Funcionalidad del secoiridoide oleaceína y su derivado o-metilado como moduladores del epigenoma en enfermedades inflamatorias inmunomediadas

DEPARTAMENTO DE FARMACOLOGÍA FACULTAD DE FARMACIA UNIVERSIDAD DE SEVILLA FUNCIONALIDAD DEL SECOIRIDOIDE OLEACEÍNA Y SU DERIVADO O-METILADO COMO MODULADORES DEL EPIGENOMA EN ENFERMEDADES INFLAMATORIAS INMUNOMEDIADAS Tesis Doctoral Presentada por: Rocío Muñoz García Para optar al Grado de Doctor en Farmacia con Mención Internacional Sevilla, 2024 DEPARTAMENTO DE FARMACOLOGÍA FACULTAD DE FARMACIA UNIVERSIDAD DE SEVILLA Las Dras. Dña. Catalina Alarcón de la Lastra Romero, Profesora Catedrática de Universidad y Dña. Marina Sánchez Hidalgo, Profesora Titular de Universidad, adscritas al Departamento de Farmacología de la Facultad de Farmacia de la Universidad de Sevilla, CERTIFICAN: Que la presente Tesis Doctoral titulada “FUNCIONALIDAD DEL SECOIRIDOIDE OLEACEÍNA Y SU DERIVADO O-METILADO COMO MODULADORES DEL EPIGENOMA EN ENFERMEDADES INFLAMATORIAS INMUNOMEDIADAS” realizada por Rocío Muñoz García, para optar al grado de Doctora en Farmacia con mención internacional, ha sido llevado a cabo bajo su dirección Sevilla, 2024 Dra. Catalina Alarcón de la Lastra Romero Dra. Marina Sánchez Hidalgo DEPARTAMENTO DE FARMACOLOGÍA FACULTAD DE FARMACIA UNIVERSIDAD DE SEVILLA La Dra. María Concepción Pérez Guerrero, Profesora Titular de Universidad y Directora del Departamento de Farmacología de la Facultad de Farmacia de la Universidad de Sevilla, CERTIFICA: Que la presente Tesis Doctoral titulada “FUNCIONALIDAD DEL SECOIRIDOIDE OLEACEÍNA Y SU DERIVADO O-METILADO COMO MODULADORES DEL EPIGENOMA EN ENFERMEDADES INFLAMATORIAS INMUNOMEDIADAS” realizada por Rocío Muñoz García, ha sido dirigida por la Dra. Catalina Alarcón de la Lastra Romero y la Dra. Marina Sánchez Hidalgo, para optar al grado de Doctora en Farmacia con mención internacional, cumpliendo los requisitos para este tipo de Trabajo Sevilla, 2024 Dra. María Concepción Pérez Guerrero Esta tesis doctoral se ha llevado a cabo gracias a la financiación de las siguientes instituciones y proyectos de investigación: A. PROYECTOS DE INVESTIGACIÓN 1. Secoiridoides del olivo como nuevos nutracéuticos moduladores epigenéticos de la respuesta inmunoinflamatoria (AGL2017-89342-P). Investigador principal: Catalina Alarcón de la Lastra Romero Ministerio de Economía y Competitividad. Gobierno de España. Enero 2018- junio 2021. 2. Secoiridoides del olivo en la terapia nutricional del lupus eritematoso sistémico: implicaciones epigenéticas (PID2021-125488OB-I00) Investigador principal: Marina Sánchez Hidalgo/ Ricardo Gómez Huelgas Plan Estatal 2021-2023 - Proyectos Investigación Orientada. Ministerio de Ciencia e Innovación. Gobierno de España. Enero 2022- agosto 2025. 3. Funcionalidad del aceite de oliva virgen extra en la prevención de la artritis psoriásica: implicaciones epigenéticas (PROYEXCEL_00547). Investigador principal: Marina Sánchez Hidalgo PAIDI 2021: Proyectos I+D+i. Junta de Andalucía: Consejería de Universidad, Investigación e Innovación. Diciembre 2022- diciembre 2025. B. AYUDAS A LA MOVILIDAD 1. Ayudas complementarias de movilidad destinadas a beneficiarios del programa de formación del profesorado universitario (FPU). Convocatoria 2023. Ministerio de Ciencia, Innovación y Universidades. Gobierno de España. Abril 2023 - julio 2023. C. FINANCIACIÓN PRE-DOCTORAL 1. Ayudas para la formación de profesorado universitario FPU. Convocatoria 2018. Ministerio de Ciencia, Innovación y Universidades. Gobierno de España. Septiembre 2019 - septiembre 2023. “Defiende tu derecho a pensar, porque incluso pensar de forma errónea es mejor que no pensar” Hipatia de Alejandría Abbreviations/ Abreviaturas 3 o 5mC 5-methylcytosines A o ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid o ACh Acetylcholine/ Acetilcolina o ACN Acetonitrile o AcOH Acetic Acid o AIN American Institute of Nutrition o AKT Protein Kinase B o AOVE Aceite De Oliva Virgen Extra o APs Artritis Psoriásica o AR Artritis Reumatoide o ASC Apoptosis-associated speck-like protein containing a CARD B o BACH BTB and CNC homology 1 o BANK B Cell Scaffold Protein with Ankyrin Repeats (BANK) o BLK B-Lymphoid tyrosine kinase o Blys Human Lymphocyte-Stimulating Protein C o C Components of the Complement System o C1q Complement Component 1q o CC Colum Chomatographic o CCL Chemokine Ligand o CCR C-C Chemokine Receptor o CD Cluster of Differentiation o CD40L CD40 Ligand o cDNA Complementary DNA o CHCl3 Chloroform o CDCl3 Deuterated Chloroform o COX Cyclooxygenase/ Ciclooxigenasa o COSY Contraction Of Correlation Spectroscopy o CREM cCAMP-Responsive Element Modulator o CXCR6 C-X-C Chemokine Receptor D o DAB 3,3- Diaminobenzidine Tetrahydrochloride Abbreviations/ Abreviaturas 4 o DCFDA 2,7-dichlorodihydrofluoroscein diacetate o DCF 2’, 7’ –dichlorofluorescein o DCM Dichloromethane o dimet-OLA Dimethyl-oleacein/ Dimetil-oleaceína o DM Dieta Mediterránea o DMARD Disease-Modifying Antirheumatic Drugs o DMS Differentially Methylated Sites o DMSO Dimethyl Sulfoxide o DNMT DNA Methyltransferase/ AND Metiltransferasa o DTT Dithiothreitol o DMEM Dulbecco’s modified Eagle’s medium E o EAE Encefalomielitis Autoinmune o EDTA Ethylenediaminetetraacetic acid o EGTA ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid o EII Enfermedad Inflamatoria Intestinal o ELISA Enzyme-Linked Immunosorbent Assay/ Ensayo por Inmunoadsorción Ligado a Enzimas o eNOS Endothelial Nitric Oxide Synthase/ Óxido Nítrico Sintasa Endotelial o ERK Extracellular Signal-Regulated Kinase o ESI Electrospray Ionization o ESIMS Mass Spectra o Et2O Diethyl ether o EtOAc Ethyl Acetate o EtOH Ethanol o EVOO Extra Virgin Olive Oil o EZH2 Zeste Homolog 2 Histone Methyl Transferase Enhancer F o FBS Fetal Bovine Serum o FCPC Fast Centrifugal Partition Chromatography o FOXP3 Transcription Forkhead Box P3 G o GM-CSF Granulocyte-Monocyte Colony-Stimulating Factor H o H3 Histone 3/ Histona 3 Abbreviations/ Abreviaturas 5 o H3ac Acetylated histone H3 o H3K18ac Acetylated histone H3 in lysine 18 o H3K27me3 Acetylated histone H3 in lysine 27 o H3K9me3 Acetylated histone H3 in lysine 9 o H4 Histone 4 o HATs Histone Acetyltransferases o HDACs Histone Deacetylases/ Histona Deacetylasas o Hex Hexane o HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid o HLA Human Leukocyte Antigens o HMBC Heteronuclear Multiple Bond Correlation o HMTs Histone Methyltransferases o HO-1 Heme-oxygenase 1/ Hemo-oxigenasa 1 o HPLC High-Performance Liquid Chromatography o HRMS High-Resolution Mass Spectra o HRP Horseradish Peroxidase-Labeled o HSQC Heteronuclear Single Quantum Coherence o H&E Haematoxylin-Eosin I o IBD Inflammatory Bowel Disease o IC50 Half maximal inhibitory concentration o IF Immunofluorescence o IFI44L Interferon-Induced Protein 44-Like o IFN Interferon / Interferón o IFNAR1 Human Immunoglobulin G1 Kappa-Type Monoclonal Antibody That Binds to The Type I Interferon Receptor Subunit 1 o Ig Immunoglobulin/ Inmunoglobulina o IHC Immunohistochemistry o IkB −  Inhibitor of Nuclear Factor Kappa B/ Inhibidor del Factor Nuclear Kappa B o IL Interleukin/ Interleuquina o IL-23R Receptor of IL-23 o IMID Immune-Mediated Inflammatory Disease / Enfermedad Inflamatoria Inmunomediada o iNOS Inducible Nitric Oxide Synthase/ Óxido Nítrico Sintasa Inducible o IRAK IL-1 Receptor Associated Kinase o IRF IFN Regulatory Factor J Abbreviations/ Abreviaturas 6 o Jak Janus Kinase / Janus quinasa o JMJD3 Jumonji Domain-Containing Protein-3 L o LASP1 LIM And SH3 Protein 1 o LES Lupus Eritematosos Sistémico o LN Lupus nephritis o lncRNAs Long Non-Coding RNAs o LPS Bacterial lipopolysaccharide /Lipopolisacárido Bacteriano o LOX Lipooxygenase/ Lipooxigenasa M o MAPKs Mitogen-Activated Protein Kinases / Proteínas Quinasas Activadas por Mitógenos o MeOH Methanol o MHC Mayor histocompatibility complex o MIF Macrophage Migration Inhibitory Factor o Min Minutes o miRNA microRNA o MMP Matrix Metalloproteinases/ Metaloproteinasas de la Matriz o mPGES Prostaglandin E Synthase Microsomal / Prostaglandina E Sintasa Microsomal o mRNAs Messenger RNAs o MS Multiple Sclerosis o MT Masson´s trichrome o MW Microwave N o NADPH Nicotinamide Adenine Dinucleotide Phosphate o ncRNAs Non-Coding RNAs/ ARN no codificantes o NET Neutrophil extracellular traps o NF- κB Nuclear Factor-κB/ Factor nuclear κB o NLRP3 Nod-Like receptor family pyrin domain containing 3 o NMR Nuclear Magnetic Resonance o NO Nitric oxide/ Óxido Nítrico o NOX-1 Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase 1/ Nicotinamida Adenina Dinucleótido Fosfato (NADPH) Oxidasa -1 Abbreviations/ Abreviaturas 7 o Nrf-2 Nuclear Factor-Erythroid 2 related factor 2/ Factor Nuclear Eritroide 2 o NSAIDs Non-Steroidal Anti-Inflammatory Drugs O o OL Oleuropein/ Oleuropeína o OLA Oleacein / Oleaceína o OLE Oleocanthal/ Oleocantal P o PAS Periodic Acid-Schiff o PBMCs Peripheral Blood Mononuclear Cells/ Células mononucleares de sangre periférica o PBS Phosphate-Buffered Saline Solution o PD-1 Programmed Cell Death Protein 1 o PDC Plasmacytoid Dendritic Cell o PG Prostaglandin o PHA Phytohemagglutinin/ Fitohematoglutinina o PL Phospholipase/ Fosfolipasa o PMN Polymorphonuclear Leukocyte o PMSF Phenylmethylsulfonyl Fluoride o PsA Psoriatic Arthritis o PTPN protein tyrosine phosphatase non-receptor type o PUFAs omega-3 polyunsaturated fatty acids R o RA Rheumatoid arthritis o RANK Nuclear Factor Kappa B Ligand o RASF Human Synovial Fibroblasts of Rheumatoid Arthritis o RFX Regulatory Factor X o ROS Reactive Oxygen Species/ Especies Reactivas de Oxígeno o RT-qPCR Reverse transcription-quantitative polymerase chain reaction S o SD Standard Diet o SEM means ± standard error o siRNAs Short Interfering RNAs o SIRT-1 Sirtuin 1 o SLE Systemic Lupus Erythematosus o SLEDAI Systemic Lupus Erythematosus Disease Activity Measure Abbreviations/ Abreviaturas 8 o SPP Secreted Phosphoprotein o SRB Sulforhodamine B o STAT Signal Transducer and Activator of Transcription / Transductor de Señal y Activador de Transcripción T o Th T Helper Cells/ Linfocitos T colaboradores o TLC Analytical Thin-Layer Chromatographic o TLR Toll-Like Receptor o TMS Tetramethylsilane o TNF- Tumor Necrosis Factor Alpha / Factor de Necrosis Tumoral Alfa o TNFAIP3 TNF- Induced Protein 3 o TNFR Tumor Necrosis Factor Receptor o TNFSF Tumor Necrosis Factor Ligand Superfamily o TRAF6 (TNFR)-associated-factor 6 o Treg Regulatory T cells o TROLOX 6-hydroxy 2,5,7,8-tetramethylchroman-2-carboxylic acid o Tyk Tyrosine Kinase U o U46619 Thromboxane Receptor Agonist 9,11-Dideoxy-11,9- Epoxymethanoprostaglandin F2 o UV Ultraviolet light ABSTRACT RESUMEN Abstract/ Resumen 18 To continue investigating the immunomodulatory properties of OLA, its ability to regulate lymphocyte activation and pro-inflammatory mediator production was evaluated in phytohemagglutinin (PHA)-activated PBMCs isolated from healthy donors and patients with SLE. Treatment with OLA decreased the overproduction of pro-inflammatory cytokines induced by PHA in cells from healthy donors and SLE patients. However, OLA counteracted PHA-induced lymphocyte activation in PBMCs from healthy donors, but not in cells isolated from SLE patients. In this objective, we evaluated the role of OLA as an epigenetic modulator by examining the degree of global DNA methylation. We observed a tendency towards DNA hypomethylation in SLE patients compared to healthy donors. Although the results were not statistically significant, we also observed a trend towards increased DNA methylation in the OLA-treated group. Additionally, the role of OLA and dimet-OLA in CD4+ memory T cells activated with aCD3/aCD28 was investigated. After 72 h of treatment, our results show a reduction in INF- and IL-17 secretion in cells treated with OLA and dimet-OLA. Finally, we analysed the activity of OLA and dimet-OLA in a human synovial fibroblast cell line (SW982) and a co-culture of primary synovial fibroblasts isolated from patients with PsA and a population of CCR6+ Th memory lymphocytes from healthy donors. The study compounds will be investigated for their potential anti-inflammatory and immunomodulatory activity at the synovial tissue level in other IMIDs such as PsA. In both experimental models, we observed a decrease in the production of pro-inflammatory cytokines. Specifically, dimet- OLA and its precursor OLA significantly reduced IL-6 and TNF- levels in supernatant from the SW982 cell line stimulated with IL-1. In the same way, both compounds regulated the production of IL-6 in cell supernatant of co-cultured human synovial fibroblasts and T-cells. Notably, dimet-OLA showed greater efficacy in regulating IL-8, INF- and IL-17 secretion in the co-culture of aCD3/aCD28-activated cells, while no significant changes were observed for OLA. Despite these results, OLA was able to increase the expression of the anti-inflammatory cytokine IL-10, whereas this effect was not observed in dimet-OLA treated cells. Furthermore, it was shown that the expression of matrix metalloproteinases (MMP)-1 and MMP-3, which are involved in joint damage, was modulated by OLA and its O-methylated derivative in IL- 1-activated SW982 cells. Abstract/ Resumen 19 These results confirm previous findings from ex vivo and in vivo models and provide further insight into the effects of dimet-OLA and OLA on the regulation of T cell activation through the production of pro-inflammatory mediators, as well as the regulation of DNA methylation in patients with lupus. With our data, the present Thesis demonstrates for the first time the anti-inflammatory activity of the OLA and dimet-OLA compounds in a co-culture that resembles the real-life scenario of joint inflammation in PsA, where activated T-cells and fibroblasts coexist. Conclusion The results obtained in this Doctoral Thesis describe for the first time the anti-inflammatory, antioxidant and immunoregulatory activities of the secoiridoid of EVOO, OLA, and its O- methylated derivative, dimet-OLA, in a cross-sectional manner using different experimental models, including a cell line of human synovial fibroblasts, primary cultures of peritoneal macrophages and murine splenocytes, an in vivo model of SLE and cultures of T cells isolated from healthy donors and SLE patients and synovial fibroblasts isolated from PsA, to investigate the pro-inflammatory markers and molecular mechanisms involved, as well as their role as epigenetic modulators. 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Gutiérrez-Miranda, B.; Gallardo, I.; Melliou, E.; Cabero, I.; Álvarez, Y.; Magiatis, P.; Hernández, M.; Nieto, M.L. Oleacein Attenuates the Pathogenesis of Experimental Autoimmune Encephalomyelitis through Both Antioxidant and Anti-Inflammatory Effects. Antioxidants 2020, 9, 1161, doi:10.3390/antiox9111161. 39. Rosillo, M.Á.; Villegas, I.; Vázquez-Román, V.; Fernández-Santos, J.M.; Ortega-Vidal, J.; Salido, S.; González-Rodríguez, M.L.; Alarcón-de-la-Lastra, C. Dietary Oleacein, a Secoiridoid from Extra Virgin Olive Oil, Prevents Collagen-Induced Arthritis in Mice. Food Funct 2024, doi:10.1039/D3FO04240E. 40. Gutiérrez-Grijalva, E.P.; Ambriz-Pére, D.L.; Leyva-López, N.; Castillo-López, R.I.; Heredia, J.B. Review: Dietary Phenolic Compounds, Health Benefits and Bioaccessibility. Arch Latinoam Nutr 2016, 66, 87–100. 41. Koirala, N.; Thuan, N.H.; Ghimire, G.P.; Thang, D. Van; Sohng, J.K. 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Abstract/ Resumen 23 FUNCIONALIDAD DEL SECOIRIDOIDE OLEACEÍNA Y SU DERIVADO O- METILADO COMO MODULADORES DEL EPIGENOMA EN ENFERMEDADES INFLAMATORIAS INMUNOMEDIADAS Antecedentes del tema El concepto de enfermedades inflamatorias inmunomediadas (IMID) engloba a un grupo heterogéneo de enfermedades crónicas como la hidradenitis supurativa. artritis reumatoide (AR), espondilitis anquilosante, artritis psoriásica (APs), psoriasis, artritis idiopática juvenil, enfermedad inflamatoria intestinal (EII), uveítis y lupus eritematosos sistémico (LES), caracterizadas por la presencia de inflamación y la desregulación de la respuesta inmune [1]. Aunque la patogenia de estas enfermedades es desconocida, se ha descrito que factores genéticos y medioambientales podrían estar implicados en su desarrollo [2]. En este aspecto, la epigenética está desempeñando un papel central para intentar comprender la etiopatogenia de las IMID. Los mecanismos epigenéticos podrían describirse como las alteraciones hereditarias de la expresión génica que se producen sin que haya cambios en la secuencia de ADN [3,4]. Los principales cambios epigenéticos son la expresión de los ARN no codificantes (ncRNAs), la modificación de las histonas y la metilación del ADN, los cuales modulan la estructura de la cromatina, la accesibilidad del ADN y por tanto la expresión génica [5]. De este modo, las modificaciones epigenéticas tienen un rol esencial en la regulación de la diferenciación celular, reparación y replicación del ADN, así como la expresión de proteínas [6]. Además, las modificaciones epigenéticas están directamente relacionadas con los procesos immuno-inflamatorios. Recientemente, se ha puesto de manifiesto que alteraciones epigenéticas están relacionados con la aparición de enfermedades como LES, AR o APs [7,8]. De hecho, la modulación de estos cambios epigenéticos podría ser una alternativa interesante en el abordaje terapéutico de las mismas [5,9]. El LES se caracteriza por una inflamación crónica y la producción de autoanticuerpos que conducen a daño sistémico [10]. Del mismo modo, las manifestaciones clínicas de la APs se centran en la piel y el sistema musculoesquelético. El lupus es predominante en mujeres (ratio 9/1) [11], mientras que la APs afecta de igual manera a hombres que a mujeres [12]. Ambas enfermedades cursan con comorbilidades como complicaciones cardiovasculares, diabetes o Abstract/ Resumen 24 síndrome metabólico que empeoran la calidad de vida y aumentan la mortalidad en los pacientes que las padecen [13–15]. La falta de tratamientos eficaces y curativos para estas patologías ha conllevado a la búsqueda de alternativas terapéuticas que puedan servir como coadyuvantes a la farmacología clásica. En este campo, la terapia nutricional podría ser una herramienta eficaz en el manejo de las IMID [16,17]. Concretamente, el aceite de oliva virgen extra (AOVE), así como su fracción polifenólica y compuestos fenólicos aislados han demostrado tener una prometedora actividad inmunoreguladora y antiinflamatoria en modelos experimentales de AR, EII y LES [18–24]. Además, la fracción polifenólica del AOVE, reguló la activación linfocitaria en células T aisladas de pacientes de LES y EII [25,26]. Estudios preclínicos recientes han puesto de manifiesto los prometedores efectos beneficiosos para la salud de oleaceína (OLA), uno de los principales secoiridoides del AOVE [27,28]. OLA ha demostrado tener una potente actividad anticancerosa, siendo capaz de inducir la apoptosis y la proliferación celular [29,30]. Interesantemente, los efectos anticancerosos de OLA podrían estar mediados mediante cambios del epigenoma. En los últimos años se ha descrito su capacidad para regular la expresión de varios micro(mi)RNAs en células de melanoma y la inhibición de la enzima histona deacetilasa en células de mieloma múltiple, lo cual podría estar relacionada con la inhibición de la proliferación celular [30,31]. Adicionalmente, Marrero et al. describen a OLA como un interesante modulador de la angiogénesis [32]. Adicionalmente, OLA reguló la obesidad, la esteatohepatitis y la resistencia a la insulina en un modelo de ratones alimentados con una dieta alta en grasas [33,34] y previno la desestabilización de placas ateroscleróticas [35]. En cuanto a su actividad antiinflamatoria, Cirmi et al. observaron una fuerte inhibición del estrés oxidativo y la respuesta inflamatoria inducida por LPS en células THP-1 tratadas con OLA [36]. Del mismo modo, OLA inhibió la producción de óxido nítrico (NO), fosfolipasa (PL)A2, lipooxigensa-5 (5-LOX), ciclooxigenasa (COX)-1 and COX-2 en macrófagos RAW 264.7 [37]. Finalmente, el ámbito de las IMID, el tratamiento con OLA mejoró el desarrollo de la enfermedad en modelos Abstract/ Resumen 25 murinos de encefalomielitis autoinmune y RA sugiriendo su potencial uso terapéutico en enfermedades autoinmunes [38,39] . Sin embargo, los polifenoles son moléculas con una baja lipofilia y estabilidad química, lo que conlleva a la dificultad para atravesar membranas y un bajo perfil farmacocinético [40]. Con el objetivo de mejorar estas deficiencias, se ha descrito que la metilación de los grupos hidroxilos fenólicos (O-metilación) mejoran el transporte a través de membranas, conllevando a una mejora de la biodisponibilidad [41]. Además, estudios recientes describen que la O- metilación puede mejorar la actividad terapéutica de los compuestos fenólicos [42]. Teniendo como base lo descrito anteriormente se planteó la presente Tesis Doctoral con el objetivo general de desarrollar un estudio multidisciplinar del secoiridoide presente en el AOVE, OLA, y su derivado O-metilado, evaluando sus actividades inmunomoduladora, antioxidante y antiinflamatoria a través de la modulación del epigenoma en diversos modelos experimentales. Con el fin de alcanzar este objetivo general, se plantearon los siguientes objetivos específicos: 1. Valoración de los efectos de OLA y su derivado metilado en un modelo de inflamación ex vivo de macrófagos peritoneales y esplenocitos de ratón estimulados con lipopolisacárido bacteriano (LPS). Estudio de mediadores proinflamatorios, vías de señalización implicadas en la respuesta inflamatoria y modificaciones en el epigenoma mediante la evaluación de la acetilación y metilación de la histona H3. 