Boosting NAD level suppresses inflammatory activation of PBMCs in heart failure

Abstract

BACKGROUNDWhile mitochondria play an important role in innate immunity, the relationship between mitochondrial dysfunction and inflammation in heart failure (HF) is poorly understood. In this study we aimed to investigate the mechanistic link between mitochondrial dysfunction and inflammatory activation in peripheral blood mononuclear cells (PBMCs), and the potential antiinflammatory effect of boosting the NAD level.METHODSWe compared the PBMC mitochondrial respiration of 19 hospitalized patients with stage D HF with that of 19 healthy participants. We then created an in vitro model of sterile inflammation by treating healthy PBMCs with mitochondrial damage-associated molecular patterns (MitoDAMPs) isolated from human heart tissue. Last, we enrolled patients with stage D HF and sampled their blood before and after taking 5 to 9 days of oral nicotinamide riboside (NR), a NAD precursor.RESULTSWe demonstrated that HF is associated with both reduced respiratory capacity and elevated proinflammatory cytokine gene expressions. In our in vitro model, MitoDAMP-treated PBMCs secreted IL-6 that impaired mitochondrial respiration by reducing complex I activity. Last, oral NR administration enhanced PBMC respiration and reduced proinflammatory cytokine gene expression in 4 subjects with HF.CONCLUSIONThese findings suggest that systemic inflammation in patients with HF is causally linked to mitochondrial function of the PBMCs. Increasing NAD levels may have the potential to improve mitochondrial respiration and attenuate proinflammatory activation of PBMCs in HF.TRIAL REGISTRATIONClinicalTrials.gov NCT03727646.FUNDINGThis study was funded by the NIH, the University of Washington, and the American Heart Association.

Keywords: Cardiology; Cardiovascular disease; Drug therapy; Inflammation; Mitochondria.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Heart failure is associated with a reduced maximal respiration and elevated proinflammatory cytokine gene expressions in PBMCs.

(A) Representative OCR plot upon various inhibitor injections in a standard Seahorse Mito stress test, comparing PBMCs from healthy subjects and those with stage D HF. Oligomycin A: inhibitor of complex V. FCCP (trifluoromethoxy carbonylcyanide phenylhydrazone): uncoupling agent by permeabilizing inner mitochondrial membrane. Antimycin A: inhibitor of complex III. Rotenone: inhibitor of complex I. (B and C) Basal and FCCP-induced maximal respiration of PBMCs from healthy subjects (n = 19) and subjects with stage D HF (n = 19), respectively. OCR data normalized via log10 transformation were subjected to ordinary unpaired 2-tailed parametric test (Welch’s t test). Normal distribution was assessed by Kolmogorov-Smirnov test. (D) Relative mRNA levels of NLRP3 and proinflammatory cytokines of PBMCs of healthy subjects and those with stage D HF by RT-qPCR. NLRP3 (healthy n = 9, HF n = 9), IL-1B (n = 9, n = 9), IL-6 (n = 12, n = 11), IL-18 (n = 7, n = 9), TNF-α (n = 9, n = 9). Mean mRNA level of healthy subjects normalized to 1. mRNA data analyzed with unpaired nonparametric 2-tailed t test. All data shown in mean ± SEM.

Figure 2. MitoDAMP induces PBMC respiratory impairment and inflammatory cytokine gene expression, and the latter can be partially attenuated by inhibition of the NLRP3 inflammasome axis.

(A) Schematics of the MitoDAMP extracted from mitochondria purified from human myocardial tissue. (B) Relative mRNA levels of NLRP3 and proinflammatory cytokines of healthy PBMCs after 4-hour treatments of vehicle or MitoDAMP. Vehicle normalized to 1. P value was determined by paired 2-tailed t test. n = 4. (C) Mitochondrial ROS levels of healthy PBMCs after MitoDAMP treatment. Zero hour normalized to 100%. P value cut off of 0.05 was determined by paired 2-tailed t test. n = 3. (D) Maximal respiration of healthy PBMCs after 4-hour treatment of vehicle or MitoDAMP with or without 0.5 mM MitoTempo, n = 4. P value determined by 1-way ANOVA with multiple pairwise comparisons. (E) Percent change of NLRP3 and cytokine mRNA levels of healthy PBMCs after 4-hour treatment of MitoDAMP with 0.5 mM MitoTempo (n = 4) or 1 μM MCC950 (n = 5) relative to MitoDAMP alone. P value was determined by paired 2-tailed t test. (F) Maximal respiration of healthy PBMCs after 4-hour treatments of vehicle or MitoDAMP with or without 1 μM MCC950. n = 3. P value determined by 1-way ANOVA with multiple pairwise comparisons. All data shown in mean ± SEM.

