Estudio químico biodirigido y caracterización farmacológica del aceite de oliva virgen extra en el lupus eritematoso sistémico experimental

! ESTUDIO QUÍMICO BIODIRIGIDO Y CARACTERIZACIÓN FARMACOLÓGICA DEL ACEITE DE OLIVA VIRGEN EXTRA EN EL LUPUS ERITEMATOSO SISTÉMICO EXPERIMENTAL Tesis doctoral presentada por MARINA APARICIO SOTO Para optar al Grado de Doctor en Farmacia con Mención Internacional Sevilla, 2017 DEPARTAMENTO DE FARMACOLOGÍA FACULTAD DE FARMACIA UNIVERSIDAD DE SEVILLA ! ! Las Dras. Catalina Alarcón de la Lastra Romero, catedrática de Universidad, y Marina Sánchez Hidalgo, profesora contratada doctora, adscritas al departamento de Farmacología de la Facultad de Farmacia de la Universidad de Sevilla, INFORMAN Que la Tesis doctoral titulada “ESTUDIO QUÍMICO BIODIRIGIDO Y CARACTERIZACIÓN FARMACOLÓGICA DEL ACEITE DE OLIVA VIRGEN EXTRA EN EL LUPUS ERITEMATOSO SISTÉMICO EXPERIMENTAL” presentada por la Lda. Marina Aparicio Soto para optar al grado de Doctora en Farmacia con mención internacional ha sido llevaba a cabo bajo su dirección. Sevilla, 2017 Dra. Catalina Alarcón de la Lastra Dra. Marina Sánchez Hidalgo DEPARTAMENTO DE FARMACOLOGÍA FACULTAD DE FARMACIA UNIVERSIDAD DE SEVILLA ! ! A mis padres ! ! Für Tarek ! ! ! Este trabajo de investigación se ha llevado a cabo gracias a la financiación de las siguientes instituciones y proyectos de investigación: A. BECAS PREDOCTORALES Ayudas a la realización de proyectos de investigación científica Entidad financiadora: Instituto Danone Duración: Enero 2012- Abril 2013 Beca de formación de profesorado universitario (F.P.U) Entidad financiadora: Ministerio de Educación y Ciencia (M.E.C) Duración: Mayo 2013- Abril 2017 B. ESTANCIAS EN EL EXTRANJERO Ayudas a la movilidad del personal docente investigador (P.D.I). Tipo A Klinik für Dermatologie, Allergologie und Venerologie. Charite-Universitätsmedizin Berlin, Alemania Entidad financiadora: Universidad de Sevilla Duración: Septiembre-Diciembre 2014 Ayudas a estancias cortas en el extranjero. Programa de Formación de Profesorado Universitario (F.P.U) Klinik für Dermatologie, Allergologie und Venerologie. Charite-Universitätsmedizin Berlin, Alemania Entidad financiadora: Ministerio de Educación y Ciencia (M.E.C) Duración: Septiembre 2015-Febrero 2016 C. PROYECTOS DE INVESTIGACIÓN Plan Nacional del 2011: Estudio químico biodirigido y caracterización farmacológica del aceite de oliva virgen extra en el lupus eritematoso sistémico experimental. Investigador principal: Catalina Alarcón de la Lastra Referencia: AGL2011-26949 Entidad financiadora: Ministerio de Ciencia e Innovación Duración: 2012-2015 Abreviaturas ! ! HRP Horseradish peroxidase HTy HTy-Ac Hydroxytyrosol/ Hidroxitirosol Hydroxytyrosyl acetate/ Acetato de hidroxitirosol I IBD I3C IC ICAM-1 Inflammatory bowel disease Indole-3-carbinol Aberrant immune complexes/ Inmunocomplejos aberrantes Intercellular adhesion molecule-1 IFN Interferon / Interferón Ig IGF1R IκBα Immunoglobulin Insulin-like growth factor receptor 1 Inhibitor of NF-κB IL Interleukin / Interleucina iNOS Inducible nitric oxide synthase / Óxido nitrico sintasa Inducible i.p. IRF-1 Intraperitoneally / Intraperitoneal Interferon regulatory factor-1 IU i.v. International units Intravenous / Intravenoso J JAK/STAT Janus kinase-signal transducer and activator of Transcription/ Janus quinasas-transductor de señal y activador de la transcripción JNK c-Jun N H2-terminal kinase / C-Jun NH2-terminal cinasa Abreviaturas ! ! L LDL LES LFA LPL LPS Low-density lipoproteins Lupus eritematoso sistémico Lymphocyte function-associated antigen 1 Lipoprotein lipase Lipopolysaccharide LPO Lipid peroxides M MAPK Mitogen-activated protein kinases / Proteínas cinasas activadas por mitógenos MCP-1 Monocyte chemoattractant protein-1 MHC MIP Major histocompatibility complex Macrophage inflammatory protein MMP MOG Metalloproteinases / Metaloproteinasas Myelin oligodendrocyte glycoprotein MPC-1 Monocyte chemoattractant protein-1 mPGES-1 Microsomal prostaglandin E synthase-1 MPO Myeloperoxidase mRNA MS Messenger RNA Multiple sclerosis mTOR MTT Mammalian target of rapamycin 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium MUFA Monounsaturated fatty acid / Ácidos grasos monoinsaturados Abreviaturas ! ! N NADH NF-κB NK NLRP3 Nicotinamide adenine dinucleotide Nuclear transcription factor-kappa B / Factor nuclear kappa B Natural killer cell NLR family pyrin domain-containing 3 inflammasome/ Inflamasoma NLR3 NO Nitric oxide/ Óxido nítrico Nrf2 Nuclear factor E2-related factor 2/ Factor nuclear eritroide 2 O OA OD OL OL-aglycon Oleanolic acid Optical density Oleuropein Oleuropein aglycone P pAKT Phospho AKT PAS PBMC Periodic acid Schiff Peripheral blood mononuclear cells PBS PCNA Phosphate buffered saline Proliferating cell nuclear antigen PE Phenolic fraction PG PHA Prostaglandin/ Prostaglandina Phytohaemagglutinin/ Fitohemaglutinina Abreviaturas ! ! PI3 PI3K-Akt-mTOR Phosphatidylinositol-3 phosphoinositide 3-kinase/Akt/mammalian target of rapamycin PKC Protein kinase C PMSF Phenylmethyl sulfonyl fluoride p.o PPAR ppm Oral administration (per os) Peroxisome proliferator-activated receptor/ Receptores activados por proliferadores de peroxisomas Parts per million PUFA Polyunsaturated fatty acids R RA Rheumatoid arthritis RANKL Receptor activator of NF-κB ligand RNA RNP Ribonucleic acid Ribonucleoprotein ROS RT-qPCR Reactive oxygen species Reverse transcription polymerase chain reaction/ Reacción en cadena de la polimerasa con transcriptasa inversa S SD SDG SDS Standard diet Secoisolariciresinol diglucoside Sodium dodecyl sulfate S.E.M. Standard error SOD Superoxide dismutase Abreviaturas ! ! SRB ssDNA Sulforhodamine B Anti-single-stranded DNA STAT Signal transducer and activator of transcription T TG Triglycerides TGF Transforming growth factor Th THF TLC TLR TLR3-l TSLP Helper T cells / Células T cooperadoras Tetrahydrofuran Thin layer chromatography Toll like receptor Toll ike receptor 3 ligand/ Ligando de receptor de tipo toll 3 Thymic stromal lymphopoietin/ Linfopoyetina estromal tímica TNF Tumour necrosis factor / Factor de necrosis tumoral Tregs Regulatory T cells / Células T reguladoras U UF UGT UVB Unsaponifiable fraction Uridine diphosphate glucuronosyltransferase Ultraviolet B V VEGF VLDL VOO Vascular endothelial growth factor Low-density lipoprotein cholesterol Virgin olive oil Abreviaturas ! ! WST-8 W (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium, monosodium salt) ! ! ! ! INDICE ! ! ! Indice ! INTRODUCCIÓN / INTRODUCTION An update on diet and nutritional factors in systemic lupus erythematosus management 5 Extra virgin olive oil: a key functional food for prevention of immuneinflammatory diseases 51 JUSTIFICACIÓN Y OBJETIVOS 81 CAPÍTULO I / CHAPTER I Dietary extra virgin olive oil attenuates kidney injury in pristane-induced SLE model via activation of HO-1/Nrf2 antioxidant pathway and suppression of JAK/STAT, NF-ΚB and MAPK activation. 93 CAPÍTULO II / CHAPTER II Potential involvement of the phenolic fraction of extra virgin olive oil in pristane-induced lupus in mice by regulating PPAR-γ dependent monocyte activation 123 CAPÍTULO III / CHAPTER III The phenolic fraction of extra virgin olive oil modulates the activation and the inflammatory response of t cells from patients with systemic lupus erythematosus and healthy donors 149 CAPÍTULO IV / CHAPTER IV Naturally occurring hydroxytyrosol derivatives: hydroxytyrosyl acetate and 3,4-dihydroxyphenylglycol modulate inflammatory response in murine peritoneal macrophages. potential utility as new dietary supplements. 171 CAPÍTULO V / CHAPTER V Dietary hydroxytyrosol and hydroxytyrosyl acetate supplementation prevent pristane-induced systemic lupus erythematous in mice 197 CAPÍTULO VI / CHAPTER VI Olive oil-derived phenols exert effective anti-inflammatory properties by interference with the NF-ΚB pathway and TSLP production in human keratinocytes 221 DISCUSIÓN GENERAL / GENERAL DISCUSSION 245 CONCLUSIONES ABSTRACT ANEXOS 267 269 281 Curriculum vitae 291 ! Introduction ! AN UPDATE ON DIET AND NUTRITIONAL FACTORS IN SYSTEMIC LUPUS ERYTHEMATOSUS MANAGEMENT Marina Aparicio-Soto, Marina Sánchez-Hidalgo, Catalina Alarcón-de-la-Lastra Department of Pharmacology, Faculty of Pharmacy, University of Seville, Spain This chapter reproduces the following article: M. Aparicio-Soto, M. Sánchez-Hidalgo, C. Alarcón-de-la-Lastra. An update on diet and nutritional factors in systemic lupus erythematosus management. Nutr Research Reviews (2016). ABSTRACT Systemic lupus erythematosus (SLE) is a chronic inflammatory and autoimmune disease characterised by multiple organ involvement and a large number of complications. SLE management remains complicated owing to the biological heterogeneity between patients and the lack of safe and specific targeted therapies. There is evidence that dietary factors can contribute to the geoepidemiology of autoimmune diseases such as SLE. Thus, diet therapy could be a promising approach in SLE owing to both its potential prophylactic effects, without the side effects of classical pharmacology, and its contribution to reducing comorbidities and improving quality of life in patients with SLE. However, the question arises as to whether nutrients could ameliorate or exacerbate SLE and how they could modulate inflammation and immune function at a molecular level. The present review summarises preclinical and clinical experiences to provide the reader with an update of the positive and negative aspects of macro and micronutrients and other nutritional factors, including dietary phenols, on SLE, focusing on the mechanisms of action involved. Keywords: diet, immunomodulation, lupus, nutrient, SLE. Introduction ! ! References • Aparicio-Soto, M., Sánchez-Hidalgo, M., Cárdeno, A., Rosillo, M., Sánchez-Fidalgo, S., Utrilla, J., Martín-Lacave, I., and Alarcón-de-la-Lastra, C. (2016). 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Alarcónde-la-Lastra. Extra virgin olive oil: a key functional food for prevention of immuneinflammatory diseases. Food Funct. (2016). 7, 4492-4505. RESUMEN En la actualidad se considera que una dieta desequilibrada constituye uno de los principales factores que contribuyen a la aparición de enfermedades inflamatorias y autoinmunes tanto en países desarrollados como en países en vías de desarrollo. Por el contrario, la dieta mediterránea ha sido especialmente asociada con una disminución en la incidencia de ciertas patologías relacionadas con la inflamación crónica y el sistema inmune. En este sentido, el aceite de oliva, la principal fuente de lípidos de la dieta mediterránea, presenta excepcionales cualidades nutricionales, así como una especial composición particularmente destacable en el caso del aceite de oliva virgen extra (AOVE), obtenido a partir de la aceituna únicamente a través de procedimientos mecánicos o físicos bajo una serie de condiciones que no alteran la composición natural del aceite. El AOVE ha sido ampliamente descrito como un alimento funcional de primera línea con múltiples propiedades beneficiosas, que podrían ser de interés en el tratamiento de diversas enfermedades inmunoinflamatorias. En el presente trabajo, se recogen los resultados clave que aportan evidencias sobre los efectos beneficiosos del AOVE y sus componentes minoritarios, prestando especial interés a los mecanismos por los cuales pueden ejercer dichos efectos beneficiosos en el manejo de diversas enfermedades inmunoinflamatoria como la artritis reumatoide, el lupus eritematoso sistémico, la enfermedad inflamatoria intestinal y la esclerosis. ! Introduction ! EXTRA VIRGIN OLIVE OIL: A KEY FUNCTIONAL FOOD FOR PREVENTION OF IMMUNEINFLAMMATORY DISEASES Marina Aparicio-Soto, Marina Sánchez-Hidalgo, Mª Ángeles Rosillo, Mº Luisa Castejón, Catalina Alarcón-de-la-Lastra Department of Pharmacology, Faculty of Pharmacy, University of Seville, Spain This chapter reproduces the following article: M. Aparicio-Soto, M. Sánchez-Hidalgo, Mª Ángeles Rosillo, Mº Luisa Castejón C. Alarcónde-la-Lastra. Extra virgin olive oil: a key functional food for prevention of immuneinflammatory diseases. Food Funct. (2016). 