2. Estudio del efecto protector de dietas enriquecidas con OLA y su derivado O- metilado, en un modelo murino de LES inducido por pristano. Evaluación del daño a nivel renal y cardiovascular. Estudios histopatológicos, evaluación de las vías de señalización implicadas en el desarrollo del LES y modificaciones en la expresión de miRNAs. 3. Evaluación de los efectos de OLA en células mononucleares de sangre periférica (PBMC) procedentes de una cohorte de controles sanos y pacientes de LES. Análisis de la activación linfocitaria, evaluación de marcadores proinflamatorios y cambios epigenéticos a través de la metilación del ADN. Abstract/ Resumen 26 4. Valoración de los efectos de OLA y dimetil-oleaceína (dimet-OLA) en un modelo in vitro de fibroblastos sinoviales humanos (SW982) estimulados con IL-1. Evaluación de marcadores proinflamatorios 5. Estudio del papel inmunomodulador de OLA y dimet-OLA en células T CD4+ de memoria y en un co-cultivo de células T colaboradoras de memoria CCR6+ provenientes de controles sanos y fibroblastos sinoviales procedentes de pacientes con APs. Resultados Inicialmente, con el fin de evaluar la actividad antiinflamatoria y antioxidante de OLA y su derivado O-metilado, evaluamos la capacidad de estos compuestos para contrarrestar la respuesta inflamatoria aguda y el estrés oxidativo en macrófagos peritoneales murinos estimulados con LPS. Tras 18 h de tratamiento, OLA y dimet-OLA redujeron los niveles de NO y citocinas proinflamatorias como interleuquina (IL)-17, IL-6, interferón gamma (IFN-), IL-1 y factor de necrosis tumoral alfa (TNF-) además de la producción de especies reactivas de oxígeno (ROS) intracelulares. Del mismo modo, ambos compuestos inhibieron de forma significativa la expresión de enzimas claves en la respuesta inflamatoria como la ciclooxigenasa (COX)-2, la prostaglandina E sintasa microsomal (mPGES)-1 y la óxido nítrico sintasa inducible (iNOS). Adicionalmente, se estudiaron las posibles vías de señalización asociadas a los efectos de beneficiosos de ambos compuestos. Los resultados obtenidos mostraron una reducción de la activación de las proteínas quinasas activadas por mitógenos (MAPKs), Janus quinasa/transductor de señal y activador de transcripción (JAK/STAT) y del inflamasoma junto con la inducción del eje factor nuclear eritroide-2/Hemo-oxigenasa-1 (Nrf- 2/HO-1) y la prevención de la degradación de la proteína inhibidora del factor nuclear kappa B (IkB−). Además, OLA y dimet-OLA regularon mecanismos epigenéticos implicados en la producción de citocinas en esplenocitos murinos activados por LPS, a través de modificaciones postraduccionales de histonas, como la desacetilación de histonas (H3K18ac) y la metilación de la histona H3 (H3K27me3 y H3K9me3). Por ello, OLA y dimet-OLA se presentan como nuevas estrategias terapéuticas moduladores del epigenoma en la respuesta Abstract/ Resumen 27 inmunoinflamatoria. Sin embargo, a pesar de los prometedores resultados de dimet-OLA se necesitan más estudios para una mejor compresión de las consecuencias de la metilación de OLA en su actividad farmacológica y sus propiedades farmacocinéticas. En el objetivo 2, evaluamos el papel preventivo de dietas enriquecidas al 0,01% (p/p) con OLA y dimet-OLA en el desarrollo de LES en un modelo murino experimental de LES inducido por pristano. Tras 24 semanas de suplementación dietética, se observó una mejoría de la nefropatía lúpica apreciándose una patente restauración de la arquitectura renal en los ratones tratados con una dieta rica en OLA. Además, también se observó una disminución en la deposición de inmunocomplejos (Inmunoglobulina (Ig)G e IgM). Por otro lado, el tratamiento dietético con dimet-OLA también fue capaz de reducir la acumulación de inmunocomplejos en tejido renal, aunque se observó hipercelularidad en los glomérulos, así como la tiroidización de algunos túbulos renales. Además, la suplementación dietética con ambos compuestos generó modificaciones epigenéticas como la regulación de la expresión renal de miRNAs (miRNA-146a, miRNA-26, miRNA-24-3p y miRNA-123b). Adicionalmente, en el grupo suplementado con OLA también se pudo observar una regulación de la expresión de la enzima ADN metiltransferasa (DNMT)-1. Con el objetivo de profundizar en la regulación de vías de señalización claves para el desarrollo del LES, nuestros resultados demostraron que OLA y dimet-OLA bloquearon en mayor o menor medida la activación de las vías de señalización JAK/STAT, factor nuclear kappa B (NF−B), inflamasoma y MAPKs. Asimismo, la suplementación dietética con OLA también moduló la expresión del eje Nrf- 2/HO-1. Adicionalmente, en este estudio se evaluó el efecto de OLA y su derivado O-metilado sobre la función endotelial, en tejido aórtico. OLA y dimet-OLA modularon la expresión de la proteína nicotinamida adenina dinucleótido fosfato (NADPH) oxidasa 1 (NOX-1) en aorta torácica, además, dimet-OLA contrarrestó la disfunción endotelial inducida por pristano mediante la modulación de la relajación dependiente del endotelio a acetilcolina (ACh), mientras que la dieta enriquecida con OLA no fue capaz. A pesar de que, a nivel renal, el tratamiento con OLA parece ser más efectivo, dimet-OLA presenta una interesante actividad cardioprotectora en ratones lúpicos inducidos con pristano. Estos resultados nos presentan la suplementación dietética con OLA y dimet-OLA como una herramienta útil en el abordaje INTRODUCTION INTRODUCCIÓN Introduction/ Introducción 37 1. Immune-Mediated Inflammatory diseases (IMIDs) IMIDs encompass a diverse group of diseases characterized by the onset of chronic inflammation and the aberrant regulation of the immune system. Within this group, we can find diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), multiple sclerosis (MS), or psoriatic arthritis (PsA), among others. Besides, IMIDs may be related to complications and comorbidities derived from chronic inflammation, such as cardiovascular risk factors, metabolic syndrome or diabetes [1]. Therefore, these diseases are very limiting to patients, who experience a reduction in their quality of life. Generally, IMIDs are classified according to the organs affected (Table 1), nevertheless, Schett et al. point out that a classification based on the molecular mechanism allows a better understanding of the IMIDs and also the mechanisms that cause them [2]. In this way, the correlation between IMIDs that affects several organs in addition to the different mechanisms which involve the same organ in diverse IMIDs could be elucidated (Table 1). Table 1. IMIDs classification based on organ involvement and molecular mechanism [2] Principal Organs Affected Cytokines Molecular Mechanism SLE Kidney, skin Interleukin (IL)-17, interferon gamm (IFN-) tumor necrosis factor alpha (TNF-) PsA Joints, skin TNF- IL-17, IL-23 RA Joints TNF- IL-6 IBD Gut TNF-, IL-23 MS Nervous system IL-23, INF- Psoriasis Skin TNF- IL-17, IL-23 Ankylosing spondylitis Spine TNF- IL-17 SLE: Systemic Lupus Erythematosus, PsA: Psoriatic Arthritis, RA: Rheumatoid Arthritis, IBD: Inflammatory Bowel Disease, MS: Multiple Sclerosis Until date, the lack of effective treatments and the complexity of the therapeutic approach to IMIDs has led to the search for new alternative non-pharmacological therapies as a complement to the classical treatments. From this point of view, a diet-based treatment is proposed as an Introduction/ Introducción 38 interesting option to support the treatment of IMIDs. Dietary interventions in MS conclude that a balanced diet can improve the quality of life of patients by improving the symptoms of the disease [3]. On the other hand, there are also numerous studies in patients with RA and IBD, which demonstrate the improvement of the disease after dietary modifications [4,5]. Although the etiopathogenesis of IMIDs is unclear, genetic predisposition and exposure to environmental factors (ultraviolet, smoking, diet) play a key role in the development of these diseases. Furthermore, increased evidence showed that epigenetic changes could influence the development of IMIDs along with the factors mentioned previously [6–9]. In the search for new and more specific treatments for IMIDs, epigenetics has become one of the fields of great interest, as it can help elucidate the mechanisms involved in the etiopathogenesis of these autoimmune diseases. Moreover, dietary components have been reported to play a regulatory role in the epigenome, offering a promising alternative in the treatments of IMIDs through epinutraceuticals compounds that can exert beneficial effects by regulating epigenetic modifications. In this work, we will focus on the etiopathogenesis, treatment and epigenetic profile of SLE and PsA. 1.1 Systemic lupus erythematosus SLE is a chronic immune-inflammatory systemic disease characterized by the aberrant regulation of innate and adaptive immune response, the deposition of immune complex and the production of autoantibodies leading to systemic damage [10]. SLE appears mainly in childbearing age women with a ratio male/female 1/9 [11,12]. Therefore, the symptomatology of SLE is very heterogeneous, covering a wide variety of manifestations that include renal damage, arthritis, hematologic, and dermatological symptoms (butterfly rash), among others. Although the symptoms are very diverse, the organ most affected by SLE is the kidney, which can lead to nephritis and renal failure. In addition, other diseases such as type II diabetes or osteoporosis may be associated with SLE as comorbidities [13,14]. The heightened occurrence of SLE among offspring of SLE patients and its manifestation in monozygotic twins suggests that genetics plays a vital role in the predisposition to suffer from this disease. Traditionally, genetic involvement in SLE has been associated with the human Introduction/ Introducción 39 leukocyte antigens (HLA) chromosomal region, especially HLA-DR2 and HLA-DR3, as well as genes that encode some components of the complement system (C) [15,16]. Indeed, homozygous deregulation of the complement component 1q (C1q) has been described as the most influential generic risk factor in SLE [17]. Furthermore, alteration in complement components such as C1q, C2, and C4 is related to reduced ability to recognize immune complex and poor clearance of cell debris [18]. It has also been described that the genetic predisposition to develop SLE is related to other genes associated with the innate immune response and IFN regulatory factor (IRF) 5, IL-1 receptor associated kinase (IRAK) 1, secreted phosphoprotein (SPP) 1, signal transducer and activator of transcription (STAT) 4, Toll-like receptor (TLR) 7 and TNF- induced protein 3 (TNFAIP3) or the activation or suppression of T or B cells ((B Cell Scaffold protein with ankyrin repeats (BANK)-1, B-Lymphoid tyrosine kinase (BLK), LYN, OX40L ligand, protein tyrosine phosphatase non-receptor type (PTPN) 22 and programmed cell death protein 1 (PD-1)) [19]. Regarding the environmental factors that can influence the susceptibility to SLE, exposure to ultraviolet light (UV) radiation promotes the production of pro-inflammatory cytokines and reactive oxygen species, damaging DNA [20]. In fact, UV exposure is a primary risk factor for dermal lesions in SLE [21,22] and could lead to exacerbations in cases of pre-existing SLE [20]. On the other hand, estrogens are presented as a risk factor for SLE; thus, SLE is more prevalent in women. Furthermore, the high presence of estrogen during puberty or pregnancy usually leads to periods of exacerbation in women [23]. Other environmental factors such as smoking are able to produce oxidative stress and DNA damage [24]. A poor approach to infections such as those caused by cytomegalovirus, Epstein-Barr, or Hepatitis B can also induce the development of SLE [25,26]. A notable observation is that patients with PsA have a higher risk of developing SLE than healthy controls; both diseases have similarities in treatment and pathology, but further studies are needed to elucidate the mechanisms involved in this relationship [27,28]. The gut microbiota also appears to be altered in SLE patients [29]. In fact, a disbalance in gut microbiota may be related to the pathogenesis of SLE [30]. Recent studies have described that patients with SLE have a less diverse gut microbiota than healthy controls and have also linked this observation to the activity of the disease [31,32]. The evidence shows a Introduction/ Introducción 40 lower presence of the genus Lactobacillus and a decrease in the ratio of Firmicutes/Bacteroidetes in patients with SLE compared to healthy controls [33]. Along with the above, nutrition is also a key environmental factor in SLE. In effect, increased sugar and fructose consumption has been shown to be one of the reasons why SLE has a higher incidence in afro-american women. Furthermore, a diet with a high carbohydrate intake and a low fat and protein intake could be considered a risk factor for developing SLE [34]. On the other hand, a good diet can significantly improve the quality of life of patients and reduce symptoms. In this way, adherence to a Mediterranean diet rich in fruits, vegetables, and extra virgin olive oil (EVOO) may have cardioprotective effects in patients with SLE, also reducing SLE activity [35]. Indeed, an EVOO- enriched diet attenuated lupus nephritis (LN) in SLE pristane-induced mice [36]. In this way, diets supplemented with vitamin D or omega-3 reduce the inflammatory response and oxidative stress, improve lipid profiles, and reduce disease activity [37]. Although more studies are still necessary to fully elucidate the pathogenesis of SLE, great advances have been made in the last few decades. Cytokines play a primary role in the immunoinflammatory response and in the pathogenesis of SLE. Specifically, T helper (Th) 1, Th2 and Th17 cytokine levels are usually increased in patients with SLE, and differences can also be observed in cytokine production between patients with active disease and patients in remission [38]. Aberrant regulation of T cells is one of the most important processes in the development of SLE. Amarilyo et al. described that IL-17 deficient mice did not develop pristane-induced SLE, evidencing the importance of IL-17 in SLE [39]. Furthermore, IL-17 levels have been described to be related to SLE activity [40]. Besides, Koga et al. showed increased levels of Th17 cells and consequently IL-17 in serum from patients with SLE with kidney affectation [41]. Cluster of differentiation (CD)4+ T cells have been detected in renal tissue or skin contributing to tissue damage [42]. Regulatory T cells (Treg) are responsible for maintaining the homeostasis, inhibiting the response to autoantigens and maintaining self-tolerance, blocking exaggerated immune responses [43]. IL-2 levels are essential for Treg activity, so if IL-2 production is downregulated, the number of Tregs decreases. In SLE, IL-2 production is depleted, leading to impaired Tregs [44]. Besides, IL-2 is related to the modulation of IL-17 production, so in SLE IL-2 levels are negatively regulated, causing IL-17 overproduction [42]. Moreover, the high rate of Introduction/ Introducción 41 immune cell apoptosis and its poor clearance could be related to the pathogenesis of LN. Cellular debris would be detected and presented as autoantigens, promoting the activation of T cells and the production of autoantibodies by B cells, leading to the immunoinflammatory response and tissue damage [45]. B cells can also behave as antigen-presenting cells, activating T cells that in turn produce pro-inflammatory cytokines, creating a loop that extends the immune response [46]. Figure 1 shows a summary of the possible factors that influence the etiopathogenesis of SLE [47]. Figure 1. Ethiopatogenesis of SLE. Adapted From: Kaul, A.; Gordon, C.; Crow, M.K.; Touma, Z.; Urowitz, M.B.; van Vollenhoven, R.; Ruiz-Irastorza, G.; Hughes, G. Systemic Lupus Erythematosus. Nat Rev Dis Primers 2016, 2, 16039, doi:10.1038/nrdp.2016.39. [47]. TLR: Toll like receptor; PDC: Plasmacytoid Dendritic Cell; PMN: Polymorphonuclear Leukocyte; NET: Neutrophil extracellular traps; MHC: Mayor histocompatibility complex; CD40; CD 40 Ligand; IFN: Interferon. Introduction/ Introducción 42 The complexity and lack of knowledge of the aetiology of SLE makes it difficult to develop new effective therapeutic strategies. Traditionally, the most commonly used drugs for the treatment of SLE are non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and antimalarials. Approximately 88% of patients with SLE are treated with glucocorticoids, which are very powerful and effective immunosuppressants and anti-inflammatory drugs, but they present a wide variety of adverse reactions that can ultimately cause organ damage [48–50]. Other drugs such as methotrexate, azathioprine or mycophenolate are also used as pharmacological therapies for SLE [51]. In general, patients with SLE do not take a single treatment, but rather combine therapeutic strategies to achieve a synergistic effect between drugs [52]. In recent years, the search for new treatments has focused on biological drugs. To date, only belimumab and anifrolumab are approved for the treatment of SLE, but there are several monoclonal antibodies in clinical trials for the treatment of SLE. Table 2 lists the new biological treatments under investigation for SLE. Belimumab was the first monoclonal antibody approved by the FDA for the treatment of SLE [53]. Its mechanism of action is based on binding to the soluble form of the human lymphocytestimulating protein (BLyS), which is increased in SLE patients [54]. This union inhibits its binding to BLyS receptors present in B cells and decreases the survival of autoreactive B cells. All this leads to the depletion of autoantibody production. In 2021, the FDA approved anifrolumab for the treatment of severe to moderate SLE [55]. This was followed by the approval from the European Medicines Agency (2022). Anifrolumab is a human immunoglobulin (Ig) G1 kappa-type monoclonal antibody that binds to the type I interferon receptor subunit 1 (IFNAR1) and inhibits the type I IFN signaling pathway [56]. In addition, anifrolumab also reduces IFNAR1 levels on the cell surface. As a consequence, plasma cell differentiation is decreased and normalizes peripheral T lymphocyte subsets, restoring the balance between adaptive and innate immunity that is altered in SLE. Introduction/ Introducción 43 Table 2. New SLE biological treatments DRUG STATE MECHANISM FINDING REFERENCES Atacicept Phase IIb Phase II Inhibition of BlyS and A proliferationinducing ligand (APRIL) Efficacy in active SLE patients Reduced Disease activity Acceptable safety profile Long-term safety profile Durable efficacy. [57] [58] Ustekinumab Phase II Phase III IL-12 and IL- 23 inhibitor Ameliorated joint and cutaneous manifestations and SLEDAI-2K score Safety