UNIVERSIDAD DE MURCIA · 2020. 3. 13. · FACULTAD DE BIOLOGÍA Departamento de Biología Celular e...
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Transcript of UNIVERSIDAD DE MURCIA · 2020. 3. 13. · FACULTAD DE BIOLOGÍA Departamento de Biología Celular e...
UNIVERSIDAD DE MURCIA
ESCUELA INTERNACIONAL DE DOCTORADO
ELUCIDATING THE ROLE OF NAMPT AND PARP1
IN CHRONIC INFLAMMATORY SKIN DISEASES
Estudio del papel de NAMPT y PARP1 en las
enfermedades inflamatorias crónicas de la piel
D. Francisco Javier Martínez Morcillo
2019
UNIVERSIDAD DE MURCIA
FACULTAD DE BIOLOGÍA
Departamento de Biología Celular e Histología
ELUCIDATING THE ROLE OF NAMPT AND PARP1 IN CHRONIC
INFLAMMATORY SKIN DISEASES
Estudio del papel de NAMPT y PARP1 en las enfermedades
inflamatorias crónicas de la piel
Memoria presentada por Francisco Javier Martínez Morcillo para
optar al grado de Doctor por la Universidad de Murcia
(Tesis Doctoral con Mención Europea)
Murcia, septiembre 2019
The experimental work collected in this Doctoral Thesis has been supported by the Ministry of
Economy and Competitiveness (grant BIO2014-52655-R), co-funded with FEDER.
Francisco Javier Martínez Morcillo holds a predoctoral fellowship FPU from the University of Murcia
(2016).
Participation in publications during the PhD
Tyrkalska, S. D., Pérez-Oliva, A. B., Rodríguez-Ruiz, L., Martínez-Morcillo, F. J., Alcaraz-Pérez, F.,
Martínez-Navarro, F. J., … Mulero, V. (2019). Inflammasome Regulates Hematopoiesis through
Cleavage of the Master Erythroid Transcription Factor GATA1. Immunity, 51(1), 50-63.e5.
https://doi.org/10.1016/j.immuni.2019.05.005
Martínez-Morcillo, F.J., Martínez-Navarro, F.J., de Oliveira, S., Candel, S., Martínez-Menchón, T.,
Corbalán-Vélez, R., … Mulero, V. (2019). Hydrogen peroxide in neutrophil inflammation: Lesson from
the zebrafish. Antioxidant & Redox Signaling (under review)
Contribution to scientific conferences during the PhD
García-Moreno, D., Bernabé, M., Alcaráz-Pérez, F., Gómez-Abenza, E., Gabellini, C., Martínez-
Morcillo, F.J., … Mulero, V. In vivo genome editing technologies in zebrafish to study immunity and
inflammation. The 13th ISDCI (International Society of Developmental and Comparative
Immunology) Congress, Murcia (Spain). 28 June-3 July 2015. Poster.
Martínez-Morcillo, F.J., Martínez-Navarro, F.J., Corbalán-Vélez, R., Martínez-Menchón, T., Peñalver-
Meseguer, J., García-Moreno, D., Mulero, V. Impact of oxidative stress in psoriasis: A role for XHD
and NRF2. 39 Congreso de la Sociedad Española de Inmunología, Alicante (Spain). 5-7 May 2016.
Poster.
García-Moreno, D., Martínez-Morcillo, F.J., Martínez-Navarro, F.J., Martínez-Menchón, T.,
Corbalán-Vélez, R., Peñalver-Meseguer, J., Mulero, V. A link between NAD+ biosynthesis and
psoriasis. 39 Congreso de la Sociedad Española de Inmunología, Alicante (Spain). 5-7 May 2016.
Poster.
Ibañez-Molero, S., Gómez-Abenza, E., Martínez-Morcillo, F.J., García-Moreno, D., Mulero, V.
Identification of new therapeutical targets for melanoma using the zebrafish. Cell Symposia:
Hallmarks of Cancer, Ghent (Belgium). 11-13 December 2016. Poster
Martínez-Morcillo, F.J., Martínez-Navarro, F.J., Martínez-Menchón, T., Corbalán-Vélez, R., Peñalver-
Meseguer, J., García-Moreno, D., Mulero, V. A crucial role of NAD+ metabolites in the regulation of
skin inflammation. 40 Congreso de la Sociedad Española de Inmunología, Zaragoza (Spain). 25-27
May 2016. Poster.
Martínez-Navarro, F.J., Martínez-Morcillo, F.J., Martínez-Menchón, T., Corbalán-Vélez, R., Peñalver-
Meseguer, J., García-Moreno, D., Mulero, V. A preclinical zebrafish psoriasis model reveals that
vitamin B6 and H2S are involved in skin inflammation. 40 Congreso de la Sociedad Española de
Inmunología, Zaragoza (Spain). 25-27 May 2016. Poster.
Martínez-Navarro, F.J., Martínez-Morcillo, F.J., Valera-Pérez, A., Corbalán-Vélez, R., Martínez-
Menchón, T., Peñalver-Meseguer, J., … Mulero, V. Modelling chronic inflammatory and infectious
diseases using the zebrafish. Congreso Nacional de Biotecnología (Biotec 2017), Murcia (Spain). 18-
21 June 2017. Oral communication.
Martínez-Morcillo, F.J., Martínez-Navarro, F.J., Corbalán-Vélez, R., Martínez-Menchón, T., Pérez-
Oliva, A. B., Zon, L.I., … Mulero, V. Pharmacological inhibition of Nampt ameliorates skin
inflammation in a preclinical zebrafish model. 11th Zebrafish Disease Models (ZDM11), Leiden
(Holland). 10-13 July 2018. Oral communication.
Martínez-Morcillo, F.J., Martínez-Navarro, F.J., Corbalán-Vélez, R., Martínez-Menchón, T., Pérez-
Oliva, A. B., Zon, L.I., … Mulero, V. Nampt enzymatic activity inhibition restores epithelial integrity
and skin inflammation in a preclinical zebrafish model. III Jornadas Científicas del Instituto Murciano
de Investigación Biosanitaria-Arrixaca, Murcia (Spain). 19-20 November 2018. Oral communication.
A mi familia, especialmente a mis padres y
mis hermanos y a Idoya
AGRADECIMIENTOS
Agradecimientos
En primer lugar, quiero dar las gracias a mi director de tesis Víctor y a mi co-directora Diana.
Víctor ha sido, es y será un referente y un mentor para mí. A lo largo de mis años como estudiante
de grado, me enseñó a valorar el razonamiento y la actitud crítica en biología por encima de
cualquier otra competencia. Sin duda, fue uno de los profesores más comprometidos y entregados,
con la plena convicción de que la formación de sus alumnos merece todo su esfuerzo y dedicación.
Diana ha sido un pilar fundamental, es una persona maravillosa que siempre ha estado dispuesta a
ayudarme pacientemente en mis dificultades en el laboratorio y también en las personales. Ella me
ha transmitido su pasión por la genética. Durante mi tesis, Víctor y Diana me han enseñado a analizar
cada resultado con mucha atención. Su sabiduría y experiencia me han orientado y motivado para
centrar mis esfuerzos en los resultados realmente relevantes, con los que hemos ocupado largas
horas de discusiones.
Se podría decir que Inma FUENTES ha sido para mí una madre de adopción, y es que gracias
a sus innumerables consejos y ayuda, por supuesto, no faltaron varios cientos de ¨ ¡Vamos a veeeer!
¨, me he convertido en un investigador consciente de la importancia de la precisión, la limpieza y,
sobre todo, la concienciación en la bioseguridad. En lo personal, Inma siempre ha sido un apoyo
emocional y buena consejera, tanto fue así, que incluso cuando no le correspondía, me ayudó en
todo lo que estaba en su mano. Víctor puede estar tranquilo en confiar en ella como jefa del labo.
Pedro, siempre con una sonrisa en la cara, echa una mano en lo que puede, incluso recomendando
una buena ruta por los Pirineos… ¡lo que nos costó la dichosa ¨ Senda de Camille ¨!, Amanda puede
dar buena cuenta de ello. Aunque pasamos momentos difíciles, nuestra amistad está por encima de
ello. Han sido muchos los momentos que hemos compartido, gracias por ser tan graciosa y risueña.
María Luisa sin duda tiene el espíritu investigador, me encanta su curiosidad, su ambición y
sus ganas de trabajar con las ideas más locas. Tienes un grupo de estudiantes fantástico: Miriam,
Elena Martínez, Elena Naranjo, David, Inma, Manolo, Isadora… Jesús y Paqui son otra pieza esencial
de tu equipo, unas personas con una inteligencia desbordante, que analizan cada detalle de los
seminarios del grupo. La simbiosis Víctor-María Luisa es lo mejor que les ha ocurrido a varias
generaciones de investigadores de una calidad inmejorable. Cuando hice las prácticas de grado en
su grupo, me enamoré de su forma de trabajar y decidí que costase lo que costase, haría mi tesis
con ellos.
Si algo se me viene a la cabeza cuando pienso en Alfonsa es la excelencia. Conozco a muy
pocas personas que sean capaces de expresar sus ideas de una forma tan clara y concisa y de
desarrollar su trabajo de una manera tan impecable. También tengo que agradecer a Ana Belén su
Agradecimientos
interés por intentar ayudarme y su impresionante capacidad de trabajo y nivel de exigencia, lo que
comparte con Víctor. Me habría encantado haber colaborado con ella en algún proyecto.
De Joaquín puedo decir que es un trabajador incansable, al que me ha encantado enseñar,
no podría haber tenido más suerte en haber sido tu compañero y más tranquilidad en saber que tú
serás el que lidere el futuro de la línea que compartimos. Sinceramente pienso que eres un
profesional excepcional que tendrá una carrera brillante. Por suerte, tengo el placer de considerarte
un amigo.
FJ y yo hemos sido un apoyo mutuo, lo que nos ha permitido desarrollar nuestras tesis desde
los inicios. Tras haber compartido habitación, incluso cama, en algunos congresos, siempre
recordaré tu predisposición para ayudar en la informática y la tecnológica a todo el que lo ha
necesitado.
Sofía también ha sido una amiga especial para mí, una persona muy inteligente, ambiciosa
y trabajadora, que le encanta pasarlo bien siempre que puede. Fueron muchas las fiestas que
compartimos y seguimos compartiendo. Sinceramente, fue la persona que más eché de menos
cuando se fue, me hubiese encantado acompañarte en tu tesis, para ser testigo de hasta dónde eres
capaz de llegar.
Poco puedo decir de Isa que cualquiera que la conozca no sepa, su bondad, alegría y
compañerismo le preceden, me ha encantado trabajar contigo en esta última etapa, eres toda una
profesional. Irene y Lola, las dos nuevas estudiantes de doctorado. Estoy seguro de que vais a ser
muy felices en el laboratorio, tenéis todo el apoyo necesario para alcanzar vuestras metas. De iLola,
como me gusta llamarla, echaré de menos su complicidad y cariño. Giusi, Carlotta e Isabela
conforman un trío especial, he disfrutado de todos los momentos que hemos compartido tanto
dentro como fuera del laboratorio, son unas chicas geniales. Espero que seáis muy felices allá donde
estéis. Luis ¨el panameño¨, ha sido muy divertido compartir con él comidas aderezadas con su
carácter brasileño y sus variadas feijoadas. Elena, todo un terremoto, me ha encantado compartir
contigo esa complicidad y alegría que desprendes. Jorge, aunque quien lo conoce sabe que le
encanta ser quisquilloso siempre que puede, impresiona su capacidad para la ciencia, me ha
encantado compartir con él interesantes discusiones, siempre con los temas más actuales como
punto de referencia. No me olvido de dar las gracias a todos lo que recuerdo que han pasado por
nuestro laboratorio: Ana Valera, Prabhu Azu, Belén, Juan Francisco, Martina, Pablo, Pilar, Gülçin,
Alejandro, Gloria, Bárbara, Gian Marco, Sergio, Laura, Aurora, Cecilia, Irene Campillo, Gloria, Fatma,
Dani…
Agradecimientos
Victoria, Nuria, Sylwia y Mari Carmen os agradezco mucho todo lo que me habéis ayudado
y aportado, sobre todo en mis primeros pasos en el laboratorio y, especialmente Victoria con el
papeleo de la tesis. Siempre recordaré los buenos momentos de risas por las tardes en el despacho.
Os eché mucho de menos cuando os fuisteis.
Pili, aunque recientemente has superado algunas adversidades, afrontas la vida con paz y
amor, remarcando la gran persona que eres. Siempre me encantaste como profesora, pero también
como compañera. Por otro lado, puedo decir sin temor a equivocarme que Chiara marcó un antes y
un después en su paso por nuestro laboratorio, nunca he conocido a una persona tan bondadosa
como ella, simplemente es irrepetible e inmejorable. Allá donde está, despierta en sus compañeros
un amor hacia su persona que perdura toda la vida.
También me gustaría dar las gracias al IMIB, al Hospital Virgen de la Arrixaca y al Servicio de
Dermatología, principalmente a Raúl y Teresa. Este trabajo no podría haberse completado sin la
profesionalidad de los Servicios de Apoyo a la Investigación y el CAID, en concreto Toñi, Juana y
Alejandro. Todo ello también ha sido posible gracias al Departamento de Biología Celular e
Histología, en especial, aquellos que cimentaron la investigación de este departamento. No puedo
evitar acordarme de Pepe Meseguer, un hombre polivalente capaz de destacar en todo aquello que
se propone, sin importar que se trate de dirigir una facultad, aprender a tocar el piano, o incluso
estudiar la carrera de Bellas Artes y hacer una tesis seguidamente.
Al laboratorio de Matthias Hammerschmidt, un hombre con un conocimiento e inteligencia
increíble. Fue de vital importancia su apoyo e interés para poder realizar mi estancia, sus discusiones
durante horas con Joy y Julia me sirvieron para enfocar mis investigaciones desde otro punto de
vista. Todos ellos me recibieron con mucha amabilidad, haciendo de mi estancia una experiencia
inolvidable.
A mis padres, Manolo y Jose, y mis hermanos, José Manuel, Miguel Ángel y mi melliza María.
Tengo que daros las gracias por su apoyo incondicional y por entender el poco tiempo que les he
podido dedicar estos años. No ha sido fácil para ninguna parte, pero han sabido comprenderme y
poner todo de su parte.
Y finalmente a Idoya. quién me iba a decir a mí, que la conocería en un curso de cultivo de
células animales… Es todo un ejemplo de esfuerzo y dedicación. A tu lado termino esta etapa, con
la ilusión de seguir compartiendo contigo mi vida. Te quiero.
Agradecimientos
En definitiva, aunque no han sido pocas las dificultades y los fracasos que se afrontan
durante una tesis, gracias por apoyarme y haber creído en mí, a todos os estaré eternamente
agradecido por hacer de estos años los más felices de mi vida.
TABLE OF CONTENTS
Table of contents
3
TABLE OF CONTENTS 1
ABBREVIATIONS 7
SUMMARY 13
INTRODUCTION 17
1. Immunity 19
1.1 Natural barriers 19
1.2 Innate immunity 19
1.3 Adaptive immunity 20
2. Inflammation 21
2.1 Acute inflammation 21
2.2 Chronic inflammation 23
2.3 Chronic inflammatory diseases 24
2.3.1 Relevance of NFkB in chronic inflammatory diseases. A potential target
for therapeutics? 24
2.3.2 Psoriasis and atopic dermatitis 24
2.3.3 Oxidative stress 27
3. NAD+ metabolism 28
3.1 NAD+ biosynthetic pathways 28
3.1.1 NAMPT family, structure and function 29
3.1.1.1 The role of NAMPT in inflammation 31
3.2 NAD+-consuming enzymes 32
3.2.1 PARP1 family, structure and function 34
3.2.1.1 The role of PARP1 in inflammation and parthanatos 36
4. The zebrafish 38
4.1 Zebrafish in nature 38
4.2 The zebrafish as a vertebrate research model 39
4.3 Zebrafish skin structure 40
4.4 Skin integrity disruption in zebrafish mutants 41
4.4.1 penner (lgl2) mutant 41
4.4.2 spint1a mutant 42
4.4.3 clint1 mutant 42
4.4.4 psoriasis/m14 mutant 43
4.4.5 epcam mutant 43
Table of contents
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OBJECTIVES 45
MATERIALS AND METHODS 49
1. Animals 51
2. Chemical treatments 51
3. Imaging of zebrafish larvae 51
4. Whole-mount immunohistochemistry 52
5. TUNEL assay 53
6. Comet assay 53
7. Western blot 54
8. Total NAD+ & NADH determination 54
9. Gene Expression Omnibus (GEO) database 55
10. Immunohistochemistry in human skin samples 55
11. HPLC-MS 55
12. Statistical analysis 56
RESULTS 57
1. NAD+ and its precursors contribute to skin inflammation 59
2. Pharmacological inhibition of Nampt induce muscle inflammation 61
3. Pharmacological inhibition of Nampt diminishes oxidative stress and skin inflammation, and
restores epithelial integrity in a zebrafish psoriasis model 63
4. Parp1 activity inhibition rescues skin inflammation of spint1a mutant larvae 67
5. FK-866 and olaparib block keratinocyte hyperproliferation in spint1a mutant larvae 69
6. spint1a mutants displays higher DNA damage, which is induced by Parp1 inhibition while
reduced by Nampt inhibition 70
7. FK-866 and olaparib treatments do not trigger apoptosis in the skin of spint1a mutants 72
8. Parp1 pharmacological inhibition reduces cell death in the skin of spint1a mutant larvae 73
9. ROS scavenging molecules rescue skin inflammation of spint1a mutant larvae 73
10. Nitric oxide synthesis inhibition aggravated skin inflammation of spint1a mutant larvae 76
11. Parthanatos cell death inhibition rescues skin inflammation of spint1a mutant larvae 76
12. Pharmacological inhibition of Parp1 or Nampt rescue zebrafish psoriasis mutant 78
13. Altered expression profile of genes encoding key NAD+ metabolic enzymes in atopic
dermatitis and psoriasis 79
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14. The expression profile of genes encoding NAD+ metabolic enzymes correlate with
inflammatory gene markers 82
15. Altered expression profile of genes encoding enzymes related to parthanatos in atopic
dermatitis and psoriasis 83
16. The expression of genes encoding key components involved in parthanatos and PAR
metabolism correlate with inflammatory gene markers of psoriasis and atopic dermatitis 86
17. Human serum levels of NAD+ and SAM are altered in psoriasis patients 87
18. NAMPT and PAR are overexpressed in the nucleus of human keratinocytes from psoriatic
lesions 89
DISCUSSION 91
1. NAD+ metabolism and H2O2 release by keratinocytes 93
2. spint1a mutant 93
2.1 Novel features of the spint1a mutant: oxidative stress and DNA damage 93
2.2 Effects of enzymatic inhibition of Nampt and Parp1 in spint1a mutant 94
2.3 Effects of ROS scavengers 95
2.4 Effect of AIFM1 inhibitor 96
3. Human psoriasis and atopic dermatitis 96
3.1 Transcriptomic datasets 96
3.2 NAD+ metabolites in serum 97
3.3 Immunohistochemistry in skin samples 98
CONCLUSIONS 99
REFERENCES 103
RESUMEN EN CASTELLANO 119
1. Introducción 121
2. Objetivos 122
3. Resultados 122
4. Discusión 125
5. Conclusiones 127
ABBREVIATIONS
Abbreviations
9
µM Micromolar
a.u. Arbitrary units
AIFM1 Apoptosis-inducing factor gene
ARH1 Autosomal recessive hypercholesterolemia protein gene
ARH3 ADP-Ribosylhydrolase 3 gene
atp1b1a ATPase Na+/K+ transporting subunit β1a gene
BER Base excision repair
CHT Caudal hematopoietic tissue
clint1 Clathrin interactor 1 gene
CXCL8 Chemokines like CXC motif chemokine ligand 8 gene
DAMPs Damage-associated molecular patterns
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
dpf Days post-fertilization
dsBs Double-strand DNA breaks
ENPP Ectonucleotide pyrophosphatase/phosphodiesterase 1 gene
GFP Green fluorescent protein gene
hai1 or spint1a Hepatocyte growth factor activator inhibitor 1 gene
hpf Hours post-fertilization
HPLC-MS High-performance liquid chromatography-mass spectrometry
HR Homologous recombination
IDO1 Indoleamine 2,3-dioxygenase gene
IL1B Interleukin 1 beta gene
LPS Lipopolysaccharide
MACROD1 Mono-ADP ribosylhydrolase 1 gene
Abbreviations
10
MACROD2 Mono-ADP ribosylhydrolase 2 gene
MIF Migration inhibitory factor gene
mM Milimolar
MMP Matrix metalloproteinase
NA Nicotinic acid
NAC N- acetylcysteine
NAD+ Nicotinamide adenine dinucleotide
NADSYN1 NAD+ synthetase gene
NAM Nicotinamide
NAMPT Nicotinamide phosphoribosyl transferase gene
NAPRT NA phosphoribosyltransferase gene
NFKB Nuclear factor κappa B gene
NHEJ Non-homologous end joining
nM NanoMolar
NMMA NG-monomethyl-L-arginine
NMN Nicotinamide mononucleotide
NNMT Nicotinamide-N-methyltransferase gene
NP N-phenylmaleimide
NR Nicotinamide riboside
NRK Nicotinamide riboside kinase gene
NUDT16 Nudix Hydrolase 16 gene
pH2Ax Phosphorylated histone variant H2AX
PAMPs Pathogen-associated molecular patterns
PAR Poly(ADP-Ribose)
PARP Poly(ADP-Ribose) polymerases
Abbreviations
11
PBEF Pre-B-cell colony-enhancing factor gene
PBS Phosphate buffer saline
PBST Phosphate buffer saline with 0.1% tween-20
PFA Paraformaldehyde
PNP Purine nucleoside phosphorylase gene
PRRs Pattern recognition receptors
QPRT Quinolinate phosphoribosyltransferase gene
RA Rheumatoid arthritis
ROS Reactive oxygen species
S.E.M. Standard error of the mean
SAH S-adenosylhomocysteine
SAM S-adenosylmethionine
SSBR Single-strand-break repair
ssBs Single-strand DNA breaks
TARG1 Terminal ADP-ribose protein glycohydrolase 1 gene
TCR T-cell receptor
TDO2 Tryptophan 2,3-dioxygenase gene
TH Helper T lymphocyte CD4+
TNFA Tumor necrosis factor α gene
SUMMARY
Summary
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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. 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 NFB 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
β1a (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
Summary
16
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.
