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

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. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0546-x) contains supplementary material, which is available to authorized users.


Background
The human intestine houses a dense and diverse microbial community, which is estimated to contain at least 500 different species of bacteria. These microbes, collectively referred to as the commensal microbiota, play important roles in human health, such as processing of nutrients, regulation of fat storage [1,2], and protection against pathogens [3]. Salmonella typhimurium (S. typhimurium), an intestinal pathogenic bacterium, is one of the most common non-typhoidal Salmonella (NTS), which is the leading cause of acute food-borne disease [4]. Salmonellosis results in diarrhea, vomiting, and fever in the majority of people after infection. Approximately 400 persons die each year because of severe Salmonellosis, particular young children, the elderly, and the immunocompromised persons. S. typhimurium penetrates intestinal cells and macrophages to induce intestinal inflammation using two type III secretion systems (T3SSs) [5]. S. typhimurium induces NF-kB activation and the secretion of pro-inflammatory cytokines, such as interleuckin-8 (IL-8) [6] and tumor necrosis factor alpha (TNF-α) [7]. The excessive inflammatory response may help S. typhimurium eliminate other competitive microbiota in the host [8].
Although several studies demonstrated the suppression of inflammation using probiotics, the TGF-βmediated signaling caused by probiotics administration on S. typhimurium-induced inflammation in human intestinal cells remains unclear. In this study, we examined the role of TGF-β signaling molecules in human intestinal Caco-2 cells infected with S. typhimurium in the absence or presence of the probiotic L. acidophilus alone or combination with the prebiotic inulin, a non-digestible oligosaccharide that enhances the growth of probiotics. We found that TGF-β and MIR21 expression is induced, whereas SMAD7 expression was decreased in human colorectal Caco-2 cells pretreated with L. acidophilus compared to cells infected with S. typhimurium alone. SMAD3/4 transcriptional activity was further elevated in L. acidophilus-treated cells, consistent with reduced NF-kB transcriptional activity and inflammatory cytokines induction, including IL-8 and TNF-α. Therefore, our findings elucidate the effect of TGF-β signaling on probiotics or synbiotics, which may identify a new treatment strategy for inflammation caused by Salmonella infection using probiotics.

S. typhimurium culture and infection
The enteric pathogen S. typhimurium (BCRC 10747) was grown on a blood agar plate (BAP, trypticase soy agar with 5 % sheep blood) (CMP Ltd.) at 37°C overnight. The bacterial colonies were collected with disposable L-shaped cell spreaders in PBS (Biological Industries). The collected bacteria were washed, centrifuged at 1,5000 × g for 15 min at 4°C and resuspended in PBS. The concentration of S. typhimurium was measured using a spectrophotometer as the optical density (OD) at 600 nm. For bacterial infection, Caco-2 cells (1×10 6 cells/well) were incubated with various concentrations S. typhimurium, ranging from 1 × 10 4 CFU/ml to 1 × 10 10 CFU/ml in antibiotic-free DMEM for 1 h at 37°C. The cells were washed with PBS and recovered with DMEM media containing gentamicin (50 μg/ml, Sigma-Aldrich) for the following experiments.

Transfection and luciferase reporter assay
To test the SMAD and NF-κB transcriptional activity, the Caco-2 cells in a 384-well plate were transfected with 50 ng of plasmid containing NF-κB-responsive reporter vector pGL4.32 (Promega) or SMAD responsive reporter vector pGL4.48 (Promega), which contains the NF-κB and SMAD-binding sites, respectively. The pGL4.50 (Promega) constitutively expressing luciferase was used as a normalization control. At 20 h post transfection, the cells were infected with S. typhimurium in the absence of presence of probiotic or synbiotic for 1 h. The cells were washed once with PBS and recovered with DMEM media containing gentamicin (50 μg/ml, Sigma-Aldrich). The luminescence of the luciferase-based reporter was measured with DMEM medium containing 200 μM D-luciferin (Promega). The results were read using a Fluoroskan Ascent FL reader (Thermo Fisher Scientific).