profile acceptable: No malignancies or opportunistic infections detected [59] [60] Tofacitinib Phase I Inhibitor Janus Kinase (JAK) Safe in SLE Improvement of premature atherosclerosis biomarkers in SLEBG [61] Deucravacitinib Phase II Tyrosine Kinase (TYK) 2 inhibitor Ameliorated SLE activity and joint manifestations [62] Obinutuzumab Phase II Anti-CD20 monoclonal antibody Ameliorate renal damage [63] Bortezomib Proteosome inhibitor Reduced SLEDAI-2K scores Reduced proteinuria Safety: special attention to infections, and hypogammaglobulinemia [64] Litifilimab Phase II Anti-binding of blood dendritic cell antigen 2 Ameliorated joint manifestations [65] [66] Introduction/ Introducción 50 Table 3. Comparative SLE vs PsA SLE PsA Prevalence 43.7 per 100 000 persons[139] 133 per 100 000 persons [140] Male/female 1/9 [12] Equally [141] Age Incidence childbearing age (15-44 years) [25] 40-50 years [142] Manifestations Renal damage, arthritis, hematologic, and dermatological affectation [13,22,143,144] musculoskeletal symptoms (arthritis, dactylitis, axial disease, enthesis) and non musculoskeletal manifestations such as uveitis or metabolic syndrome [124,142] Arthritis No erosive [145] Erosive [96,124] Skin manifestations Malar Rash (Butterfly Rash), UV hypersensitivity [22,146] Psoriasis [147] Comorbidities Type II diabetes, osteoporosis, cardiovascular diseases [13,14] Metabolic syndrome, obesity, hyperlipidemia, cardiovascular diseases, IBD [95,148–150] Autoantibodies Yes [10,151] Yes [152,153] Introduction/ Introducción 51 2. Epigenetic and IMIDs Epigenetic is described as the emergence of reversible and heritable changes that can regulate gene expression without altering the DNA sequence [154]. Although nowadays the scope of epigenetic modifications is not completely known, it has been shown that they are fundamental factors in the regulation of the inflammatory response, cell growth and the differentiation of immunocompetent cells [155,156]. In the search for new and more specific treatments for IMIDs, epigenetics has become one of the fields of great interest, as it can help elucidate the mechanisms involved in the etiopathogenesis of these autoimmune diseases. The main epigenetic modifications are (Figure 3): - DNA methylation: is the most studied epigenetic mechanism. DNA methylation regulates gene expression by inhibiting the binding of transcription factors to DNA and the recruitment of proteins that participate in gene repression [157]. This epigenetic change occurs through the methylation of cytosines by the enzymes DNA methyltransferases (DNMT) 1 and 3. A methyl group is transferred to the 5’- carbon in the cytosine residues. DNMT-1 is responsible for maintaining methylation patterns during cell replication, while DNMT-3 participates in de novo DNA methylation [158]. In mammals, 5-methylcytosines (5mC) are usually found in CpG islands, formed by cytosine-guanidine dinucleotides. CpG islands are usually located near the promoter regions of genes, so DNA methylation decreases DNA accessibility, contributing to genetic silencing [159]. Notably, DNA methylation has been described to be related to histone acetylation [160]. - Histone Modifications: Histones can be modified at their N-terminal amino residues; these modifications affect the interaction between histones and DNA and consequently alter chromatin structure [161]. The main histone modifications are acetylation and methylation, both of which are reversible and catalysed by enzymes with opposite actions. Acetylation is catalysed by histone acetyltransferases (HATs), which transfer an acetyl group to a lysine residue and neutralize the positive charge of the lysine. This decreases the interaction between DNA and histone, leading to open chromatin and Introduction/ Introducción 52 increasing the accessibility of DNA to transcription factors; thus, histone acetylation is related to active gene transcription expression [162,163]. Histone deacetylases (HDACs) remove acetyl groups and increase the interaction between histones and DNA, resulting in a closed and inaccessible chromatin structure, leading to gene silence [164]. Moreover, Histone methylation can affect to lysine or arginine residues and is catalysed by histone methyltransferases (HMTs) [165]. Usually, histone methylation is associated with repressed gene expression through chromatin remodelling. However, depending on the type and position of the methylated residue, histone methylation can be associated with gene expression or repression [165,166]. - Non-Coding RNAs (ncRNAs). The ncRNAs are functional RNA molecules which do not get translated into proteins [167]. The most studied ncRNAs are long non-coding RNAs (lncRNAs), short interfering RNAs (siRNAs) and microRNAs (miRNAs). lncRNAs (more than 200 nucleotides (nt)) could regulate gene expression at the transcriptional and post-transcriptional levels through interactions with RNA, proteins and DNA, splicing and changes in chromatin structure, among others [168,169]. Moreover, miRNAs are short noncoding RNA molecules of 18-24 nucleotides that negatively regulate their messenger RNAs (mRNAs) targets. MiRNAs bind to the 3ʹ- untranslated region of the miRNA target, leading to its degradation or/and the prevention of its translation into proteins [170]. Therefore, this epigenetic mark is related, like DNA methylation, to gene silencing. In the same line, siRNAs (20-25 nt) interfere with gene expression by reducing it [171]. Both DNA methylation and histone modifications are reversible processes, catalysed by specific enzymes. This indicates that cells can change their epigenetic expression patterns in response to different stimuli, while allowing their manipulation with therapeutic intent [172]. Epigenetic mechanisms are essential for the normal development and function of the immune system; therefore, alterations in the control of these mechanisms can lead to immunological dysfunction and, in some cases, the development of autoimmunity [155]. Introduction/ Introducción 53 Figure 3. Principal epigenetic mechanism. Me: methyl group, Ac: Acetyl group 2.1 Epigenetic in SLE In recent years, the study of epigenetic changes proposes new mechanisms that could influence SLE development (Table 4). Actually, global DNA hypomethylation seen in patients with lupus may be related to deficient DNMT activity. In fact, the oxidant response can decrease the activity of DNMT1 leading to hypomethylation in FOXP3 promoter, blocking the transcription forkhead box P3 (FOXP3) and CD4+ CD25+ Treg cell differentiation [173]. Liang et al. observed decreased levels of DNMT1 in CD4+ T cells from SLE patients [174]. Moreover, it has also been observed that CD4+ T cells from active SLE patients have down-regulated levels of DNMT-1 leading to DNA hypomethylation [175]. The results of Ehtesham et al. showed that the promoter of MMP-9 was hypomethylated in PBMCs from SLE patients, which could result in an increase in the expression of MMP-9 and could be used as a new biomarker in the diagnosis of SLE [176]. Several researchers have detected DNA hypomethylation in the interferon-induced protein 44-like (IFI44L) promoter compared to healthy controls [177–179]. Therefore, they conclude that DNA methylation levels on the IFI44L promoter could also be used as a key epigenetic marker in SLE. In this vein, Zhao et al. found differences in DNA methylation levels between patients without kidney damage and Introduction/ Introducción 54 patients with kidney affectation. In the latter, DNA methylation was decreased on the IFI44L promoter [180]. Although DNA hypomethylation has generally been detected in the promoters of different genes that participate in SLE development. Interestingly, Hong et al. described the opposite, CD3z hypermethylation is appreciated as a factor and risk for SLE [181]. Epigenetic changes are not isolated events; they are interrelated. It has recently been shown that overexpression of the zeste homolog 2 histone methyl transferase enhancer (EZH2) in CD4+ T lupus cells could be mediated by decreased expression of miRNA-26a and miRNA-101, inducing hypermethylation of histone H3 in lysine 27 (H3K27) and altered DNA methylome. In addition, EZH2 up-regulation mediates CD4+ T cell adhesion [182]. Numerous reports notice H3 and H4 histone modifications in cells from SLE patients. Recently, Abbasifard et al. observed overexpression of HDAC1 in blood from SLE patients and proposed it as a possible epigenetic marker for the diagnosis of SLE [183]. These results are in agreement with previous studies that describe a deregulation of HAT/HDAC enzymes that results in global hypoacetylation of H3 and H4 in CD4+ T cells from patients with lupus [184]. However, in human SLE monocytes and T cells, global hyperacetylation of H3 and H4 has been reported [185,186]. Deregulation in histone acetylation levels in B cells has also been described. Gautam et al. detected global hypoacetylation of H3 and H4 in B cells from SLE patients compared to healthy controls [187]. Unlike the significant DNA hypomethylation observed in SLE, the pattern of histone acetylation remains unclear. Hu et al. also describe a relation between the level of H3 acetylation and disease activity [184]. Additionally, hypermethylation of H3K4 and H3K27 in dendritic cells has also been described [188]. As mentioned above, the deregulation of pro-inflammatory cytokines production is one of the main axes in the pathogenesis of SLE. Post-translational histone modifications influence this aberrant cytokine production. The cCAMP-responsive element modulator (CREM) is responsible for the regulation of cytokine production. In SLE patients, the modulator (CREM) is overexpressed, resulting in a decrease in IL-2 levels and consequently the overproduction of IL-17, which promotes SLE [189]. In addition, hyperacetylation of H3K18 and hypomethylation of H3K27me3 in the IL-17A-IL17F promoters induce an imbalance between Introduction/ Introducción 55 these cytokines [190,191]. On the contrary, in the IL-2 promoter Hedrich et al. observed an increase in H3K27 methylation and low levels of H3K18 acetylation silencing IL-2 expression [192]. TNFAIP3 is involved in the regulation of inflammatory responses by modulating the nuclear factor-κB (NF-κB) pathway. TNFAIP3 suppression is related to the overexpression of IL- 17 and IFN-, key biomarkers in SLE. Zhao et al. found decreased levels of TNFAIP3 in CD4+ T cells from lupus patients. They concluded that this fact could be mediated by H3K4me3 demethylation in the promoter region of TNFAIP3 [193]. Otherwise, histone modifications can work together with DNA methylation to regulate gene expression. CREM promoted DNA methylation in the IL-2 promoter inducing DNMT3a, conversely, CREM decreased DNA methylation in the IL-17 promoter and reduced HDAC1 and DNMT3, as consequence the IL-17 levels increases, and IL-2 expression is suppressed [190– 192]. The Regulatory factor X (RFX)1 regulates the expression of CD11a and CD70 through the recruitment of HDAC1 and DNMT1. In CD4+ T cells from patients with SLE, RFX1 is downregulated, resulting in the overexpression of CD11a and CD70 by H3 hyperacetylation in the promoter region of CD11a and CD70 [194,195]. Down-regulation of RFX1 triggered autoactivation and increased auto-antibody production. Zhou et al. have found elevated levels of H3K4me2 and H3ac at the tumor necrosis factor ligand superfamily (TNFSF)7 promoter in CD4+ T SLE cells, causing overexpression of CD70 that is related to autoimmunity [185]. In contrast, increased Jumonji domain-containing protein-3 (JMJD3) expression decreased H3K27me3 levels in the CD11a promoter region, triggering gene activation. They showed that RFX1 inhibits Th17 cell differentiation through increased H3 acetylation, decreased DNA methylation and H3K9 trimethylation [194–196]. With respect to miRNAs, altered expression of several miRNAs has been detected in blood and T cells from SLE patients. It has been suggested that miRNA-146a could be a protecting factor against LN. Zhu et al. showed decreased miRNA-146a levels and up-regulated expression of tumor necrosis factor receptor (TNFR)-associated-factor 6 (TRAF6) in PBMCs from SLE patients. The reduction in miRNA-146a was associated with disease activity and serum levels of IL-6, IL- 1, TNF- and IL-8, which were higher in patients with active LN [197]. However, miRNA-124 is under expressed in patients with active LN [198]. These results are in line with those of Zhu et al. Introduction/ Introducción 56 since the main target of miRNA-124 is TRAF6's mRNA [197]. Therefore, if miRNA-124 is downregulated, miRNA-124 cannot block TRAF6's mRNA and TRAF6 expression increases [198]. Moreover, reduced expression of miRNA-126, the main goal of which is suppression of INF-, has been detected in plasma from SLE patients compared to healthy controls. Additionally, miRNA-126 is correlated with DNA methylation [199]. Other authors have studied circulating miRNAs in plasma from patients with LN, observing alterations in their expression, although their role in LN is not very clear [200]. MiRNA-654 was increased in serum from SLE patients, inducing macrophage migration inhibitory factor (MIF) and consequent activation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) [201]. MiRNA-654 depletion may be of interest in attenuating SLE [201]. Similar results were observed for miRNA-873 [202] and Yan et al. noticed higher levels of miRNA-124-3p and miRNA-377-3p in serum and PBMCs from SLE patients [203]. Up-regulated miRNA-155 is correlated with antibody overproduction, altered T cell differentiation, and kidney damage. As expected, miRNA-155 expression increased in PBMCs from SLE patients [204]. Although most altered miRNAs have been studied in the blood or immune cells of SLE patients, there are also changes in miRNA expression in kidney tissue. Huang et al. assessed the role of the negative regulation of miRNA-133 in LN. MiRNA-133 modulates cell proliferation and apoptosis in human mesangial cell though LIM and SH3 Protein 1 (LASP1) inhibition. However, in lupus patients, miRNA-133 is decreased, which could contribute to the appearance of LN [205]. MiRNA-130b modulates the type I IFN pathway by blocking STAT-1 activation. Surprisingly, miRNA-130b levels were down-regulated in renal tissue, contrary to its expression in serum [206]. On the other hand, Du et al. identified down-regulated miRNA‑98‑5p, which in normal conditions inhibits the activation of BTB and CNC homology 1 (BACH1) and the production of TNF‑α and IL‑6 [207]. It is noteworthy that miRNAs have also been found as biomarkers of SLE in body fluids such as urine [208]. It is proposed as a promising alternative to invasive biopsies. Recent studies described a decrease in miRNA-21 expression and increased miRNA-146a/ miRNA-155 levels in urine samples, relating them to proteinuria, Systemic Lupus Erythematosus Disease Activity Measure (SLEDAI) score, and kidney damage [209–211]. Introduction/ Introducción 57 Table 4. Epigenetic changes in SLE DNA METHYLATION SAMPLE EPIGENETIC MODIFICATION REFERENCES CD4+ T cells Hypomethylation of IL17F promoter region [190] ↓DNMT1 [174,175] ↓DNA methylation [174,196] Recruitment of DNMT1 [194] ↓DNA methylation on IL-10 and ILL-13 promoter region [212] PBMCs ↓DNA methylation in MMP-9 promoter ↑ expression MMP-9 [176] ↑ DNA methylation in HDAC6 promoter [213] ↓IFI44L methylation promoter [178,179] Blood ↑CD3z gene methylation [181] ↓IFI44L methylation promoter [177,180] HISTONE MODIFICATIONS SAMPLE EPIGENETIC MODIFICATION REFERENCES Blood ↑HDAC1 [183] CD4+ T cells ↓Global acetylation of H3 and H4 [186] ↓ H3K4me3 in TNFAIP3 ↓TNFAIP3 expression [193] ↑H3ac CD11a and CD70 promoter region Recruitment of HDAC1 DNMT1 ↑CD11a and CD70 expression [194,195] ↑H3K4me2 and H3ac TNFSF7 promoter overexpression of CD70 [185] ↓H3K27me3 levels CD11a promoter ↓Th17 cell differentiation ↑H3 acetylation ↓H3K9me3 [193–195] ↓H3K27me3 ↑ H3K18ac ↑HDAC ↓DNA methylation ↑ IL-17A expression [191] Dendritic cells ↑ H3K4me3 and H3K27me3 [188] B cells ↓Global acetylation of H3 and H4 [187] PBMCs ↓HDAC6 [213] Introduction/ Introducción 58 Monocytes Global H3 and H4 hyperacetylation [185,186] miRNA expression SAMPLE EPIGENETIC MODIFICATION REFERENCES PBMCs ↓ miRNA-146 TRAF6 [197,214] ↓miRNA-125a [215] ↑ miRNA-199a-1 and miRNA-125b-5p [200] ↑ miRNA-155 and miRNA-21 [204] Serum ↓miRNA-124 ↑ TRAF6 [198] ↑ miRNA-654 [201] PBMCs and Serum ↑ miRNA-124-3p and miRNA-377-3p [203] B cells ↑ miRNA-145 [216] ↑ miRNA-22, miRNA-18b, miRNA-345 and miRNA- 365, [217] ↓ miRNA-30a [218] ↓ miRNA-1246 [219] CD4+ T cells ↑ miRNA-873 [202] Plasma ↑ miRNA-126 [199] Human Mesangial Cells ↓miRNA-133 [205] ↓miRNA‑98‑5p [207] ↓miRNA-130b [206] Urine ↓let-7a and miRNA-21 [210] ↑miRNA-146a [211] ↑miRNA-155 [209] Furthermore, new therapeutic targets for SLE, such as HDAC inhibitors, have been discovered thanks to advances in the study of epigenetic changes in SLE [220–222]. Introduction/ Introducción 59 2.2 Epigenetic in PsA In the last years, some researchers have highlighted the involvement of epigenetic changes in the etiopathogenesis of PsA (Table 5). This field is not well known and much remains to be elucidated, but it is presented as a promising strategy to understand the pathogenesis of PsA and look for new targets. Kim et al. demonstrated global DNA hypomethylation in PBMCs from PsA patients compared to patients treated with methotrexate or healthy donors [223]. Similar to other autoimmune diseases like lupus, psoriasis, or rheumatoid arthritis, DNA methylation patterns have been found to be closely linked to the activity of dermal manifestations in PsA. Indeed, the regulation of these patterns could be an interesting tool for monitoring the disease [224]. On the other hand, 17 differentially methylated sites (DMS) have been found in the blood of patients with PsA compared to healthy controls. Different DMS have also been described comparing patients with PsA and patients with psoriasis [225]. Similar results were observed in sperm cells from patients with PsA [226]. Regarding posttranslational histone modifications, Ovejero-Benito et al. reported hypoacetylation of H4 and hypermethylated H3K4 in PBMCs from PsA patients in comparison to healthy donors [227]. HDAC inhibitor drugs are presented as valuable alternatives for the treatment of other autoimmune diseases such as SLE or RA [228,229]. In psoriatic disease, they have been shown to have anti-inflammatory effects in preclinical studies. Sodium Butyrate, a HDACS inhibitor, reduces IL-17 mRNA levels and increases IL-10 and FOXP3 expression in vivo Imiquimod-induced psoriasis-like skin inflammation model [230]. Among epigenetic changes, the variation in the expression of different microRNAs is the most studied in patients with PsA. 