The following reagents were used: NRH (14), NR (Laurus Labs), Olaparib (LC laboratories), 5-iodotubercidin (5-IT) (Selleckchem), nitrobenzylthioinosine (NBTI), ABT-702 (Cayman Chemical), adenosine 5’-(α,β-methylene) diphosphate (APCP) (Tocris), BMS 345541 (Selleckchem). NAM, NAD, NMN, ADH, Diaphorase, adenosine, phorbol myristate acetate (PMA), FK866, lypopolysaccharide (LPS) were obtained from Sigma-Aldrich.

BMDM were isolated from 3-6 months C57BL/6 mice.

Immunoblottings for BMDMs were performed as previously described by Tarrago et al. (23). Cells were homogenized and lysed in NETN buffer (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA and 0.5% Nonidet P-40) supplemented with 50 mM β-glycerophosphate, 5 mM NaF and a protease inhibitor cocktail (Roche). For PARylation analysis 100 μM tannic acid was included in the lysis buffer. After 20 minutes of incubation on ice, the samples were centrifuged at 12,000 rpm for 10 minutes at 4°C. Protein concentrations in the supernatants were determined by Bio-Rad protein assay. Lysates were separated by SDS–PAGE, and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore). Enhanced chemiluminescence detection was performed using SuperSignal West Pico or Femto Chemiluminescence Substrate (Thermo Scientific). Films were scanned and densitometry was performed using ImageJ. The following antibodies and their dilutions were used for immunoblotting: anti-mouse pp65 (S536) (Cell Signaling Technology; 3033, 1:1,000) anti-mouse p65 (Cell Signaling Technology; 8242, 1:1,000), actin (Cell Signaling Technology; 8457, 1:5,000), PAR (Trevigen, 4335, 1:1,000).

Detection of NAD⁺ was performed using a in house cycling assay. The use of the cycling assay has potential limitations related with the sensitivity and the specificity of the measurements. To mitigate these limitations, we have previously extensively validated our assay for specificity and sensitivity. Our cycling assay is very specific for NAD⁺ and NADH and does not detect any of the other nucleotides that we have tested including NADP⁺, Nicotinamide, nicotinic acid, NAAD (nicotinic acid adenine dinucleotide), NAADP (nicotinic acid adenine dinucleotide phosphate), NGD (nicotinamide guanine dinucleotide), NHD (hypoxanthine dinucleotide), cADPR (cyclic-ADP-ribose), ATP, ADP, AMP, cAMP, and GTP. Importantly, we used an acid extraction that degrades NADH to only measure NAD⁺. Furthermore, in control experiments we have performed the cycling assay and correlated the results with side-by-side measurements with HPLC-MS. Extensive validation of the cycling assay has been previously described (20, 23, 24). In summary, to determine intracellular NAD⁺ levels, 3×10⁶ cells were homogenized in 10% trichloroacetic acid (TCA). Samples were centrifuged at 12,000 rpm for 2 minutes at 4°C. The supernatants were collected, and the pellets were resuspended in 0.2 N NaOH for protein determination. TCA was removed with organic solvents (three volumes of 1,1,2-trichloro-1,2,2-trifluroethane: one volume of trioctylamine) in a ratio of two volumes of organic solvent to one volume of sample. After phase separation, the top aqueous layer containing NAD⁺ was recovered and the pH was corrected by the addition of 1 M Tris (pH 8.0). For the cycling assay, samples were diluted in 100 mM sodium phosphate buffer (pH 8) in a volume of 100 μl per well and added to white 96-well plates. Next, 100 μl of reaction mix (0.76% ethanol, 4 μM flavin mononucleotide, 27.2U/ml alcohol dehydrogenase (ADH), 1.8 U/ml diaphorase and 8 μM resazurin) was added to each well. Then, 96-well plates were read in a fluorescence plate reader (Molecular Devices, SpectraMax Gemini XPS) in an excitation wavelength of 544 nm and an emission wavelength of 590 nm. We have previously demonstrated that the NAD⁺ cycling assay is as sensitive and specific as the ultra-performance liquid chromatography (UPLC)-mass spectroscopy (MS) assay.

Cells were lysed and RNA was isolated using Qiagen RNeasy kit. cDNA was synthesized using the ABI High Capacity cDNA Reverse Transcription kit. Real-time qPCR was performed using commercially available TaqMan gene expression probes (Applied Biosystems), according to the manufacturer’s instructions on a Bio-Rad CFX384 thermal cycler. The relative mRNA abundance of target genes was calculated by the 2^(−ΔΔCq) method. Expression changes were calculated relative to control cells or treatments. The Taqman probes used are described in Tables 1 and 2 .

