Dose-response studies of methylated and nonmethylated CpG ODNs from Bifidobacterium longum subsp. infantis for optimizing Treg cell stimulation.

Abstract

Background: Allergen immunotherapy (AIT) is the most effective treatment for atopic allergic diseases, aiming to induce regulatory T cells (Treg) that modify the immune response to specific allergens, which leads to long-term tolerance and reduced symptoms. Enhancing Treg activity is crucial for improving immunotherapy outcomes. In a previous murine model study, we examined the effects of a synthetic methylated DNA oligodeoxynucleotide (ODN) from the Bl-T2 m5C motif of Bifidobacterium longum subsp. infantis. The ODN that contains the methylated BI-T2 m5C motif (methylated ODNA) sequence conjugated with ovalbumin induced Treg production, whereas ODN that contains the unmethylated BI-T2 m5C motif (unmethylated ODNB) induced proinflammatory responses, which demonstrated the potential of methylated ODNs for AIT. Objective: In building on these results, this study explored the effects of methylated and nonmethylated DNA motifs from B. longum subsp. infantis on inflammation and Treg induction, while investigating the dose-response relationships of methylated Cytosine-phosphate-Guanine (CpG) ODNs for optimal Treg stimulation in clinical applications. Methods: Serum levels of IL-17A, IL-4, IL-10, and transforming growth factor beta (TGF-β) were measured by enzyme linked immunosorbent assay (ELISA), and flow cytometry assessed splenic Treg populations in BALB/c mice receiving graded doses of methylated or unmethylated ODNs. Mice were immunized intraperitoneally with a single 100-μg dose (plan A) or multiple 25 μg (plan B) or 100 μg (plan C) doses. Calf thymic DNA served as a positive control, with phosphate-buffered saline solution and alum as negative controls. Results: Methylated ODNs significantly increased CD25⁺FOXP3⁺ Tregs compared with unmethylated ODNs and controls. Plan A (100 μg) elevated serum IL-10, which indicated effective Treg induction, whereas plan B (four 25 μg doses) did not activate Tregs. Plan C (multiple 100 μg doses) reduced Treg responses, which highlighted a critical dosing threshold for optimal Treg induction. Conclusion: This study demonstrated the potential of methylated DNA motifs as therapeutic agents in AIT. The dose-response relationships of methylated CpG ODNs from B. longum pave the way for clinical applications that target Treg activity in allergic diseases.