Figure Legends
Figure 1. NAD+ enhancement shifts macrophage
polarization into an anti-inflammatory character.
(a) LMT503 structure. (b-g) BMDM originated from wild-type B6 mice were
treated with 200 ng/ml LPS for 4 h followed by treatment with 10 μM or
100 μM LMT503 for 18 h. (b) NQO1 activity measured by OD change per min.
Gene expression of (c) inflammatory markers, (d) anti-inflammatory
markers, (e) Sirtuins 1, 3, 6, and (f) GATA3 in BMDMs. (g) IL-1β
expression in culture supernatant of BMDMs stimulated with 200 ng/ml LPS
3 h followed by 5 mM ATP 1h, afterwards 10 μM or 100 μM LMT503 for 18 h.
(h) Human monocyte THP-1 cells or (i) human peripheral blood derived
monocytes were treated with 200 ng/ml LPS 4 h followed by treatment with
10 μM LMT530 for 18 h. ns; not significant, *, p <
0.05, **, p < 0.01, ***, p < 0.001,
one-way ANOVA with multiple comparison.
Figure 2. NAD+ enhancement ameliorates inflammation in
a murine DSS-induced colitis model.
C57BL/6 female mice were given 2% DSS in drinking water for 5 days.
Mice were then provided with normal drinking water afterwards (n =
9/group, n = 5 for healthy control). After DSS treatment, 50 mg/kg
LMT503 or 30 mg/kg tofacitinib was then orally given to mice every day
starting from Day 5.
(a) Body weight, (b) disease activity index (***, p <
0.001 one-way ANOVA with multiple comparison vs. vehicle control group),
(c) colon length, and (d) bacterial translocation (CFU/g) in the MLN.
(e) H&E staining of mouse colon and histological score.
(f) Cytokine levels (TNF, MCP-1, and IL-1β) of colon homogenates.
*p<0.05, **p<0.01, ***p<0.001 one-way
ANOVA with multiple comparison.
Figure 3. NAD+ enhancement reduces infiltration of
innate inflammatory cells in colon of DSS-induced colitis mouse.
Percentage and absolute number of (a, b)
Ly6G+CD11b+ neutrophils, (c, d)
Ly6C+CD11b+ monocytes, and (e, f)
F4/80+CD11b+ macrophages from colon
lamina propria were analyzed (n = 5/group). ns, not significant;
*p < 0.05, **p < 0.01, ***p< 0.001 one-way ANOVA with multiple comparison.
Figure 4. Metabolic reprograming of colon macrophages shift to an
anti-inflammatory character.
C57BL/6 female mice were given 2% DSS in drinking water for 5 days.
Mice were then provided with normal drinking water (n = 6/group). Mice
were given 50 mg/kg LMT503 orally everyday starting from Day 5. At day
10, macrophages isolated from colon lamina propria were analyzed for
sub-population and gene expression.
(a) Colon macrophage population. CD206 and CX3CR1 were
analyzed among CD11b+ gated cells.
(b) CX3CR1hi cell population and cell
number among CD11b+ gated cells.
(c) CX3CR1int cell population and cell
number among CD11b+ gated cells.
(d) Inflammatory cytokines, (e) anti-inflammatory cytokines, and (f)
chemokine expression in colon macrophages. ns, not significant,
*p < 0.05, **p < 0.01, ***p< 0.001 one-way ANOVA with multiple comparison.
Figure 5. Treatment with LMT503 does not increase vulnerability toSalmonella Typhimurium infection.
At day 13 of DSS-induced colitis, mice (n = 6/group) were infected
orally with 106 CFU of Salmonella Typhimurium
(UK-1). Bacteria count (CFU) and cytokine levels in colon tissues were
analyzed at 3 days after infection.
(a) Body weight, (b) UK-1 (CFU/g) in the spleen, Peyer’s Patches, and
mesenteric lymph nodes. (c) Cytokine levels in colon homogenates.
Figure 6. CX3CR1+ macrophages are
critical for immune regulation via metabolic reprograming.
For macrophage depletion, CX3CR1-DTR mice werei.p. injected with 200 ng diphtheria toxin at Days 1, 4, and 7.
At day 10, colon length and cytokines were analyzed (n = 6/group).
(a) Body weight, (b) disease activity index (*** p <
0.001, two-way ANOVA with multiple comparison), (c) body weight at day
10, (d) disease activity index at day 10, (e) colon length, and (f, g)
cytokine gene expressions of colon homogenates. ns, not significant;
*p <0.05, **p <0.01,
***p <0.001 one-way ANOVA with multiple comparison.