7, 4492-4505. ABSTRACT Nowadays, it is clear that unhealthy diet is one of the prime factors that contribute to the rise of inflammatory diseases and autoimmunity in both developed and developing countries. The Mediterranean diet has been associated with a reduced incidence of certain pathologies related to chronic inflammation and immune system. Olive oil, the principal source of dietary lipids of the Mediterranean diet, possesses a high nutritional quality and a particular composition, which is especially relevant in the case of Extra Virgin Olive Oil (EVOO). EVOO is obtained from olives solely by mechanical or other physical preparation under conditions that do not alter natural composition. EVOO is described as a key bioactive food with multiple beneficial properties and it may be effective in the management of some immune-inflammatory diseases. 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Macrophages commit postnatal endothelium-derived progenitors to angiogenesis and restrict endothelial to mesenchymal transition during muscle regeneration. Cell Death Dis 5, e1031. • Zvaifler, N. (1965). A speculation on the pathogenesis of joint inflammation in rheumatoid arthritis. Arthritis Rheum 8, 289-293. Justificación y objetivos ! ! 84 expresada en gramos de ácido oleico, es como máximo un 0.8%. El AOVE está constituido por una fracción mayoritaria saponificable, que constituye entre un 90-99% del total del aceite y por una fracción minoritaria compuesta por dos subfracciones: fracción insaponificable (FI) y fracción polifenólica (FP). Los principales componentes de la fracción saponificable son ácidos grasos, de los cuales el ácido graso monoinsaturado (MUFA) ácido oleico (n-9) constituye hasta un 80% del total de la composición lipídica. Además, la fracción saponificable contiene ácidos grasos poliinsaturados, en un porcentaje del 3 al 22%, principalmente acido linoleico, y ácidos grasos saturados, en un porcentaje del 8 al 26%, principalmente acido palmítico. Por otra parte, la FI está constituida principalmente por hidrocarburos, principalmente escualeno aunque también contiene esteroles, alcoholes y dioles terpénicos y alifáticos (eritrodiol, uvaol, ácido oleanólico entre otros), vitaminas (α, β, γ y δ tocoferoles), pigmentos (clorofila y carotenos) y compuestos volátiles responsables de las propiedades organolépticas del AO (Ghanbari et al., 2012). Figura 1. Tipos de aceite de oliva según reglamentos de la Comunidad Europea La FP del AOVE es una mezcla compleja de compuestos fenólicos que contribuyen a las propiedades organolépticas del aceite. Su contenido varia considerablemente en función de la variedad del fruto y de las condiciones de cultivo y procesamiento del mismo. Al menos 36 compuestos fenólicos distintos han sido ACEITUNA( ORUJO( ACEITE(DE(OLIVA( VIRGEN( ACEITE(DE(OLIVA( VIRGEN(EXTRA( ACEITE(DE(OLIVA( LAMPANTE( Sabor,(aroma(u(otras(cualidades( defectuosas( No(apto(para(consumo(humano( Acidez(>2(º( ( Excelentes(cualidad(organolépMcas( Acidez(<(0.8(º( ACEITE(DE(OLIVA( REFINADO( Acidez(<(0.3(º( ( PROCESO'DE' REFINADO' ' ACEITE(DE( OLIVA( Mezcla(de(aceite(de(oliva(virgen(y( aceite(de(oliva(refinado( Acidez(<((1(º( ( Buenas(cualidad(organolépMcas( Acidez(<(0.8(º( Justificación y objetivos ! ! 85 identificados en la FP del AOVE, principalmente ácidos fenólicos y otros compuestos bioactivos como el hidroxitirosol (HTy), oleuropeína, oleaceína, oleuropeína aglicona, oleocantal, apigenina, (+)-pinoresinol y (+)-1-acetoxipinoresinol, entre otros (Pérez et al., 2014). Hasta hace relativamente poco tiempo, las propiedades beneficiosas del AO habían sido atribuidas casi exclusivamente a su alto contenido en MUFAs, particularmente, ácido oleico. En este sentido, recientes estudios han puesto de manifiesto que los MUFAs son capaces de modular diversos parámetros inmunológicos, desempeñando un importante papel en la regulación de la respuesta inmune, por lo que podrían ser de gran interés en el tratamiento de enfermedades inmunoinflamatorias y en la regulación general del sistema inmune (Carrillo et al., 2012; Sales-Campos et al., 2013). Sin embargo, también existen evidencias contradictorias sobre el papel de los MUFA en la respuesta inflamatoria y la modulación del sistema inmune (Cury-Boaventura et al., 2008; Hatanaka et al., 2006). Este hecho ha propiciado la búsqueda de otros posibles componentes del AO con actividad antiinflamatoria e inmunomoduladora. Existen evidencias que demuestran que los componentes minoritarios presentes en el AOVE también contribuyen a los beneficios asociados al consumo del mismo. En este sentido, AOs de baja calidad (aceites de oliva refinados) presentan una menor capacidad antioxidante y antiinflamatoria, posiblemente debido a la falta de estos componentes minoritarios (Estruch and Salas-Salvadó, 2013). Por lo tanto, los compuestos fenólicos del AOVE parecen ser responsables de algunas de sus propiedades beneficiosas, incluyendo sus actividades antiaterogénica, hipoglucemiante, antiinflamatoria, antitumoral, antiviral e inmunomodulatoria, que aparecen estar relacionadas solo parcialmente con la capacidad antioxidante intrínseca de estos compuestos (Rigacci and Stefani, 2016). Particularmente, en estudios realizados en humanos ,AOVEs ricos en compuestos fenólicos han demostrado mejores actividades antioxidante y antiinflamatoria comparados con AO de bajo contenido en compuestos fenólicos (Covas, 2007; Medina-Remón et al., 2016). Asimismo, recientes publicaciones de nuestro grupo de investigación han confirmado los efectos antiinflamatorios e inmunomoduladores de las fracciones FP y FI, así como de compuestos fenólicos aislados presentes en el AOVE en enfermedades inmunoinflamatorias en particular la enfermedad inflamatoria intestinal y la artritis reumatoide y algunos tipos de cáncer como el cáncer colorrectal así como en modelos de inflamación in vitro (Cardeno et al., 2014; Rosillo et al., 2015a; Rosillo et al., 2015b; Sanchez-Fidalgo et al., 2013; Sánchez-Fidalgo et al., 2010). Estos resultados sugieren que la ingesta de AOVE a través de la dieta, así como la suplementación dietética con Justificación y objetivos ! ! 86 fracciones del AOVE o con compuestos fenólicos podría constituir una interesante estrategia para el tratamiento nutricional de patologías relacionadas con la inflamación y el sistema inmune, como es el caso del LES. A pesar del esfuerzo investigador llevado a cabo en los últimos años, aún se desconoce el potencial antiinflamatorio e inmunomodulador del AOVE y de sus componentes bioactivos en el LES. Previamente, diversos estudios preclínicos y clínicos han puesto de manifiesto que el consumo regular de AOVE dentro de la dieta mediterránea posee diversas propiedades beneficiosas que podrían ser de interés en el manejo del LES, debido a su papel cardioprotector, antiinflamatorio, antiartrítico e inmunomodulador (Covas et al., 2015; Miggiano and Gagliardi, 2005; Perez-Jimenez et al., 2005). No obstante, hasta el momento no existían estudios experimentales concluyentes que validasen los posibles beneficios concretos del AOVE o de sus componentes bioactivos en el desarrollo y progresión de esta enfermedad autoinmune, así como de los mecanismos bioquímicos y moleculares involucrados. En vista a estos antecedentes, el objetivo general de la presente tesis doctoral ha sido investigar el funcionalismo del AOVE en el LES experimental. Para ello, nos hemos centrado en una serie de objetivos específicos: 1. Valoración del potencial inmunomodulador de una dieta elaborada con AOVE en un modelo experimental in vivo de LES inducido por pristano (2,6,10,14- tetrametilpentadecano) en ratones BALB/c. Estudio de las rutas bioquímicas y mecanismos de señalización molecular implicados más significativos. 2. Caracterización del protagonismo de la FP del AOVE seleccionado así como los mecanismos y vías de señalización implicadas en modelos in vitro de: 2a. Monocitos y macrófagos de individuos sanos 2b. Células mononucleares de sangre periférica de pacientes con LES y controles sanos. 3. Determinación del papel modulador y antiinflamatorio de compuestos fenólicos presentes en la FP del AOVE, hidroxitirosol (HTy) y acetato de hidroxitirosol (HTy- AC): 3a. Valoración de la actividad antiinflamatoria e inmunomoduladora de HTy, HTy-Ac y otros compuestos fenólicos del AOVE en un modelo in vitro de macrófagos peritoneales murinos. Justificación y objetivos ! ! 87 3b. Valoración del potencial inmunomodulador de dietas enriquecidas con HTy o HTy-Ac en un modelo experimental in vivo de LES inducido por pristano (2,6,10,14-tetrametilpentadecano) en ratones BALB/c. Dilucidar las rutas bioquímicas y mecanismos de señalización molecular implicados. 3c.!Determinación del posible efecto antiinflamatorio de HTy e HTy-Ac en un modelo experimental in vitro de queratinocitos humanos aislados de piel de individuos sanos. Estudio de la modulación génica, producción de citoquinas y vías de señalización implicadas. Bibliografía • Alarcón de la Lastra, C., Barranco, M.D., Motilva, V., and Herrerías, J.M. (2001). Mediterranean diet and health: biological importance of olive oil. 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Racial differences in treatment preferences among lupus patients: a two-site study. Clin Exp Rheumatol 32, 680-688. • Xiong, J., He, Z., Zeng, X., Zhang, Y., and Hu, Z. (2014). Association of vitamin D receptor gene polymorphisms with systemic lupus erythematosus: a meta-analysis. Clin Exp Rheumatol 32, 174-181. ! ! 90 ! ! ! CHAPTER I DIETARY EXTRA VIRGIN OLIVE OIL ATTENUATES KIDNEY INJURY IN PRISTANEINDUCED SLE MODEL VIA ACTIVATION OF HO-1/NRF2 ANTIOXIDANT PATHWAY AND SUPPRESSION OF JAK/STAT, NF-κB AND MAPK ACTIVATION Published in The Journal of Nutritional Biochemistry (2016). 27, 278-288. ! ! ! ! Capítulo I ! ! 93 EL ACEITE DE OLIVA VIRGEN EXTRA DISMINUYE EL DAÑO RENAL EN UN MODELO MURINO DE LUPUS ERITEMATOSO SISTEMICO INDUCIDO POR PRISTANO. Marina Aparicio-Soto1, Marina Sánchez-Hidalgo1, Ana Cárdeno1, María Ángeles Rosillo1, Susana Sánchez-Fidalgo1, José Utrilla2, Inés Martín-Lacave2, Catalina Alarcón-de-la-Lastra1 1Departamento de Farmacología, Facultad de Farmacia, Universidad de Sevilla, España. 2Departamento de Citología e Histología, Facultad de Medicina, Universidad de Sevilla, España. Este capítulo reproduce el siguiente artículo: M. Aparicio-Soto, M. Sánchez-Hidalgo, A. Cárdeno, M. Rosillo, S. Sánchez-Fidalgo, J. Utrilla, I. Martín-Lacave, C. Alarcón-de-la-Lastra, Dietary extra virgin olive oil attenuates kidney injury in pristane-induced SLE model via activation of HO-1/Nrf2 antioxidant pathway and suppression of JAK/STAT, NF-κB and MAPK activation, J Nutr Biochem (2016). 