Abstract
Rational probiotic combinations should be supported by evidence of enhanced effects of the combined formulation and validated by in vivo efficacy. In this study, we investigated the immunostimulatory potential of IBP35, a 1:1 combination of Lactiplantibacillus plantarum IDCC 3501 and Ligilactobacillus salivarius IDCC 3551, using in vitro assays and in vivo experiments. In RAW 264.7 macrophages, IBP35 significantly increased nitric oxide production in a dose-dependent manner, achieving levels 25%-43% higher than those induced by either strain alone (p < 0.01-0.001). IBP35 also increased TNF-α and IL-6 secretion by 2.0-2.5-fold compared with individual strains (p < 0.001), indicating enhanced macrophage activation. IBP35 was evaluated using a cyclophosphamide (CPA)-induced immunosuppressed mouse model. CPA administration markedly reduced splenic natural killer (NK) cell activity (46.43% ± 10.64% vs. 62.47% ± 9.53% in normal controls, p < 0.05), whereas high-dose IBP35 significantly increased NK cell activity compared with the CPA group, reaching a level comparable to the normal control group (61.3% ± 7.93%, p < 0.05 vs. CPA). In CPA-treated mice, IBP35 significantly enhanced ConA-stimulated cytokine production, increasing IFN-γ, IL-2, and TNF-α levels by 2.0-3.0-fold relative to the CPA group (p < 0.01-0.001). Macrophage lysosomal enzyme activity was also significantly elevated (124% of control, p < 0.001). Histological analysis revealed that IBP35 attenuated CPA-induced splenic atrophy and lymphoid depletion. In healthy mice, IBP35 modulated immune responses without inducing excessive inflammation. Collectively, these results indicate that IBP35 modulates both innate and adaptive immune readouts and supports immune function under immunosuppressed conditions.