The NADase CD38 is a central regulator in gouty inflammation and a novel druggable therapeutic target

doi:10.21203/rs.3.rs-3459547/v1

Download PDF

Research Article

The NADase CD38 is a central regulator in gouty inflammation and a novel druggable therapeutic target

https://doi.org/10.21203/rs.3.rs-3459547/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 15 Mar, 2024

Read the published version in Inflammation Research →

You are reading this latest preprint version

Objectives: Cellular NAD⁺declines in inflammatory states associated with increased activity of the leukocyte-expressed NADase CD38. In this study, we tested the potential role of therapeutically targeting CD38 and NAD⁺in gout.

Methods: We studied cultured mouse wild type and CD38 knockout (KO) murine bone marrow derived macrophages (BMDMs) stimulated by monosodium urate (MSU) crystals and used the air pouch gout synovitis model.

Results: MSU crystals induced CD38 in BMDMs in vitro, associated with NAD⁺ depletion, and IL-1b and CXCL1 release, effects reversed by pharmacologic CD38 inhibitors (apigenin, 78c). Mouse air pouch inflammatory responses to MSU crystals were blunted by CD38 KO and apigenin. Pharmacologic CD38 inhibition suppressed MSU crystal-induced NLRP3 inflammasome activation and increased anti-inflammatory SIRT3-SOD2 activity in macrophages. BMDM RNA-seq analysis of differentially expressed genes (DEGs) revealed CD38 to control multiple MSU crystal-modulated inflammation pathways. Top DEGs included the circadian rhythm modulator GRP176, and the metalloreductase STEAP4 that mediates iron homeostasis, and promotes oxidative stress and NF-kB activation when it is overexpressed.

Conclusions: CD38 and NAD⁺ depletion are druggable targets controlling the MSU crystal- induced inflammation program. Targeting CD38 and NAD⁺ are potentially novel selective molecular approaches to limit gouty arthritis.

CD38

NAD+

macrophages

gout

inflammation

In gout, flares of severely painful inflammatory arthritis intersect with metabolism, and macrophage activation [1–3]. Gout represents a major public health problem, and severe flares commonly lead to physical incapacity, emergency department visits, and hospitalization [1]. Moreover, recent gout flare is a major risk factor for non-lethal and lethal myocardial infarction and stroke as subsequent cardiovascular events [4].

Gout flares are principally prevented and treated with nonselective, frequently toxic drugs (colchicine, NSAIDs, and corticosteroids). Gout flares are a phenotype of the acute inflammatory response to deposits of monosodium urate (MSU) crystals. Interaction of MSU crystals with resident cells induces acute inflammation in large part via induction of NF-κB and NLRP3 inflammasome activation, and consequent release of inflammatory cytokines by macrophage lineage cells [5, 6]. Evolving strategies to treat flares with biologic agents that selectively target IL-1b and the NLRP3 inflammasome have illustrated the impact of the current understanding of the molecular mechanisms involved in gouty inflammation [7].

This study focuses on nicotinamide adenine dinucleotide (NAD⁺) metabolism as a potentially selective molecular target niche for gouty inflammation. NAD⁺ is a necessary cofactor and key metabolite in pathways involved in cellular energy homeostasis and adaptive responses of cells to bioenergetic stressors including inflammation and aging [8, 9]. Intracellular NAD⁺ levels are significantly affected by environmental stimuli (7–9) and diverse cell stressors [8, 9]. For example, intracellular depletion of (NAD⁺) is promoted by aging and inflammatory diseases [8, 9]. In turn, NAD⁺ depletion promotes aging-related tissue damage and inflammation, partly mediated by priming of NLRP3 inflammasome activation in macrophages [10, 11]. As such, boosting cellular NAD⁺ levels are an emerging, broadly investigated, and selective molecular approach to diseases of aging and inflammation [8, 9].

Changes in cellular NAD⁺ levels are related to NAD⁺ biosynthesis and/or degradation. Most NAD⁺ is synthesized through the salvage pathway from nicotinamide (NAM), the by-product of NAD⁺ degradation [8, 9], which can be promoted by Cluster of differentiation 38 (CD38) [8, 9, 12]. CD38 has both NAD⁺ glycohydrolase and ADP-ribosyl cyclase activities, and thereby catalyses production of ADP-ribose (ADPR), cADPR, and NAM [8, 9, 12]. That said, CD38 NAD⁺ glycohydrolase “NADase” function represents > 90% of CD38 enzyme activity [9, 12]. CD38 is the principal NADase in mammalian tissues [9, 12], evidenced by tissue NAD⁺ levels being 10- to 20-fold higher in CD38 knockout (KO) compared to wild type (WT) mice [12]. In addition, overall NADase activity is attenuated in CD38KO mice [12].

CD38 is ubiquitously expressed in all immune cells including macrophages [13–15]. CD38 expression increases in diseases associated with inflammatory conditions [13–15]. A recent study showed that MSU crystals up-regulate CD38 and reduce intracellular NAD⁺ levels in human and mouse macrophages in vitro [16]. NAD⁺ degradation by CD38 is strongly linked to reduced activities of NAD⁺-dependent protein deacetylase family of sirtuins (SIRTs) [8, 9]. In this regard, MSU crystal-induced inflammation is more severe in SIRT1^+/− compared to WT mice [17]. Activation of SIRT1 suppresses MSU crystal-induced inflammatory responses [17, 18]. Since CD38 is responsible for NAD⁺ decline in inflammatory macrophages [13–15], we investigated the role of CD38 and NAD⁺ in macrophage responses to MSU crystals and in a model of acute gouty synovitis.

