Schistosomiasis Induces Persistent DNA Methylation and Tuberculosis-Specific Immune Changes

Abstract

Epigenetic mechanisms, such as DNA methylation, determine immune cell phenotype. To understand the epigenetic alterations induced by helminth coinfections, we evaluated the longitudinal effect of ascariasis and schistosomiasis infection on CD4+ T cell DNA methylation and the downstream tuberculosis (TB)-specific and bacillus Calmette-Guérin-induced immune phenotype. All experiments were performed on human primary immune cells from a longitudinal cohort of recently TB-exposed children. Compared with age-matched uninfected controls, children with active Schistosoma haematobium and Ascaris lumbricoides infection had 751 differentially DNA-methylated genes, with 72% hypermethylated. Gene ontology pathway analysis identified inhibition of IFN-γ signaling, cellular proliferation, and the Th1 pathway. Targeted real-time quantitative PCR after methyl-specific endonuclease digestion confirmed DNA hypermethylation of the transcription factors BATF3, ID2, STAT5A, IRF5, PPARg, RUNX2, IRF4, and NFATC1 and cytokines or cytokine receptors IFNGR1, TNFS11, RELT (TNF receptor), IL12RB2, and IL12B (p < 0.001; Sidak-Bonferroni). Functional blockage of the IFN-γ signaling pathway was confirmed, with helminth-infected individuals having decreased upregulation of IFN-γ-inducible genes (Mann-Whitney p < 0.05). Hypomethylation of the IL-4 pathway and DNA hypermethylation of the Th1 pathway was confirmed by Ag-specific multidimensional flow cytometry demonstrating decreased TB-specific IFN-γ and TNF and increased IL-4 production by CD4+ T cells (Wilcoxon signed-rank p < 0.05). In S. haematobium-infected individuals, these DNA methylation and immune phenotypic changes persisted at least 6 mo after successful deworming. This work demonstrates that helminth infection induces DNA methylation and immune perturbations that inhibit TB-specific immune control and that the duration of these changes are helminth specific.

Copyright © 2018 by The American Association of Immunologists, Inc.

Figures

Figure 1. Global CD4+ T cell DNA methylation changes during helminth infection

The Illumina DNA methylEPIC array was performed on CD4+ T cells from helminth infected and uninfected individuals. n = 5 per group. A) Volcano plot showing the hypo (blue dots) and hyper (red dots) methylation (β values) in helminth infected individuals compared to uninfected controls. B) Gene ontology pathways enriched in genes with DNA hypo-methylation after helminth infection. C) Gene ontology pathways enriched in genes with DNA hyper-methylation after helminth infection.

Figure 2. Targeted DNA methylation of transcription factors, cytokines and surface receptors depending on helminth infection

Methyl-specific endonuclease digestion followed by RT-PCR was performed on isolated DNA from CD4+ T cells from ascaris-infected, Schistosome-infected infected or uninfected children. Successful deworming was confirmed by negative PCR and microscopy two and six months after anti-helminth therapy. n = 6 per group. A) Bar graph depicts the median and range of the percent DNA methylation depending on helminth status. B) Heatmap of the CD4+ T cell DNA methylation signature depending on helminth status. (S.h = Schistosoma haematobium)

Figure 3. Pathway analysis identified inhibition of the Th1 and IFN-γ signaling pathway by helminth infection

Methyl-specific endonuclease digestion followed by RT-PCR was performed on isolated DNA from CD4+ T cells from helminth infected or uninfected children. Genes outlined in pink expressed increased DNA methylation in helminth-infected children compared to uninfected age-matched controls. n = 6 per group. A) Th1 Signaling Pathway. B) IFN-γ signaling pathway.

Figure 4. Helminths decrease IFN-γ-inducible gene up-regulation

Peripheral blood mononuclear cells (PBMCs) from recent TB-exposed children were stimulated overnight with IFN-γ (50ng/mL) followed by CD4+ T cell selection and RNA isolation. Fold up-regulation was determined by comparing gene expression non-stimulated gene expression to IFN-γ gene expression. n = 8 helminths and 4 controls. A) IFN-γ-inducible gene expression of specific genes. The # symbol denotes Sidak-Bonferroni p

Figure 5. Helminths decrease the Mycobacterium tuberculosis (Mtb)-specific Th1 CD4+ T cell function with associated increases in Th2

Peripheral blood mononuclear cells (PBMCs) from recent TB-exposed children were stimulated overnight with Mtb-specific peptides (ESAT-6 and CFP-10) peptide pools. n = 18 helminth-uninfected and 8 helminth-infected. A) Flow cytometry-gating strategy. B) Representative dot plots of non-stimulated, ESAT-6 and CFP-10 stimulated or BCG stimulated CD4+ T cells. C) The overall Mtb-specific CD4+ T cell response is illustrated as a pie graph with TNF-α (yellow arcs) and IFN-γ (red arcs) and IL-4 (green arcs). Each pie slice corresponds to a discreet functional subpopulation, outlined in D. The Th1: Th2 ratio was determined by the ratio of IFN-γ and TNF-α compared to IL-4. D) The bar graph represents the absolute frequency of CD4+ T cells producing, IFN-γ, TNF-α or IL-4. The # symbol denotes Wilcoxon P<0.05 between helminth uninfected and infected groups. Data represent 18 helminth-uninfected and 8 active helminth-infected individuals.

Figure 6. Persistent Th2:Th1 perturbations after schistosomiasis

Children with or without helminth infection had Peripheral blood mononuclear cells (PBMCs) collected at baseline and six-months later. Children with Ascaris or Schistosoma haematobium were treated with albendazole or praziquantel, respectively. Successful deworming was confirmed by microscopy and PCR at two and six months. The overall Mtb-specific CD4+ T cell response is illustrated as a pie graph with TNF-α (yellow arcs) and IFN-γ (red arcs) and IL-4 (green arcs). The Th1: Th2 ratio was determined by the ratio of IFN-γ and TNF-α compared to IL-4. Data represent 6 per helminth group and 18 un-infected controls.