Helicobacter ISSN 1523-5378 doi: 10.1111/hel.12105
Anti-inflammatory Properties of Gastric-derived Lactobacillus plantarum XB7 in the Context of Helicobacter pylori Infection Thien Thiraworawong,* Jennifer K. Spinler,†,‡ Duangporn Werawatganon,§ Naruemon Klaikeaw,¶ Susan F. Venable,†,‡ James Versalovic†,‡ and Somying Tumwasorn** *Interdisciplinary Program of Medical Microbiology, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand, †Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX 77030, USA, ‡Department of Pathology, Texas Children’s Hospital, Houston, TX 77030, USA, §Department of Physiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand, ¶Department of Pathology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand, **Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
Keywords Lactobacillus plantarum, Helicobacter pylori, interleukin-8, stomach, Sprague–Dawley rat, probiotic. Reprint requests to: Somying Tumwasorn, Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand. E-mail: [email protected]
Abstract Background: Helicobacter pylori colonization of the gastric epithelium induces interleukin-8 (IL-8) production and inflammation leading to host cell damage. We searched for gastric-derived Lactobacillus with the ability to suppress H. pylori-induced inflammation. Materials and methods: Conditioned media from gastric-derived Lactobacillus spp. were tested for the ability to suppress H. pylori-induced IL-8 production in AGS gastric epithelial cells. IL-8 protein and mRNA levels were measured by ELISA and qPCR, respectively. The changes on host cell signaling pathway were analyzed by Western blotting and the anti-inflammatory effect was tested in a Sprague–Dawley rat model. Results: Conditioned media from L. salivarius B101, L. rhamnosus B103, and L. plantarum XB7 suppressed IL-8 production and IL-8 mRNA expression in H. pylori-induced AGS cells without inhibiting H. pylori growth. Conditioned media from LS-B101, LR-B103, and LP-XB7 suppressed the activation of NF-jB in AGS cells, while strain LP-XB7 also suppressed c-Jun activation. The antiinflammatory effect of LP-XB7 was further assessed in vivo using a H. pyloriinfected Sprague–Dawley rat model. Strain LP-XB7 contributed to a delay in the detection and colonization of H. pylori in rat stomachs, attenuated gastric inflammation, and ameliorated gastric histopathology. Additionally, the administration of LP-XB7 correlated with the suppression of TNF-a and CINC-1 in sera, and suppression of CINC-1 in the gastric mucosa of H. pylori-infected rats. Conclusions: These results suggest that L. plantarum XB7 produces secreted factors capable of modulating inflammation during H. pylori infection, and this probiotic Lactobacillus strain shows promise as an adjunctive therapy for treating H. pylori-associated disease.
Helicobacter pylori is the main cause of gastric-associated disease world-wide. Persistence of H. pylori colonization results in gastric inflammation which can lead to a more severe diseases, such as peptic ulcers and gastric cancer in a minority of infected individuals [1–3]. The controversy surrounding H. pylori, that is, whether it is the direct causative agent or secondary agent after predisposing factors, stems from its specialized coevolution with humans to inhabit the harsh environment of the stomach. While its role in pathogenesis cannot be denied, the possibility that H. pylori could be protective to colonized humans is still debatable [4–6]. Infection
144
with H. pylori strains induces inflammatory cytokine production by host epithelium resulting in host cell damage and inflammation [7–13]. The pro-inflammatory cytokine, interleukin-8 (IL-8), plays a major role in gastric mucosal inflammation [14–16] by recruiting and activating neutrophils [17,18]. Both H. pyloriinfected and gastric cancer patients have been reported to show increased levels of IL-8 in the epithelium of the stomach corpus and antrum [19], and H. pylori can induce IL-8 production in gastric epithelial cell lines [20,21]. Additionally, previous studies demonstrated that major transcription factors responsible for IL-8
© 2014 John Wiley & Sons Ltd, Helicobacter 19: 144–155
Gastric Inflammation Suppressed by L. plantarum
Thiraworawong et al.