Reagents

All chemicals were from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. Apigenin and 78c were obtained from MedChemExpress (Monmouth Junction, NJ). CD38 (# ab108403, RRID:AB_10890803) and acetylated SOD2 (K68) (#ab137037, RRID:AB_2784527) antibodies were from Abcam (Waltham, MA). NLRP3 (#15101, RRID:AB_2722591), caspase-1 (#24232, RRID:AB_2890194), SIRT3 (#5490, RRID:AB_10828246) and SOD2 (#13141, RRID:AB_2636921) antibodies were from Cell Signaling Technology, Inc (Danvers, MA). Cleaved caspase-1 (p10) antibody (#AG-20B-0044-C100, RRID:AB_2490253) from Adipogen Life Sciences (San Diego, CA).

Mice. CD38KO (RRID:IMSR_JAX:003727) and WT mice were used for the study. All animal procedures were humanely performed with institutionally peer reviewed, approved protocols at VA San Diego.

Subcutaneous air pouch model. Subcutaneous pouches with synovium-like membrane lining were generated by repeated injection of sterile air in 8–10-week-old mice, and after 7 days were injected with MSU crystals (3mg), in 1 ml of sterile, endotoxin-free PBS. Where indicated, apigenin (20 mg/kg/day) or nicotinamide riboside (NR) (400 mg/kg/day) were orally administered (gavage) to mice daily 3 days before injection of MSU crystals. Mice were euthanized 6 hours after MSU crystal injection. Pouch fluids were harvested, and numbers of cells and cytokines in pouch fluid were determined. Air pouch tissues were fixed (in 10% formalin), embedded and sectioned for histological analysis of infiltrating inflammatory cells by staining with Hematoxylin and Eosin Stain kit (#H-3502, Vector Lab, Newark, CA).

Cell culture. Bone marrow cells isolated from 10-week-old WT or CD38KO mice were cultured in RPMI containing 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml) and 20% L929 conditioned media for 7 days to generate BMDMs. After priming with GM-CSF, 20 ng/ml for 24 hours, cells were washed with PBS and subjected to experiments in RPMI containing 1% FBS.

Quantitative RT-PCR

Total RNA was extracted using the RNeasy Mini kit (#74004) from Qiagen, (Germantown, MD) followed by reverse transcription to generate cDNA using the First Strand cDNA Synthesis kit (#11483188001, MilliporeSigma, Burlington, MA). The cDNAs were subjected to quantitative PCR analysis for expression of CD38, NLRP3, Steap4, Olr1 and Gpr176 using TaqMan Gene Expression Assay probe sets (ThermoFisher, Waltham, MA) for CD38 (Mm01220906_m1), NLRP3 (Mm00840904_m1), Steap4 (Mm00475405_m1), Ch25h (Mm00515486_s1), Olr1 (Mm00454582_m1), Gpr176 Mm01277657_m1) and endogenous control HPRT (Mm03024075_m1). The data were analyzed using the ΔΔCT method.

RNA sequencing (RNA-seq) and data analysis: After priming with GM-CSF (20 ng/ml) for 24 hours, WT BMDMs in the presence or absence of apigenin and CD38KO BMDMs were stimulated with MSU crystals for 6 hours. Cells were collected and subjected to RNA-seq analysis, performed by LC Sciences (Houston, TX). Kyoto Encyclopedia of Genes and Genomes (KEGG) was used for pathway enrichment analysis of differentially expressed genes (DEGs). STRING application-Protein Query was employed to generate a node network of functional enriched DEGs with defined molecular functions (Gene Ontology (GO)) for each cluster.

Western Blotting

Cells were lysed in RIPA buffer containing protease inhibitor cocktails (Roche, Mannheim, Germany). Cell lysates (10–15 µg) were separated by gradient 4–20% SDS-PAGE and transferred onto Immobilon® PVDF membranes (MilliporeSigma, Burlington, MA), probed with primary followed by near-infrared secondary antibodies. The membranes were scanned on the LI-COR Odyssey imaging system (LI-COR Biotech, Lincolin, NB).

Measurement of NAD⁺ and NADH

Liquid chromatography–mass spectrometry (LC–MS) was used to measure NAD⁺ in de-identified human whole blood samples (detailed description in the supplemental materials). Macrophage intracellular NAD⁺ and NADH (the reduced form of NAD⁺) content were measured using a NAD⁺/NADH quantification colorimetric kit (#K337, BioVision, Milpitas, CA). NAD⁺ to NADH ratio was determined.

Cytokine Analyses. IL-1β and CXCL1 (KC) were measured using DuoSet ELISA (#DY401 and #DY453, R&D Systems, Minneapolis, MN).

Statistical analyses

GraphPad PRISM 9 (San Diego, CA) was used for statistical analyses. All data were subjected to the normality test. For normally distributed data, unpaired student t-test (comparing 2 groups, 2-sided, alpha = 0.05) or two-way ANOVA with Tukey multiple comparisons test (comparing 2 \(\ge\)groups with 2 independent variables, alpha=0.05) were performed. The data were expressed as mean\(\pm\)SD or mean\(\pm\)SEM. P< 0.05 was considered statistically significant.

