Lactobacillus acidophilus attenuates Salmonella-induced intestinal inflammation via TGF-β signaling

Abstract

Background: Salmonella is a common intestinal pathogen that causes acute and chronic inflammatory response. Probiotics reduce inflammatory cytokine production and serve as beneficial commensal microorganisms in the human gastrointestinal tract. TGF-β (transforming growth factor β)/SMAD and NF-κB signaling play important roles in inflammation in intestinal cells. However, the involvement of the signaling in regulating inflammation between Salmonella and probiotics is not fully understood.

Methods: L. acidophilus and prebiotic inulin were used to treat human intestinal Caco-2 cells prior to infection with Salmonella. The cells were harvested to examine the cytokines and MIR21 expression with immunoblotting and real-time PCR. NF-κB and SMAD3/4 reporter vectors were transfected into cells to monitor inflammation and TGF-β1 signaling, respectively.

Results: In this study, we showed that the probiotic L. acidophilus decreased Salmonella-induced NF-κB activation in human intestinal Caco-2 cells. Expression of the inflammatory cytokines, TNF-α and IL-8, in L. acidophilus-pretreated cells was also significantly lower than that in cells infected with Salmonella alone. Moreover, TGF-β1 and MIR21 expression was elevated in cells pretreated with L. acidophilus or synbiotic, a combination of inulin and L. acidophilus, compared to that in untreated cells or cells infected with S. typhimurium alone. By contrast, expression of SMAD7, a target of MIR21, was accordingly reduced in cells treated with L. acidophilus or synbiotics. Consistent with TGF-β1/MIR21 and SMAD7 expression, SMAD3/4 transcriptional activity was significantly higher in the cells treated with L. acidophilus or synbiotics. Furthermore, TGF-β1 antibody antagonized the SMAD3/4 and NF-κB transcriptional activity modulated by L. acidophilus in intestinal cells.

Conclusion: Our results suggest that the TGF-β1/MIR21 signaling pathway may be involved in the suppressive effects of L. acidophilus on inflammation caused by S. typhimurium in intestinal Caco-2 cells.

Fig. 1

Effects of L. acidophilus on S. typhimurium-induced NF-κB activation. a Human intestinal Caco-2 cells were transfected with a luciferase reporter vector for NF-κB or CMV (normalization control) overnight and then infected with various concentrations of S. typhimurium as indicated, ranging from 10⁴ to 10¹⁰ CFU/ml in antibiotic-free DMEM for 1 h at 37 °C. The cells were recovered with DMEM media containing gentamicin (50 μg/ml) and NF-κB transcriptional activity was measured at 6 or 24 h post-infection. b For treatment with L. acidophilus, transfected cells were treated with 1 % inulin or L. acidophilus (L. acidophilus:S. typhimurium = 2:1) or synbiotics (L. acidophilus and 1 % inulin) 1 h prior to infection of S. typhimurium as mentioned above. The cells were added with DMEM medium containing D-luciferin and antibiotic at 6 h post-infection to measure NF-κB activity. The data were analyzed with Prism 5, and the results are shown as the means ± SEM from three independent experiments

Fig. 2

Effects of L. acidophilus and S. typhimurium on IL-8 and TNF-α mRNA expression. Human intestinal Caco-2 cells were treated with 1 % inulin or L. acidophilus (L. acidophilus:S. typhimurium = 2:1) or synbiotics (L. acidophilus and 1 % inulin) 1 h prior to infection with S. typhimurium in DMEM without antibiotic for 1 h. The cells were harvested at 6 h post-infection for mRNA isolation. The isolated mRNA was further used to determine the expression of (a) IL-8 and (b) TNF-α by quantitative PCR (qPCR). The data are shown as the means ± SEM from three independent experiments

Fig. 3

Effects of L. acidophilus and S. typhimurium on TGF-β and MIR21 expression. Human intestinal Caco-2 cells were treated with 1 % inulin or L. acidophilus (L. acidophilus : S. typhimurium = 2:1) or synbiotics 1 h prior to infection with S. typhimurium in DMEM without antibiotic for 1 h. The cells were harvested at 6 h post-infection for protein extraction. a The cells were lysed to extract the proteins and assess TGF-β expression by immunoblotting. b The treated cells were harvested at 6 h post-infection for total RNA isolation. The isolated RNA was further used to determine the expression of MIR21 by quantitative PCR (qPCR). The results are from three independent experiments, and the data are shown as the means ± SEM. NS: not significant

Fig. 4

Effects of L. acidophilus and S. typhimurium on SMAD7 expression and SMAD3/4 transcriptional activity. a Human intestinal Caco-2 cells were treated with 1 % inulin or L. acidophilus (L. acidophilus:S. typhimurium = 2:1) or synbiotics (L. acidophilus and 1 % inulin) 1 h prior to treatment with S. typhimurium in DMEM without antibiotic for 1 h. The cells were harvested at 6 h post-infection for protein extraction. SMAD7 protein expression was determined by immunoblotting. b Human intestinal Caco-2 cells were transfected with luciferase reporter plasmid for SMAD3/4 or CMV (normalization control) overnight and then treated or infected as above. The cells were added DMEM medium containing D-luciferin at 6 h post-infection to read the signal with a Luminometer. The quantitative results are shown as the means ± SEM from three independent experiments. NS: not significant

Fig. 5

Effects of TGF-β on L. acidophilus regulated SMAD3/4 and NF-κB transcriptional activity. Human intestinal Caco-2 cells were transfected with luciferase reporter plasmid for a SMAD3/4 or b NF-κB overnight and then treated with L. acidophilus (L. acidophilus:S. typhimurium = 2:1) or L. acidophilus mixed with anti-TGF-β antibody (1 μg/ml) 1 h prior to infection with S. typhimurium. The cells were recovered and added DMEM medium containing D-luciferin at 6 h post-infection to read the signal with a Luminometer. The results are normalized with the cells harboring luciferase constitutively expressed vector (CMV) and are shown as the means ± SEM from three independent experiments