INTRODUCTION
Introduction
19
1. Immunity
Immunity is an intrinsic characteristic of the life, from the simplest organism to the most
complex, which responds to numerous hazards threaten to destabilize organism homeostasis, or
even life. In 2005, an incredible breakthrough in immunity took place, the discover suggested an
adaptive immune-like system in Bacteria and Archaea (Mojica et al., 2005). Initially designated as
`Short Regularly Spaced Repeats´ (SRSRs) (Mojica et al., 2000), two years later become known as
`Clustered regularly-interspaced short palindromic repeat´ (CRISPR) sequences. The system
cooperates with CRISPR-associated (Cas) proteins mediating an adaptative and heritable resistance
against foreign DNA. Today, CRISPR-Cas technology has become the most important tool for
genomic editing (García-Martínez et al., 2018).
Generally, animal immune system consists of different cell types, molecules and tissues that
orchestrate an integrative defense response against microbial or danger substances (immune
response). The immune system can be divided into anatomical and physiological barriers, innate
immunity and adaptive immunity (Turvey & Broide, 2010). The interplay between innate and
adaptive immunity is a highly regulated process which main purpose is to fight against non-self, but
is not less important to avoid exacerbated responses resulting in damage or attack its own
structures, causing autoimmune diseases (Chaplin, 2010).
1.1 Natural barriers
Anatomical and physiological barriers suppose the first line of defense, due to its direct contract
with pathogens and the environment are vital. Skin is the main physical barrier, in which cellular
junctions maintain integrity. Mucociliated structures present in respiratory airways, exert another
important protective role. On the other hand, anti-microbial substances are present in mucus of
gastrointestinal and genitourinary epitheliums (Figure 1) (Chaplin, 2010; Turvey & Broide, 2010).
1.2 Innate immunity
Innate immunity support and expand the protection provided by natural barriers. This system
employs cells such as macrophages and neutrophils, with an important phagocytic activity, mast
cells, dendritic cells, eosinophils and natural killer (NK) cells. Additionally, cells that integrate natural
barriers can also participate in an innate immune response. Innate immunity relies on pattern
recognition receptors (PRRs) able to identify pathogen-associated molecular patterns (PAMPs) to
trigger a quick immune response. PAMPs are widespread and conserved molecular structures
present in microbes, they activate the release of pro-inflammatory cytokines and chemokines that
recruit other immune cells. Some PRR can also be activated by damage-associated molecular
patterns (DAMPs) released during tissue damage caused by infection and sterile inflammation.
Introduction
20
Additionally, the humoral innate immunity comprises complement proteins, mannose binding
lectin, lipopolysaccharide (LPS) binding protein, C-reactive protein and different antimicrobial
peptides. There is a crosstalk between innate and adaptive system, one example is the antigen
presentation develop by dendritic cells to fully activate T and B cells (Figure 1) (Chaplin, 2010; Turvey
& Broide, 2010).
1.3 Adaptive immunity
Adaptive immunity is the third line of defense that allow a specific and memory response to
antigens. The cell-mediated response is carried out by T- and B-lymphocytes that express in their
surface T-cell receptor (TCR) and immunoglobulin, respectively. Genetic reorganization of gene
elements encoding T and B receptors virtually allows the specificity for any antigen. Clonal selection
potentiates antigen-reactive lymphocytes that proliferate and are converted into effectors and
memory cells. Extracellular microbes are targeted by B cells, in contrast to intracellular microbes,
whose clearance is mediated by T cells. After clearance, memory cells rest in lymphoid organs, a
second exposure to the antigen will trigger an immediate and potent immune response. The
humoral components of adaptive immunity are antibodies created by B cells. These antibodies
bound to antigens boost phagocytic activity mediated by innate immune cells. Furthermore, there
is a complex crosstalk in which cytokines and other molecules intervene to integrate both immune
systems (Figure 1) (Chaplin, 2010; McComb et al., 2013). Owing to infinite antigen recognition of
TCR, sometimes these cells can develop a misdirected immune response binding self-antigens,
potentially provoking autoimmune diseases. Despite having tolerance mechanisms to avoid self-
reactivity, eventually it can happen (McComb et al., 2013).
Figure 1. Integrative view of immunity components (Adapted from Abbas & Lichtman, 2003).
Introduction
21
2. Inflammation
Inflammation is protective reaction of the organism activated upon infection or tissue
stress/damage (both can activate PRRs), which main purpose is to recover tissue homeostasis (Kotas
et al., 2015).
2.1 Acute inflammation
Inflammation mechanism is initiated by epithelial cells, tissue resident macrophages and
mast cells, they release cytokines such as interleukin-1β (IL-1β), tumor necrosis factor α (TNFα) or
IL6 and chemokines like CXC motif chemokine ligand 8 (CXCL8) that immediately recruit neutrophils,
and other leukocytes, at the affected tissue (Dempsey et al., 2003; Furue & Kadono, 2017) (Figure
3). Additionally, the release of vasoactive amines (histamine and serotonin) mediated by mast
cell/platelets, complement activation, or release of prostaglandins and leukotrienes (Germolec et
al., 2018; Medzhitov, 2008), among others, activate endothelium of blood vessels increasing its
permeability and adhesion markers, causing visual manifestation of flushing (rubor), heat (calor) and
swelling (tumor). Swelling compress nerves causing pain (dolor). These different changes lead to
loss of function at the lesion site (function laesa). Induced because of reduced oxygen influx and
glycolytic shift, acidosis is another component of inflammation (Dempsey et al., 2003; Kuprash &
Nedospasov, 2016) (Figure 2).
Figure 2. Classical and new clinical signs of inflammation (Adapted from Kuprash & Nedospasov, 2016).
Introduction
22
Coordinated events mentioned, are collectively termed as acute inflammation phase that is
followed by the resolution phase (Figure 3). Due to the main role of neutrophils in acute
inflammation, their clearance from the inflamed area ensures homeostasis restoration. Tissue-
resident and recruited macrophages orchestrate resolution phase, they secrete important anti-
inflammatory cytokines like IL10, transforming growth factor-β (TGFβ) (Pasparakis et al., 2014),
lipoxins, protectins and resolvins (Medzhitov, 2008). Acute inflammation can take from hours to
days, but under some conditions, resolution phase of inflammation fails, leading to a chronically
activated inflammatory response (Kourtzelis et al., 2017; Kotas et al., 2015).
Figure 3. Inflammatory response from onset to homeostasis. Inflammation response triggers the release of proinflammatory mediators (1) that recruit leukocytes (2) to eliminate the challenge (3). Inflammation resolution switches proinflammatory mediators (4) by anti-inflammatory and proresolving molecules (5) inducing neutrophil apoptosis (6), later phagocyted by recruited monocytes (efferocytosis) (7 and 8). Macrophages reprogramming enhance production of proresolving mediators (9) helping reverse migration of nonapoptotic cells to blood (10) or lymphatics (11). Homeostasis stage reassembly tissue-resident macrophages and dendritic cells, besides, adaptive immune cells are recruited, in order to effectively respond to additional agent exposition (12 and 13). Abbreviations: DC, dendritic cells; MØ, macrophages (Adapted from Sugimoto et al., 2019).
Introduction
23
2.2 Chronic inflammation
Chronic inflammation can derive from two different scenarios. If neutrophils are unable of
achieve pathogen/initiating stimulus clearance, the situation is taken over by macrophages and T
cells. Consequently, both try to defeat the hazard, if fail, persistent pathogens are contained in
granulomas that avoid pathogen dissemination. Likewise, uncontrolled tissue damage generated in
autoimmune response or if the host fails to generate a resolution response, both initiates a chronic
inflammatory response (Medzhitov, 2008; Soehnlein et al., 2017).
In chronic inflammatory diseases, intense immune cells activity result in elevated
inflammatory mediators released at the inflamed area and in systemic circulation (Nasef et al.,
2017). In an established site of chronic inflammation, blood flow and capillary permeability allow
immune cell recruitment and persistence. Macrophages represent the dominant cell type, they
contribute in the cytokine synthesis [IL-1β, TNFα, cytokine granulocyte colony- stimulating factor
(G-CSF) and granulocyte–macrophage colony-stimulating factor (GM-CSF)], leading to the extension
of neutrophil lifespan. Consequently, inhibition of neutrophil apoptosis prevents them to deplete
cytokines and chemokines through decoy receptors of apoptotic cells and avoid the release of
molecules that facilitate inflammation resolution (Soehnlein et al., 2017). On the other hand,
lymphocytes recruitment is another hallmark of chronic inflammation. B and T cells participate in
antibodies and cytokine production. Additionally, they directly act as effectors, damaging tissues
and facilitating immune cell recruitment. The interaction between lymphocytes and macrophages
in the process of antigen presentation induces the release of proinflammatory cytokines and
chemokines, synergistically stimulating each other and perpetuating the immune response
(Germolec et al., 2018).
Recently, neutrophils have been recognized as important contributors not only in acute
inflammation but also in chronic inflammatory conditions. They participate in the switch from
neutrophils to monocytes recruitment and cooperate with platelets, macrophages and monocytes
(Soehnlein et al., 2017). Neutrophils contain different granules in their cytosol with a variety of
peptides and proteins. Upon phagocytosis, they are ready for a rapid release of reactive oxygen
species (respiratory burst), able to destroy microbes or other inflammatory inciting agents.
However, this potent machinery can attack self-tissues under malfunctioning conditions (Germolec
et al., 2018). An important complication of inflammatory response is its potential damage to nearby
tissue that can develop fibrosis. A reduced fibrosis degree is physiological, not dangerous. However,
persistent or severe inflammation could provoke extensive fibrosis affecting tissues/organs
function. It is known that specific combination of cytokines can induce epithelial cells transition to
fibroblast (Chaplin et al., 2010).
Introduction
24
2.3 Chronic inflammatory diseases
Treatments for chronic inflammatory diseases that block the synthesis or action of
proinflammatory mediators including nonsteroidal anti-inflammatory drugs (NSAIDs) and
antibodies against TNFα, had remarkable success. However, present and future studies tend to be
focused in the manipulation of the inflammatory resolution phase (Feehan & Gilroy, 2019).
2.3.1 Relevance of NFkB in chronic inflammatory diseases. A potential target for
therapeutics?
The nuclear factor κappa B (NFkB) is a master transcriptional factor that regulate several
genes involved in immunity and inflammation. This is the reason why changes in its transcriptional
activity or expression are widely used as biomarker of chronic inflammation diseases (Germolec et
al., 2018). NFkB can be activated by canonical and non-canonical signaling pathways. The latter
regulates immunity. While defects on its activation leads to immune deficiencies, uncontrolled
activation is found in autoimmune and inflammatory diseases (Sun, 2017).
Given its pro-inflammatory role, it could be a dangerous pathogenic factor under certain
conditions, triggering and maintaining inflammation. However, it is known that NFkB is required for
homeostasis in epithelial cells (including skin and intestine). PRRs (and other environmental factors)
activate NFkB in epithelial cells, mediating a cytoprotective effect on them. At the same time, the
synthesis of cytokines and chemokines that will act on immune cells is activated. The immune cells
are recruited and activated both by the factors released by the epithelial cells, and subsequently by
the recognition of the inflammatory stimulus, mediating the clearance of the hazard. The lack of
NFkB signaling in epithelial cells alters epithelial homeostasis leading to inflammation. As expected,
persistent NFkB activation promote detrimental inflammatory response activation (Wullaert et al.,
2011).
Therefore, a strict regulation of NFkB is crucial to provide homeostasis to the epithelia and
develop an adequate immune response. The manipulation of the activity of NFkB could be a very
useful therapeutic approach, but its pleiotropic functions in different cellular tissues make it
complicated.
2.3.2 Psoriasis and atopic dermatitis
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 (Furue & Kadono,
2017). Unfortunately, prevalence seems to be increasing over time. While atopic dermatitis
incidence is common in infancy, ameliorated in adolescence and reappears during the thirties, the
Introduction
25
prevalence of psoriasis increases after the age of forty. Moreover, even though both diseases have
a genetic predisposition, environmental factors act as triggers of the pathology (Dainichi et al.,
2018). Despite being relapsing and disabling diseases that affect both physically and mentally, none
of them is usually life threatening, however, the cytokines and chemokines produced in the lesion
reach the blood and consequently, these patients suffer from comorbidities. In psoriasis, most
common comorbidities caused by systemic inflammation (specially IL-1β and TNFα) are
cardiovascular complications, metabolic syndrome (such as obesity, dyslipidemia, atherosclerosis
and type 2 diabetes mellitus) and autoimmune diseases (psoriasis complication develops psoriatic
arthritis). Comorbidities development is progressive, being a process known as the “inflammatory
skin march” (Furue & Kadono, 2017). Although some studies found similar results in atopic
dermatitis, according to some authors, most common comorbidities are allergic rhinitis, asthma (as
a result of TH2-mediated IgE overexpression) and, with low confidence, inflammatory bowel disease
and rheumatoid arthritis (Weidinger et al., 2018).
Atopic dermatitis is characterized by redness eczematous skin lesions that provoke intense
pruritus (itch, because of histamine release) and consequently induce epidermal barrier disruption
due to scratching. Indeed, skin lesions vicinity shows hyperinnervation, increasing itchy feeling.
Additionally, in these regions microbiota is altered (dysbiosis), with a principal colonization by
Staphylococcus aureus. At the site of inflammation, there is an important presence of Langerhans
cells (skin dendritic cells) and T helper cells (helper T lymphocyte CD4+). Predominantly, TH2 (release
cytokines IL4, IL10 and IL13) and TH12 (produce IL22) immune responses contribute to the pathology
(Weidinger et al., 2018) (Figure 4).
Psoriasis vulgaris is the most widespread psoriasis form, affecting the skin and joints with a
very relevant genetic susceptibility. Histological manifestation is keratinocyte hyperproliferation
and increased tissue thickness (acanthosis), characterize by the development of silvered-covered
erythematous scaly plaques. These lesions provoke pain, burning and itching sensation, being
usually occurring on the scalp, trunk and extensor surfaces of extremities. Both innate and adaptive
immune systems contribute to the pathology in the dermis and epidermis. Remarkably,
keratinocytes, neutrophils, dendritic cell and T cells (TH17 immune responses synthesize IL17 and
IL23) play a crucial role (Greb et al., 2016) (Figure 4).
Generally, first treatment applied to both patients is topical therapies followed by
phototherapy. In moderate-severe patients systemic immunosuppressants can be an alternative,
but no in the long-term use due to side effects. Finally, neutralizing antibodies against characteristic
cytokines are the most expensive treatments, however, they lead to a partial/almost total rescue of
Introduction
26
the inflammation. Of note, inhibition of IL4 or IL13 in atopic dermatitis and IL17, IL23 or TNFα in
psoriasis are the most efficient treatments (Dainichi et al., 2018; Greb et al., 2016; Weidinger et al.,
2018).
Figure 4. Atopic dermatitis versus psoriasis epithelial immune microenviroment. (Top) Scheme showing epidermis from psoriasis, atopic dermatitis and healthy skin (Modified from Salimi & Ogg, 2014). (Bottom) External agents trigger inflammation in genetically predispose epithelium. Cytokines release by keratinocytes stimulate dendritic/Langerhans cells that drive specific T cell immune response. Additional cytokines and chemokines close the inflammatory feed-back loop that result in the skin lesion (Adapted from Dainichi et al., 2018).
Introduction
27
2.3.3 Oxidative stress
Oxidative stress refers to a situation in which the excessive accumulation of oxidizing
molecules, cannot be assimilated by the different antioxidant systems of the organism. These
oxidizing molecules are known as reactive oxygen species (ROS) and reactive nitrogen species (RNS)
(Burton & Jauniaux, 2011). Some ROS and RNS are hydrogen peroxide (H2O2), nitric oxide (NO●),
superoxide radical anion (O2●) and peroxynitrite (ONOO― resultant from nitric oxide and superoxide
anion reaction). They can react with different molecules, causing protein and lipid modification and
DNA damage (Poprac et al., 2017). ROS production predominantly takes place in the mitochondria
and by NADPH oxidases (NOX1-5 and DUOX1-2, catalyze the synthesis of H2O2) (Ameziane-El-Hassani
et al., 2016).
Independently of H2O2 role in respiratory burst of neutrophils, recently, H2O2 has emerged
as a mediator that informs the host about tissue damage. In contrast to high reactive ROS that hardly
ever reach far from the source cell, H2O2 can diffuse (Niethammer et al., 2016). Using zebrafish as a
model of acute inflammation upon tailfin amputation, it was described that H2O2 is produced by
Duox1 at the wound, orchestrating the recruitment of leukocytes (Niethammer et al., 2009). Rapid
leukocyte recruitment is dependent of Lyn intracellular protein residues oxidation (belongs to Src
kinases family). Similarly, this damage signaling in conserved in drosophila and plants (Niethammer
et al., 2016).
Further studies in zebrafish found that H2O2 synthesis is dependent on early extracellular
ATP release that activate a signaling cascade involving intracellular Ca2+ as a second messenger (de
Oliveira et al., 2014). Of note, Duox1-derived H2O2 induce cytokine expression like Cxcl8, an
important mediator in the late phases of neutrophil recruitment (de Oliveira et al., 2015). Indeed,
this mechanism activates NFB, P38 MAPK and AP1 proinflammatory transcription factors (de
Oliveira et al., 2014; de Oliveira et al., 2015). Therefore, H2O2 not only induce leukocyte infiltration
in the very beginning, but also ensure the persistence of immune cells for wound healing and tissue
repair. As evidenced by posterior studies, the control of this mechanism is indispensable for
epithelium homeostasis, whose deregulation develops skin chronic inflammation. The research
performed in zebrafish larvae demonstrated that the lack of signaling through Tnfa or Tnfr2, triggers
Duox1-derived H2O2 production in keratinocytes, activating local inflammatory responses. Strikingly,
it has also been found an increased expression of DUOX1 in skin lesion samples of patients suffering
from psoriasis and lichen planus, suggesting that oxidative stress could be a potential therapeutic
target for skin inflammatory diseases (Candel et al., 2014).
Introduction
28
3. NAD+ metabolism
Since nicotinamide adenine dinucleotide (NAD+) was discovered by Harden and Young more
than 100 years ago (Harden and Young, 1906), several generations of researches have tried to
understand the biological relevance of this molecule. NAD+ being the most important hydrogen
carrier in redox reactions in the cell, is a pleiotropic molecule participating in over 500 enzymatic
reactions. Today it is known that mitochondrial
function and metabolism, redox reactions,
circadian rhythm, immune response and
inflammation, DNA repair, cell division, protein-
protein signaling, chromatin, and epigenetics are
processes in which NAD+ has been shown to
regulate or be implicated. Recently, NAD+ has
been recognized as the molecule of the life, given
that the increase of NAD + levels in elder or sick
animals can raise their health and enlarge
lifespan (Rajman et al., 2018) (Figure 5).
Figure 5. Cellular processes regulated by or dependent on NAD+ (Adapted from Rajman et al., 2018).
3.1 NAD+ biosynthetic pathways
NAD+ level is very dynamic, either in the cytoplasm or in the organelles, depending on diet,
exercise and time of day (Rajman et al., 2018). Commonly, cells keep intracellular NAD+
concentrations between 0.2 and 0.5 mM, due to the relevance of this molecule, its levels are tightly
regulated by Preiss-Handler pathway [utilizes nicotinic acid (NA)], de novo pathway [employs dietary
tryptophan (Trp) or alternatively quinolinic acid (QA)] and NAD+ salvage pathway [mainly uses
nicotinamide (NAM) but nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) can
also act as precursors] (Cantó et al., 2015) (Figure 6). Different tissues preferentially employ a
distinct pathway regarding available precursors (Marletta et al., 2015). While Preiss-Handler
pathway has been described to exert an important NAD+ biosynthesis ability, under NA exposure, in
liver and kidney, de novo pathway is active in liver and kidney and possibly brain (Houtkooper et al.,
2010).
Therefore, NAD+ can be synthesized by different precursors, the most important is the
dietary uptake niacin (also known as vitamin B3) that comprise NAM and NA (Sun et al., 2012). NAM
is also the product of NAD+-consuming enzymes, that is why, most mammalian tissues rely on NAM
to maintain NAD+ pool, via the NAD+ salvage pathway (Cantó et al., 2015; Rajman et al., 2018). The
Introduction
29
rate-limiting enzyme in the the NAD+ salvage pathway is NAM phosphoribosyltransferase (NAMPT)
that converts NAM into NMN (Revollo et al., 2004). After that, NMN adenylyltransferases (NMNAT
1-3) transform NMN into NAD+. There are three isoforms of NMNAT: NMNAT1 is present in nucleus;
NMNAT2 is in the cytosol and Golgi apparatus; and NMNAT3 is expressed in mitochondria and
cytosol. The rationale for specific differential expression of NMNATs suggests the maintenance of
an independent NAD+ pool in every cell compartment (cytosol, nucleus and mitochondria) (Cantó et
al., 2015).