Decreased blood NAD⁺ levels and increased CD38 expression in PBMCs of gout patients

In limited, seminal studies, LC-MS analysis of whole blood samples from 6 healthy controls and 10 gout patients with acute gout flare revealed that the mean NAD⁺ levels were significantly decreased in gout patients (3.28\(\pm\)1.29 µM) compared to healthy controls (6.76\(\pm\)0.88) (Fig. 1A), suggesting systemic NAD⁺ decline in gout patients. Western blot analysis of cell lysates of PBMCs from 3 healthy controls and 3 gout patients showed increased CD38 expression in gout patients compared to healthy controls (Fig. 1B). This prompted us to study the role of CD38 in MSU crystal-induced inflammatory responses in macrophages in vitro and in the mouse air pouch gout model in vivo.

Induction of CD38 expression by MSU crystals and associated effects on NAD ⁺ /NADH and macrophage cytokine expression

After stimulation of mouse BMDMs with MSU crystals for 6 and 24 hours, CD38 was upregulated at mRNA and protein levels, respectively (Fig. 2A, 2D). Intracellular levels of NAD⁺/NADH, a key measure of cellular redox state reflecting both total metabolic function and the cellular health status [19], were significantly reduced at both time points (Fig. 2B, 2E), associated with induction of IL-1b and the chemokine CXCL1 at both mRNA and protein levels (Fig. 2C, 2F). These results suggested an association of decreased intracellular NAD⁺/NADH with the inflammatory responses to MSU crystals in macrophages.

Effects of CD38 expression on macrophage responses to MSU crystals

We assessed the effects of inhibition of CD38 by apigenin, a natural flavonoid CD38 inhibitor [20], in macrophages stimulated with MSU crystals in vitro. As shown in Fig. 3, apigenin prevented induction of CD38 (A) and reduction of intracellular levels of NAD⁺/NADH (B). The results also aligned with attenuation of IL-1b and CXCL1 release (C and D) induced by MSU crystals. We then tested specificity of the effects of CD38 inhibition, using either a more selective CD38 pharmacological inhibitor 78c or CD38KO BMDMs. IL-1b and CXCL1 induction by MSU crystals were blunted in BMDMs that either received 78c treatment or were genetically deficient in CD38 (Supplemental Fig. 1).

Intracellular NAD⁺ depletion is permissive for NLRP3 inflammasome activation in macrophages [10, 11]. As seen in Fig. 3, MSU crystals induced upregulation of NLRP3 expression at both mRNA and protein levels, effects diminished by apigenin (E and F). In addition, MSU crystal-induced expression of cleaved caspase-1 was attenuated by apigenin (Fig. 3F).

Mitochondrial reactive oxygen species (ROS) centrally mediate NLRP3 inflammasome activation [21], and mitochondrially located SIRT3 plays an important role in regulating oxidative stress by deacetylation of substrates involved in ROS production [22]. Hence, we examined levels of NAD⁺-dependent SIRT3 and acetylation of superoxide dismutase (SOD2), an antioxidant enzyme located in mitochondria. Decreased SIRT3 expression and increased acetylation of SOD2 were observed in BMDMs stimulated with MSU crystals, which were reversed by apigenin (Fig. 3G). These results suggested that apigenin inhibited NLRP3 inflammasome activation at least partly via SIRT3-SOD2 signalling.

Apigenin suppressed MSU crystal-induced inflammation in vivo

In the murine subcutaneous air pouch model of gouty synovitis, pouch leukocyte infiltration was markedly decreased in response to MSU crystals by apigenin treatment (Fig. 4A). In addition, histology of the air pouch lining showed decreased swelling and cell infiltration in MSU crystal-injected mice that received apigenin treatment (Fig. 4B). Moreover, MSU crystal-induced IL-1b and CXCL1 release in the pouch were significantly inhibited by apigenin (Fig. 4C and 4D). Similarly, MSU crystal-induced leukocyte infiltration and release of IL-1b and CXCL1 were attenuated in CD38KO compared to WT mice (Supplemental Fig. 2).

Effects of CD38 pharmacologic inhibition or genetic knockout on gene expression in MSU crystal-stimulated macrophages

We performed RNA-seq to identify differentially expressed genes (DEGs) and effects of CD38 deficiency in macrophages stimulated with MSU crystals. Of the 507 genes upregulated (log₂FC\(\ge\)1) by MSU crystals in WT BMDMs (WT+MSU vs WT non-treated), 401 and 207 were downregulated (log₂FC\(\le -\)1) by apigenin in WT (WT + MSU+Api vs WT+MSU) and CD38KO (CD38KO+MSU vs WT+MSU) BMDMs, respectively, of which 154 genes were common (Fig. 5A, mean log₂FC values of DEGs, and supplemental Figure S3, heatmap of DEGs with biological replicates). Many of the shared changes were in inflammatory genes.

STRING enrichment analysis of the 154 DEGs displayed 3 major clusters (Supplemental Figure S4A) with molecular function (GO) in chemokine and cytokine activities, chemokine and cytokine receptor binding, nuclear receptor activity, transcription coactivator or cofactor binding, growth factor activity and growth factor receptor binding (cluster 1), Bradykinin receptor activity (cluster 2) and glutamate-cysteine ligase activity (cluster 3) (Supplemental Figure S4B). KEGG pathway enrichment analysis of these 154 DEGs revealed several signalling pathways implicated in inflammation. These included IL-17, TNF, chemokine, NF-kB, C-type lectin receptor, Toll-like receptor, JAK/STAT, MAPK, PI3K/Akt, and NOD-like receptor signalling pathways, as well as ferroptosis and osteoclast differentiation (Fig. 5B).

