{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,16]],"date-time":"2026-03-16T23:07:40Z","timestamp":1773702460130,"version":"3.50.1"},"reference-count":27,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2025,7,14]],"date-time":"2025-07-14T00:00:00Z","timestamp":1752451200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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\u2019-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\u2019-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.<\/jats:p>","DOI":"10.3390\/ijms26146755","type":"journal-article","created":{"date-parts":[[2025,7,15]],"date-time":"2025-07-15T09:45:52Z","timestamp":1752572752000},"page":"6755","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Methylated CpG ODNs from Bifidobacterium longum subsp. infantis Modulate Treg Induction and Suppress Allergic Response in a Murine Model"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3272-2513","authenticated-orcid":false,"given":"Dongmei","family":"Li","sequence":"first","affiliation":[{"name":"Department of Microbiology & Immunology, Georgetown University Medical Center, Washington, DC 20057, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Idalia","family":"Cruz","sequence":"additional","affiliation":[{"name":"Department of Oncology, Animal Models, Shared Resources, Georgetown University Medical Center, Washington, DC 20057, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-4135-468X","authenticated-orcid":false,"given":"Samantha N.","family":"Peltak","sequence":"additional","affiliation":[{"name":"Department of Microbiology & Immunology, Georgetown University Medical Center, Washington, DC 20057, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5735-9485","authenticated-orcid":false,"given":"Patricia L.","family":"Foley","sequence":"additional","affiliation":[{"name":"Department of Oncology, Animal Models, Shared Resources, Georgetown University Medical Center, Washington, DC 20057, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5038-7202","authenticated-orcid":false,"given":"Joseph A.","family":"Bellanti","sequence":"additional","affiliation":[{"name":"Department of Microbiology & Immunology, Georgetown University Medical Center, Washington, DC 20057, USA"},{"name":"Department of Pediatrics, Georgetown University Medical Center, Washington, DC 20057, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,7,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"546","DOI":"10.1038\/374546a0","article-title":"CpG motifs in bacterial-DNA trigger direct B-cell activation","volume":"374","author":"Krieg","year":"1995","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1016\/j.addr.2008.12.003","article-title":"CpG oligonucleotides as immunotherapeutic adjuvants: Innovative applications and delivery strategies","volume":"61","author":"Salem","year":"2009","journal-title":"Adv. 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