PROBIOTICS-INDUCED EPIGENETIC MODIFICATIONS IN IMMUNE CELLS AS A NOVEL STRATEGY TO ASSESS THEIR ACTIVITY

The human body is densely populated by complex bacterial communities, which are called microbiota. Since microbiota has an important role in the physiology of the host, there is an increasing interest in the use of probiotic bacteria to ensure the well-being of the person. The intestinal microbiota, among its various functions, stimulates and influences host’s immune system, through diverse and complex mechanisms. Recently it is emerging the ability of gut microbes to induce DNA epigenetic modifications, such as DNA methylation and histones modification that can deeply influence gene expression and cellular phenotype. While the benefits of probiotics are well known, an objective method to evaluate their actual effects is still missing. This project aims to identify the epigenetic modifications caused by probiotic supplementation and identify markers in circulating immune cells to be used to objectively evaluate their efficacy. Experiments were conducted using a probiotic with established immuno-modulatory properties, Bifidobacterium animalis ssp lactis, and a probiotic strain devoid of significative effects on immune system, Lactobacillus rhamnosus. We decided to investigate the impact on DNA methylation in mucosal dendritic cells (DCs) since these cells are strategic in immune response development. By performing a whole genome analysis (RRBS) oral supplementation with Bifidobacterium animalis ssp lactis caused a significative modification in cytosine methylation profile in mucosal DCs. RRBS data highlighted that B. animalis oral supplementation significantly impacts on DNA cytosines methylation, affecting the methylation of at least 3000 individual cytosines, and 707 cytosine-rich regions present on promoters or genes. Moreover, 152 analyzed regions resulted more methylated, while 555 regions resulted less methylated compared to control mice group. By bioinformatic analysis we selected the promotorial area of genes and identified a cluster of promoters with modified methylation level belonging to the immune responses. Since the methylation level of CpG islands in gene promoters can modify gene’s transcription rate, we quantified by qRT-PCR the specific mRNA and quantified the methylation status of these promoter by bisulfite DNA conversion and sequencing. We observed augmented mRNA levels of zbtb33, while mRNA sec14l and tspan6 were reduced, in agreement with the reduced methylation of zbtb33 and the increase in cytosines methylation of sec14l promoters. These data were confirmed at protein level by FACS analysis of mucosal DCs showing increased CD80+ Tspan6+ and CD80+ Kaiso+ cells and reduced CD80+ Sec14l+, in mice supplemented with Bifidobacterium animalis ssp lactis but not with Lactobacillus rhamnosus. On circulating DCs we detected a 15% increase only in CD11c+ CD80+ Tspan6+ in mice supplemented with Bifidobacterium animalis ssp lactis but not with Lactobacillus rhamnosus. We attempted to extend our findings to human mucosal DCs cells using an in vitro reductionist system, co-culturing epithelial cells, probiotic and monocyte-derived DCs. In this system, we confirmed that exposure of epithelial cells to B. animalis increase number of CD80+ Sec14l, CD80+ Tspan6+ and CD80+ Kaiso+ cells. In conclusion, we report for the first time that the administration of B. animalis is able to induce appreciable epigenetic modification on a specific and relevant immune cells such as DCs resulting in appreciable variations in both gene expression and cellular phenotype. These findings can be used as a basis for further study the impact of probiotics on host’ epigenetic to better understand their mechanisms of action. Further analysis on subset of circulating DCs are required to identify the more suited subpopulation representing gut-derived mucosal DCs carrying epigenetic/phenotypic trait induced by probiotics.