Figure 6. NAD+ biosynthetic pathways generate NAD+ from different precursors. Main NAD+-consuming enzymes are also shown (Adapted from Cantó et al., 2015).
3.1.1 NAMPT family, structure and function
Initially identified as a cytokine that promote pre-B-cell colony formation, NAMPT received
the name of pre-B-cell colony-enhancing factor (PBEF) (Samal et al., 1994). Some years later, studies
in bacteria found that nadV gene confers NAD+ independence, allowing to use NMN as a precursor.
Introduction
30
The homologous gene in human was PBEF, suggesting that PBEF could be a NAM phosphoribosyl
transferase (Martin et al., 2001). NAMPT have received an additional name, visfatin. Visfatin is
released from visceral fat, playing a hormone-like role (adipocytokine) in human osteoblast and
mimicking insulin effect, due to the interaction with its receptor (Xie et al., 2007). However,
leukocytes turn out to be the main responsible for NAMPT release in the bloodstream (Friebe et al.,
2011).
NAMPT belongs to the type II nicotinic acid phosphoribosyl transferase (NAPRTase) family,
a member of phosphoribosyl transferase (PRTase) superfamily (Wang et al., 2014). NAMPT gene
structure is composed of 11 exons and 10 introns, which encodes 491 amino acids (Zhang et al.,
2011). The human protein has 2 isoforms (Chappie et al., 2005). The expression of NAMPT is
ubiquitous in human tissues, highlighting its relevance in cell metabolism (Garten et al., 2015).
NAMPT (55.5 KDa) act as a homodimer in solution and folds in a α/β barrel structure with 23 β-
strands and 15 α-helices. Furthermore, NAMPT structure contains three domains, A, B and C (Khan
et al., 2006). Protein structure is highly conserved between human, mouse and rat (Chappie et al.,
2005; Takahashi et al., 2010) (Figure 7).
According to ZFIN database, in zebrafish there are two nampt genes: nampta (ZDB-GENE-
110420-1) and namptb (ZDB-GENE-030131-2931). Both are predicted to be orthologous to human
NAMPT. Nampta is highly conserved in the protein sequence (Score 88.24), whereas Namptb exhibit
a less amino acid sequence identity (Score 61.12).
Figure 7. The crystal structures of human NAMPT. (A) NAMPT monomer bound to NMN (in green) or FK866 (in black; NAMPT inhibitor) in the same region (B) NAMPT homo-dimerize
Introduction
31
(Mol1/Mol2) with a head-to-tail configuration. NMN and FK866 binding sites are also shown (Adapted from Khan et al., 2006).
NAMPT enzymatic activity catalyzes the reaction between NAM and 5’-phosphoribosyl-1’-
pyrophosphate (PRPP) to yield NMN and pyrophosphate (PPi) (Takahashi et al., 2010). In 2003, a
noncompetitive highly specific NAMPT pharmacological inhibitor was identified, FK866 (also known
APO866). This work found that HepG2 liver carcinoma cells underwent a progressive NAD+ depletion
that resulted in apoptosis, upon FK866 treatment. Strikingly, ATP content was maintained some
days, probably to accomplish apoptosis, a cellular process that requires energy (Hasmann &
Schemainda, 2003). According to the FK866 binding site in the enzyme, FK866 interfere with NAMPT
substrate NAM (Khan et al., 2006). At present, FK-866 has been employed in three clinical trials to
treat cutaneous T-cell lymphoma, advanced melanoma and refractory B-cell chronic lymphocytic
leukemia [ClinicalTrials.gov website, National Institutes of Health (NIH)].
3.1.1.1 The role of NAMPT in inflammation
As previously mentioned, NAMPT acts as a growth factor, stimulating the proliferation of
pre-B-cells (Samal et al., 1994), human vascular smooth muscle cells (Van Der Veer et al., 2005) and
human osteoclast (Xie et al., 2007), among others. Furthermore, NAMPT play a cytokine role, its
expression is upregulated in several acute and chronic inflammatory diseases. Jia et al. (2004) were
the first in demonstrate this facet. They described that IL-1β induced NAMPT expression in human
neutrophils, similarly, LPS provokes the expression and the release of NAMPT in vitro. More
importantly, NAMPT prevents neutrophils apoptosis upon its induction in response to inflammatory
stimuli. Strikingly, NAMPT expression was shown to be increased in neutrophils from sepsis patients
(Jia et al., 2004).
NAMPT has also been linked to rheumatoid arthritis (RA). RA synovial fibroblast
overexpressed NAMPT after TLR ligands activation (polyinosinic-polycytidylic acid [poly(I:C) and LPS]
and joints RA-associated cytokines, such as IL-1β and TNFα. Physiologic concentrations of
recombinant human NAMPT induced IL-6, matrix metalloproteinase 1 (MMP-1) and MMP-3
expression in RA synovial fibroblast. Similarly, in primary human monocyte it induces IL-6 and TNFα.
All these experiments in vitro were supported by the enhanced detection of NAMPT in serum and
synovial fluid that correlated with inflammation status (Brentano et al., 2007). In a model of
collagen-induced arthritis in mice, that recapitulates human RA, NAMPT levels were upregulated in
serum and arthritic tissue. NAMPT inhibitor FK866 was able of revert this inflammatory phenotype
(Busso et al., 2008).
In acute lung injury, NAMPT expression was upregulated at mRNA and protein level (Ye et
al., 2005). Furthermore, posterior studies found that recombinant human NAMPT administrated
Introduction
32
intratracheally in mice acted as a chemoattractant molecule for neutrophils and induced the
expression of leukocytes chemoattractant molecules (Hong et al., 2008). Recently, NAMPT has been
found to be augmented in the serum, colon and leukocytes of inflammatory bowel disease, that
comprise ulcerative colitis and Crohn‘s disease, being an indicator of disease severity (Neubauer et
al., 2019). In the same way, NAMPT blocking by FK866 decreased murine colitis model severity and
PARP1 mucosal activity. Furthermore, FK866 induced monocyte/macrophage anti-inflammatory
phenotype. Finally, NAMPT inhibition reduced cytokines production from human IBD-derived
immune cells (Gerner et al., 2017).
Psoriasis is a skin chronic inflammatory disease in which NAMPT could also be involved. A
study compared gene expression in skin samples of healthy donors and psoriasis subjects, divided
into lesional and non-lesional skin. The research found NAMPT overexpression only in lesional skin
samples, being crucial to distinguish it from non-lesional psoriatic and healthy samples (Xie et al.,
2014). Additionally, in peripheral blood derived mononuclear cell samples, NAMPT was identified as
a universal biomarker of chronic inflammation. Chronic inflammation diseases such as psoriasis,
inflammatory bowel disease and RA shared this biomarker as opposed to healthy controls (Mesko
et al., 2010). In summary, NAMPT has been suggested to be an inflammatory biomarker in acute
lung injury, RA, inflammatory bowel disease and psoriasis, among others inflammatory diseases
(cancer, type 2 diabetes mellitus, nonalcoholic fatty liver disease and obesity) (Garten et al., 2015).
3.2 NAD+-consuming enzymes
NAD+ metabolites uptake and biodistribution comprise crucial processes that ensure
NAD+-dependent enzymes to accomplish their biological functions. Serum concentrations of NAD+
precursors are usually low to have an impact on NAD+ biosynthesis. Some investigations suggest
that NAM in blood would be converted into NMN through extracellular NAMPT (eNAMPT) or by
intracellular NAMPT, if NAM enters the cell (NAM is also able to cross mitochondrial membrane). In
the case of both NA and NR, they have specific transporters (Cantó et al., 2015; Rajman et al., 2018).
Recently, Slc12a8 gene has been described as a specific NMN transporter highly expressed in the
mouse small intestine (Grozio et al., 2019). This gene was previously identified as a susceptibility
psoriasis gene that contains single-nucleotide polymorphisms (SNPs) formerly found to be
associated with psoriasis (Hewett et al., 2002). On the other hand, NMN and NAD+ can be
transformed into NR that is the preferred molecule to be transported into the cell; only neurons
seem to be able to uptake NAD+.
The major influence in NAD+ levels is performed by CD38. CD38 and CD157 are two ADP-
ribosyl cyclase/cyclic ADP-ribose hydrolase that cleave NAD+ into NAM and adenosine
diphosphoribose (ADPR) or cyclic ADPR (cADPR). cADPR is a secondary messenger involved in Ca2+
Introduction
33
signaling, cell cycle control and insulin signaling. Additionally, they both can perform cADPR
hydrolase activity (Cantó et al., 2015). As organisms aged, NAD+ levels decrease, because of an
impaired NAD+ synthesis and increasing NADase activity, mainly mediated by CD38. CD38-deficient
mice exhibit boost NAD+ levels (10-30 fold) in several tissues and extended life span. CD38 has also
been described to exert NMNase activity in vitro and in vivo (Camacho-Pereira et al., 2016;
Yamamoto-Katayama et al., 2002). CD73 is another glycohydrolase that can sequentially convert
NAD+ into in NMN and NMN into NR. Once NAM and NR are inside the cell, they will are incorporated
in NAD+ pool. NR is converted into NMN by nicotinamide riboside kinases (NRKs) (Rajman et al.,
2018) (Figure 8).
Figure 8. NAD+ metabolism. NAD+ metabolites fate in the extra-cellular space and in different cellular compartments (Adapted from Rajman et al., 2018).
Sirtuins are another protein family that consume NAD+ in order to develop their enzymatic
activity. In mammals, there are seven sirtuin enzymes distributed in the cytoplasm, mitochondria
and nucleus. Energy shortage situations trigger sirtuin activation that result in improve metabolic
efficiency. Sirtuins enzymatic activity consist on removing specific covalent modification in lysine
residues such as acetyl, crotonyl, malonyl, glutaryl, succinyl, myristoyl and lipoyl, releasing NAM and
Introduction
34
O-(residue)-ADP-ribose (Jiang et al., 2017). Among other biological functions, sirtuins have been
shown to be involved in the regulation of chromosomal integrity, DNA damage repair, metabolism,
aging, health span, and longevity (Cantó et al., 2015).
Finally, Poly(ADP-Ribose) polymerases (PARP) area also NAD+-consuming enzymes. PARP
enzymatic activity consists on the transference of ADP-ribose molecules (linear or branching PAR)
to proteins or itself using NAD+ as a substrate (auto-poly-ADP-ribosylation) (Qi et al., 2019).
3.2.1 PARP1 family, structure and function
Sharing a common catalytic domain, PARPs are encoding for 18 genes and they are mainly
implicated in DNA repair and chromatin organization, gene transcription, inflammation and cell
death or stress responses among other (Cantó et al., 2015; Qi et al., 2019). PARP1, PARP2, PARP5a
and PARP5b synthesize branching PAR chains, whereas PARP9 and PARP13 do not possess enzymatic
activity. In contrast, and regardless uncharacterized PARP18, remaining PARP enzymes exhibit mono
ADP-ribosylation activity. Mono(ADP-ribose)ylation (MARylation) is much more frequent than poly
ADP-ribosylation, the latter being induced in response to stress (Qi et al., 2019).
Given that poly(ADP-ribose)ylation (PARylation) affect several molecules, regulating their
biological functions, it is needed to reverse this process. PARylated targets are hydrolyzed by
enzymes that cleave the bond protein-ADP-ribose (MacrodD1, MacroD2 and TARG1) and the PAR
chain (ARH1-3 and PARG). However, NUDIX and ENP family proteins hydrolyze specifically
MARylation. Importantly, PARG exhibit a rapid degradation of PAR following DNA damage (or other
stress-responses), a crucial role for completion of DNA repair (Qi et al., 2019).
Human PARP1 gene include 23 exons and the predominant isoform consist of 1014 amino
acids. From N-terminal to C-terminal, PARP1 (116 kDa) contains three zinc finger domains (Zn1, Zn2,
Zn3) in which there is a nuclear localization signal and a caspase 3 cleavage site. Zn1 and Zn2 are
DNA strands break sensors; a BRCA-C-terminus (BRCT) domain, which bears the major sites of
automodification and mediates protein-protein contacts; a Trp-Gly-Arg (WGR) domain, with DNA
binding activity; and finally a regulatory helical domain (HD) and a ADP-ribosyl transferase (ART)
domain, two subdomains that form the catalytic domain (CAT) (Langelier et al., 2013) (Figure 9).
Introduction
35
Figure 9. Schematic representation of human PARP1 domains and its crystal structures. (Adapted from Langelier et al., 2013).
Main PARP biological function is to orchestrate spatio-temporal reparation of DNA damage,
that is why, PARP1 is predominantly localized in the nucleus (Cantó et al., 2015) and is responsible
for approximately 90 % of PAR biosynthesis (Qi et al., 2019). PARP1-3 are recruited and activated
upon single- and double-strand DNA breaks (ssBs and dsBs) (Qi et al., 2019). ssBs or small base
lesions are repaired by single-strand-break repair (SSBR) or base excision repair (BER), respectively.
This mechanism induces PARP1-mediated PARylation of histone H1 and H2B or interaction with free
PAR or autoPARylated PARP1 (Schreiber et al., 2006). This event provokes nucleosomes dissociation
and chromatin loosening, in order to facilitate access to DNA and recruitment of XRCC1 (scaffold
protein for SSBR/BER factors) (Aguilar-Quesada et al., 2007; Schreiber et al., 2006).
Regarding dsBs repair, PARP1 is thought to be somehow implicated, but is dispensable. This
kind of lesion requires homologous recombination (HR) and non-homologous end joining (NHEJ)
pathways. While, HR employs a homologous dsDNA template sequence and is relevant during S-G2
cell cycle stage, NHEJ is the predominant pathway and is an error-prone system because the lack of
homologous template (Qi et al., 2019; Schreiber et al., 2006).
BRCA1 and BRCA2 play a fundamental role in HR, mediating the recruitment of other repair
proteins. Inactivator mutations in other indispensable HR proteins induce a phenotype named
`BRCAness´, in which HR pathway is damaged (Nesic et al., 2018).
According to the involvement of PARP1 in BER, its pharmacological inhibition leads to the
persistence of ssBs. Consequently, during DNA replication these lesions produce dsBs. As previously
Introduction
36
mentioned, HR deals with dsBs, but in a BRCA1/2 loss-of-function mutation or BRCAness genetic
background, NHEJ takes over. The result is a phenomenon called synthetic lethality, characterized
by a progressive accumulation of genetic mutations leading to cell death. Medicine takes advantage
of synthetic lethality to treat cancer with hampered HR. On the other hand, combination of PARP
inhibitors with chemotherapy, independently of HR, has gain interest. PARP inhibitors might act as
a sensitizing factor to potentiate DNA alteration induced by chemotherapy drugs, instead of repair
the lesions (Guha, 2011).
PARP inhibitors resemble NAM molecule, interfering with NAD+ binding. Until now, several
drugs have been developed, among them, olaparib (Lynparza), the first PARP inhibitor approved by
the US Food and Drug Administration and by the European Medicines Agency to treat pretreated or
platinum sensitive ovarian cancer with both germ line and somatic BRCA1/2 mutations. Additionally,
niraparib (Zejula) and rucaparib (Rubraca), both approved in the USA, and talazoparib and veliparib
currently participating in several clinical trials (Kujolj et al., 2017; Nesic et al., 2018).
PARP inhibitors act through the collapse of replication forks resulting in dsBs generation.
Once PARP is recruited in a ssB, the inhibitors prevent PARP enzymatic activity, entrapping and
accumulating inactive PARP on DNA and triggering dsBs formation during replication. Therefore,
PARP inhibitors induce G2/M cell cycle arrest and perturbations during mitosis that lead sister
chromatid scattering in metaphase cells. These alterations eventually provoke cell death, especially
in those cells in which HR pathway is damaged, promoting synthetic lethality. Different PARP
inhibitors show a different ability to induce PARP trapping rather than enzymatic activity inhibition
(Kujolj et al., 2017).
3.2.1.1 The role of PARP1 in inflammation and parthanatos
PARP1 has been described to play a relevant part in some acute and chronic inflammatory
diseases such as neurological diseases (Parkinson´s disease), sepsis, arthritis, colitis, diabetes and
myocardial infarction (Kunze et al., 2019). An important reason for this relationship, is the caspase-
independent regulated cell death driven by PARP1, Parthanatos. Parthanatos term derives from
‘par’ (form PAR polymer), and ‘Thanatos,’ the personification of death in Greek mythology (Fatokun
et al., 2014). Under physiological conditions, DNA damage provoked by cellular metabolism is
successfully handled by PARP1. However, alkylating DNA damage, oxidative stress, hypoxia,
hypoglycemia or activation of inflammatory pathways can trigger PARP1 hyperactivation. Excessive
PARylation deplete cellular NAD+ and ATP, although it does not directly imply cell death. However,
the accumulation of PAR polymers and PARylated proteins reach the mitochondria causing
depolarization of the membrane potential and apoptosis-inducing factor (AIFM1) release (Fatokun
et al., 2014; Galluzzi et al., 2018). AIFM1 is released into the cytosol, where it recruits macrophage
Introduction
37
migration inhibitory factor (MIF) to the nucleus. In the nucleus AIFM1-MIF nuclease activity execute
a large-scale DNA fragmentation resulting in cell death (Wang et al., 2016) (Figure 10).
Additionally, PARP1 has been described to play an active role in apoptosis. After apoptosis
activation, PARP1 can be cleaved by Caspase 3. Then, split PARP1 DNA-binding domain bind DNA,
blocking DNA repair and replication (McCann, 2017).
Figure 10. Representation of parthanatos mechanism. Extensive DNA damage induces PARP1 overactivation ultimately leading to cell death (Adapted from Wang et al., 2016).
PARP inhibition was shown to have a beneficial effect in the viability and reduction of
proinflammatory cytokines (such as TNFα) and chemokine production in LPS-stimulated murine
macrophages. Likewise, PARP enzymatic activity blocking, reduced parthanatos in murine
macrophages and thymocytes challenged with peroxynitrite. Peroxynitrite is a DNA-damage
inducer, and consequently, a PARP1 activator. In vivo experiments exhibit protection against
ischemia/reperfusion (I/R) injury of the intestine in mice, besides, rats intraperitoneally injected
with LPS displayed a reduced TNFα and IL1β serum concentrations and mortality upon PARP
inhibition, pointing out a PARP role in shock and systemic inflammation (Jagtap et al., 2002).
Multiple studies have shown anti-inflammatory effects and diminution in PAR staining, immune cell
Introduction
38
infiltration, and severity of chemical induced-colitis obtained upon PARP inhibitors treatment (Di
Paola et al., 2005; Sánchez-Fidalgo et al., 2007; Mazzon et al., 2002; Zingarelli et al., 2003). In the
same line, IL-10 gene deficiency in mice is an inflammatory bowel disease model, PARP inhibitor 3-
aminobenzamide significantly rescue the phenotype with an improved colonic permeability and
reduced histological inflammation and protenin nitrotyrosination (hallmark of peroxynitrite-
mediated tyrosine protein residues oxidation) (Jijon et al, 2000).
Pharmacological and genetic inhibition of PARP counteracts mitochondrial dysfunction of
embryonic fibroblast exposed to peroxynitrite. Furthermore, PARP inhibitors prevent development
of established collagen-induced arthritis in mice, showing anti-inflammatory effect reducing protein
and gene expression of TNFα, IFNɣ, IL-6, IL-1β and IL-12 and chemokines and Th1 autoimmune
response. In contrast, the anti-inflammatory cytokine IL-10 was induced. On the other hand, there
was an impaired nitrotyrosine staining at inflamed joints after the treatment (Gonzalez-Rey et al.,
2007; Szabó et al., 1998). Finally, PARP inhibition through chemical inhibitors or gene silencing was
anti-inflammatory (reduced NFB and AP-1 transcriptional activity) and anti-proliferative, in
fibroblast-like synoviocytes from RA patients stimulated with TNFα (García et al., 2008). Strikingly,
PARP1 has been described to be a coactivator of NFB. In fact, enzymatic or DNA binding activity of
PARP1 are not needed (Hassa & Hottiger, 2002).
Generally, PARP inhibitors can reduce cell death, and consequently, inflammation and
DAMP exposure. Moreover, this reduction decreases tissue damage and proinflammatory cytokines
release, limiting immune cell recruitment (Kunze et al., 2019).
4. The Zebrafish
4.1 Zebrafish in nature
Zebrafish (Danio rerio H.) is a teleost fish that belongs to the family Cyprinidae and reaches
a maximum length of 6 centimeters in the adult stage (Mayden et al., 2007). Although the average
lifespan is around 42 months, in their natural environment they do not reach this age (Spence et al.,
2008).
In the natural environment, the zebrafish is present in the calm waters of the rivers or in
smaller water courses (near the wetlands and rice-fields), distributed from the southwest to the
northeast of the Indian peninsula (Sarasamma et al., 2017). In these areas, small insects and
crustaceans support their omnivorous diet. Environmental temperature ranges from 6-38 oC in
winter and summer, respectively, highlighting zebrafish temperature resistance. Consequently,
spawning season starts just before the beginning of monsoon season, after which, temperature and
food availability are maximum. During this period, the zebrafish offspring show the fastest lifelong
Introduction
39
growth rate. According to mating, zebrafish is promiscuous, largely influenced by photoperiod
(Spence et al., 2008).
