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

ABSTRACT: Objectives: In gout, flares of severely painful inflammatory arthritis intersect with metabolism, circadian rhythm, and macrophage activation. NAD+ is a necessary cofactor and key metabolite in cellular bioenergy homeostasis, and NAD+ suppresses the NLRP3 inflammasome and inflammation. However, cellular NAD+ declines in inflammatory states, associated with increased activity of the leukocyte-expressed NADase CD38. Gouty arthritis is principally prevented and treated with nonselective and frequently toxic drugs (colchicine, NSAIDs, corticosteroids). Hence, 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 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 in macrophages. BMDM RNA-seq analysis of 176 differentially expressed genes (DEGs) revealed CD38 control of multiple MSU crystal-modulated inflammation pathways. The 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. Conclusion: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE214587 | GEO | 2024/05/08

REPOSITORIES: GEO

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Project description:Objective. To identify novel monosodium urate (MSU) crystal-induced mRNAs by transcript profiling of isolated murine air pouch membranes. Methods. Nine hours after injecting crystals into air pouches, membranes were meticulously dissected away from the adjacent soft tissues. mRNA expression differences between inflamed and control membranes were determined by oligonucleotide microarray analysis. Induction of selected mRNAs was validated by real-time relative quantitative reverse transcriptase PCR (qPCR) in pouch membranes and murine peritoneal macrophages. Results. Eleven of the 12 most highly upregulated mRNAs related to innate immunity and inflammation. They included mRNAs encoding histidine decarboxylase (the enzyme that synthesizes histamine), interleukin (IL)-6, the cell surface receptors PUMA-g and TREM-1, and the polypeptides Irg1 and PROK-2. MSU crystals induced dramatic rises in these mRNAs in the pouch membrane within 3-8 hours after the surge in pro-inflammatory cytokine (IL-6, IL-1beta and TNFalpha) and immediate early gene (Egr-1) transcription, which occurred 1h after crystal injection. MSU crystals induced these mRNAs in cultured macrophages with similar kinetics but lower fold changes. In keeping with their downregulation by MSU crystals according to the microarrays, qPCR confirmed that TREM-2 and granzyme D mRNAs decreased 79% and 94%, respectively, in MSU crystal inflamed membranes. Conclusions. This analysis disclosed several genes previously not implicated in MSU crystal inflammation. Their rise after the early surge in cytokine mRNAs suggests that they may, for instance, amplify or perpetuate inflammation. Transcript profiling of the isolated air pouch membrane promises to be a powerful tool to identify genes acting at different stages of inflammation. Experiment Overall Design: Mouse strain: female Balb/C, 6-8 weeks old Experiment Overall Design: Number of mice= 6. Experiment Overall Design: Air pouches raised on back for 7 days by injecting 2ml of sterile air. Experiment Overall Design: On day 7, injection of 1ml PBS or 2mg MSU crystals in 1ml PBS (n=3 each) Experiment Overall Design: 9 h after injection, dissection of the air pouch membranes from the adjacent soft tissues, RNA extraction with RNeasy columns. Pooling of RNA from the 3 control pouches or the 3 MSU crystal pouches. Processing and labeling of 5ug aliquots in the U Penn microarray core according to standard Affy protocols. Hybridization to Affy Mo430_2 oligonucleotide microarrays.

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

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