Methylated CpG ODNs from Bifidobacterium longum subsp. infantis Modulate Treg Induction and Suppress Allergic...

In our previous studies, methylated CpG oligodeoxynucleotides (ODN) derived from Bifidobacterium longum subsp. infantis have demonstrated immunomodulatory effects through the induction of regulatory T cells (Tregs). To define the structural determinants underlying this effect, we synthesized four CpG ODNs varying in methylation degree, CpG motif placement, and backbone length. These include (1) ODN-A (2m-V1), a 20-nucleotide CpG oligodeoxynucleotide incorporating two 5-methylcytosines at positions 4 and 12 within centrally placed CpG motifs; (2) ODN-B (um-V2), a 20-nucleotide CpG oligodeoxynucleotide with a backbone structure identical to ODN-A but unmethylated; (3) ODN-C (2m'-V3), a 20-nucleotide CpG oligodeoxynucleotide with a backbone structure identical to ODN-A, but with two 5-methylcytosines shifted to positions 7 and 15; (4) ODN-D (3m-V4), a 27-nucleotide CpG oligodeoxynucleotide with an extended backbone structure, this time with three 5-methylcytosines at positions 3, 11, and 19. Using a murine model of an OVA-induced allergy, we show that methylated ODN-A (2m-V1) and ODN-D (3m-V4) markedly reduce serum anti-OVA IgE, clinical symptoms, eosinophilic infiltration, and Th2/Th17 responses, while promoting splenic Treg expansion and IL-10 production. In contrast, unmethylated ODN-B (um-V2) and a positionally altered methylated ODN-C (2m'-V3) both failed to suppress allergic inflammation, and, in contrast, enhanced the Th2/Th17 response and induced robust in vitro Toll-like receptors TLR7/8/9 expression in native splenocytes. These findings suggest that both methylation and motif architecture critically influence the immunologic profile of CpG ODNs. Our results provide mechanistic insights into CpG ODN structure/function relationships and support the therapeutic potential of select methylated sequences for restoring immune tolerance in allergic diseases.