4.2 The zebrafish as a vertebrate research model
Animal models that accurately recapitulate human diseases, at cellular and molecular level,
are a necessity to develop life sciences. Biomedical research has relied on mouse models for many
years, due to genomic, anatomical and physiological similarity. However, and despite differences
between fish and human, zebrafish was started to be employed for the study of embryology and
development as soon as thirties. Today, zebrafish has become in a reference model to research from
immunity & inflammation, toxicology and cancer, to congenital and hereditary disease, even
psychological and behavioral abnormalities (Lieschke & Currie, 2007). This change in the paradigm
is due to the several advantages that zebrafish offers:
• Low size that reduces maintenance cost and required space for living.
• High resistance to physical and chemical conditions, especially against toxic
compounds and infections.
• High promiscuity, fecundity and great female spawning, about 300 eggs per week.
• Offspring reach sexual maturation in 3-4 months.
• Large eggs externally developed facilitating manipulation.
• Zebrafish embryos are transparent during the first stages of life, which allows
obtaining images and cell tracking in fluorescent transgenic lines (Figure 11).
• Almost all major organ systems are completely functional by 36 hours post-
fertilization (hpf) and fish hatching between 48 to 72 hpf (Figure 11).
• Excellent model for drugs discovery by high-throughput screening at very low cost,
due to compounds can easily penetrate their bodies by bath immersion.
• New gene-editing technologies and easy embryo genetic manipulation potentiate
reverse genetic approaches to develop diseases models by transgenesis and specific
mutations.
• Fully sequenced genome and high homology to human genome, representing a
powerful organism for human disease modelling.
All the mentioned advantages and the growing community of zebrafish will no doubt encourage
science to accept the zebrafish as an alternative and complementary tool for the improvement of
the quality of human life (Lieschke & Currie, 2007; MacRae & Peterson, 2015; Meyers, 2018;
Sarasamma et al., 2017; Trede et al., 2004; Veldman & Lin, 2008)
Introduction
40
Figure 11. Lifecycle and embryonic development of zebrafish (Adapted from http://www.mun.ca/biology/desmid/brian/BIOL3530/DEVO_03/devo_03.html).
4.3 Zebrafish skin structure
Zebrafish skin is composed of epidermis, dermis and hypodermis as terrestrial vertebrates.
In contrast, the epithelium only contains living cells; peridermal keratinocytes, the single cell layer
comprising the superficial stratum, are only individually renewed when dead. This is possible due to
most cells in intermediate region remain undifferentiated, ready to a rapid division. In this
intermediate stratum there are several specialized cell types: mucous goblet cells, which synthesis
antimicrobial mucus; club cells, in charge of releasing alarm substances in order to activate defense
behavior (Speedie & Gerlai, 2008); and sensory cells. Basal keratinocytes form the next layer (basal
stratum) are responsible for the anchoring to the basement membrane (Le Guellec et al., 2004).
The dermis is organized in the superficial region or stratum laxum and the basal region or
stratum compactum. The stratum laxum contains a loose collagenous matrix with an important
vascularization around the scales. Fibroblast, nerves and pigment cells are the cellular components
in this stratum. In exception to some fibroblasts, the main component of the stratum compactum is
a dense plywood-like organized collagen matrix (Le Guellec et al., 2004).
Introduction
41
The hypodermis or subcutis contains a loose and vascularized collagenous matrix, bordered
by attached fibrocytes-like cells, which harbor chromatophores and adipose cells. The deeper region
is more compact, resembling a basement membrane (Le Guellec et al., 2004).
4.4 Skin integrity disruption in zebrafish mutants
Skin epithelial integrity depends on a stable association of dermis and epidermis mediated
by interconnecting anchoring structures. Cellular attachment structures are specifically designed
and located according to their function. Outer peridermal cells are bound through tight junctions
(the most apical junctional component) in a horizontally oriented network, very convenient to deal
with mechanical stress (Brandner & Schulzke, 2015; Kiener et al., 2008). Adherens junctions and
desmosomes are below tight junctions in order to guarantee a cell-adhesion deeper (Campbell et
al., 2017). E-cadherin is the most relevant component in adherens junctions (Stemmler, 2008).
Finally, hemidesmosomes and focal adhesion, only present at basal keratinocytes, are responsible
for epidermis anchoring basement membrane, involving transmembrane proteins integrins
(Jefferson et al., 2004; Miyazaki, 2006; Walko et al., 2015). Improper attachment lead to skin
blistering, a condition known as epidermolysis bullosa. There is currently no cure, and there are four
types of epidermolysis bullosa: simplex, junctional, dystrophic and kindler syndrome. A
heterogeneous set of mutations affecting to several genes can provoke this disease (Salera et al.,
2019). Recently, the study of microRNAs (miRNAS) shed light on the posttranscriptional regulation
of gene expression of several proteins implicated in cellular junctions. The loss of miRNAs regulation
could lead to pathologies such as cancer and inflammatory disease (Zhuang et al., 2016). In recent
years, some zebrafish mutants that manifest epidermis integrity disruption have been described.
4.4.1 Penner (lgl2) mutant
The first was the penner (pen)/lethal giant larvae 2 (lgl2) mutant, isolated from a
mutagenesis screen (Eeden et al., 1996). In wild type larvae, hemidesmosomes are completely
functional at 5.5 dpf, but lgl2 mutant did not exhibit hemidesmosomes at that stage. Despite the
presence of normal tight junctions, adherens junctions, desmosomes and basement membrane,
mutants develop skin integrity disruption, migratory and overgrowth basal keratinocytes and
rounded up cells in the fin fold (Sonawane et al., 2005). Posterior studies indicate that the
localization of integrin α6 (main hemidesmosome component) at the plasma membrane of basal
keratinocytes is facilitated by Lgl2 and prevented by E-cadherin (beyond 3.5 dpf), in the developing
zebrafish fin fold (Sonawane et al., 2009).
Introduction
42
4.4.2 spint1a mutant
An insertional mutagenesis screen in zebrafish (Amsterdam et al., 1999) lead to the
characterization of the hepatocyte growth factor activator inhibitor 1 (hai1; also known as spint1a)
mutant (hi2217). HAI (in mammals) is an inhibitor of Hepatocyte growth factor activator (HGFA) and
Matriptase 1 (also known as ST14). Both proteins can proteolytically activate some proteins and
zymogens. Moreover, Matriptase 1 is able to degrade extracellular matrix proteins (Carney et al.,
2007; Mathias et al., 2007).
As soon as 24 hpf, mutants showed basal keratinocytes aggregates on the yolk sac and yolk
extension. These keratinocytes became mobile, presenting a fibroblastoid behavior, contact loss
with neighbor cells and cytoplasmic protrusions. Additional mesenchymal-like properties were
revealed due to the cytoplasmic distribution of E-cadherin, instead of occupying the cell membrane
to participate in adherens junctions. By the same time, epidermal aggregates contained a high
number of acridine orange-positive keratinocytes. Cell death strongly attracted leukocytes, which
remained recruited after cell death ceased, pointing out inflammation as a secondary consequence
of the phenotype. Indeed, leukocytes ablation does not rescue the mutant phenotype. Macrophages
were recruited at 26 hpf, before neutrophils, which started to accumulate in the aggregate from 36
hpf (Mathias et al., 2007). Leukocyte behavior indicated abnormal periods of high and directed
motility interrupted by pauses and malfunction in reverse migration between skin and vasculature
(Carney et al., 2007; Mathias et al., 2007). More importantly, cell death could not be prevented by
inhibition of p53, mRNA microinjection of the anti-apoptotic Bcl- 2 protein or treatment with
caspase inhibitor, signifying an unidentified cell death programmed. Authors hypothesize that
keratinocyte death could be a consequence of the detachment from basement membrane, a
process called anoikis. Furthermore, the aggregates display an increased proliferation at 48 hpf due
to the previous acquisition of mesenchymal-like properties and the contact inhibition. This
phenotype of the Spint1a-deficient larvae was fully rescued by inactivation of matriptase1a.
4.4.3 clint1 mutant
Additional screenings looking for abnormal tissue distribution of neutrophils in the
insertional mutagenesis screen in zebrafish (Amsterdam et al., 1999), provided a mutant in the
clathrin interactor 1 (clint1) (hi1520). Clint1 is an adaptor protein that can bind SNARE proteins,
participating in clathrin-mediated vesicular trafficking. At 48 hpf mutants displayed keratinocyte
hyperproliferation and cell death and leukocyte recruitment in aggregates. Importantly, Clint1-
deficeint larvae presented an increased il1b expression. Leukocyte ablation, treatment with caspase
inhibitors or il1b blocking failed to prevent epidermal aggregation or hyperproliferation. Similar to
penner mutant, clint1 mutant exhibit defects in hemidesmosome formation, besides, Lgl2 and Clint1
Introduction
43
synergistically regulate epithelial homeostasis. Furthermore, keratinocytes showed mesenchymal-
like properties. Leukocyte trafficking, although pause periods alternated with high motility,
presented a reverse migration between epidermal aggregates and blood vessels (Dodd et al., 2009).
4.4.4 psoriasis/m14 mutant
The m14 mutant was isolated from an insertional mutagenesis screen (Solnica-Krezel et al.,
1994). Webb and colleagues characterized and gave the name psoriasis to the mutant due to
phenotypic features, besides, they provided a list of possible genes affected by the mutation.
Mutant larvae developed heart edema at 48 hpf, basal keratinocytes aggregates from 2.5 dpf,
hyperproliferation and cell death within or near epidermal aggregates. Some experiments suggested
that there was a secreted factor encoded by the psoriasis gene that acted non–cell-autonomously.
Moreover, keratinocytes displayed disrupted differentiation and loss of cell polarity, given that they
fail to express or down-regulate epidermal-specific keratins (Webb et al., 2008). Additionally, a
posterior study determined that cell-cell connections between basal keratinocytes were loosen with
a basement membrane discontinuous, allowing basal cells to invade underneath regions. Loss-of-
function mutation in atp1b1a (beta subunit of a Na,K-ATPase pump) was responsible of the psoriasis
phenotype. Surprisingly, this study demonstrated that inhibition of PI3K-AKT-mTORC1-NFB-MMP9
pathway, activated in basal keratinocytes, restored polarity defects in epidermis. In a similar way,
hypotonic stress blockade diminished keratinocyte hyperplasia and invasiveness. Leukocyte
recruitment at skin was also described, but ablation of the myeloid lineage did not show any
alteration of epidermal malignancy (Hatzold et al., 2016).
4.4.5 epcam mutant
epcam (tacstd) mutants (hi2151 and hi2836) were obtained from an insertional screening
(Amsterdam et al., 2004). EpCAM (epithelial cell adhesion molecule) functions as a surface adhesion
molecule, with intracellular α-actin binding sites, present in many epithelia. epcam mutants
enveloping layer cells showed increased levels in tight junctions, and reduced E-cadherin in the
basolateral membrane, suggesting a role in cell-cell adhesion collaborating with E-cadherin. Mutant
larvae displayed basal keratinocytes aggregates and delay in otolith development. Moreover,
mutant embryos exhibited leukocyte recruitment in the skin at 24 hpf in absence of cell death, only
upon 48 hpf, apoptosis increased moderately. Impaired barrier function and enhanced infection
susceptibility in the periderm layer turned out to be responsible of innate immune cells recruitment
(Slanchev et al., 2009).
OBJECTIVES
Objectives
47
The specific objectives of the present work are:
1. Characterize the role played by NAD+ and PAR metabolism in chronic skin
inflammation.
2. Study the influence of pharmacological inhibition of Nampt and Parp1 in oxidative
stress, skin neutrophil infiltration, skin inflammation, PARylation, keratinocyte
proliferation, DNA damage and cell death in zebrafish models of chronic skin
inflammation.
3. Evaluate the effect of ROS scavengers and prevention of parthanatos cell death in
zebrafish models of chronic skin inflammation.
4. Study the involvement of NAD+ and PAR metabolism in human chronic skin
inflammatory diseases.
MATERIALS AND METHODS
Materials and methods
51
1. Animals
Wild-type zebrafish (Danio rerio) lines AB, TL and WIK obtained from the Zebrafish International
Resource Center (ZIRC) were used and handled according to the zebrafish handbook (Westerfield,
2000). The transgenic zebrafish line Tg(lyz:dsRED)nz50 (lyz:dsRED for simplicity) contains the genomic
regulatory regions of the zebrafish lysozyme C gene, neutrophil-specific promoter, that controls the
expression of the red fluorescent protein DsRED2 (Hall et al., 2007), and Tg(NFkB-RE:eGFP)sh235Tg
(NF-kB:eGFP for simplicity) (Kanther et al., 2011) were provided by Profs. Phil Crosier and Stephen
A. Renshaw, respectively. The mutant zebrafish lines Tg(spint1a)hi2217 (spint1a/hai mutant for
simplicity) (Amsterdam et al., 1999) and the psoriasism14 (atpb1a mutant for simplicity) (Webb et al.,
2008) were isolated from insertional and ehyl methanesulfonate induced mutagenesis screens,
respectively. Both lines were provided by Prof. Matthias Hammerschmidt.
The experiments performed comply with the Guidelines of the European Union Council (Directive
2010/63/EU) and the Spanish RD 53/2013. Experiments and procedures were performed as
approved by the Bioethical Committees of the University of Murcia (approval numbers #75/2014,
#216/2014 and 395/2017) and Ethical Clinical Research Committee of The University Hospital Virgen
de la Arrixaca (approval number #8/13).
2. Chemical treatments
Zebrafish embryos were manually dechorionated at 24 hpf. Larvae were treated from 24 hpf to
48 hpf or 72 hpf by chemical bath immersion at 28 oC. Incubation was carried out in 6-well plate
containing 20-25 larvae/well in egg water (including 60 µg/mL sea salts in distilled water)
supplemented with 1% dimethyl sulfoxide (DMSO).
3. Imaging of zebrafish larvae
Live imaging of 72 hpf larvae was obtained employing buffered tricaine (200 µg/mL) dissolved in
egg water. Images were capture with an epifluorescence LEICA MZ16FA stereomicroscope set up
with green and red fluorescent filters. All images were acquired with the integrated camera on the
stereomicroscope and were analyzed to determine number of leukocytes (lyz:dsRED) and their
distribution in the larvae. The transcriptional activity of NF-κB was visualized and measured with the
zebrafish line NF-kB:eGFP.
H2O2 imaging was quantified employing the live cell fluorogenic substrate acetyl-
pentafluorobenzene sulphonyl fluorescein (Cayman Chemical) (Candel et al., 2014; de Oliveira et al.,
2014). Briefly, about 20 embryos of 72 hpf were rinse with egg water and collected in a well of a 24-
well plate with 50 µM of the substrate in 1% DMSO for 1 hour.
Materials and methods
52
ImageJ software was employed to determine mean intensity fluorescence of a common region
of interest (ROI) placed in the dorsal skin for H2O2 production quantification. Similarly, a ROI located
in muscle or skin was used to obtain mean intensity fluorescence of NF-kB:eGFP transgenic line.
4. Whole-mount immunohistochemistry
BrdU incorporation assay was used to determine cell proliferation. Embryos of 48 hpf were
incubated in 10 mM of BrdU dissolved in egg water for 3 hours at 28 oC followed by a one-hour wash
out with egg water and fixation in 4% PFA overnight at 4oC or 2 hours at room temperature (RT). For
the rest of immunofluorescence techniques, embryos/larvae were directly fixed in 4%
paraformaldehyde (PFA), as indicated above. Embryos/larvae were then washed with phosphate
buffer saline (PBS) with 0.1% tween-20 (PBST) 3 times for 5 minutes. In order to dehydrate the
sample progressively, 25%/75% methanol (MeOH)/PBST, 50%/50% MeOH/PBST and 75%/25%
MeOH/PBST and 100% MeOH were employed each for 5 minutes. At this point, embryos were
stored at -20 oC until immunofluorescence.
To proceed with the immunofluorescence, samples were re-hydrated in decreasing solutions of
MeOH/PBST, as previously described, and then washed 3 times for 5 minutes with PBST. For BrdU
staining, next step consist on apply 30 minutes a solution of 2N HCl in PBS supplemented with 0.5 %
Triton X-100 (PBSTriton), followed by 3 wash with PBSTriton for 15 minutes. Blocking step was
carried out for at least 2 hours at RT with a PBSTriton solution supplemented with 10 % fetal calf
serum (FBS) and 0.1 % DMSO. The primary antibody incubation in blocking solution was done
overnight at 4oC or 3-4 hours at RT. After that, larvae were wash 6 times for 5 minutes. Incubation
in secondary antibody in blocking solution was performed for 2-3 hours in dark. From then on, the
protocol followed in darkness. In order to remove unbound secondary antibody, embryos were
washed 3 times for 10 minutes with PBT. In this step, the sample was ready for 2-(4-Amidinophenyl)-
6-indolecarbamidine (DAPI) staining, a DAPI solution (1:1000) in PBT for 20 minutes followed by a
wash out step of 3 times for 10 minutes with PBT. Finally, embryos were transferred to 80%
glycerol/20% PBST and stored in dark at 4 oC until imaging.
The following primary antibodies were used: rabbit anti-BrdU (Abcam, ab152095, 1:200), mouse
anti-p63 (Santa Cruz Biotechnology, sc-7255, 1:200), rabbit Anti-Active Caspase-3 (Bd Bioscience,
#559565, 1:250) and rabbit anti-H2AX.XS139ph (phospho Ser139) (GeneTex, GTX127342, 1:200).
Secondary antibodies were goat anti-rabbit Alexa Fluor-488 (Molecular probes, CAT#A11008,
1:1000) and goat anti-mouse Cyanine 3 (Life technologies, A10521, 1:1000). Images for BrdU
staining were taken using a Zeiss Confocal (LSM710 META), the other stains were acquired by ZEISS
Apotome.2. All images were processed using ImageJ software.
Materials and methods
53
5. TUNEL Assay
Emrbyos/larvae were fixed and dehydrate as described above. Afterwards, embryos/larvae were
rinsed with pre-cooled (-20 oC) 100% acetone and then incubated 100% acetone at -20 oC for 10
minutes. Samples were then washed 3 times for 10 minutes with PBST and incubated in a solution
of 0.1% TritonX-100 and 0.1% sodium citrate (10%) in PBS for 15 minutes to further permeabilize
the embryos/larvae. Next step consisted on rinse specimens 2 times for 5 minutes in PBST. Following
completely removed PBST from samples, it was added 50 µL of fresh TUNEL reaction mixture
composed by 5 µL of enzyme solution mixed with 45 µL of labeling solution (In Situ Cell Death
Detection kit, POD, ROCHE, version 15.0) for 1 hour at 37 oC, followed by 5 wash with PBST for 5
minutes. Blocking step was carried out for at least 1 hours at RT with blocking buffer. To proceed
with TUNEL assay, blocking buffer was removed and added 50 µL Converted-POD (anti-fluorescein
antibody conjugated to peroxidase) for 1 hour at room temperature or overnight at 4 oC on rocker.
Embryo were rinsed 4 times for 30 minutes in PBST and incubated in 1 mL of 3,3´-Diaminobenzidine
(DAB) solution for 30 minutes in the dark and transferred to a 24 well-plate. From then on, the
protocol followed in darkness. Two µL of a fresh 0.3% H2O2 solution was added to initiate peroxidase
reaction that was monitored 10-20 minutes followed by rinsing and a wash out step of 2 times for 5
minutes with PBST. Finally, embryos were transferred to 80% glycerol/20% PBST and stored in dark
at 4 oC until imaging. Images were acquired by ZEISS Apotome.2 and processed using ImageJ
software.
6. Comet assay
Zebrafish embryos at 48 hpf were anesthetized in tricaine (200 µg/mL) dissolved in egg water
and the end of the fin fold was amputated with a scalpel. Tissues collected from around 60 embryos
were pooled, then spin and resuspended in 1 mL PBS. Liberase at 1:65/volume of PBS (Roche,
cat # 05401119001) was added and tissues were incubated at 28 oC for 35 minutes, pipetting up and
down every 5 minutes. To stop the reaction, FBS was added to a final concentration of 5% in PBS.
From now on, samples were kept on ice. Disaggregated fin folds were filtered through a 40 µM filter
and washed using PBS + 5% FBS. Cell suspension was centrifuged at 650xg for 5 minutes and
resuspended in 50 µL of PBS + 5% FBS. In order to determine cell number, Trypan Blue-treated cell
suspension was applied to Neubauer chamber and cell were counted in an inverted microscope.
Around 15.000 cells were employed to perform the Alkaline Comet Assay according to the
manufacturer's protocol (Trevigen). Briefly, cells were added in low melting point agarose at 37 oC
at a ratio of 1:10 (v/v) and then were placed onto microscope slides. After adhesion at 4 °C for 30
Materials and methods
54
minutes in dark, slides were immersed in lysis buffer (precooled at 4 °C) overnight at 4 °C. Next, DNA
was unwound in alkaline electrophoresis solution pH>13 (200 mM NaOH, 1 mM EDTA) at room
temperature for 20 min in dark, followed by electrophoresis run in the same buffer at 25 V (adjusting
the current to 300 mA) for 30 minutes. Slides were washed twice in distilled water for 5 minutes
and in 70% ethanol for 5 minutes, then they were dried at 37 °C for 30 minutes. Finally, DNA was
stained with SYBR™ Green I Nucleic Acid Gel Stain 10,000X (Invitrogen) and images were taken using
a Nikon Eclipse TS2 microscope with 10x objective lens. Quantitative analysis of the tail moment
(product of the tail length and percent tail DNA) was obtained using CASPLAB software. More than
100 randomly selected cells were quantified per sample. Values were represented as the median of
the tail moment of treated cells relative to the median of the tail moment of untreated cells.
