Lactobacillus acidophilus ameliorates H. pylori-induced gastric inflammation by inactivating the Smad7 and NFκB pathways

Background H. pylori infection may trigger Smad7 and NFκB expression in the stomach, whereas probiotics promote gastrointestinal health and improve intestinal inflammation caused by pathogens. This study examines if probiotics can improve H. pylori-induced gastric inflammation by inactivating the Smad7 and NFκB pathways. Results Challenge with H. pylori increased IL-8 and TNF-α expressions but not TGF-β1 in MKN45 cells. The RNA levels of Smad7 in AGS cells increased after H. pylori infection in a dose-dependent manner. A higher dose (MOI 100) of L. acidophilus pre-treatment attenuated the H. pylori-induced IL-8 expressions, but not TGF-β1. Such anti-inflammatory effect was mediated via increased cytoplasmic IκBα and depletion of nuclear NFκB. L. acidophilus also inhibited H. pylori-induced Smad7 transcription by inactivating the Jak1 and Stat1 pathways, which might activate the TGF-β1/Smad pathway. L. acidophilus pre-treatment ameliorated IFN-γ-induced Smad7 translation level and subsequently reduced nuclear NF-κB production, as detected by western blotting. Conclusions H. pylori infection induces Smad7, NFκB, IL-8, and TNF-α production in vitro. Higher doses of L. acidophilus pre-treatment reduce H. pylori-induced inflammation through the inactivation of the Smad7 and NFκB pathways.


Background
Helicobacter pylori infection is considered a major factor inducing chronic gastritis, peptic ulcer, and even gastric cancer in humans [1][2][3]. In mice and human studies, the gastric mucosa of H. pylori-infected subjects show up-regulated NF-B pathway and Th1 type cytokine responses [4][5][6][7][8][9], which may disturb the integrity of the gut epithelial barrier [10]. Accordingly, the inactivation of the NF-B pathway and its downstream immune cascades may be helpful in preventing serious H. pylori-induced complications.
Probiotics are known to inhibit enteric pathogens likes Salmonella, Shigella, and Citrobacter rodentium in both in vitro and animal models [11][12][13]. Their potential clinical benefits in preventing or resolving gastrointestinal diseases have been emphasized [14,15]. There are several mechanisms through which they provide gut protection, including decreasing the luminal pH value by producing lactic acid [16,17] or by competing with gut pathogens for host surface receptors [18].
Nonetheless, Coconnier et al. have shown that probiotics may inhibit H. pylori growth independent of pH and lactic acid levels [19] while Tien et al. report that Lactobacillus casei may down-regulate Shigella flexneriinduced pro-inflammatory cytokines, chemokines, and adherence molecules by inhibiting the NF-B pathway [12]. Another critical mechanism involving probiotics relates to changes in host immune responses to infection via reduced TNF-α and IL-8 but increased IL-10 [20,21]. Regarding the brief contact between the flora of probiotics and the gastric epithelium, an intake of probiotics by H. pylori-infected patients has anti-inflammation benefits resulting from a change in host immune responses.
Transforming growth factor (TGF)-β1 negatively regulates Th1 cell such that TGF-β1/deficient mice spontaneously develop gastritis [22,23]. It is well accepted that the TGF-β1 signaling pathway is positively regulated by receptor-associated Smad 2/3, but negatively by Smad7 [24,25]. H. pylori infection is reportedly associated with increased expression of gastric Smad7, but controversial results in TGF-β1 levels [26,27]. These suggest that the TGF-β1/Smad signaling pathway plays an important role in gut inflammation. However, the exact mechanism of probiotics reducing H. pylori-induced gastric inflammation remains unclear. Thus, this study aimed to examine whether probiotics could regulate the Smad-and NFBmediated signaling pathways to reduce the down-stream inflammatory cytokine production after H. pylori infection.

Cell lines and culture condition
This study was approved by the Ethical Committee of National Cheng Kung University Hospital (ER-98-208). Two human gastric epithelial cancer cell lines (MKN45 and AGS) were obtained from the Health Science Research Resources Bank in Japan and maintained in RPMI 1,640 medium (GIBCO BRL, Grand Island, NY) and F-12 medium (GIBCO BRL, Grand Island, NY) containing 10% FBS at 37°C in a humidified atmosphere (95%) with 5% CO 2 . The cells were sub-cultured every second day. Prior to the bacterial infection study, the cells were incubated in antibiotic-free RPMI 1,640 medium containing 10% FBS overnight at 37°C in 5% CO 2 .

