Epigenetic modifications in il-10 gene locus are pivotal for the understanding of il-10 competence in B cells

The homeostasis of the immune system is a finely regulated process, that depends on the equilibrium between the effector and the regulatory compartment, if a misbalance occurs the result could be either excessive inflammation or exaggerate immunosuppression. B cells are traditionally known for their role in production of antibodies and therefore are considered pro-inflammatory cells, however in the last decades, B cells with immune-suppressive functions have been identified, and were termed Regulatory B cells (Breg). Despite great effort of several groups in this field, nowadays, there is no agreement about their origin, nor about their phenotype, and a specific transcription factor, like for example FoxP3 for regulatory T cells, has not been identified yet. Breg mainly exert their immune-suppressive function through the secretion of the anti-inflammatory cytokine Interleukin-10 (IL-10), that is currently used as marker for their detection. A major point in the debate regarding these particular population of B cells is whether the capacity to produce IL-10 is intrinsic property of the cell, or if rather B cells belonging to diverse subset acquire the capacity to produce IL-10 after they are instructed by the surrounding microenvironment. With the aim to clarify this point, my PhD project focused on IL-10 competent B cells, a particular subset able to produce IL-10 after a short-term stimulation, in the specific, after 5 hours of treatment with LPS, PMA and ionomycin. The initial hypothesis was that IL-10 competent B cells quickly respond to stimulation because they are in a suspended state for its production, and we wanted to clarify which are the possible mechanisms at the basis, with a major focus on epigenetic. The initial observation was that B cells stimulated through CD40, treated with the demethylating agent 5-azacytidine, and then briefly re-stimulated with LPS, present a higher expression of il-10 mRNA, compared to cells that did not received the chemical compound. That was the starting point that led us to investigate the methylation status of CpGs among the il-10 gene locus. Starting from a pure and viable population of IL-10 competent B cells, isolated with the combination of an IL-10 secretion assay and cell-sorting, we were able to perform different type of analysis. In first instance, a methylation analysis was performed, taking advantage of a bisulphite-based method, that allows to determine the percentage of methylation at specific sites through sequencing. A specific methylation signature was found, in particular, IL-10 competent B cells are less methylated, compared to the whole population, on two conserved non coding sequence (CNS) located upstream from the transcription starting site of the gene. Interestingly, we found also that IL-10 competent B cells has an active histones signature, with an enrichment of acetylation in the CNS studied also for DNA methylation. The second step was to test whether B cells that cannot produce immediately IL-10, can do it in a longer time frame, to this purpose the non-competent B cells were re-stimulated, and not surprisingly, they started producing IL-10 after 48 hours, and of note, these cells did not acquire the DNA methylation signature IL-10 competent B cells. Collectively, the data presented in this thesis support the hypothesis that, among the total B cell population, IL-10 competent B cells are a particular functional subset that, differently from the rest of the population, can respond quickly to a stimulus triggering with the production of IL-10. In the specific, we found that the IL-10 competent B cells can be recognized among the total population as two CNS of their il-10 gene locus are almost devoid of DNA methylation, contrarily from all other B cells. Finally, IL-10 competent B cells are an effective functional immune-suppressive subset, while all other IL-10 producing B cells acquire this capacity during time and depending on the characteristics of surrounding microenvironment