Boosting NAD+ blunts TLR4-induced type I IFN in control and systemic lupus erythematosus monocytes
Jing Wu, Komudi Singh, Amy Lin, Allison M. Meadows, Kaiyuan Wu, Vivian Shing, Maximilian Bley, Shahin Hassanzadeh, Rebecca D. Huffstutler, Mark S. Schmidt, Luz P. Blanco, Rong Tian, Charles Brenner, Mehdi Pirooznia, Mariana J. Kaplan, Michael N. Sack
Jing Wu, Komudi Singh, Amy Lin, Allison M. Meadows, Kaiyuan Wu, Vivian Shing, Maximilian Bley, Shahin Hassanzadeh, Rebecca D. Huffstutler, Mark S. Schmidt, Luz P. Blanco, Rong Tian, Charles Brenner, Mehdi Pirooznia, Mariana J. Kaplan, Michael N. Sack
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Clinical Research and Public Health Inflammation Metabolism

Boosting NAD+ blunts TLR4-induced type I IFN in control and systemic lupus erythematosus monocytes

Abstract

BACKGROUND Fasting and NAD+-boosting compounds, including NAD+ precursor nicotinamide riboside (NR), confer antiinflammatory effects. However, the underlying mechanisms and therapeutic potential are incompletely defined.METHODS We explored the underlying biology in myeloid cells from healthy volunteers following in vivo placebo or NR administration and subsequently tested the findings in vitro in monocytes extracted from patients with systemic lupus erythematosus (SLE).RESULTS RNA-Seq of unstimulated and LPS-activated monocytes implicated NR in the regulation of autophagy and type I IFN signaling. In primary monocytes, NR blunted LPS-induced IFN-β production, and genetic or pharmacological disruption of autophagy phenocopied this effect. Given that NAD+ is a coenzyme in oxidoreductive reactions, metabolomics was performed and identified that NR increased the inosine level. Inosine supplementation similarly blunted autophagy and IFN-β release. Finally, because SLE exhibits type I IFN dysregulation, we assessed the NR effect on monocytes from patients with SLE and found that NR reduced autophagy and IFN-β release.CONCLUSION We conclude that NR, in an NAD+-dependent manner and in part via inosine signaling, mediated suppression of autophagy and attenuated type I IFN in myeloid cells, and we identified NR as a potential adjunct for SLE management.TRIAL REGISTRATION ClinicalTrials.gov registration numbers NCT02812238, NCT00001846, and NCT00001372.FUNDING This work was supported by the NHLBI and NIAMS Intramural Research divisions.

Authors

Jing Wu, Komudi Singh, Amy Lin, Allison M. Meadows, Kaiyuan Wu, Vivian Shing, Maximilian Bley, Shahin Hassanzadeh, Rebecca D. Huffstutler, Mark S. Schmidt, Luz P. Blanco, Rong Tian, Charles Brenner, Mehdi Pirooznia, Mariana J. Kaplan, Michael N. Sack

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Figure 3

NR administration to human monocytes suppresses LPS-mediated type 1 IFN signaling.

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NR administration to human monocytes suppresses LPS-mediated type 1 IFN ...
(A) IFNB mRNA level in monocytes was measured by RT-PCR at 30, 60, and 90 minutes after LPS stimulation (n = 2–4 replicates). (B) Representative immunoblot of phospho-TBK1 and phospho-IRF3 levels in human monocytes preincubated overnight with vehicle or NR (0.5 mM) followed by LPS stimulation (10 ng/mL for 90 minutes). (C) Quantification of phospho-TBK1 or phospho-IRF3 level in vehicle- or NR-incubated monocytes normalized to total TBK1 or IRF3 levels (n = 3–4 replicates from 3 experiments). (D) LPS-stimulated (10 ng/mL for 8 hours) IFN-β production measured by ELISA from human monocytes incubated with vehicle or NR (0.5 mM) for 24 hours (n = 5 replicates/treatment; representative results from 3 experiments). (E) TNF-α production measured by ELISA from human monocytes incubated with vehicle or NR for 16 hours (n = 5 replicates/treatment). (F) Representative immunoblot of phospho-STAT2 and phospho-STAT1 levels in human monocytes preincubated overnight with vehicle or NR followed by LPS stimulation. (G) Quantification of phospho-STAT2 or phospho-STAT1 level in vehicle- or NR-incubated monocytes normalized to total STAT2 or STAT1 level (n = 5–6 replicates from 3 experiments). (H) CXCL10 mRNA level was measured by RT-PCR at different time points upon LPS stimulation. (I) CXCL10 production was measured by ELISA from human monocytes incubated with vehicle or NR for 24 hours (n = 6 replicates/treatment; representative results from 3 experiments). Data were analyzed by unpaired 2-tailed Student’s t test. All data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.