Mitochondrial oxygen consumption rates (OCR) were measured by high-resolution respirometry using an Oxygraph-2k instrument (Oroboros Instruments, Austria). 1-1.5 million BMDM cells were resuspended in 2ml of fresh DMEM 2% FBS and loaded into oxygraph chambers. Different respiratory parameters were assessed. Basal OCR was recorded after signal stabilization. Oligomycin addition (0.25 nM final concentration) allowed the evaluation of OCR uncoupled to ATP synthesis, referred here as leak respiration. The maximal OCR was measured in the presence of the optimum concentration of carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), determined after FCCP titration. Data acquisition and analysis were carried out using DatLab6 software (Oroboros Instruments, Austria). Data were represented relative to the non-treated control for each cell preparation.

Data are mean ± SD analyzed by unpaired two-sided t-test or one-way ANOVA. Analyses were performed using Microsoft Excel 2010 and GraphPad Prism 9.2.0 software. Differences were considered statistically significant when P<0.05. *P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001.

Recently, NRH has been characterized as a potent NAD⁺ precursor in cell lines such as AML-12, NIH 3T3, 293T and HepG3 (14, 16). However, its effect on immune cells has not been described. In BMDM treatment with NRH for 6 hours induced a dose-dependent increase in NAD⁺ to levels 6-7 times higher than control non-treated cells ( Figure 1A ). In contrast, 6 hours supplementation with NMN, NR or NAM did not produce significant increases in NAD⁺ levels in BMDM for most of the concentrations tested ( Figure 1A ). We also observed that 500 μM NRH induced a rapid increase in intracellular NAD⁺ levels in BMDM, with a small increase detected as soon as 30 min ( Figure 1B ). Increases in NAD⁺ levels induced by NRH were similar at 6 and 16 hours of treatment, indicating that the increase is sustained for many hours ( Figures 1B, C ). Other NAD⁺ precursors did not induce NAD⁺ boosting in BMDM after 16 hours supplementation ( Figure 1C ). Moreover, we found that NRH also increased NAD⁺ levels in B (Ramos, JVM-13) and T (Jurkat) lymphoblasts ( Supplementary Figures 1A–C ). In B and T cells we found that NR could also increase NAD⁺ levels significantly. However, in all cell types tested, NRH was a more potent NAD⁺ precursor ( Supplementary Figures 1A–C ).

We then investigated the functional consequences of this large increase in NAD⁺ levels in BMDM. To determine the impact of NRH supplementation on parameters such as cell viability and oxygen consumption we performed a longer treatment with NRH (20 hours). Supplementation with 500 μM NRH increased BMDM cell viability, as determined by staining with alamarBlue, unlike other NAD⁺ precursors ( Figure 1D ). This increase in cell viability indicates that in BMDM NRH is not showing cytotoxic effects during our treatment period but, on the contrary, it appears to be improving cell survival. To confirm these results, we also determined cell viability and survival by cell counting and cellular visualization after 20 hours of supplementation with 500 μM NRH ( Supplementary Figures 1D, E ), or lipopolysaccharide (LPS) treatment ( Supplementary Figure 1D ). We did not observe a deleterious effect of NRH or LPS on the cell viability during the treatment period ( Supplementary Figures 1D, E ). We also investigated whether NRH supplementation altered oxygen consumption rates (OCR). Treatment of BMDM with 500 μM NRH for 20 hours did not significantly change the basal, leak, or maximum respiration in comparison to non-stimulated cells ( Figures 1E, F and Supplementary Figure 1F ). As a control, we treated BMDM with 1 or 10 ng/ml LPS and found that both concentrations decreased maximum respiration, differently than NRH ( Figure 1F ).

Next, we determined whether NRH treatment modified the expression of NAD⁺ synthesizing and degrading enzymes expressed by macrophages. To investigate the NRH effect on gene expression we treated BMDM with NRH for 6 hours, a time where NAD⁺ levels reach maximum increase in these cells. Treatment of BMDM with NRH for 6 hours increased mRNA expression of Cd38 (NADase) and Nampt (NAD synthesizing enzyme) ( Figure 1G ), likely promoting an increase in the turnover of NAD metabolism. However, since NRH bypasses both CD38 and NAMPT, the consequences of these enzymes to NRH metabolism are likely small. These two enzymes are known to be induced by agents that promote a pro-inflammatory (M1-like) phenotype in macrophages, like LPS (20). In view of these observations, we next investigated the role of NRH in macrophage activation and inflammatory responses.

