Identification of sUA as an endogenous inhibitor for CD38

(A) Preliminary screening of 8-OG binding proteins by mass spectrum (MS)-based proteomics, and effect of 8-OG (50 μM) on CD38 activity. (n = 3 experiments/technical replicates).

(B) Hydrolase and cyclase activities of recombinant human CD38 (hCD38) in the presence of sUA, using nicotinamide 1, N6-ethenoadenine dinucleotide (ε-NAD+), and nicotinamide guanine dinucleotide (NGD) as substrates, respectively (n = 3 experiments/technical replicates).

(C and D) Effect of different substrate concentrations on sUA inhibition of recombinant hCD38 hydrolase (C) and cyclase (D) activities (n = 3 experiments/technical replicates).

(E) Effect of different sUA concentrations on hydrolase and cyclase activities (FU/min/μg protein) in tissues from 8- to 12-week-old WT mice (n = 3 mice).

(F and G) Reversibility of inhibition of recombinant hCD38 hydrolase (F) and cyclase

(G) activities by sUA. After 30-min pre-incubation as indicated, samples were diluted 100-fold in reaction buffer with or without 500 μM sUA for enzyme assay (n = 3-5 experiments/technical replicates).

Data are mean ± s.d. (B-E) or mean ± s.e.m. (A, F, and G). See also Figure S1.

CD38 inhibition is restricted to sUA in purine metabolism

(A and B) Effect of sUA precursors and metabolite on hydrolase and cyclase activities of recombinant hCD38 (n = 3 experiments/technical replicates for each ligand).

(C) Major pathways of purine metabolism.

(D) Effect of sUA analogs on hydrolase and cyclase activities (n = 3 experiments/technical replicates). THP-1 cells were used to detect the effects of oxypurinol, caffeine, 1-MU, and 1,3-DMU on hydrolase activity, recombinant hCD38 was used in the remaining detections.

(E) Effect of uracil and 1,3-dihydroimidazol-2-one (1,3-DHI-2-one) on hydrolase and cyclase activities of recombinant hCD38 (n = 3 experiments/technical replicates).

(F) A structural comparison reveals the functional group for CD38 inhibition. The concentrations of all ligands are from 5 to 500 μM. Data are mean ± s.e.m. See also Figure S1.

sUA physiologically limits NAD+ degradation via CD38 inhibition

WT and CD38 KO mice (10- to 12-week-old) received oral administration of saline, OA, or OA plus inosine (Ino) twice (1-day moderate sUA supplementation).

(A-D) Effect of 1-day sUA supplementation on whole blood NAD+ (A), NMN (B), cADPR (C), and plasma sUA (D) levels in WT and CD38 KO mice (WT-Saline: n = 8 mice, WT-OA: n = 9 mice, WT-OA + Ino: n = 9 mice, KO-Saline: n = 6 mice, KO-OA: n = 8 mice, KO-OA + Ino: n = 8 mice).

(E) Effect of 1-day or 3-day release on whole blood NAD+, NMN, cADPR, and plasma sUA levels in WT mice that received 1-day supplementation (WT-Saline: n = 6 mice, WT-OA: n = 8 mice, WT-OA + Ino: n = 8 mice).

(F) Effect of sUA (100, 200, or 500 μM) and other ligands (analogs at 500 μM, 78c, a CD38 inhibitor, at 0.5 μM) on NAD+ degradation by recombinant hCD38 (n = 5 independent samples).

Data are mean ± s.e.m. Significance was tested using 2-way ANOVA (A-D), or 1-way ANOVA (E and F) with Tukey’s multiple comparisons test. NS, not significant.

See also Figures S1-S5 and S7.

sUA physiologically prevents excessive inflammation by interacting with CD38

WT and CD38 KO mice (10- to 12-week-old) received 1-day moderate sUA supplementation, plasma sUA was increased to the minimum physiological levels of humans in OA plus inosine groups. Then, the mice were stimulated with cLPS (2 mg/kg) or MSU crystals (2 mg/mouse) for 6 h.