Protein extraction and western blot
Human colorectal epithelia Caco-2 cells were briefly rinsed in PBS (Biological Industries) and lysed with RIPA buffer ( ). The cell lysates were centrifuged at 13200 × g for 30 min and the supernatant was collected to determine the protein concentration using Bradford Reagent (Sigma-Aldrich). The cell lysates were resolved by SDS-PAGE and transferred onto nitrocellulose membranes using a wet electrophoretic transfer system (Bio-Rad). The membrane was blocked with 5 % skim milk and then incubated with primary antibodies against SMAD7 (sc-11392) (Santa Cruz Biotech), TGF-β (abcam) and actin (Sigma-Aldrich) to determine the protein expression using the ChemiDoc XRS Imaging System (Bio-Rad) and analysis with Image Lab4.1 (Bio-Rad).

Data analysis
Data were statistically analyzed by Prism 5.0 (Graph-Pad) using one-way ANOVA followed by Tukey's multiple comparison test to compare the effects between each group. All results are expressed as the mean ± SEM from at least 3 individual experiments. The lev el of significance was set at P < 0.05 (P-value ≦ 0.05 considered significant (*), P-value ≦ 0.01 considered highly significant (**), and P-value ≦ 0.001 considered extremely significant (***)).

Results
L. acidophilus attenuates S. typhimurium-induced inflammatory response in Caco-2 cells NF-κB transcribes global genes involved in inflammation, such as TNF-α [22] and IL-8 [23]. To explore the effect of probiotic on S. typhimurium-induced inflammation, an NF-κB responsive reporter assay was used to optimize the infected concentration of S. typhimurium for human intestinal cells. Human intestinal Caco-2 cells were transfected with NF-κB responsive reporter vector for 16 h and then incubated with S. typhimurium, ranging from 1 × 10 6~1 × 10 10 CFU/ml (MOI 1 to 10000), in antibiotic-free media for 1 h (Fig. 1). The infected Caco-2 cells were recovered with media containing gentamicin to monitor NF-κB transcriptional activity at 6 h or 24 h post-infection. NF-κB transcriptional activity was significantly increased 2.5-and 3-fold in Caco-2 cells infected with S. typhimurium at 6 h post-infection in a concentration of 1 × 10 6 CFU/ml (p≦ 0.05) and 1 × 10 7 CFU/ml (p≦ 0.01), respectively (Fig. 1a). Therefore, the optimal ratio for S. typhimurium to human colorectal epithelia Caco-2 cells is an MOI 10 for this study. Moreover, S. typhimurium entered host cells at 3 h after infection (Additional file 1: Figure S1), suggesting that S. typhimurium triggered inflammatory response may be mainly resulted from extracellular stimuli, most likely are the lipopolysaccharides of S. typhimurium. Because L. acidophilus is one of the most commonly used probiotics in the food supply, we used L. acidophilus as a probiotic for the following experiments. Further, inulin is a well-known prebiotic that is used to improve the growth and viability of probiotics in fermented milk [24]. Therefore, we employed human intestinal Caco-2 cells harboring NF-κB responsive reporter vector to infect with S. typhimurium (MOI 10) without or with inulin or L. acidophilus (MOI 20) or combination (synbiotic). Both L. acidophilus (MOI 20) and synbiotic significantly suppressed NF-κB transcriptional activity in cells infected with S. typhimurium (p≦ 0.05) (Fig. 1b), whereas inulin alone did not alter S. typhimurium-induced NF-κB transcriptional activity. Furthermore, the mRNA level of IL-8 and TNF-α, inflammatory cytokines and transcripts of NF-κB, were significantly reduced in L. acidophilus and synbiotic pretreated Caco-2 cells compared to the cells infected with S. typhimurium alone (Fig. 2) (p≦0.05), suggesting that the L. acidophilus and synbiotic decrease NF-kB activation, which in turn inhibits inflammatory cytokine induction and reduces the inflammation in human intestinal cells during S. typhimurium infection.

L. acidophilus induces TGF-β and MIR21 expression
TGF-β, an important suppressor of inflammation, is induced by L. acidophilus treatment in a mouse model [18], whereas TGF-β expression did not change in human gastric epithelial cells [9]. The role of TGF-β in L. acidophilus-treated human colorectal epithelia Caco-2  cells is not fully understood. To determine whether TGF-β is involved in the anti-inflammation of L. acidophilus in Caco-2 cells during S. typhimurium infection, TGF-β expression in treated cells was assessed by immunoblotting (Fig. 3a). Both L. acidophilus and synbiotic increased TGF-β expression in human intestinal Caco-2 cells during S. typhimurium infection. Moreover, TGF-β enhances MIR21 maturation in primary cultured fibroblasts [19]; therefore, we further evaluated MIR21 expression in the treated Caco-2 cells (Fig. 3b). Consistently, MIR21 expression was increased in cells with L. acidophilus or synbiotic, but not in the other treated cells, suggesting that the TGF-β/MIR21 axis may be involved in the anti-inflammatory effects of L. acidophilus in Caco-2 cells post-infected with S. typhimurium.

