the meal challenge, monocytes were isolated from 10 male and 10 female volunteers (n=20 per time point). B, Plasma Na+, (C) monocytic basal oxygen consuption rate (OCR) in relation to baseline, and (E) TMREhigh monocytes in relation to baseline at baseline, 3 hours, and 8 hours after pizza. D, Relation between fold-change in monocytic basal OCR and difference in plasma Na+ as single time point comparison between 3 hours after pizza and baseline. B, C, and E, Data were analyzed by Friedman test with Benjamini-Hochberg false discovery rate (FDR) correction. D, Relationship between the change in plasma Na+ and the change in basal OCR (of 3 hours compared with baseline) was determined by Pearson correlation and linear regression analysis. TMRE indicates tetramethylrhodamine ethyl ester.