Six-transmembrane epithelial antigen of prostate 4 (Steap4), a metalloreductase involved in iron homeostasis, was found to be the top DEG upregulated in WT BMDMs (log₂FC = 8.2) but was downregulated in CD38KO (log₂FC=\(-\)4.4) and apigenin-treated WT (log₂FC=\(-\)7.2) BMDMs stimulated with MSU crystals. Notably, some genes involved in cholesterol and lipid metabolism and signalling such as cholesterol 25-hydroxylase (Ch25h) and oxLDL receptor 1 (Olr1), also known as lectin-like oxidized low-density lipoprotein receptor-1 (Lox-1), were upregulated in MSU crystal-stimulated WT BMDMs, but were downregulated in MSU crystal-stimulated CD38KO BMDMs and WT BMDMs in the presence of apigenin. Gene expression changes of Steap4, Ch25h and Olr1 were validated by qRT-PCR analysis of BMDMs stimulated with MSU crystals in the presence of apigenin (Fig. 5C-E).

Of the 527 downregulated (log₂FC\(\le -\)1) genes by MSU crystals in WT BMDMs, 64 and 48 genes were upregulated by apigenin in WT BMDMs and CD38KO BMDMs, respectively, of which 13 genes were common shown in Fig. 6A (mean log₂FC values of DEGs) and Supplemental Fig. 3B (heatmap of DEGs with biological replicates). Gpr176, a Gz-linked orphan G protein-coupled receptor (GPCR), was the top DEG downregulated by MSU crystals (log₂FC<-4) but was upregulated by apigenin or CD38 deficiency. This result was validated by qRT-PCR in BMDMs stimulated with MSU crystals in the presence of apigenin (Fig. 6B).

Nicotinamide Riboside (NR) supplementation attenuated MSU crystal-induced inflammatory responses

Mice received oral administration of NR, a bioavailable NAD⁺ precursor that has exerted anti-inflammatory effects in clinical studies [23–25], also exhibited suppression of MSU crystal-induced leukocyte infiltration and release of IL-1b and CXCL1 (Fig. 7A-D). In addition, NR significantly inhibited MSU crystal-induced gene expression of CD38, Steap4, Ch25h and Olr1 and reversed MSU crystal-induced reduction of Gpr176 (Fig. 7E-I).

In this study, we confirmed induction of CD38 by MSU crystals, and elucidated the association with significantly reduced intracellular levels of NAD⁺/NADH, in mouse BMDMs in vitro. These effects were reversed by apigenin, a natural flavonoid with ability to block CD38 NADase activity [20]. Importantly, inhibition of CD38 either by two pharmacological inhibitors (apigenin or the more specific compound 78c) or CD38 genetic knockout suppressed MSU crystal-induced release of IL-1b and CXCL1 in BMDMs. Moreover, apigenin-treated or CD38KO mice had blunted inflammatory responses to MSU crystals in vivo.

CD38 induction by MSU crystals was likely mediated by activation of transcription factors NF-kB and STAT, whose binding sites are in the CD38 gene promoter. Both NF-kB and JAK/STAT signalling pathways were highlighted in our KEGG pathway enrichment analysis of genes upregulated by MSU crystals but downregulated by both apigenin and CD38 deficiency. Inhibition of MSU crystal-induced NRLP3 gene expression by apigenin may also act on by inactivation of NF-kB signalling, since NF-kB-dependent signals regulate NLRP3 expression [26]. Shim et al. showed that intracellular NAD⁺ decline can provide a non-transcriptional priming signal for NLRP3 inflammasome activation by causing mitochondrial retrograde transport [11]. A lower concentration of intracellular NAD⁺ can lead to increased acetylation of a-tubulin via inhibition of NAD⁺-dependent SIRT2 activity [27]. Our data suggest that reduced intracellular NAD⁺/NADH contributed to NLRP3 inflammasome activation by MSU crystals, evidenced by apigenin inhibition of CD38 expression, preserved intracellular levels of NAD⁺/NADH, and ameliorated caspase-1 activation and IL-1b release induced by MSU crystals in macrophages.

Several studies have shown that SIRT3-mediated activation of SOD2 signalling inactivates NLRP3 inflammasome activation [28–30]. We observed reduction of SIRT3 expression and increased acetylation of SOD2 in macrophages stimulated with MSU crystals, an effect limited by apigenin treatment. Hence, apigenin may act on activation of SIRT3-SOD2 signalling to suppress MSU crystal-induced NLRP3 inflammasome activation by inhibiting generation of mitochondrial ROS. CD38 consumes NAD⁺ to form NAM, ADPR and to a less extent cADPR [8, 9]. Both ADPR and cADPR can act as second messengers controlling cell functions through calcium (Ca²⁺) mobilization [8, 9]. Ca²⁺ mobilization also plays a critical role in NLRP3 inflammasome activation [31]. CD38 regulates NLRP3 inflammasome activation through cADPR-mediated Ca²⁺ release in vascular smooth muscle cells in diabetic mice [32]. Whether CD38 regulates NLRP3 inflammasome activation by MSU crystals through ADPR or cADPR-mediated Ca²⁺ mobilization in macrophages remains to be determined.