L. acidophilus increases TGF-β/SMAD signaling in intestinal cells
MIR21 targets 3'-UTR of SMAD7 and attenuates gene expression [19], which may augment TGF-β signaling for SMAD3/4 transcriptional activity, thereby playing an important role in this positive feedback loop to suppress inflammation. The protein expression of SMAD7 in S. typhimurium infected cells with or without L. acidophilus was determined by immunoblotting (Fig. 4a). The expression of SMAD7 was significantly increased in cells infected with S. typhimurium compared to the uninfected cells, whereas the cells pretreated with L. acidophilus or synbiotic had a reduction in the increased expression of SMAD7 (Fig. 4a). We further evaluated the SMAD3/ 4 transcriptional activity with the cells harboring SMAD3/4 responsive reporter vector. L. acidophilus induced SMAD3/4 activity in intestinal cells with either live or UV-inactivated S. typhimurium or LPS (Additional file 2: Figure S2). Moreover, SMAD3/4 transcriptional activity was increased 7-fold and 2.5-fold in cells administered with L. acidophilus and synbiotics, respectively (Fig. 4b). Pretreatment with synbiotics resulted in significantly less activation of SMAD3/4 compared to the cells treated with L. acidophilus alone, suggesting that the combination of inulin and L. acidophilus has additional effects on TGF-β/SMAD signaling. In addition, the cells pretreated with TGF-β antibody modestly diminished the effects of L. acidophilus on SMAD3/4 and NF-κB transcriptional activity (Fig. 5), supporting the hypothesis that L. acidophilus activates TGF-β signaling to suppress inflammation caused by S. typhimurium in intestinal cells.

