For centuries, natural products and their derivatives have provided a rich source of compounds for the development of new immunomodulators in the treatment of human diseases. Natural immune modulators may provide the key to control and ultimately defeat disorders affecting the immune system, by either up- or down-regulating the immune response with few adverse side effects. Many of these compounds are currently undergoing clinical trials, particularly as anti-oxidative, anti-microbial, and anti-cancer agents. However, the functions and mechanisms of action of natural products, and how they interact with the immune system, has yet to be extensively explored. In recent years, the increasing body of knowledge regarding the role of macrophages in the steady-state and in the context of inflammation has opened diverse new avenues of investigation and possibilities for therapeutic intervention targeting the inherent plasticity of macrophages for the treatment of acute and chronic inflammatory disease. Toll-Like Receptors (TLRs), including TLR4, play a crucial role in inflammatory-based diseases, therefore TLR4 signalling has been identified as a therapeutic target for pharmacological intervention. This work aims to screen and investigate the potential of phytoextracts and phytochemicals to affect TLR4 signalling in a macrophage-like cell model. Specifically, extracts from green (GCE) and roasted (RCE) coffee beans as well as pure chlorogenic acid (5-CQA) were tested. Also, the anti-inflammatory effect of palmitoylethanolamide (PEA), and its synthetic analogue RePEA, was assessed. Human monocyte-derived macrophages, deriving from the differentiation of a monocytic cell line (THP-1) and/or from human primary CD14+ monocytes, were employed as an in vitro model. MTT was used to determine cytotoxic effects of the treatments. TLR4 activation was stimulated by exposure of cells to bacterial endotoxin LPS (E. coli), in presence or absence of treatments. Different readouts were evaluated: endpoint pro-inflammatory cytokines production, as well as phosphorylation and nuclear translocation of intracellular signalling mediators. These parameters were measured applying different cellular and molecular techniques, mainly enzyme-linked immunosorbent assay (ELISA), High Content Analysis (immunofluorescence microscopy), and Western blot. Alongside, we employed different commercially available stable transfected cells as tools to investigate the mechanism of action of our tested extract or molecule, THP1-XBlue™, RAW-Blue™ and HEK-Blue™ cells, respectively. Key findings include a dramatic, dose-dependent, inhibitory effect of both green and roasted coffee extracts towards interferon-β (IFN-β) release, upon LPS stimulation. Consistently, chlorogenic acid, a major polyphenolic component of coffee extracts, showed a comparable biological activity. Additionally, novel evidence towards the immunomodulatory effects and mechanism of action of coffee extracts and chlorogenic acid as modulators of TLR4-related pro-inflammatory signalling have been provided. Alongside, PEA capability of reducing TNF-α release from LPS-stimulated microglial cells, was corroborated in our macrophage model also, confirming its anti-inflammatory potential. Moreover, RePEA, a synthetic PEA analogue designed to be degraded slower, was revealed to be more active than its parent compound. These experimental data demonstrate that natural products may act as lead molecules for the development of safe and effective immunomodulators. Taken together, these findings help to validate the above-mentioned natural molecules, 5-CQA and PEA, but also RePEA, as potential novel candidates for further preclinical investigations for the treatment of inflammatory-based disorders.

Per secoli, i prodotti naturali e i loro derivati hanno fornito una ricca fonte di composti per lo sviluppo di nuovi immunomodulatori. Gli immunomodulatori naturali possono fornire la chiave per controllare e, infine, sconfiggere i disturbi che colpiscono il sistema immunitario, stimolando o inibendo la risposta immunitaria con pochi effetti collaterali negativi. Molti di questi composti sono attualmente in fase di sperimentazione clinica, in particolare come agenti antiossidanti, antimicrobici e antitumorali. Tuttavia, la funzione e il meccanismo di azione dei prodotti naturali, e il modo in cui interagiscono con il sistema immunitario, devono ancora essere ampiamente esplorati. Negli ultimi anni, la conoscenza del ruolo dei macrofagi nel contesto dell'infiammazione ha aperto numerose nuove strade, tra cui la possibilità di intervento terapeutico mirato sfruttando la plasticità intrinseca dei macrofagi per il trattamento di patologie infiammatorie acute e croniche. I recettori Toll-simili (TLR), incluso TLR4, svolgono un ruolo cruciale nelle malattie a base infiammatoria; pertanto, la via di segnalazione di TLR4 è stata identificata come bersaglio terapeutico per l'intervento farmacologico. Questo lavoro mira a valutare il potenziale dei fitoestratti e delle sostanze fitochimiche di influenzare la via di segnalazione di TLR4 in un modello cellulare di macrofago. Nello specifico, sono stati testati estratti ottenuti da chicchi di caffè verde (GCE) e tostato (RCE), nonché acido clorogenico puro (5-CQA). Inoltre, è stato valutato l'effetto antinfiammatorio della palmitoiletanolamide (PEA) e del suo analogo sintetico RePEA. Come modello in vitro sono stati impiegati macrofagi originati da monociti umani, derivanti dalla differenziazione di una linea cellulare (THP-1) e/o da monociti umani primari. Il saggio MTT è stato utilizzato per determinare gli effetti citotossici dei trattamenti. L'attivazione di TLR4 è stata stimolata mediante esposizione delle cellule all'endotossina batterica LPS (E. coli) in presenza o assenza di trattamento. Sono stati valutati diversi parametri: produzione di citochine pro-infiammatorie, ma anche attivazione e traslocazione nucleare dei mediatori di segnalazione intracellulare. Questi parametri sono stati misurati applicando differenti tecniche di biologia cellulare e molecolare, principalmente test di immunoassorbimento enzimatico (ELISA), immunofluorescenza e immunofissazione (Western blot). Inoltre, abbiamo impiegato diverse cellule transfettate stabilmente disponibili in commercio come strumenti per studiare il meccanismo d'azione del nostro estratto o della nostra molecola, rispettivamente le cellule THP1-XBlue™, RAW-Blue™ e HEK-Blue™. I risultati chiave includono un marcato effetto inibitorio, dose-dipendente, degli estratti di caffè verde e tostato verso il rilascio di interferone-β (IFN-β) dopo la stimolazione con LPS. Coerentemente, l'acido clorogenico, un importante componente polifenolico degli estratti di caffè, ha mostrato un'attività biologica comparabile. Complessivamente, i risultati ottenuti forniscono nuove prove sugli effetti immunomodulatori e sul meccanismo d'azione degli estratti di caffè e dell'acido clorogenico, in particolare come modulatori della via di segnalazione pro-infiammatoria mediata da TLR4. Inoltre, la capacità della PEA di ridurre il rilascio di TNF-α da parte delle cellule microgliali stimolate con LPS è stata corroborata anche nel nostro modello di macrofagi, confermando il suo potenziale antinfiammatorio. Inoltre, RePEA, un analogo sintetico della PEA progettato per essere degradato più lentamente, si è rivelato più attivo del suo composto originario. Questi risultati aiutano a convalidare le suddette molecole naturali, 5-CQA e PEA, ma anche RePEA, come potenziali nuovi candidati per ulteriori indagini precliniche per il trattamento delle malattie autoimmuni e infiammatorie.

