Pharmacological Inhibition of Nicotinamide Phosphoribosyltransferase/Visfatin Enzymatic Activity Identifies a New Inflammatory Pathway Linked to NAD

Nicotinamide phosphoribosyltransferase (NAMPT), also known as visfatin, is the rate-limiting enzyme in the salvage pathway of NAD biosynthesis from nicotinamide. Since its expression is upregulated during inflammation, NAMPT represents a novel clinical biomarker in acute lung injury, rheumatoid arthritis, and Crohn's disease. However, its role in disease progression remains unknown. We report here that NAMPT is a key player in inflammatory arthritis. Increased expression of NAMPT was confirmed in mice with collagen-induced arthritis, both in serum and in the arthritic paw. Importantly, a specific competitive inhibitor of NAMPT effectively reduced arthritis severity with comparable activity to etanercept, and decreased pro-inflammatory cytokine secretion in affected joints. Moreover, NAMPT inhibition reduced intracellular NAD concentration in inflammatory cells and circulating TNFα levels during endotoxemia in mice. In vitro pharmacological inhibition of NAMPT reduced the intracellular concentration of NAD and pro-inflammatory cytokine secretion by inflammatory cells. Thus, NAMPT links NAD metabolism to inflammatory cytokine secretion by leukocytes, and its inhibition might therefore have therapeutic efficacy in immune-mediated inflammatory disorders

APO866 reduces intracellular NAD in PEC <i>in vivo</i> and inhibits TNFα production after LPS challenge.

<p>(a) Mice were treated with thioglycollate to elicit PEC, and then received 10 mg/kg APO866 by ip injection. PEC were obtained by lavage after different time points and intracellular NAD was determined. Data are mean+sem of 3 mice per group. (b) Mice were treated with thioglycollate to elicit PEC, and then received 10 mg/kg APO866 or placebo by ip injection 7 h before ip challenge with LPS. Serum TNFα at 90 min (mean+sem of 3 mice per group is shown. PEC were obtained by lavage and intracellular NAD was determined. Data are mean+sem of 3 mice per group. This panel is representative of at least 4 experiments performed. <i>P</i><0.05 9 h versus 0 h in panel a, or APO866 versus placebo in panel b.</p

Induction of NAMPT expression in collagen-induced arthritis.

<p>Sera (a) and tissue extracts of paws (b) from CIA at day 14 (n = 8) and from non-arthritic, non-immunized, naïve (n = 7) mice were prepared and analyzed by NAMPT ELISA. *<i>P</i><0.05 arthritic versus naïve in panel a and b. (c) NAMPT immunohistochemistry was performed on paw joints, using a specific rat anti-mouse NAMPT antibody (panels 1 and 2). Staining specificity was confirmed using an irrelevant isotype-matched antibody as primary antibody (panels 3 and 4). Synovial lining layer (SLL), synovial membrane (S) and pannus (P). Original magnifications: x100 for panels 1 and 3; x400 for panels 2 and 4.</p

Exogenous nicotinamide mononucleotide, NAMPT-catalyzed reaction end product, reverts the inhibitory effects of APO866.

<p>Mouse PEC were incubated in the presence or absence of 200 nM APO866 and 10 mM nicotinamide mononucleotide (NMN). Cells were further stimulated with LPS and intracellular NAD levels and TNFα and IL-6 secretion were determined. Data are mean±SEM of triplicates.</p

Inhibition of NAMPT enzymatic function with APO866 reduces intracellular NAD concentration and pro-inflammatory cytokine production in mouse and human inflammatory cells, without affecting viability.

<p>(a) Mouse PEC and (b) human monocytes were cultured for 4 h with increasing doses of APO866, and then stimulated overnight with SAC or LPS, respectively. At the end of the culture, the culture supernatants were tested for TNFα, IL-1β and IL-6 content by ELISA, cell viability was assessed using the Live/Dead kit, and intracellular NAD concentration was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002267#s4" target="_blank">Methods</a>. Data are mean ± SEM of triplicates. This panel is representative of at least 3 experiments performed.</p

Clinical, histological and biochemical effects of NAMPT inhibition on established arthritis.

<p>Test mice (n = 20) were twice daily treated ip with 10 mg/kg of APO866 from the first day onward of appearance of clinical arthritis (clinical score >1) during 14 days. Placebo mice (n = 20) received vehicle only. (a) Representative photographs of paws of CIA mice APO866-treated or placebo-treated. Groups of animals were compared with respect to variation of their clinical scoring (b), and of their weight (c) by statistical analysis using the two-way ANOVA. (d) Histological features of arthritic joints: representative knee and paw histology from placebo and APO866-treated mice after 14 days of treatment. In the placebo group (pictures 1 and 2), the synovial membrane (noted S on picture) was significantly thicker than in treated animals (pictures 3 and 4). An effect on the articular cartilage (C) was also observed, with a decreased loss of Safranin-O staining in the treated group (compare panel 1 and 3 for knees and 2 and 4 for paws). Original magnification ×40. (e) A semi-quantitative histological evaluation was performed on the knee sections using a 4 points (0–3) scoring system to evaluate inflammatory infiltrate and synovial hyperplasia. (f) Circulating SAA levels: Sera from placebo- and APO866-treated CIA mice at day 14 (n = 8 and n = 7, respectively) were prepared and analyzed by SAA ELISA according to the manufacturer's instructions. (g) Cytokine levels in paw extracts: At the end of the experiment, IL-1β, IL-6, MCP-1, and IL-10 levels in paw extracts were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002267#s4" target="_blank">Methods</a>. *<i>P</i><0.05 APO866 versus placebo in panels b, e and g.</p

Effect of NAMPT inhibition with APO866 on established collagen-induced arthritis.

<p>(a) Dose-response effect of APO866: test mice were treated twice daily ip with APO866 2, 5, or 10 mg/kg (n = 10 in each group) during 15 days. Placebo mice received vehicle only (n = 10). (b) Severity of arthritis in CIA mice receiving APO866 10 mg/kg ip twice daily or etanercept 15 mg/kg every three days (n = 10 in each group) over 15 days. Mice groups were compared by two-way ANOVA. *<i>P</i><0.05 APO866 or etanercept versus placebo in panel a and b.</p