27, 278-288. RESUMEN El lupus eritematoso sistémico (LES) es una enfermedad autoinmune caracterizada por una amplia y diversa afectación del organismo. Recientes estudios han puesto de manifiesto que el aceite de oliva virgen extra (AOVE) puede presentar efectos preventivos en el desarrollo de esta enfermedad inmunoinflamatoria. Sin embargo, en la actualidad se desconoce el posible efecto beneficioso del AOVE en el LES. En el presente estudio se evalúan los efectos de una dieta elaborada con AOVE en un modelo murino de LES inducido por pristano. Para ello se utilizaron ratones de 3 meses de edad, los cuales fueron inyectados con pristano o solución salina y alimentados con dietas elaboradas con aceite de girasol o AOVE. Tras 24 semanas de desarrollo de la enfermedad, los animales fueron sacrificados y bazos y riñones fueron extraídos. La expresión renal de diversas proteínas inflamatorias involucradas en el daño renal fue evaluada mediante western blot y los niveles de metaloproteinasa (MMP)-3 en suero y de citoquinas inflamatorias en riñón y esplenocitos fueron determinadas por ELISA. Nuestro estudio reveló que aquellos animales alimentados con dietas elaboradas con AOVE presentaban una significativa reducción del daño renal inducido por pristano así como menores niveles de MMP-3 en suero y prostaglandina E2 en riñón. Además, la dieta elaborada con AOVE mejoró la expresión de diversas proteínas implicadas en el daño renal . Estos resultados demuestran el interés del AOVE como un alimento funcional de primera categoría, que puede ejercer un papel preventivo/paliativo en el manejo del SLE. Chapter I ! ! • Urpi-Sarda, M., Casas, R., Chiva-Blanch, G., Romero-Mamani, E.S., Valderas-Martínez, P., Arranz, S., Andres-Lacueva, C., Llorach, R., Medina-Remón, A., Lamuela-Raventos, R.M., et al. (2012). Virgin olive oil and nuts as key foods of the Mediterranean diet effects on inflammatory biomakers related to atherosclerosis. Pharmacol Res 65, 577-583. • van Vollenhoven, R.F., Parodis, I., and Levitsky, A. (2013). Biologics in SLE: towards new approaches. Best Pract Res Clin Rheumatol 27, 341-349. • Wang, G., Pierangeli, S.S., Papalardo, E., Ansari, G.A., and Khan, M.F. (2010). Markers of oxidative and nitrosative stress in systemic lupus erythematosus: correlation with disease activity. Arthritis Rheum 62, 2064-2072. • Wang, J.X., Tang, W., Yang, Z.S., Wan, J., Shi, L.P., Zhang, Y., Zhou, R., Ni, J., Hou, L.F., Zhou, Y., et al. (2007). Suppressive effect of a novel water-soluble artemisinin derivative SM905 on T cell activation and proliferation in vitro and in vivo. Eur J Pharmacol 564, 211-218. • Wong, C.K., Ho, C.Y., Li, E.K., and Lam, C.W. (2000). Elevation of proinflammatory cytokine (IL-18, IL-17, IL-12) and Th2 cytokine (IL-4) concentrations in patients with systemic lupus erythematosus. Lupus 9, 589-593. • Wong, C.K., Wong, P.T., Tam, L.S., Li, E.K., Chen, D.P., and Lam, C.W. (2009). Activation profile of intracellular mitogen-activated protein kinases in peripheral lymphocytes of patients with systemic lupus erythematosus. J Clin Immunol 29, 738-746. • Xie, C., Ma, L., Liu, J., Li, X., Pei, H., Xiang, M., and Chen, L. (2013). SKLB023 blocks joint inflammation and cartilage destruction in arthritis models via suppression of nuclear factor-kappa B activation in macrophage. PLoS One 8, e56349. • Xing, Q., Wang, B., Su, H., Cui, J., and Li, J. (2012). Elevated Th17 cells are accompanied by FoxP3+ Treg cells decrease in patients with lupus nephritis. Rheumatol Int 32, 949-958. • Yu, C., Gershwin, M.E., and Chang, C. (2014). Diagnostic criteria for systemic lupus erythematosus: A critical review. J Autoimm 48-49,10-13, ! ! ! CHAPTER II POTENTIAL INVOLVEMENT OF THE PHENOLIC FRACTIONOF EXTRA VIRGIN OLIVE OIL IN PRISTANE-INDUCED LUPUS IN MICE BY REGULATING PPAR-γ DEPENDENT MONOCYTE ACTIVATION ! ! ! ! Capítulo II ! 123 POSIBLE IMPLICACIÓN DE LA FRACIÓN POLIFENÓLICA DEL ACEITE DE OLIVA VIRGEN EXTRA EN UN MODELO MURINO DE LUPUS A TRAVÉS DE LA REGULACIÓN DE LA ACTIVACIÓN MONOCITARIA DEPENDIENTE DE PPAR-γ Marina Aparicio-Soto1, Sergio Montserrat de la Paz2, Marina Sánchez-Hidalgo1, Ana Cárdeno1, Beatriz Bermúdez2, Francisco J. Garcia Muriana2, Catalina Alarcón-de-la-Lastra1 1Department of Pharmacology, Faculty of Pharmacy, University of Seville, Spain 2Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa, CSIC, Seville, Spain. Este capítulo reproduce el siguiente artículo: M. Aparicio-Soto, S. Montserrat de la Paz, M. Sánchez-Hidalgo, A. Cárdeno, B. Bermúdez, FJG. Muriana, C. Alarcón-de-la-Lastra, Potential involvement of phenolic fraction of extra virgin olive oil in pristane-induced lupus in mice by regulating PPAR-γ dependent monocyte activation RESUMEN El lupus eritematoso sistémico (LES) es una enfermedad autoinmune caracterizada por la pérdida del equilibrio y control de la regulación celular del organismo. Monocitos y macrófagos tienen un papel fundamental en la regulación del proceso inflamatorio y la inmunidad innata. Recientes estudios han sugerido que el aceite de oliva virgen extra (AOVE) podría presentar efectos preventivos y paliativos en enfermedades inmunoinflamatorias. En el presente trabajo se evaluaron los efectos de una dieta elaborada con AOVE en macrófagos peritoneales procedentes de ratones con LES inducido por pristano. Además se evaluó el efecto de la fracción polifenólica (FP) del AOVE en un modelo in vitro de monocitos humanos y macrófagos derivados de monocitos. Para ello, ratones BALB/c fueron inyectados con pristano o solución salina y alimentados durante 6 meses con dietas experimentales elaboradas con AOVE o aceite de girasol. Tras el periodo experimental, los animales fueron sacrificados y los macrófagos peritoneales fueron extraídos para la determinación de mediadores inflamatorios. Para el estudio In vitro de los efectos de la FP se utilizaron monocitos y macrófagos derivados de monocitos extraídos de sangre periférica de donantes. La activación de monocitos fue determinada mediante citometría de flujo, reacción en cadena de polimerasa reversa y ELISA. La expresión de marcadores relacionados con el proceso inflamatorio en macrófagos fue determinada mediante citometría de flujo. La dieta elaborada con AOVE disminuyó significativamente la producción de oxido nítrico (NO) y citoquinas en Capítulo II ! macrófagas peritoneales procedentes de ratones con LES. La FP del AOVE disminuyó la producción de NO y citoquinas inflamatorias junto con la expresión génica y proteica de marcadores inmunoinflamatorios en monocitos humanos. Además, la FP moduló la expresión de marcadores anti y proinflamatorios en macrófagos humanos. Estos resultados ponen de manifiesto el papel beneficioso del consumo de AOVE, como un alimento funcional de primera línea, y el especial protagonismo de la FP en dichos efectos beneficiosos, pudiendo ejercer efectos preventivos y moduladores en el tratamiento de enfermedades inmunoinflamatorias, como el LES. Chapter II ! POTENTIAL INVOLVEMENT OF THE PHENOLIC FRACTION OF EXTRA VIRGIN OLIVE OIL IN PRISTANE-INDUCED LUPUS IN MICE BY REGULATING PPAR-γ DEPENDENT MONOCYTE ACTIVATION Marina Aparicio-Soto1, Sergio Montserrat de la Paz2, Marina Sánchez-Hidalgo1, Ana Cárdeno1, Beatriz Bermúdez2, Francisco J. Garcia Muriana2, Catalina Alarcón-de-la-Lastra1 1Department of Pharmacology, Faculty of Pharmacy, University of Seville, Spain 2Laboratory of Cellular and Molecular Nutrition, Instituto de la Grasa, CSIC, Seville, Spain. This chapter reproduces the following article: M. Aparicio-Soto, S. Montserrat de la Paz, M. Sánchez-Hidalgo, A. Cárdeno, B. Bermúdez, FJG. Muriana, C. Alarcón-de-la-Lastra, Potential involvement of phenolic fraction of extra virgin olive oil in pristane-induced lupus in mice by regulating PPAR-γ dependent monocyte activation ABSTRACT Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a loss of balance control of cellular immunoregulation. Monocytes and macrophages are critical effectors and regulators of inflammation and innate immune response. Recent studies suggested that extra virgin olive oil (EVOO) might possess preventive effects on immuneinflammatory diseases management. The present work evaluates the impact of an EVOO diet on peritoneal macrophages from pristane-induced SLE model mice as well as the effect of the EVOO Phenolic fraction (PE) on human monocytes and monocyte-derived macrophages. BALB/c mice received a pristane or saline solution injection and were fed with sunflower oil diet or EVOO diet. After 24 weeks, mice were sacrificed and peritoneal macrophages cultured to evaluate inflammatory markers. In vitro studies with primary human monocytes and monocyte-derived macrophages were performed. Monocyte and macrophage activations were measured by flow cytometry and diverse parameters implicated in the inflammatory process were determined by reverse transcription polymerase chain reaction (RT-qPCR) and Enzyme-Linked ImmunoSorbent Assay (ELISA). EVOO diet significantly reduced nitric oxide (NO) and inflammatory cytokines on peritoneal macrophages from SLE mice. PE decreased NO, cytokine production together with gene and protein expression of immune-inflammatory markers on human monocytes. Moreover, PE treatment downregulated the gene expression of M1 markers and up regulated M2 molecules on M0 macrophages. Altogether, our findings outline that EVOO Chapter II ! and particularly its PE have a significant therapeutic potential for the treatment of immuneinflammatory diseases such as SLE, through the modulation of monocytes/macrophages over-activation, decreasing NO and cytokine production as well as several immuneinflammatory markers, thereby offering a new promising therapeutic strategy in SLE management whose clinical use in SLE patients needs to be further investigated. Keywords: EVOO, SLE, immunomodulation, macrophages, monocytes. References • Alarcon de la Lastra, C., Barranco, M.D., Motilva, V., and Herrerias, J.M. (2001). Mediterranean diet and health: biological importance of olive oil. 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J Biol Chem 270, 9558-9563. • Zhang, Y., Zhao, M., Sawalha, A.H., Richardson, B., and Lu, Q. (2013). Impaired DNA methylation and its mechanisms in CD4(+)T cells of systemic lupus erythematosus. J Autoimmun 41, 92-99. ! CHAPTER IV NATURALLY OCCURRING HYDROXYTYROSOL DERIVATIVES: HYDROXYTYROSYL ACETATE AND 3,4-DIHYDROXYPHENYLGLYCOL MODULATE INFLAMMATORY RESPONSE IN MURINE PERITONEAL MACROPHAGES. POTENTIAL UTILITY AS NEW DIETARY SUPPLEMENTS . Published in Journal of Agricultural and Food Chemistry (2015). 63, 836-846 ! ! ! Capítulo IV ! ! 171 COMPUESTOS POLIFENÓLICOS DERIVADOS DEL HIDROXITIROSOL PRESENTES EN EL ACEITE DE OLIVA VIRGEN EXTRA MODULAN LA RESPUESTA INFLAMATORIA EN MACRÓFAGOS PERITONEALES MURINOS. POTENCIAL USO COMO SUPLEMENTOS DIETÉTICOS. Marina Aparicio-Soto1, Susana Sánchez-Fidalgo1, Alejandro González-Benjumea2, Inés Maya2, José G. Fernández-Bolaños2, Catalina Alarcón-de-la-Lastra1. 1 Departamento de Farmacología, Facultad de Farmacia, Universidad de Sevilla, España 2 Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, España Este capítulo reproduce el siguiente artículo: M. Aparicio-Soto, S. Sánchez-Fidalgo, A. Gonzalez-Benjumea, I. Maya, J. Fernández- Bolaños, C. Alarcón-de-la-Lastra. Naturally occurring hydroxytyrosol derivatives: hydroxytyrosyl acetate and 3,4-dihydroxyphenylglycol modulate inflammatory response in murine peritoneal macrophages. Potential utility as new dietary supplements. J Agric Food Chem. (2015). 63, 836-846. RESUMEN En el presente trabajo se evalúaron los efectos de dos compuestos polifenólicos presentes en el aceite de olive virgen extra (AOVE): hidroxitirosil acetato (2) y 3,4-dihidroxifenilglicol (3), así como dos nuevos acil derivados de 3: 4-(1',2'-dibutanoiloxietil)benceno-1,2-diol (7) y 4-(1',2'-dilauroiloxietil)benceno-1,2-diol (8), en comparación con hidroxitirosol (HTy, 1), en un modelo de inflamación en macrófagos peritoneales murinos estimulados con lipopolisácarido bacteriano (LPS). Los compuestos 2, 3, 7 y 8 mostraron una marcada actividad antioxidante, previniendo la formación de especies reactivas de oxigeno y disminuyendo los niveles de nitritos y la expresión de la enzima oxido nítrico sintasa inducible (iNOS). Sin embargo, solo 2 y 3 disminuyeron la expresión de la enzima ciclooxigenasa inducible (COX-2) mientras que los derivados diacetilados 7 y 8 demostraron una menor actividad antiinflamatoria que el compuesto original 3. En conclusión, los compuestos fenólicos 2 y 3 presentan una marcada actividad antiinflamatoria en un modelo de inflamación en macrófagos peritoneales murinos, pudiendo contribuir a los efectos beneficiosos atribuidos al consumo de AOVE y constituyendo una interesante alternativa al hIdroxItIrosol. debido a su mejor perfil farmacocinético y farmacodinámico. ! ! Chapter IV ! ! NATURALLY OCCURRING HYDROXYTYROSOL DERIVATIVES: HYDROXYTYROSYL ACETATE AND 3,4-DIHYDROXYPHENYLGLYCOL MODULATE INFLAMMATORY RESPONSE IN MURINE PERITONEAL MACROPHAGES. POTENTIAL UTILITY AS NEW DIETARY SUPPLEMENTS Marina Aparicio-Soto1, Susana Sánchez-Fidalgo1, Alejandro González-Benjumea2, Inés Maya2, José G. Fernández-Bolaños2, Catalina Alarcón-de-la-Lastra1. 1 Department of Pharmacology, Faculty of Pharmacy, University of Seville, Spain 2 Department of Organic Chemistry, Faculty of Chemistry, University of Seville, Spain This chapter reproduces the following article: M. Aparicio- Soto, S. Sánchez-Fidalgo, A. Gonzalez-Benjumea, I. Maya, J. Fernández- Bolaños, C. Alarcón-de-la-Lastra. Naturally occurring hydroxytyrosol derivatives: hydroxytyrosyl acetate and 3,4-dihydroxyphenylglycol modulate inflammatory response in murine peritoneal macrophages. Potential utility as new dietary upplements. J Agric Food Chem. (2015). 