Investigation of the effect of gut dysbiosis on histone post-translational modifications in neutrophils

Abstract

The interaction between neutrophils and intestinal microbiota is important for homeostasis and dysbiosis, as occurring in Inflammatory Bowel Disease (IBD). Short-Chain Fatty Acids (SCFAs) are metabolites generated by commensal bacteria and promote immunomodulatory effects. These effects occur, in part, due to the ability of SCFAs to promote Post-Translational Modifications (PTMs) of histones in leukocytes. The major histone PTMs already described in neutrophils are methylations, citrullination and acetylation, but there are no reports of other types of acylations including butyrylation or crotonylation in neutrophils. Our hypothesis is that reduction in the availability of SCFAs in the gastrointestinal tract and, consequently, in the bloodstream, changes the pattern of histone acylation in neutrophils, altering the gene expression and the effector function of these cells. In this study, we will analyse the epigenetic modulation of human neutrophils in healthy volunteers submitted or not to treatment for induction of intestinal dysbiosis. This study will be carried out with samples obtained from healthy male volunteers, with normal or overweight body weight (BMI between 18.5 and 29.9), nonsmokers, with no recent history of antibiotic or corticosteroid therapy, without a diagnosis of metabolic or inflammatory disease, without signs of acute infection and good general health. Such volunteers will be divided into a control group and a treated group. The treated group will be submitted to oral antibiotic therapy for 3 days. Blood and stool samples will be collected. Stool samples will be used for microbiota evaluation and quantification of SCFAs. Blood samples will be used for the detection of SCFAs in the serum and isolation of neutrophils to be analyzed. Purified neutrophils will be stimulated in vitro for and processed for different analyzes: (i) production of cytokines; (ii) phagocytic and killing capacity; (iii) evaluation of the transcriptome by RNAseq; (iv) evaluation of the accessibility profile of transcription factors to different chromatin regions by ATAC-seq; (v) evaluation of histone acylation by Western Blotting. In this study, we will advance in the understanding of the interactions between microbiota/SCFAs and neutrophils. (AU)