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Am J Clin Nutr 2013, 98, 480–487, doi:10.3945/ajcn.112.052241. JUSTIFICATION AND AIMS JUSTIFICACIÓN Y OBJETIVOS Justification and Aims/ Justificación y Objetivos 87 Dentro del grupo de las enfermedades inflamatorias inmunomediadas (IMID) encontramos patologías como la artritis reumatoide (AR), la enfermedad inflamatoria intestinal, el lupus eritematoso sistémico (LES) y la artritis psoriásica (APs) las cuales tienen en común su carácter immunoinflamatorio y la ausencia de un tratamiento curativo seguro y eficaz [1,2]. Las IMID comparten mecanismos moleculares implicados en su desarrollo, aun así, cada una tiene su propia idiosincrasia y su manejo es distinto. Aunque en los últimos años se ha observado un repunte en los casos de IMID en países de América del Sur y Asia, por lo general, son más prevalentes en países occidentales [3]. El LES se caracteriza por una alteración de la respuesta inmune innata y adquirida [4] que conlleva a la producción de autoanticuerpos antinucleares, citocinas pro-inflamatorias, deposición de inmunocomplejos aberrantes e hiperactividad linfocítica desencadenando daño sistémico, siendo los órganos más afectados los riñones, la piel, las articulaciones y el sistema nervioso [5]. A menudo, la inespecificidad de los síntomas iniciales y su confusión con otras patologías conlleva a un retraso en el diagnóstico del LES [6]. Aunque actualmente no se comprende del todo su etiología, se sabe que la influencia de factores genéticos y ambientales como infecciones, el consumo de ciertos medicamentos, la radiación ultravioleta o la dieta, podrían influir en su aparición. De hecho, la prevalencia del LES varía en función del género, la edad y el origen étnico, lo cual podría ser debido a la influencia de los factores genéticos y el entorno en el desarrollo y evolución del LES [7]. En los pacientes con APs coexisten las lesiones dérmicas propias de la psoriasis y la inflamación articular, en ausencia del factor reumatoide [8]. Al igual que en el LES, se desconoce la etiología de la APs, aunque también se ha descrito que factores genéticos y medioambientales participan en su desarrollo. Generalmente, en los pacientes con APs, aparecen antes las manifestaciones cutáneas y posteriormente la afectación articular [9]. El daño articular de la APs es difícil de identificar al mostrar similitudes con otras enfermedades como artritis reactiva, osteoartritis, AR o espondilitis anquilosante [10] y pueden transcurrir varios años desde la aparición de las lesiones dérmicas a la primera manifestación articular. Todo ello conllevaría al retraso en el diagnóstico de la APs que Justification and Aims/ Justificación y Objetivos 88 podría resultar en el avance de la enfermedad con la consecuente pérdida en la calidad de vida de los pacientes [11]. La epigenética, la cual estudia los cambios hereditarios y reversibles en la expresión génica que no involucran cambios en la propia secuencia de ADN [12], está tomando relevancia en la búsqueda de una mejor compresión de la patogenia del LES y la APs. Los principales mecanismos epigenéticos son la expresión de ARN no codificantes (ncRNA), la metilación del ADN y las modificaciones postraduccionales de las histonas, los cuales participan en la homeostasis celular controlando la mayoría de los procesos biológicos como el mantenimiento de fenotipos o la replicación y diferenciación celular [13,14]. Estos procesos son reversibles y están catalizados por enzimas y cofactores específicos. La alteración de estos mecanismos epigenéticos puede conllevar a la aparición de enfermedades [13]. Esto indica que las células son capaces de cambiar sus patrones de expresión epigenéticos en respuesta a diferentes estímulos. De hecho, hay evidencias de la influencia de estas modificaciones epigenéticas en el desarrollo de IMID como el LES y APs [15–18], y como algunas investigaciones se centran en la manipulación del epigenoma con intención terapéutica [19–22]. Debido a la heterogeneidad de ambas patologías, el abordaje terapéutico es complejo y multidisciplinar. Actualmente los tratamientos aprobados son inespecíficos y se centran en minimizar la sintomatología, prevenir exacerbaciones y demorar el avance de la enfermedad. Aunque las investigaciones actuales se centran en la búsqueda de fármacos más específicos y eficaces, hasta la fecha no existe un tratamiento curativo. En la búsqueda de nuevas estrategias terapéuticas, la terapia nutricional ha demostrado generar efectos positivos sobre la salud, presentándose como una interesante alternativa para la prevención y tratamiento de IMID como el LES y APs [23,24]. Adicionalmente, el tratamiento dietético puede inducir cambios metabólicos y fisiológicos implicados en la activación de proteínas, inducción de la apoptosis o regulación de procesos inflamatorios y autoinmunidad mediante modificaciones en los patrones de expresión Justification and Aims/ Justificación y Objetivos 89 epigenéticos en respuesta a diferentes estímulos, permitiendo al mismo tiempo su manipulación con intención terapéutica. Estudios recientes demuestran que la adherencia a la Dieta Mediterránea (DM), la cual se caracteriza por su aporte de ácido grasos poliinsaturados, minerales y vitaminas mediante el consumo diario de frutas y verduras, aceite de oliva, e ingesta moderada de productos lácteos, pescado y carne [25,26], podría ejercer un papel protector modulando la respuesta inmunoinflamatoria [27]. En concreto, los efectos beneficiosos del aceite de oliva virgen extra (AOVE) como principal aporte graso de la DM han sido atribuidos a sus compuestos polifenólicos de entre los cuales destacan los secoiridoides [28,29]. Asimismo, tanto el AOVE como algunos de sus componentes bioactivos han demostrado tener propiedades moduladoras del epigenoma [30,31]. Las propiedades organolépticas y sensoriales del AOVE, como la astringencia, el amargor y el picor se deben principalmente la presencia polifenoles como los secoiridoides, los cuales están presentes en el AOVE en concentraciones superiores a 10 mg/kg [29]. Los secoiridoides proceden del metabolismo secundario de los terpenos y estructuralmente están compuestos por un alcohol feniletílico (3,4-DHPEA o p-HPEA) unido al ácido elenólico o sus derivados y en generalmente están glicosilados [32]. El Ligustrósido, la oleuropeína (OL) junto con sus correspondientes agliconas y el oleocantal (OLE) y la oleaceína (OLA) son algunos de los polifenoles que forman parte de esta familia. OLA, es junto a OLE, uno de los secoiridoides más abundantes del AOVE y corresponde a la forma dialdehídica de la decarboximetil aglicona de OL (3,4-DHPEA-EDA) [33]. La proporción de OLA en el fruto del olivo es mucho menor que en el AOVE debido a la acción de las enzimas -glicosilasas endógenas que se encargan de hidrolizar a OL para generar OLA durante el proceso de molturación de las aceitunas, aumentando así la concentración de OLA en el AOVE [34]. La riqueza y composición polifenólica en el fruto del olivo y consecuentemente en el AOVE puede varían en función del tipo de cultivo, las condiciones medioambientales, la etapa de maduración del fruto y los procesos de extracción utilizados [35,36]. Justification and Aims/ Justificación y Objetivos 90 En los últimos años, nuestro grupo de investigación ha explorado las propiedades inmunoreguladoras del AOVE y sus compuestos fenólicos minoritarios en células inmunes y en diferentes modelos experimentales de IMID. El tratamiento con dietas enriquecidas con AOVE o sus compuestos polifenólicos aislados (OLE, OL, hidroxitirosol) fue capaz de revertir el daño articular en un modelo murino de artritis reumatoide inducido por colágeno [37–40], prevenir la nefritis lúpica en ratones Balb/C inducidos por pristano [41–43] y reducir el daño y la inflamación a nivel intestinal en un modelo experimental de colitis ulcerosa inducida por dextrano sulfato de sodio [44,45]. Del mismo modo, también demostraron actividad inmunomoduladora, antiinflamatoria y antioxidante en ensayos ex vivo e in vitro de células inmunes murinas y humanas [46–52] . En los últimos años numerosos estudios preclínicos han demostrado las propiedades antiinflamatorias [53–56], cardioprotectoras [57–60], anticancerosas [61–63], antimicrobianas [64–66], neuroprotectoras [67–71] y antioxidantes [72–74] de OL y OLE. Sin embargo, A diferencia de OL y OLE cuyos efectos beneficiosos para la salud han sido ampliamente estudiados, por nuestro grupo de investigación como se ha comentado y por otros autores, la información existente hasta la fecha de las actividades farmacológicas de OLA es más limitada. En cuanto a OLA, previamente se han puesto de manifiesto propiedades antiinflamatorias en células THP-1 y RAW 264.7 mediante la supresión de mediadores pro-inflamatorios [75,76]. En el ámbito de las enfermedades autoinmunes, en un modelo experimental murino de encefalomielitis autoinmune (EAE), la administración de OLA indujo un significativo efecto neuroprotector mejorando la afectación motora y los síntomas de la enfermedad mediante mecanismos antiinflamatorios y antioxidantes [77]. Curiosamente, este mismo modelo, OLA fue capaz de contrarrestar la disfunción de la barrera intestinal asociada a la EAE [78]. Además, la suplementación dietética con OLA redujo el daño artrítico en ratones DBA-1/J inducidos con colágeno. OLA presentó actividad antiinflamatoria e inmunomoduladora regulando la producción de mediadores pro-inflamatorios, así como la activación vías de señalización implicadas en la AR [79]. Estos resultados presentan a OLA como un interesante nutracéutico en el tratamiento de las IMID. Justification and Aims/ Justificación y Objetivos 91 En un estudio llevado a cabo por Wang et al. en adipocitos diferenciados de células madre derivadas de tejido adiposo humano (hASC) y hASC diabéticas (d-hASC), OLA inhibió significativamente la formación de lípidos en dichos adipocitos [80]. Además, a través de un análisis de microarrays pusieron de manifiesto que el tratamiento con OLA podría reducir significativamente los genes relacionados con el metabolismo de los lípidos y modular el metabolismo de la glucosa sugiriendo que el tratamiento con OLA podría ser interesante en el manejo de los desórdenes metabólicos [80]. En este contexto, OLA también fue capaz de regular la inflamación en adipocitos mediante la inhibición de la vía del factor nuclear κB [81] y modular la obesidad, adipogénesis y la resistencia a la insulina asociados a una dieta alta en grasa en ratones [82,83]. Otros autores han estudiado sus efectos cardioprotectores en la prevención de la desestabilización de las lesiones ateroscleróticas, la deposición de grasas en macrófagos derivados de monocitos humanos y la consecuente formación de células espumosas [84–87]. Por otro lado, Di Pietro et al. describieron las propiedades antimicrobianas de OLA frente a diferentes cepas de bacterias Gram – y Gram + resistentes a múltiples antibióticos, así como su actividad antifúngica frente a Candida spp. [64]. Adicionalmente, en diferentes ensayos in vitro OLA ha mostrado inducir interesantes efectos anticancerosos a través de la inhibición de la proliferación celular, inducción de apoptosis y reducción de la angiogénesis [88–91]. Por otro lado, OLA podría ejercer sus efectos mediante la modulación del epigenoma. Investigaciones recientes demuestran su capacidad para regular la expresión de diferentes micro(mi)RNAs en células de melanoma y preadipocitos humanos del síndrome de Simpson-Golabi-Behmel [81,92]. Del mismo modo el tratamiento con OLA fue capaz de reducir la transcripción y traducción de las enzimas histonas deacetilasas (HDACs) en células de mieloma múltiple pudiendo conllevar a una reducción en la acetilación global [91], presentando a OLA como un interesante compuesto epinutracéutico. El principal hándicap de los polifenoles del AOVE es su termolabilidad y fotolabilidad, así como su pobre perfil farmacocinético como consecuencia de la dificultad para atravesar las membranas biológicas y su baja solubilidad en fluidos acuosos [93]. Con el objetivo de Justification and Aims/ Justificación y Objetivos 98 55. Elmazoglu, Z.; Bek, Z.A.; Sarıbaş, G.S.; Özoğul, C.; Goker, B.; Bitik, B.; Aktekin, C.N.; Karasu, Ç. 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Methylation of Flavonoids: Chemical Structures, Bioactivities, Progress and Perspectives for Biotechnological Production. Enzyme Microb Technol 2016, 86, 103–116, doi:10.1016/j.enzmictec.2016.02.003. Justification and Aims/ Justificación y Objetivos 101 95. Wen, X.; Walle, T. Methylated Flavonoids Have Greatly Improved Intestinal Absorption and Metabolic Stability. Drug Metabolism and Disposition 2006, 34, 1786–1792, doi:10.1124/dmd.106.011122. 96. Wen, L.; Jiang, Y.; Yang, J.; Zhao, Y.; Tian, M.; Yang, B. Structure, Bioactivity, and Synthesis of Methylated Flavonoids. Ann N Y Acad Sci 2017, 1398, 120–129, doi:10.1111/nyas.13350. CHAPTER I IMMUNOMODULATORY EFFECTS OF OLEACEIN AND ITS NEW O- METHYLATED DERIVATIVE ON LPS-STIMULATED MURINE IMMUNE CELLS: MOLECULAR PATHWAYS AND EPIGENETIC IMPLICATIONS IN HISTONE MARKERS Partially Published and modified from: Muñoz-García R, Sánchez-Hidalgo M, Montoya T, Alcarranza M, Ortega- Vidal J, Altarejos J, Alarcón-de-la-Lastra C. Effects of Oleacein, a New Epinutraceutical Bioproduct from Extra Virgin Olive Oil, in LPS-Activated Murine Immune Cells. Pharmaceuticals (Basel). 2022 Oct 28;15(11):1338. https://doi.org/10.3390/ph15111338 CAPÍTULO I EFECTOS INMUNOMODULADORES DE OLEACEÍNA Y SU NUEVO DERIVADO O-METILADO EN CÉLULAS INMUNES MURINAS ESTIMULADAS POR LPS: VÍAS MOLECULARES E IMPLICACIONES EPIGENÉTICAS EN MARCADORES DE HISTONAS ´ Este capítulo recoge resultados publicados en: Muñoz-García R, Sánchez-Hidalgo M, Montoya T, Alcarranza M, Ortega-Vidal J, Altarejos J, Alarcón-de-la-Lastra C. Effects of Oleacein, a New Epinutraceutical Bioproduct from Extra Virgin Olive Oil, in LPS-Activated Murine Immune Cells. Pharmaceuticals (Basel). 2022 Oct 28;15(11):1338. https://doi.org/10.3390/ph15111338 Chapter I/ Capítulo I 107 ABSTRACT The present study aimed to assess the antioxidant and anti-inflammatory effects of oleacein (OLA), an extra virgin olive oil secoiridoid in murine peritoneal macrophages induced by bacterial lipopolysaccharide (LPS). As the O-methylation process could increase the hydrophobicity and biological activity without modifying therapeutic indices, a new O- methylated OLA derivative , dimethyl-oleacein (dimet-OLA), was synthetized and also assayed. Besides, we studied epigenetic changes in histone markers and related cytokine production in murine spleen cells after LPS stimulation. Reactive oxygen species (ROS) was evaluated by fluorescence analysis, and nitric oxide (NO) levels were measured using the Griess reaction. Furthermore, the production of pro-inflammatory cytokines and protein expression was analysed by enzyme-linked immunosorbent assay (ELISA) and Western Blot respectively. Cells were treated with OLA or dimet-OLA (25-12,5µM) and then activated with LPS (5 μg/mL) for 18 or 24 h. OLA and its new derivative could modulate LPS- stimulated inflammatory response and oxidative stress in murine peritoneal macrophages by suppressing ROS, NO and pro-inflammatory cytokines (tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-1β, interferon gamma (IFN-γ), IL-17 and IL-18) production. Both compounds also inhibited the expression of cyclooxygenase (COX-2), prostaglandin E synthase microsomal (m-PGES1) and inducible NO synthase (iNOS) proteins. The mechanism underlying these effects could be related to the regulation of Janus kinase/signal transducer and activator of transcription (JAK/STAT), mitogen-activated protein kinases (MAPKs) and inflammasome signalling pathways, the activation of nuclear factor erythroid 2-related factor/heme oxygenase (Nrf2/HO-1) axis and the prevention of nuclear factor κB inhibitor (IB-) degradation. Furthermore, OLA and dimet-OLA modulated LPS-induced epigenetic modifications throughout histone modifications and inhibited related proinflammatory cytokines secretion (IL-6, IL-1 and IL-17) in LPS-activated spleen cells. Taken together, our results present OLA and dimet-OLA as interesting new compounds with antiinflammatory and antioxidant activity. In addition, dimet-OLA enhanced the lipophilicity of its precursor OLA. However, for a better understanding of the beneficial effects and improvement of the OLA pharmacokinetic profile through the methylation process, further in vivo studies are required. Chapter I/ Capítulo I 114 2.2 Isolation and Synthesis of (–)-Oleacein and (–)-Dimethyl-oleacein 2.2.1 Isolation and Identification of (–)-Oleacein A sample of 300 g of EVOO was obtained from olive drupes. The EVOO sample was extracted following the protocol described by the International Olive Council (IOC, 017) as reported by Diez-Bello et al. [49]. The resulting dry extract (1.5 g) was chromatographed with the FCPC instrument using a solvent System composed of Hex:AcOEt:EtOH:H2O (2:3:2:3, v/v/v/v). 17 fractions were collected and monitored by analytical HPLC using a step gradient with mixtures of methanol (MeOH)/ACN (1:1, v/v, solvent A) and H2O/AcOH (99.8:0.2, v/v, solvent B) at a flow rate of 0.7 mL/min (min). The gradient program consisted of a linear gradient from 4 to 50% A in 40 min; a linear gradient from 50 to 60% A in 5 min; a linear gradient from 60 to 100% in 15 min; 100% A for 10 min; and other 12 min to return to the initial conditions. As a result, 200 mg of crude OLA was obtained, which was re-purified by semi-preparative HPLC using as solvent ACN/AcOH (99.8:0.2, v/v, solvent A) and H2O/AcOH (99.8:0.2, v/v, solvent B) at a flow rate of 5 mL/min. The separation was carried out with a linear gradient from 20 to 25% A. Finally, pure OLA (32 mg) was obtained and structurally characterized by 1HNMR and 13C-NMR (Figures S2 and S3), and optical rotation; []D = -4,5 (c = 1.0, CHCl3). The spectroscopy data of this compound agreed with those reported in the literature for OLA [50]. 2.2.2 Semysinthesis of (–)-Dimethyl-Oleacein 2.2.2.1 Isolation of (–)-Oleuropein from Olive Tree Wood A wood sample of Olea europaea L. obtained from pruning material of a tree growing in the province of Jaén, Spain, was used to isolate (–)-oleuropein. A sample of wood chips (50 g) was extracted with EtOAc (600 mL) for 2 h at reflux. Solvent was evaporated under reduced pressure at 40 °C to give the corresponding dry extract (2.5 g). EtOAc extract was stored under argon atmosphere at −20 °C until use. Ethyl acetate extract (1.5 g) was chromatographed by column chromatography using silica gel 60 as the stationary phase and a mixture of DCM-EtOH (9:1, v/v) as a developing solvent. Thirty-two fractions of 50 mL each were collected and monitored by TLC using DCM:EtOH (9:1, v/v) as eluent and UV Chapter I/ Capítulo I 115 radiation (254 nm) to visualize spots. As a result, 0.2 g of (–)-oleuropein (purity by HPLC: 90%) were isolated. For analytical HPLC analysis, separation was carried out by a step gradient with mixtures of MeOH/AcOH (99.8:0.2, v/v, solvent A) and H2O/AcOH (99.8:0.2, v/v, solvent B). The gradient program consisted in a linear gradient from 20 to 80% A in 55 min, a linear gradient from 70 to 100% A in 5 min and other 10 min to return to initial conditions. The purity of (–)-oleuropein was determined by an external standard method using the HPLC peak area at 280 nm. Previously, a calibration curve (y = 2825.9 x – 19483; R2 = 0.9986) was constructed (Graphic S1) with six concentrations (0.1–1.0 mg•mL−1 in MeOH) of pure (–)-oleuropein (Table S1), available in authors’ lab. 2.2.2.2 Synthesis of (–)-Dimethyl-oleuropein from (–)-Oleuropein A solution of diazomethane in Et2O (35 mL), freshly prepared from N-methyl-N-nitrosourea (3.5 g) (see Supplementary Material) and aq. 50% KOH, was added dropwise to a solution of (–)-oleuropein (200 mg) solved in MeOH (8 mL). The reaction was under stirring at room temperature with TLC monitoring. After 1 h, solvent was removed under reduced pressure to give a light yellow solid (230 mg). The purification was carried out by FCPC using a quaternary biphasic solvent system composed of Hex/EtOAc/EtOH/H2O (2:5:2:5, v/v/v/v) at a flow of 7 mL/min and a rotation speed of 1200 r.p.m. As a result, 160 mg (reaction yield: 79%) of (–)-dimethyl-oleuropein was obtained as a white solid: ESIMS: m/z 591 [M+Na]+ (Figure S12); 1H NMR (400 MHz, CDCl3) (Figure S10): δ (ppm) 7.46 (s, 1H, H-9), 6.78 (d, 1H, J7’,8’= 8.8 Hz, H-7’), 6.72 (m, 2H, H-4’, H-8’), 6.04 (q, 1H, J7’,8’= 7.0 Hz, H-5), 5.75 (br s, 1H, H-7), 4.81 (d, 1H, J7’,8’= 7.7 Hz, H-1’’), 4.24 (dt, 1H, J1’a,2’= 7.2 Hz, J1’a,1’b= 10.8 Hz, H-1’), 4.10 (dt, 1H, J1’b,2’= 7.1 Hz, J1’b,1’a= 10.8 Hz, H-1’), 3.92 (dd, 2H, J6’a,5’= 4.4 Hz, J6’a,5’= 9.2 Hz, H-6’’a), 3.86 (s, 3H, C3’’-OCH3), 3.83 (s, 3H, C4’’-OCH3), 3.69 (s, 3H, C11-OCH3), 3.77 (m, 5H, H-2’’, H-3’’, H- 4’’, H-5’’, H-6’’b), 2.84 (t, 2H, J2’,1’= 7.2 Hz, H-2’), 2.76 (dd, 1H, J2a,3= 4.2 Hz, J2a,2b= 13.9 Hz, H- 2a), 2.38 (dd, 1H, J2b,3= 9.4 Hz, J2b,2a= 13.8 Hz, H-2b), 1.63 (d, 3H, J6,5= 6.7 Hz, H-6). 13C NMR (100 MHz, CDCl3) (Figure S11): δ (ppm) 171.7 (C-1), 168.9 (C-11), 153.7 (C-9), 149.1 (C3’’), 147.9 (C4’’), 130.2 (C-3’), 128.8 (C-4), 124.5 (C-5), 121.01 (C-8’), 112.4 (C-4’), 111.5 (C-7’), 108.5 (C-10), 100.2 (C-1’’), 95.0 (C-7), 77.4 (C-5’’), 76.3 (C-3’’), 73.4 (C-2’’), 70.1 (C-4’’), 65.7 (C-1’), Chapter I/ Capítulo I 116 62.0 (C-6’’), 56.1 (C4’’-OCH3)*, 56.0 (C3’’-OCH3)*, 51.6 (C11-OCH3), 40.4 (C-2), 34.7 (C-2’), 30.7 (C-3), 13.5 (C-6) (*these signals may be interchanged); [α]D 25: –152.7 (c 0.9, MeOH). NMR data of this compound were previously reported in the literature [51]. The purity of (–)- dimethyl-oleuropein (92%) was determined using the oleuropein’s calibration curve described above (Graphic S1). 