To determine whether NRH regulates activation markers in macrophages, we treated BMDM with increasing doses of NRH (300–1000 μM) for 6 hours, and then assessed gene expression of several cytokines, chemokines, and enzymes by RT-qPCR. NRH treatment induced a marked dose-dependent increase in the mRNA expression of genes that are markers of a macrophage M1 phenotype, such as Ccl5, Il1, Il6, Cxcl1 ( Figure 2A ) (25). In addition, the expression of Cd38 and Nos2, two enzymes that are also markers of M1 macrophage phenotype (25), were significantly increased by most of the NRH concentrations tested ( Figure 2A ). We also examined the effect of 500 μM NRH on the expression of other genes, such as Ccl2, Il12, and Tnfa, and found that NRH caused at least a fivefold increase in the expression level of these cytokines/chemokines ( Figure 2B ). In contrast, Myc expression, a marker of the macrophage M2 phenotype (26), was not increased after NRH treatment, showing a trend for decreased expression ( Figure 2B ). We also examined Irg1 expression in macrophages supplemented with NRH. Irg1 (ACOD1) is an enzyme involved in itaconate production and is induced by agents that promote inflammation in macrophages, like LPS (27, 28). Consistent with other markers of inflammation, Irg1 expression was also markedly increased by NRH supplementation ( Figure 2B ).

We next investigated whether treatment with other NAD precursors at 500-1000 μM for 6 hours induced similar effects on gene expression as observed with NRH supplementation. Of all the precursors tested only NMN supplementation increased the expression of some cytokines/chemokines, Cd38 and Nos2. This NMN effect was noted especially at 1000 μM, but it was smaller and less consistent compared to NRH ( Figure 3A ). Although no significant increase in NAD⁺ levels were detected by NMN supplementation at the time point tested ( Figures 1A, C ), we cannot exclude the possibility that NMN transiently increases NAD⁺ and regulates gene expression through this mechanism.

When gene expression of activation markers was measured 16 hours after treatment with NAD⁺ precursors, we observed that only NRH supplementation was able to sustain an effect on gene expression. The expression of several cytokines/chemokines, Cd38, Nos2, and Irg1 were still increased after 16 hours of treatment with NRH ( Figure 3B ). This prolonged effect on gene expression is consistent with the NRH effect on NAD⁺ levels ( Figure 1C ), which is still increased after 16 hours.

To further confirm the role of NRH on NAD⁺ boosting and regulation of gene expression in immune cells, we investigated the effect of NRH in THP-1 cells, a human monocytic leukemia cell line. This cell line can be differentiated into a macrophage-like phenotype using agents like phorbol 12-myristate 13-acetate (PMA) (29). When THP-1 cells were supplemented with NAD⁺ precursors, we found that only NRH was able to significantly increase NAD⁺ levels ( Figure 4A ). In THP-1 cells treated with PMA, NRH supplementation also caused a significant increase in NAD⁺ levels, but this increase was smaller compared to non-PMA-treated THP-1 cells ( Figure 4B ).

To investigate the reason behind the differences observed in non-treated and PMA-treated THP-1 cells, we measured the mRNA levels of NAD metabolism enzymes under these two conditions. When PMA-treated THP-1 cells were compared to non-treated cells, there was a significant decrease in mRNA expression of enzymes such as CD38, CD157, and NMNAT3. In contrast, gene expression of CD73 and the putative NMN transporter SLC12A8 (30) were increased, while other enzymes and transporters did not significantly change ( Figure 4C ). Since there were several changes in the pattern of gene expression for NAD metabolism enzymes between THP-1 untreated and PMA-treated cells, some of those changes may be contributing to the lower NAD⁺ boosting effect of NRH in PMA-treated THP-1 cells.

When cytokine gene expression was measured in non-treated THP-1 cells, NRH did not show a significant effect on increasing gene expression ( Figure 4D ), even though it promoted a large increase in NAD⁺ levels in these cells ( Figure 4A ). However, there was a trend for both NRH and NMN, but not NR, to increase pro-inflammatory gene expression. THP-1 cells express the enzymes and transporters necessary for NRH-induced NAD boosting, in addition to CD38 (which degrades NMN), CD157/BST1 (which degrades NR), and the putative NMN transporter. Since CD157/BST1 appears to be abundant in THP-1 cells (31), this could be a possible reason why NR is less effective than NMN in THP-1 cells.