Bacteria and culture condition
Bacterial strain (HP238) isolated from a clinical patient was used. The HP238 expressed CagA, VacA, and BabA proteins in previous studies [28,29]. The bacteria were maintained on a Brucella agar plate containing 10% horse serum and incubated under micro-aerophilic conditions (10% CO 2 , 5% O 2 and 85% N 2 ) for 24-48 hours. The bacteria were then transferred to PBS before infecting the cells. Growth density was measured spectrophotometrically at 600 nm. The infectious dose of bacteria was 1 × 10 8 bacteria/ml at an OD of 1.
The MKN45 cells were infected with a multiplicity of infection (MOI) 1-100 for various time periods. A probiotic strain, one contained in AB-yogurt, Lactobacillus acidophilus (LA5 ® , originated from the Chr. Hansen, Denmark, provided by the President Corp., Tainan, Taiwan) was used. The bacteria were maintained on a Brucella agar, incubated in anaerobic conditions, and then harvested and suspended in phosphate-buffered saline (PBS) before infection. The viable density of L. acidophilus was 1 × 10 8 bacteria/ml at an OD of 1.
MKN45 cells viability after exposure to H. pylori and L. acidophilus The cytotoxicity of MKN45 cell exposure to H. pylori and L. acidophilus was determined by percentage of lactate dehydrogenase (LDH) leakage (Cytotoxicity Assay, Promega Co., Madison, WI, USA) and by assessing viable cell counts using non-stained trypan blue. The culture supernatant and remaining MKN45 cells were collected after incubation with variable doses (MOI 1-1000) of L. acidophilus and H. pylori (MOI 100) for 8 and 4 hours, respectively. Cell cultures without bacterial infection served as controls. The procedures were performed according to the instruction manuals and post-infection cells with non-stained trypan blue staining were directly counted.

Enzyme-linked immuno-sorbent assay (ELISA) for cytokines
To determine the optimal dose and incubation time of various bacteria, bacteria (H. pylori and L. acidophilus) were cultured with MKN45 cells (MOI 1-100) in an antibiotic-free RPMI 1,640 medium (5 ml) containing 10% FBS at 35°C in micro-aerophilic conditions for up to 8 hours. In the experimental study, L. acidophilus were added to MKN45 cells and incubated for 8 hours under the same conditions. After PBS washing and removal of the bacilli, an equal volume of H. pylori was added and the cells were incubated for another 4 hours. The final culture supernatant was centrifuged at 12,000 rpm for 5 min to remove bacteria and cell debris. Concentrations of TNF-α, IL-8 (R & D System, Minneapolis, MN), and TGF-β1 (eBioscience, San Diego, CA) were measured by ELISA according to the manufacturer's instructions. The absorbance of each micro-plate was read on a spectro-photometer using 450 nm as the primary wave length and 570 nm as the reference wave length. All tests were done in triplicate.

Preparation of cytoplasmic and nuclear extracts
The MKN45 and AGS cells were pre-treated with L. acidophilus for 8 hours followed by various doses of H. pylori for 1 hour; then cytoplasmic and nuclear extracts were isolated by a Nuclear Extract Kit (Active Motif, Japan). Briefly, cells were washed with ice-cold saline containing phosphatase inhibitors and pelleted. The cell pellets were then re-suspended in a hypotonic buffer and incubated for 15 min on ice. They were lysed by the addition of detergent and vortexed vigorously for 10 s. After the nuclei were pelleted and re-suspended in complete lysis buffer, the tube was vigorously shaken at 4°C for 30 min on a shaking platform. The nuclear extracts were then centrifuged and the supernatants were aliquoted and stored at -80°C.

Statistical analysis
The Student's t test and paired t test were used, as appropriate, for parametric differences. One-way analysis of variance (ANOVA) with Bonferroni's correction was applied for the multiple testing of data. The Mann-Whitney U test was used for the difference between non-parametric data while Pearson's c2 test was used for non-parametric proportion difference. All tests were two-tailed and a P < 0.05 was considered statistically significant.

Results
Cell viability after incubation with H. pylori and L. acidophilus The cytotoxicity and viability of MKN45 cells incubated with H. pylori (MOI 100) and L. acidophilus (MOI 1-1000) were determined by assessing the percentage leakage of LDH and non-stained trypan blue at the 4 th and 8 th hours, respectively (Table 1). Plasma membrane damage assessed by the percentage of LDH leakage from MKN45 after H. pylori incubation (18.1%) was not different to those of control cells (18.0%). Moreover, the viable cell count calculated by non-stained trypan blue did not markedly decrease. When L. acidophilus was incubated with MKN45 cells for 8 hours, the cytotoxicity and viable cell count at MOI 1-100 were not significantly affected. However, LDH leakage and cell death slightly increased as incubation with MOI 1,000 for 8 hours. Therefore, the optimal dose of bacteria used for the experimental study was limited to MOI 100.

H. pylori stimulated IL-8 and TNF-a but not TGF-b1 production in vitro
In MKN45 cells incubated with H. pylori (MOI 100) at various time periods, the IL-8 level increased from the 4 th to the 8 th hour after co-incubation, as determined by ELISA ( Figure 1A). For TNF-α, the post-incubation level rose after the 4 th hour and maintained a plateau until the 8 th hour ( Figure 1B). However, the TGF-β1 level did not increase after H. pylori incubation for 4 hours (data not shown).
In contrast, L. acidophilus did not induce IL-8, TNFα, and TGF-β1 expressions of MKN45 at least within the 8-hour co-incubation period.  doses of L. acidophilus pretreatment for 8 hours (Figure 2). Compared to the control group, L. acidophilus pre-treatment with higher bacterial colony count (MOI 100) reduced H. pylori-induced IL-8 expressions in MKN45 cells (P < 0.05). The TGF-β1 level did not change (P > 0.05).

