Elucidating the role of Nampt and Parp1 in chronic inflammatory skin diseases

Resumo

Psoriasis and atopic dermatitis, or eczema, are two non-contagious skin chronic inflammatory diseases, which global prevalence is 0.1-3 % and 2-20 %, respectively. The aetiology is still undetermined, even though both diseases have a genetic predisposition, numerous environmental factors act as triggers of the pathology, being allergic and immunological causes also involved. Nicotinamide adenine dinucleotide (NAD+) being the most important hydrogen carrier in redox reactions in the cell, is a pleiotropic molecule participating in over 500 reactions. NAD+ regulates vital cellular processes such as mitochondrial function and metabolism, redox reactions, immune response, inflammation and DNA repair, among others. NAMPT, the rate-limiting step enzyme in the NAD+ salvage pathway, has been associated to oxidative stress and inflammation, being identified as a universal biomarker of chronic inflammation, including psoriasis. Experiments with transgenic zebrafish lines that enable in vivo immune cell-tracking and NF B transcriptional activity monitoring, let us to demonstrate that NAD+ and its precursors critically regulated H2O2 keratinocyte release and skin inflammation. Consistently, pharmacological inhibition of Nampt by FK-866, that induces NAD+ depletion, efficiently counteracted H2O2 synthesis by keratinocytes in wild type animals. By using psoriasis model spint1a mutant, we found that Spint1a-deficient zebrafish exhibited increased skin H2O2 production and DNA damage. In this model, FK-866 reduced H2O2 production by keratinocytes, skin inflammation, neutrophil infiltration, keratinocyte proliferation and DNA damage, collectively restoring epithelial integrity. Notably, all these effects could be reversed by exogenous supplementation of NAD+. As NAD+ depletion mediated by FK-866 must have an impact on the enzymatic activity of enzymes that depend on NAD+ as a cofactor, in this work several specific inhibitors of various enzymes that consume NAD+ were used. Inhibition of the enzymatic activity of Parp1 by olaparib, veliparib or talazoparib, recapitulated the effects of FK-866. Both FK-866 and olaparib were also able to restore epithelial integrity in another zebrafish model of psoriasis, the ATPase Na+/K+ transporting subunit ?1a (atp1b1a) mutant. In Spint1a-deficient zebrafish, olaparib treatment additionally induced DNA damage while reduced cell death and PARylation. Strikingly, ROS scavengers were also able to rescue spint1a mutant phenotype. This fact together with increased number of DNA lesions in mutant embryos, led us to hypothesized parthanatos, a PARP1 dependent cell death upon extensive DNA damage, as the programmed cell death occurring in dermal aggregates. In agreement with our hypothesis, an inhibitor of AIFM1 translocation from mitochondria to nucleus, a critical step in parthanatos mechanism, also recapitulated the effects of Nampt and Parp1 inhibition in Spint1a-deficient animals. In human transcriptomic data comparing skin from healthy subjects and psoriasis or atopic dermatitis patients, we found an altered expression profile of genes encoding key enzymes involved in NAD+ salvage pathway, Preiss-Handler pathway and de novo pathway in lesional compared with non-lesional or healthy samples. In a similar way, we found an altered expression profile of genes encoding enzymes involved in PAR metabolism and parthanatos. The expression profile of genes involved in NAD+ and PAR metabolism and parthanatos correlated with inflammatory gene markers of each disease, according to specific cytokines implicated in their TH lymphocyte responses. Additionally, HPLC-MS analysis of serum samples of psoriasis patients indicated that responders to phototherapy (PUVA) exhibited reduced levels of NAD+, NAD+/NADH ratio and SAM before the treatment that were normalized to control group after the treatment, potentially being useful as serum biomarkers to predict the response of psoriasis patients to phototherapy. Finally, we observed increased expression of NAMPT at protein level and PAR accumulation in the nucleus of epidermal keratinocytes from psoriatic lesions. Collectively, our results point out to NAD+ and PAR metabolism as new potential therapeutic targets to treat psoriasis and probably other skin chronic inflammatory diseases.