In contrast, PMA-treated THP-1 cells, show an increase in expression levels of several cytokines upon NRH supplementation, as seen in BMDM ( Figure 4E ). Supplementation with other NAD⁺ precursors did not significantly increase gene expression. These different patterns of responses observed in macrophages and monocytes indicate that the NAD metabolome and the machinery to regulate inflammatory responses varies between immune cell types, highlighting the importance of characterizing the role of these precursors in several cell types.

We next set out to determine whether the effect of NRH in inducing a pro-inflammatory phenotype in macrophages was related to its effect on NAD⁺ boosting. For that reason, we investigated whether the transporters and enzymes that have been shown to regulate entry and conversion of NRH into NAD⁺ in cells also regulate the NRH effect on gene expression.

NRH, like NR, appears to be transported into the cell mainly by equilibrative nucleoside transporters (ENTs) (14, 32). Once inside the cell, NRH appears to be phosphorylated by adenosine kinase (ADK) into NMNH and subsequently converted to NADH by NMNATs (14, 17, 33). We first confirmed whether these enzymes were expressed in resting and NRH-treated macrophages. mRNA expression of Adk, Ent1, Ent2, Nmnat1 and Nmnat3 was observed in resting BMDM and supplementation with 500 μM NRH for 6 hours did not significantly change expression of most of these genes in BMDM. The only exception was Adk, which showed decreased, but still detectable, expression ( Figure 5A ).

The effect of NRH in NAD⁺ boosting has been shown to be blocked by the ENT inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI) and the ADK inhibitors 5-iodotubercidin (5-IT) and ABT-702 in other cells (14). Pre-treatment of BMDM with all three inhibitors before NRH supplementation for 6 hours completely prevented the NAD⁺ boosting induced by NRH, while they did not affect NAD⁺ levels in control untreated cells ( Figure 5B ). NRH supplementation also induced a marked increase in poly-ADP-ribosylation (PAR), which is a measurement of PARP activity. This effect was blocked by 5-IT and NBTI, indicating that these inhibitors prevent the NRH-induced NAD ⁺ boosting and activation of downstream effectors such as PARP ( Figure 5C ).

By measuring mRNA expression, we found that 5-IT, ABT-702, and NBTI blocked the NRH-induced increase in the expression of Il1, Il6, Cxcl1, Cd38, Irg1 and Nos2, but had mostly no effect in the absence of NRH ( Figure 5D and Supplementary Figure 2A ). Importantly, we measured the levels of the chemokine CXCL1 in the cell culture media of cells treated with NRH for 6 hours ( Figure 5E ) and 16 hours ( Supplementary Figure 2B ). Supplementation with NRH increased protein levels of CXCL1 in the media ( Figure 5E and Supplementary Figure 2B ), and this effect was blocked by pre-treatment with the inhibitors 5-IT and NBTI ( Figure 5E ). The CXCL1 levels were higher at 16 hours than at 6 hours, indicating a time-dependent accumulation of this chemokine in the media. These results together indicate that the NRH effect on NAD⁺ levels and pro-inflammatory responses in macrophages occurs through an ENT-ADK-dependent pathway.

To further explore the mechanisms by which NRH regulates macrophage cytokine/chemokine expression we pretreated BMDM with compounds that inhibit other enzymes involved in NAD metabolism. We wanted to determine whether these compounds regulate NAD⁺ levels or prevent pro-inflammatory gene expression when added before supplementation with NRH. In resting BMDM addition of the NAMPT inhibitor FK866 decreased NAD⁺ levels, but it did not affect the NAD⁺ boosting induced by NRH ( Supplementary Figure 2C ). Inhibition of PARP (with Olaparib), CD73 (with APCP), or addition of the immunosuppressive drug adenosine had also no effect on both basal and NRH-dependent NAD⁺ boosting ( Supplementary Figure 2C ). Except for the immunosuppressive effect of adenosine on NRH-induced Il6 expression, and the effect of FK866 on Il6 expression, the above compounds did not significantly alter the NRH-induced regulation of gene expression ( Supplementary Figure 2D ). These results indicate that the effect of NRH on NAD⁺ boosting and cytokine gene expression occurs independently of NAMPT, PARP, or CD73.