63, 836-846. ABSTRACT This work evaluated the effects of extra virgin olive oil (EVOO) phenols, hydroxytyrosyl acetate (2) and 3,4-dihydroxyphenylglycol (3), as well as two new acyl derivatives of 3, 4- (1,2-di(butanoyloxy)ethyl)benzene-1,2-diol (7) and 4-(1,2-di(lauroyloxy)ethyl)benzene-1,2- diol (8), on LPS-stimulated murine peritoneal macrophages in comparison with hydroxytyrosol (HTy, 1). Compounds 2, 3, 7 and 8 showed a strong reactive oxygen species (ROS)-scavenging activity, reducing significantly nitrites levels with a significant decrease on iNOS expression [2 (50 µM, 0.44±0.03; 100 µM, 0.44±0.01; p<0.01); 3 (50 µM, 0.37±0.03; 100 µM, 0.37±0.01; p<0.001); 7 (50 µM, 0.45±0.06; p<0.01)]. However, only 2 and 3 downregulated COX-2 expression [2 (50 µM, 0.72±0.04, p<0.05; 100 µM, 0.54±0.06, p<0.01); 3 (50 µM, 0.56±0.05, p<0.05; 100 µM, 0.37±0.04; p<0.001)] and prevented IKBα degradation [2 (100 µM, 1.63±0.14, p<0.01); 3 (100 µM, 1.82±0.09; p<0.01)]; the diacylated compounds 7 and 8 showing worse anti-inflammatory activity than the parent 3. In conclusion, 2 and 3 phenolic derivatives could play an important role in the anti-inflammatory effect of EVOO. 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CHAPTER VI OLIVE OIL-DERIVED PHENOLS EXERT EFFECTIVE ANTI-INFLAMMATORY PROPERTIES BY INTERFERENCE WITH THE NF-ΚB PATHWAY AND TSLP PRODUCTION IN HUMAN KERATINOCYTES ! ! ! ! Capítulo VI ! ! 221 COMPUESTOS FENÓLICOS DEL ACEITE DE OLIVA VIRGEN EXTRA MODULAN LA RESPUESTA INFLAMATORIA A TRAVÉS DE LA VÍA DEL NF-κB EN QUERATINOCITOS HUMANOS Marina Aparicio-Soto1, Margitta Worm2, Marina Sánchez-Hidalgo1, José G. Fernández- Bolaños3, Catalina Alarcón-de-la-Lastra1, Magda Babina2 1Departamento de Farmacología, Facultad de Farmacia, Universidad de Sevilla, España 2Departamento de Dermatología y Alergología, Clínica Universitaria Charité, Berlín, Alemania 3Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, España Este capítulo reproduce el siguiente artículo: Marina Aparicio-Soto, Margitta Worm, Marina Sánchez-Hidalgo, José G. Fernández- Bolaños, Catalina Alarcón-de-la-Lastra, Magda Babina. Olive oil-derived phenols exert effective anti-inflammatory properties by interference with the NF-κB pathway and TSLP production in human keratinocytes. RESUMEN El aceite de oliva virgen extra (AOVE) es rico en una serie de compuestos fenólicos, incluyendo hidroxitirosol (HTy) y acetato de hidroxitirosol (HTy-Ac), que han presentado un amplio rango de efectos beneficiosos. Sin embargo, los posibles beneficios de estos compuestos fenólicos no han sido aún estudiados en el proceso inflamatorio de la piel. El presente estudio fue diseñado para evaluar los potenciales efectos de HTy y HTy-Ac en un modelo de inflamación in vitro con queratinocitos humanos y esclarecer los mediadores y vías de señalización implicados. Para ello, queratinocitos humanos fueron obtenidos de piel de individuos sanos y pre-tratados con HTy-Ac o HTy. Las células fueron estimuladas con interleucina (IL)-1β o con ligando de receptor de tipo Toll 3 (TR3-l) a distintos tiempos experimentales. Los niveles de la citoquina linfopoyetina estromal tímica (TSLP) fueron evaluados por ELISA, mientras que los niveles de expresión génica de TSLP, sus isoformas y diversos genes proinflamatorios fueron determinados mediante la reacción en cadena de la polimerasa con transcriptasa inversa (RT-qPCR). La expresión del factor nuclear kappa B (NF-κB) fue estudiada mediante Western blot. Nuestro estudio puso de manifiesto que HTy y HTy-Ac disminuyeron significativamente la producción y expresión génica de TSLP, junto con los niveles de otros genes proinflamatorios (TNF-α, IL-6, IL-8 y COX-2) en queratinocitos Capítulo VI ! ! humanos, posiblemente a través de la modulación de la actividad de la vía del NF-κB, mediante de la prevención de la degradación de la proteína inhibitoria IκBα y la inhibición de la translocación nuclear de la proteína p65. Estos resultados nos permiten concluir que ambos compuestos fenólicos mejoran el balance inflamatorio en este modelo de queratinocitos humanos a través de la regulación de la producción y expresión génica de TSLP y otros mediadores inflamatorios mediante la modulación de la vía del NF-κB, pudiendo constituir una nueva alternativa para el abordaje de enfermedades inflamatorias de la piel, como es el caso del LES. Chapter VI ! ! OLIVE OIL-DERIVED PHENOLS EXERT EFFECTIVE ANTI-INFLAMMATORY PROPERTIES BY INTERFERENCE WITH THE NF-ΚB PATHWAY AND TSLP PRODUCTION IN HUMAN KERATINOCYTES Marina Aparicio-Soto1, Margitta Worm2, Marina Sánchez-Hidalgo1, José G. Fernández- Bolaños3, Catalina Alarcón-de-la-Lastra1 ,Magda Babina2 1Department of Pharmacology, Faculty of Pharmacy, University of Seville, Spain 2Allergy Center Charité, CCM, Department of Dermatology and Allergy, Charité- Universitätsmedizin Berlin, Germany 3 Department of Organic Chemistry, Faculty of Chemistry, University of Seville, Spain This chapter reproduces the following article: Marina Aparicio-Soto, Margitta Worm, Marina Sánchez-Hidalgo, José G. Fernández- Bolaños, Catalina Alarcón-de-la-Lastra, Magda Babina. Olive oil-derived phenols exert effective anti-inflammatory properties by interference with the NF-ΚB pathway and tslp production in human keratinocytes. ABSTRACT Extra virgin olive oil (EVOO) is rich in diverse phenolic compounds, including hydroxytyrosol (HTy) and hydroxytyrosyl acetate (HTy-Ac) that have present a wide range of beneficial properties. However, the potential benefit from these EVOO bioactive compounds in the inflammatory balance of the skin have not yet been elucidated. The present study was designed to evaluate the potential effects of HTy and HTy-Ac in an in vitro model of human keratinocytes and clarify the mediators and molecular mechanism involved. Human keratinocytes were isolated from skin of healthy donors. HTy-Ac or HTy were added at concentrations ranging 12.5 to 100 µM and 30 minutes later, cells were stimulated with interleukin (IL)-1β or Toll-like receptor 3 ligand (TR3-l) for different time points. Levels of thymic stromal lymphopoietin (TSLP) were evaluated by enzyme-linked immunosorbent assay. TSLP and TSLP isoforms mRNA levels as well as other genes related to the inflammatory response were evaluated by quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR). The expression of nuclear transcription factor-kappa B (NF-κB) pathway was determined by Western blot. EVOO phenols significantly reduced TSLP production and TSLP and its isoforms mRNA levels, as well as proinflammatory gene levels (TNF-α, IL-6 and IL-8) in human keratinocytes. Moreover, HTy and HTy-Ac counteracted induced NF-κB activity through prevention of IκBα degradation and p65 nuclear Chapter VI ! ! translocation. Thus, these EVOO polyphenols, HTy-Ac and HTy, efficiently improve the inflammatory process in human keratinocytes by decreasing TSLP and other inflammatory mediators through interference with the NF-κB pathway. Keywords: EVOO, inflammation, olive oil, skin, TSLP References § Albanesi, C., Scarponi, C., Giustizieri, M.L., and Girolomoni, G. (2005). Keratinocytes in inflammatory skin diseases. Curr Drug Targets Inflamm Allergy 4, 329-334. § Aparicio-Soto, M., Sánchez-Fidalgo, S., González-Benjumea, A., Maya, I., Fernández- Bolaños, J.G., and Alarcón-de-la-Lastra, C. (2015). 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Nat Prod Rep 24, 1225-1244. § Chen, M.C., Chang, W.W., Kuan, Y.D., Lin, S.T., Hsu, H.C., and Lee, C.H. (2012). Resveratrol inhibits LPS-induced epithelial-mesenchymal transition in mouse melanoma model. Innate Immun 18, 685-693. § Cho, J.W., Lee, K.S., and Kim, C.W. (2007). Curcumin attenuates the expression of IL- 1beta, IL-6, and TNF-alpha as well as cyclin E in TNF-alpha-treated HaCaT cells; NF- kappaB and MAPKs as potential upstream targets. Int J Mol Med 19, 469-474. § Christmann, R.B., Mathes, A., Affandi, A.J., Padilla, C., Nazari, B., Bujor, A.M., Stifano, G., and Lafyatis, R. (2013). Thymic stromal lymphopoietin is up-regulated in the skin of patients with systemic sclerosis and induces profibrotic genes and intracellular signaling that overlap with those induced by interleukin-13 and transforming growth factor β. Arthritis Rheum 65, 1335-1346. § Chung, E.Y., Kim, B.H., Hong, J.T., Lee, C.K., Ahn, B., Nam, S.Y., Han, S.B., and Kim, Y. (2011). Resveratrol down-regulates interferon-γ-inducible inflammatory genes in macrophages: molecular mechanism via decreased STAT-1 activation. J Nutr Biochem 22, 902-909. § Cultrone, A., de Wouters, T., Lakhdari, O., Kelly, D., Mulder, I., Logan, E., Lapaque, N., Doré, J., and Blottière, H.M. (2013). The NF-κB binding site located in the proximal region of the TSLP promoter is critical for TSLP modulation in human intestinal epithelial cells. Eur J Immunol 43, 1053-1062. § Di Piazza, M., Nowell, C.S., Koch, U., Durham, A.D., and Radtke, F. (2012). Loss of cutaneous TSLP-dependent immune responses skews the balance of inflammation from tumor protective to tumor promoting. Cancer Cell 22, 479-493. § Dong, J., Segawa, R., Mizuno, N., Hiratsuka, M., and Hirasawa, N. (2016). Inhibitory effects of nicotine derived from cigarette smoke on thymic stromal lymphopoietin production in epidermal keratinocytes. Cell Immunol 302, 19-25. Chapter VI ! ! § Facchini, A., Cetrullo, S., D'Adamo, S., Guidotti, S., Minguzzi, M., Borzì, R.M., and Flamigni, F. (2014). Hydroxytyrosol prevents increase of osteoarthritis markers in human chondrocytes treated with hydrogen peroxide or growth-related oncogene α. PLoS One 9, 1-9.. § Fernandez-Bolanos, J., Rodriguez, G., Rodriguez, R., Heredia, A., Guillen, R., and Jimenez, A. (2002). Production in large quantities of highly purified hydroxytyrosol from liquid-solid waste of two-phase olive oil processing or "Alperujo". Journal of agricultural and food chemistry 50, 6804-6811. § Fernández-Bolaños, J., Rodríguez, G., Rodríguez, R., Heredia, A., Guillén, R., and Jiménez, A. (2002). Production in large quantities of highly purified hydroxytyrosol from liquid-solid waste of two-phase olive oil processing or "Alperujo". J Agric Food Chem 50, 6804-6811. § Fornasa, G., Tsilingiri, K., Caprioli, F., Botti, F., Mapelli, M., Meller, S., Kislat, A., Homey, B., Di Sabatino, A., Sonzogni, A., et al. (2015). Dichotomy of short and long thymic stromal lymphopoietin isoforms in inflammatory disorders of the bowel and skin. J Allergy Clin Immunol 136, 413-422. § González-Correa, J.A., Navas, M.D., Lopez-Villodres, J.A., Trujillo, M., Espartero, J.L., and De La Cruz, J.P. (2008). Neuroprotective effect of hydroxytyrosol and hydroxytyrosol acetate in rat brain slices subjected to hypoxia-reoxygenation. Neurosci Lett 446, 143- 146. § Grandjean-Laquerriere, A., Antonicelli, F., Gangloff, S.C., Guenounou, M., and Le Naour, R. (2007). UVB-induced IL-18 production in human keratinocyte cell line NCTC 2544 through NF-kappaB activation. Cytokine 37, 76-83. § Guo, W., An, Y., Jiang, L., Geng, C., and Zhong, L. (2010). The protective effects of hydroxytyrosol against UVB-induced DNA damage in HaCaT cells. Phytother Res 24, 352- 359. § Guttman-Yassky, E., Lowes, M.A., Fuentes-Duculan, J., Whynot, J., Novitskaya, I., Cardinale, I., Haider, A., Khatcherian, A., Carucci, J.A., Bergman, R., et al. (2007). Major differences in inflammatory dendritic cells and their products distinguish atopic dermatitis from psoriasis. J Allergy Clin Immunol 119, 1210-1217. § Hussain, Z., Katas, H., Mohd Amin, M.C., and Kumolosasi, E. (2014). Efficient immunomodulation of TH1/TH2 biomarkers in 2,4-dinitrofluorobenzene-induced atopic dermatitis: nanocarrier-mediated transcutaneous co-delivery of anti-inflammatory and antioxidant drugs. PLoS One 9, 1-17. § Hussain, Z., Katas, H., Mohd Amin, M.C., Kumolosasi, E., Buang, F., and Sahudin, S. (2013). Self-assembled polymeric nanoparticles for percutaneous co-delivery of hydrocortisone/hydroxytyrosol: an ex vivo and in vivo study using an NC/Nga mouse model. Int J Pharm 444, 109-119. § Kalali, B.N., Köllisch, G., Mages, J., Müller, T., Bauer, S., Wagner, H., Ring, J., Lang, R., Mempel, M., and Ollert, M. (2008). Double-stranded RNA induces an antiviral defense status in epidermal keratinocytes through TLR3-, PKR-, and MDA5/RIG-I-mediated differential signaling. J Immunol 181, 2694-2704. § Killeen, M.J., Linder, M., Pontoniere, P., and Crea, R. (2014). NF-κβ signaling and chronic inflammatory diseases: exploring the potential of natural products to drive new therapeutic opportunities. Drug Discov Today 19, 373-378. § Kumari, V., Babina, M., Hazzan, T., and Worm, M. (2014). TSLP induction by skin irritation is independent of TNF-α, but supported by IL-1. Br J Dermatol 172, 951-960 § Lee, H.C., and Ziegler, S.F. (2007). Inducible expression of the proallergic cytokine thymic stromal lymphopoietin in airway epithelial cells is controlled by NFkappaB. Proc Natl Acad Sci U S A 104, 914-919. § Mateos, R., Trujillo, M., Pereira-Caro, G., Madrona, A., Cert, A., and Espartero, J.L. (2008). New lipophilic tyrosyl esters. Comparative antioxidant evaluation with hydroxytyrosyl esters. J Agric Food Chem 56, 10960-10966. § Meyer-Hoffert, U. (2009). Reddish, scaly, and itchy: how proteases and their inhibitors contribute to inflammatory skin diseases. Arch Immunol Ther Exp (Warsz) 57, 345-354. Results & general discussion ! 