2.2.2.3 Synthesis of (–)-Dimethyl-Oleacein from (–)-Dimethyl-oleuropein A mixture of (–)-dimethyl-oleuropein (100 mg), water (5 mL) and dimethyl sulfoxide DMSO (0.4 mL) was subjected to MW irradiation at 180 °C. After 9 minutes (min), the crude was extracted with Et2O and the solvent removed under reduced pressure to give a brown oil (67 mg). The purification was achieved by FCPC using Hex:EtOAc:EtOH:H2O (1:1:1:1, v/v/v/v) as biphasic solvent system, yielding 42 mg (reaction yield: 64%) of (–)-dimet-OLA as a yellow oil: ESIMS: m/z 371 [M+Na]+ (Figure S8); HRMS (ESI/Q-TOF) m/z 349.1645 [M+H]+, corresponding to C19H24O6 (Figure S9); 1H NMR (400 MHz, CDCl3) (Figure S3): δ (ppm) 9.62 (br s, 1H, H-8), 9.22 (d, 1H, J9,7b= 1.8 Hz, H-9), 6.78 (d, 1H, J5’’,6’’= 7.9 Hz, H-5’’), 6.71 (m, 2H, H-2’’,H-6’’), 6.61 (q, 1H, J5,6= 7.0 Hz, H-5), 4.20 (m, 2H, H-1’), 3.85 (s, 3H, C3’’- OCH3), 3.84 (s, 3H, C4’’-OCH3), 3.60 (m, 1H, H-3), 2.95 (dd, 1H, J7b,3= 8.7 Hz, J7b,7a= 18.3 Hz, H-7b), 2.82 (t, 2H, J2’,1’= 7.0 Hz, H-2’), 2.73 (dd, 1H, J7a,3= 5.5 Hz, J7a,7b= 18.3 Hz, H-7a), 2.68 (dd, 1H, J2b,3= 8.4 Hz, J2b,2a= 16.0 Hz, H-2b), 2.60 (dd, 1H, J2a,3= 6.6 Hz, J2a,2b= 16.0 Hz, H-2a), 2.06 (d, 3H, J6,5= 7.1 Hz, H-6). 13C NMR (100 MHz, CDCl3) (Figure S4): δ (ppm) 200.4 (C-8), 195.2 (C- 9), 172.0 (C-1), 154.3 (C-5), 149.0 (C-3’’), 148.0 (C-4’’), 143.4 (C-4), 130.3 (C-1’’), 121.0 (C-2’’), 112.2 (C-6’’), 111.4 (C-5’’), 65.2 (C-1’), 56.0 (C3’’-CH3, C4’’-OCH3), 46.3 (C-7), 36.9 (C-2), 34.7 (C-2’), 27.3 (C-3), 15.3 (C-6). The full assignment of 1H and 13C NMR resonances was supported by DEPT (Figure S4), HSQC (Figure S5), HMBC (Figure S6) and COSY (Figure S7) NMR spectra. [α]D 25: –1.1 (c 1.1, CHCl3). The purity of (–)-dimet-OLA (97%) was determined by an external standard method using the HPLC peak area at 280 nm. Previously, a calibration curve was constructed (Graphic S2) with six concentrations (0.01–0.03 mg•mL−1 in ACN) of pure (–)-OLA (Table S2), available in authors’ lab, following the same Chapter I/ Capítulo I 117 chromatographic conditions described above. The equation formulated and the coefficient of determination (R2) were y = 5117.1x – 25442; R2 = 0.9993. 2.3 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay The ABTS assay was carried out according to the method described by Re et al. [52]. 6- hydroxy 2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was used as a positive antioxidant standard. Initially, the ABTS stock solution was prepared solving 7 mM of ABTS and 2.45 mM of potassium persulfate in H2O. The solution was kept overnight for 16 h protected from light at 4 °C to produce de radical ABTS+. Then, the ABTS+ solution was diluted with ethanol until an absorbance of 1.00 at 734 nm. OLA, dimet-OLA and Trolox samples (final concentrations 200–1.6 M) were mixed with ethanol (70 L) and ABTS+ diluted solution (100 L). Finally, the absorbance was read at 734 nm for 6 min after mixing. The ABTS assay results were expressed as half maximal inhibitory concentration (IC50), the concentration (M) of that was able to inhibit 50% of ABTS+ radicals. All determinations were performed in triplicate. 2.4 Animals 8-10 weeks old Swiss mice (females 20-30 g) were purchased from the Animal Production Center of the University of Seville (Seville, Spain). Animals were randomly assigned to cages. Cages were maintained at constant temperature (20-25°C) and humidity (40-60%) with a 12-hour light/dark cycle. Mice had ad libitum access to water and rodent chow (PanlabA04, Panlab®, Seville, Spain). All experiments were performed in accordance with the Guidelines of the European Union regarding animal experimentation (Directive of the European Counsel 2012/707/EU) and using a protocol approved by the Animal Ethics Committee of the University of Seville (CEEA-US 2018-11/2 and CEEA-US 2019-6) and by the Consejeria de Agricultura, Pesca y Desarrollo (Junta de Andalucía, 23/07/2018/119), according to the RD 53/1 February 2013, at the Animal Laboratory Center of the Faculty of Pharmacy (University of Seville, Spain). Chapter I/ Capítulo I 118 2.5 Culture of Murine Cells 1 mL of sterile thioglycolate medium (3,8% w/v) (BD Difco®, Le Pont de Claix, France) was injected to female Swiss mice. 72 h after the injection, mice were sacrificed, and peritoneal macrophages were isolated by peritoneal washes with sterile phosphate-buffered saline solution (PBS). Then, cells were washed twice with PBS, centrifuged (1200 r.p.m, 10 min, 4°C) and resuspended in supplemented RPMI 1640 medium (10% Fetal bovine serum (FBS) and 1% penicillin-streptomycin). Murine cells were cultured in 24-wells culture plates (1x 106 cells/mL) for 2 h at 37 °C with a 5% CO2 humidified atmosphere in order to cell adhesion. Then, RPMI 1640 medium was retired and fresh RPMI1640 (0%FBS) in presence or absence of DMSO, OLA (25 and 12.5 M) and dimet-OLA (25 and 12.5 M) was added. After a pre-treatment of 30 min, LPS (5 g/mL) from Escherichia coli (Sigma-Aldrich®, St. Louis, MO, USA) was added as stimuli for 18 h. LPS was added to all wells except the unstimulated control cells. DMSO was added to the LPS-DMSO group as a vehicle control in which the compounds were dissolved. After incubation time, supernatants and cell samples were stored at -80 °C. At the same time, in order to prepare a splenocytes single cell-suspension, spleens were harvested, crushed, and passed through a nylon cell strainer (70 m) (BD Falcon®, USA) using supplemented RPMI 1640 medium (2 mM glutamine ,10% FBS, 1 mM sodium pyruvate, 1% penicillin and streptomycin, and 50M 2-mercaptoethanol). Spleen cells were centrifuged (1200 r.p.m, 10 min, 4 °C) and incubated twice with red blood lysis buffer (BD®, Biosciences, Bloomfield Hills, MI, USA) in order to remove erythrocytes (10 min and 5 min respectively). Then, splenocytes were pelleted (1200 r.p.m, 6 min, 4 °C) and washed with PBS. The pelleted cells (1200 r.p.m, 5 min, 4 °C) were resuspended in supplemented RPMI 1640 (2 mM glutamine ,10% FBS, 1 mM sodium pyruvate, 1% penicillin and streptomycin, and 50M 2-mercaptoethanol) and seeded in 24-well plates (3 x 106 cells/mL) in presence or absence of OLA (25 and 12.5 M), dimet-OLA (25 and 12.5M), DMSO and/or LPS (5 g/mL). After 24 h of incubation at 37 °C with a 5% CO2 humidified atmosphere, cellular pellets and supernatants were stored at -80 °C. Chapter I/ Capítulo I 119 2.6 Sulforhodamine B (SRB) assay: Cell Viability To ensure that cell survival was not compromised by OLA and dimet-OLA treatment, the SRB assay was conducted in murine peritoneal macrophages. Cells were cultured in 96-well plates (100000 cells/well) and incubated in the presence of OLA/dimet-OLA (200–1.56 M) during 18 h. Then, 50 L of 50% (w/v) cold trichloroacetic acid (Sigma–Aldrich®, St. Louis, MO, USA) was added to each well to fix the cells. After 60 min of incubation at 4°C, plates were washed with distilled H2O (5 times). Next, 50 L of 0.4% w/v SRB (Sigma–Aldrich®, St. Louis, MO, USA) in 1% acetic acid (Panreac®, Barcelona, Spain) solution was added and stained for 30 min at room temperature, protected from light. Plates were washed 5 times with 1% acetic acid solution and air-dried. Finally, 100 L of 10 mmol/L Tris base, pH 10.5 (Sigma–Aldrich®, St. Louis, MO, USA) was added to each well, then the absorbance was read using a multiwell plate reader (Biotek®, Bad Friedrichshall, Germany) at 492 nm. The absorbance of non-treated cells was considered as 100% of cell survival. Cell survival was expressed as a percentage by comparing the absorbance obtained with those of control cells (non-treated cells). The toxicity of the vehicle DMSO was also evaluated. 2.7 Nitrite Production Nitrite production in macrophages supernatants was assessed through the Griess reaction (Sigma- Aldrich®, St. Louis, MO, USA) [53]. Nitrite levels was used as an index of NO production. Murine peritoneal macrophages were isolated as described above and seeded in 24-well plates (1 x 106 cells/mL). Cells were pre-treated with OLA (25 and 12.5 M) or dimet- OLA (25 and 12.5 mM) for 30 min and activated with LPS (5 g/mL) for 18 h. Then, Griess reagent was added to fresh culture supernatant in a 1:1 ratio and the absorbance was measured at 540 nm using an ELISA reader (BioTek®, Bad Friedrichshall, Germany). Nitrite levels were determined through extrapolation from a standard curve with sodium nitrite and represented as a percentage compared with LPS-DMSO cells. Chapter I/ Capítulo I 120 2.8 Intracellular ROS: 2,7-dichlorodihydrofluoroscein diacetate (DCFDA) assay Intracellular ROS production in peritoneal macrophages was evaluated using the DCFDA assay kit (Abcam®, Cambridge, UK) according to the manufacturer’s instructions. DCFDA penetrate into the cells and is deacetylated by intracellular esterases to a non-fluorescent molecule. Then is oxidized by ROS into 2’, 7’ –dichlorofluorescein (DCF), a fluorescent compound. Cells were cultured (2.5×104 cells/well) in a 96-well dark bottom plate and pretreated with OLA (25 and 12.5 M) or dimet-OLA (25 and 12.5 M) for 30 minutes. Then, macrophages were stimulated with LPS (5 g/mL) for 18 h. After that, 25 M of DCFDA was added to each well and the cells were incubated at 37 °C for 45 min. Intracellular ROS production was measured using 485 and 535 nm as excitation and emission wavelengths respectively in a fluorescence microplate reader (Biotek®, Bad Friedrichshall, Germany). H2O2 (Sigma-Aldrich®, Barcelona, Spain) was used as a pro-oxidant standard. The results were expressed as a percentage compared with the H2O2 group. 2.9 Enzyme-Linked Immunosorbent Assay (ELISA): Determination of Pro-Inflammatory Cytokines Levels The production of pro-inflammatory cytokines was evaluated on culture macrophages and spleen cells culture supernatants using interferon gamma (IFN-), IL-6 (Diaclone®, Besancon Cedex, France), tumor necrosis factor alpha (TNF- ), IL-1, (R&D System®, Minneapolis, MN, USA) and IL-17 (Peprotech®, London, UK), ELISA kits, according to manufacturer’s instructions. 2.10 Histone Extraction Histones were isolated from spleen cells following the protocol described by Hajji et al. [54] with modifications. Splenocytes were lysed with lysis buffer (50 mM sodium bisulfite, 10 mM Tris pH 6.5, 8.6% sucrose, 10 nM MgCl2, 1% Triton X-100). 1 mL of lysis buffer was added to the cells for 15 min at 4 °C. After incubation, spleen cells were pelleted (3500x g, 10 min, 4 °C) and rewashed with lysis buffer. Then, 1 mL of Tris-Ethylenediaminetetraacetic acid (EDTA) (10 mM Tris pH 7.4 and 13 mM EDTA) was added to the pellet and the Chapter I/ Capítulo I 121 samples were centrifuged another time (3500x g, 10 min, 4 °C). Subsequently, 100 mL of 0.2M sulfuric acid was added and incubated by at least 1 h. Samples were then centrifuged (15,000xg, 1 h, 4 °C) and the resultant supernatants were collected and incubated with acetone (overnight, -20°C) to precipitate histones. Samples were centrifuged at (15,000xg, 10 min, 4 °C) and the pelleted histones were diluted with H2O. The Bradford’s method was used to determine the protein concentration [55] and the histone modifications were evaluated by Western Blot. 2.11 Western Blot Analysis Murine peritoneal macrophages were scrapped and lysed using ice-cold lysis buffer containing a cocktail of protease and phosphatase inhibitors buffer (5 mmol/L ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 8 mmol/L MgCl2, 250 mmol/L NaCl, 50 mmol/L Tris–HCl (pH 7.5), 0.5 mmol/L EDTA, 0.01 mg/mL leupeptin, 0.01 mg/mL pepstatin 0.01, mg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF)). Protein content was evaluated following the Bradford method [55]. -globulin was used as a standard. Aliquots of 5 g (histones) or 15 g (macrophages) of total protein were separated on 15% or 10% by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Then the proteins were transferred to a nitrocellulose membrane (BioRad®, Hercules, CA, USA) and incubated with specific primary antibodies overnight at 4 °C (Table 1). After washing, the membranes were incubated for 1.5-2 h with blocking buffer (1% casein (Sigma-Aldrich®, St. Louis, MO, USA) and 5% Nonident-P40 substitute (Sigma-Aldrich®, St. Louis, MO, USA) in PBS buffer) containing antirabbit horseradish peroxidase-labeled (HRP) secondary antibody (Cell Signaling®, Danvers, MA, USA) or anti mouse HRP secondary antibody (Dako®, Atlanta, GA, USA). As loading control, total H3, ERK, P38, JNK (Cell Signaling®, Danvers, MA, USA) and -actin (Abcam®, Cambridge, UK) were used. Immunodetection was conducted with enhanced chemiluminescence light detection kit (Biorad®, Hercules, CA, USA). The immunosignals were captured using Amersham Imager 600 from GE Healthcare® (Buckinghamshire, UK) and quantified using Java (ImageJ®, Softonic). Finally, Chapter I/ Capítulo I 122 results were normalized with the DMSO-LPS group cells. All antibodies used for the evaluation of protein expression by western blot are summarized in Table 1. Table 1. Antibodies used in Western Blot Antibody Dilution Identification anti-rabbit IgG Cell Signaling Technology® (Danvers, MA, USA) 1:1000 Cat#7074; RRID: AB_2099233 anti-mouse IgG Dako®, Atlanta, GA, USA 1:2000 Cat#A4416; RID: AB_258167 −actin Abcam® (Cambridge, UK) 1:10000 Cat#ab49900; RRID: AB_867494 COX-2 Cell Signaling Technology® (Danvers, MA, USA) 1:1000 Cat#12282; RRID: AB_2571729 mPGES-1 Abcam® (Cambridge, UK) 1:1000 Cat#ab62050; RRID: AB_2269175 iNOS Cell Signaling Technology® (Danvers, MA, USA) 1:1000 Cat#13120. RRID: AB_2687529 IL-18 Abcam® (Cambridge, UK) 1:1000 Cat#ab71495; RRID: AB_1209302 NLRP3 Cell Signaling Technology® (Danvers, MA, USA) 1:1000 Cat#15101; RRID: AB_2722591 Caspase-1 Cell Signaling Technology® (Danvers, MA, USA) 1:1000 Cat# 24232; RRID: AB_2890194 Apoptosis-associated speck-like protein containing a CARD (ASC) Cell Signaling Technology® (Danvers, MA, USA) 1:1000 Cat#67824. RRID: AB_2799736 Caspase-11 Novus Biologicals® Littleton, CO, USA 1:2000 Cat#NBP1-45453; RRID: AB_10008917 HO-1 Enzo Life Sciences® (Madrid, Spain) 1:1000 Cat#ADI-SPA-896-D; RRID: AB_2039233 pERK Cell Signaling Technology® (Danvers, MA, USA) 1:1000 Cat#4370; RRID: AB_2315112 pP38 Cell Signaling Technology® (Danvers, MA, USA) 1:1000 Cat#4511; RRID: AB_2139682 pJNK Cell Signaling Technology® (Danvers, MA, USA) 1:1000 Cat#4668; RRID: AB_823588 ERK Cell Signaling 1:1000 Cat#9107; RRID: AB_10695739 Chapter I/ Capítulo I 123 Technology® (Danvers, MA, USA) P38 Cell Signaling Technology® (Danvers, MA, USA) 1:1000 Cat#8690; RRID: AB_10999090 JNK Cell Signaling Technology® (Danvers, MA, USA) 1:1000 Cat#9252; RRID: AB_2250373 Nrf-2 Cell Signaling Technology® (Danvers, MA, USA) 1:1000 Cat#12721; RRID: AB_2715528 IB- Cell Signaling Technology® (Danvers, MA, USA) 1:1000 Cat#4812; RRID: AB_10694416 STAT-3 Cell Signaling Technology® (Danvers, MA, USA) 1:1000 Cat#9145; RRID: AB_2491009 pJAK2 Cell Signaling Technology® (Danvers, MA, USA) 1:1000 Cat#3771; RRID: AB_330403 Total H3 Cell Signaling Technology® (Danvers, MA, USA) 1:1000 Cat# 3638; RRID: AB_1642229 H3K18Ac Cell Signaling Technology® (Danvers, MA, USA) 1:1000 Cat# 13998; RRID: AB_2783723 H3K27me3 Cell Signaling Technology® (Danvers, MA, USA) 1:1000 Cat# 9733; RRID: AB_2616029 H3K9me3 Cell Signaling Technology® (Danvers, MA, USA) 1:1000 Cat# 13969, RRID:AB_2798355 2.12 Statistical Analysis All values in the figures and text are expressed as arithmetic means ± standard error (SEM). Data were evaluated with GraphPad Prism_Version 5.01 software. The statistical significance of any difference in each parameter among the groups was evaluated by oneway analysis of variance (ANOVA), using Tukey–Kramer multiple comparisons test as post hoc test. p values of <0.05 were considered statistically significant. Chapter I/ Capítulo I 130 Figure 5. OLA and dimet-OLA treatment counteract COX-2 and mPGES-1 overexpression in murine peritoneal macrophages induced by LPS. (A) Western blot analysis of COX-2 and mPGES-1 protein expression in OLA treated cells (25 and 12.5 μM); (B) Western blot analysis of COX-2 and mPGES- 1 protein expression in OLA (25 μM) and dimet-OLA (25 and 12.5 μM) treated cells. Cells were pretreated (30 min) with the study compounds and incubated in absence or presence of LPS for 18 h. Control cells were also treated with DMSO (solvent control) and LPS or nontreated. Densitometry was performed after normalization to the loading control (β-actin housekeeping gene). Data are represented as mean ± SEM (n = 6). +++ P< 0.001 vs. control cells (no stimulated); ** P< 0.01, *** P< 0.001 vs. LPS−DMSO cells. 3.8 Effect of OLA and Dimet-OLA in the Regulation of JAK/STAT Pro-Inflammatory Signalling Pathway Among the signalling pathways involved in inflammatory and immune responses, the JAK/STAT axis has demonstrated to play a crucial role. Indeed, its abnormal activation may be related to the pathogenesis of immune-inflammatory diseases [61]. Therefore, we assessed the impact of OLA and dimet-OLA on the phosphorylation and activation of the JAK/STAT proteins in LPS-stimulated macrophages. As described by Castejón et al. [10], increased phosphorylation of JAK2 and STAT-3 proteins was detected in LPS activated macrophages (+P<0.05, ++ P<0.01, +++ P<0.001 vs. unstimulated control cells). Meanwhile, Chapter I/ Capítulo I 131 our results demonstrated a decrease in JAK/STAT proteins phosphorylation in presence of OLA and dimet-OLA treatment (* P<0.05, ** P<0.001, ** P<0.001 vs. LPS-DMSO cells). Figure 6. OLA and its methylated derivative modulated JAK/STAT activation in LPS-activated peritoneal macrophages. (A) Western blot analysis of pJAK2/pSTAT-3 protein expression in OLA (25 and 12.5 μM) and dimet-OLA (25 and 12.5 μM) treated cells. Cells were pretreated for 30 min with the OLA or dimet-OLA and stimulated with LPS for 18 h. Control cells were also treated with DMSO (solvent control) and LPS or nontreated. Densitometry was performed after normalization to the control (β-actin). Data are represented as mean ± SEM (n = 6). + P< 0.05, ++ P< 0.01, +++ P<0.001 vs. control cells (no stimulated); * P<0.05, ** P<0.01, *** P<0.001 vs. LPS−DMSO control-treated cells. 3.9 OLA and Dimet-OLA Down-regulated MAPKs Activation in Murine Macrophages Stimulated with LPS Additionally, we determined whether OLA and dimet-OLA could regulate the phosphorylation of the MAPKs family. The activation of the MAPKs signalling pathway results in the expression of numerous genes that regulate the inflammatory response [62]. As shown in Figure 7, a significant decrease in ERK and P38 protein phosphorylation was found in cells treated with OLA (*P<0.05, ** P<0.01, *** P<0.001 vs. LPS-DMSO cells). Moreover, after dimet-OLA incubation, the phosphorylation of pERK and pJNK proteins was modulated (Figure 7B) (* P< 0.05 vs. LPS−DMSO control cells). Interestingly, dimet- Chapter I/ Capítulo I 132 OLA regulated the phosphorylation of pJNK, contrary to its precursor OLA. However, OLA significantly reduced P38 phosphorylation, while no changes in P38 phosphorylation were detected in dimet-OLA treated cells. Figure 7. Effects of OLA and dimet-OLA on MAPKs family activation. (A) Western blot analysis of pJNK/pERK/pP38 protein expression in OLA treated cells (25 and 12.5 μM); (B) Western blot analysis of pJNK/pERK/pP38 protein expression in OLA (25 μM) and dimet-OLA treated cells (25 and 12.5 μM). Cells were pretreated for 30 min with the OLA or dimet-OLA and stimulated with LPS for 18 h. Control cells were also treated with DMSO (solvent control) and LPS or nontreated. Densitometry was performed after normalization to the control (ERK, JNK, P38). Data are represented as mean ± SEM (n = 6). + P< 0.05, ++ P< 0.01, +++ P<0.001 vs. control cells (no stimulated); * P<0.05, ** P<0.01, *** P<0.001 vs. LPS−DMSO control-treated cells. Chapter I/ Capítulo I 133 3.10 Effect of OLA and its Methylated Derivative in the Degradation of IB- induced by LPS in Murine Peritoneal Macrophages. The activation of murine macrophages with LPS leads to the degradation of IB- (Figure 8). (+P<0.05 vs. unstimulated control cells). Nevertheless, LPS-induced degradation of IB- was prevented by the treatment with OLA and dimet-OLA (* P<0.05 vs. LPS-DMSO cells). This effect was only observed when cells were pretreated with high concentrations of both compounds (25 μM). Figure 8. OLA and dimet-OLA treatment prevent IkB- degradation in LPS-induced macrophages. (A) IkB- protein expression in OLA treated cells (25 and 12.5 μM); (B) Western blot analysis of IkB- protein in OLA (25 μM) and dimet-OLA treated cells (25 and 12.5 μM). Murine macrophages were pretreated for 30 min with the OLA or dimet-OLA and then activated with LPS for 18 h. Cells were also treated with DMSO (solvent control) and LPS or nontreated. Densitometry was performed after normalization to the control (β-actin, ERK, JNK, P38). Data are represented as mean ± SEM (n ≥ 6). + P< 0.05 vs. control cells (no stimulated); * P<0.05 vs. LPS−DMSO control-treated cells. 