(2022). Modulation of the Innate Immune Response by Targeting Toll-like Receptor 4 Signalling: Exploring the Role of Natural Products. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2022).

Modulation of the Innate Immune Response by Targeting Toll-like Receptor 4 Signalling: Exploring the Role of Natural Products

ARTUSA, VALENTINA
2022

Abstract

For centuries, natural products and their derivatives have provided a rich source of compounds for the development of new immunomodulators in the treatment of human diseases. Natural immune modulators may provide the key to control and ultimately defeat disorders affecting the immune system, by either up- or down-regulating the immune response with few adverse side effects. Many of these compounds are currently undergoing clinical trials, particularly as anti-oxidative, anti-microbial, and anti-cancer agents. However, the functions and mechanisms of action of natural products, and how they interact with the immune system, has yet to be extensively explored. In recent years, the increasing body of knowledge regarding the role of macrophages in the steady-state and in the context of inflammation has opened diverse new avenues of investigation and possibilities for therapeutic intervention targeting the inherent plasticity of macrophages for the treatment of acute and chronic inflammatory disease. Toll-Like Receptors (TLRs), including TLR4, play a crucial role in inflammatory-based diseases, therefore TLR4 signalling has been identified as a therapeutic target for pharmacological intervention. This work aims to screen and investigate the potential of phytoextracts and phytochemicals to affect TLR4 signalling in a macrophage-like cell model. Specifically, extracts from green (GCE) and roasted (RCE) coffee beans as well as pure chlorogenic acid (5-CQA) were tested. Also, the anti-inflammatory effect of palmitoylethanolamide (PEA), and its synthetic analogue RePEA, was assessed. Human monocyte-derived macrophages, deriving from the differentiation of a monocytic cell line (THP-1) and/or from human primary CD14+ monocytes, were employed as an in vitro model. MTT was used to determine cytotoxic effects of the treatments. TLR4 activation was stimulated by exposure of cells to bacterial endotoxin LPS (E. coli), in presence or absence of treatments. Different readouts were evaluated: endpoint pro-inflammatory cytokines production, as well as phosphorylation and nuclear translocation of intracellular signalling mediators. These parameters were measured applying different cellular and molecular techniques, mainly enzyme-linked immunosorbent assay (ELISA), High Content Analysis (immunofluorescence microscopy), and Western blot. Alongside, we employed different commercially available stable transfected cells as tools to investigate the mechanism of action of our tested extract or molecule, THP1-XBlue™, RAW-Blue™ and HEK-Blue™ cells, respectively. Key findings include a dramatic, dose-dependent, inhibitory effect of both green and roasted coffee extracts towards interferon-β (IFN-β) release, upon LPS stimulation. Consistently, chlorogenic acid, a major polyphenolic component of coffee extracts, showed a comparable biological activity. Additionally, novel evidence towards the immunomodulatory effects and mechanism of action of coffee extracts and chlorogenic acid as modulators of TLR4-related pro-inflammatory signalling have been provided. Alongside, PEA capability of reducing TNF-α release from LPS-stimulated microglial cells, was corroborated in our macrophage model also, confirming its anti-inflammatory potential. Moreover, RePEA, a synthetic PEA analogue designed to be degraded slower, was revealed to be more active than its parent compound. These experimental data demonstrate that natural products may act as lead molecules for the development of safe and effective immunomodulators. Taken together, these findings help to validate the above-mentioned natural molecules, 5-CQA and PEA, but also RePEA, as potential novel candidates for further preclinical investigations for the treatment of inflammatory-based disorders.
PERI, FRANCESCO
BRUNO, ANTONINO
Composti naturali; Immunità innata; Infiammazione; Estratti di caffè; Acido clorogenico
Natural products; Innate immunity; Inflammation; Coffee Extracts; Acido clorogenico
BIO/14 - FARMACOLOGIA
English
11-mag-2022
TECNOLOGIE CONVERGENTI PER I SISTEMI BIOMOLECOLARI (TeCSBi)
34
2020/2021
open
(2022). Modulation of the Innate Immune Response by Targeting Toll-like Receptor 4 Signalling: Exploring the Role of Natural Products. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2022).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/375443
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