Lactobacillus rhamnosus GG Activation of Dendritic Cells and Neutrophils Depends on the Dose and Time of Exposure

Abstract

This study evaluates the ability of Lactobacillus rhamnosus GG (LGG) to activate DC and neutrophils and modulate T cell activation and the impact of bacterial dose on these responses. Murine bone marrow derived DC or neutrophils were stimulated with LGG at ratios of 5 : 1, 10 : 1, and 100 : 1 (LGG : cells) and DC maturation (CD40, CD80, CD86, CD83, and MHC class II) and cytokine production (IL-10, TNF-α, and IL-12p70) were examined after 2 h and 18 h coculture and compared to the ability of BCG (the present immunotherapeutic agent for bladder cancer) to stimulate these cells. A 2 h exposure to 100 : 1 (high dose) or an 18 h exposure to 5 : 1 or 10 : 1 (low dose), LGG : cells, induced the highest production of IL-12 and upregulation of CD40, CD80, CD86, and MHC II on DC. In DCs stimulated with LGG activated neutrophils IL-12 production decreased with increasing dose. LGG induced 10-fold greater IL-12 production than BCG. T cell IFNγ and IL-2 production was significantly greater when stimulated with DC activated with low dose LGG. In conclusion, DC or DC activated with neutrophils exposed to low dose LGG induced greater Th1 polarization in T cells and this could potentially exert stronger antitumor effects. Thus the dose of LGG used for immunotherapy could determine treatment efficacy.

Figures

Figure 1

LGG and BCG induced dose and time dependent effects on DC cytokine production by direct or indirect stimulation via neutrophils. LGG was assessed at 5 : 1, 10 : 1, and 100 : 1 ratios and BCG at 5 : 1 ratios. (a) Production of IL-10, TNF-α, and IL-12p70 after 18 h of continuous coculture of DCs with LGG/BCG (white bar), 2 h of exposure of DCs (black bar) or neutrophil (striped bar) to LGG/BCG, followed by 18 h of bacteria free incubation and 18 h of DC coculture with neutrophils pretreated with LGG/BGC for 2 h (crisscross bar). “∗” indicates a significant difference (p < 0.05) compared to low dose (5 : 1). For BCG “∗” indicates a significant difference from neutrophils and for TNF-α significance with respect to DC. Data are presented as the mean ± SEM. (b) Neutrophils were prestimulated with low (5 : 1) and high (100 : 1) dose of LGG for 2 h before they were cocultured with DC for 18 h in the presence and absence of a COX-2 inhibitor, NS398 (crisscross bar); COX-2 inhibitor solvent control (black bar); IL-10 neutralizing antibody (IL-10 Ab) (striped bar); and the isotype control for the antibody (double striped bar). The impact on PGE2, IL-10, and IL-12p70 secretion is shown. “†” indicates a significant difference from the respective controls (p < 0.05). Data are presented as the mean ± SEM.

Figure 2

T cell activation is dependent on the dose of LGG used to stimulate DC or neutrophils. IFNγ (a) and IL-2 (b) production by T cells after 5 days of coculture with DC, neutrophil, or DC-neutrophil (stimulated with low or high dose of LGG for 2 h and then DC for 18 h). Data are presented as the mean ± SEM. “∗” indicates a significant difference compared to their respective no LGG controls (p < 0.05). “#” indicates significant difference between high and low dose of LGG treatment (p < 0.05).