activation by H. pylori involved nuclear factor-jB (NFjB) and/or activator protein 1 (AP-1) [22–25]. Triple therapy, consisting of a proton-pump inhibitor (PPI) and two antibiotics, is the standard treatment for H. pylori infection. This drug combination works to decrease the acidity of the stomach, subsequently increasing antibiotic activity, resulting in an eradication rate less than 80% [26–28]. Eradication decreases to below 60% in cases of clarithromycin-resistant H. pylori [29,30]. Probiotics have been proposed as a promising adjunct therapy to triple therapy and have the potential to diminish side effects of antibiotics, increase the H. pylori eradication rate, and decrease the severity of host cell damage [3,31]. L. johnsonii La1 attenuates H. pylori-associated gastritis in mice [32], and contributes to a decrease in MIP-2, a pro-inflammatory chemokine that is a potent murine neutrophil-chemoattractant [33]. L. salivarius WB1004 inhibits the production of H. pylori-stimulated IL-8 by gastric epithelial cells in vitro and prevents H. pylori colonization in the stomach of mice pre-inoculated with L. salivarius [34]. Viable L. gasseri OLL2716 suppressed H. pylori-induced IL-8 production from MKN45 cells in vitro, and in vivo from biopsies of human gastric mucosa [35]. Recently, L. bulgaricus has been reported to specifically inhibit H. pylori SS1-LPS-induced IL-8 production by the inhibition of Toll-like receptor (TLR) 4 pathway in SGC7901 human gastric adenocarcinoma cells [36]. The evolution of gastric-derived Lactobacillus to inhabit the harsh environment of the stomach provides a selective advantage against H. pylori-associated gastric disease. In the current study, we examined the ability of eleven Lactobacillus spp. previously isolated from the human gastric mucosa to suppress H. pylori-induced IL8 production from gastric epithelial cells. In addition, we characterized the signaling pathway affected in
gastric epithelial cells by IL-8 suppressing Lactobacillus strains. Based on the above results, the most promising candidate Lactobacillus plantarum XB7 was chosen and tested for anti-inflammatory effects in a Sprague– Dawley rat model of H. pylori infection. Strain LP-XB7 contributed to a delay in the detection and colonization of H. pylori in rat stomachs, attenuated gastric inflammation, and ameliorated gastric histopathology.
Methods Bacterial Strains and Culture Conditions Eleven Lactobacillus spp., listed in Table 1, were previously isolated from gastric biopsies of dyspeptic patients and chosen for this study [37]. Conditioned media from these Lactobacillus spp., except L. salivarius B37, have previously been shown to inhibit LPS-induced TNF production in THP-1 monocytoid cells [37]. Five of the eleven isolates were previously reported to suppress H. pylori-stimulated IL-8 production in AGS gastric adenocarcinoma epithelial cells [38]. Lactobacillus spp. were routinely cultured on deMan-Rogosa-Sharpe (MRS) agar (Oxoid, Hampshire, UK) under anaerobic conditions (10% CO2, 10% H2, and 80% N2) at 37 °C for 24 hours. Helicobacter pylori ATCC 43504 (ATCC, Manassas, VA, USA) was cultured on Columbia agar (Oxoid) supplemented with 7% (v/v) horse serum (Gibco New Zealand Ltd, Auckland, New Zealand) and 7% (v/v) sheep blood under microaerophilic conditions (6–12% O2, 5–8% CO2) at 37 °C for 48–72 hours.
Preparation of Lactobacillus-Conditioned Media The Lactobacillus-conditioned media (LCM) were prepared as previously described [39]. Briefly, overnight
Table 1 Gastric-derived Lactobacillus spp. used in this study
Isolate
% Identitya
Patient sex
Patient age (year)
Reference
L. L. L. L. L. L. L. L. L. L. L.
100.0 99.5 96.8 99.0 100.0 99.5 100.0 100.0 99.5 99.0 99.3
Female Male Male Female Male Female Female Female Male Male Male
45 44 53 48 34 59 58 74 67 72 60
Panpetch Panpetch Panpetch Panpetch Panpetch Panpetch Panpetch Panpetch Panpetch Panpetch Panpetch
plantarum B6 salivarius B37 murinus B57 salivarius B60 plantarum B70 salivarius B74 plantarum B90 salivarius B101 rhamnosus B103 casei group B106 plantarum XB7
[37] [37] [37] et al. [37] [37] et al. [37] et al. et al. et al.
2011 [38]
2011 [38] 2011 [38] 2011 [38] 2011 [38]
a
Nucleotide sequences were analyzed using the Ribosomal Database Project II (RDP-II; http://rdp.cme.msu.edu).