7. Western blot
Zebrafish embryos at 72 hpf were anesthetized in tricaine (200 µg/mL) dissolved in egg water
and the end of the fin fold was amputated with a scalpel. Tissues collected from around 120 embryos
were pooled, then spin and resuspended in 80 µL of 10 mM Tris pH 7.4 + 1% Sodium Dodecyl Sulfate
(SDS). Samples were then incubated at 95 oC for 5 min with 1400 rpm agitation, followed by
maximum speed centrifugation for 5 min. Supernatants were frozen at – 20 oC until proceeding. BCA
kit was employed to quantify protein using BSA as a standard. Fin lysates (10 μg) in SDS sample
buffer were subjected to electrophoresis on a polyacrylamide gel and transferred to PVDF
membranes. The membranes were incubated for 1 h 30 min with TTBS containing 5% (w/v) skimmed
dry milk powder and immunoblotted in the same buffer 16 h at 4 oC with the mouse monoclonal
antibody to human poly(ADP-ribose) (1/400, ALX-804-220, Enzo). The blot was then washed with
TTBS and incubated for 1 h at room temperature with secondary HRP-conjugated antibody diluted
2500-fold in 5% (w/v) skimmed milk in TTBS. After repeated washes, the signal was detected with
the enhanced chemiluminescence reagent and ChemiDoc XRS Biorad.
8. Total NAD+ & NADH determination
Zebrafish embryos at 72 hpf were anesthetized in tricaine (200 µg/mL) dissolved in cold PBS in
order to amputate the tail at the end of the yolk sac extension with a scalpel. Tissues from around
120 embryos were pooled and collected in lysis buffer provided by the kit (Total NAD and NADH
Assay Kit, ab186032) according to the manufacturer's protocol (Abcam). Tissues were homogenized
and centrifuged at 1400 rpm for 5 minutes at 4 oC. Supernatants were collected and centrifuged at
Materials and methods
55
maximum speed for 10 minutes at 4 oC. Supernatants were employed to protein quantification with
BCA kit using BSA as a standard. To proceed with Total NAD and NADH determination, 50 µg of
protein were employed.
9. Gene Expression Omnibus (GEO) database
Human psoriasis (accession number: GSD4602) and atopic dermatitis (accession number:
GSE57225) transcriptomic data collected in the GEO database were used to analyze the differential
gene expression and correlation analysis.
10. Immunohistochemistry in human skin samples
Skin biopsies from healthy donors (n = 5) and psoriasis patients (n = 6) were fixed in 4% PFA,
embedded in Paraplast Plus, and sectioned at a thickness of 5 µm. After being dewaxed and
rehydrated, the sections were incubated in 10 mM citrate buffer (pH 6) at 95 oC for 30 min and then
at room temperature for 20 min to retrieve the antigen. Afterwards, steps to block endogenous
peroxidase activity and nonspecific binding were performed. Then, sections were immunostained
with a 1 1/100 dilution of mouse monoclonal antibodies to NAMPT (sc-166946, Santa Cruz
Biotechnology) a poly (ADP-ribose) (ALX-804-220; Enzo Life Sciences) followed by 1/100 dilution of
biotinylated secondary antibody followed by ImmunoCruz® goat ABC Staining System (sc-2023,
Santa Cruz Biotechnology) according to manufacturer’s recommendations. Finally, after DAB
staining solution was added, sections were dehydrated, cleared and mounted in Neo-Mount. No
staining was observed when primary antibody was omitted. Sections were finally examined under a
Leica microscope equipped with a digital camera Leica DFC 280, and the photographs were
processed with Leica QWin Pro software.
11. HPLC-MS
Human serum samples were filtered with AMICON™ ULTRA 0.5 mL centrifugal filters 3 KDa
cutoff (UFC500396; EMD Millipore) and supplemented with N-Acetyl-Glutamine at 1 mM as an
internal standard. Samples were injected in an Agilent 6550 Q-TOF Mass Spectrometer (Agilent
Technologies, Santa Clara, CA, USA) using an Agilent Jet Stream Dual electrospray (AJS-Dual ESI)
interface. The mass spectrometer was operated in the positive mode. Standards were analyzed in
the range 1000-1 nM. The peak area data of standards were used for the calculation of the
calibration curve, from which the concentration of both compounds in samples was obtained.
Materials and methods
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12. Statistical analysis
Data were analyzed by analysis of variance (ANOVA) and a Tukey multiple range test to
determine differences between groups with gaussian data distribution. Non-parametric data were
analyzed by Kruskal-Wallis test and Dunn's multiple comparisons test. The differences between two
samples were analyzed by the Student t-test. The contingency graphs were analyzed by the Chi-
square (and Fisher’s exact) test and correlation studies with Pearson's correlation coefficient.
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1. NAD+ and its precursors contribute to skin inflammation
In the absence of recruitment stimuli, most neutrophils of 72 hpf zebrafish embryos were located
between the dorsal aorta and axial vein, a region denominated caudal hematopoietic tissue (CHT)
(Murayama et al., 2006) (Figure 12A).
In order to determine if NAD+ metabolism has a role in the regulation of skin inflammation, we
decided to perform functional experiments in the transgenic zebrafish line lyz:dsRED. Manually
dechorionated lyz:dsRED larvae were treated by bath immersion with different concentrations of
NAD+ from 24 hpf to 72 hpf. Compound incubation resulted in a statistically significant increased
neutrophil dispersion from CHT compared to control (Figure 12B). Despite the altered pattern of
neutrophil distribution, some of which were present in the skin, both the integrity of the skin and
its morphology were not affected (Figure 12B´).
Figure 12. NAD+ treatment alters neutrophil distribution pattern. Schematic representation of a 3 dpf zebrafish larvae. Red dots represent neutrophils mostly positioned at CHT, highlighted with a green box (A). Quantification of the percentage of neutrophils out of the CHT in 3 dpf embryos treated 48 hours with NAD+ (0.25, 0.5 and 1 mM), considering the red box describe in A as the limits of the CHT (B). Representative merge images (brightfield and red channels) of lyz:dsRED zebrafish larvae of every group are shown (B´). The mean ± S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test *p≤0.05, ****p≤0.0001.
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Given the role of H2O2 in driving neutrophil mobilization to acute (Niethammer et al., 2009) and
chronic (Candel et al., 2014) insults, we used a H2O2 specific fluorescent probe to know if this
molecule was implicated in the observed phenotype. Fluorescent probe indicated that NAD+
treatment was able to enhance H2O2 production by skin keratinocytes in a dose-dependent manner,
compared to the control group (Figure 13A). Similar results were obtained with NAM, a well-known
NAD+ booster (Rajman et al., 2018). However, while NMN precursor was unable to increase skin
oxidative stress by itself, NAM and NMN combination synergistically induce the same effect as NAM
alone (Figure 13B). Nevertheless, no differences in neutrophil redistribution were observed (Figure
13A´ & B´). These results might suggest that NAD+ levels could regulate oxidative stress in the skin.
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Figure 13. NAD+ and their precursors induce skin oxidative stress. For H2O2 imaging, embryos of 72 hpf treated 48 hours with NAD+ metabolites were incubated in 50 µM of acetyl-pentafluorobenzene sulphonyl fluorescein solution for 1 hour. Quantification of fluorescence intensity for NAD+-mediated (A) and NAM-/NMN-mediated (B) induction of H2O2 in the zebrafish skin. Representative merge images (brightfield and red channels) of lyz:dsRED zebrafish larvae of every group are shown (A´ & B´). The mean ± S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test ****p≤0.0001.
2. Pharmacological inhibition of Nampt induce muscle inflammation
Once demonstrated the influence of NAD+ in the regulation of skin oxidative stress and
neutrophil scattering, we wondered if the depletion of cellular NAD+ mediated by the well-
characterize NAMPT inhibitor FK-866 (Hasmann & Schemainda, 2003) could also have an impact on
these parameters. In order to study inflammation in zebrafish larvae, we used the transgenic line
NF-kB:eGFP, which accurately report NFKB transcriptional activity by GFP expression (Figure 14A´).
Strikingly, FK-866 treatment at 100 µM for 48 hours promoted a considerable muscle inflammation
compared to control group, probably caused by disruption of cellular bioenergetics (Figure 14A &
A´).
Figure 14. High concentrations of FK-866 triggers muscle inflammation. Quantification of fluorescence intensity in 3 dpf embryos treated 48 hours with increasing doses of FK-866 (1, 10 and 100 µM) (A). Representative images (green channel) of NF-kB:eGFP zebrafish larvae of every group are shown (A´). The mean ± S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test ****p≤0.0001.
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Despite the NAD+ induction of skin oxidative stress, no differences were observed regarding NFKB
transcriptional activity in any tissue. As expected, NAD+ effectively restored muscle homeostasis
when embryos were co-incubated with FK-866 (Figure 15A & A´). This result clearly demonstrates
the specific effect of the inhibitor.
Figure 15. NAD+ reverses the induction of NFKB in the muscle by high concentrations of FK-866. Quantification of fluorescence intensity in 72 hpf embryos treated 48 hours with 1 mM NAD+ in the presence or absence of 100 µM FK-866 (A). Representative merge images (brightfield and green channel) of NF-kB:eGFP zebrafish larvae of every group are shown (A´). The mean ± S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test ****p≤0.0001.
Considering the augmented NFKB transcriptional activity in muscle and its role driving
inflammatory response, we wanted to determine if neutrophil distribution was affected in that
condition. The experiments in lyz:dsRED zebrafish line with FK-866 inhibitor revealed robust
neutrophil infiltration in the inflamed tissue treated with 100 µM, compared with untreated
embryos or exposed to lower concentrations (Figure 16A). At the highest inhibitor concentration
tested, neutrophils were regularly distributed in muscle (Figure 16C). H2O2 production by skin
keratinocytes was dramatically changed upon inhibitor treatment (Figure 16B). Of note, the lowest
FK-866 concentration was able to provoke an almost complete abolishment of H2O2 release.
Collectively, these results suggest that not only NAD+ levels vitally regulate oxidative stress in the
skin and neutrophil pattern distribution, but also critically low levels of NAD+ trigger muscle
inflammation.
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Figure 16. FK-866 reduces oxidative stress in the skin. Neutrophil distribution in zebrafish embryos of 3 dpf treated 48 hours with FK-866 (A), quantification of skin H2O2 production (B) and representative merge images (green and red channel) of lyz:dsRED zebrafish larvae of every group are shown (C). The mean ± S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test *p≤0.05, ****p≤0.0001.
3. Pharmacological inhibition of Nampt diminishes oxidative stress and skin inflammation, and
restores epithelial integrity in a zebrafish psoriasis model
Recently, various zebrafish mutants in key genes that encode or regulate interconnecting
anchoring structures that mediates stable association of dermis and epidermis have been described.
These mutants with defective skin epithelial integrity show persistent leukocyte skin infiltration, a
hallmark of psoriasis and other skin inflammatory diseases (Carney et al., 2007; Dodd et al., 2009;
Hatzold et al., 2016; Mathias et al., 2007; Slanchev et al., 2009; Sonawane et al., 2005).
The influence of NAD+ metabolism on oxidative stress and inflammation in wild type zebrafish,
encouraged us to study its effect on the zebrafish psoriasis model with an hypomorphic mutation of
spint1a (allele hi2217), which encodes the serine protease inhibitor, kunitz-type, 1a. In order to
study neutrophil distribution on this mutant, we used the lyz:dsRED transgenic line.
Studies in spint1a mutant demonstrated increased H2O2 release in the skin compared with
control animals (Figure 17A), suggesting a putative contribution of oxidative stress to the mutant
phenotype. As previously described for wild type embryos, pharmacological inhibition of Nampt
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remarkably decreased H2O2 production in a dose-dependent manner (Figure 17A). Surprisingly, H2O2
levels in treated larvae were much lower than in their untreated counterparts, demonstrating the
crucial dependence of H2O2 production by NAD+ metabolism and a probable physiological role for
H2O2 in the skin in steady state conditions.
Spint1a deficient larvae show neutrophil infiltration in the skin (Carney et al., 2007; Mathias et
al., 2007). We observed that 40% neutrophils were out of the CHT in the mutants compared to 10%
in wild type embryos (Figure 17B). Mutant embryos treated with FK-866 inhibitor at 10 and 50 µM
displayed a strong reduction of neutrophil dispersion (Figure 17B & C). However, at 100 µM of FK-
866 resulted in infiltration of neutrophils into the muscle, as observed in wild type animals (Figure
17B & C). More importantly, epithelial integrity was almost completely restored in mutant larvae
treated with 10 µM FK-866 (Figure 17C).
Figure 17. Pharmacological inhibition of Nampt diminishes oxidative stress and skin inflammation and restores epithelial integrity in spint1a mutant. Neutrophil distribution of zebrafish embryos of 3 dpf treated 48 hours with FK-866 (10, 50 and 100 µM) (A), quantification of skin H2O2 production (B) and representative merge images (brightfield and red channel) of lyz:dsRED zebrafish larvae of every group are shown (C). The mean ± S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test **p≤0.01, ***p≤0.001, ****p≤0.0001.
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Additional experiments in spint1a mutant; lyz:dsRED larvae confirmed that NAD+
supplementation exerted a negative effect . On the one hand, NAD+ aggravates the mutant
phenotype, causing further skin morphology alterations and neutrophil scattering pattern
compared with control (Figure 18A´). It is worthy to mention that spint1a mutant were more
susceptible to NAD+ than wild type embryos (Figure 12). In addition, NAD+ treatment neutralized the
beneficial effects of FK-866 on spint1a mutant, worsening the phenotype (Figure 18A & A´).
Therefore, this result supports that the beneficial effects of FK-866 on the skin were mediated by
reducing skin NAD+ availability.
Figure 18. NAD+ intensifies spint1a mutant phenotype and interferes with FK-866 beneficial effects in the skin. Neutrophil distribution of 3 dpf zebrafish embryos treated 48 hours with 1 mM NAD+ in the presence or absence of 10 µM FK-866 (A). Representative merge images (brightfield and red channel) of lyz:dsRED zebrafish larvae of every group are shown (A´). The mean ± S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test ***p≤0.001, ****p≤0.0001.
At this point, we wondered if we were manipulating NAD+ levels in larvae as expected by the
treatments applied to zebrafish embryos. For that purpose, we quantified by ELISA total NAD+ and
NADH levels. The results indicated that FK-866 and NAD+ supplementation, decreased and increased
NAD+ and NADH levels, respectively, as expected. Furthermore, no statistically significant
differences between wild type and spint1a mutant embryos were found (Figure 19).
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Figure 19. Total NAD+ and NADH levels were efficiently modulated by treatments with FK-866 and NAD+. Wild type and spint1a mutant embryos of 72 hpf treated for 2 days with 10 µM FK-866 and 1 mM NAD+ were used for total NAD+ and NADH determination by ELISA. The mean ± S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test **p≤0.01.
In order to study NFKB transcriptional activity on spint1a mutant, we used the reporter line NF-
kB:eGFP. In agreement with previous studies in which increased NFKB transcriptional activity
(upregulation of il1b and mmp9, among other NFKB target genes) was found in spint1a mutant
larvae (LeBert et al., 2015), we demonstrated that this pathway was induced in the skin of mutants
compared with wild type animals (Figure 20A & A´). Importantly, mutant embryos treated with FK-
866 inhibitor at 10 µM displayed a statistically significant reduction in the activation of this signaling
pathway (Figure 20A & A´).
Figure 20. FK-866 rescues enhanced skin NFKB transcriptional activity on spint1a mutants. Quantification of fluorescence intensity of wild type and spint1a mutant embryos of 72 hpf treated for 2 days with 10 µM FK-866 (A). Representative images (green channel) of NF-kB:eGFP zebrafish larvae of every group are shown (A´). The mean ± S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test **p≤0.01, ****p≤0.0001.
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In conclusion, it has been demonstrated that NAD+ metabolism remarkably influences the
spint1a mutant phenotype. While NAD+ supplementation exacerbated mutant phenotype, FK-866-
mediated NAD+ depletion counteracts increased oxidative stress and NFKB transcriptional activity in
the skin and neutrophil infiltration, characteristic of the spint1a mutant. More importantly, FK-866
treatment dramatically improves skin epithelial integrity.
4. Parp1 activity inhibition rescues skin inflammation of spint1a mutant larvae
Due to the pleiotropic roles of the NAD+ molecule that participates in more than 500 enzymatic
reactions and regulates several cellular processes (Rajman et al., 2018), we analyzed the main
enzymes whose activity could be affected by the depletion of NAD+. Given that major influence in
NAD+ levels in the organism is performed by CD38 (Cantó et al., 2015), we decided to inhibit its
enzymatic activity by a specific inhibitor named 78c (Haffner et al., 2015). Similarly, we were
interested in sirtuin activity inhibition, since sirtuins are well established NAD+-consuming enzymes
(Cantó et al., 2015). For that purpose, we employed a selective inhibitor of SIRT1 over SIRT2 and
SIRT3, called EX 527 (Hixon et al., 2007). However, upon different concentrations and experimental
settings tested, we found that neither cd38 nor sirtuin enzymatic activity inhibition were able to
rescue the skin inflammation of spint1a mutant larvae (data not shown).
Next, we studied another NAD+-consuming protein family, Poly(ADP-Ribose) polymerases
(PARPs). Surprisingly, Parp1 inhibition mediated by olaparib, efficiently blocked spint1a mutant
phenotype (Figure 21). Olaparib treatment at 100 µM for 48 hours diminished neutrophil dispersion
(Figure 21A and A´), skin inflammation (Figure 21B and B´) and restored epithelial integrity
compared with control animals (Figure 21A´). Consistently, additional experiments using the Parp1
inhibitors veliparib and talazoparib showed similar results (Figure 21C and C´). Parp1 inhibitors show
different catalytic inhibition and PARP trapping efficiency. Although talazoparib and olaparib display
similar enzymatic activity inhibition potency, talazoparib is close to 100 times more effective at
trapping PARP1 with DNA. In the case of veliparib, this inhibitor is the less potent in both activities
(Kukolj et al., 2017). This observation could be the rational for the different concentrations able to
rescue skin inflammation in spint1a mutant larvae: 500 µM veliparib > 100 µM olaparib >1 µM
talazoparib.
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Figure 21. Parp1 pharmacological inhibitors reduce neutrophil scattering and skin inflammation and reestablish epithelial integrity. Wild type and spint1a mutant embryos of 72 hpf treated for 2 days with olaparib (A & B), veliparib or talazoparib (C). Quantification of neutrophil dispersion out of the CHT (A & C) and NFKB transcriptional activity in the skin (B). Representative images (brightfield and red channel in A´ & C´; green channel in B´) of lyz:dsRED and NF-kB:eGFP zebrafish larvae of every group are shown. The mean ± S.E.M. for each group is also shown. P values were calculated using one-way ANOVA and Tukey multiple range test *p≤0.05, ****p≤0.0001.
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In other to determine Parp activity in skin and the effect of the inhibitors, PAR levels were
determined by western blot in the end of the fin fold (skin-enriched sample). The results indicated
that spint1a mutant showed increased PAR levels (Cortes et al., 2004) compared with control or
inhibitors treated animals. Both FK-866 and olaparib were able to reduce PAR levels in the skin of
spint1a mutants (Figure 22).
Figure 22. The high levels of PAR in the skin of spint1a mutant are counteracted by FK-866 and olaparib. Representative western blot with anti-PAR and anti-Gapdh of fin fold lysates (highlighted in red) from 3 dpf wild type and spint1a mutant zebrafish embryos treated for 48 hours with 10 µM FK-866 or 100 µM Olaparib.
5. FK-866 and olaparib block keratinocyte hyperproliferation in spint1a mutant larvae
spint1a mutant phenotype starts with basal keratinocytes aggregation, mesenchymal-like
properties acquisition and cell death, finally leading to uncontrolled proliferation (Carney et al.,
2007; Mathias et al., 2007). All together lead to integrity disruption of the skin epithelia. Given that
Nampt or Parp1 inhibition in spint1a mutant embryos alleviated the phenotype, it is tempting to
speculate that they interfere with these alterations.
Since proliferation in the spint1a mutant occurs at 48 hpf we decided to carry out a BrdU
incorporation assay at that age. As described in the characterization of the mutant, spint1a mutant
showed increased keratinocyte proliferation compared with wild type siblings (Figure 23A & A´).
Surprisingly, as early as 24 hours of treatment, either Nampt or Parp1 pharmacological inhibition
significantly reduced keratinocyte proliferation (Figure 23A & A´).
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Figure 23. Nampt or Parp1 pharmacological inhibition significantly reduces keratinocyte hyperproliferation in spint1a mutant. Quantification of BrdU positive cells from 48 hpf wild type and spint1a mutant zebrafish embryos treated for 24 hours with 10 µM FK-866 or 100 µM olaparib (A). Representative merge images maximum intensity projection of a confocal Z stack from zebrafish larvae of every group are shown (A´). WIHC with anti-BrdU (green), anti-p63 (red, basal keratinocyte marker) were counterstained with DAPI (blue). The mean ± S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test ****p≤0.0001.
6. spint1a mutants displays higher DNA damage, which is induced by Parp1 inhibition while
reduced by Nampt inhibition
The main PARP1 biological function is to orchestrate spatio-temporal reparation of DNA damage,
being indispensable for SSBR and participating in HR (Schreiber et al., 2006). PARP inhibitors
entrapping and accumulating inactive PARP1 on DNA trigger dsBs formation during replication
(Kujolj et al., 2017).