Discussion
Human immunity plays an important role in the development of more serious clinical diseases after S. typhimurium infection because of increased pro-inflammatory cytokine expression in the intestine [25]. S. typhimurium infection activates NF-κB in intestinal epithelium cells and subsequently up-regulates gene transcription of proinflammatory cytokines, such as IL-8 [26] and TNF-α [22]. The present study demonstrates that S. typhimurium The previous study demonstrated that L. acidophilus pretreatment decreases S. typhimurium induced inflammation [7]. The finding is consistent with the results showing that anti-inflammatory effects are achieved by probiotics in clinical setting [27]. In normal intestinal mucosal cells, the TGF-β signal may negatively regulate NF-κB activation by stimulating the negative regulator, IκBα [16]. Silencing SMAD7 can restore TGFβ/SMAD3 signaling and result in the suppression of inflammatory cytokine production in patients with inflammatory bowel   [28]. Our results reveal that L. acidophilus induces TGF-β/MIR21 expression and down-regulates SMAD7 expression, which may decrease NF-κB-activated inflammation caused by S. typhimurium in human intestinal Caco-2 cells.
A prebiotic was first defined as a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon and improving host health [29]. Preliminary research on prebiotics demonstrated potential effects on calcium and other mineral absorption [30], immune system effectiveness [31], bowel pH, reduction of colorectal cancer risk [32], and inflammatory bowel disorders, such as Crohn's disease and ulcerative colitis [33]. Additionally, Roberfroid stated that only two particular prebiotics then fully meet this definition, including trans-galactooligosaccharide and inulin [29]. Inulin, a long-chain prebiotic, stimulates the growth of probiotics [29]. Our results showed that combination of probiotic and inulin as synbiotic had a suppressive effect on the S. typhimurium-induced inflammatory cytokine production in human intestinal Caco-2 cells, including IL-8 and TNF-α. However, synbiotics showed a smaller effect on TGF-β and MIR21 expression, and SMAD3/4 transcriptional activity compared to the cells treated with L. acidophilus alone during infection with S. typhimurium. Previous report indicates that prebiotic oligosaccharides reduce proinflammatory cytokines in intestinal Caco-2 cells via activation of PPARγ and peptidoglycan recognition protein 3 [34]. These results suggest that inulin may trigger additional signaling events to prevent S. typhimurium-induced inflammation in a TGF-β independent manner.
S. typhimurium is an intracellular bacterium that enters cells after 2 h of infection. We infected Caco-2 cells with S. typhimurium for only 1 h and recovered the cells with media containing gentamicin. Therefore, the effects on the NF-κB activation and inflammatory cytokines induction might be resulted from lipopolysaccharides of S. typhimurium. On the other hand, our current study showed that L. acidophilus significantly induced TGF-β/SMAD activity in intestinal cells. In addition to TGF-β/MIR21 regulation on SMAD7 expression, L. acidophilus inactivates Jak1/Stat1 signaling to decrease SMAD7 expression, suggesting that Jak1/Stat1 may be involved in the regulation of L. acidophilus on S. typhimurium-induced SMAD7 expression [9]. It is necessary to further elucidate the detailed mechanisms of TGF-β/SMAD regulation in human intestinal cells treated with L. acidophilus.
Microbes play a pivotal role in intestinal homeostasis, such as the uptake of nutrients and the generation of essential elements. However, pathogen infection may disrupt the homeostasis of intestinal microbes and cause acute gastroenteritis or chronic inflammatory diseases. Numerous clinical reports show that probiotics play beneficial roles on reducing incidence of gastrointestinal infectious diseases, including acute diarrhea, necrotizing enterocolitis and death [35][36][37]. Our study showed that TGF-β1/MIR21 signaling pathway may be involved in the suppressive effects of L. acidophilus on inflammation in intestinal cells during S. typhimurium infection. Previous reports also indicate that prebiotics activate immune system in animals during enteric pathogen infection [31]. However, our current study show that prebiotic (inulin) has no effect on diminishing inflammation in intestinal cells during S. typhimurium infection, suggesting inulin may preferentially modulate immune cells to reduce inflammation in vivo. Although the cell culture model may not be able to precisely reflect actual infection due to complicated nature of microbiota and host defense system in vivo, our study provide a potential mechanism of L. acidophilus on inflammation suppression in intestinal cells during S. typhimurium infection.

Conclusion
Our current study indicates that L. acidophilus induces TGF-β/MIR21 expression to reduce NF-κB-activated inflammation caused by S. typhimurium in human intestinal Caco-2 cells. The TGF-β/MIR21 expression might be used as a marker to evaluate anti-inflammatory effects of other strains of Lactobacillus, which requires further study to determine the potential application.

Additional files
Additional file 1: Figure S1. Time required for S. typhimurium to enter host cells. Human intestinal Caco-2 cells were incubated with S. typhimurium (sal, 1×10 7 CFU per well) for 1, 2, or 3 h in a six-well plate. The cells were subsequently washed with PBS, and the medium was replaced with medium containing with antibiotics for 6 h. The cells were then lysed with 1 % Triton X-100 (in PBS) to obtain the intracellular S. typhimurium. Then, the lysates were incubated overnight on LB agar to determine the time required for S. typhimurium to enter cells. (TIFF 234 kb) Additional file 2: Figure S2. Effect of L. acidophilus on SMAD3/4 transcriptional activity with active or inactive S. typhimurium. Human intestinal Caco-2 cells were transfected with luciferase reporter plasmid containing the promoter of the SMAD-binding site or CMV promoter overnight. The cells were then pretreated with L. acidophilus (MOI: 20) 1 h prior to infection with S. typhimurium (MOI: 10) or UV-inactivated S. typhimurium (MOI: 10) in antibiotic-free DMEM for 1 h at 37°C. Then, the cells were washed twice with PBS and added to medium containing Dluciferin to monitor SMAD activity. The cells were washed twice with PBS, added to DMEM medium containing D-luciferin and antibiotics for signal measurement with a Luminometer. Data were analyzed with Prism 5, and the results are shown as the means ± SEM from three independent experiments. (TIFF 100 kb) Abbreviations S. typhimurium: Salmonella typhimurium; L. acidophilus: Lactobacillus acidophilus; TGF-β: Transforming growth factor beta; NF-κB: Nuclear Factor-kappa B; CFU: Colony-forming unit; IL-8: Chemokine (C-X-C motif)