(A-C) Effect of sUA at physiological levels on serum levels of IL-1β (A), IL-18 (B), and TNF-α (C) in mice with cLPS-induced systemic inflammation (WT-OA: n = 6 mice, WT-OA + cLPS: n = 11 mice, WT-OA + Ino + cLPS: n = 12 mice, KO-OA: n = 6 mice, KO-OA + cLPS: n = 8 mice, KO-OA + Ino + cLPS: n = 8 mice).

(D-H) Effect of sUA at physiological levels on IL-1β (D), IL-6 (E), and CXCL1 (F) levels and recruitment of viable cells (red blood cells excluded) (G) and neutrophils (H) in peritoneal lavage fluid from the mice with MSU crystal-induced peritonitis (WT-OA: n = 6 mice, WT-OA+MSU: n = 12 mice, WT-OA+Ino+MSU: n = 13 mice, KO-OA: n = 6 mice, KO-OA+MSU: n = 10 mice, KO-OA+Ino+MSU: n = 10 mice).

Data are mean ± s.e.m. Significance was tested using 2-way ANOVA with Tukey’s multiple comparisons test. NS, not significant.

See also Figures S3 and S5-S9.

Effect of sUA or other ligands on CD38 activity. Related to Figures 1-3

(A) Effect of different ε-NAD+ concentrations on sUA inhibition of hydrolase activity of THP-1 cells (n = 3 experiments/technical replicates).

(B) Hydrolase activity of THP-1 cells in the presence of sUA (0 to 500 μM) (n = 3 experiments/technical replicates).

(C and D) Reversibility of inhibition of hydrolase (A549 cells) (C) and cyclase (THP-1 cells) (D) by sUA (n = 3 experiments/technical replicates).

(E-G) Effect of sUA precursors and metabolite on hydrolase (E and F) and cyclase (G) activities. (n = 3 experiments/technical replicates)

(H and I) Effect of uracil and 1,3-dihydroimidazol-2-one (1,3-DHI-2-one) on hydrolase and cyclase activities of WT lung tissues. (n = 3 experiments/technical replicates)

(J) Endogenous sUA concentrations in the final reaction buffer for enzyme assays. sUA levels in initial homogenate or membrane fractions were measured, then the endogenous sUA concentrations in the final reaction buffer were calculated based on loading dilution (n = 3 biologically independent samples).

(K) Comparison between Ki values and mean levels of tissues sUA. Ki values were also shown in Fig. 1E, and tissue sUA levels were from WT mice that received 1-day treatment of saline (also shown in Supplemental Fig. 2A).

(L and M) Hydrolase and cyclase activities of lung tissues from WT mice in the presence of OA (0 to 5 mM) (n = 3 experiments/technical replicates).

(N) Effect of OA administration on plasma sUA levels in WT mice that received oral administration of inosine. In saline group, the mice received oral administration and intraperitoneal injection of saline. In OA p.o. group, the mice received oral administration of inosine (1.5 g/kg) and OA (1.5 g/kg) (the same treatment in our models), and intraperitoneal injection of saline. In OA i.p. group, the mice received oral administration of inosine (1.5 g/kg), and intraperitoneal injection of OA (0.25 g/kg). Four hours after treatment, plasma sUA was measured (n = 5 mice per group).

Data are mean ± s.d. (A, B, L, and M) or mean ± s.e.m. (C-J, and N). Significance was tested using 1-way ANOVA with Tukey’s multiple comparisons test (N).

Effect of moderate sUA supplementation on tissue NAD+, NMN, and sUA levels. Related to Figure 3

WT and CD38 KO mice (10- to 12-week-old) received oral administration of saline, OA, or OA plus inosine (Ino) twice daily for 1, 3, or 7 days. Four hours after the last treatment, the mice were sacrificed. In 1-day model, the mice were treated from the evening of day 0 to the morning of day 1.

(A) Effect of 1-day sUA supplementation on tissue NAD+, NMN, and sUA levels (n = 5 male mice per group).