The findings described above demonstrate that NRH supplementation can produce a pro-inflammatory phenotype in resting macrophages. Therefore, next we wanted to explore the effects of NRH in combination with inflammatory stimuli such as LPS. When BMDM were treated with NRH alone for 6 hours, NRH promoted a nearly 8-fold increase in NAD⁺ levels. However, when a low concentration of LPS (1 ng/ml) was added 1 hour after NRH addition, NRH promoted only a fourfold increase in NAD⁺ levels, even though LPS alone at this concentration and time did not change NAD⁺ levels ( Figure 6A ). This difference was not due to a decrease in cell viability when LPS was added together with NRH, since the viability of NRH or NRH+LPS-treated macrophages for 20 hours was similar and higher than non-stimulated cells ( Figure 6B ). Also, LPS treatment alone did not decrease cell viability ( Figure 6B and Supplementary Figure 1D ).

It is known that LPS treatment promotes changes in the expression of many enzymes involved in NAD metabolism (20, 34, 35). Thus, we next investigated the effect of LPS on the mRNA expression of enzymes/transporters involved in NRH-induced NAD⁺ boosting. Interestingly, while expression of Adk, Ent1, and Nmnat1 did not change after 6 hours of treatment with LPS, there was a major decrease in expression of Ent2 and Nmnat3 ( Figure 6C ). The same pattern of expression was observed when LPS was added in combination with NRH ( Figure 6C ). These results suggest that LPS may be inhibiting the expression of enzymes that are important for the NRH-induced NAD⁺ boosting effect.

LPS is a potent inducer of the macrophage M1 phenotype, even at lower concentrations. 6 hours treatment of BMDM with 1 ng/ml LPS strongly induced expression of many cytokines such as Ccl5, Cxcl1 and Il6, and the enzymes Cd38, Nos2 and Irg1 in comparison to untreated control cells ( Figure 6D ). When NRH and LPS were added together, there was no additive effect on the increase in expression of Cd38, Nos2 and Irg1 compared to LPS alone. However, the combination of NRH+LPS treatment increased the expression of cytokines/chemokines to higher levels than LPS alone ( Figure 6D ), suggesting that NRH-induced NAD⁺ boosting can potentiate the effect of inflammatory stimuli on cytokine gene expression. The decrease in NAD⁺ levels promoted by high doses of LPS (100 ng/ml) (9, 10) may represent a negative feedback mechanism for its cytokine-producing effect that, as demonstrated here, can be at least partially bypassed by boosting NAD with NRH.

An important pathway that regulates gene expression of various pro-inflammatory genes including cytokines and chemokines in immune cells is the NF-κB pathway (36). To better understand the mechanism by which NRH regulates cytokine/chemokine gene expression we investigated if supplementation with NRH activates the NF-κB pathway in BMDM. Phosphorylation plays an important role in the activation of several components of the NF-κB pathway. The increase in phosphorylation of the subunit p65 at S536 has been used as a marker of NF-kB activation. Treatment of BMDM for 30 and 90 minutes with NRH caused a small increase in p65 levels and a marked increase in phosphorylation of S536 on NF-κB p65 subunit, suggesting that NRH supplementation is activating the NF-κB pathway ( Figure 7A ).

These results prompted us to test whether the NF-κB pathway was involved in the NRH effect on BMDM. The canonical pathway for NF-κB activation involves the multi-subunit IκB kinase (IKK) complex, which when activated by different stimuli phosphorylates IκBα, an NF-κB inhibitor. Phosphorylation of IκBα triggers ubiquitin-dependent IκBα degradation, releasing NF-κB to translocate to nuclei and activate gene expression (36). As shown in Figure 7B , pre-treatment of BMDM with 5 μM BMS-345541, a highly selective inhibitor of the catalytic subunit of IKK (37), blocked the effect of NRH on pro-inflammatory genes, suggesting a role for IKK on this pathway. Since the effect of NRH on gene expression in BMDM appears to be dependent on its NAD⁺-boosting effect ( Figure 5 ), we then investigated if pre-treatment with BMS-345541 interferes with the NAD⁺-boosting effect of NRH. Surprisingly, IKK inhibition with BMS-345541 blocked the NAD⁺-boosting induced by NRH, suggesting that IKK may regulate components of the NRH pathway ( Figure 7C ).

To understand why IKK inhibition regulated the NRH-induced NAD⁺ boosting we examined the expression of enzymes involved in NRH-induced NAD⁺ boosting. Interestingly, BMS-345541 pre-treatment decreased the expression of Nmnat1, Nmnat3, and Ent2 by 90% and the expression of Ent1 by 40% in the presence of NRH ( Figure 7D ). All together, these results suggest that in BMDM NRH activates the NF-κB pathway and pro-inflammatory gene expression, and that the effects of NRH in NAD⁺ boosting and gene expression are regulated by IKK ( Figure 8 ).