3.11 Inhibitory Effect of OLA and dimet-OLA in the Activation of Inflammasome in LPS- Induced Peritoneal Macrophages NLRP3 inflammasome activation triggers pyroptosis and IL-18/IL-1 maturation [63]. Moreover, it has been reported that an aberrant inflammasome activation is involved in the development of several diseases like Alzheimer, cancer or inflammatory diseases [64–66]. In Chapter I/ Capítulo I 134 fact, the inflammasome suppression is therefore a promising approach in the search for new therapeutic targets [65,66]. In order to investigate, the effect of OLA and dimet-OLA in inflammasome activation, the protein expression of canonical and non-canonical inflammasome signalling pathway was evaluated by Western Blot. As figure 9 showed, after 18h of LPS stimulation we detected increased expression of NLRP-3 and caspase 1-proteins in LPS-induced peritoneal macrophages (Figure 9A-D) (+ P<0.05, +++ P<0.001 vs. unstimulated control cells), compared to untreated cells. Consequently, elevated levels of IL- 1 and IL-18 were also found to be upregulated in LPS-treated cells (Figure 3E and Figure 9A, B) (+ P<0.05, +++ P<0.001 vs. unstimulated control cells). The treatment with OLA and its methylated derivative, dimet-OLA, effectively suppressed the overactivation of canonical inflammasome pathway, reducing NLRP-3 and caspase 1 protein expression (Figure 9A-D) (* P<0.05, ** P<0.01 *** P<0.001 vs. LPS-DMSO cells), as well as the maturation of IL-18 and IL-1 pro-inflammatory cytokines (Figure 3B and Figure 9A, B) (* P<0.05, ** P<0.01, *** P<0.001 vs. LPS-DMSO cells). Additionally, the expression of ASC protein was also modulated in OLA or dimet-OLA treated murine macrophages (Figure 8A, B) (* P<0.05, ** P<0.01, *** P<0.001 vs. LPS-DMSO cells). On the other hand, the non-canonical inflammasome pathway is initiated by caspase 11, which is also associated with pyroptosis and the maturation of IL-18/IL-1β [67]. Accordingly, in cells treated with OLA, a significant decrease in caspase 11 activation was observed (* P<0.05, ** P<0.01, *** P<0.001 vs. LPSDMSO cells). Conversely, its derivative dimet-OLA was not able to reduce the activation of caspase 11 induced by LPS (Figure 9D). Chapter I/ Capítulo I 135 Figure 9. Effects of OLA and dimet-OLA in canonical and non-canonical inflammasome activation. (A) NLRP3. ASC and IL-18 Western Blot analysis in OLA treated cells (25 and 12.5 μM); (B) NLRP3, ASC and IL-18 Western Blot analysis in OLA (25 μM) and dimet-OLA treated cells (25 and 12.5 μM). (C) caspase 1 and caspase 11 Western Blot analysis in OLA treated cells (25 and 12.5 μM); (D) Western blot analysis of caspase 1/caspase 11 expression in OLA (25 μM) and dimet-OLA treated cells (25 and 12.5 μM). Peritoneal macrophages were pretreated for 30 min with the OLA, dimet-OLA or DMSO and then stimulated for 18 h whit LPS. Control cells were also treated with LPS or nontreated. Densitometry was performed after normalization to the control (β-actin). Data are represented as mean ± SEM (n = 6). + P< 0.05, +++ P<0.001 vs. control cells (no stimulated); * P<0.05, ** P<0.01, *** P<0.001 vs. LPS−DMSO controltreated cells. Chapter I/ Capítulo I 136 3.12 Effects of OLA and Dimet-OLA in Nrf-2/HO signaling pathway in Murine Macrophages Stimulated by LPS Moreover, to evaluate the antioxidant effects of OLA and dimet-OLA, the protein expression of Nrf-2 and HO-1 was measured by Western Blot. We found a significant up-regulation in Nrf-2 and HO-1 protein expression in comparison to LPS-DMSO cells in OLA treated cells (* P<0.05, ** P<0.01 vs. LPS-DMSO cells) (Figure 10A). These findings, together with the modulation of intracellular ROS production (Figure 4B), may support the potential role of OLA in modulating the antioxidant response. On the contrary, in cells treated with dimet- OLA, no changes were observed in the protein expression of Nrf-2/HO-1 axis, although it was able to reduce the intracellular levels of ROS (Figure 10B, Figure 4B). Figure 10. Effects of OLA and dimet-OLA treatments on Nrf-2/HO-1 axis in murine peritoneal macrophages induced by LPS. (A) Nrf-2 and HO-1 protein expression in OLA treated cells (25 and 12.5 μM); (B) Nrf-2 and HO-1 Western Blot analysis in OLA (25 μM) and dimet-OLA treated cells (25 and 12.5 μM). Cells were with OLA, dimet-OLA (30 min) and then stimulated for 18 h with LPS. Control cells were also treated with LPS or DMSO or nontreated. Densitometry was performed after normalization to the control (β-actin). Data are represented as mean ± SEM (n = 6) * P<0.05, ** P<0.01 vs. LPS−DMSO control-treated cells; # P< 0.05, ## P<0.01 vs. OLA (25 μM). Chapter I/ Capítulo I 137 3.13 OLA and dimet-OLA Induced Histone Modifications in LPS-activated Splenocytes To investigate the potential epigenetic mechanisms responsible for the effects of OLA and dimet-OLA, we assessed changes in histone H3 methylation and acetylation, as well as the production of related cytokines. LPS incubation promote the acetylation of H3, leading to an increase in the expression of H3K18ac in LPS-stimulated spleen cells (+++ P<0.001 vs. unstimulated control cells). On the contrary, the treatment with our compounds resulted in decreased levels of H3K18ac (Figure 11A). Notably, only de highest concentration of OLA (25 M) induced significant changes (** P<0.01 vs. LPS-DMSO cells), whereas dimet-OLA was effective at 25 and 12.5 M (** P<0.01, *** P<0.001 vs. LPS-DMSO cells) (Figure 11A). Moreover, we noticed that only OLA at 25 M, increased H3K27 and H3K9me3 histone methylation levels (Figure 11D, E) (* P<0.05 vs. LPS−DMSO-treated cells). We also evaluated the production of pro-inflammatory cytokines which were related to these histone modifications in their promoter regions. OLA was able to regulate the overproduction of IL- 1, IL-6 and IL-17 induced by LPS in splenocytes (* P<0.05, ** P<0.01, *** P<0.001 vs. LPS−DMSO-treated cells). In the same way, dimet-OLA also reduced IL-6 levels along with Il-1 and IL-17, although for IL-17 we only found significantly differences at the 25 M concentration (** P<0.01, *** P<0.001 vs. LPS−DMSO-treated cells). Chapter I/ Capítulo I 138 Figure 11. Effect of OLA and dimet-OLA treatment on histone acetylation (D) and methylation (E) and the inhibition of the cytokine-related response (A-C). Spleen cells were treated with OLA or dimet-OLA (25 and 12.5 M) and stimulated with LPS for 24 h. Histones were isolated from cells with acid extraction and were evaluated by Western blot. Densitometry was performed following normalization to the control (H3 housekeeping gene). Cytokine levels were measured in cell supernatants using ELISA assays. Results are presented as mean ± SEM of (n ≥ 6). + P< 0.05, ++ P< 0.01, +++ P< 0.001 vs. control cells (no stimulated); * P< 0.05, ** P< 0.01, *** P< 0.001 vs. LPS-DMSO-treated cells. Chapter I/ Capítulo I 139 4. DISCUSSION In this work, the anti-inflammatory and antioxidant effects of OLA and its O-methylated derivative, dimet-OLA, were demonstrated in murine immune cells stimulated with LPS. Nowadays, certain studies have proved the therapeutic potential of OLA [31,33,36,68,69] but its methylated derivative has not been studied yet. Specifically, Cirmi et al. described that OLA treatment was able to counteract the inflammatory response induced by LPS in THP-1 cells, reducing the expression of COX-2, in addition to NO and PGE2 levels [36]. Furthermore, Czerwinska et al. revealed the potent antioxidant activity of OLA, even improving that of its precursor oleuropein [70]. However, it is the first time that the synthesis of dimet-OLA and its protective effects were assessed in murine cells. Olive secoiridoids such as oleuropein, oleocanthal, or OLA are known to have a wide variety of beneficial health activities [21,35,71–74]; however, polyphenols are unstable molecules, sensitive to light and temperature, that present difficulties in crossing biological membranes and low bioavailability values [37]. Recent studies have shown that the methylation of natural drugs can improve their stability and bioavailability. Hydroxyl group methylation (orthomethylation) results in more lipophilic compounds, preventing the degradation and ameliorating the permeation of molecules across membranes [41]. Thus, the methylation process could enhance its therapeutic potential [42]. In this context, methylated flavonoids exhibited better antioxidant properties by inhibiting Nicotinamide- Adenine-Dinucleotide-Phosphate (NADPH) oxidase [75]. Yao et al. presented how Pterostilbene and 4′-Methoxyresveratrol, two methylated derivatives of resveratrol, modulated the LPS-induced inflammatory response in RAW 264.7 macrophages through the inhibition of NF-κB and MAPKs signalling pathways [1]. Further, a methylated derivate of chrysin improved its anti-inflammatory effect by the reduction of IL-6, IL-8, COX-2 and PGE2 production on CaCo-2 cells [43]. In this line, our research group previously explored the activity of a methylated derivative of oleocanthal, presenting it as an interesting epigenome-regulating compound. Collectively, these promising results suggest that the orthomethylation of polyphenols such as secoiridoids is able to ameliorate their biological Chapter III/ Capítulo III 242 3.9 Role of OLA As a Regulator of DNA Methylation in Activated PBMCs To further investigate the potential role of OLA as a modulator of the epigenome, we examined the % of global methylation in PHA-stimulated PBMCs isolated from healthy controls and patients with SLE. Although the variations obtained are not significant due to the great variability presented by the patient samples, a lower degree of methylation can be observed in the unstimulated PBMCs of patients with SLE compared to healthy controls (Figure 9). In fact, global DNA hypomethylation had previously been found in patients with SLE [39]. On the other hand, we can observe that treatment with OLA tends to increase the % of methylation in both healthy controls and patients with lupus (Figure 9). Figure 9. Global DNA methylation status in PHA-activated PBMCs isolated from SLE patients and healthy controls. The % methylated DNA was evaluated by Methylamp™ Global DNA Methylation Quantification Ultra Kit. PBMCs were incubated with PHA (10 g/mL) and treated with OLA (100-50 M) for 24 h. Control groups were untreated or treated with PHA. Data are expressed as means ± SEM (n = 7). Statistical analysis was performed by ANOVA followed of Tukey–Kramer multiple comparisons test. No significative differences were found. Chapter III/ Capítuo III 243 3.10 OLA and dimet-OLA Modulated Cytokine Secretion Induced by aCD3/aCD28 in a Co- Culture of CCR6+ Memory Th Cells and PsA Fibroblast Additionally, we also quantified the levels of various cytokines involved in the inflammatory response in culture supernatant from a co-culture setup comprising CCR6+ memory Th cells and synovial fibroblasts isolated from PsA patients. As showed in Figure 10, levels of IL-6, IL- 8, IFN- and IL-17 pro-inflammatory cytokines were increased in cell supernatant after 72 h of aCD3/aCD28 stimulation (+ P<0.05, ++ P<0.01 vs Control cells (no stimulated) cells). Nevertheless, OLA and dimet-OLA (12.5 M) were able to significantly reduce IL-6 levels (* P<0.05 vs aCD3/aCD28-DMSO cells). Interestingly in this study, a trend in IL-8 reduction was detected in dimet-OLA treated. In addition, dimet-OLA treatment also resulted in a significantly greater reduction than its precursor in the secretion of IFN- and IL-17 levels (* P<0.05 vs aCD3/aCD28-DMSO cells) (# P<0.05 vs OLA-treated cells). Notwithstanding, OLA showed a tendency to up-regulate the expression of IL-10, while no changes were seen in cells treated with dimet-OLA. Chapter III/ Capítulo III 244 Figure 10. Evaluation of IL-6 (A), IL-8 (B), IL-10 (C). IFN- (D) and IL-17 (E) secretion in a coculture of memory CCR6+ T cells and PsA synovial fibroblast. aCD3/aCD28 cell activator was used to induce the cell and then OLA and dimet-OLA treatment were added for 72 h (12.5 M). Control cells were also activated, treated with DMSO or nontreated. Data are expressed as means ± SEM (n = 3). Statistical analysis was performed by T-student test. + P<0.05, ++ P<0.01 vs. control cells (no stimulated); * P<0.05 vs. aCD3/aCD28-DMSO; ## P<0.05, # P<0.01 vs. dimet-OLA treated cells. Chapter III/ Capítuo III 245 4.DISCUSSION SLE and PsA are disorders included in the immune-mediated inflammatory diseases, which are characterized by the alteration of the immune system with the key role of T cells. These disorders display a wide range of clinical manifestations and can be challenging to diagnose due to their heterogeneity. Several authors have highlighted the immunomodulatory role of EVOO the regulation of T cell activation. In this way, our research group has previously described that the unsaponifiable fraction of EVOO was found to modulate the activation of T cells from patients with inflammatory bowel disease. This was observed in both blood and bowel samples [40]. Moreover, Álvarez-Laderas et al. observed that a phenolic extract isolated from EVOO reduces T cell proliferation and activation [41]. Specifically, we have previously demonstrated the immunomodulatory effects of OLA and dimet-OLA in immune murine cells and a murine model of pristane-induced SLE (Chapter I and II), nevertheless, to date there is limited knowledge regarding the effectiveness of these compounds on T cell regulation. PBMCs are a cellular fraction present in the blood that mainly includes lymphocytes and monocytes [42]. In this work, we explored the effects of OLA on T cell activation through stimulation with PHA. PHA binds to T cell surface carbohydrates and is generally used as an inducer of T cell proliferation leading to increased cytokine production [43]. In SLE patients, there is an increase in circulating cytokines that contribute to the inflammatory process [44]. Ding et al. found evidence of elevated serum IL-6 in patients with SLE, which appears to be positively correlated with disease activity [45]. Likewise, an increase in TNF- production is usually associated with SLE activity, with a higher concentration of TNF- detected in the blood and kidneys of patients diagnosed with active SLE [46,47]. However, it should be noted that some authors have reported a reduction in TNF- levels in patients with very severe SLE [48], indicating the need for further studies in order to fully understand the role of TNF- in SLE pathogenesis. Chapter III/ Capítulo III 246 With regards to IL-1, it has been reported that increased levels of this cytokine in SLE patients serum could be associated with lupus nephritis [49], and studies in MRL/lpr mice linked increased expression of IL-1 with the progression and severity of SLE [50,51]. In this sense, Aparicio-Soto et al. observed an increase in the levels of IL-6, IL-1 and TNF- in PBMCs isolated from SLE patients compared to healthy donors, which was increased in both cases after incubation with PHA [28]. Consistent with this study, our results showed an increase in the production of IL-6, IL-1 and TNF- in PHA-stimulated PBMCs isolated from healthy donors and SLE patients. Interestingly, OLA treatment drastically reduced the production of these pro-inflammatory cytokines in both cases. However, there were no significant differences in cytokine production between unstimulated PBMCs from healthy controls and SLE patients. This may be attributed to the fact that many of our patients were administered glucocorticoids or antimalarials, which can result in decreased secretion of proinflammatory cytokines[52,53]. Furthermore, T cell activation constitutes a crucial initial stage in the initiation the adaptive immune response. Although the role of the surface marker CD69 in the regulation of the immune response is controversial [54]. Vitales-Noyola et al. described an increase in CD4CD69+ T cells in patients with lupus [55]. Our results demonstrated that after 24 h of stimulation with PHA, there was an increase in the expression of the surface marker CD69 in CD4+ lymphocytes from healthy donors and patients with SLE according to Aparicio-Soto et al. [28]. On the contrary, OLA treatment (100 M) was able to regulate CD69 expression in cells from healthy donors and no significant changes were observed in OLA treated cells from patients with SLE. Similarly, the polyphenolic fraction from EVOO regulated CD69+CD4+ lymphocytes activation in SLE patients[28]. We also studied epigenetic changes in PHA-stimulated PBMCs isolated from healthy controls and SLE patients. Although the etiopathogenesis of SLE is still unclear, there is increasing evidence that epigenetics plays an essential role in the development of SLE [56]. Specifically, in this work, we have focused on DNA methylation. In fact, global DNA hypomethylation in CD4+ lymphocytes from SLE active patients has previously been described [57], relating it to Chapter III/ Capítuo III 247 disease activity. Similarly, a reduction in DNA methylation in genes responsible for IL-17A expression has also been observed, leading to an increase in its production [58]. Although the DNA methylation changes observed in this work were not statistically significant, we found a trend towards hypomethylated DNA in unstimulated PBMCs from SLE patients when compared with healthy donors. On the other hand, in OLA-treated cells, we found a trend to increase DNA methylation, suggesting a role for OLA as an epigenome modulator, in accordance with recent studies [37,59]. Regarding memory CD4 + T cells, Xu et al. indicated that they are the main generators of IL- 17A in the synovial fluid of patients with PsA [60]. In this way, we attempt to assess the effect of OLA and dimet-OLA on activated CD4+ memory T cells. Following aCD3/aCD28 stimulation for 72 h, an increase in pro-inflammatory cytokines IFN-γ and IL-17 production was observed in memory CD4+ T cells. Nevertheless, OLA and dimet-OLA (12.5 μM) treatments led to a substantial reduction in the levels of these both cytokines in CD4+ memory T cells. These results are preliminary but show a promising effect of OLA and dimet-OLA on IL-17 regulation which is closely related to the pathogenesis of PsA and SLE. We also evaluated the impact of OLA and dimet-OLA on IL-10 production; however, no significant alterations were observed. The role of IL-10 in immune-inflammatory diseases is controversial; although it is widely described as an anti-inflammatory cytokine, increased levels of IL-10 have been detected in SLE patients being associated with up-regulated autoantibody production and disease progression [61,62]. In this context, Amend et al. suggested the dual activity of IL-10 in SLE, which can act as a protective factor or exacerbate the disease [63]. On the other hand, Mavropoulos et al. reported reduced levels of IL-10- producing cells in patients with PsA and psoriasis, which were inversely related to disease severity [64]. Therefore, the implications of IL-10 in immune-inflammatory diseases is not fully dilucidated. Recent studies documented an increase in Th17 cell infiltration within the joint synovium in patients with PsA [6]. Activated fibroblasts lead to the recruitment and activation of Th17 cells through the secretion of mediators, thereby facilitating the inflammatory process in the joints Chapter III/ Capítulo III 248 of PsA patients [65]. Particularly, an increase in IL-6 expression is observed in PsA, being more marked in the synovium than in dermal lesions [66]. This may be related to the ability of synovial fibroblasts to produce IL-6; in fact, in