In this study, more genes upregulated or downregulated by MSU crystals in WT BMDMs were reversed by apigenin treatment (401 or 207 genes, respectively) than by CD38 genetic knockout (64 or 48 genes, respectively). Because apigenin is a selective but not specific CD38 inhibitor, to focus on CD38-targeting effects of apigenin, we performed subsequent analyses with those MSU crystal-induced DEGs (upregulated and downregulated) that were commonly reversed by apigenin and by CD38 knockout. Inhibition of CD38 suppressed several other inflammatory signalling pathways activated by MSU crystals, especially pathways for IL-17 signalling and Th17 cell differentiation, which were also enriched in our previous genome-wide DNA methylation analyses of PBMCs of gout patients [33]. chemokine and cytokine receptor binding and activity, and nuclear receptor activity.

Steap4 was identified as the DEG most robustly induced by MSU crystals that was blunted by both apigenin and CD38 knockout in macrophages. STEAP4 is a metalloreductase that reduces both Fe³⁺ to Fe²⁺ and Cu²⁺ to Cu⁺, which are prerequisites for the transport of these metals into cells [34]. Increased expression of STEAP4 increases iron and/or copper import into cells [34]. STEAP4 is linked to inflammation and innate immune response, and its expression is induced by inflammatory cytokines including IL-1b, TNFa, and IL-6 [35]. Copper levels are elevated in inflamed tissues. Increased STEAP4 enhances cellular copper uptake, activating E3-ligase activity of XIAP, resulting in sustained NF-κB activation [35]. Overexpression of STEAP4 can cause iron accumulation in mitochondria leading to enhanced ROS production and mitochondrial dysfunction, which can further amplify inflammation [34, 36].

Notably, ferroptosis signalling pathway was identified in KEGG pathway enrichment analysis of 154 genes upregulated by MSU crystals but downregulated by apigenin and CD38 knockout. Ferroptosis is an iron-dependent cell death pathway with unique characteristics including lipid peroxide accumulation, mitochondrial cristae loss, and mitochondrial membrane rupture and condensation [37]. Ferroptosis is triggered by accumulation of iron and lipid peroxidation and is linked to NLRP3 inflammasome activation [37]. Given the ability of MSU crystals to robustly induce Steap4 expression and promote macrophage mitochondrial cristae loss [38], it is conceivable that ferroptosis via mitochondrial iron accumulation is involved in MSU crystal-induced NLRP3 inflammasome activation.

STEAP4 also plays a critical role in osteoclastogenesis [39]. Enhanced monocyte/macrophage differentiation to osteoclasts, and increased osteoclastogenesis at the tophus-bone interface are believed to contribute to bone erosion in gouty arthritis (40). Notably, osteoclast differentiation was one of enriched BMDM signalling pathways upregulated by MSU crystals but downregulated by apigenin and CD38 genetic knockout in the KEGG pathway analysis.

This study also revealed that apigenin and CD38 genetic knockout suppressed induction by MSU crystals of Ch25h and Olr1, genes involved in cholesterol and fatty acid metabolism and signalling in macrophages. Ch25h is a cholesterol 25-hydroxylase that converts cholesterol to 25-hydroxycholesterol (25-HC), an oxysterol acting as an innate immune mediator that can amplify inflammatory responses in macrophages [41]. OLR-1 is a transmembrane glycoprotein that binds a broad spectrum of structurally distinct ligands such as oxLDL, phosphatidylserine (PS), apoptotic bodies, advanced glycation end-products (AGEs), and Hsp60 [42, 43]. Following uptake of its ligand, OLR-1 induces inflammatory signalling pathways leading to production of ROS, secretion of inflammatory cytokines and induction of apoptosis [42, 43]. These data implicate that MSU crystal-induced inflammatory responses could be mediated through impaired iron homeostasis and dysregulated cholesterol and lipid metabolism and signalling in macrophages, all targetable by inhibition of CD38.

Gpr176, the DEG most robustly downregulated by MSU crystals in macrophages, was conversely upregulated by both apigenin and CD38 knockout. GPR176 is an orphan GPCR involved in normal circadian rhythm behaviour [44]. Studies have shown that expression of GPR176 is enriched in the suprachiasmatic nucleus (SCN), the brain's circadian pacemaker, and that GRP176 governs daily rhythms in behaviour and physiology [44]. Expression of GPR176 in the SCN is under the control of the core clock components Cry1 and Cry2 [44]. Circadian disruption can lead to dysregulation of immune responses and inflammation which can further interfere with circadian rhythms [45]. Gout flares, disproportionately frequent at night, appear significantly influenced by circadian rhythm, which regulates NLRP3 inflammasome activation, and phagocyte behaviour, and is subject to altered epigenomic modulation in gout patients [46, 47]. Hence, downregulation of GRP176 by MSU crystals, by altering circadian rhythm behaviour in macrophages, has the potential capacity to modulate inflammatory responses to MSU crystals.

