NAMPT-derived NAD+ fuels PARP1 to promote skin inflammation through parthanatos

Several studies have revealed a correlation between chronic inflammation and NAD+ metabolism but the precise mechanism involved is unknown. Here we report that the genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis, reduced oxidative stress, inflammation, and keratinocyte DNA damage, hyperproliferation and cell death in zebrafish models of chronic skin inflammation, while all these effects were reversed by NAD+ supplementation. Similarly, genetic and pharmacological inhibition of poly ADP-ribose (PAR) polymerase 1 (Parp1), overexpression of PAR glycohydrolase, inhibition of apoptosis-inducing factor 1, inhibition of NADPH oxidases and reactive oxygen species (ROS) scavenging, all phenocopied the effects of Nampt inhibition. Pharmacological inhibition of NADPH oxidases/NAMPT/PARP/AIFM1 axis decreased expression of pathology-associated genes in human organotypic 3D skin models of psoriasis. Consistently, an aberrant induction of both NAMPT amounts and PARP activity was observed in lesional skin from psoriasis patients. In conclusion, hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD+, mediates skin inflammation through parthanatos cell death. Highlights NAMPT inhibition alleviates inflammation in zebrafish and human epidermis organoid models of psoriasis. NADPH oxidase-derived ROS mediates keratinocyte DNA damage and Parp1 overactivation. Inhibition of parthanatos cell death phenocopies the effects of NAMPT inhibition in zebrafish and human psoriasis models. NAMPT and PAR metabolism is altered in psoriasis patients.

2 Summary 27 Several studies have revealed a correlation between chronic inflammation and 28 NAD + metabolism but the precise mechanism involved is unknown. Here we report that 29 the genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase 30 (Nampt), the rate-limiting enzyme in the salvage pathway of NAD + biosynthesis,  Despite the altered pattern of neutrophil distribution, some of which were present in the 132 skin, both the integrity of the skin and its morphology were not affected ( Figure 1C).  Figures 1F, 1G). Nevertheless, no differences in neutrophil 141 redistribution were observed ( Figure 1G). 142 We next wondered if the depletion of cellular NAD + stores by the well-143 characterized NAMPT inhibitor FK-866 (Hasmann and Schemainda, 2003) could also 144 have an impact on skin oxidative stress and inflammation using the H2O2 probe and the  Figures 1H, 1J). Despite the NAD + ability to induce 149 skin oxidative stress when used at 1 mM, it was unable to activate NFKB transcriptional 150 activity in any tissue. Importantly, NAD + effectively restored muscle NFKB activity in 151 FK-866-treated larvae ( Figures 1I, 1J), confirming the specificity of the inhibitor. 152 The inflammatory effect of FK-866 in muscle was confirmed by the robust 153 neutrophil infiltration ( Figure 1K, 1M). Furthermore, H2O2 production by skin 154 keratinocytes was almost abolished by 1 µM of FK-866 ( Figure 1L, 1M). Collectively, 155 6 these results suggest that not only NAD + metabolite levels regulate oxidative stress in 156 the skin and neutrophil infiltration, but also that low levels of NAD + trigger muscle 157 inflammation.

159
Inhibition of Nampt alleviates oxidative stress and skin inflammation in a zebrafish 160 model of psoriasis 161 The influence of NAD + metabolism on skin oxidative stress and inflammation in 162 wild type zebrafish, encouraged us to study its effect on the zebrafish psoriasis model 163 with an hypomorphic mutation of spint1a (allele hi2217), which encodes the serine 164 protease inhibitor, kunitz-type, 1a. Spint1a-deficient larvae showed increased H2O2  decreased and increased, respectively NAD + /NADH levels ( Figure 2K). However, no 187 statistically significant differences between NAD + /NADH levels in wild type and 188 Spint1a-deficient larvae were found ( Figure 2K). Collectively, these results indicate that 189 7 Spint1a-deficient animals were more susceptible to NAD + supplementation than their 190 wild type siblings and that the beneficial effects of FK-866 on the skin were mediated 191 by reducing skin NAD + availability.

193
NADPH oxidase-derived ROS promote skin inflammation in Spint1a-deficient larvae 194 The higher levels of ROS in the skin of Spint1a-deficient larvae, together with 195 their drastic reduction by pharmacological inhibition of Nampt, led us to hypothesize 196 that Nampt-derived NAD + was fuelling NADPH oxidases. We therefore used N-            Figure 6D). However, NAMPT was widely overexpressed in the spinous layer and in a 343 few basal keratinocytes and dermal cells in psoriasis lesional skin ( Figure 6D).

344
Curiously, NAMPT immunoreactivity was mainly found in the nucleus of keratinocytes 345 but a fainter immunoreactivity was also observed in their cytoplasm ( Figure 6D).

346
Consistently, although PAR immunoreactivity was found in the nuclei of scattered     (Figure 7). This is consistent with the results obtained with Nampt 431 inhibition, which resulted in depleted NAD + levels, but was also able to rescue skin  In conclusion, we report that hyperactivation of Parp1 in response to ROS-     Table S1. were acquired by ZEISS Apotome.2. All images were processed using ImageJ software.           NAD + biosynthetic pathways generate NAD + from different precursors, de novo pathway employs dietary tryptophan (Trp) or alternatively quinolinic acid (QA), NAD + Salvage pathway mainly uses nicotinamide (NAM) but nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) can also act as precursors. However, Preiss-Handler pathway utilizes nicotinic acid (NA). NAD + is consumed by CD38 yielding NAM and adenosine diphosphoribose (ADPR) or cyclic ADPR (cADPR). NNMT also reduces NAD + pool mediating the reaction between NAM and S-adenosylmethionine (SAM) to produce N-methylnicotinamide (1-MNA) and S-adenosylhomocysteine (SAH). Finally, PARP1 synthesizes PAR by using NAD + as a cofactor. PAR is degraded to ADPR mediated by different PAR hydrolases which cleave specific chemical linkages (exo-or endoglycosidically). Metabolic intermediates: N-formylkynurenine (NFK), nicotinic acid adenine dinucleotide (NAAD) and nicotinic acid mononucleotide (NAMN). NAD + transporter: connexin 43 (CX43). Transcriptomic data from human psoriasis (GDS4602) samples from the Gene Expression Omnibus (GEO) database. Non-lesional and lesional psoriasis skin were compared with heathy skin samples. Each dot represents one individual and the mean ± S.E.M. for each group is also shown. P values were calculated using one-way ANOVA and Tukey multiple range test (A) and t-Test (B). ns, not significant. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.