248 In the chapter II, we evaluated the impact of EVOO diet on peritoneal macrophages from pristane-induced SLE model mice as well as the effect of the EVOO Phenolic fraction (PE) on human monocytes and monocyte-derived macrophages In the past, most of SLE pathogenesis studies considered the adaptive immune system to be the primary cause of autoimmunity, focusing on the primary abnormalities of B and T lymphocyte functions. However, nowadays studies are also centered on the innate immunity and on how the SLE autoimmune response is initiated and maintained (Li et al., 2010). In this sense, monocytes and macrophages play a pivotal role in the innate immune system with widespread immunological function (Unanue, 1978) and abnormalities in it phenotype and functions have been associated with a variety of autoimmune disorders, including SLE (Katsiari et al., 2010). Oxidative stress plays a substantial role not only in the pathogenesis of autoimmune rheumatic diseases and their complications, but also on specific disease activity. Particularly, a deregulation of redox homeostasis may lead to overproduction of proinflammatory cytokines and nitric oxide (NO) leading to a condition of oxidative stress which plays an important role in SLE (Sukkar and Rossi, 2004). Furthermore, several lines of evidence suggested mechanisms through which the activity of inducible nitric oxide synthase (iNOS) is pathogenic on SLE, altering enzyme activity, increasing the immunogenicity of self antigens, modifying immune tolerance and leading to glomerular and vascular pathology (Oates and Gilkeson, 2006). Thus, iNOS and control oxidative stress targeted therapies may provide the means to reduce the pathogenic consequences of SLE. In this regard, we have shown that EVOO diet decreased NO production on LPS- stimulated peritoneal macrophages from pristane induced BALB/c mice (Chapter II. Figure 1A). Additionally, PE treatment significantly reduced NO with a significant decrease of iNOS protein and NOS-2 gene expression on stimulated human monocytes (Chapter II. Figure 2A, 2B and 2C). These findings are in accordance with those previously described on LPS-activated murine macrophages and acute experimental colitis in mice, where PE inhibited LPS-induced iNOS protein expression through NF-κB and MAPK (Cardeno et al., 2014b; Sanchez-Fidalgo et al., 2013). Modulation of proinflammatory mediators in macrophages is considered one of the strategies to develop therapeutic compounds against several inflammatory diseases. In this regard, Toll like receptor (TLR)-4 plays important roles in the process of macrophage activation and polarization and its activation by LPS triggers the release of cytokines such as TNF-α, IL-1β and IL-6 (Chen et al., 2015). Our results revealed that dietary EVOO significantly reduced TNF-α, IL-6 and IL-17 production on LPS-stimulated peritoneal Results & general discussion ! ! 249 macrophages from pristane-induced lupus mice in comparison with those animals fed with SD suggesting that this EVOO-mediated suppression of cytokines may be beneficial for attenuating pristane-induced lupus (Chapter II. Figure 1B). Moreover, PE decreased TLR4 mRNA levels as well as the gene expression of TNF-α, IL-1β and IL-6 proinflammatory cytokines on LPS-activated human monocytes (Chapter II. Figure 3). Despite the limited available data related to peroxisome proliferator-activated receptors (PPAR)-γ and SLE, some studies have shown the possible therapeutic benefits of PPAR-γ agonists on this disease (Venegas-Pont et al., 2009). Thus, we further studied PPAR-γ signaling pathway, which activation inhibits the expression of inflammatory cytokines and directs the differentiation of immune cells towards anti-inflammatory phenotypes (Chinetti et al., 2000; Martin, 2009). According to our results, human monocytes were responsive to LPS stimulation by decreasing PPAR-γ levels, whereas PE treatment was able to significantly increased PPAR-γ protein expression as well as PPAR-γ mRNA levels on human monocytes suggesting a possible modulation of macrophage activation and polarization by PE EVOO (Chapter II. Figure 2D and 2E). Macrophages are key modulator and effector cells in the immune response because their activation influences and responds to other arms of the immune system. In this regard, macrophages can be classified into M0, M1 and M2 subsets: M1 macrophages [activated by LPS or interferon (IFN)-γ] express a spectrum of proinflammatory molecules, such as IL-1β, IL-6, NO and iNOS whereas M2 macrophages (activated by IL-4) express a wide array of anti-inflammatory molecules (Perez-Jimenez et al., 2005). Our results showed that LPS and IFN-γ increased the expression of M1 markers on M0 macrophages whereas IL- 4 up regulated the gene expression of M2 markers. On the contrary, PE treatment downregulated the gene expression of M1 markers and up regulated M2 molecules on M0 macrophages suggesting that the PE from EVOO prevents the classical macrophages activation by LPS and IFN-γ regulating inflammatory cytokine production and polarized M0 macrophages into M2 macrophages contributing to its anti-inflammatory activities (Chapter II. Figure 4). Finally, we explored whether PE could modify monocyte subset distribution. Human monocytes can be classified into two general categories based on their expression of cellsurface markers. Most monocytes are CD14++CD16+, which are referred as “classical” monocytes and considered as proinflammatory cells because after LPS stimulation they exhibit IL-6 and IL-10 profile and produce high levels of C-C chemokine receptor type 2 (CCR2), which promotes monocyte migration through inflammation sites. “Non-classical” monocytes (CD14+ CD16++) monocytes express low levels of CD14 (a co-receptor with Results & general discussion ! 250 TLR4 for LPS) resulting in a lower cytokine and chemokine response to LPS as well as lower levels of CCR2, decreasing their ability to migrate in response to inflammatory stimuli (Moser et al., 2009; Yang et al., 2014). Moreover, “non-classical” monocytes have also modulatory effects on ‘”classical” monocytes and T cells and appear to be decreased and non-functional in SLE patients (Burbano et al., 2014). In this sense, PE caused a significant decrease of “classical” monocytes but a significant increase of “non-classical” monocytes on LPS human monocytes. Furthermore, PE treatment produced a significant reduction of CCR2+ cells and CCR2 gene expression levels on LPS-treated classical monocytes (Chapter II. Figure 5). Altogether, our findings outline that EVOO and particularly its PE might have a significant therapeutic potential for the treatment of immune-inflammatory diseases such as SLE, through the modulation of monocytes/macrophages over-activation, decreasing NO and cytokine production as well as several immune-inflammatory markers, thereby offering a new promising therapeutic strategy in SLE management whose clinical use in SLE patients needs to be further investigated. In the chapter III, we determined the in vitro effects of the PE from EVOO on peripheral blood mononuclear cells (PBMC) from patients with SLE and healthy donors. Several elements of the immune system are potential targets for therapeutic intervention in patients with SLE. In this sense, IL-6 is implicated in the development and progression of SLE in mice and it is identified as one of the major genetic risk factors for SLE in humans (Jain et al., 2016), thus its modulation seems to be a potential therapeutic approach in SLE management (Wallace et al., 2016). In the present study, we found a remarkable increase of IL-6 production in patients with SLE compared with healthy subjects. Moreover, IL-6 levels were significantly increased in both groups after phytohaemagglutinin (PHA) stimulation, whereas PE treatment was able to prevent this induced cytokine production (Chapter III. Figure 1A). In addition, some studies have provided evidence for the role of IFN in SLE pathogenesis (Hron and Peng, 2004; Santiago-Raber et al., 2003). Similarly, IFN levels are also directly correlated with SLE disease activity and it appears increased in sera from patients with SLE (Bakshi et al., 2015). Our study demonstrates that the in vitro treatment with PE positively regulated the induced production of IFN-γ on stimulated PBMC from patients with SLE and healthy donors, contributing to equilibrate this induced immuneinflammatory imbalance (Chapter III. Figure 1B). In this context, studies on lupus prone mice and SLE patients have documented that high concentrations of TNF-α are correlated with the severity of kidney damage, SLE disease activity and anti-dsDNA antibodies Results & general discussion ! ! 251 production (Mao et al., 2014; Umare et al., 2014). In our study, there were significant differences in TNF- α production between patients with SLE and healthy donors: the levels of this proinflammatory cytokine appeared significantly increased in unstimulated and PHA-stimulated cells from patients with SLE compared with healthy donors. Moreover, PE treatment was able to decrease in a significant way this induced production in both groups (Chapter III. Figure 1C). The overproduction of IL-1β is involved in the pathogenesis of SLE and other autoimmune diseases (Umare et al., 2014). In the present study we did not find differences in IL-1β production after PHA stimulation on PBMC, but these results are in accordance with previous studies, where PHA stimulation did not alter the production of this cytokine (De Groote et al., 1992; Le Meur et al., 1999). However, we found that IL-1β production in patients with SLE was significantly lower than in healthy donors, which may be due to the inactive cohort selected for this study. Remarkably, PE treatment was capable of decreasing the production of this cytokine in both groups (Chapter III. Figure 1D). IL-10 is an important immunoregulatory cytokine with pleiotropic effects, which plays a crucial role in immune-inflammatory diseases. Particularly, IL-10 inhibits T cell function by suppressing the expression of proinflammatory cytokines such as TNF-α, IL-1 or IL-6 (Ding et al., 1993). High IL-10 expression seems to have a causal role in SLE pathogenesis and development of lupus nephritis and its polymorphisms contribute, at least in part, to the genetics involved in SLE (Abdallah et al., 2016; Zhang et al., 2013). Our study shows that PHA produces a significant increase of IL-10 levels that was significantly elevated in patients with SLE when compared with healthy donors. Moreover, PE treatment showed distinctive activities between groups: in healthy donors, IL-10 levels were enhanced after PE treatment whereas in patients with SLE its levels were significantly decreased (Chapter III. Figure 1E). Therefore, since several studies in SLE have shown that anti-IL-10 treatment can decrease disease activity in terms of clinical features and biologic markers, PE from EVOO could be a promising approach for SLE management (Abdallah et al., 2016). T cell activation is a fundamental step for the onset of the adaptive immune response. In this regard, CD69 is thought to be among the earliest surface markers of T-cells activation, and its changes are correlated with SLE disease activity (Draborg et al., 2014). In fact, active SLE patients show increased CD69 expression levels in comparison with inactive patients and healthy subjects (Chavez-Rueda et al., 2005). In the present study, we describe that the PE from EVOO significantly decreased CD69 induced expression levels on CD4+ T lymphocytes from SLE patients, with a similar but dose-dependent effect on CD4+ T lymphocytes from healthy donors. Moreover, after PHA stimulation, CD69 Results & general discussion ! 