Figure 3. Secreted IL-6 from MitoDAMP stimulation impairs mitochondrial respiration by reducing complex I activity.

(A) Secreted IL-6 protein level by ELISA of healthy PBMCs after 2-hour (n = 3) or 4-hour (n = 6) treatment of vehicle or MitoDAMP. P value determined by paired 2-tailed t test. (B) Maximal respiration of healthy PBMCs after 4-hour treatments of vehicle or MitoDAMP with or without 100 μM LMT28, a specific inhibitor of the IL-6 receptor b (GP 130). n = 4. (C) Maximal respiration of healthy PBMCs after 4-hour treatments of vehicle or increasing concentrations of human recombinant IL-6. n = 4. B and C analyzed with 1-way ANOVA with multiple pairwise comparisons. (D) Representative Seahorse plot of baseline and post-drug treatment OCR of healthy PBMCs after 4-hour treatments of vehicle or IL-6 (1 ng/mL). FCCP; uncoupling agent by permeabilizing inner mitochondrial membrane; Rotenone (Rot): complex I inhibitor; Antimycin A (AA): complex III inhibitor; TMPD/Ascorbate: exogenous electron donor for complex IV. n = 3. (E) Quantitation of D. (F) Complex I activity of healthy PBMCs after 4-hour treatments of vehicle or IL-6 (1 ng/mL). n = 5. Vehicle normalized to 1. E and F analyzed by paired 2-tailed t test. All data shown in mean ± SEM.

Figure 4. NR attenuates MitoDAMP-induced PBMC respiratory impairment and proinflammatory cytokine production in vitro.

(A) Secreted IL-6 protein by ELISA of healthy PBMCs after 4-hour treatment of vehicle or 1 mM NR in the presence of MitoDAMP. P value determined by paired 2-tailed t test. (B) Percent change of NLRP3 and cytokine mRNA levels of healthy PBMCs after 4-hour treatment of MitoDAMP with 1 mM NR relative to MitoDAMP only. P value was determined by paired 2-tailed t test. n = 4. (C) Maximal respiration of healthy PMBCs after 4-hour treatment of vehicle, MitoDAMP, or MitoDAMP with 1 mM NR. n = 4. P value determined by ordinary 1-way ANOVA with multiple pairwise comparisons. B and C shown in mean ± SEM.

Figure 5. NR enhances mitochondrial respiration and reduces proinflammatory cytokine production in HF.

(A and B) Ratios of basal and maximal respiration of healthy or HF PBMCs after 4-hour 1 mM NR treatments relative to vehicle, respectively. n = 8 and n = 10, respectively. (C) Whole blood NAD+ level of subjects with stage D HF before or after 5 to 9 days of oral NR administration. (D and E) Basal and maximal respiration of PBMCs from subjects with stage D HF before and after oral NR administration, respectively. (F) Relative mRNA levels of NLRP3 and inflammatory cytokines of PBMCs from subjects with stage D HF before and after NR administration. Post-NR mRNA level normalized to 1. n = 4. (A and B) P values determined by unpaired 2-tailed t test, and data shown in mean ± SEM. (C–F) P values determined by paired 2-tailed t test.

Figure 6. Study design of oral NR administration in patients with HF.

Figure 7. Model of DAMP-induced monocyte activation.

The priming signal involves interaction of MitoDAMP with TLRs to stimulate the expression of inflammasome components and proinflammatory cytokines via activation of transcription factor NFKB. The secreted IL-6 in the priming step feeds back in an autocrine manner to impair mitochondrial respiration by inhibiting complex I activity and inducing mitochondrial ROS production, which leads to the assembly of the NLRP3 inflammasome to active caspase 1. Caspase-1, in turn, cleaves pro–IL-1B to IL-1B. Secreted IL-1B feeds back to further potentiate the NFKB axis.