Consumption of Bifidobacterium animalis subsp Consumption of Bifidobacterium animalis subsp. lactis BB-12 reduces TNF-α secretion from LPS-stimulated PBMCs, but does not alter serum TNF-α levels Hannah VanEvery1, Huicui Meng1, Jennifer A. Fleming1, Yujin Lee1, Zhaoyong Ba2, Emily J. Furumoto2, Robert F. Roberts2, Penny M. Kris-Etherton1, Connie J. Rogers1 1Department of Nutritional Sciences, 2Department of Food Science, Pennsylvania State University, University Park, PA, 16802. Abstract Results Results Increasing evidence suggests that probiotic bacteria can modulate inflammatory responses. However the immune response varies by species and strain of organism, as well as delivery matrix of the probiotic. Furthermore the tissue compartment and assay method by which inflammatory mediators are quantified in clinical trials is a debated issue. The goal of the current study was to evaluate the effect of one probiotic species, Bifidobacterium animalis subsp. lactis BB-12 at a dose of log 10 ± 0.5 CFUs/day on both plasma- and in vitro cultured peripheral blood mononuclear cells (PBMC)-derived inflammatory mediators in a partially blinded, 4-period crossover, free-living study. Healthy adults (n=30) aged 18-40 years were recruited, and received 4 treatments in a random order: 1) yogurt smoothie alone; smoothie with organism added 2) before or 3) after fermentation, or 4) organism given in capsule form. At baseline and after each treatment, plasma was collected and PBMCs were isolated and stimulated in vitro with LPS as an inflammatory stimulus. Inflammatory cytokines were quantified in each compartment at baseline and after each treatment. Participants who consumed yogurt smoothies with BB-12 added post-fermentation had a significant reduction in TNF-α secretion from LPS-stimulated PBMCs compared to baseline (p=0.039). However, there was no treatment effect observed in plasma TNF-α levels. Additionally, plasma TNF-α levels were not correlated with TNF-α produced by cultured PBMCs in the presence of stimulation. These results demonstrate that quantification of inflammatory mediators in the plasma of healthy adults may not be adequate to detect the effect of probiotic consumption on inflammatory responses. The assessment of a nutritional intervention on in vitro cultured PBMCs in response to LPS stimulation may be a more sensitive assay method to detect probiotic-induced changes in inflammatory responses. These findings demonstrate that selection of the tissue compartment and the assay method to evaluate inflammatory response is a critical variable in clinical trial design. TNF-α Secretion from BB-12 or LPS stimulated PBMCs Correlation Between In Vitro and In Vivo TNF-α p = 0.0032 r = -0.3161 p = 0.1627 * p = 0.0328 r = 0.1676 p = 0.4929 * Aims To determine if BB-12 will alter innate and/or adaptive immune responses of human subjects. To determine the appropriate tissue compartment and assay to quantify changes in inflammatory mediators in this model. Figure 3. The correlation between plasma TNF-α and TNF-α secreted from LPS-stimulated PBMCs. Serum TNF-α data was not normally distributed, so it was transformed using the logarithm function, which yielded a normally distributed data set. No significant correlation was found using the Pearson Correlation Coefficient test at baseline (A) or after treatment with YS-Post (B). Study Design Figure 1. The effect of treatment on TNF-α secretion from BB-12 or LPS stimulated PBMCs. PBMCs were stimulated with 10μg/ml BB-12 or LPS, and culture supernatants were collected post 4h stimulation. (A) TNF-α secretion from BB-12 stimulated PBMCs and (B) TNF-α secreted from LPS stimulated PBMCs were assessed by ELISA. Data are presented as LSMEANS±SEM (N=30). Repeated measure ANOVA demonstrated significant differences in TNF-α secretion from BB-12 stimulated PBMCs (p=0.0032) and TNF-α (p=0.0328) secretion from LPS stimulated PBMCs among baseline and treatments. Bonferroni’s post-hoc test found lower TNF-α (p=0.0025) secretion from LPS stimulated PBMCs following YS-Post treatment compared to baseline. Summary Subjects who consumed a yogurt smoothie with BB-12 added post fermentation had significantly lower TNF-α from LPS stimulated PBMCs. Plasma TNF-α was not correlated with TNF-α from LPS stimulated PBMCs at any time point. No treatment effect was observed on plasma levels of TNF-α. Randomized, 4-period cross-over free-living design A B C D Gut transit time ≥ 24h 18-40 years old BMI:22-40 kg/m2 Screening Period 1 Period 2 Period 3 Period 4 All subjects free-living (4 weeks) Randomization Sample collection 2 week compliance break change to next treatment Treatment groups include: A: yogurt smoothie without added BB-12 (YS); B: yogurt smoothie with BB-12 added post fermentation (YS-Post); C: yogurt smoothie with BB-12 added pre-fermentation (YS-Pre); D: a tablet containing BB-12 (TAB). Immune Outcomes: TNF-α secretion from PBMCs in response to LPS stimulation. Serum TNF-α quantification. Evaluation of treatment effects on TNF-α. Comparison of these two assay methods and tissue compartments in regards to TNF-α quantification. 4 weeks Plasma TNF-α Before and After YS-Post Treatment Conclusions p = 0.2509 Probiotic consumption may alter inflammatory mediators, however, these effects may not be robust enough to detect in plasma. The assessment of probiotic consumption on in vitro cultured PBMCs in response to LPS stimulation may be a more sensitive and accurate assay method to detect probiotic-induced changes in inflammatory responses. These findings demonstrate that selection of the tissue compartment and the assay method to evaluate inflammatory response is a critical variable in clinical trial design. Acknowledgements Funding provided by the Dairy Research Institute (CJR) and the CTSI TL1 Training Program (HLV). Figure 2. The effect of treatment on serum TNF-α. Serum was isolated from peripheral blood samples drawn at baseline and after treatment with YS-Post. TNF-α was assed by Quest Diagnostics using a high-sensitivity ELISA assay. Serum TNF-α data was not normally distributed, so it was transformed using the logarithm function, which yielded a normally distributed data set. A paired t-test demonstrated no significant differences in serum TNF-α among baseline and post YS-Post treatment. Contact information: Connie Rogers Email: cjr102@psu.edu Tel: 814-867-3716 Hannah VanEvery Email: hqv5028@psu.edu Tel: 515-231-5640