ABSTRACT
Introduction: Atopic dermatitis (AD) is an inflammatory skin disease which may arise due to the imbalance between Type 1 T helper (Th1), Type 2 T helper (Th2), regulatory T cell (Treg) cell-related immune responses. Evidence suggests that appropriate stimulation with probiotics may correct the skewed immune response in children with AD. The aim of this study was to determine the effects of the yoghurt culture lactobacillus delbrueckii subsp. bulgaricus on the secretion of Th1/Th2/Treg-type cytokines by peripheral blood mononuclear cells (PBMCs) from children with AD.
Methods: L. Bulgaricus was cultivated on MRS agar broth. The PBMCs from 20 children with AD were separated by Ficoll-Hypaque centrifugation and co-cultured with different intensities of ultraviolet to kill bacteria in RPMI-1640, plus 10% fetal calf serum for 48/72 hours. The levels of interleukin (IL)-10, IL-4, IL-12, and interferon gamma (IFN-γ) were measured in the supernatant of PBMCs by enzyme-linked immunosorbent assay. RNA extraction and real time reverse transcription polymerase chain reactions were used to measure Foxp3 mRNA expression.
Results: L. bulgaricus significantly upregulated the secretion of IL-10, IL-12, and IFN-γ, whereas it reduced the secretion of IL-4 by PBMCs at both incubation times of 48 and 72 hours and both bacteria-to-PBMCs ratios 100:1/50:1, compared to controls (p<0.05). There were no significant differences between incubation times of 48 and 72 hours regarding the secretion levels of IL-12, IFN-γ, and IL-4; however, the secretion level of IL-10 by L. bulgaricus-stimulated PBMCs at the incubation time of 72 hours, and in the presence of a bacteriato-PBMCs ratio of 100:1, was significantly higher than the incubation time of 48 hours and in the presence of a bacteria-to-PBMCs ratio of 50:1 (p<0.0001 and p<0.001, respectively). Neutralisation of IL-10 by the anti-IL-10 antibody increased the secretion levels of IL-12 and IFN-γ at the incubation time of 72 hours, compared to 48 hours, and at a bacteria-to-PBMCs ratio of 100:1, compared to 50:1. The expression level of Foxp3 mRNA by L. bulgaricus-stimulated PBMCs, as correlated to IL-10 secretion, at an incubation time of 72 hours and in the presence of a bacteria-to-PBMCs ratio 100:1, was significantly higher than 48 hours and in the presence of a bacteria-to-PBMCs ratio of 50:1 (p<0.01 and p<0.05, respectively).
Discussion: These data show that L. bulgaricus may modulate the secretion of Th1, Th2, and Treg-related cytokines in AD patients. It seems that the activity of Treg cells, after 72 hours of stimulation or at a bacteria-to-PBMCs ratio of 100:1 by L. bulgaricus, dominates the effector T cells. The Th1 and Th2 cells do not secrete more cytokines after the 72 hours incubation time and at a bacteria-to-PBMCs ratio of 100:1, compared to 48 hours and a bacteria-to-PBMCs ratio of 50:1. Therefore, the potential therapeutic use of L. bulgaricus for treatment of AD should be considered for further investigation.
