Oral Porphyromonas gingivalis Infection Induces Epigenetic Changes that Promote Persistence of Cardiovascular Disease Risk

Abstract

In the last two decades, periodontitis has become recognized as a risk factor for cardiovascular disease (CVD), a term that encompasses many circulatory disorders, often underpinned by atherosclerosis. One of the major questions regarding this association has been whether elimination of periodontitis promotes reversal of CVD risk. Studies in edentulous populations have shown that residual CVD risk persists, even with clinically acceptable elimination of periodontitis. Owing to the strong inflammatory components of both diseases, our lab hypothesizes that persistence of CVD risk is in part a function of epigenetic reprogramming of hematopoietic stem cells in the bone marrow during periodontitis, promoting a pro-atherogenic phenotype in the immune system long-term. To simulate persistence of CVD risk, a bone marrow transplant approach was used. Bone marrow was transplanted into healthy, irradiated pro-atherogenic mice from two syngeneic donor cohorts: mice orally infected with Porphyromonas gingivalis (Pg) or sham infected. In previous experiments using this transplant approach, mice receiving BM from Pg-infected donors displayed increased atherosclerotic lesion burden, providing a strong underpinning to performing epigenetic analysis. Using whole genome bisulfite sequencing of donor bone marrow progenitor DNA, with cross-referencing of transcriptome analysis from recipient bone marrow-derived macrophages, differentially methylated and expressed genes were identified. Systemic lipid phenotype analyses were also performed as initial studies into differences in phenotype between transplant recipient groups. Mice receiving bone marrow from Pg-infected donors displayed a number of notable differences in plasma lipid profiles. These include increased total triglycerides, esterified cholesterol, very low-density density lipoprotein (VLDL) triglycerides, and decreased high density lipoprotein cholesterol. Transcriptome analysis showed differential expression in oxidative mitochondrial metabolism and cell stress management. When cross-referenced to whole genome bisulfite sequencing data from progenitors, nine genes differentially methylated and expressed were identified as candidate genes for future phenotypic studies. PON2, APOE, CBR2, NPC2, ATP5O, ATPF1 were found to be hypermethylated and downregulated, while SLC23A2, ACC1, and SORL1 were found to be hypomethylated and upregulated. Based on their epigenetic and expression profiles, enrichment of these genes suggests a pro-oxidative phenotype, contributing to increased cellular oxidized LDL, in addition to increased triglyceride and cholesterol accumulation. Impaired cholesterol trafficking, as well as catabolism of triglyceride rich lipoproteins may explain the VLDL/triglyceride/esterified cholesterol phenotype data. Presently, our experiments have suggested that macrophages originating from the bone marrow of mice infected by Pg exhibit a more pro-atherogenic phenotype, which may be guided by changes in DNA methylation in upstream progenitors during hematopoiesis. If our hypothesis is supported by future experiments, this will necessitate the development of CVD therapeutics targeting epigenetic processes. Our findings may have broader applicability to other CVD risk factors which are also known to leave increased risk after removal, such as diabetes and smoking.