Lactobacillus saerimneri and Lactobacillus ruminis: novel human-derived probiotic strains with immunomodulatory activities

Abstract

Human-derived lactobacilli were isolated from fecal samples of healthy volunteers. Forty-six isolates from different volunteers were selected and investigated for their immunomodulatory properties. Conditioned medium from each isolate was assessed for its effect on tumor necrosis factor (TNF) production in lipopolysaccharide-activated THP-1 monocytes. Of 46 Lactobacillus isolates, 12 significantly inhibited TNF production in varying magnitude. Lactobacillus strain TH58 displayed the most potent TNF-inhibitory activity (70% inhibition). In contrast, Lactobacillus strain TH14 exhibited immunostimulatory property by activating TNF production in THP-1 monocytes. Lactobacillus TH14 induced nuclear factor-kappaB (NF-kappaB) activation in the absence of lipopolysaccharide stimulation, whereas Lactobacillus TH58 had no effect on NF-kappaB signaling, irrespective of lipopolysaccharide stimulation. Strain TH58 was identified as Lactobacillus saerimneri and strain TH14 as Lactobacillus ruminis by sequence analysis of their 16S rRNA genes. This is the first report of a human isolate of L. saerimneri with TNF-inhibitory activity and L. ruminis, an indigenous species to humans, with TNF stimulatory activity. Our data suggest the potential use of these two strains as immunoprobiotic candidates.

Fig. 1

Effects of Lactobacillus conditioned media on TNF production in LPS-activated THP-1 cells. THP-1 cells were coincubated with Lactobacillus conditioned media and E. coli - derived lipopolysaccharide. TNF secretions were determined by human TNF ELISA. The results were from three independent experiments in triplicate. MRS: deMan-Rogosa-Sharpe, media control; Asterisks denote significantly different from media control; ***, p<0.001; **, p<0.01; *, p<0.05; Error bars indicate standard deviations.

Fig. 2

Immunomodulatory effects of Lactobacillus TH14 and TH58 conditioned media on TNF production in THP-1 cells. Bioassays for TNF secretion were performed without or with LPS activation. The results represent three independent experiments. MRS: deMan-Rogosa-Sharpe, media control; Asterisks denote significantly different from media control; ***, p<0.001; **, p<0.01; Error bars indicate standard deviations.

Fig. 3

The viability of THP-1 cells determined by staining with Annexin V-FITC and Propidium Iodide (PI) followed by flow cytometric analysis. Viable cells are negative for both Annexin V-FITC and PI. Cells that are in early apoptosis are Annexin V-FITC positive and PI negative. Cells that are in late apoptosis or already dead are both Annexin V-FITC and PI positive. The percentages of cell viability under each plot are representative of three experiments. Control: RPMI cell culture medium; MRS: deMan-Rogosa-Sharpe, bacterial media control; TH58: Lactobacillus TH58 conditioned medium.

Fig. 4

Effect of Lactobacillus TH 14 conditioned medium on the proliferation of THP-1 cells. THP-1 cells were incubated with TH14 conditioned medium in the absence or presence of LPS. Cell suspensions were stained with trypan blue, counted on a hemocytometer and the number of viable cells in each sample was calculated. RPMI: cell culture medium; MRS: deMan-Rogosa-Sharpe, bacterial media control; TH14: Lactobacillus TH14 conditioned medium. The number of viable cells is shown as mean value with standard deviation from three different experiments.

Fig. 5

Effect on NF-κB activation by Lactobacillus TH14 and TH58 in THP-1 cells. THP-1 cells were incubated with TH14 or TH58 conditioned media in the absence or presence of LPS. Nuclear extracts were prepared and assayed for NF-κB levels. The results were from two independent experiments in triplicate. MRS: deMan-Rogosa-Sharpe, media control; Asterisk denotes significantly different from media control; ***, p<0.001; Error bars indicate standard deviations.