Our demonstration of the capacity of oral supplementation of the NAD⁺ precursor NR to attenuate inflammatory responses to MSU crystals was noteworthy. Clinical trials have shown that chronic oral supplementation of NR is well-tolerated, has boosted human NAD⁺ metabolism in a dose-dependent manner [48, 49], and exerts anti-inflammatory effects [23–25]. Therefore, translational strategies to boost NAD⁺ levels using oral NR supplementation warrant further clinical investigations for preventing and limiting gout flares.

In conclusion, CD38 deficiency through either pharmacologic inhibition or genetic knockout attenuated inflammatory responses to MSU crystals in macrophages in vitro and in the air pouch gouty synovitis model in vivo. CD38 inhibition acts by preserving intracellular NAD⁺ content, which is associated with NAD^±-dependent sirtuin signalling. Seminal studies suggested that gout patients may have lower systemic NAD⁺ and increased PBMC CD38 compared to healthy controls. Larger-scale analyses would be of interest but were beyond the scope of this study. Gout incidence and prevalence increase with age. In this context, CD38 expression and activity are increased during aging associated with NAD^± decline [8, 9]. Thus, inhibition of CD38 is identified as a novel candidate druggable approach to prevent and treat gouty inflammation, by correcting an aging-associated change.

Acknowledgement

We would like to thank Dr. Jacob Karsh, University of Ottawa, Ottawa, Canada for providing the human whole blood samples from healthy controls and Dr. Ann-Charlotte Åkerblad, Sobi Pharmaceuticals, Stockholm, Sweden for providing the whole blood samples from gout patients in early flare from the Anakinra in Gout (anaGO) clinical trial of gout flare treatment. We are grateful to Dr. S.L. Bridges at University of Alabama, Birmingham, Alabama, USA for providing us with frozen human peripheral blood mononuclear cell (PBMC) pellets of healthy controls and gout patients from the UAB RADAR cohort. All samples were de-identified. We would also like to thank the mass spectrometry core, University of South Alabama, for the LC-MS technical support.

Funding: This work was supported by the US Rheumatology Research Foundation (Innovation Research Award to RL-B) and the US Department of Veterans Affairs (Merit Review grant 1I01BX002234 to RL-B).

Author Contributions: Conception and design: PGA, RT, RL-B. Data acquisition: PGA, PO, HQ, TY, MG, MM, and HQ. Data analysis and interpretation: PGA, PO, HQ, TY, MG, MM, RT and RL-B. Drafting: PGA, RT, RL-B. All authors approved the final version. Dr. Liu-Bryan had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Conflict of interests: The authors have no competing interests to declare that are relevant to the content of this article.

Data availability statement: The data supporting the findings of this study are available within the article and its supplementary information files or from the corresponding author upon reasonable request. RNA-seq data is available in the Gene Expression Omnibus (GEO) database #GSE214587.

Supplemental materials: Supplemental data and method are available online.