rheumatic diseases with inflammatory arthritis, such as RA, synovial fibroblasts have been reported to generate significant amounts of IL-6 [67]. Additionally, CCR6+ cells are present in the synovium of RA patients, producing IL-17, TNF- and INF− and contributing to synovial activation [68]. Shi et al. noticed a high presence of CCR6+ T cells in the synovial membrane of PsA patients compared to healthy donors. They also suggest that blocking the CCR6+/Chemokine (C-C motif) ligand (CCL)20 axis improved joint and dermal damage in mice, suggesting CCR6+ T cells modulation as a potential therapeutic target in the management of PsA [69]. Moreover, the activation of fibroblasts-like synoviocytes also contributes to the activation of CCR6+ memory Th cells leading to an inflammatory loop in the synovium. Through the co-culture of CCR6+ memory Th cells and synovial fibroblasts, we studied the immunomodulatory and anti-inflammatory effects of OLA and dimet-OLA in a more realistic environment that mimics the process that occurs in the joints of patients with rheumatic diseases. Moreover, IL-8 would also be produced, which attracts the arrival of more immune cells [70]. This mechanism, explored in the RA patients joints, could be proposed as an interesting interpretation of the synovial inflammatory process of PSA; however, further studies are needed to better understand the interaction between Th17 cells and synovial fibroblasts in the joints of patients with PsA. Consistent with the above mentioned, our results indicate a significant increase in IL-6 secretion both in IL-1-activated SW982 fibroblast and in co-culture of CCR6+ memory Th cells and primary fibroblast from PsA patients. Interestingly, OLA and its methylated derivative significantly reduced the levels of this pro-inflammatory cytokine in both cellular models. Additionally, the treatment of IL-1-activated fibroblast-like synoviocytes with OLA and dimet-OLA resulted in a reduction of TNF- levels, which is related to osteoclastogenesis and erosion in PsA [71]. Surprisingly, dimet-OLA was able to reduce IL-8, IFN-, and IL-17 production induced by aCD3/aCD28, whereas OLA treatment did not produce significant changes but tended to upregulate the IL-10 secretion which is a protective anti-inflammatory factor [72]. On the other hand, the production of MMPs leads to collagen degradation, thus Chapter III/ Capítuo III 249 promoting endothelial cell and synovial fibroblast infiltration [73]. According to previous reports, we found MMP-1 and MMP-3 overexpressed in the IL-1-−stimulated SW982 cell line [12,22]. However, dimet-OLA and its precursor were able to counteract this effect, significantly reducing the expression of these markers in cell supernatants. All together, these results suggest that OLA and dimet-OLA could be interesting tools for the management of inflammatory arthritis in PsA. The anti-inflammatory effects of the polyphenolic fraction of EVOO and oleuropein in synovial fibroblasts induced by IL-1 have already been explored [12,22], but this is the first time that the beneficial properties of OLA and its O-methylated derivative have been evaluated both in the SW982 cell line and in primary fibroblasts of patients with PsA. To conclude, in this chapter we describe for the first time the immunomodulatory and antiinflammatory activities of OLA and dimet-OLA in different populations of T cells and synovial fibroblasts. At first, OLA and dimet-OLA (12.5 M) were able to suppress the oversecretion of pro-inflammatory cytokines (INF- and IL-17), after 72 h of treatment, in aCD3/aCD28-activated memory CD4+ T Cells. Furthermore, dimet-OLA and its natural precursor regulate the expression of MMP-1, MMP-3, IL-6 and TNF- in a cell line of fibroblast-like synoviocites (SW982) that counteracts the induction by IL-1 We also evaluated the beneficial effects of OLA in PBMCs isolated from healthy donors and SLE patients. OLA significantly reduced the production of IL-6, TNF- and IL-1, both in healthy donors and in PBMCs from SLE patients but was only able to reduce CD69+ expression in PHA-activated PBMCs from healthy donors. Interestingly, we detect a trend to DNA hypomethylation in SLE patients compared to healthy donors that was enhanced by OLA treatment. Finally, we explore whether OLA and dimet-OLA counteract the inflammatory loop in a co-culture of CCR6+ Memory Th cells from buffycoats and synovial fibroblasts from PsA patients. More noteworthy results were achieved with dimet-OLA in decreasing the generation of pro-inflammatory cytokines. However, OLA was able to stimulate IL-10 secretion. In general, our findings suggest that both OLA and dimet-OLA hold promise in the management of immune-mediated diseases by reducing the production of pro-inflammatory Chapter III/ Capítulo III 250 mediators. Additionally, OLA has the potential to regulate the epigenome through DNA methylation, which presents an interesting avenue for further research. ACKNOWLEDGMENTS Rocío Muñoz García gratefully acknowledges support from a Postgraduate National Program of FPU fellowship and the financial support of the Spanish Ministry of Universities. The results presented in this chapter have been achieved by funding the following projects: AGL2017-89342-P, PID2021-125488OB-I00, PROYEXCEL_00547. Rocío Muñoz García gratefully acknowledges Drs. Joaquín Altarejos and Juan Ortega-Vidal (University of Jaen, Spain) for providing the OLA and dimet-OLA, Dr. Erik Lubberts as principal investigator of the Immunology and Inflammatory Arthritis research group (Department of Rheumatology and Immunology, Erasmus Medical Center, Rotterdam, The Netherlands) for allowing her to carry out a stay in his laboratory funded by the FPU mobility grant program, and Dr. Jose Manuel Lucena Soto (Hospital Virgen del Rocío, Seville, Spain) for his contribution to the work with PBMCs from SLE patients. REFERENCES 1. Azuaga, A.B.; Ramírez, J.; Cañete, J.D. Psoriatic Arthritis: Pathogenesis and Targeted Therapies. Int J Mol Sci 2023, 24, 4901, doi:10.3390/ijms24054901. 2. 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Heterogeneous Expression Pattern of Interleukin 17A (IL-17A), IL- 17F and Their Receptors in Synovium of Rheumatoid Arthritis, Psoriatic Arthritis and Osteoarthritis: Possible Explanation for Nonresponse to Anti-IL-17 Therapy? Arthritis Res Ther 2014, 16, 426, doi:10.1186/s13075-014-0426-z. 18. Vecellio, M.; Hake, V.X.; Davidson, C.; Carena, M.C.; Wordsworth, B.P.; Selmi, C. The IL-17/IL- 23 Axis and Its Genetic Contribution to Psoriatic Arthritis. Front Immunol 2021, 11, 596086, doi:10.3389/fimmu.2020.596086. 19. De Vito, R.; Fiori, F.; Ferraroni, M.; Cavalli, S.; Caporali, R.; Ingegnoli, F.; Parpinel, M.; Edefonti, V. Olive Oil and Nuts in Rheumatoid Arthritis Disease Activity. Nutrients 2023, 15, 963, doi:10.3390/nu15040963. 20. Caso, F.; Navarini, L.; Carubbi, F.; Picchianti-Diamanti, A.; Chimenti, M.S.; Tasso, M.; Currado, D.; Ruscitti, P.; Ciccozzi, M.; Annarumma, A.; et al. Mediterranean Diet and Psoriatic Arthritis Activity: A Multicenter Cross-Sectional Study. Rheumatol Int 2020, 40, 951–958, doi:10.1007/s00296-019-04458-7. 21. Pocovi-Gerardino, G.; Correa-Rodríguez, M.; Callejas-Rubio, J.L.; Ríos-Fernández, R.; Martín- Amada, M.; Cruz-Caparros, M.G.; Rueda-Medina, B.; Ortego-Centeno, N. Beneficial Effect of Mediterranean Diet on Disease Activity and Cardiovascular Risk in Systemic Lupus Erythematosus Patients: A Cross-Sectional Study. Rheumatology (United Kingdom) 2021, 60, 160– 169, doi:10.1093/rheumatology/keaa210. General Results and Discussion/ Resultados Generales y Discusión 259 The purpose of the present Doctoral Thesis was to evaluate the beneficial effects of oleacein (OLA), one of the main secoiridoids of extra virgin olive oil (EVOO), and its O-methylated derivative dimethyl-oleacein (dimet-OLA) in immune-inflammatory diseases. Particularly, the immunomodulatory, anti-inflammatory, antioxidant and cardioprotective properties of both OLA and dimet-OLA have been examined across multiple experimental models including primary murine cells, an in vivo model of systemic lupus erythematosus (SLE), a human synovial fibroblast cell line and finally primary human cells. All this has provided a comprehensive understanding of the benefits of these compounds and the potential mechanisms of action. In Chapter I, we have investigated the ability of OLA and dimet-OLA to modulate the inflammatory response induced by bacterial lipopolysaccharide (LPS) in peritoneal macrophages and murine splenocytes, exploring the molecular mechanisms involved and the possible epigenetic modifications. Although previous research has reported the antiinflammatory properties of OLA in the LPS-induced THP-1 cell line [1], this work improves our knowledge of OLA modulatory effects on the signalling pathways involved in the inflammatory response and, for the first time, described the beneficial effects of its methylated derivative dimet-OLA. In line with previously reported evidence [2–4], Chapter I describes that LPS stimulation produces a strong increase in the production of intracellular reactive oxygen species (ROS) and several pro-inflammatory cytokines such as interleukin (IL)-1IL- 6, IL-17, tumour necrosis factor alpha (TNF)-, and interferon gamma (IFN)−. Pre-treatment with OLA and dimet-OLA resulted in a significant reduction in pro-inflammatory cytokine levels and ROS production. Both compounds exhibited similar activity in the suppression of the pro-inflammatory markers mentioned above (Chapter I, Figure 3A-E, Figure 4B). However, only the lower concentration of dimet-OLA (12.5 M) was not able to significantly reduce IL-17 secretion (Chapter I, Figure 3C). Moreover, the treatment with OLA and dimet- OLA suppressed the LPS-induced overexpression of key enzymes associated to the inflammatory process such as ciclooxigenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1) (Chapter I, Figure 5A, B) and inducible nitric oxide synthase (iNOS) General Results and Discussion/ Resultados Generales y Discusión 260 (Chapter I, Figure 4C, D). This finding could lead to the decrease of NO (Chapter I, Figure 4A) and Prostaglandin E2 (PGE2) production [5]. To elucidate the possible molecular mechanisms involved in the beneficial effects of OLA or dimet-OLA in acute inflammation, we evaluated whether our compounds were able to inhibit the Janus kinase/signal transducer and activator of transcription (JAK/STAT) axis and mitogen-activated protein kinases (MAPKs) family and also the release of nuclear factor kappa-B (NF-B) complex. It is well known that these routes regulate the expression of genes that are crucial for the inflammatory response [6–8]. According to the literature, after 18 h of LPS stimulation, our data showed a significant increase in the phosphorylation of these proteins and therefore, in the activation of JAK2/STAT3 axis (Chapter I, Figure 6A, B) and the members of the MAPKs family (Chapter I, Figure 7A, B). Interestingly, treatment with both OLA and dimet-OLA was able to significantly reduce the phosphorylation of JAK/STAT proteins (Chapter I, Figure 6A, B). On the other hand, while OLA decreased P38 and ERK phosphorylation (Chapter I, Figure 7A), dimet-OLA treatment exerted its effect through the dephosphorylation of JNK and ERK MAPKs (Chapter I, Figure 7B). In addition, OLA and dimet-OLA could exert their anti-inflammatory effects by inhibiting NF-B, since they are able to prevent the degradation of NF-κB inhibitor (IB-), avoiding this cascade from being activated (Chapter I, Figure 8A, B). We also investigated whether OLA and its O-methylated derivative regulated the multimeric inflammasome complex, which has been previously described to be triggered in LPS-stimulated macrophages [2,9]. Our data showed that OLA and dimet-OLA counteracted the canonical nucleotide-binding domain, leucine-rich– containing family, pyrin domain–containing-3 (NLRP3) inflammasome activation (Chapter I, Figure 9A-D) induced by LPS in peritoneal macrophages, leading to decreased IL- 1 (Chapter I, Figure 3E) and IL-18 levels (Chapter I, Figure 9A, B). Nevertheless, a significant reduction in caspase 11 expression, leading to the suppression of non-canonical inflammasome, was only described in those cells treated with OLA (Chapter I, Figure 9D). General Results and Discussion/ Resultados Generales y Discusión 261 Moreover, we evaluated the protective antioxidant role of OLA and dimet-OLA through the nuclear factor erythroid 2-related factor 2/ heme-oxygenase 1 (Nrf-2/HO-1) axis. The Nrf- 2/HO-1 axis acts as a protector in oxidative stress and is also linked to anti-inflammatory mechanisms [10]. OLA was able to mitigate LPS-induced oxidative stress in murine macrophages by enhancing the Nrf2/HO-1 axis (Chapter I, Figure 10A). These results, together with those obtained in the ABTS test and the suppression of intracellular ROS production (Chapter I, Figure 4B), proved the promising antioxidant activity of OLA. On the other hand, dimet-OLA had no impact on the regulation of the Nrf-2/HO-1 axis (Chapter I, Figure 10B), however, it was able to reduce intracellular ROS production (Chapter I, Figure 4B), exhibiting intracellular antioxidant activity. Finally, we tried to elucidate the role of OLA and dimet-OLA as post-translational histone modification modulators. Particularly, we were focused on the study of H3 acetylation and methylation in murine spleen cells, which could influence the overproduction of proinflammatory cytokines [11,12]. Specifically, we observed that after 24 h of LPS stimulation (5 g/mL) there was an increase in the H3K18 acetylation (Chapter I, Figure 11D) and a decrease in the methylation of both H3K27 and H3K9me3 (Chapter I, Figure 11E, F), which could be associated with enhanced gene expression [13]. In addition, we observed upregulated secretion of IL-6, IL-1 and IL-17 cytokines in LPS-stimulated splenocytes (Chapter I, Figure 10A-C). Interestingly, treatment with OLA and dimet-OLA regulated H3K18 acetylation (Chapter I, Figure 11D), but only in cells treated with OLA found changes in H3 methylation (Chapter I, Figure 11E, F). These epigenetic modifications could be related to cytokine production, as determined by ELISA, where both OLA and dimet-OLA were able to attenuate the overproduction of pro-inflammatory cytokines induced by LPS in murine splenocytes (Chapter I, Figure 8A-C). To conclude, in Chapter I, we described, for the first time, the beneficial effects of OLA and its O-methylated derivative, dimet-OLA, in LPS-induced murine peritoneal macrophages. Both compounds were able to modulate the oxidative stress and the acute inflammatory General Results and Discussion/ Resultados Generales y Discusión 262 response in LPS-activated murine peritoneal cells. These effects could be related to the blockade of iNOS, COX-2, and mPGES-1 proteins as well as the attenuation of NO, ROS, IL- 6, IL-1β, IFN−, IL-17, and TNF- production. Our findings have further clarified the potential mechanisms of action of OLA and dimet-OLA, inhibiting JAK/STAT, MAPKs and inflammasome, as well as the prevention of IkB- degradation and the induction of Nrf- 2/HO-1 axis. Furthermore, OLA and dimet-OLA induced changes in histone H3 acetylation leading to down-regulation of pro-inflammatory cytokines in LPS-activated splenocytes. In addition, OLA also regulated the level of histone H3 methylation. Our results are consistent with previous works that report the anti-inflammatory and antioxidant effects of EVOO secoiridoids, (oleocanthal (OLE) and oleuropein (OL)), in murine macrophages [14,15]. Additionally, Montoya et al. reported that the O-methylation of OLE enhances its pharmacological activity and regulates the epigenome through histone modification in murine splenocytes [16]. Although the O-methylation of phenolic compounds has been proposed as an interesting tool to increase the chemical and metabolic stability of these compounds, even improving biological activity [17,18], in Chapter I it can be seen that dimet- OLA presents an anti-inflammatory activity similar to that of its precursor, only improving its effects in inhibiting the phosphorylation of JNK protein. Even so, in the following chapters, the activity of dimet-OLA has further been explored along with the beneficial effects of OLA. SLE is a chronic autoimmune disease that involves loss of tolerance to self-antigens, production of autoantibodies, deposition of immunocomplexes, and chronic inflammation [19]. The search for alternative treatments for SLE highlights the nutritional intervention as a promising tool in the management of SLE as an adjunct to pharmacological treatment. In this context, the aim of Chapter II was to evaluate the preventive role of OLA and its O- methylated derivative in the development of endothelial dysfunction and lupus nephritis (LN) in a pristane-induced SLE-like murine model. The SLE-like model was induced by pristane injection and then, mice were fed with a diet enriched with OLA or dimet-OLA (0.01% w/w) for 24 weeks. This in vivo SLE model more closely emulates human lupus than spontaneous strains [20] and is characterized by proteinuria, autoantibodies production, General Results and Discussion/ Resultados Generales y Discusión 263 glomerulonephritis, and arthritis. The renal damage was initially evaluated through histopathological analysis. The stain of kidney sections (Hematoxylin-Eosin, Masson's Trichomic and Periodic Schiff Acid) showed compressed glomerular tuft, partial glomerular, interstitial fibrosis and haemorrhagic areas in SD-pristane animals (Chapter II, Figure 3B, F, J) compared to naïve mice which presented a normal renal architecture (Chapter II, Figure 3A, E, I). In addition, the presence of membranous nephritis in the SD-pristane group was demonstrated by the deposition of IgG and IgM immune complexes throughout the renal corpuscle, as evidenced by immunohistochemistry and immunofluorescence (Chapter II, Figure 4B, F, J, N). Nevertheless, the nutritional supplementation with OLA restored renal architecture, which was similar to that of mice in the naïve group (Chapter II, Figure 3C, G, K). Additionally, OLA supplemented diet decreased the deposition of IgG and IgM, which was only noticeable at the glomerular level; no extraglomerular deposits were observed in OLA-treated mice (Chapter II, Figure 4C, G, K, Ñ). Similarly, in dimet-OLA animals group mice the histopathological study revealed some signs of thyroidization in renal tubules and glomerular hypercellularity (Chapter II, Figure 3D, H, L). There was also a reduction in IgG and IgM deposition in dimet-OLA animals, but it was not as noticeable as in animals treated with OLA (Chapter II, Figure 4D, H, L, O). In the last years, it has been suggested that the persistent inflammatory process, which characterises SLE, may be linked to endothelial dysfunction in patients with SLE [21]. Moreover, previous reports found cardiovascular manifestations such as hypertension and endothelial dysfunction in SLE-pristane induced mice [22,23]. Our results showed upregulated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-1 protein expression and aberrant endothelial nitric oxide synthase (eNOS) phosphorylation in aortic tissue from SD-pristane mice, which could lead to endothelial dysfunction (Chapter II, Figure 1B, D). In particular, OLA and dimet-OLA supplemented diets were able to reduce NOX-1 production, however, no significant changes were observed in peNOS (Thr495) phosphorylation (Chapter II, Figure 1B, D). Although OLA dietary treatment modulated the protein expression of NOX-1, it was not able to counteract the altered vascular relaxation response to ACh induced by pristane (Chapter II, Figure 1C). On the other hand, dimet-OLA General Results and Discussion/ Resultados Generales y Discusión 264 supplementation significantly improved the vascular relaxation response to Ach (Chapter II, Figure 1A). Similar results were observed in pristane-induced mice after six month of OLE supplemented diet which reversed endothelial dysfunction and LN [24]. This evidence suggests that the O-methylation of OLA could improve its cardioprotective effect, improving SLE-induced endothelial dysfunction. Epigenetics is an emerging focus of research in the study of SLE