Treg-inducing capacity of genomic DNA of Bifidobacterium longum subsp. infantis

Abstract

Background: Allergic and autoimmune diseases comprise a group of inflammatory disorders caused by aberrant immune responses in which CD25⁺ forkhead box P3-positive regulatory T cells (Treg) cells that normally suppress inflammatory events are often poorly functioning. This has stimulated an intensive investigative effort to find ways of increasing Tregs as a method of therapy for these conditions. Commensal microbiota known to have health benefits in humans include the lactic acid-producing, probiotic bacteria B. longum subsp. infantis and Lactobacillus rhamnosus. Mechanistically, several mechanisms have been proposed to explain how probiotics may favorably affect host immunity, including the induction of Tregs. Analysis of emerging data from several laboratories, including our own, suggest that DNA methylation may be an important determinant of immune reactivity responsible for Treg induction. Although methylated CpG moieties in normal mammalian DNA are both noninflammatory and lack immunogenicity, unmethylated CpGs, found largely in microbial DNA, are immunostimulatory and display proinflammatory properties. Objective: We hypothesize that microbiota with more DNA methylation may potentiate Treg induction to a greater degree than microbiota with a lower content of methylation. The purpose of the present study was to test this hypothesis by studying the methylation status of whole genomic DNA (gDNA) and the Treg-inducing capacity of purified gDNA in each of the probiotic bacteria B. longum subsp. infantis and L. rhamnosus, and a pathogenic Escherichia coli strain B. Results: We showed that gDNA from B. longum subsp. infantis is a potent Treg inducer that displays a dose-dependent response pattern at a dose threshold of 20 µg of gDNA. No similar Treg-inducing responses were observed with the gDNA from L. rhamnosus or E. coli. We identified a unique CpG methylated motif in the gDNA sequencing of B. longum subsp. infantis which was not found in L. rhamnosus or E. coli strain B. Conclusion: Although the literature indicates that both B. longum subsp. infantis and L. rhamnosus strains contribute to health, our data suggest that they do so by different mechanisms. Further, because of its small molecular size, low cost, ease of synthesis, and unique Treg-inducing feature, this methylated CpG oligodeoxynucleotide (ODN) from B. longum would offer many attractive features for an ideal novel therapeutic vaccine candidate for the treatment of immunologic diseases, such as the allergic and autoimmune disorders, in which Treg populations are diminished.

Figure 1.

Flow cytometric analysis of the effects of gDNA on T regulatory cell induction. Representative flow cytometric assessments expressed as percentages of CD4⁺CD25^high/FOXP3⁺ (T regulatory) cells induced by different concentrations of gDNAs. (A) Three controls for gDNA-Treg response: un-activated CD4⁺ cells (A.1); the CD4⁺ cells activated by αCD3/αCD28 (A.2) as control for CD4⁺ cells treated with gDNAs alone; and αCD3/αCD28 + IL-2/TGF-β control (A.3) for gDNA stimuli with IL-2/TGFβ. (B) Sampled CD4⁺CD25^high/FOXP3⁺ percentages with high dose (50 µg) and low dose (0.08 µg) of each bacterial gDNA stimulus without two cytokines (top panel) and with two cytokines (bottom panel). (C and D) The overall dose-response results of Treg induction by bacterial gDNA stimulation. The bacterial gDNA-Treg response (percentage) at gDNA concentration ranging from 0.0625 µg ∼ 50 µg without (C) and with IL-2/TGF-β (D) were averaged from three individual CD4⁺ samples derived from three healthy subjects (n = 3). B. longum represents B. longum subsp. infantis in all figures. The dash lines in C & D represent baseline values of anti-CD3 and anti-CD28 antibody (AB) and AB+ cytokine (AB-CK) treatments, respectively. Statistical analyses versus AB co. (C) and AB-CK co. (D) were performed by two-ANOVA analysis. Data are expressed as mean ± SEM; “*”: p < 0.05; “**”: p < 0.01 and “***”: p < 0.001.

Figure 2.

Inhibitory effects of gDNAs on Treg cell differentiation (A) and CD4⁺ cell expansion (B). The total numbers of induced CD25^highFOXP3⁺ Treg cells and CD4⁺ cells are shown which resulted from bacterial gDNA (s) at different concentrations (n = 3). The gDNA concentrations and cytokines treatments used were the same as those shown in Fig. 1. The controls used for statistical analyses included gDNA alone and gDNA+ cytokines. Data were performed by two-ANOVA analysis and are expressed as mean ± SEM. “*”: p < 0.05; “**”: p < 0.01 and “***”: p < 0.001. C. Representative photomicrographs showing CD4⁺ cell expansion in response to high (50 µg), intermediate (20 µg) and low (0.08 µg) doses of each bacterial gDNA on day 7 cultures.

Figure 3.

The percentages of stimulated CD25^highFOXP3⁺ cells following single or double stimulation with B. longum subsp. infantis gDNA with or without IL-2/TGF-β. (A) Representative flow cytometric analyses of 20 or 50 µg of B. longum gDNA at single (on day 1) or double stimulation (on day 1 and day 4) were compared. (B) The double gDNA treatment at 20 µg of B. longum gDNA (2^nd_DNA) induced less Treg than single DNA (1^st_DNA), resulted in no significance versus AB control in terms of Treg percentages (n = 3). With IL-2/TGF-β, 50 µg of B. longum gDNA (2^nd_DNA_CK) shown the same inhibitory effects as 20 µg of B. longum gDNA observed above. Data are represented as mean ± SEM; statistical analyses were performed by one-tail t-test. *p < 0.05 and **p < 0.01. C. Representative photomicrographs showing morphological characteristics of gDNA-treated CD4⁺ cells following one or two B. longum gDNA stimulations with or without IL-2/TGF-β treatment.

Figure 4.

Results of the genomic DNA sequencing studies of B. longum subsp. Infantis, L. rhamnosus and E. coli genomes. (A) Schematic representation of the molecular structures of common base modifications in bacterial DNAs. (B) Scatter plot results of DNA modifications in L. rhamnosus (B1 & B2) and E. coli (B3 & B4) with per-strand coverage (left panel) and with distribution of modified DNA bases in variable modQV (modification quality value) (right panel). At each genomic position, modQV was computed as the −10 log (P-value) for each modified base position, based on the distributions of interpulse durations (IPD ratios) versus to in silico kinetic reference values from all reads covering this position. The color specified the nucleotide bases that have been detected positive for the modification. Adenine is colored in red, guanine is colored in blue, cytosine is colored in green, and thymine is colored in purple. (C) Scatter plot results of DNA modifications (C1 & C2), m5C motif analysis (C3) and distribution of identified m5C motifs in B. longum (C4). The descending greyish marker on the left of the Fig. indicates the possible range where m5C and m4C contexts are located. (C4) The distribution of three types of m5C motifs (RGCGGCGCC, YGCGGCGCC and CCCTCGAG) are displayed by colored dots.