PROJECT SUMMARY: After apparently successful treatment, Tuberculosis (TB) survivors have a 16.9% of death, a rate 3-fold higher than age and sex matched controls. In our previous work, we identified that six months after the completion of successful TB therapy, survivors retained detrimental epigenetic scars that continue to perturb host immunity and inflammation. In this proposal, using currently enrolling cohorts, we will test our proposal that these detrimental epigenetic scars induce pathologic inflammation and decrease immune responsiveness, leading to increased risk of post-TB mortality. Our previous work demonstrated that TB induces more than a thousand DNA methylation perturbations in pathways related to inflammation, TNF, IL-6 and IFN signaling pathways. Functionally, these DNA methylation perturbations are associated with increased inflammation and decreased immune responsiveness. Other infections (HIV, CMV, schistosomiasis) also induce long-lasting epigenetic scars, yet, to date, no study has identified which of these epigenetic scars are associated with post-TB mortality. Therefore, in Aim 1, we will follow TB patients for 30 months after completion of successful therapy to identify which DNA methylation marks are associated with post-TB mortality. While most immune cells only live a few days or weeks, our previous work demonstrated that DNA methylation perturbations persist at least 6 months after successful therapy. Animal models and our preliminary data support the hypothesis that detrimental epigenetic scars occur in progenitor and stem cells. To test this hypothesis, we will use cutting-edge single cell epigenomic (scATAC) and transcriptomics (scRNA) sequencing and pseudotime bioinformatic techniques to link progenitor exhausted populations with terminally exhausted populations. Our in vitro preliminary data, demonstrate that activation of the TCA metabolic pathway mediates the induction of detrimental epigenetic marks and that inhibiting the TCA activation can mitigate infection induced DNA hyper-methylation and restore immune responsiveness. Therefore, using our established guinea pig TB model, we will evaluate if inhibitors of the TCA cycle can block or reverse detrimental epigenetic scars and restore mycobacterial immunity. The elucidation of the specific epigenetic marks that subvert immune homeostasis and increase the risk of post-TB mortality is a necessary step in the development of adjunctive host directed therapy to decrease the current high mortality rates that occur after successful TB therapy.