In order to study DNA damage, we took two different approaches. One approach consists on
analyzing the presence of phosphorylated histone variant H2AX (pH2Ax), which label dsBs. This
epigenetic mark is mediated by the activation of HR-related proteins, independently of PARP1
(Schreiber et al., 2006). The other approach was the comet assay, which under alkaline conditions
can detect both ssBs and dsBs, allowing a deeper analysis (Pu et al., 2015).
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Strikingly, the results indicated that spint1a mutants exhibited higher DNA damage, assayed by
either pH2Ax staining (Figure 24A and A´) or by comet assay (Figure 24B). In line with previous
studies, Parp1 inhibitor olaparib induced DNA damage in spint1a mutants and their wild type siblings
(Figure 24). Remarkably, olaparib-induced DNA lesions were higher in spint1a mutant suggesting an
increased susceptibility to this compound (Figure 24A). Another interesting result was the reduced
pH2Ax staining mediated by FK-866 treatment (Figure 24A). Taking together, the DNA damage
analysis results suggest that spint1a mutants accumulate DNA breaks, what probably increase their
susceptibility to DNA stressors. The reduction in DNA damage mediated by FK-866 treatment could
be explained by the indirect inhibition of Parp activity and the subsequent rescue of the spint1a
mutant phenotype.
Figure 24. DNA damage analysis reveals increased susceptibility and a higher number of lesions
in the skin of spint1a mutant larvae. Quantification of pH2Ax positive cells from 48 hpf wild type and spint1a mutant zebrafish embryos treated for 24 hours with 10 µM FK-866 or 100 µM olaparib (A). Similarly, around 60 fish fin folds were amputated and disaggregated into cells for comet assay analysis in alkaline conditions (B). Representative merge images of maximum intensity projection of
an apotome Z stack from zebrafish larvae of every group are shown (A´). WIHC with anti-pH2Ax (green), anti-p63 (basal keratinocyte marker, red) were counterstained with DAPI (blue). The mean ± S.E.M. (A) and median (B) for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test (A) and Kruskal-Wallis test and Dunn's multiple comparisons test (B) *p≤0.05, **p≤0.01, ****p≤0.0001.
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7. FK-866 and olaparib treatments do not trigger apoptosis in the skin of spint1a mutants
Independently of synthetic lethality phenomena caused by PARP inhibitors when HR pathway is
hampered (Kukolj et al., 2017), PARP inhibitors has also been described to induce apoptosis in
proliferating cells (Schreiber et al., 2006). Apoptotic factors regulated by p53 trigger cytochrome c
release from mitochondria and subsequent caspases cascade activation, ultimately cleaving caspase
3, among other targets (Schreiber et al., 2006).
Initial studies of spint1a mutants, although they found keratinocyte cell death, they were unable
to block it targeting caspases or pro-apoptotic factors, suggesting an unidentified programmed cell
death (Carney et al., 2007; Mathias et al., 2007). Similarly, cleaved caspase 3 staining in 2 dpf spint1a
mutant embryos or wild type larvae was negative (Figure 25A & A´). In addition, spint1a mutants
treated with FK-866 or olaparib did not show keratinocyte apoptosis (Figure 25A & A´), even at 3 dpf
upon 48 hours of treatment (data not shown).
Figure 25. Pharmacological inhibition of Parp1 or Nampt does not induce apoptosis. Quantification of cleaved caspase 3 positive cells from 48 hpf wild type and spint1a mutant zebrafish embryos treated for 24 hours with 10 µM FK-866 or 100 µM Olaparib (A). Representative merge images of maximum intensity projection of an apotome Z stack from zebrafish larvae of every group are shown (A´). WIHC with anti-cleaved casp3 (green), anti-p63 (basal keratinocyte marker, red) were counterstained with DAPI (blue) (A´). The mean ± S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test.
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8. Parp1 pharmacological inhibition reduces cell death in the skin of spint1a mutant larvae
Since Parp1 inhibition lead to the accumulation of DNA lesions, it is eventually expected to induce
cell death (Schreiber et al., 2006). TUNEL was a second approach to study cell death; this technique
consists on the labelling of exposed 3´OH ends of DNA fragments. Therefore, TUNEL labels ssBs and
dsBs (Zingarelli et al., 2003).
Although apoptosis was not detected, even in cell aggregates, the same experimental setting for
TUNEL staining indicated that cell death occurred at the edge of the embryo tail fin of wild type and
mutant embryos during this phase of development (Figure 26A´). However, the spint1a mutant
larvae displayed a higher number of TUNEL+ cells, while olaparib treatment significantly decreased
cell death (Figure 26A). Somehow, this unexpected result could point out Parp1 as responsible for
cell death in spint1a mutant.
Figure 26. Olaparib blocks cell death in the skin of spint1a mutants. Quantification of TUNEL+ cells from 48 hpf wild type and spint1a mutant zebrafish embryos treated for 24 hours with 100 µM Olaparib (A). Representative images of zebrafish larvae of every group are shown (A´). The mean ± S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test **p≤0.01, ***p≤0.001.
9. ROS scavenging molecules rescue skin inflammation of spint1a mutant larvae
The results mentioned above showed higher levels of ROS in the skin of spint1a mutants, which
together the accumulation of DNA lesions, suggests that oxidative stress could be playing an
important role in the development of the phenotype. Importantly, it has been hypothesized that
cell death might be responsible for immune cell recruitment in spint1a mutants (Carney et al., 2007).
In first place, we decided to inhibit Duox1-derived H2O2. The inhibition of this enzyme by the
NADPH oxidase inhibitor dibenziodolium chloride (DPI) successfully diminishes skin oxidative stress
and rescues a zebrafish skin chronic inflammatory model (Candel et al., 2014). However, upon
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different concentrations and experimental settings tested for this inhibitor, we concluded that
Duox1 does not contribute to the spint1a mutant skin phenotype (data not shown). Similarly,
genetic inactivation of Duox1 in skin keratinocytes by overexpressing a dominant negative form of
Duox1 (de Oliveira, et al., 2014), also failed to rescue skin inflammation on spint1a mutants (data
not shown).
Then, we tested the effect of ROS scavengers. Firstly, we employed N- acetylcysteine (NAC), a
reduced glutathione (GSH) precursor, the main cellular antioxidant molecule (Wu et al., 2015).
Mutant embryos treated from 1 dpf to 3 dpf with 100 µM NAC displayed a statistically significant
reduction in the neutrophil dispersion of spint1a mutant larvae (Figure 27A), coupled with an
improvement in skin morphology compared to untreated mutant embryos (Figure 27A´). NAC
beneficial effects stimulated us to analyze the effectiveness of other antioxidants. We next tested
mito-TEMPO, which is an antioxidant that specifically accumulates in the mitochondria imitating
superoxide dismutase activity against superoxide and alkyl radical (Ni et al., 2016). 100 µM mito-
TEMPO applied during 48 hours to spint1a mutant embryos diminished neutrophils dispersion
(Figure 27B) and restored epithelial integrity (Figure 27B´). Finally, we assayed a last ROS scavenger
named tempol, a nitroxide antioxidant that acts against the peroxynitrite decomposition
compounds, nitrogen dioxide and superoxide radical anion (Mustafa et al., 2015). Similarly, to NAC
and mito-TEMPO, as low as 100 nM tempol reduced neutrophil skin infiltration (Figure 27B) and
restored skin integrity (Figure 27B´). NFKB transcriptional activity of spint1a mutant treated with
mito-TEMPO and tempol showed that although tempol displayed a statistically significant reduction
in the activation of the pathway, surprisingly, mito-TEMPO did not rescue this feature of the spint1a
mutant phenotype (Figure 27C and C´). Collectively, these results strongly suggest that Duox1 or
other NADPH-oxidases susceptible to be inhibited by DPI does not contribute to spint1a mutant
phenotype. However, the general ROS scavenger NAC and mito-TEMPO and tempol that target
superoxide anion, among other ROS, demonstrated the influence of oxidative stress on spint1a
mutant phenotype.
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Figure 27. The antioxidants NAC, mito-TEMPO and tempol rescue skin neutrophil recruitment and skin morphology of spint1a mutant larvae. Wild type and spint1a mutant embryos of 72 hpf treated for 2 days with 100 µM NAC (A), 100 µM mito-TEMPO and 100 nM tempol (B & C). Quantification of neutrophil dispersion out of CHT (A & B) and NFKB transcriptional activity in the skin (C). Representative images (brightfield and red channel in A´ & B´; green channel in C´) of lyz:dsRED and NF-kB:eGFP zebrafish larvae of every group are shown. The mean ± S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test. ns, not significant. ****p≤0.0001.
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10. Nitric oxide synthesis inhibition does not affect skin inflammation of spint1a mutant larvae
Stimulated by the outstanding results regarding ROS scavengers, we wanted to investigate if
the reactive nitrogen specie, nitric oxide (NO), may also contribute to the mutant pathology. NO is
synthesize from L-arginine and NADPH by nitric oxide synthetase (endothelial, neuronal or
inducible/inflammatory NOS) and, if it is present, rapidly react with superoxide anion to from
peroxynitrite. NG-monomethyl-L-arginine (NMMA) is an inhibitor of the 3 NOS isoforms (Vallance &
Leiper, 2002). Unexpectedly, NMMA treatment at 1 mM for 48 hours failed to diminish skin
neutrophil infiltration or epithelial integrity compared with control animals (Figure 28A & A´).
Figure 28. NMMA does not alter skin neutrophil infiltration in spint1a mutant larvae. Measurement of neutrophil distribution of 3 dpf zebrafish wild type and spint1a mutant embryos treated 48 hours with 1 mM NMMA (A). Representative merge images (brightfield and red channel) of lyz:dsRED zebrafish larvae of every group are shown(A´). The mean ± S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test ns, not significant, ****p≤0.0001.
11. Parthanatos cell death inhibition rescues skin inflammation of spint1a mutant larvae
According to this work and published data, zebrafish spint1a mutant phenotype is
characterized by skin oxidative stress (involving H2O2), skin inflammation, keratinocyte aggregation,
hyperproliferation and subsequent cell death (unidentified) and DNA damage (Carney et al., 2007;
Mathias et al., 2007).
On the one hand, regarding oxidative stress, hydrogen peroxide can react with metals yielding
hydroxyl radical (●OH) (Poprac et al., 2017). Peroxynitrite and hydroxyl radical are recognized as DNA
damage inducers, provoking ssBs (Szabó et al., 1998). ROS scavengers assayed, were able to rescue
skin inflammation of the mutants. Similarly, FK-866 strongly diminished H2O2 production at the skin.
These results, therefore, indicate a crucial role for ROS in the development of the phenotype.
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On the other hand, Parp1 inhibition also improved mutant phenotype in relation to epithelial
integrity, skin NFKB transcriptional activity, neutrophil infiltration, keratinocyte proliferation and,
more importantly, reduced PAR activity and cell death. These last results, coupled with the
uncharacterized keratinocyte cell death, led us to hypothesize that parthanatos would be
responsible for keratinocyte cell death in spint1a mutants. Parthanatos is a PARP1-dependent cell
death in which extensive DNA damage induces PARP1 overactivation. Accumulation of PAR
polymers and PARylated proteins reach the mitochondria causing depolarization of the membrane
potential and apoptosis-inducing factor (AIFM1) release (Fatokun et al., 2014; Galluzzi et al., 2018).
AIFM1 is released into the cytosol, where it recruits macrophage migration inhibitory factor (MIF)
to the nucleus. In the nucleus AIFM1-MIF nuclease activity execute a large-scale DNA fragmentation
resulting in cell death (Wang et al., 2016).
To test this hypothesis, we employed a chemical inhibitor of AIFM1 translocation, named N-
phenylmaleimide (NP) (Susin et al.,1996). Mutant embryos treated from 1 dpf to 3 dpf with 10 nM
NP displayed a statistically significant reduction in neutrophil dispersion (Figure 29A), besides a
restoration of skin integrity compared to untreated mutant embryos (Figure 29A´).
Figure 29. Pharmacological inhibition of Aifm1 translocation reduces skin neutrophil infiltration and improves skin morphology. Measurement of neutrophil distribution of 3 dpf zebrafish embryos treated 48 hours with 10 nM NP(A). Representative merge images (brightfield and red channel) of lyz:dsRED zebrafish larvae of every group are shown (A´). The mean ± S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test ****p≤0.0001.
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12. Pharmacological inhibition of Parp1 or Nampt rescue zebrafish psoriasis mutant
Once demonstrated the effectiveness of FK-866 and olaparib in spint1a mutant, we decided to
use another mutant which presented a similar skin inflammatory phenotype. Our first choice was
clint1a mutant (Dodd et al., 2009). In a similar way to spint1a mutant, at 48 hpf clint1a mutants
displayed keratinocyte hyperproliferation and aggregates, cell death and leukocyte recruitment to
the skin. However, upon several attempts, we found that FK-866 and olaparib were both unable to
restore skin homeostasis in clint1a mutant larvae (data not shown).
Next, we utilized zebrafish psoriasis mutant, whose phenotype features are shared with spint1a
and clint1a mutants; that is, keratinocyte aggregation and hyperproliferation from 2.5 dpf, cell
death within or near epidermal aggregates, and immune cells infiltration to disrupted skin
epithelium (Hatzold et al., 2016; Webb et al., 2008). Unfortunately, we did not have psoriasis mutant
in lyz:dsRED background. Therefore, we applied a phenotype assessment method to determine
different outputs to inhibitor treatment. Consistently with spint1a mutant results, 50 µM FK-866
and 100 µM olaparib applied during 1.5 days to zebrafish embryos significantly improved skin
epithelial integrity (Figure 30A & A´). This result might suggest that although spint1a and psoriasis
mutant phenotype is caused by mutations affecting different genes, apparently with very different
biological function, they develop a similar phenotype that share similar altered metabolic pathways.
Figure 30. FK-866 and olaparib improves skin epithelial integrity in psoriasis mutants.
Determination of the skin phenotype of 2.5 dpf zebrafish embryos treated 1.5 days with 50 µM FK-866 or 100 µM olaparib (A). Representative bright field images of zebrafish larvae of every group are shown(A´). The mean ± S.E.M. for each group is shown. P values were calculated using Chi-square and Fisher´s exact test *p≤0.05, ****p≤0.0001.
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13. Altered expression profile of genes encoding key NAD+ metabolic enzymes in atopic
dermatitis and psoriasis
Psoriasis and atopic dermatitis, or eczema, are two non-contagious skin chronic inflammatory
diseases (Furue & Kadono, 2017). Although environmental factors act as triggers of the pathologies
(Dainichi et al., 2018), psoriasis and atopic dermatitis have a strong genetic component affecting
individual susceptibility (Greb et al., 2016; Weidinger et al., 2018). In order to study if NAD+
metabolism is affected in psoriasis and atopic dermatitis, probably paying a role in the pathologies,
we analyzed transcriptomic data of enzymes implicated in NAD+ metabolism.
Primarily, we studied NAD+ salvage pathway (carried out by NAMPT and NMNAT1-3), generally
used to keep intracellular NAD+ levels in a great variety of tissues (Cantó et al., 2015; Rajman et al.,
2018). This pathway is fueled by Preiss-Handler pathway (participating NAPRT and NADSYN), de
novo pathway (mediated by IDO1, TDO2 and QPRT, among others) (Cantó et al., 2015) and
nicotinamide riboside (NR) conversion (catalyzed by PNP and NRK1/2) (Nikiforov et al., 2015).
Additionally, we were interested in CD38, due to its NADase activity influencing NAD+ levels in the
organism (Cantó et al., 2015) and the NAM-consuming enzyme nicotinamide-N-methyltransferase
(NNMT), which catalyzes the reaction between NAM and S-adenosylmethionine (SAM) to yield N-
methylnicotinamide (1-MNA) and S-adenosylhomocysteine (SAH) (Nikiforov et al., 2015).
In this work we employed a freely available transcriptomic data obtained from human psoriasis
where skin from healthy subjects and psoriasis patients (non-lesional and lesional skin) were
compared (Figure 31A). Analysis revealed an important differential expression profile of genes
encoding NAD+ metabolic enzymes. In general, in psoriatic lesions, transcript levels were
upregulated compared with non-lesional psoriatic skin and healthy samples. Importantly, the mRNA
levels of the gene encoding NAMPT, the rate-limiting enzyme in NAD+ salvage pathway, was
increased, in contrast with the reduction in NMNAT3 levels. Curiously, NRK2 transcript levels also
augmented in lesional skin, at the same level than non-lesional skin, compared with control group.
Preiss-Handler pathway seemed to show and intensified activity, due to the upregulation of mRNA
levels of genes encoding its components NAPRT and NADSYN in psoriasis lesional skin compared to
healthy epithelium. Finally, genes coding for NAD+ biosynthetic enzymes involved in de novo
pathway were also altered: while IDO1 and TDO2 were strongly induced, QPRT slightly decreased
compared to healthy skin (Figure 31A).
We also analyze transcriptomic data from a study of atopic dermatitis. This study compared
skin from healthy donors with skin punch biopsies from lesions of patients suffering from atopic
dermatitis. Atopic dermatitis tissue revealed induced mRNA levels of NAMPT and reduced levels of
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NMNAT3 compared with healthy skin, probably indicating a variation in NAD+ salvage pathway.
However, genes related to Preiss-Handler pathway did not displayed a distinctive expression profile
to control group. Finally, IDO1 and TDO2 involved in NAD+ de novo biosynthesis exhibited increased
transcript levels in comparison with healthy skin (Figure 31B).
Altogether, the data from psoriasis and atopic dematitis gene expression might indicate
increased activity of NAD+ salvage pathway with a common reduction of NMNAT3 expression, which
is in charge of controlling NAD+ levels in the mitochondria (Rajman et al., 2018). Additionally, de
novo biosynthesis of NAD+ could be also enhanced in both diseases, in those tissue in which it is
active. Curiously, Preiss-Handler pathway is only altered in psoriasis, while no differences were
found in atopic dermatitis analysis. In contrast, NNMT expression profile showed enhanced
induction specifically in atopic dermatitis and both shared high levels of CD38 transcript levels
(Figure 31C).
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Figure 31. Differential expression profiles of genes encoding key NAD+ metabolic enzymes in atopic dermatitis and psoriasis. Transcriptomic data from human psoriasis (GDS4602) (A) and atopic dermatitis (GSE57225) (B) samples from the Gene Expression Omnibus (GEO) database. (C) Venn diagram showing common and specific gene expression in lesional skin. The mean ± S.E.M. for each group is 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. Indoleamine 2,3-dioxygenase, IDO1; NA phosphoribosyltransferase, NAPRT; NAD+ synthetase, NADSYN1; NR kinase 1/2, NRK1/2; Purine nucleoside phosphorylase, PNP; Quinolinate phosphoribosyltransferase, QPRT; Tryptophan 2,3-dioxygenase, TDO2.
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14. The expression profile of genes encoding NAD+ metabolic enzymes correlate with
inflammatory gene markers
Once demonstrated that in atopic dermatitis and psoriasis genes encoding some enzymes
related to NAD+ metabolism, especially those involved in NAD+ salvage pathway and NAD+ de novo
biosynthesis, were deregulated, we wondered if their expression could be correlated with
inflammatory gene markers. Specific inflammatory gene markers of each disease were employed to
carry out correlation analysis. For psoriasis, we focused on TH 17 immune response, that synthesizes
IL17A and IL23A (Greb et al., 2016). Furthermore, we included TNFA and IL1B. In the case of atopic
dermatitis, we used cytokines related to TH2 (IL4, IL10 and IL13) and TH12 (IL22) immune responses
(Weidinger et al., 2018), in addition to IL6 and TNFA.
In relation to psoriasis, NAMPT and PNP robustly correlated its expression with IL1B and IL17A,
similarly to IDO1 and TDO2 expression, but with lower significance (Figure 32A). In addition, NRK1
expression slightly correlated with IL23A and importantly, NAPRT showed an inverse correlation
with the expression of this cytokine (Figure 32A). In atopic dermatitis, NAMPT correlated with IL4
and IL6; IDO1 and TDO2 were also linked to IL6 transcript levels and other cytokines (Figure 32B).
Remarkably, NNMT and CD38 mRNA levels showed very important association with those of genes
encoding several cytokines, what could mean relevant correlation with disease severity. In contrast,
NMNAT3 transcript levels displayed an inverse correlation with numerous genes coding for
inflammatory cytokines, probably pointing out NMNAT3 expression as a protective factor (Figure
32B).
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Figure 32. The expression of genes encoding key NAD+ metabolic enzymes correlate with those of specific inflammatory gene markers of psoriasis and atopic dermatitis. Transcriptomic data in lesioned skin from human psoriasis (GDS4602) (A) and atopic dermatitis (GSE57225) (B) samples from the Gene Expression Omnibus (GEO) database were employed to study their correlation with inflammatory marker genes expression. Linear regression for each group is shown. P values were calculated using Pearson's correlation coefficient. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.
15. Altered expression profile of genes encoding enzymes related to parthanatos in atopic
dermatitis and psoriasis
In this work it was demonstrated that Parp inhibition rescues skin inflammation in spint1a and
psoriasis mutant larvae, reducing PAR activity and cell death. Furthermore, chemical inhibition of
Aifm1 translocation provided an improvement in the spint1a mutant phenotype. All these results
together support the idea that keratinocyte cell death that takes place in these animal models and
in human skin chronic inflammatory disorders is mediated by overactivation of PARPs. In order to
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determine if parthanatos components and other related proteins are altered in skin chronic
inflammatory diseases, we employed transcriptomic data experiments mentioned above.
We mainly focused on PARP1, AIFM1 and MIF, three indispensable parthanatos components
(Wang et al., 2016). Additionally, we analyzed the expression of genes encoding different PAR
hydrolases that regulate protein PARylation, and consequently control the levels of free PAR and
PAR bound to proteins, key mediators of mitochondria membrane potential depolarization and
subsequent AIFM1 release (Fatokun et al., 2014).
