Saviano, Anella (2023) Preclinical studies on compounds with nutraceutical activity to be used in the prevention and/or modulation of inflammatory and autoimmune pathologies targeting the COX-2/mPGES-1 and Treg/Th17 axis. [Tesi di dottorato]

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Abstract

The immune response against pathogens and non-pathogens is regulated by key signaling pathways. Appropriate control of the immune response is required to prevent either hyperresponsive or inadequate responses, which are harmful to the host body and may lead to immune-related diseases. The modulating effects of compounds on the immune response via signaling networks cause different expression levels of cytokines, chemokines, acute phase proteins, anti-apoptotic proteins, cell-adhesion molecules, and other inflammatory mediators. In various cellular events, mechanisms for intracellular cell signaling play an important and specific role in chronic inflammatory diseases. Inhibition or modulation of these pathways is a possible target to provide a better alternative compared to current treatment strategies. Currently, several chemical immunomodulators are used to treat various inflammatory disorders. However, safer, and more effective drugs are needed to replace commercial drugs because most of them have side effects. Several plants have demonstrated the ability to regulate immune signaling networks and prevent the occurrence of inflammatory disorders. In this thesis, we provide (Chapter 1) a current state of the art about the anti-inflammatory and immunomodulatory properties of natural-derived compounds (including nutraceuticals, functional food, and dietary supplements) targeting microsomal prostaglandin E synthase-1 (mPGES-1) and the T helper (Th)-17 and regulatory T (Treg) cells axis, in order to provide a scientific rationale for their potential therapeutic use. In the context of inflammation and immunity, there are several (but also fragmented and observational) studies reporting that Mangifera indica L. (a plant that belongs to the Anacardiaceae family) and/or its main active component mangiferin (a xanthone present at significant levels in different parts of the mango fruit) display anti-inflammatory/immunomodulatory activity. According to previous reports on the traditional use of this medicinal plant, the main biological activity seems to be correlated with the modulation of cyclooxygenase (COX)s and 5-lipoxygenase (5-LOX) enzymatic pathways and through the inactivation of NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome and NF-κB activity. Other mechanisms proposed include inhibition of Th17 (CD4+/IL-17+) responses and induction of regulatory Treg (CD4+/FOXP3+) via suppression of mTOR signalling. Intriguingly, analysing the current literature, it emerges that all studies performed to date have two main common denominators: i) the anti-inflammatory/immunomodulatory activity and ii) a low bioavailability of mangiferin. Moreover, most of the current data obtained by using either the pure compound or the extract do not take into account the possible pharmacokinetic differences as well as the different solubilization methods. These factors make it very complex to match data that are obtained in different preclinical settings. To overcome these limitations and aimed to dissect the anti-inflammatory and immunomodulatory activity ascribed to this medicinal plant, we have used a Mangifera indica L. extract (MIE) at 90% purity in mangiferin in a mouse model of gouty arthritis (Chapter 2) and in a CD4+CD45RBhigh T cells transfer model of colitis, also evaluating its protective effects on irritable bowel syndrome (IBS) and Crohn’s disease (CD) patients (Chapter 3). We have found, preliminary, that mangiferin and the fully characterized extract used in our studies have a similar pharmacokinetic profile and anti-inflammatory activity but different cytotoxic effects. Taking advantage of these findings, we have used the extract (MIE) to define its mechanism/s of action and, demonstrated that treatment with MIE revealed a dose-dependent reduction in joint inflammatory scores with maximal inhibition observed at 10 mg kg-1. MIE significantly reduced leukocyte infiltration and activation and the expression of different pro-inflammatory cyto-chemokines in inflamed tissues. Furthermore, biochemical analysis revealed that MIE modulated COX-2/mPGES-1 and mPGDS-1/PPARγ pathways. Flow cytometry analysis also highlighted a prominent modulation of inflammatory monocytes (CD11b+/CD115+/LY6Chi), and Treg cells (CD4+/CD25+/FOXP3+) after MIE treatment. Successively, we have depicted the protective role of this plant extract on IBDs demonstrating that treatment with MIE significantly reduced clinical severity of colitis and intestinal inflammation. Specifically, we observed a significant modulation of infiltrating Th1 (CD4+/IFN-γ+), Th17 (CD4+/IL-17+) and Treg (CD4+/CD25+/FOXP3+) cells to the gut, coupled with modulation of several pro/anti-inflammatory cytokines on colonic lamina propria, such as IL-6, IL-17A, IL-22 and IL-10, in MIE treated animals compared to control group. Moreover, MIE mitigated the gut permeability and tight junction functionality coupled to main intestinal metabolites. Mechanistically, MIE co-treatment (1 μg ml-1) significantly ameliorated the levels of COX-2, and even more, DP2 receptor on stimulated-HDBEC. Of note, MIE significantly reduced TNF-α and, in part, IL-17 levels on IBD stratified sera. Taken together, the results of this study demonstrate the beneficial activity of MIE on the immunological perturbance during the onset of colitis and on the systemic inflammatory reaction typical of IBD patients, paving the way for its rationale use as nutraceutical and/or functional food.

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