Effect of bioactive compounds on the immune system. A multiplatform metabolomics approach

Resumo

Natural Bioactive compounds are ubiquitous, as they can be found in bacteria, fungi, plants or animals. However, bacteria and plants are an important source of these bioactive substances. They are defined as bioactive primarily on the basis of their biological activities, which can be assigned to their unique chemical structures, unusual chemical groups, as well as structural elements. Among these specific properties, the role of bioactive compounds in modulating the immune system has attracted a great deal of attention. It is known that several human diseases of non-infectious origin have been related with altered intestinal microbiota (dysbiosis) and an unbalanced immune response such as obesity, type 1 and 2 diabetes, inflammatory bowel disease, rheumatoid arthritis, lupus systemic erythematosus and colorectal cancer. It is also known the potential to positively modulate the intestinal microbiota by using bacteria or bacterial substrates (probiotics). They can modulate the host immune system, re-establishing homeostasis, by the expression of specific receptors or signaling pathways, the suppression of inflammatory responses NF-kB or promoting the secretion of anti-inflammatory mediators. Among the effector molecules present in the microbiota with beneficial effects on human health, the protein molecules are worthy of note. On the other hand, an important group of phytochemicals that have demonstrated the ability to modulate the immune system response are phenolic compounds. They are a group of natural substances found in vegetables, fruits, grains and vegetal beverages. Among these compounds, dihydrochalcones are phenolic compounds of growing interest. They are a group of flavonoids found mainly in apples with antioxidant, anti-cancer, and anti-inflammatory effects. In this regard, the phenolic compound structure such as the presence of sugar residues, the position, and degree of glycosylation determine their immunomodulatory properties. The structural and molecular mechanism related with the beneficial effects promoted by different bioactive compounds on human health, constitute an important field of research. Therefore, the development of the analysis of massive data sets, such as untargeted metabolomics approaches wold be of great importance. In this context, the present Doctoral Thesis aims to interpret the in vitro effect of bioactive compounds on the immune system, focusing the study of cell metabolism through the application of multiplatform untargeted metabolomics techniques. To address this issue, it has been structured in two main blocks: 1. The immunomodulatory evaluation effect of a peptide encrypted in an extracellular glycosidase from Bifidobacterium longum NCIMB 8809, called HM14. The immunomodulatory role of peptides derived from intestinal bacteria through a multiomics approach was evaluated. To this end, the anti-inflammatory effect of HM14, an extracellular glycosidase encrypted peptide from Bifidobacterium longum NCIMB 8809, on peripheral blood mononuclear cells (PBMCs) from healthy donors and from a cohort of patients with Crohn's disease was conducted by performing multiplex analysis of cytokines. Moreover, the molecular mechanisms of action of HM14 were evaluated firstly in PBMCs through a multiplatform untargeted metabolomics approach by gas chromatography (GC)-mass spectrometry (MS) and liquid chromatography (LC)–high-resolution mass spectrometry (HRMS). Finally, the molecular mechanism of action of HM14 was assessed on monocyte-derived dendritic cells (MoDCs) using a transcriptomics approach and on CD4+ T-cells. Based on the outcomes of this first part, it was possible to conclude that the human gut metaproteome is source for a vast array of potential bioactive compounds such as immunomodulatory peptides, that can be identified through an in silico pipeline. One of these peptides, HM14, exerted its anti-inflammatory effect by acting on a yet uncharacterized innate immune receptor. Presentation of HM14 to naïve T-cells, in vitro, stimulated iTreg expansion in healthy donors and reduced proinflamatory IL-12 in PBMCs isolated from Crohn’s patients. In all in vitro settings, HM14 increased the production of anti-inflammatory IL-10 by immune cells. Metabolomics analysis also supported a differential behaviour between immune cells exposed to HM14 and untreated cells, suggesting that HM14 was able to interact with cells in a specific way, which differs from other bacterial proteinaceous molecules. HM14 triggered an increase in protein biosynthesis rate, glycolytic intermediaries, lipid remodelling and enhanced the NAD+ metabolic pathways. So that, this study opens new insights directed to the understanding on how these millions of immunomodulatory peptides originated from the gut metaproteome affect the way our microbiota interacts with our immune and potentially with our nervous system. 2. The immunomodulatory evaluation effect of apples and their constituents (leaf, peel and pulp) from a structure-activity approach. The immunomodulatory role of apples and their constituents through metabolomics was evaluated. To this end, a deep characterization of the phenolic profile of a local variety of apples (leaf, peel and pulp) was performed by using LC-HRMS for the subsequent isolation and quantification of dihydrochalcone-rich fractions by using LC-preparative and LC coupled to tandem mass spectrometry (LC-QqQ-MS). Finally, the immunomodulatory role of whole apple extracts (leaf, apple, peel and pulp), dihydrochalcone-rich fractions (FI-FV) and pure dihydrochalcones (phlorizin (PZ) and phloretin (PT)) was evaluated on THP-1 derived human macrophages through a non-targeted multiplatform metabolomic approach (based on GC-MS and LC-HRMS). Based on the outcomes of this second part, it was possible to conclude that apples were a valuable source of bioactive compounds and more specifically of phenolic compounds that could exert beneficial effects on human health. This study has provided an untargeted multiplatform metabolomics approach (LC/GC-MS) to understand metabolic effects promoted by: four apple extracts (leaf, apple, peel and pulp), dihydrochalcone-rich isolated fractions (FI-FV); and pure standards of dihydrochalcones (PZ and PT), under normal and inflammatory conditions, on human THP-1 derived macrophages. The immunomodulatory activity of phenolic mixtures could not be predicted from the activity of pure compounds. Moreover, it was not possible to attribute the effect observed in both extracts and fractions to the effect reported by the pure dihydrochalcones. Different phenolic compound ratios promoted alterations in the metabolism of THP-1 derived macrophages, since both fractions and extracts showed statistically significant differences. Apple extracts appeared to counteract the molecular mechanisms triggering a pro-inflammatory phenotype in THP-1 derived macrophages, since statistically significant differences were observed between extracts-treated THP-1 cells and those treated with LPS. Those differences were mainly reported on glycerophospholipid, glycerolipid, sphingolipid, galactose and alanine, aspartate and glutamate metabolism. Dihydrochalcone-rich fractions triggered metabolic patterns that differed from those triggered by LPS, by modulating glycerophospholipid, glycerolipid and sphingolipid metabolism; galactose, fructose and mannose, and glyoxylate and dicarboxylate metabolism; valine, leucine and isoleucine biosynthesis; and pantothenate and CoA biosynthesis. Pure dihydrochalcone standards (PZ and PT) were capable of inhibiting the response triggered by LPS, promoting differential metabolic patterns mainly in glycerophospholipid and sphingolipid metabolism. Furthermore, PT seemed to exert greater immunomodulatory capacity than PZ, which showed limited metabolic alterations in comparison to untreated THP-1 derived macrophages. This study highlights the sustainable use of apple and its by-products, either for the isolation of bioactive compounds or for the preparation of therapeutically useful extracts, could be a valuable resource for pharmaceuticals and/or food applications for managing inflammatory disorders.