Psoriasis transcriptomic data revealed strong increased mRNA levels of PARP1, AIFM1 and MIF
in lesional tissue compared with healthy skin and non-lesional psoriatic skin (Figure 33A). In addition,
it was observed slight decreased and increased AIFM1 and MIF levels, respectively, in non-lesional
skin from psoriasis patients (Figure 33A).
In the case of atopic dermatitis samples, mild increased transcript levels of PARP1 and AIFM1
compared to control group (Figure 33B). However, no differential MIF transcript levels were found
(Figure 33B).
Although no alteration in the expression profile of gene encoding PARG was found in psoriasis
or atopic dermatitis, both disorders exhibited decreases levels of MACROD1, MACROD2 and TARG1
mRNAs (Figure 33C). These genes encode proteins that are in charge of cleaving the bond protein-
ADP-ribose, releasing PAR chains (Qi et al., 2019). Furthermore, in psoriatic lesional skin ARH3
(exoglycosilase enzymatic activity) levels increased, while those of NUDT16 and ENPP1 decreased
(Figure 33C). These two genes encode two proteins that cleave the PAR chain from target proteins,
leaving them monoPARylated (Qi et al., 2019). In addition, both diseases shared enhanced ARH1
transcript levels, whose product cleaves the terminal bond but only for targets PARylated on
arginine (Qi et al., 2019) (Figure 33C).
In conclusion, the results indicate that both diseases, more pronounced in psoriasis, display a
reduced ability to remove PARylation from proteins, due to PAR hydrolases downregulation, what
under certain conditions could facilitate parthanatos.
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Figure 33. Differential gene expression profile of parthanatos components and PAR hydrolases in psoriasis and atopic dermatitis. Transcriptomic data from human psoriasis (GDS4602) (A) and atopic dermatitis (GSE57225) (B) samples from the Gene Expression Omnibus (GEO) database. (C) Venn diagram showing common and specific gene expression in lesional skin. The mean ± S.E.M. for each group is 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≤0.0001. ADP-Ribosylhydrolase 3, ARH3; Autosomal Recessive Hypercholesterolemia Protein, ARH1; Ectonucleotide Pyrophosphatase/Phosphodiesterase 1, ENPP; Mono-ADP Ribosylhydrolase 1, MACROD1; Mono-ADP Ribosylhydrolase 2, MACROD2; Nudix Hydrolase 16, NUDT16; Terminal ADP-Ribose Protein Glycohydrolase 1, TARG1.
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16. The expression of genes encoding key components involved in parthanatos and PAR
metabolism correlate with inflammatory gene markers of psoriasis and atopic dermatitis
After confirmation that genes encoding parthanatos components and other factors implicated
in PAR metabolism are deregulated in atopic dermatitis and psoriasis, we wanted to determine if
their expression could be correlated with inflammatory marker genes. In the same line that NAD+
metabolism previously analysed, we took the same specific inflammatory gene marker genes to
carry out the correlation analysis.
Correlation analysis with psoriasis transcriptomic data indicated strong correlation between
the mRNA levels of AIFM1 and those of the inflammatory genes IL1B and IL17A (Figure 34A).
Furthermore, ARH1 expression slightly correlated with IL1B, IL17A and TNFA (Figure 34A). In
contrast, MACROD1 and MACROD2 inversely correlated with IL1B and IL17A and IL1B and IL23A,
respectively (Figure 34A). In atopic dermatitis, PARP1 and AIFM1 positively correlated their
expression with IL13. Similarly, MIF and ARH3 correlated with IL6 and TNFA (Figure 34B). Strikingly,
MACROD1 transcript levels showed robust inverse association with those of several cytokine genes
(Figure 34B). Therefore, in both diseases parthanatos components and other genes related to PAR
metabolism showed associated expression with those of genes encoding cytokines specific of every
disease. Oppositely, MACROD1 and MACROD2 displayed an inverse correlation with inflammatory
marker genes, what probably could indicate a protective role against the pathologies.
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Figure 34. Correlation of the expression of genes involved in parthanatos and PAR metabolism with specific inflammatory gene markers of psoriasis and atopic dermatitis. Transcriptomic data in lesional skin from human psoriasis (GDS4602) (A) and atopic dermatitis (GSE57225) (B) samples from the Gene Expression Omnibus (GEO) database were employed to study their correlation with inflammatory gene marker expression levels. Linear regression for each group is shown. P values were calculated using Pearson's correlation coefficient. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.
17. Human serum levels of NAD+ and SAM are altered in psoriasis patients
We have shown that NAD+ metabolism play a fundamental role in the regulation of oxidative
stress and skin inflammation in a zebrafish preclinical model of psoriasis. In addition, transcriptomic
data displayed altered expression profile of genes encoding key NAD+ metabolic enzymes in atopic
dermatitis and psoriasis. Therefore, we set out to investigate if serum levels of metabolites relative
to NAD+ metabolism could be altered in psoriasis patients. For this purpose, we analyzed by high-
performance liquid chromatography-mass spectrometry (HPLC-MS) serum samples of psoriasis
patients before and after being treated with phototherapy. Phototherapy treatment consist on
psoralen drugs combined with UVA light exposure (PUVA) (Greb et al., 2016).
Metabolites determination by HPLC-MS indicated that those patients who responded properly
to the treatment exhibit reduced levels of NAD+ and SAM before the treatment, compared with
healthy subject and samples from patients that did not improved with phototherapy. Surprisingly,
NAD+ and SAM amounts were normalized to control group after the treatment (Figure 35). It is
worthy to mention that NADH levels of responders patients before and after phototherapy tended
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to be slightly enhanced in comparison with healthy sample, it was not statistically significant, though
(Figure 35). However, the relation between NAD+ and NADH levels clearly showed an imbalance
between them, following the same pattern of recovery described for NAD+ and SAM upon the
treatment (Figure 35).
Reduction in NAD+ levels together with SAM, could suggest increased activity of NNMT, which
catalyzes the reaction between NAM and SAM to yield 1-MNA and SAH. NAM elimination by NNMT
may negatively affect NAD+ pool of salvage pathway. However, no differences were found in NAM
and SAH or 1-MNA levels before or after the treatment. NAD+, NAD+/NADH and/or SAM might be
potential serum biomarkers to predict the response of psoriasis patients to phototherapy.
Figure 35. Recovery of reduced levels of NAD+ and SAM in serum of psoriasis patients treated with PUVA. Measurement by HPLC-MS of serum metabolites of psoriasis patients before and after receiving a phototherapy treatment. The mean ± S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test *p≤0.05, **p≤0.01.
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18. NAMPT and PAR are overexpressed in the nucleus of human keratinocytes from psoriatic
lesions
Encouraged by transcriptomic data of psoriasis lesions indicating overexpression of genes
encoding proteins implicated in the metabolism of NAD+ and PAR, we decided to check if these
alterations could be detected at protein level in lesional skin of psoriasis patients.
Immunohistochemical analysis of samples from healthy skin and psoriasis lesions using an
antibody to human NAMPT, clearly demonstrated that NAMPT was weakly expressed in healthy
epidermis. However, NAMPT was widely overexpressed in the spinous layer and in a few basal
keratinocytes and dermal cells in psoriasis lesional skin. Curiously, the drastic overexpression of
NAMPT in psoriasis epidermis was mainly present in the nucleus of keratinocytes, but it was also
detected in the cytoplasm (Figure 36A).
On the other hand, immunohistochemistry for detection of PAR in skin samples from healthy
subjects and psoriasis lesions indicated that in control group PAR was naturally present in epidermis
and less pronouncedly in dermis. Strikingly, PAR displayed an apparently random pattern in the
spinous layer and dermis of psoriasis skin where not every cell in this layer showed increased levels
of PAR, which was accumulated in the nucleus (Figure 36B). The keratinocyte nuclear staining is
consistent with the subcellular location of PARPs.
These results strongly suggest a role for NAMPT and PARP in psoriasis. Further studied would
be necessaries to definitively demonstrated them as potential therapeutic strategies to treat
psoriasis and probably atopic dermatitis or other skin chronic inflammatory diseases.
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Figure 36. NAMPT and PAR are overexpressed in human psoriatic lesions. Representative images of sections from healthy and psoriatic skin biopsies that have been immunostained with an anti-NAMPT monoclonal antibody (sc-166946) or anti-poly (ADP-ribose) monoclonal antibody (ALX-804-220) and then slightly counterstained with hematoxilin.
DISCUSSION
Discussion
93
1. NAD+ metabolism and H2O2 release by keratinocytes
NAD+ metabolism play a fundamental role in maintaining organism homeostasis. NAMPT, the
rate-limiting step enzyme in the NAD+ salvage pathway, has been associated to oxidative stress and
inflammation (Garten et al., 2015), being identified as a universal biomarker of chronic
inflammation, including psoriasis (Mesko et al., 2010). Experiments with transgenic zebrafish lines
that enable immune cell-tracking, let us to demonstrate the contribution of NAD+ to skin
inflammation. Previous studies with an acute colitis model in mice, demonstrated that NAD+ fueled
inflammation. NAD+ depletion with FK-866 ameliorated disease severity, reducing NAD+-consuming
enzymes PARP1/SIRT/CD38 expression and activity, besides, favoring an anti-inflammatory
phenotype in monocytes/macrophages. Furthermore, NAMPT inhibition reduced cytokines
production from human IBD-derived immune cells (Gerner et al., 2017).
The ability of NAD+ and its precursors to induce oxidative stress can be explained by their capacity
to boost NADPH intracellular levels, susceptible to be oxidized by NADPH-oxidases in order to
catalyze the synthesis of H2O2. Consistently, pharmacological inhibition of NAMPT, efficiently
counteracted H2O2 synthesis by keratinocytes. Curiously, high dose FK-866 supplementation showed
that critically low levels of NAD+ trigger muscle inflammation. Beyond emphasizing the relevance of
NAD+ in the cell, the results could be explicated by the dependence of the mitochondrial electron
transport chain and other mitochondrial metabolic processes of the NAD+/NADH redox couple.
Excessively low NAD+ levels could cause disruption of the cellular bioenergetics. In fact, several
studies have associated boosted NAD+ levels with improved mitochondrial function under stress
(Cantó et al., 2015).
2. spint1a mutant
2.1 Novel features of the spint1a mutant: oxidative stress and DNA damage
In this work, we characterized new features of spint1a mutant. In first place, zebrafish mutant of
3 dpf exhibited increased H2O2 release in the skin, a well-known signal for leukocyte recruitment to
acute (Niethammer et al., 2009) and chronic (Candel et al., 2014) insults. H2O2 could be released
during cell death in aggregates, which in turn could direct chemotaxis of neutrophils. Additionally,
the putative contribution of oxidative stress to the mutant phenotype was confirmed by ROS
scavenger and pharmacological inhibition of Nampt, since both were able to rescue the spint1a
mutant phenotype. We corroborated that FK-866 remarkably decreased H2O2 production in a dose-
dependent manner, as previously observed in wild type zebrafish. Surprisingly, mutants treated with
FK866 showed H2O2 levels below the untreated control larvae, demonstrating the crucial
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dependence of H2O2 production by NAD+ and a probable physiological role for H2O2 in the skin in
steady state conditions. In second place, we determined that spint1a mutants exhibited higher DNA
damage, evaluated by either pH2Ax staining or by comet assay, as soon as 48 hpf. In addition,
spint1a mutants exhibited an increased susceptibility to olaparib, a DNA damage inducer (Kukolj et
al., 2017). This new facet of spint1a mutants might be provoked by the oxidative stress mediated by
H2O2 released by keratinocytes and probably other ROS. This study demonstrated that FK-866
counteracted DNA damage: being, its ability to block H2O2 synthesis crucial for this activity.
Furthermore, ROS scavengers also rescued skin inflammation in spint1a mutants, further analysis of
DNA damage in larvae treated with ROS scavengers should provide a similar result to Nampt
inhibition.
2.2 Effects of enzymatic inhibition of Nampt and Parp1 in spint1a mutant
Globally, in spint1a mutant psoriasis model, pharmacological inhibition of Nampt reduced
oxidative stress, skin inflammation and neutrophil infiltration, and keratinocyte aggregation,
hyperproliferation and DNA damage. 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. It was shown that inhibition of the
enzymatic activity of Parp1 by olaparib, veliparib or talazoparib, recapitulated the effects of FK-866
on the spint1a mutant, with the exception of H2O2 production by skin keratinocytes that was not
assayed. It is noteworthy that the oxidative stress analysis of mutant embryos treated with olaparib
could provide two opposite results. On the one hand, all the parameters analyzed indicate that the
mutant phenotype has been rescued. Consequently, it would be expected that oxidative stress
would also do so. However, on the other hand, the ability of pharmacological inhibition of
keratinocyte death by parthanatos, in principle, would not lead to a reduction of oxidative stress,
although blocking cell death could also prevent the release of cell debris from dying cells, reducing
oxidative stress. Definitively, this experiment could shed light onto the mechanism.
Additionally, olaparib increased DNA damage, but at the same time it reduced cell death and
PARylation. Moreover, neither olaparib nor FK-866 treatments promoted keratinocyte apoptosis at
2 dpf or 3 dpf upon 48 hours of treatment. These results contrast previous studies where PARP
inhibitors were described to induce apoptosis in proliferating cells (Schreiber et al., 2006).
Therefore, olaparib induces an unsustainable situation where keratinocytes accumulate DNA
damage and do not suffer apoptosis or another form of cell death. However, the mutant larvae
treated have a wild type phenotype, although this does not mean that a sustained treatment might
Discussion
95
result in the death of the larvae. Whatever the outcome, our results support that the rescue of the
mutant phenotype is due to the inhibition of programmed cell death parthanatos.
The blockage of keratinocyte proliferation in the spint1a mutant treated with FK-866 and
olaparib can be understood as a consequence of mutant phenotype rescue, but at the same time,
one can see it as a cause. In fact, various treatment for psoriasis target proliferation of keratinocytes
and immune cells, such as Acitretin, PUVA (Zhang & Wu, 2018), Methotrexate and Cyclosporine,
among others (Greaves & Weinstein, 1995). The depletion of NAD+, a key cofactor in numerous
metabolic processes, might resulted in an energy deficit blocking proliferation (Hasmann &
Schemainda, 2003), despite the fact that keratinocytes have mesenchymal properties and have lost
contact inhibition (Carney et al., 2007; Mathias et al., 2007). Indeed, it is known that cancer cells are
more sensitive to the loss of NAMPT, a distinctive feature that was proposed to be used clinically
with other anticancer agents (Garten et al., 2015). However, no significant morphological
differences in size were found between treated and non-treated siblings. In the case of olaparib
treatment, Parp1 inhibitors have been shown to induce the collapse of replication forks resulting in
dsBs generation. The detection of DNA damage triggers G2/M cell cycle arrest (Kujolj et al., 2017).
Although the possibility that enzymatic inhibitors are causing a blockage in cell proliferation cannot
be definitively ruled out, the experiments shown in this work strongly support that the recovery of
the mutant phenotype is due to the inhibition of oxidative stress that ultimately lead to parthanatos.
2.3 Effects of ROS scavengers
The newly characterized features of spint1a mutant phenotype described in this work, higher
levels of H2O2 in the skin and the accumulation of DNA lesions, prompted us to target oxidative
stress. After failing with chemical and genetic strategies to demonstrate the contribution of Duox1
to the mutant phenotype, we tested the effectiveness of ROS scavengers. N- acetylcysteine, mito-
TEMPO and tempol all rescued the mutant phenotype. However, regarding NFB transcriptional
activity in the skin, we cannot explain why mito-TEMPO did not rescue this feature of the spint1a
mutant phenotype.
Consistently, analyses in serum from psoriasis patients indicated increased levels of oxidative
stress markers, decreased levels of antioxidants molecules (Lin & Huang, 2016) and reduced activity
of the main antioxidant enzymes, such as superoxidase dismutase and catalase (Houshang et al.,
2014). Additionally, higher levels of oxidized guanine species, a marker of DNA/RNA damage, was
found in peripheral blood serum samples of psoriasis patients (Borska et al., 2017). In conclusion,
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psoriasis is associated with systemic oxidative stress and, therefore, the spint1a mutant is an
excellent model of this disease.
2.4 Effect of AIFM1 inhibitor
According to this work and published data, zebrafish spint1a mutant phenotype rescue might be
caused by prevention of parthanatos cell death in the susceptible skin epithelium. In order to block
parthanatos, we targeted a required step to fulfil the programmed cell death, AIFM1 release from
mitochondria, once its membrane potential is depolarized (Fatokun et al., 2014). As hypothesized,
inhibition of AIFM1 translocation rescued the mutant phenotype.
As previously described, PARP1 is known to play a relevant role in some acute and chronic
inflammatory diseases such as neurological diseases (Parkinson´s disease), sepsis, arthritis, colitis,
diabetes and myocardial infarction. Prevention of cell death might reduce inflammation and DAMP
exposure. Moreover, this reduction decreases tissue damage and proinflammatory cytokines
release, limiting immune cell recruitment (Kunze et al., 2019). In this line, it has been hypothesized
that cell death might be responsible for immune cell recruitment in spint1a mutants (Carney et al.,
2007).
Further studies are needed to completely point out parthanatos as the programmed cell death
occurring in keratinocytes responsible for spint1a mutant phenotype and its dependence of NAD+
and PAR metabolism. Transcriptomic analyses upon FK-866 and olaparib treatment can be very
informing in order to determine the effect of inhibitors, as a whole. Ideally, this experiment should
be focused on skin epithelia to avoid genetic contamination of irrelevant tissues.
3. Human psoriasis and atopic dermatitis
3.1 Transcriptomic datasets
It is known that psoriasis and atopic dermatitis have a strong genetic influence affecting
individual susceptibility (Greb et al., 2016; Weidinger et al., 2018). For that reason, we studied
human transcriptomic data, finding a differential expression profile of genes encoding NAD+
metabolic enzymes. Specially we found altered expression of genes involved in NAD+ salvage
pathway, Preiss-Handler pathway and de novo pathway in lesional skin compared with non-lesional
samples or healthy skin. In a similar way to NAD+ metabolism, we found an altered expression profile
of key enzymes involved in PAR metabolism and parthanatos.
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The expression profile of genes involved in NAD+ and PAR metabolism correlated with
inflammatory gene markers specific for each disease, according to specific cytokines implicated in
their TH immune responses. Remarkably, we found positive correlation, what could inform about an
association of the altered gene with disease severity. In contrast, we also uncovered inverse
correlation with numerous genes coding for inflammatory cytokines, probably pointing out the
expression of those genes as a protective factor.
It is worthy to mention that lesions of both diseases are characterize by an important cellular
immune infiltration and growth of other cell types like nerves or blood vessels. Some genes whose
expression is altered in lesioned skin, are specifically expressed in different tissues other than the
skin. Therefore, it would be worthy to analyze the expression of this molecules by
immunohistochemisry and/or single cell RNA seq.
3.2 NAD+ metabolites in serum
HPLC-MS analysis of serum samples of psoriasis patients indicated that responders exhibited
reduced levels of NAD+, NAD+/NADH ratio and SAM before PUVA treatment that were normalized
to control group after the treatment. Similarly, a study in multiple sclerosis (MS), a chronic
neuroinflammatory disease that affect the central nervous system (Dendrou et al., 2015), found
reduced serum levels of NAD+, higher levels of NADH and a lower NAD+/NADH ratio in patients
affected by MS. Furthermore, lower levels of NAD+ correlated with disease severity (Braidy et al.,
2013).
As previously mentioned, reduction in NAD+ levels together with SAM, could suggest increased
activity of NNMT. We found NNMT overexpression in lesional skin of atopic dermatitis patients, as
reported for models of injury associated with inflammation. Indeed, inflammation is thought to
drive its expression. Likewise, NNMT expression increased in numerous types of cancer, in which its
genetic inhibition counteracts cancer aggressiveness. Probably, this effect could be due to
dependence of methyltransferases on SAM as a methyl group donor. Studies in cancer lines showed
that DNA methylation was controlled by NNMT expression (Pissios, 2017). Therefore, increased
expression of NNMT might result in depletion of SAM, inducing epigenetic changes in atopic
dermatitis and subsequently, affecting the expression of genes related to inflammation.
In conclusion, further studies could elucidate if NAD+, NAD+/NADH and/or SAM might be
potential serum biomarkers to predict the response of psoriasis patients to phototherapy.
Discussion
98
3.3 Immunohistochemistry in skin samples
In order to finish this work, we confirmed the altered expression of NAMPT at protein level and
PAR accumulation in the nucleus of epidermal keratinocytes from psoriatic lesions. These results
strongly suggest a role for NAMPT and PARP in human psoriasis. It would be interesting to also
analyze whether AIFM1 is translocated to the nuclei of keratinocytes of lesional skin to confirm that
parthanatos is involved, and a putative therapeutic target, in psoriasis.
In conclusion, we report a critical role for NAD+ and PAR metabolism to control H2O2 keratinocyte
release and skin inflammation. Particularly, we show that pharmacological inhibition of Nampt and
Parp1 efficiently rescue zebrafish psoriasis models. Additionally, by the first time, this work points
out parthanatos as the mechanism responsible for keratinocyte cell death in spint1a zebrafish model
of chronic skin inflammation, paving the way for the study of the influence of keratinocyte cell death
through parthanatos to psoriasis pathology. Finally, human evidences support the alteration of
NAD+ and PAR metabolism in psoriasis encouraging new research to definitively characterize them
as potential therapeutic targets to treat psoriasis and probably other chronic skin inflammatory
diseases.
