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

Comparison of Gene Expression Profiles of Candida albicans Azole-Resistant Clinical Isolates and Laboratory Strains Exposed to Drugs Inducing Multidrug Transporters

Azole resistance in Candida albicans can be due to upregulation of multidrug transporters belonging to ABC (ATP-binding cassette) transporters (CDR1 and CDR2) or major facilitators (CaMDR1). Upregulation of these genes can also be achieved by exposure to fluphenazine, resulting in specific upregulation of CDR1 and CDR2 and by exposure to benomyl, resulting in specific CaMDR1 upregulation. In this study, these two different states of gene upregulation were used to determine coregulated genes that often share similar functions or similar regulatory regions. The transcript profiles of a laboratory strain exposed to these drugs were therefore determined and compared with those of two matched pairs of azole-susceptible and -resistant strains expressing CDR1 and CDR2 (CDR strains) or CaMDR1 (MDR isolates). The results obtained revealed that, among 42 commonly regulated genes (8.6% of all regulated genes) between fluphenazine-exposed cells and CDR isolates, the most upregulated were CDR1 and CDR2 as expected, but also IFU5, RTA3 (which encodes putative membrane proteins), HSP12 (which encodes heat shock protein), and IPF4065 (which is potentially involved in stress response). Interestingly, all but HSP12 and IPF4065 contain a putative cis-acting drug responsive element in their promoters. Among the 57 genes (11.5% of all regulated genes) commonly regulated between benomyl-exposed cells and MDR isolates, the most upregulated were CaMDR1 as expected but also genes with oxido-reductive functions such as IFD genes, IPF5987, GRP2 (all belonging to the aldo-keto reductase family), IPF7817 [NAD(P)H oxido-reductase], and IPF17186. Taken together, these results show that in vitro drug-induced gene expression only partially mimics expression profiles observed in azole-resistant clinical strains. Upregulated genes in both drug-exposed conditions and clinical strains are drug resistance genes but also genes that could be activated under cell damage conditions

TAC1, Transcriptional Activator of CDR Genes, Is a New Transcription Factor Involved in the Regulation of Candida albicans ABC Transporters CDR1 and CDR2

The ABC transporter genes CDR1 and CDR2 can be upregulated in Candida albicans developing resistance to azoles or can be upregulated by exposing cells transiently to drugs such as fluphenazine. The cis-acting drug-responsive element (DRE) present in the promoters of both genes and necessary for their upregulation contains 5′-CGG-3′ triplets that are often recognized by transcriptional activators with Zn(2)-Cys(6) fingers. In order to isolate regulators of CDR1 and CDR2, the C. albicans genome was searched for genes encoding proteins with Zn(2)-Cys(6) fingers. Interestingly, three of these genes were tandemly arranged near the mating locus. Their involvement in CDR1 and CDR2 upregulation was addressed because a previous study demonstrated a link between mating locus homozygosity and azole resistance. The deletion of only one of these genes (orf19.3188) was sufficient to result in a loss of transient CDR1 and CDR2 upregulation by fluphenazine and was therefore named TAC1 (transcriptional activator of CDR genes). Tac1p has a nuclear localization, and a fusion of Tac1p with glutathione S-transferase could bind the cis-acting regulatory DRE in both the CDR1 and the CDR2 promoters. TAC1 is also relevant for azole resistance, since a TAC1 allele (TAC1-2) recovered from an azole-resistant strain could trigger constitutive upregulation of CDR1 and CDR2 in an azole-susceptible laboratory strain. Transcript profiling experiments performed with a TAC1 mutant and a revertant containing TAC1-2 revealed not only CDR1 and CDR2 as targets of TAC1 regulation but also other genes (RTA3, IFU5, and HSP12) that interestingly contained a DRE-like element in their promoters. In conclusion, TAC1 appears to be the first C. albicans transcription factor involved in the control of genes mediating antifungal resistance

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

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