L. acidophilus reduced H. pylori-induced NF-B by increasing IBa
The study determined that MKN45 cells (MOI 100) incubated with H. pylori led to a peak increase of nuclear NF-B production within one hour. Thus, nuclear NF-B levels of MKN45 cells co-incubated with H. pylori, after prior pre-treatments by various MOIs (1-100) of L. acidophilus were tested for anti-inflammatory effects of probiotics. Pre-treatment of L. acidophilus increased cytoplasmic IBα but decreased the nuclear NF-B levels induced by H. pylori in a dose-dependent manner ( Figure 3). Because IBα level could be mediated by activating the TGF-β1/ Smad signaling pathway, the role Smad7 played in L. acidophilus restoring TGF-β1/Smad activity after H. pylori challenge was tested.

L. acidophilus inhibited H. pylori-and IFN-g-induced Smad7 expression
The Figure 4A shows that pre-treatment with high-dose L. acidophilus (MOI 100) for 8 h prevented H. pyloriinduced Smad7 production by semi-quantitative RT-PCT. Compared to positive controls (AGS cells co-incubated with H. pylori at MOI 100), L. acidophilus pretreatment as high as MOI 100 significantly reduced the H. pyloriinduced Smad7 production at the RNA level (P < 0.05) via inactivation of Jak1 and Stat1 transcriptions. L. acidophilus pre-treatment also inhibited the expression of IFN-γinduced Smad7 protein (P < 0.05) in vitro, with a subsequent increase in cytoplasmic IBα (P < 0.01) and a decrease in nuclear NF-B (P < 0.01) ( Figure 4B).

Discussion
Human immunity plays an important role in the development of more serious clinical diseases after H. pylori infection because of increased pro-inflammatory cytokine expressions on the patients' gastric mucosa [6,8]. H. pylori infection can activate NF-B in gastric epithelium cells and subsequently up-regulate IL-8 gene transcription [4]. Consistent with previous human studies [ [6][7][8][9], the present study reveals that H. pylori infection can induce TNF-α and IL-8 pro-inflammatory cytokine expressions in vitro.
In agreement with the animal study reported by McCarthy et al. [35], the present study illustrates that yogurt-containing probiotics, L. acidophilus does not stimulate proinflammatory cytokines after an 8-hour incubation with MKN45 cells. This suggests that probiotics can exert anti-inflammatory effects in vitro. Accordingly, it will be interesting to test how the inflammatory cascades can be counteracted by probiotics.
The antimicrobial activity of Lactobacillus against enteric pathogens is, in part, due to the accumulation of lactic acid [17,21]. The ability of lactic acid production varies in the Lactobacillus spp. and L. acidophilus is a low lactic acid-production strain [34]. Experimentally, L. acidophilus decreases the viability of H. pylori in vitro independent of pH and lactic acid levels [19]. The pH value of each suspension in this study is around 6.8-7.0 (data not shown). Other mechanisms like immunomodulation should therefore contribute largely to the anti-inflammatory effects of L. acidophilus.
The current study demonstrates that L. acidophilus pre-treatment can decrease the H. pyloriinduced nuclear NF-B expression in the 1 st hour and IL-8 in the 4 th hour, after co-culture with H. pylori and MKN45 cells. Furthermore, the TNF-α level is also decreased although its value is quite low (data not shown). This study further confirms that such suppression occurs in a dosedependent manner and is mediated through the stabilization of IBα. The finding is compatible with the results of Tien et al. showing that anti-inflammatory effects can only be achieved at an adequate bacteria count in probiotics [12]. Data from the present study indicate that L. acidophilus can counteract H. pyloriinduced gastric inflammation specifically by mediation through the IBα/NF-B pathway in a dose-dependent manner.
In normal intestinal mucosal cells, the TGF-β1 signal may negatively regulate NF-B activation by stimulating the negative regulator, IBα [36]. H. pylori infection reportedly may inhibit the TGF-β1 signal pathway via activation of the gastric Smad7 expression [26]. This study also declares that both H. pylori and L. acidophilus do not affect the TGF-β1 production of gastric epithelial cells, which again confirm that L. acidophilus regulates TGFβ1/Smad3 downstream activity by restoring Smad7. The present study is the first to demonstrate that L. acidophilus can down-regulate Smad7 production to restore the TGFβ1/Smad activity and to ameliorate the H. pyloriinduced gastric inflammation in vitro ( Figure 5).
Smad7 pathway. Inhibiting Smad7 can restore the TGF-β1/Smad3 signaling and result in the suppression of inflammatory cytokine production in patients with inflammatory bowel diseases [37,38]. The data here reveals that probiotics contained in yogurt can inhibit Smad7 to diminish H. pylorirelated gastric inflammation. Such probiotics can be quite promising for the improvement of H. pylori infection control.

Conclusions
Yogurt-containing L. acidophilus can improve H. pyloriinduced gastric inflammation through the inactivation of the Smad7 and NF-B mediated pathways. Intake of L. acidophilus-containing yogurt may improve gastric inflammation in H. pylori-infected patients.