Analysis of the Interaction between Microbiome and Host in Epigenetic and Transcriptional Tissue Changes in Patients with Grade C Periodontitis

Abstract

Stage 3-4 Grade C periodontitis in young people (Perio4C) is a rapidly progressive disease triggered by an imbalance in the host's immune response to the subgingival dysbiotic microbiota, which may be related to the interaction of periodontal pathogens in the biofilm with the cells of the epithelium of the gingival sulcus and adjacent connective tissue, which, by undergoing epigenetic modifications, overexpress or fail to express important genes for tissue homeostasis. However, the impact of epigenetic modifications on the etiopathogenesis of this disease is still unknown, as is the direct relationship between specific pathogens and local gene regulation. Thus, this study seeks to assess the impact of dysbiotic colonization of individuals with Perio4C on the methylation pattern of genes related to both epithelial cell adhesion and adjacent connective tissue defense, as well as on the final product of protein secretion by cells in this tissue. For this, samples of gingival crevicular fluid (FCG), subgingival biofilm and gingival tissue will be collected from 20 patients with Perio4C and 20 patients with periodontal health (SP), and for patients with Perio4C the samples will be collected from the same dental site. will be collected from both a healthy site (PS < 4mm) and a disease-affected site (PS > 7mm). Tissue biopsy will be submitted to laser microdissection (LCM) to separate the epithelium and connective tissue, and DNA and RNA will be extracted from both structures to assess the methylation pattern of the TJP1, PLAK, DESP, CADH1 genes, for the epithelium, and IL10, IL1², IL6, 1L17, TNF±, for the connective tissue. Bacterial DNA will be extracted from the biofilm and the 16S rRNA gene will be used for sample sequencing and species identification using the Illumina MiSeq platform. The quantification of cytokines IL-1², IL-4, IL-6, IL-8, IL-10, IL-17, TNF-±, INF-³ of FCG samples will be performed using Luminex/MAGpix technology. For statistical tests a significance level of 5% will be used.