Probiotic Lactobacillus Strains Stimulate the Inflammatory Response and Activate Human Macrophages

Abstract

Lactobacilli have been shown to promote health functions. In this study, we analyzed the mechanism by which four different strains of probiotics affected innate immunity, such as regulation of ROS, cytokines, phagocytosis, bactericidal activity, signaling by NF-κB pp65, and TLR2 activation. The production of ROS was dependent on the concentration and species of Lactobacillus. The results obtained from the tested strains (Lactobacillus rhamnosus GG, L. rhamnosus KLSD, L. helveticus IMAU70129, and L. casei IMAU60214) showed that strains induced early proinflammatory cytokines such as IL-8,TNF-α, IL-12p70, and IL-6. However, IL-1β expression was induced only by L. helveticus and L. casei strains (after 24 h stimulation). Phagocytosis and bactericidal activity of macrophages against various pathogens, such as S. aureus, S. typhimurium, and E. coli, were increased by pretreatment with Lactobacillus. The nuclear translocation NF-κB pp65 and TLR2-dependent signaling were also increased by treatment with the probiotics. Taken together, the experiments demonstrate that probiotic strains of Lactobacillus exert early immunostimulatory effects that may be directly linked to the initial inflammation of the response of human macrophages.

Figure 1

ROS production by human macrophages in response to probiotic heat-killed lactobacillus was measured by luminol-enhanced chemiluminescence. (a) Mixtures were prepared using 1 ml of Hank's solution, containing luminol 0.8 × 10⁻⁴ M, 4 U HRP, and lactobacillus bacteria at different MOI (1 : 1 and 1 : 500; bacteria : macrophages). The kinetics of the oxidative response induced in human macrophages by lactobacillus was monitored (b). The values shown represent four experiments that were performed in duplicate. Asterisks indicate the level of statistical significance between conditions. Significant difference: p < 0.05.

Figure 2

Cytokine production in human macrophages that were stimulated using heat-killed lactobacillus. The supernatants of stimulated macrophages were analyzed using ELISA to determine the level of expression of the cytokines IL-8, TNF-α, IL-6, IL-12, IL-1β, and IL-10. The macrophages were treated with zymosan (10 μg/ml) or heat-killed strains of lactobacillus at a MOI of 1 : 500 or untreated (s/e). Cytokine concentrations were determined at 6 h (□) and 24 h (■) after stimulation. The results are shown as the means ± of the standard deviations and are representative of four independent experiments. Significance was set at p < 0.05 (∗) in the comparison between 6 and 24 h of stimulation. p > 0.05 (∗∗).

Figure 3

Effect of heat-killed lactobacillus on phagocytosis in human macrophages. Cells (1 × 10⁴) were treated with heat-killed strains of lactobacillus at a MOI of 1 : 500 for 1 h before zymosan particles, S. aureus, S. typhimurium, or E. coli (each at a MOI of 1 : 10) were added. Phagocytosis was allowed to proceed for 1 h at 37°C in an atmosphere containing 5% C0₂, before phagocytosis was terminated. The cells were then fixed, and the nuclei were stained with DAPI. Macrophages, untreated (a) and treated (b) cells. Fluorescence photomicroscopy in macrophages treated with different stimuli was performed using an epifluorescence Zeiss Axioskop2 microscope equipped with a Zeiss Axiocam (Zeiss AG, Oberkochen, DE). This figure shows the representative results with the strain (L. casei IMAU60214). (c) Heat-killed lactobacillus enhanced the phagocytic index of human macrophages. The ingestion of cells was determined in macrophages that were incubated in medium alone or preincubated with lactobacillus strains. The following formula was used: the percentage of cells undergoing phagocytosis × the number of particles per cells. The cells were incubated with zymosan, S. typhimurium, S. aureus, and E. coli, as indicated. Asterisks indicate a significant difference between conditions (p > 0.05).

Figure 4

Effect of lactobacillus on the bacterial activity of macrophages against the pathogens S. typhimurium, S. aureus, and E. coli. Human macrophages were infected with (a) S. typhimurium, (b) S. aureus, or (c) E. coli and then incubated for 90 min at 37°C to allow the bacteria to be internalized. External bacteria were then killed by applying gentamicin for 30 min at 37°C. Samples were taken immediately before gentamicin treatment (0 h) and every 120 min thereafter to determine viable counts following Triton X-100 lysis. The results are expressed as CFU per milliliter with means ± SD from no fewer than four separate experiments that were performed in duplicate using macrophages obtained from different human donors. Asterisks indicate a significant difference between conditions (p < 0.05).

Figure 5

Immunofluorescence analysis of NF-kB activation. Human macrophages were stimulated for 30 min with heat-killed lactobacillus at a MOI of 1 : 500, zymosan (positive control) or left unstimulated. (a) Nuclear translocation was defined as blue fluorescence (Hoechst staining), and the NF-kB pp65 subunit was identified using green fluorescence (Dylight 488). (b) The percentage of nuclei that were positive for pp65. The results represent three independent experiments.

Figure 6

Transfection assay of HEK293-hTLR2 cells showing IL-8 was secreted in response to strains of lactobacillus. The recognition of lactobacillus strains was evaluated using a model in which HEK293-hTLR2 cells were transfected, and then blocking assays were performed using anti-hTLR2-IgA antibodies at a concentration of 10 μg/ml. p < 0.05 (∗).