Combined prenatal Lactobacillus reuteri and ω-3 supplementation synergistically modulates DNA methylation in neonatal T helper cells

Abstract

Background: Environmental exposures may alter DNA methylation patterns of T helper cells. As T helper cells are instrumental for allergy development, changes in methylation patterns may constitute a mechanism of action for allergy preventive interventions. While epigenetic effects of separate perinatal probiotic or ω-3 fatty acid supplementation have been studied previously, the combined treatment has not been assessed. We aimed to investigate epigenome-wide DNA methylation patterns from a sub-group of children in an on-going randomised double-blind placebo-controlled allergy prevention trial using pre- and postnatal combined Lactobacillus reuteri and ω-3 fatty acid treatment. To this end, > 866000 CpG sites (MethylationEPIC 850K array) in cord blood CD4+ T cells were examined in samples from all four study arms (double-treatment: n = 18, single treatments: probiotics n = 16, ω-3 n = 15, and double placebo: n = 14). Statistical and bioinformatic analyses identified treatment-associated differentially methylated CpGs and genes, which were used to identify putatively treatment-induced network modules. Pathway analyses inferred biological relevance, and comparisons were made to an independent allergy data set.

Results: Comparing the active treatments to the double placebo group, most differentially methylated CpGs and genes were hypermethylated, possibly suggesting induction of transcriptional inhibition. The double-treated group showed the largest number of differentially methylated CpGs, of which many were unique, suggesting synergy between interventions. Clusters within the double-treated network module consisted of immune-related pathways, including T cell receptor signalling, and antigen processing and presentation, with similar pathways revealed for the single-treatment modules. CpGs derived from differential methylation and network module analyses were enriched in an independent allergy data set, particularly in the double-treatment group, proposing treatment-induced DNA methylation changes as relevant for allergy development.

Conclusion: Prenatal L. reuteri and/or ω-3 fatty acid treatment results in hypermethylation and affects immune- and allergy-related pathways in neonatal T helper cells, with potentially synergistic effects between the interventions and relevance for allergic disease. Further studies need to address these findings on a transcriptional level, and whether the results associate to allergy development in the children. Understanding the role of DNA methylation in regulating effects of perinatal probiotic and ω-3 interventions may provide essential knowledge in the development of efficacious allergy preventive strategies. Trial registration ClinicalTrials.gov, ClinicalTrials.gov-ID: NCT01542970. Registered 27th of February 2012-Retrospectively registered, https://ichgcp.net/clinical-trials-registry/NCT01542970 .

Keywords: Allergy prevention; CD4+ T cells; Combined intervention; Cord blood; DNA methylation; Lactobacillus reuteri; MethylationEPIC 850K; Postnatal; Prenatal; ω-3 fatty acids.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1

Graphical representations of multidimensional scaling (MDS) analyses are shown in panels A–C for the double active (Lω) and single active treatment groups (LP, Pω), respectively, in comparison to the double placebo group (PP). Red dots represent the active treatment group, blue dots the placebo group. In panels D-F, volcano plots illustrate the distribution of DMCs when comparing each of the active treatment groups to the double placebo group (PP). DMCs are defined as having an MMD of > 5% along with an FDR-corrected p value of < 0.1, and are depicted as red dots. DMC—differentially methylated CpG, MDS—multidimensional scaling, MMD—mean methylation difference, L—Lactobacillus reuteri, ω—ω-3 fatty acids, P—placebo

Fig. 2

A graphic representation of overlapping DMGs between the different comparisons. The sets represent the different comparisons, and the intersectional sizes show the number of overlapping genes between the comparisons. Dark blue represents the double-treated group, light blue the L. reuteri single-treated group and coral blue the ω-3 single-treated group. DMGs—differentially methylated genes, L—Lactobacillus reuteri, ω—ω-3 fatty acids, P—placebo

Fig. 3

Visualisation of the consensus module created with the DMGs from the differential methylation comparison between the double-treated L.reuteri/ω-3 (Lω) and the double placebo (PP) group. Nodes represent genes and connecting lines represent protein–protein interactions (STRING combined score > 0.7) within the network. Red nodes illustrate hypermethylated genes, blue nodes indicate hypomethylated genes and both colours denote mixed methylation patterns. Black lines enclose biologically relevant clusters identified by bioinformatic pathway enrichment analyses. Nodes marked with * are also present in an independent allergy data set (described later in the paper). DMGs—differentially methylated genes, L—Lactobacillus reuteri, ω—ω-3 fatty acids, P—placebo

Fig. 4

Venn diagrams illustrating the degree of overlap and uniqueness for the obtained genes from the three network modules for the double active treatment (Lω vs. PP) and the single active treatment (LP vs. PP, Pω vs. PP) comparisons. In A the number of common genes from the overlap are displayed for each comparison is shown, with corresponding gene names denoted in italics, and selection of related pathways in bold. No relevant pathways were revealed for the shared genes of the LP vs. PP and Pω vs. PP comparisons. In B the number of genes that are unique for each comparison are displayed along with a selection of corresponding related pathways. No pathways of relevance were revealed for the unique genes from the Pω vs. PP comparison. L—Lactobacillus reuteri, ω—ω-3 fatty acids, P—placebo

Fig. 5

River plots illustrating the methylation status for overlapping genes being shared between the double-treated Lω group and A the single-treated probiotic (LP vs. PP), and B ω-3 treated (Pω vs. PP) group. Panel C illustrates comparisons between the single treated groups (LP and Pω vs. PP) and D shows the comparison between all treatment groups. Genes are represented as flows of size proportional to number of genes within their methylation status in a particular set, and differences between sets are shown as connections between flows. The respective gene names are denoted next to each flow. Red represents hypermethylation, blue hypomethylation and green a mixed methylation pattern. The number of genes in the respective flows are denoted in the graph. L—Lactobacillus reuteri, ω—ω-3 fatty acids, P—placebo

Fig. 6

Enrichment analyses relating DMCs from A. differential DNA methylation analyses and B. network analyses for all three comparisons, to DMCs from a study investigating DNA methylation patterns in adults with seasonal allergic rhinitis. Panel A shows treatment DMC overlaps with allergy DMCs. Panel B shows consensus module gene associated DMC overlaps with allergy DMCs. The number of overlapping DMCs is shown at the bottom of the bars (n). A one-sided Fisher’s exact test was used for the enrichment analyses. Computed p values represent the enrichment significance, while the fold enrichment illustrates how over-represented the overlap is, compared to what would be expected by chance for the given background