CONCLUSIONS
Conclusions
101
The results obtained in this work lead to the following conclusions:
1. NAD+ and its precursors critically regulate H2O2 keratinocyte release and skin
inflammation.
2. Spint1a-deficient zebrafish exhibit increased H2O2 skin production and DNA damage.
3. Pharmacological inhibition of Nampt and Parp1 effectively decreases oxidative stress,
neutrophil infiltration and inflammation, PARylation, and keratinocyte
hyperproliferation, DNA damage and cell death, in the spint1a and atpb1a zebrafish
models of chronic skin inflammation.
4. ROS scavengers rescue skin inflammation in the spint1a zebrafish model of chronic skin
inflammation.
5. Parthanatos has been identified as the cell death mechanism of keratinocytes of spint1a
zebrafish model of chronic skin inflammation. In addition, pharmacological inhibition of
parthanatos rescues skin inflammation in this model.
6. The expression profile of genes encoding key enzymes involved in NAD+ metabolism,
PAR metabolism and parthanatos is altered in atopic dermatitis and psoriasis. In
addition, their expression strongly correlated with disease specific inflammatory
markers.
7. NAMPT and PAR are robustly expressed in the nucleus of human keratinocytes from
psoriatic lesions.
8. NAD+, NAD+/NADH and SAM are potential serum biomarkers to predict the response of
psoriasis patients to phototherapy.
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RESUMEN EN CASTELLANO
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1. Introducción
La psoriasis y la dermatitis atópica, o ezcema, son dos enfermedades no contagiosas de
inflamación crónica en piel, cuya prevalencia es 0,1-3 % y 2-20 %, respectivamente (Furue & Kadono,
2017). Su etiología no ha sido determinada, aunque ambas enfermedades tienen una predisposición
genética (Greb et al., 2016; Weidinger et al., 2018), numerosos factores ambientales pueden
desencadenar la patología (Dainichi et al., 2018). A pesar de que son enfermedades recurrentes e
incapacitante que afectan tanto física como mentalmente, raramente amenazan la vida. Sin
embargo, las citoquinas y quimioquinas producidas en el entorno de la lesión alcanzan la sangre y,
en consecuencia, los pacientes pueden desarrollar diferentes comorbilidades (Furue & Kadono,
2017). NAD+ es el transportador de hidrógenos en reacciones redox más importante en la célula;
además, participa en más de 500 reacciones enzimáticas. NAD+ regula procesos celulares vitales,
como el funcionamiento y el metabolismo mitocondrial, reacciones redox, ritmo circadiano,
respuesta inmunitaria e inflamación, división celular, señalización proteína-proteína, cromatina,
daño en ADN y epigenética (Rajman et al., 2018). Los niveles de NAD+ son muy dinámicos, pero sus
niveles están estrechamente regulados por la ruta Preiss-Handler, la ruta de novo y la ruta de
salvamento (Cantó et al., 2015). Diferentes tejidos emplean una ruta biosintética diferente
dependiendo de los precursores disponibles (Marletta et al., 2015), pero en general, la mayoría de
los tejidos de mamíferos dependen de NAM para mantener los niveles de NAD+ a través de la ruta
de salvamento (Cantó et al., 2015; Rajman et al., 2018). NAMPT, la enzima limitante de la ruta de
salvamento de NAD+, ha sido asociada a estrés oxidativo e inflamación (Garten et al., 2015), siendo
identificada como un biomarcador universal de inflamación crónica, incluyendo psoriasis (Mesko et
al., 2010). Un estudio que comparó el perfil de expresión génica en muestras de piel de donantes
sanos y pacientes de psoriasis, en los que se tomó piel de lesiones y piel no afectada por psoriasis,
encontró que la sobreexpresión de NAMPT en la lesión psoriásica era clave para diferenciarla de la
piel no afectada por psoriasis y de la de pacientes sanos (Xie et al., 2014).
En ausencia de estímulos de reclutamiento, la mayoría de los neutrófilos en larvas de pez
cebra de 3 dpf se encuentran localizados entre la aorta dorsal y la vena axial, una región que se
denomina tejido hematopoyético caudal (CHT, de sus siglas en inglés) (Murayama et al., 2006). El
peróxido de hidrógeno desarrolla un papel importante en la movilización de neutrófilos en daños
agudos (Niethammer et al., 2009) y crónicos (Candel et al., 2014), para la visualización de los
cambios en la producción de H2O2 en diferentes condiciones, empleamos una sonda fluorescente
sensible a H2O2. Por otro lado, para analizar la distribución de neutrófilos se utilizó la línea
transgénica lyz:dsRED de pez cebra que expresa bajo un promotor específico de leucocitos una
proteína fluorescente (Hall et al., 2007), permitiendo el seguimiento de células inmunitarias in vivo.
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De forma similar, se utilizó la línea transgénica NF-kB:eGFP que monitoriza la actividad del factor
transcripcional NFB a través de la expresión de una proteína fluorescente verde (Kanther et al.,
2011) para visualizar así la inflamación en cualquier tejido.
2. Objetivos
Los objetivos específicos de este trabajo son:
1. Caracterización de papel que juega el metabolismo de NAD+ y PAR en la inflamación
crónica en la piel.
2. Estudio de la influencia de la inhibición farmacológica de Nampt y Parp1 en el estrés
oxidativo, la infiltración de neutrófilos en la piel, la inflamación en piel, la PARilación, la
proliferación de queratinocitos, el daño en ADN y la muerte celular en modelos de pez
cebra de inflamación crónica en piel.
3. Evaluar los efectos de moléculas antioxidantes y de la inhibición de la muerte celular
parthanatos en modelos de pez cebra de inflamación crónica en piel.
4. Estudiar la participación del metabolismo de NAD+ y PAR en las enfermedades
inflamatorias crónicas de la piel humana.
3. Resultados
Ensayos en peces cebra silvestres nos permitió demostrar que NAD+ y sus precursores regulan
la liberación de H2O2 por los queratinocitos y la inflamación en piel de forma dosis dependiente. A
pesar de que el patrón de distribución de neutrófilos quedó alterado, algunos de los cuales estaban
presentes en la piel, tanto la integridad de la piel como su morfología no se vieron afectados. En
vista a los resultados que sugerían un papel proinflamatorio de NAD+, decidimos reducir los niveles
de este metabolito a través de un inhibidor específico de NAMPT. Consecuentemente, la inhibición
farmacológica de Nampt por FK-866, que induce depleción de NAD+, contrarrestó la síntesis de H2O2
por los queratinocitos. Sin embargo, FK-866 a alta dosis desencadenó una importante inflamación
en el músculo, la cual pudo revertirse con la aplicación de NAD+. Como cabía esperar, en este
contexto de activación transcripcional de NFB en el músculo, se encontró una importante
infiltración de neutrófilos.
A continuación, decidimos emplear en nuestras investigaciones el modelo de psoriasis
mutante en spint1a. Spint1a es un inhibidor de la Matriptasa 1, la cual puede activar
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proteolíticamente varias proteínas y zimógenos, además de degradar ciertas proteínas de la matriz
extracelular. Este mutante presenta disrupción epitelial debido a que sus queratinocitos se agregan,
adquieren propiedades mesenquimales y tienen una alta proliferación. Adicionalmente, los
agregados de queratinocitos son susceptibles a la muerte celular, lo que desencadena el
reclutamiento de células inmunitarias que desarrollan una respuesta inflamatoria (Carney et al.,
2007; Mathias et al., 2007). En este trabajo describimos por primera vez que el mutante spint1a
presenta una mayor producción de H2O2 en la piel y daño en ADN. En este modelo, la depleción de
NAD+ a través del inhibidor FK-866 mostró una importante reducción de la síntesis de H2O2; de
hecho, los niveles registrados de H2O2 fueron más bajos de los que presentaban los animales
silvestres, indicando la gran dependencia de su síntesis a partir del metabolismo de NAD+ y un
posible papel fisiológico en condiciones normales. Por otro lado, la inhibición farmacológica de
Nampt redujo considerablemente la inflamación en piel, la dispersión de neutrófilos, la proliferación
de queratinocitos y el daño en ADN, restaurando así la integridad epitelial. De acuerdo con los
resultados en larvas silvestres, el tratamiento de los organismos mutantes con NAD+ agravó su
fenotipo y bloqueaba los efectos beneficiosos ejercidos por FK-866.
La depleción de NAD+ mediada por FK-866 debe tener un impacto en la actividad enzimática
de enzimas que dependen de NAD+ como cofactor. Por ello, analizamos las principales enzimas cuya
actividad se podría ver comprometida. Dado que la mayor influencia en los niveles de NAD+ en el
organismo están mediados por CD38 (Cantó et al., 2015), decidimos emplear un inhibidor específico
llamado 78c (Haffner et al., 2015). De forma similar, empleamos un inhibidor de la actividad
enzimática de sirtuinas, denominado EX 527 (Hixon et al., 2007), puesto que son unas importantes
consumidoras de NAD+. Sin embargo, tras diferentes concentraciones y diseños experimentales
aplicados, concluimos que las actividades enzimáticas de Cd38 o sirtuina no contribuían al fenotipo
mutante spint1a. Seguidamente, estudiamos otra familia de proteínas consumidoras de NAD+, las
Poli(ADP-ribosa) polimarasas (PARPs), demostrando que la inhibición de Parp1 por olaparib,
veliparib o talazoparib, mostraba los mismos efectos que FK-866. En este punto nos interesamos
por otro modelo de psoriasis que presenta disrupción epitelial, el mutante atp1b1a (Hatzold et al.,
2016; Webb et al., 2008), en el que determinamos que tanto FK-866 como olaparib eran capaces de
restaurar la integridad epitelial. Curiosamente, aunque los mutantes están afectados en diferentes
genes, ambos deben compartir rutas metabólicas alteradas.
En los peces cebra silvestres y deficientes en Spint1a, olaparib indujo daño en ADN. De
acuerdo con investigaciones previas, los inhibidores de PARPs atrapan y acumular Parp1 inactiva en
el ADN, desencadenando la formación de daños de doble cadena durante la replicación (Kujolj et
al., 2017). Sin embargo, aunque la acumulación de lesiones en el ADN eventualmente conduce a la
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muerte celular (Schreiber et al., 2006), la inhibición farmacológica de Parp1 no conllevó un aumento
en la apoptosis, analizada con un anticuerpo que detecta el fragmento de Caspasa 3 activa
(Schreiber et al., 2006). De hecho, olaparib redujo la cantidad de células TUNEL+ en los embriones
mutantes. Por otro lado, también encontramos una disminución en la PARilación, lo que demuestra
que el inhibidor actuó sobre la actividad enzimática de Parp1 como se esperaba.
Teniendo en cuenta que anteriormente habíamos detectado una mayor cantidad de estrés
oxidativo en mutantes spint1a y junto a la acumulación de lesiones en ADN, pensamos que el estrés
oxidativo podría están jugando un papel relevante en el desarrollo del fenotipo. En primer lugar,
decidimos inhibir Duox1, una reconocida NADPH-oxidasa cuya inhibición ha demostrado reducir
exitosamente el estrés oxidativo la inflamación en un modelo de psoriasis (Candel et al., 2014). Sin
embargo, su inhibición génica y farmacológica aplicando diferentes concentraciones y diseños
experimentales no revirtió el fenotipo mutante. Por lo tanto, optamos por emplear compuestos
eliminadores de ROS, encontrado que tanto N-acetilcisteína, un precursor de glutatión reducido
(Wu et al., 2015), mito-TEMPO, un antioxidante que se acumula en la mitocondria y tiene actividad
similar a las superóxido dismutasa (Ni et al., 2016) y tempol, un antioxidante que actúa sobre los
compuestos de descomposición del peroxinitrito (Mustafa et al., 2015), rescataron el fenotipo
mutante.
En vista a los resultados anteriores hipotetizamos que parthanatos podría ser la muerte
celular que se había detectado en los agregados cutáneos de los mutantes spint1a. En condiciones
normales el daño en ADN provocado por el metabolismo celular u otros factores es gestionado por
PARP1. Sin embargo, en situaciones de excepcional daño en ADN se desencadena la hiperactivación
de Parp1 que PARila a diferentes proteínas y moléculas produciendo una acumulación de polímeros
de PAR que pueden alcanzar la mitocondria y desestabilizar su potencial de membrana, induciendo
la liberación de AIFM1 que recluta a MIF del citosol para que juntas, lleven a cabo una fragmentación
del ADN nuclear a gran escala (Wang et al., 2016). De acuerdo con nuestra hipótesis, un inhibidor
de la traslocación de AIFM1 de la mitocondria al núcleo, un paso fundamental para que se desarrolle
parthanatos, recuperó la disrupción epitelial en las larvas deficientes en Spint1a al igual que la
inhibición de Nampt y Parp1 y la aplicación de moléculas antioxidantes.
Una vez que determinamos que parthanatos era el mecanismo responsable de la muerte
celular de los agregados de queratinocitos, cuya muerte contribuía al reclutamiento de célula
inmunitarias, la inflamación en piel y la disrupción del epitelio presente en el fenotipo mutante,
quisimos saber si el metabolismo de NAD+, PAR y parthanatos estaban alterados en las
enfermedades de inflamación crónica en piel que afectan a humanos. Para ello analizamos datos
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transcriptómicos de humano que comparaban piel de donantes sanos con piel procedente de
lesiones y de tejido sano de pacientes de psoriasis. De forma similar estudiamos datos de pacientes
que sufrían dermatitis atópica. En ambos casos encontramos un perfil de expresión alterado en
genes que cifran enzimas clave en las rutas de NAD+ de salvamento, Preiss-Handler y de novo en
tejidos lesionados en comparación con tejidos sin lesión o sanos. Cabe destacar que, en las dos
enfermedades, NAMPT se encuentra sobreexpresado respecto a los sujetos control. De forma
similar, encontramos un perfil de expresión alterado en genes que cifran enzimas implicadas en el
metabolismo de PAR y parthanatos. Especialmente de interés es el hecho de que en ambas
enfermedades PARP1 y AIFM1 se encontraban sobreexpresadas mientras que varios genes
implicados en la eliminación de polímeros de PAR de proteínas se encontraban menos expresados,
lo que podría favorecer la muerte celular parthanatos.
Para ampliar el estudio transcriptómico realizamos correlaciones del perfil génico de las
lesiones con varios genes marcadores inflamatorios como TNFA e IL1B y otros más específicos de
cada enfermedad. Para psoriasis, nos centramos en las citoquinas IL17A e IL23A características de
una respuesta inmunitaria TH17 (Greb et al., 2016) y para dermatitis atópica utilizamos las citoquinas
IL4, IL10 e IL13 derivadas de una respuesta inmunitaria TH2 y la citoquina IL22 representativa de una
respuesta inmunitaria TH12. El análisis indicó que el perfil de expresión de genes involucrados en el
metabolismo de NAD+, PAR y parthanatos correlacionaron tanto positiva como negativamente con
genes marcadores inflamatorios de cada enfermedad, de acuerdo con su respuesta inmunitaria TH.
En busca de alteraciones del metabolismo de NAD+ en pacientes de psoriasis, analizamos en
HPLC-MS varios metabolitos en el suero sanguíneo. Estos metabolitos se midieron en pacientes
antes y después de recibir un tratamiento de fototerapia (PUVA) y se comparó con sujetos control.
Los análisis indicaron que los pacientes que presentaban una buena respuesta al tratamiento
mostraban menores niveles de NAD+, relación NAD+/NADH y SAM antes del tratamiento que se
normalizaban tras este. Este estudio sugiere que podrían ser potencialmente utilizados como
biomarcadores séricos para predecir la respuesta a la fototerapia. Finalmente, confirmamos una
acumulación a nivel proteico de NAMPT y PAR en el núcleo de queratinocitos en lesiones psoriásicas.
Colectivamente, el metabolismo de NAD+ y PAR podrían representar nuevas dianas terapéuticas
para la psoriasis y probablemente otras enfermedades de inflamación crónica en piel.
4. Discusión
Se sabe que el metabolismo de NAD+ juega un papel fundamental en el mantenimiento de la
homeostasis del organismo. En este trabajo hemos demostrado la contribución de NAD+ a la
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inflamación en piel. De acuerdo con un estudio previo en un modelo murino de colitis aguda, se
determinó que NAD+ estimulaba la inflamación y que la inhibición farmacológica de NAMPT reducía
la severidad de la enfermedad (Gerner et al., 2017).
Por otro lado, la habilidad de NAD+ y sus precursores para inducir estrés oxidativo puede ser
explicada por su capacidad para aumentar los niveles de NADPH, sustrato de NADPH oxidasas que
sintetizan H2O2. Hemos descrito que una mayor producción de H2O2 en la piel, es una característica
del fenotipo del mutante spint1a. Se sabe que este factor media el reclutamiento de leucocitos en
daños tisulares agudos (Niethammer et al., 2009) y crónicos (Candel et al., 2014). Su liberación
durante la muerte celular en los agregados de queratinocitos podría desencadenar la quimiotaxis
de neutrófilos. Se confirmó que tanto moléculas antioxidantes, como la inhibición farmacológica de
Nampt, eran capaces de rescatar el fenotipo mutante. Adicionalmente, se determinó que los
embriones mutantes en spint1a exhibían un mayor daño en ADN, el cual podría estar provocado por
el estrés oxidativo detectado en el epitelio cutáneo. De hecho, FK-866 redujo el daño en ADN en el
mutante, siendo su habilidad para bloquear la síntesis de H2O2 crucial para esta actividad.
En general, la inhibición farmacológica de Nampt y Parp1 disminuyeron eficientemente el
estrés oxidativo, la infiltración de neutrófilos y la inflamación en la piel, la PARilación, la
hiperproliferación de queratinocitos, el daño en ADN y la muerte celular en los modelos de pez cebra
de inflamación crónica en piel spint1a y atp1b1a. Concretamente, aunque olaparib es un agente
inductor de daño en ADN (Kukolj et al., 2017), consiguió reducir la muerte celular y no indujo
apoptosis, en contraste a estudios previos en los que inhibidores de PARPs indujeron apoptosis en
células proliferativas (Scherieber et al., 2006). En cuanto al bloqueo de la hiperproliferación de
queratinocitos, podría ser consecuencia del rescate del fenotipo mutante, de un déficit energético
por la depleción de NAD+ (Hasmann & Schemainda, 2003) o un arresto en el ciclo celular (Kukolj et
al., 2017). Sin embargo, la relación de Nampt con el estrés oxidativo junto a la de Parp1 con la
muerte celular programada parthanatos y, sobre todo, el rescate del fenotipo mutante con las
moléculas antioxidantes y el inhibidor de la traslocación de AIFM1 de la mitocondria, en su conjunto
sugieren que parthanatos contribuye al fenotipo spint1a. La inhibición de la muerte celular reduciría
la inflamación y la exposición a DAMPs, lo que disminuiría el daño tisular, la liberación de citoquinas
proinflamatorias y el reclutamiento de células inmunitarias (Kunze et al., 2019).
Finalmente, este trabajo demuestra que a nivel de expresión génica tanto el metabolismo
NAD+, PAR y parthanatos esta alterado en dermatitis atópica y psoriasis. Estas alteraciones podrían
contribuir a la fuerte influencia genética que intervieneen la susceptibilidad de los individuos en
ambas enfermedades (Greb et al., 2016; Weidinger et al., 2018), en especial, aquellos genes cuya
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expresión correlacionan con la severidad de la enfermedad. Por otro lado, determinamos que NAD+,
NAD+/NADH y SAM son potenciales biomarcadores séricos para predecir la respuesta de pacientes
con psoriasis a la fototerapia. De forma similar, un estudio en esclerosis múltiple encontró niveles
séricos alterados de NADH y NAD+, donde niveles reducidos de NAD+ correlacionaban con la
severidad de la enfermedad (Braidy et al., 2013). Para finalizar el trabajo, se confirmó la
sobreexpresión de NAMPT a nivel proteico y la acumulación de PAR en el núcleo de queratinocitos
de lesiones psoriásicas.
5. Conclusiones
Los resultados obtenidos en este trabajo condujeron a las siguientes conclusiones:
1. NAD+ y sus precursores regulan la liberación de H2O2 por los queratinocitos y la inflamación
en piel.
2. Peces cebra deficientes en Spint1a exhiben una mayor producción de H2O2 y daño en ADN.
3. La inhibición farmacológica de Nampt y Parp1 disminuyen eficientemente el estrés
oxidativo, la infiltración de neutrófilos y la inflamación en la piel, la PARilación, la
hiperproliferación de queratinocitos, el daño en ADN y la muerte celular en los modelos de
pez cebra de inflamación crónica en piel spint1a y atp1b1a.
4. Las moléculas antioxidantes rescatan la inflamación en la piel en el modelo de pez cebra de
inflamación crónica en piel spint1a.
5. Parthanatos ha sido identificado como el mecanismo de la muerte celular de los
queratinocitos del modelo de pez cebra de inflamación crónica en piel spint1a. Además, la
inhibición farmacológica de parthanatos rescata la inflamación en piel en este modelo.
6. El perfil de expresión de genes que codifican enzimas clave implicadas en el metabolismo
del NAD+, PAR y parthanatos esta alterado en dermatitis atópica y psoriasis. Además, su
expresión correlaciona con genes marcadores inflamatorios específicos de cada
enfermedad.
7. NAMPT y PAR se expresan de forma robusta en el núcleo de queratinocitos humanos en
lesiones psoriásicas.
8. NAD+, NAD+/NADH y SAM son potenciales biomarcadores séricos para predecir la respuesta
de pacientes con psoriasis a la fototerapia.