(B) Effect of 3-day sUA supplementation on tissue NAD+, NMN, and sUA levels (n = 5 male mice per group).

(C-F) Effect of 3-day sUA supplementation on plasma sUA (C), whole blood NAD+ (D), NMN (E), and cADPR (F) levels (WT-Saline: n = 6 mice, WT-OA: n = 8 mice, WT-OA+Ino: n = 8 mice, KO-Saline: n = 6 mice, KO-OA: n = 8 mice, KO-OA+Ino: n = 8 mice).

(G-I) Effect of 7-day sUA supplementation on tissue NAD+ and sUA levels (n = 5 male mice per group).

Data are mean ± s.e.m. Significance was tested using 2-way ANOVA (A-H) with Tukey’s multiple comparisons test or 1-way ANOVA (I) with Dunnett’s multiple comparisons test.

Not OA or inosine but sUA limits NAD+ degradation under inflammatory conditions. Related to Figure 3 and 4

(A) Effect of sUA on intracellular NAD+ levels of A549 cells (n = 5 biologically independent samples).

(B) Effect of sUA pre-incubation on intracellular NAD+ levels of THP-1 cells. Naïve THP-1 cells were incubated with sUA (0-10 mg/dL) for 2h, then the cells were washed twice with PBS and stimulated with MSU crystals (200 μg/mL), cLPS (20 μg/mL), zymosan (50 μg/mL) or ATP (2 mM) for 6 h (n = 6 biologically independent samples). (C-F) Effect of 1-day sUA supplementation on plasma sUA (C) and whole blood NAD+ (D), NMN (E), and cADPR (F) levels in WT and CD38 KO mice under inflammatory conditions. The mice received 1-day sUA supplementation. Two hours after the last treatment, the mice were intraperitoneally stimulated with sterile PBS or cLPS (2 mg/kg) for 6 h (WT-OA: n = 6 mice, WT-OA+cLPS: n = 11 mice, WT-OA+Ino+cLPS: n = 12 mice, KO-OA: n = 6 mice, KO-OA+cLPS: n = 8 mice, KO-OA+Ino+cLPS: n = 8 mice). (G-J) Effect of 1-day treatment of OA or inosine (Ino) on plasma sUA (G) and whole blood NAD+ (H), NMN (I), and cADPR (J) levels in WT mice under inflammatory conditions. The mice received 1-day treatment of OA or Ino (from the evening of day 0 to the morning of day 1). Two hours after the last treatment, the mice were intraperitoneally stimulated with sterile PBS or cLPS (2 mg/kg) for 6 h (n = 6 mice in Saline+cLPS group, n = 5 mice in other groups).

Data are mean ± s.e.m. Significance was tested using 1-way ANOVA with Dunnett’s (A and B) or Tukey’s (G-J) multiple comparisons test, or 2-way ANOVA with Tukey’s multiple comparisons test (C-F).

sUA inhibits NMN degradation via CD38. Related to Figure 3

(A) Effect of sUA on recombinant hCD38-mediated NMN (200 μM) degradation in medium (n = 8 independent samples).

(B) Effect of sUA (100, 200, and 500 μM) or 78c (0.5 μM) on intracellular NAD+ levels of WT BMDMs treated with NMN. WT BMDMs were primed with100 ng/mL ultrapure LPS for 8 h (n = 6 biologically independent samples).

(C-E) Effect of sUA on extracellular NMN degradation in WT (C) or CD38 KO BMDMs in the absence (D) or presence (E) of recombinant hCD38 (10 ng/mL). BMDMs were primed with 100 ng/mL ultrapure LPS for 8 h before metabolic assays. (n = 6 biologically independent samples in C and D, n = 8 biologically independent samples in E)

Data are mean ± s.e.m. Significance was tested using 1-way ANOVA with Tukey’s multiple comparisons test.

Moderate sUA supplementation fails to prevent high-dose cLPS-induced systemic inflammation. Related to Figures 3 and 4

WT mice received 1-day treatment of OA or OA plus inosine (Ino), 2 h after the last treatment, the mice were intraperitoneally stimulated with sterile PBS or cLPS (20 mg/kg) for 4 h (n = 5 mice per group).

