Dissecting the Role of DNA Methylation in Tuberculosis-Induced Immunological Exhaustion Using CRISPR/Cas9-Based Epigenome Editing

Tuberculosis (TB) infection results in 10 million cases and 1.5 million deaths a year, making TB the world’s leading infectious cause of mortality, and one of the top ten causes of mortality overall. We have recently shown that TB patients display generalized immunological exhaustion and dampened T Cell-mediated immune responses, and that these deficits are correlated with DNA hypermethylation at immune signaling pathway genes (e.g. IL12RB2 and IFN-γ) in TB patient T Cells relative to healthy household controls. Despite these correlations, the molecular mechanisms by which DNA hypermethylation can illicit inhibited host immunity remain unclear.

To clarify the role of focal DNA hypermethylation in TB-induced immunological exhaustion, we applied epigenome editing using dCas9-DNMT3A/3L to target DNA hypermethylation to specific loci in the IL12RB2 and IFN-γ genes based on differentially methylated regions (DMR) identified in TB patient-derived immune cells. Our preliminary studies in HEK293T cells reveal that IL12RB2 and IFN-γ gene expression decreases in response to targeted DNA hypermethylation and suggest that forced DNA methylation at these loci may be directly causal for TB-mediated immune exhaustion, even after eradication of M. tuberculosis from patients. Additionally, to understand how global levels of DNA methylation alter immune cell phenotype, we used CRISPRa (dCas9-VPR) and CRISPRi (dCas9-KRAB) to up- and downregulate, respectively, the DNMT3A and TET1 genes.

Ongoing work is aimed at investigating the immune phenotype disruption using flow cytometry-based multi-dimensional immune profiling and mycobacterial growth inhibition assays. We expect that the precision control of DNA methylation in immune cells will illuminate how specific changes in DNA methylation perturb immune function and open new avenues for a generalized therapeutic intervention for immune exhaustion, which is a hallmark of other infections and several human cancers.