pathogenesis. In Chapter II, we evaluated the expression of several microRNAs (miRNAs) that are closely related to the development of SLE. The enrichment of diets with OLA and its O-methylated derivative significantly improved the down-regulation of miRNAs induced by pristane injection (Chapter II, Figure 5). Particularly, OLA and dimet-OLA dietary interventions were able to restore miRNA-146a levels in pristane-induced mice preventing LN (Chapter II, Figure 5D). In fact, miRNA-146a is considered a protective marker against LN [25]. Similarly, both experimental diets increased miR-23b expression in kidney tissue from SLE-like animals (Chapter II, Figure 5C), which could be related to modulation of IL-17 secretion [26], a key cytokine involved in SLE pathogenesis. Furthermore, we detected an increase in miR-24-3p in OLA and dimet-OLA treated animals when compared to SD-pristane mice (Chapter II, Figure 5A). Increased miRNA-24-3p has also been proposed as a cardiovascular protective factor [27,28]. On the other hand, reduced miR-126 is related to the development of SLE by IFN- suppression [29]. We found down-regulated miRNA-126 levels in kidney tissue from pristane-induced mice; however, OLA and dimet-OLA treatment counteract this alteration (Chapter II, Figure 5B). Although its ability to regulate miRNA expression has been previously described for OLA [30,31], this is the first time it has been described for its methylated derivative. Therefore, the protective effects of OLA and dimet-OLA in LN could be due to their potential to modify miRNA expression. Finally, we explored the expression of DNA methyltransferase (DNMT) -1, which was reduced in SD-pristane mice and can lead to DNA hypomethylation (Chapter II, Figure 5E). Surprisingly, only OLA-treated mice showed significant up-regulation of DNMT-1 expression (Chapter II, Figure 5E). General Results and Discussion/ Resultados Generales y Discusión 265 Furthermore, we evaluated the production of conventional pro-inflammatory markers in kidney homogenate from SLE-pristane-induced mice. Our data suggest overexpression of the COX-2/mPGES-1 axis and iNOS protein in SD-pristane mice (Chapter II, Figure 6). In kidneys from animals fed with OLA supplemented diet, we observed a reduction in the protein expression of COX-2, mPGES-1 and iNOS (Chapter II, Figure 6). Conversely, dietary intervention with dimet-OLA down-regulated the pristane-induced overproduction of COX- 2 and mPGES-1 whereas the iNOS protein maintained inalterable (Chapter II, Figure 6). To further examine the protective effects of OLA and dimet-OLA in the SLE-like pristaneinduced model, we examined whether they could regulate the signalling pathways involved in the SLE pathogenesis (Chapter II, Figure 7). Several authors have described the implication of JAK/STAT axis in LN. In fact, treatment with JAK/STAT inhibitors improved renal function in mice [32,33]. Additionally, the triggering of MAPKs family has recently been revealed in murine models of lupus [34,35]. In this line, we found an increase in the phosphorylation of the JAK3-STAT-3 axis and MAPKs proteins that could be related to the overproduction of pro-inflammatory markers. Moreover, our results also reported the degradation of cytosolic - and the translocation of p50 and p65 NF-B subunits to the nucleus, enabling the production of pro-inflammatory mediators. On the contrary, dietary supplementation with OLA and its methylated derivative prevented the up-regulation of all these signalling pathways in renal tissue. This resulted in a reduced phosphorylation of the JAK3/STAT-3 (Chapter II, Figure 7A) axis and JNK/ERK MAPKs (Chapter II, Figure 7D) protein expression which was accompanied by an inhibition of nuclear translocation of the p65 and p50 subunits (Chapter II, Figure 7C). Furthermore, dietary enrichment with OLA results in enhanced expression of Nrf-2, a protective agent against oxidative stress (Chapter II, Figure 7B). At last, we also evaluated the role of OLA and its methylated derivative in the regulation of the inflammasome (Chapter II, Figure 8), which has been shown to be overactivated in murine models of lupus and renal biopsies from SLE patients [36–38]. Both OLA and dimet-OLA dietary supplementation significantly down-regulated the activation of canonical NLRP3 inflammasome induced by pristane, leading to the reduction of NLRP3 and caspase 1 proteins General Results and Discussion/ Resultados Generales y Discusión 266 (Chapter II, Figure 8A, B). Nonetheless, only OLA experimental diet animals modulate IL-18 production and non-canonical inflammasome induction (Chapter II, Figure 8C, D). In summary, Chapter II presents OLA and its O-methylated derivative as interesting epigenome modulators tools in the prevention of SLE progression in pristane-induced BALB/c mice. OLA and dimet-OLA reduced the deposition of immune complexes in kidney tissue. However, the effect of OLA was notably more pronounced and also restored renal architecture. Furthermore, OLA and dimet-OLA were able to regulate the altered levels of miRNAs (miRNA-126, miRNA-24-3p, miRNA-146a, miRNA-23b) in kidney tissue, but only OLA modulates the expression of DNMT-1. The protective effects of OLA and dimet-OLA could be linked to their ability to inactivate crucial inflammatory pathways involved in SLE pathogenesis such as JAK/STAT, NF-κB, and MAPKs. In addition, OLA also was able to enhance Nrf-2 protein expression. Recently, our research group has shown that a diet enriched with EVOO or some of its secoiridoids (OLE, OL) prevents the development of pristane-induced SLE [24,39,40], obtaining results consistent with those observed in Chapter II. Although OLA appears to be more effective in the renal tissue, dimet-OLA demonstrated a stronger cardiovascular protective effect by improving pristane-caused endothelial dysfunction. However, more research is required to investigate the beneficial cardiovascular properties of dimet-OLA and OLA. Finally, in Chapter III, the immunomodulatory effect of dimet-OLA and its precursor OLA was studied on a Cluster of Differentiation (CD)3/aCD28-stimulated CD4+ memory T helper (Th) cells from healthy donors. In addition, we also evaluated the ability of OLA to counteract lymphocyte activation in phytohemagglutinin (PHA)-induced peripheral blood mononuclear cells (PBMCs) isolated from healthy donors and SLE patients. Similarly, we tested the potential beneficial properties of both compounds in a human synovial fibroblast cell line (SW982). Finally, in order to mimic the inflammatory process occurring in the affected joints of patients with psoriatic arthritis (PsA), we tested the effects of OLA and its methylated General Results and Discussion/ Resultados Generales y Discusión 267 derivative in a co-culture of C-C chemokine receptor (CCR)6+ memory Th cells isolated from healthy donors and synovial fibroblasts isolated from PsA patients. The treatment with OLA and dimet-OLA was able to counteract the increase in IL-17 and IFN- γ levels induced by aCD3/aCD28 in memory CD4+ T cells after 72 h of incubation (Chapter III, Figure 2A, C). On the other hand, no significant changes were found in IL-10 secretion (Chapter III, Figure 2B). Moreover, OLA strongly counteracted the increased production of IL-6, IL-1 and TNF- in PHA-stimulated PBMCs isolated from healthy donors and SLE patients (Chapter III, Figure 7). In Chapter III, we also described that OLA reduced PHA- dependent lymphocyte activation in PBMCs isolated from healthy patients (Chapter III, Figure 8), reducing the levels of the surface marker CD69 which is involved in the regulation of the immune response [41,42]. However, we did not observe this regulation in cells from SLE patients (Chapter III, Figure 8). This could be due to the fact that some of our patients are treated with antimalarials or corticosteroids, which can reduce lymphocyte response. Recently, epigenetic has been described as a key driver in inflammatory diseases [43]. Indeed, the regulation of epigenomic changes could be an interesting tool for the control and treatment of autoimmune diseases. In Chapter III, we also assess the role of OLA on the regulation of global DNA methylation in PHA-induced PBMCs. Although the DNA methylation changes observed in this study did not reach statistical significance, we observed a trend to hypomethylated DNA in unstimulated PBMCs from patients with SLE compared to healthy donors, as previously described for T cells [44]. Nevertheless, OLA treatment showed a tendency to reverse this hypomethylation (Chapter III, Figure 9). Notably, these results are consistent with those obtained in Chapter II, where it was described that dietary treatment with OLA increased DNMT-1 expression at the renal level, resulting in an increase in DNA methylation (Chapter II, Figure 5E). The human synovial fibroblast cell line (SW982) is used to test drugs against synovitis [45,46]. In this line, the anti-inflammatory effects of oleuropein and a polyphenolic extract of EVOO in IL-1-induced cells have already been described [47,48]. However, in Chapter III this effect CONCLUSIONS CONCLUSIONES Conclusions/Conclusiones 277 En base a los resultados obtenidos en la presente Tesis Doctoral, podemos concluir: 1. OLA y su derivado O-metilado, dimet-OLA, contrarrestaron la respuesta inflamatoria y el estrés oxidativo inducido por LPS en macrófagos peritoneales murinos mediante la inhibición de la secreción de NO, IFN- IL-6, IL-17, TNF- e IL-1 y la generación de ROS, así como la disminución de la expresión proteica de las enzimas pro-inflamatorias COX-2, mPGES-1 e iNOS. Además, ambos compuestos regularon en mayor o menor medida la activación de las vías de señalización MAPK, JAK/STAT e inflamasoma, la inducción del eje Nrf-2/HO-1 y la prevención de la degradación del IkB-. Además, OLA y dimet-OLA indujeron modificaciones postraduccionales de la histona H3 que condujeron a una marcada reducción en los niveles de dichas citocinas en esplenocitos activados con LPS. Dimet-OLA, compuesto de mayor lipofilia que OLA, presentó un perfil antiinflamatorio parecido a su precursor natural sugiriéndose como una nueva herramienta farmacológica de interés para su valoración en modelos in vivo de inmunoinflamación. 2. La terapia nutricional con dietas enriquecidas con OLA o dimet-OLA tuvo un carácter preventivo en el desarrollo de nefritis lúpica y disfunción endotelial en un modelo experimental murino de LES inducido por pristano. Estos efectos protectores pueden estar asociados con la regulación de las vías de señalización protagonizadas por Nrf-2, inflamasoma, NF−B, JAK/STAT y MAPK y la modulación del epigenoma mediante de la metilación del ADN y la expresión de microRNA. Aunque OLA mostró un mejor perfil protector a nivel renal, nuestros resultados evidenciaron un interesante efecto cardioprotector de dimet-OLA. Por ello, la suplementación dietética con OLA y dimet- OLA podría considerarse como una alternativa para el control de enfermedades inmunomediadas que cursan con inflamación como es el SLE. 3. El tratamiento con OLA redujo los niveles de IL-17 e IFN- en células T CD4+ de memoria aisladas de pacientes sanos activadas con aCD3/aCD28. Del mismo modo, dimet-OLA redujo la producción de IFN- en células T CD4+ de memoria. Adicionalmente, OLA contrarrestó la sobreproducción de IL-1 IL-6 y TNF- inducida por PHA tanto en PBMC procedentes de donantes sanos como de pacientes con LES, aunque sorprendentemente solo pudo reducir de manera significativa la expresión del marcador CD69 en células T activadas de donantes sanos. Conclusions/Conclusiones 278 4. Tanto OLA como su o-metil derivado redujeron significativamente la producción de citocinas proinflamatorias (TNF- e IL-6) y metaloproteinasas de la matriz (MMP-1 y MMP-3) en un modelo in vitro de fibroblastos sinoviales humanos (SW982) activados con IL-1. Además, dimet-OLA minimizó el bucle inflamatorio en un co-cultivo de células Th de memoria CCR6+ aisladas de donantes sanos y fibroblastos sinoviales de pacientes con artritis psoriásica (APs) mediante la regulación de la producción de citocinas proinflamatorias (IL-6, IL-8, IFN- and IL-17). Del mismo modo, OLA disminuyó significativamente los niveles de IL-6 en este co-cultivo y aumentó la producción de la citocina antiinflamatoria IL-10. Estos resultados preliminares nos permiten indagar en el papel inmunomodulador y antiinflamatorio que OLA y dimet-OLA, podrían ejercer, como una novedosa aproximación terapéutica, en el manejo de IMID con afectación sinovial, como es la APs. Por todo ello, los resultados obtenidos en esta Tesis Doctoral nos muestran de manera preliminar el posible papel protector y preventivo de OLA y su derivado O-metilado, dimet- OLA, como nuevos moduladores del epigenoma en la respuesta inmunoinflamatoria que media el desarrollo de enfermedades inmunomediadas como son el LES y la APs. No obstante, serían necesarios estudios de intervención nutricional en pacientes que confirmen estos prometedores datos preclínicos. ANNEXE I: CURRICULUM VITAE ANEXO I: CURRICULUM VITAE Curriculum Vitae 283 1. PERSONAL INFORMATION Name: Rocío Muñoz García ORCID: 0000-0001-5346-3920 e-mail: [email protected] 2. EDUCATION Bachelor of Pharmacy Faculty of Pharmacy, University of Seville, Seville, Spain. September 2013- July 2018 3. SKILLS 3.1 Languages Spanish: native spiker English: B2. Preliminary English Test (PET) passed with distinction (Level B2). Cambridge University, UK. French: B1. Certificate by the Language Institute of the University of Seville. 3.2 Knowledge areas Areas: functional foods, epigenetic, immunology, inflammation, medicinal plants, systemic lupus erythematosus, rheumatoid arthritis and psoriatic arthritis. Cellular and Molecular Biology: Cell culture, ELISA, flow cytometry, histology, RT-qPCR, and Western Blott. Animal models: systemic lupus erythematosus, arthritis, inflammatory bowel disease and inflammation. Statistical Programs: GraphPad Prism® Curriculum Vitae 290 Pharmacy Laboratory. Double Degree in Pharmacy and in Optics and Optometry/Degree in Pharmacy/Degree in Pharmacy (2019). 48 hours 2021-22 Pharmacology and Pharmacotherapy I. Double Degree in Pharmacy and in Optics and Optometry/ Degree in Pharmacy. 15 hours Pharmacology and Pharmacotherapy III and Clinical Pharmacy. Double Degree in Pharmacy and in Optics and Optometry/Degree in Pharmacy/Degree in Pharmacy (2019). 9 hours Pharmacology and Pharmacotherapy I. Degree in Pharmacy (2019). 36 hours 2020-21 Pharmacology and Pharmacotherapy I. Double Degree in Pharmacy and in Optics and Optometry/ Degree in Pharmacy (2019). 36 hours Pharmacy Laboratory. Double Degree in Pharmacy and in Optics and Optometry/ Degree in Pharmacy. 24 hours 5.2 Teaching publications Calderón-Montaño J.M., Jiménez-González V., Gutiérrez-Alcántara M.R., Muñoz-García R., Guillén-Mancina E., Jiménez-Alonso J.J., Antonio José León González; Alcarranza M., Burgos- Morón E., Montoya T., Castejón-Martínez ML., García-GIL S., Díaz-Ortega P., Pérez Guerrero M.C., Caro-Clara C.M., Martín-Cordero C., Sánchez-Hidalgo M., Motilva V., Quílez-Guerrero A.M. García-Giménez M.D., Talero E., Villegas I. Determinación cualitativa del contenido en alcaloides tropánicos y evaluación del efecto antiespasmódico de un extracto de Atropa belladonna L., Determinación cualitativa del contenido en alcaloides tropánicos y evaluación del efecto antiespasmódico de un extracto de Atropa belladonna L. 30/05/2022. Available online Curriculum Vitae 291 at:<https://ra.sav.us.es/index.php/realidad-virtual/141-alcaloides-tropanicos>.DOI: https://doi.org/10.35466/RA2022n6799 Calderón-Montaño J.M., Talero E., Jiménez-González V., Gutiérrez-Alcántara M.R., Burgos- Morón E., Montoya T., Muñoz-García R., Jiménez-Alonso J.J., Pérez Guerrero M.C., Sánchez- Hidalgo M., Caro-Clara C.M., Alarcón-de-la-Lastra C., De-la-Puerta R., Motilva V. Evaluación de la actividad antiinflamatoria in vitro de una molécula en desarrollo preclínico. 09/03/2021. Available online at: <https://ra.sav.us.es/index.php/realidad-virtual/115-laboratorio-cultivos-celulares>. DOI: 10.35466/RA2021n5752. Registry number ID:5752 5.3 Teaching Innovation Projects Qualitative determination of tropane alkaloid content and evaluation of the effect antispasmodic from an extract of Atropa belladonna L. III Plan Propio de Docencia 2021. University of Seville June 2021- June 2022 Design and implementation of the structured objective clinical examination as an evaluation system of professional clinical competencies in the Faculty of Pharmacy III Plan Propio de Docencia 2021 University of Seville January 2021 - January 2022 Evaluation of the in vitro anti-inflammatory activity of a molecule in development preclinical. III Plan Propio de la Universidad de Sevilla University of Seville March 2020- March 2021 Curriculum Vitae 292 6. Others a. Resources and tools for creating digital materials for teaching. Institute of educational sciences, University of Seville, Seville, November 2022. 8 hours b. Biosafety module for doctoral students. Institute of educational sciences, University of Seville, Seville, October 2022. 5 hours c. Morphological Mouse Phenotyping Course (Online). Autonomic University of Barcelona, July 2022. 37 hours d. Research ethics. criteria for a positive evaluation. Institute of educational sciences, University of Seville, Seville, June 2022. 10 hours e. Safety in the use of cabines. Institute of educational sciences, University of Seville, Seville, December 2021. 2 hours f. Competencies level I teaching staff: integration of risk prevention specific labor and competencies of the teaching staff of the University of Seville. Institute of educational sciences, University of Seville, Seville, September 2021. 15 hours g. Research 2.0 for doctoral students: The network and the dissemination of research. Institute of educational sciences, University of Seville, Seville, September 2021. 4 hours h. Protection of industrial and intellectual property for doctoral students. Institute of educational sciences, University of Seville, Seville, September 2021. 4 hours i. Ethics in sciences for doctoral students. Institute of educational sciences, University of Seville, Seville, November 2020. 3 hours j. Internationalization and mobility in doctorate. Institute of educational sciences, University of Seville, Seville, October 2020. 3 hours k. Preparation and presentation of communications at conferences for doctoral students. Institute of educational sciences, University of Seville, Seville, October 2020. 4 hours l. Knowledge transfer models for doctoral student. Institute of educational sciences, University of Seville, Seville, October 2020. 4 hours m. Office 365 Excel 2016. Institute of educational sciences, University of Seville, Seville, May- June 2020. 20 hours Curriculum Vitae 293 n. Author profiles: Fundamentals in your research career (Online). Institute of educational sciences, University of Seville, Seville, May-June 2020. 10 hours o. Publish with impact for doctoral students (Online). Institute of educational sciences, University of Seville, Seville, June 2020. 2 hours p. Search and management of information for doctoral students: science area (Online). Institute of educational sciences, University of Seville, Seville, May 2020. 2 hours q. Mendeley, Reference Manager (Online). Institute of educational sciences, University of Seville, Seville, May 2020. 8 hours r. Basic training in computer security (Online). Institute of educational sciences, University of Seville, Seville, March 2020. 5 hours s. Course on Preparation of a Research Plan. Faculty of Pharmacy, University of Seville, Seville, October 2019. 2 hours t. Writing workshop in English for writing articles. Institute of educational sciences, University of Seville, Seville, October 2019. 12 hours u. Training course in experimental animal welfare for the accreditation of the functions of animal care. Euthanasia and performance of experimental procedures. Species rodents. I Edition. Functions A+B+C. University of Cádiz. 75 hours v. Occupational Risk Prevention Course. Faculty of Pharmacy, University of Seville, Seville, November-December 2018. 10 hours ANNEXE II: PUBLISHED PAPERS ANNEXE II: TRABAJOS PUBLICADOS Anexxe II/ Anexo II 297 Anexxe II/ Anexo II 298 Anexxe II/ Anexo II 299