Terkeltaub R. What makes gouty inflammation so variable? BMC Med. 2017;15:158.
Pourcet B, Duez H. Circadian Control of Inflammasome Pathways: Implications for Circadian Medicine. Front Immunol. 2020;11:1630.
Liu L, Zhu L, Liu M, Zhao L, Yu Y, Xue Y et al. Recent Insights Into the Role of Macrophages in Acute Gout. Front Immunol. 2022;13:955806.
Cipolletta E, Tata LJ, Nakafero G, Avery AJ, Mamas MA, Abhishek A. Association Between Gout Flare and Subsequent Cardiovascular Events Among Patients With Gout. JAMA. 2022;328:440-450.
Busso N, So A. Mechanisms of inflammation in gout. Arthritis Res Ther 2010; 12:206.
Cronstein BN, Sunkureddi P. Mechanistic aspects of inflammation and clinical management of inflammation in acute gouty arthritis. J Clin Rheumatol 2013;19:19-29.
Galozzi P, Bindoli S, Doria A, Oliviero F, Sfriso P. Autoinflammatory Features in Gouty Arthritis. J Clin Med. 2021;10:1880.
Covarrubias AJ, Perrone R, Grozio A, Verdin E. NAD (+) metabolism and its roles in cellular processes during ageing. Nat Rev Mol Cell Biol. 2021;22:119-141.
Zeidler JD, Hogan KA, Agorrody G, Peclat TR, Kashyap S, Kanamori KS et al. The CD38 glycohydrolase and the NAD sink: implications for pathological conditions. Am J Physiol Cell Physiol. 2022;322:C521-C545.
He M, Chiang HH, Luo H, Zheng Z, Qiao Q, Wang L et al. An Acetylation Switch of the NLRP3 Inflammasome Regulates Aging-Associated Chronic Inflammation and Insulin Resistance. Cell Metab. 2020;31:580-591.e5.
Shim DW, Cho HJ, Hwang I, Jung TY, Kim HS, Ryu JH et al. Intracellular NAD⁺ Depletion Confers a Priming Signal for NLRP3 Inflammasome Activation. Front Immunol. 2021;12:765477.
Aksoy P, White TA, Thompson M, Chini EN. Regulation of intracellular levels of NAD: a novel role for CD38. Biochem Biophys Res Commun. 2006;345:1386-92.
Piedra-Quintero ZL, Wilson Z, Nava P, Guerau-de-Arellano M. CD38: An Immunomodulatory Molecule in Inflammation and Autoimmunity. Front Immunol. 2020 Nov 30;11:597959.
Li W, Li Y, Jin X, Liao Q, Chen Z, Peng H et al. CD38: A Significant Regulator of Macrophage Function. Front Oncol. 2022;12:775649.
Amici SA, Young NA, Narvaez-Miranda J, Jablonski KA, Arcos J, Rosas L et al. CD38 Is Robustly Induced in Human Macrophages and Monocytes in Inflammatory Conditions. Front Immunol. 2018;9:1593.
Wen S, Arakawa H, Tamai I. CD38 activation by monosodium urate crystals contributes to inflammatory responses in human and murine macrophages. Biochem Biophys Res Commun. 2021;581:6-11.
Liu L, Zhu X, Zhao T, Yu Y, Xue Y, Zou H. Sirt1 ameliorates monosodium urate crystal-induced inflammation by altering macrophage polarization via the PI3K/Akt/STAT6 pathway. Rheumatology (Oxford). 2019;58:1674-1683.
Wang J, Chen G, Lu L, Zou H. Sirt1 inhibits gouty arthritis via activating PPARγ. Clin Rheumatol. 2019;38:3235-3242.
Clement J, Wong M, Poljak A, Sachdev P, Braidy N. The Plasma NAD⁺ Metabolome Is Dysregulated in "Normal" Aging. Rejuvenation Res. 2019;22:121-130.
Escande C, Nin V, Price NL, Capellini V, Gomes AP, Barbosa MT et al. Flavonoid apigenin is an inhibitor of the NAD+ ase CD38: implications for cellular NAD+ metabolism, protein acetylation, and treatment of metabolic syndrome. Diabetes. 2013;62:1084-93.
Tschopp J. Mitochondria: Sovereign of inflammation? Eur J Immunol. 2011;41:1196-202.
Ozden O, Park SH, Kim HS, Jiang H, Coleman MC, Spitz DR et al. Acetylation of MnSOD directs enzymatic activity responding to cellular nutrient status or oxidative stress. Aging (Albany NY). 2011;3:102-7.
Elhassan YS, Kluckova K, Fletcher RS, Schmidt MS, Garten A, Doig CL et al. Nicotinamide Riboside Augments the Aged Human Skeletal Muscle NAD+ Metabolome and Induces Transcriptomic and Anti-inflammatory Signatures. Cell Rep. 2019;28:1717-1728.e6.
Zhou B, Wang DD, Qiu Y, Airhart S, Liu Y, Stempien-Otero A et al. Boosting NAD level suppresses inflammatory activation of PBMCs in heart failure. J Clin Invest. 2020;130:6054-6063.
Wu J, Singh K, Lin A, Meadows AM, Wu K, Shing V et al. Boosting NAD+ blunts TLR4-induced type I IFN in control and systemic lupus erythematosus monocytes. J Clin Invest. 2022;132:e139828.
Bauernfeind FG, Horvath G, Stutz A, Alnemri ES, MacDonald K, Speert D et al. Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. J Immunol. 2009;183:787-91.
Misawa T, Takahama M, Kozaki T, Lee H, Zou J, Saitoh T et al. Microtubule-driven spatial arrangement of mitochondria promotes activation of the NLRP3 inflammasome. Nat Immunol. 2013;14:454-60.
Traba J, Geiger SS, Kwarteng-Siaw M, Han K, Ra OH, Siegel RM et al. Prolonged fasting suppresses mitochondrial NLRP3 inflammasome assembly and activation via SIRT3-mediated activation of superoxide dismutase 2. J Biol Chem. 2017;292:12153-12164.
Zheng J, Shi L, Liang F, Xu W, Li T, Gao L et al. Sirt3 Ameliorates Oxidative Stress and Mitochondrial Dysfunction After Intracerebral Hemorrhage in Diabetic Rats. Front Neurosci. 2018;12:414.
Dong X, He Y, Ye F, Zhao Y, Cheng J, Xiao J et al. Vitamin D3 ameliorates nitrogen mustard-induced cutaneous inflammation by inactivating the NLRP3 inflammasome through the SIRT3-SOD2-mtROS signaling pathway. Clin Transl Med. 2021;11:e312.
Murakami T, Ockinger J, Yu J, Byles V, McColl A, Hofer AM et al. Critical role for calcium mobilization in activation of the NLRP3 inflammasome. Proc Natl Acad Sci U S A. 2012 Jul 10;109:11282-7.
Li JP, Wei W, Li XX, Xu M. Regulation of NLRP3 inflammasome by CD38 through cADPR-mediated Ca²⁺ release in vascular smooth muscle cells in diabetic mice. Life Sci. 2020;255:117758.
Wang Z, Zhao Y, Phipps-Green A, Liu-Bryan R, Ceponis A, Boyle DL et al. Differential DNA Methylation of Networked Signaling, Transcriptional, Innate and Adaptive Immunity, and Osteoclastogenesis Genes and Pathways in Gout. Arthritis Rheumatol. 2020;72:802-814.
Scarl RT, Lawrence CM, Gordon HM, Nunemaker CS. STEAP4: its emerging role in metabolism and homeostasis of cellular iron and copper. J Endocrinol. 2017;234:R123-R134.
Liao Y, Zhao J, Bulek K, Tang F, Chen X, Cai G et al. Inflammation mobilizes copper metabolism to promote colon tumorigenesis via an IL-17-STEAP4-XIAP axis. Nat Commun. 2020;11:900.
Xue X, Bredell BX, Anderson ER, Martin A, Mays C, Nagao-Kitamoto H et al. Quantitative proteomics identifies STEAP4 as a critical regulator of mitochondrial dysfunction linking inflammation and colon cancer. Proc Natl Acad Sci U S A. 2017;114:E9608-E9617.
Huang, Y., Xu, W. & Zhou, R. NLRP3 inflammasome activation and cell death. Cell Mol Immunol 2021;18:2114–2127.
McWherter C, Choi YJ, Serrano RL, Mahata SK, Terkeltaub R, Liu-Bryan R. Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling. Arthritis Res Ther. 2018;20:204.
Zhou J, Ye S, Fujiwara T, Manolagas SC, Zhao H. Steap4 plays a critical role in osteoclastogenesis in vitro by regulating cellular iron/reactive oxygen species (ROS) levels and cAMP response element-binding protein (CREB)activation. J Biol Chem. 2013;288:30064-30074.
Dalbeth N, Smith T, Nicolson B, Clark B, Callon K, Naot D et al. Enhanced osteoclastogenesis in patients with tophaceous gout: urate crystals promote osteoclast development through interactions with stromal cells. Arthritis Rheum. 2008;58:1854-65.
Gold ES, Diercks AH, Podolsky I, Podyminogin RL, Askovich PS, Treuting PM et al. 25-Hydroxycholesterol acts as an amplifier of inflammatory signaling. Proc Natl Acad Sci U S A. 2014;111:10666-71.
Drouin M, Saenz J, Chiffoleau E. C-Type Lectin-Like Receptors: Head or Tail in Cell Death Immunity. Front Immunol. 2020;11:251.
Taye A, El-Sheikh AA. Lectin-like oxidized low-density lipoprotein receptor 1 pathways. Eur J Clin Invest. 2013;43:740-5.
Doi M, Murai I, Kunisue S, Setsu G, Uchio N, Tanaka R et al. Gpr176 is a Gz-linked orphan G-protein-coupled receptor that sets the pace of circadian behaviour. Nat Commun. 2016;7:10583.
Nathan P, Gibbs JE, Rainger GE, Chimen M. Changes in Circadian Rhythms Dysregulate Inflammation in Ageing: Focus on Leukocyte Trafficking. Front Immunol. 2021;12:673405.
Choi HK, Niu J, Neogi T, Chen CA, Chaisson C, Hunter D et al. Nocturnal risk of gout attacks. Arthritis Rheumatol. 2015;67:555-62.
Wang Z, Zhao Y, Phipps-Green A, Liu-Bryan R, Ceponis A, Boyle DL et al. Differential DNA Methylation of Networked Signaling, Transcriptional, Innate and Adaptive Immunity, and Osteoclastogenesis Genes and Pathways in Gout. Arthritis Rheumatol. 2020;72:802-814.
Martens CR, Denman BA, Mazzo MR, Armstrong ML, Reisdorph N, McQueen MB et al. Chronic nicotinamide riboside supplementation is well-tolerated and elevates NAD(+) in healthy middle-aged and older adults. Nat Commun. 2018;9:1286.
Dellinger RW, Santos SR, Morris M, Evans M, Alminana D, Guarente L et al. Repeat dose NRPT (nicotinamide riboside and pterostilbene) increases NAD⁺ levels in humans safely and sustainably: a randomized, double-blind, placebo-controlled study. NPJ Aging Mech Dis. 2017;3:17.