(A-C) Serum IL-1β (A), IL-18 (B), and TNG-α (C) were measured.

(D-G) Plasma sUA (D), whole blood NAD+ (E), NMN (F), and cADPR (G) levels were measured.

Data are mean ± s.e.m. Significance was tested using 1-way ANOVA with Tukey’s multiple comparisons test.

Effect of sUA or CD38 KO on IL-1β release in primed THP-1 or BMDMs. Related to Figure 4

(A) THP-1 cells were primed with 0.5 μM PMA for 3 h the day before stimulation. Primed THP-1 cells were pre-incubated with sUA (0-10 mg/dL) for 2 h, then the cells were washed twice with PBS and challenged by MSU crystals (200 μg/mL), cLPS (20 μg/mL), zymosan (50 μg/mL), and ATP (2 mM) for 4 h (n = 6 biologically independent samples).

(B) Effect of CD38 KO on IL-1β release in primed BMDMs. WT and KO BMDMs were primed with 100 ng/mL ultrapure LPS for 4 h, then primed BMDMs were challenged by ATP (5 mM, 30min), nigericin (3 μM, 2 h), MSU crystals (200 μg/mL, 6 h), cLPS (20 μg/mL, 6 h), and zymosan (50 μg/mL, 4 h). US means unstimulated. (n = 8 biologically independent samples in ATP and nigericin groups, n = 6 biologically independent samples in other groups)

(C-E) Effect of sUA pre-incubation on IL-1β release and intracellular sUA levels in primed BMDMs. WT BMDMs were primed with 100 ng/mL ultrapure LPS for 4 h. (C) The cells were pre-incubated with or without sUA (100 or 200 μM) for 2 h. Then, the cells were washed twice with PBS and were stimulated with nigericin (3 μM, 2 h), MSU crystals (200 μg/mL, 4 h), or cLPS (1 μg/mL, 4 h). (D and E) Primed BMDMs were directly incubated with sUA (100, 200, and 500 μM) or MSU crystals (100 μg/mL) for 6 h in D, 2 or 15 h in E. US means unstimulated. (n = 6 biologically independent samples in C and D, n = 3 biologically independent samples in E)

Data are mean ± s.e.m. Significance was tested using 1-way ANOVA with Dunnett’s (A and C-E) multiple comparisons test, or two-tailed unpaired t-test and Mann-Whitney test (ATP and Zymosan) (B).

OA or inosine alone does not limit cLPS-induced systemic inflammation and MSU crystal-induced peritonitis. Related to Figure 3 and 4

(A) No effect of 1- to 7-day sUA supplementation on serum IL-1β levels (n = 5 mice per group).

(B-I) WT mice received 1-day oral administration of saline, OA, or inosine (Ino), 2 h after the last treatment, the mice were intraperitoneally stimulated with sterile PBS, cLPS (2 mg/kg), or MSU crystals (2 mg/mouse) for 6 h. (B-D) Serum IL-1β (B), IL-18 (C), and TNF-α (D) levels in mice with cLPS-induced systemic inflammation were measured (n = 6 mice in Saline+cLPS group, n = 5 mice in other groups). (E-I) IL-1β (E), IL-6 (F), CXCL-1 (G), and the number of viable cells (red blood cells excluded)

(H) and neutrophils (I) in peritoneal lavage fluid from the mice with MSU crystal-induced peritonitis were measured (n = 5 mice per group).

Data are mean ± s.e.m. Significance was tested using 1-way ANOVA with Dunnett’s (A) or Tukey’s (B-I) multiple comparisons test.

Crystals precipitation in sUA stock solution. Related to Figure 4

sUA stock solutions in NaOH were prepared without pH adjustment. Crystals were immediately precipitated after dissolution in 50 mg/mL tube. Visible crystals were observed in 5 mg/mL tube after 2-month storage at 4 ℃.

Potential mechanism of the paradox in gout therapy. Related to Figure 4