252 expression levels were lower in patients with SLE when compared with healthy donors, which could be due to the influence of the pharmacological therapy on the expression of activation markers on CD4+ T cells (Chapter III. Figure 4). Current evidence suggests that a pathologic CD4+ subset characterized by impaired extracellular signal–regulated kinases (ERK) pathway signal contributes to SLE pathogenesis. In this sense, defective T cell pathway signaling occurs in SLE and it has been directly implicated in causing epigenetic abnormalities that result in lupus-like autoimmunity (Gorelik and Richardson, 2010). Moreover, different studies have shown that EVOO as well as some EVOO bioactive components are able to modulate ERK phosphorylation in different inflammatory models, including pristane induced SLE in mice (Aparicio-Soto et al., 2016; Rosillo et al., 2015). In our study, we found that ERK phosphorylation was significantly increased after PHA stimulation compared with nonstimulated cells in patients with SLE and healthy donors. In addition, PE acts as a key positive regulator of ERK1/2 phosphorylation, decreasing PHA-induced phosphorylation. On the contrary, previous studies have shown that ERK signaling pathway is decreased in T cells from SLE patients, been associated with an over-activation of T cells (Wang et al., 2016). Our data did not shown this expected decreased expression of ERK pathway, but similar results between patients with SLE and healthy donors, but these discrepancies might be due to the inactive condition of the cohort of patients along with their pharmacological treatment (Chapter III. Figure 3A). Nevertheless, given that the position of ERK in this disease seems to be controversial, further studies are needed to clarify the role of this pathway in SLE. NF-κB is a transcription factor widely accepted as a critical regulatory modulator of inflammation and innate/adaptive immunity and its inhibition can reduce the expression of many genes encoding key inflammatory mediators, such as IL-6, IL-10 and TNF-α (Tak and Firestein, 2001; Wang et al., 1995). The deregulation of NF-κB activation is considered to drive many human diseases and particularly, several studies suggested that NF-κB might play a prominent role in the onset and progression of SLE, been critically involved in B cell differentiation and autoantibodies production (Criscione and Pisetsky, 2003; Hu et al., 2015). In our study we found that the expression of IκBα, the inhibitory protein of NF-κB, was significantly increased by PE treatment after PHA stimulation in patients with SLE as well as in healthy donors, with similar results in both groups, suggesting that the activity of PE might be related to its capacity to modulate NF-κB activation and it may be an important mechanistic link to the ability of diet to offset immune deregulation (Chapter III. Figure 2A and 2B). Results & general discussion ! ! 253 Although this study can make preliminary evidences of the therapeutic mechanism of PE from EVOO in SLE, the impact of EVOO and its bioactive components in patients with SLE requires further study. Regardless, the present study proves that PE modulates cytokine production and attenuates induced activation of T cells in patients with SLE and healthy donors, probably through NF-κB signaling pathway. Moreover, we found statistically differences in the production of IL-6, TNF-α, and IL-10 in patients with SLE, whose levels were markedly higher after PHA stimulation, and in the levels of IL-1β, which production was significantly lower. Furthermore, PE showed an interesting immunomodulatory effect in IL-10 production, enhancing IL-10 levels in healthy donors and decreasing them in patients with SLE. Thus, this study does provide the first evidence that PE from EVOO has an anti-inflammatory and immunomodulatory role in human PBMC, contributing to modulate the cytokine imbalance in patients with SLE and it might therefore be considered as a potential dietary strategy in SLE management. In the chapter IV, we evaluated the effects of two EVOO phenols, hydroxytyrosol acetate (HTy-Ac, 2) and 3,4-dihydroxyphenylglycol (3), as well as two new acyl derivatives of 3, on LPS-stimulated murine peritoneal macrophages in comparison with hydroxytyrosol (HTy, 1). Balance disruption of the intracellular reduction-oxidation state has been observed in activated macrophages, which leads to oxidative stress characterized by a major shift in the cellular redox balance and usually accompanied by ROS-mediated damage (Brüne et al., 2013). Therefore, modulators of ROS production and ROS-induced signaling pathways, especially in macrophages, could represent potential targets for antiinflammatory intervention (Li et al., 2012). Our findings have shown for the first time that these phenol compounds, mainly compounds 2 and 3, showed significant antiradical and antioxidant activities and thus prevented the progression of cellular damage induced by LPS acting as effective antioxidants (Chapter IV. Figure 1). Stimulation of macrophages by LPS induces transcription of iNOS gene and generation of large amounts of NO. NO acts as an intracellular messenger, which modulates the formation of endogenous ROS including hydrogen peroxide, peroxynitrite, and other potential oxidants that orchestrate the inflammatory response (Li et al., 2012). In the present study we found that exposure of peritoneal macrophages to LPS resulted in a significant increase of nitrite/nitrate levels as a NO production indicator and an upregulation of iNOS protein expression. However, 1-3, and 7 compounds reduced significantly both nitrites production and iNOS expression, in contrast to 8, which showed a Results & general discussion ! 254 lower reduction of NO production at 50 µM but no changes in LPS-induced iNOS expression (Chapter IV. Figure 2 and 3). The cyclooxygenase (COX)-2, the inducible isoform of COX, is the key enzyme that catalyzes the two sequential steps in the biosynthesis of PG from arachidonic acid, and plays a critical role in the inflammatory response. Our data showed that COX-2 protein expression was significantly induced by LPS stimulation, but treatments of LPS-stimulated murine macrophages with 1, 2 and 3 produced a remarkable downregulation of COX-2 expression. Nevertheless, no changes were observed after treatment with 7 and 8 (Chapter IV. Figure 4). LPS can trigger groups of different TLR especially TLR-4, starting downstream signaling cascades, including MAPK and the NF-κB pathways. NF-κB, as a dimeric transcription factor exists in the cytoplasm as an inactive complex with the inhibitory protein IkBα. When cells are challenged with proinflammatory stimuli, for example LPS, IkBα undergoes phosphorylation and subsequently ubiquitination, allowing NF-κB to translocate to the nucleus. As a result, NF-κB binds to kB enhancer elements present in the promoter region of many proinflammatory genes, for instance, iNOS and COX-2 (Lee and Surh, 2012). Our results show that pretreatment with 2 and 3 significantly prevented IkBα degradation after LPS stimulus. However, no significant changes were observed in IkBα expression after 1, 7, and 8 treatments (Chapter IV. Figure 5). Others researches have shown that anti-inflammatory activity by EVOO isolated phenols is mediated, at least in part, by NF-κB signaling. For instance, 1 at 25-100 µM has been demonstrated to block NF- κB intracellular oxidative stress attenuating iNOS and COX-2 expressions in human monocytes (Zhang et al., 2009). This capacity to regulate NF-κB genes was also described in J774 murine macrophages and in N2a cells against amyloid-β-induced toxicity (Maiuri et al., 2005; St-Laurent-Thibault et al., 2011). Nevertheless, our data are in agreement with another previous study in which no significant changes after co-incubation with 1 in HT-29 human cells were observed (Cárdeno et al., 2013). Moreover, a recent paper has showed that HTy suppressed NO production by decreasing iNOS gene expression through a mechanism independent of the NF-κB signaling pathway (Takeda et al., 2014). Maybe these discrepancies could be explained in terms of different phenol concentrations and changes on cell experimental conditions. As above mentioned, LPS can induce MAPK activation. MAPK are a family of serine–threonine kinase enzymes which orchestrate the recruitment of gene transcription, protein biosynthesis and differentiation and allow cells to respond to oxidative stress and inflammatory stimuli from their extracellular environment (Munoz and Ammit, 2010). In fact, Results & general discussion ! ! 255 MAPK have been shown to play important roles in iNOS and COX-2 upregulation induced by various stimuli in mammalian cells. Nevertheless, our findings demonstrated no significant changes in p38 and JNK protein phosphorylation after treatments, suggesting that MAPK signaling pathway does not make a consistent contribution in the antiinflammatory effects of 2 and 3 in mouse peritoneal macrophages (Chapter IV. Figure 6). In conclusion, this study establishes for the first time that the natural HTy derivatives 2 and 3 present in EVOO inhibit LPS-induced oxidative stress and inflammatory response via direct downregulation of NO generation. These protective effects seem to be due to a downregulation of iNOS and COX-2 protein expression via inhibition of NF-κB signaling pathway. Acylation of the side chain of 3,4-hydroxyphenylglycol 3 by catechol protection, acylation of aliphatic hydroxyl groups, and deprotection afforded diacylated 7 and 8, which show worse anti-inflammatory activity than the parent 3. Furthermore, 8 with the longer acyl chains, showed the worst behavior on the stimulated macrophages. The implication of this study for nutrition and general health of the population recline in the possible use of natural HTy derivatives with better hydrophilic/ lipophilic balance, thus improving its pharmacodynamic and pharmacokinetic profiles, as new dietary supplements, however, further in vivo studies are necessary to fully understand the role of these EVOO phenols in the management of inflammatory-related diseases. The objective of the chapter V was to determinate the potential protective role of HTy or HTy-Ac dietary supplementation in the prevention and development of SLE in a murine pristane-induced experimental lupus model. We have demonstrated for the first time that both HTy and HTy-Ac dietary supplementation modulated pristane induced SLE on mice, preventing renal inflammatory markers through NF-κB, STAT3 and MAPK signaling pathways. SLE is a multisystem autoimmune disease in which immunological abnormalities, including increased production of proinflammatory cytokines and lymphocyte hyperactivity, are manifested in several organs, mostly in kidney and spleen (Kyttaris, 2010; Takeuchi et al., 2005). Th1 cytokines, such as TNF-α and IL-1β, are known to contribute directly to the inflammatory injury in SLE (Lourenço and Cava, 2009; Umare et al., 2014). In addition, TNF-α has a pivotal role in T cell apoptosis induction (Aringer and Smolen, 2003). Moreover, Th2 cytokines, like IL-6, have a remarkably value in lupus autoantibody production (Richards et al., 1999) whereas that IL-17 cytokines play an important role in immune response amplification via local production of chemokines and cytokines and monocytes and neutrophils recruitment, aggravating inflammation and injury in target organs (Li et al., 2015). In this study, we found lower levels of induced TNF-α, IL-1B, IL-6 and Results & general discussion ! 