No competing interests reported.

Supplmaterials.pdf

Download PDF

Journal Publication

published 15 Mar, 2024

Read the published version in Inflammation Research →

Editorial decision: Revision requested
12 Dec, 2023
Reviews received at journal
20 Nov, 2023
Reviewers agreed at journal
18 Nov, 2023
Reviewers agreed at journal
10 Nov, 2023
Reviewers invited by journal
07 Nov, 2023
Editor assigned by journal
19 Oct, 2023
Submission checks completed at journal
19 Oct, 2023
First submitted to journal
17 Oct, 2023

You are reading this latest preprint version

The NADase CD38 is a central regulator in gouty inflammation and a novel druggable therapeutic target

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and Methods

Reagents

Quantitative RT-PCR

Western Blotting

Measurement of NAD⁺ and NADH

Statistical analyses

Results

Decreased blood NAD⁺ levels and increased CD38 expression in PBMCs of gout patients

Effects of CD38 expression on macrophage responses to MSU crystals

Effects of CD38 pharmacologic inhibition or genetic knockout on gene expression in MSU crystal-stimulated macrophages

Nicotinamide Riboside (NR) supplementation attenuated MSU crystal-induced inflammatory responses

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

The NADase CD38 is a central regulator in gouty inflammation and a novel druggable therapeutic target

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and Methods

Reagents

Quantitative RT-PCR

Western Blotting

Measurement of NAD+ and NADH

Statistical analyses

Results

Decreased blood NAD+ levels and increased CD38 expression in PBMCs of gout patients

Effects of CD38 expression on macrophage responses to MSU crystals

Effects of CD38 pharmacologic inhibition or genetic knockout on gene expression in MSU crystal-stimulated macrophages

Nicotinamide Riboside (NR) supplementation attenuated MSU crystal-induced inflammatory responses

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Measurement of NAD⁺ and NADH

Decreased blood NAD⁺ levels and increased CD38 expression in PBMCs of gout patients