DNA hypermethylation during tuberculosis dampens host immune responsiveness

Abstract

Mycobacterium tuberculosis (M. tuberculosis) has coevolved with humans for millennia and developed multiple mechanisms to evade host immunity. Restoring host immunity in order to improve outcomes and potentially shorten existing therapy will require identification of the full complement by which host immunity is inhibited. Perturbation of host DNA methylation is a mechanism induced by chronic infections such as HIV, HPV, lymphocytic choriomeningitis virus (LCMV), and schistosomiasis to evade host immunity. Here, we evaluated the DNA methylation status of patients with tuberculosis (TB) and their asymptomatic household contacts and found that the patients with TB have DNA hypermethylation of the IL-2/STAT5, TNF/NF-κB, and IFN-γ signaling pathways. We performed methylation-sensitive restriction enzyme-quantitative PCR (MSRE-qPCR) and observed that multiple genes of the IL-12/IFN-γ signaling pathway (IL12B, IL12RB2, TYK2, IFNGR1, JAK1, and JAK2) were hypermethylated in patients with TB. The DNA hypermethylation of these pathways was associated with decreased immune responsiveness with decreased mitogen-induced upregulation of IFN-γ, TNF, IL-6, CXCL9, CXCL10, and IL-1β production. The DNA hypermethylation of the IL-12/IFN-γ pathway was associated with decreased IFN-γ-induced gene expression and decreased IL-12-inducible upregulation of IFN-γ. This study demonstrates that immune cells from patients with TB are characterized by DNA hypermethylation of genes critical to mycobacterial immunity resulting in decreased mycobacteria-specific and nonspecific immune responsiveness.

Keywords: Cytokines; Epigenetics; Immunology; Infectious disease; Tuberculosis.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Patients with TB exhibit an exhausted immune phenotype.

PBMCs from individuals with TB (n = 29) and their asymptomatic household contacts (HC) (n = 49) were subjected to antigenic stimulation with BCG sonicate followed by flow cytometry–based multidimensional immune profiling. CITRUS clustering was implemented on CD3, CD4, CD8, and CD56 surface markers with a minimum cluster size of 10% per node. Lines indicate the median and interquartile range, and P values were determined by nonparametric Mann-Whitney U test. (A) Citrus clustering identified Th (CD3+CD4+), CTL (CD3+CD8+), and NK (CD3–CD56+) cell populations, with CD3 signal depicted by a heatmap. Gray-shaded subclusters have an FDR of less than 1% and depict decreased IFN-γ and Ki-67 expression among Th cells (CD3+CD4+), CTLs (CD3+CD8+), and NK cells (CD3–CD56+). Representative dot plots of the MFI clusters are depicted from the yellow highlighted clusters. (B) Reanalysis of CITRUS, with exclusion of HIV-coinfected individuals, for patients with TB (n = 20) and controls (n = 40) shows CD4+, CD8+, and CD56+ lymphocyte subsets with decreased Ki-67 and IFN-γ (gray-shaded clusters have decreased IFN-γ and Ki-67, with an FDR <1%; representative dot plots of MFI clusters are depicted). Mann-Whitney U test P values describe MFI differences between controls and patients with TB.

Figure 2. Global DNA methylation perturbations persist 6 months after successful ATT.

The Illumina DNA Methylation EPIC array was performed on bulk PBMCs from asymptomatic household contacts (n = 10) and individuals with TB (n = 15; TB/HIV– = 7; TB/HIV+ = 8) at baseline. All individuals with TB had treatment success. For individuals without HIV coinfection, DNA methylation status was evaluated at baseline and 12 months later, 6 months after completion of successful ATT. (A) To ascertain the immune cell–specific DNA methylation changes, cell-specific DNA methylation reference profiles were downloaded from public archives, informative loci were identified, and cell type–specific methylation profiles were identified. (B) Cell-specific DNA methylation differences are shown for TB patients (base) and TB/HIV (HIV) patients at baseline and for TB patients 12 months after baseline (12 mo), 6 months after completion of successful ATT. All results were compared with HC data. GO pathway analysis for monocytes (CD14+), Th cells (CD3–CD4+), NK cells (CD3–CD56+), and CTLs (CD3+CD8+). (C and D) Purified Th cells (CD3+CD4+) (n = 2) and EDEC-identified Th cells (CD3+CD4+) (n = 8 TB patients; n = 10 controls) were compared with cells from controls, and common hypermethylated pathways and genes were identified.

Figure 3. The TB epigenetic landscape is similar to LCMV-induced immune exhaustion.

(A) DNA hypermethylation of genes in CD3+CD8+ CTLs in TB patients (green), and that reported by Ghoneim et al. (10) (red) and Sen et al. (4) (blue). (B) Genes and GO pathways of CD8+ CTLs with DNA hypermethylation for patients with TB from this cohort were compared with genes with DNA hypermethylation, reported by Ghoneim et al. (10), and ChARs induced by chronic LCMV–induced immune exhaustion, reported by Sen et al. (4).

Figure 4. Targeted DNA methylation confirms DNA hypermethylation of the IL-12/IFN-γ pathway.

(A) Graphic representation of the IL-12/IFN-γ pathway. (B) DNA methylation was evaluated using MSRE-qPCR in non–HIV-coinfected TB patients (n = 6) and their asymptomatic healthy household contacts (n = 5). *P < 0.01, by Mann-Whitney U test.

Figure 5. Decreased mitogen-induced responsiveness.

Fresh whole blood (0.8–1.2 mL) from patients with TB (n = 40) and asymptomatic healthy household contacts (n = 39) was stimulated overnight with and without mitogen, and the resultant plasma was evaluated for cytokine and chemokine upregulation using a custom-designed, bead-based multiplex ELISA. P values were determined by nonparametric Mann-Whitney U test. FC, fold change.

Figure 6. Decreased IFN-γ–inducible gene expression.

PBMCs (1 × 106) from patients with TB (n = 10) and asymptomatic healthy household contacts (n = 10) were cultured overnight with and without 50 ng IFN-γ. Then, RNA was isolated, and IFN-γ–inducible gene expression was evaluated by microarray. P = 0.02, by Mann-Whitney U test.

Figure 7. Decreased IL-12–inducible IFN-γ production.

PBMCs from patients with TB (n = 15) and asymptomatic healthy household contacts (n = 10) were treated overnight with PBS media control (Nil stim), or were stimulated with BCG sonicate (5 μg) or IL-12 (20 ng/mL) and BCG (minimum of 0.5 × 106 PBMCs per condition) followed by multidimensional flow cytometry. See Supplemental Figure 3 for the gating strategy and representative dot plots of CD3+CD4+ and CD3+CD8+ T cells. (A) Representative dot plots of NK cell (CD3–CD56+) production of IFN-γ and TNF-α after no stimulation or stimulation with BCG or with IL-12 plus BCG. (B) t-distributed stochastic neighbor embedding (tSNE) dimension reduction clustering of PBMCs from a control and from patients with TB demonstrated the cell-specific (CD3, CD4, CD8, and CD56) contribution of IFN-γ production after combined IL-12 and BCG stimulation, with blue indicating no protein expression, and green, yellow, and red indicating increasing protein expression. (C and D) Dot plots demonstrate the percentage of CTLs (CD3+CD8+) and NK cells (CD3–CD56+) that produced IFN-γ after stimulation with IL-12 and BCG. P = 0.0308 and 0.008, by Mann-Whitney U test, for CTLs and NK cells, respectively.