256 IL-17 in supernatants from splenocytes and peritoneal macrophages from pristane mice fed with HTy and HTy-Ac supplemented diets in comparison with pristane mice fed with standard diet (Chapter V. Figure 2), suggesting that dietary intake of HTy and HTy-Ac contribute to reduce the inflammatory response by suppressing the production of Th1, Th2 and Th17 cytokines in pristane-treated SLE mice. PGE2 plays a pivotal role in the development of inflammatory responses and tissue injuries, acting as a mediator of Th1/Th2 nature of immune response (Chae et al., 2008). Consequently, lower levels of PGE2 were found in LPS-stimulated splenocytes and peritoneal macrophages from pristane mice fed with HTy and HTy-Ac diets compared to those from pristane mice fed with standard diet (Chapter V. Figure 3A and 3B). Lupus nephritis appears in approximately 60% of lupus patients and it is one of the main causes of SLE morbidity and mortality. Accordingly with the results found in splenocytes and macrophages, HTy and HTy-Ac enriched diets reduced not only PGE2 production in kidney but also mPGES-1 protein expression, a key enzyme in PGE2 biosynthesis (Chapter V. Figure 3C). Therefore, the regulation of these proinflammatory biomarkers might represent a potential target to modulate kidney damage by HTy and HTy-Ac dietary intake. On the other hand, the generation of ROS has a wide impact in acute and chronic kidney injuries in patients with lupus nephritis. Nrf2, the major regulator of the antioxidant response, constitutes a primary cellular defence mechanism with a protective role by scavenging ROS and regulating HO-1 expression (Herrada et al., 2012; Jiang et al., 2014). It is known that HO-1 induction therapy ameliorates lupus nephritis, probably by multiple mechanisms including NO synthesis suppression, inhibition of antibody production and modulation of cytokine production (Takeda et al., 2004). In the present study, Nrf2 and HO- 1 proteins expressions were decreased in pristane group but HTy and HTy-Ac dietary supplementation could restore Nrf2/HO-1 expression, conferring a potential beneficial effect of HTy and HTy-Ac supplementation in this SLE model (Chapter V. Figure 4A). In addition, Nrf2 inhibits SLE development of lupus by suppressing NF-κB-mediated inflammatory response (Jiang et al., 2014). Therefore, we investigated whether dietary HTy and HTy-Ac might prevent the activation of NF-κB signaling pathway. In our study, kidney IκBα protein expression decreased after pristane injection. Conversely, kidneys from pristane mice fed with HTy or HTy-Ac showed similar IκBα expression levels than sham group. In accordance with these results, p65 translocation was also reduced in HTy or HTy-Ac groups compared to pristane group mice (Chapter V. Figure 4B). STAT3 inhibition represents a potential therapeutic target in SLE because its activation is critical for T:B cell cross-talk, Th17 generation and T cell migration, playing a Results & general discussion ! ! 257 central role in lupus pathogenesis and particularly in lupus nephritis (Edwards et al., 2015). Importantly, MAPK phosphorylate STAT3, leading to STAT3 activation by phosphorylation on tyrosine residues resulting in the formation of STAT dimers that translocate into the nucleus to bind specific DNA sequences (Wang et al., 2005). Our findings show that STAT3 and MAPK phosphorylation are increased in kidneys from SLE pristane mice but dietary HTy or HTy-Ac supplementation significantly reduced their phosphorylation (Chapter V. Figure 5A and 5B). Nowadays, nutritional therapy, including diet modification and the use of nutritional supplements, could be a promising way to approach SLE due to the potential prophylactic effects without the side effects of the classic pharmacological therapy and its possible contribution to reduce comorbidities and improve quality of life in SLE patients (Greco et al., 2013). Diet quality in SLE patients is important since these patients present higher risk for another diseases, which are directly influenced by diet. In this regard, phenolic compounds included in our daily diet have shown a broad spectrum of beneficial effects and bioactive properties that highlight their possible supportive role in primary and secondary prevention and management of SLE. In accordance with the results of the present study, dietary HTy and HTy-Ac supplementation significantly reduced proinflammatory cytokines on peritoneal macrophages and splenocytes with a remarkable blockage of JAK/STAT, MAPK and NF-κB pathways, showing for the first time that the dietary intake of these phenolic compounds improve different immune-inflammatory markers on a SLE mice model. However, the full biological significance of these results on SLE is a key issue and further investigations are warranted to provide insights into the influence of these phenolic compounds and their beneficial effects on human SLE. Nevertheless, our results provide preliminary evidences that these phenolic compounds could exert benefits again autoimmune and inflammatory disorders such as SLE and provide a new dietary strategy in the prevention and management of SLE. The focus of the chapter VI was to study the effects of HTy and HTy-Ac in an in vitro model of human keratinocytes and clarify the mediators and molecular mechanism involved. Apart from their well-established anti-oxidant properties, EVOO polyphenols are also found to have another beneficial properties of medical interest including antiatherogenic, antitumor, anti-inflammatory and immune-modulatory activities that appear to be only in part related to their antioxidant power (Rigacci and Stefani, 2016). Particularly, HTy and HTy-Ac have shown interesting prophylactic and preventive effects in Results & general discussion ! 264 • Takeda, Y., Takeno, M., Iwasaki, M., Kobayashi, H., Kirino, Y., Ueda, A., Nagahama, K., Aoki, I., and Ishigatsubo, Y. (2004). Chemical induction of HO-1 suppresses lupus nephritis by reducing local iNOS expression and synthesis of anti-dsDNA antibody. 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(1995). Interleukin (IL)-10 inhibits nuclear factor kappa B (NF kappa B) activation in human monocytes. IL-10 and IL-4 suppress cytokine synthesis by different mechanisms. J Biol Chem 270, 9558-9563. • Wang, R., Cherukuri, P., and Luo, J. (2005). Activation of Stat3 sequence-specific DNA binding and transcription by p300/CREB-binding protein-mediated acetylation. J Biol Chem 280, 11528-11534. • Yang, J., Zhang, L., Yu, C., Yang, X.F., and Wang, H. (2014). Monocyte and macrophage differentiation: circulation inflammatory monocyte as biomarker for inflammatory diseases. Biomark Res 2, 1. • Zhang, X., Cao, J., Jiang, L., and Zhong, L. (2009). Suppressive effects of hydroxytyrosol on oxidative stress and nuclear Factor-kappaB activation in THP-1 cells. Biological & pharmaceutical bulletin 32, 578-582. • Zhang, Y., Zhao, M., Sawalha, A.H., Richardson, B., and Lu, Q. (2013). Impaired DNA methylation and its mechanisms in CD4(+)T cells of systemic lupus erythematosus. J Autoimmun 41, 92-99. • Ziegler, S.F. (2012). Thymic stromal lymphopoietin and allergic disease. J Allergy Clin Immunol 130, 845-852. CONCLUSIONES Conclusiones ! ! 267 Los resultados obtenidos en la presente tesis doctoral nos permiten concluir: 1. Una dieta elaborada con AOVE con alto contenido en componentes fenólicos redujo significativamente las alteraciones histológicas observadas en riñón en un modelo murino de LES inducido por pristano, en comparación con aquellos animales alimentados con dieta estándar, con aceite de girasol como fuente lipídica. Igualmente, se observaron menores niveles de MMP-3 en suero y PGE2 en riñón. Además, la dieta elaborada con AOVE mejoró la expresión de diversas proteínas implicadas en el daño renal características del LES, así como la producción de citoquinas inflamatorias en esplenocitos y macrófagos peritoneales. Estos resultados sugieren que el tratamiento dietético con AOVE puede ejercer un papel preventivo/paliativo en el manejo del SLE. 2. La FP del AOVE disminuyó la producción de NO y citoquinas inflamatorias junto con la expresión génica y proteica de marcadores inmunoinflamatorios en monocitos humanos. Asimismo, la FP moduló la expresión de marcadores anti y proinflamatorios en macrófagos humanos. Estos resultados ponen de manifiesto el especial protagonismo de la FP en los efectos inmunomoduladores del AOVE, que podrían ser de especial interés en el abordaje de enfermedades inmunoinflamatorias. 3. La FP del AOVE disminuyó la frecuencia de células CD69+ y la producción de IFN γ, TNF-α, IL-6, IL-1β e IL-10 en controles sanos y pacientes con LES. También se detectaron significativos incrementos en la producción de IL-6, TNF-α, e IL-10 en dichos pacientes y en los niveles de IL-1β, cuya producción fue significativamente menor tras la estimulación con PHA comparado con controles sanos. Asimismo, la FP incrementó la producción de IL-10 en controles sanos pero la disminuyó en pacientes con LES. Asimismo, la FP incrementó la expresión de la proteína inhibitoria IκB y disminuyó la fosforilación de la proteína ERK en pacientes con LES y pacientes sanos. Estos resultados resaltan el posible papel antiinflamatorio e inmunomodulador de la FP del AOVE en la modulación de la producción de citoquinas inflamatorias y la activación de células T, probablemente a través de la vía del NF-κB. Conclusiones ! 268 4. En un modelo de macrófagos peritoneales murinos estimulados con LP, los compuestos fenólicos del AOVE, HTy e HTY-Ac exhibieron marcadas actividades antioxidantes, previniendo la formación de especies reactivas de oxigeno, y antiinflamatoria, disminuyendo los niveles de nitritos y la expresión de las proteínas iNOS y COX-2. En particular, HTy-Ac podría constituir una interesante alternativa al HTy debido a su mejor perfil farmacocinético y farmacodinámico. 5. En un modelo murino de LES inducido por pristano, la suplementación dietética con HTy o HTy-Ac redujo significativamente la producción de citoquinas proinflamatorias en riñón, macrófagos peritoneales y esplenocitos. Igualmente, las dietas con HTy e HTy-Ac minimizaron el daño renal inducido por pristano, disminuyendo la expresión de diversas proteínas implicadas en el daño renal través de diversas vías involucradas en el LES, sugiriendo que ambos compuestos fenólicos pueden representar una nueva opción en el tratamiento nutricional del LES. 6. En un modelo de inflamación en queratinocitos humanos HTy y HTy-Ac disminuyeron significativamente la producción y expresión génica de TSLP junto con los niveles de otros genes proinflamatorios (TNF-α, IL-6, IL-8 y COX-2), probablemente a través de la capacidad de ambos compuestos para modular la actividad de la vía del NF-κB,, mediante la prevención de la degradación de la proteína inhibitoria IκBα y la translocación nuclear de la proteína p65. Nuestros resultados indican que ambos compuestos fenólicos mejoran el balance inflamatorio en este modelo de inflamación, y podrían constituir una nueva posibilidad terapéutica para el abordaje de enfermedades inflamatorias de la piel, como es el caso del LES. Abstract ! ! 269 Systemic lupus erythematosus (SLE) can be defined as a chronic inflammatory and autoimmune disease that can affect multiple organ systems, including skin, joints, kidneys and the brain, among others (Noble et al., 2016). SLE is characterised by a deposition of immune complexes, formed in large amounts as antinuclear antibodies bind to the abundant nuclear material in blood and tissues, along with disturbances in both innate and adaptive immunity manifest by disorders in cytokines, apoptotic cell clearance, B-cell immunity, and T-cell signaling (Crispín et al., 2010; Lisnevskaia et al., 2014). It is also characterised by its clinical and pathogenic complexity, difficult diagnosis and the high number of complications that can affect the patients´ quality of life (Petri et al., 2012). SLE affects multiple systems and its presentation and course are highly variable, ranging from indolent to fulminant. The most common clinical manifestations include fatigue, loss of appetite and weight, cutaneous lesions (mainly malar rash), arthritis, serositis (pleuritis and/orpericarditis), renal or central nervous system involvement and hematological manifestations (cytopenias) associated with several autoantibodies, particularly antinuclear antibodies (ANA)(Petri et al., 2012). Nowadays, the overall aim of SLE therapy is to control disease activity. Mild activity can be managed with non-steroidal anti-inflammatory drugs or low-dose glucocorticoids, but more severe manifestations require more advanced treatment (Bertsias et al., 2012; Jordan and D'Cruz, 2015; Kuhn et al., 2015). Typical SLE management includes the use of antimalarial drugs (mainly hydroxychloroquine and chloroquine), immunosuppressive agents, biological agents and some adjunctive therapies following international recommendations (Kuhn et al., 2015). Nevertheless, SLE management remains complicated owing to the biological heterogeneity between patients and the lack of safe and specific targeted therapies. Thus, the search for new therapeutic targets and strategies that can act more selectively on certain routes or biological processes and improve the course of disease or reverse the outbreak phase without generating collateral damage to unaffected tissues and organs is the pillar underlying current research in SLE. Nutritional therapy, including diet modification and the use of nutritional supplements, could be a promising way to approach SLE owing to both its potential prophylactic effects, without the side effects of the classic pharmacological therapy, and its possible contribution to reducing comorbidities and improving the quality of life of patients with SLE (Greco et al., 2013). In this regard, there is evidence that dietary factors can contribute to the geoepidemiology of autoimmune diseases (Selmi, 2010). Recent studies have suggested that the traditional Mediterranean diet might confer protection from certain chronic diseases related to oxidative stress, inflammation and the immune Abstract ! ! 270 system. The beneficial effects of the Mediterranean diet have been proven in cancer, cardiovascular diseases, obesity and arthritis (Casas et al., 2014; Cárdeno et al., 2013; Toledo et al., 2013). Likewise, epidemiological data have shown a decreased prevalence of rheumatic diseases in Mediterranean countries when compared with Northern Europe, and some clinical trials have demonstrated that the Mediterranean diet improves rheumatic symptoms and decreases the use of anti-inflammatory drugs and classic pharmacotherapy-related side effects (McKellar et al., 2007; Miggiano and Gagliardi, 2005). In comparison with other healthy diets, olive oil is located at the centre of the Mediterranean diet pyramid as the principal source of dietary lipids, being described as a key bioactive food because of its high nutritional quality and its particular composition, especially relevant in the case of extra virgin olive oil (EVOO)(Bach-Faig et al., 2011; Puertollano et al., 2010). EVOO is the olive oil obtained from the fruit of the olive tree (Olea Europea L.) solely by mechanical or other physical preparation under conditions that do not alter its natural composition. Traditionally, the beneficial effects of EVOO have been ascribed to its high content of MUFA, particularly oleic acid, which has shown multiple beneficial properties (Bermudez et al., 2011; Waterman and Lockwood, 2007). However, in recent years converging evidence indicate that EVOO minority components contribute to the benefits of its consumption. In this sense, olive oil polyphenols such as hydroxytyrosol (HTy), tyrosol, hydroxytyrosyl acetate (HTy-Ac) and oleuropein may be responsible for some of the beneficial properties of EVOO activities that appear to be only in part related to the antioxidant power of these molecules (Rigacci and Stefani, 2016). Despite the broad range of evidences of the potential benefits of EVOO in immune-inflammatory related diseases, the particular effects of EVOO and its potential bioactive components as well as the specific mechanisms by which them may exert their anti-inflammatory and immunomodulatory effects in SLE are still not elucidated. Therefore, the objectives of this thesis were: 1. To determinate the potential beneficial effects of EVOO in a model of SLE induced by pristane (2,6,10,14-tetramethylpentadecane) in BALB/c mice and explore the molecular mechanism and signaling pathways involved. 2. To evaluate the role of the phenolic fraction (PE) of EVOO and elucidate molecular mechanism and signaling pathways on: 2a. Monocytes and macrophages from healthy donors 2b. Peripheral blood mononuclear cells (PBMC) isolated from patients with SLE and healthy donors. Abstract ! ! 271 3. To investigate the potential effects of HTy and HTy-Ac, phenolic compounds from EVOO: 3a. To evaluate the anti-inflammatory effects of HTy, HTy-Ac and other phenolic compounds of EVOO in an in vitro model of murine peritoneal macrophages. 3b. To investigate the effects of HTy and HTy-Ac enriched diets in a model of SLE induced by pristane (2,6,10,14-tetramethyilpentadecane) in BALB/c mice and explore the molecular mechanism and signaling pathways involved. 3c. To determinate the effects of HTy and HTy-Ac in an in vitro model of human keratinocytes isolated from skin of healthy donors. Study of the gene modulation, cytokine production and signaling pathways involved. Results and discussion 1. Dietary extra virgin olive oil attenuates kidney injury in pristane-induced SLE model via activation of HO-1/Nrf2 antioxidant pathway and suppression of JAK/STAT, NF-κB and MAPK activation [Aparicio-Soto et al. (2016) J Nutr Biochem 27, 278-88]. BALB/c Mice were randomized into four experimental groups: (1) sham sunflower diet group were fed with a diet elaborated with a marketable sunflower oil, (2) pristane sunflower diet group, fed with a diet elaborated with a marketable sunflower oil (3) sham EVOO diet group were fed with a diet made with a marketable EVOO picual variety and (4) pristane EVOO diet group, fed with a diet made with a marketable EVOO picual variety. At 16 weeks of age, SLE was induced by a pristane injection according to the procedure described by Satoh and Reeves (Satoh and Reeves, 1994). At the end of the experimental period (24 weeks), animals were euthanized and specimens including blood, spleen, thymus and kidney were collected. Our results revealed that EVOO, as the lipid component of the diet, had a therapeutic role in the development of SLE in female BALB/c in comparison with those pristane mice fed with sunflower oil diet. This beneficial effect was well correlated to an improved renal damage score, as reflected by milder proteinuria and a minor inflammatory mononuclear cells infiltration in renal interstitium and reduced paw swelling. Moreover, dietary EVOO ameliorated proinflammatory cytokines production, reducing tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and IL-17 production in lipopolysaccharide (LPS)-stimulated splenocytes in comparison with those animals fed with sunflower oil. Besides, metalloproteinase (MMP)-3 serum levels were increased in pristanetreated mice fed with sunflower diet whereas those pristane-treated animals which Abstract ! ! 272 received EVOO diet expressed similar serum MMP-3 levels to those described in control mice contributing to minimize renal damage produced by pristane induction. In addition, we demonstrated that pristane injection increased kidney phosphorylation of p38, JNK and ERK mitogen-activated protein kinases (MAPK) and signal transducer and activator of transcription 3 (STAT3) in those animals fed with sunflower diet, and it was directly related to the severity of renal damage. On the contrary, EVOO diet significantly reduced both MAPK and STAT3 activation in kidney. Moreover, EVOO diet ameliorated PGE2 production and microsomal prostaglandin E synthase (mPGES)-1 protein expression in kidney from pristane-treated mice. Our data also indicate that nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase (HO-1) protein expressions were upregulated in those mice fed with EVOO and the activation of the nuclear factor kappalight-chain-enhancer of activated B cells (NF-κB) pathway was drastically ameliorated after EVOO diet. In summary, our findings suggest that dietary EVOO has protective effects, alleviating pristane-induced SLE in mice by reducing levels of inflammatory mediators and activating HO-1/Nrf2 antioxidant pathway by blocking janus kinase (JAK)/STAT, NF-κB and MAPK-mediated inflammatory response and it may constitute a potential functional food with a preventive/palliative role in the management of SLE. 2a. Potential involvement of the phenolic fraction of extra virgin olive oil in systemic lupus erythematosus by regulating PPAR-γ dependent monocyte activation We evaluated the effects of dietary EVOO on peritoneal macrophages from pristane-induced SLE mice as well as the immune-inflammatory effects of the PE of EVOO on human primary monocytes and monocyte-derived macrophages focusing on cytokines production and molecular mechanisms. Sixty mice were randomized into four experimental groups and fed with diets elaborated with marketable sunflower oil or marketable EVOO picual variety. SLE was induced by pristane injection according to the procedure previously described (Satoh and Reeves, 1994). After 6 months of SLE induction, mice were sacrificed and peritoneal macrophages were collected and cultured. Study subjects were recruited at “Virgen del Rocío” University Hospital (Seville, Spain) for blood collection and isolation of human monocytes. Modulation of proinflammatory mediators in macrophages is considered one of the strategies to develop therapeutic compounds against several inflammatory diseases. (Chen et al., 2015). In our study, EVOO diet decreased nitric oxide (NO) production and TNF-α, IL-6 and IL-17 levels on LPS-stimulated peritoneal macrophages from pristane- Abstract ! ! 273 induced lupus mice in comparison with those animals fed with sunflower diet. Additionally, PE treatment significantly reduced NO with a significant decrease of inducible nitric oxide synthase (iNOS) protein and NOS-2 gene expression and decreased Toll-like receptor (TLR)- 4 mRNA levels as well as the gene expression of TNF-α, IL-1β and IL-6 proinflammatory cytokines on LPS-activated human monocytes. Some studies have shown the possible therapeutic benefits of peroxisome proliferator-activated receptor (PPAR)-γ agonists in SLE (Venegas-Pont et al., 2009). According to our results, PE treatment was able to significantly increased PPAR-γ protein expression as well as PPAR-γ mRNA levels on human monocytes. Additionally, PE treatment downregulated the gene expression of M1 macrophages markers and up regulated M2 molecules on M0 macrophages suggesting that the PE from EVOO prevents the classical macrophages activation by LPS and interferon (IFN)-γ regulating inflammatory cytokine production and polarized M0 macrophages into M2 macrophages contributing to EVOO anti-inflammatory activities. Besides, PE decreased “classical” monocytes but increased “non-classical” monocytes and produced a significant reduction of chemokine receptor type 2 (CCR2)+ cells and CCR2 gene expression levels on LPS-treated classical monocytes. Our findings outline that EVOO and particularly its PE have a significant therapeutic potential for the treatment of immune-inflammatory diseases such as SLE, through the modulation of monocytes/macrophages over-activation, decreasing NO and cytokine production as well as several immune-inflammatory markers. 2b. The phenolic fraction of extra virgin olive oil modulates the activation and the inflammatory response of T cells from patients with systemic lupus erythematosus and healthy donors In this study we investigated the in vitro effects of the PE from EVOO on PBMC from patients with SLE and healthy subjects, as well as the possible underlying mechanisms responsible for these potential effects. PBMC were isolated from healthy donors and patients with SLE, meeting the American College of Rheumatology criteria for SLE (Tan et al., 1982), recruited in the Connective Tissue Disease Unit of the University Hospital “Virgen del Rocío” (Seville, Spain). All patients were women without clinical activity (SLEDAI <4). We found a remarkable increase of IL-6 production in patients with SLE compared with healthy subjects. Moreover, IL-6 levels were significantly increased in both groups after phytohaemagglutinin (PHA) stimulation, whereas PE treatment was able to prevent this induced cytokine production. In addition, the in vitro treatment with PE positively regulated the induced production of IFN-γ on stimulated PBMC from patients with SLE and healthy