© 2014 John Wiley & Sons Ltd, Helicobacter 19: 144–155
145
Gastric Inflammation Suppressed by L. plantarum
cultures of Lactobacillus were adjusted to an OD600 of 0.1 in MRS broth and incubated anaerobically for 48 hours. Cell free supernatants were collected by centrifugation, filter-sterilized using 0.22 lm surfactantfree cellulose acetate membrane filters (Minisart, Sartorius Stedim Biotech GmbH, Goettingen, Germany), and concentrated by speed vacuum drying (Savant instruments, Farmingdale, NY, USA). Pellets were resuspended in an equal volume of Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco-Invitrogen, Carlsbad, CA, USA) and stored at 20 °C until further use.
Effects of LCM on H. pylori Growth by Disc Diffusion A disc diffusion assay was used to ensure that suppression of IL-8 production from AGS cells was not associated with the inhibition of H. pylori growth by LCM. Sterile 6 mm discs (Whatman, Maidstone, UK) were soaked for at least 1 hour at room temperature in LCM (5% v/v) or MRS (media control). A lawn of H. pylori 43504 was cultured as described above in the presence of LCM or MRS saturated discs and incubated microaerophilically at 37 °C for 72 hours. The zone of inhibition around each disc was measured by a vernier caliper (Mitutoyo, Japan).
Effect of LCM on IL-8 Production in Gastric Epithelial Cells Lactobacillus-conditioned media and H. pylori were cocultured with gastric epithelial cells and IL-8 production was monitored as previously described [38]. Briefly, AGS human gastric adenocarcinoma epithelial cells (ATCC CRL-1739) were maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco-Invitrogen) at 37 °C with 5%CO2 for 48 hours. Adherent cells were detached with 0.25% (v/v) Trypsin (Gibco-Invitrogen) in 1 mmol/L EDTA (Gibco-Invitrogen) and resuspended in fresh media. AGS cells (2.0 9 104 cells) were pre-incubated in a 96-well format for 24 hours. LCM (5% v/v) was added to the cells alone or in combination with viable H. pylori 43504 (6.0 9 106 CFU/well), and co-incubated for 24 hours. Cell culture supernatants were collected by centrifugation and stored at 20 °C until further use.
Effects of LCM on Cytokine Production by ELISA and Luminex Assays Supernatants from co-culture assays were tested for the effects of Lactobacillus secreted factors on IL-8
146
Thiraworawong et al.
production and other related cytokines. IL-8 concentrations in treated and untreated H. pylori-stimulated AGS cell supernatants were measured using Human CXCL8/ IL-8 DuoSet (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. To determine whether Lactobacillus secreted factors modulate cytokines other than IL-8 in this assay, supernatants were tested by Human Cytokine/Chemokine-Premixed 14-plex kit (Millipore, Billerica, MA, USA) in a Luminex 100 system (Luminex Corporation, Austin, TX, USA) for quantification of the following analytes: GM-CSF, IFN-c, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, MCP-1, and TNF-a. Analytes at concentrations exceeding the minimum detectable dose were evaluated and raw data were obtained with MasterPlex CT version 1.2.0.7 and analyzed with MasterPlex QT version 5.0.0.73 (Hitachi MiraiBio, San Francisco, CA, USA).
Analysis of IL-8 Gene Expression by qPCR To quantitate the effects of LCM on IL-8 gene expression, AGS cells (5 9 105 cells/well) were pre-incubated in a 24-well format and treated with viable H. pylori 43504 (1.5 9 108 CFU/well) alone or in combination with LCM (5% v/v), and co-incubated for 4 hours. Cell culture supernatants were removed by centrifugation and total RNA of treated AGS cells was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). IL-8 gene-specific cDNA was synthesized using the SuperScriptâ VILO cDNA Synthesis kit (Invitrogen) [40]. Quantitative PCR was carried out using LightCyclerâ FastStart DNA MasterPLUS SYBR Green I (Roche, Germany) in a LightCyclerâ 2.0 instrument (Roche) according to the manufacturer’s instructions under the following settings: 45 cycles of: denaturation at 95 °C for 10 seconds, annealing at 65 °C for 10 seconds, and extension at 72 °C for 25 seconds. Gene targets were amplified using gene specific primers: IL-8 forward (5′ACA CTG CGC CAA CAC AGA AAT TA-3′) and IL-8 reverse (5′-TTT GCT TGA AGT TTC ACT GGC ATC-3′); GAPDH forward primer (5′-GCA CCG TCA AGG CTG AGA AC-3′), GAPDH reverse primer (5′-ATG GTG GTG AAG ACG CCA GT-3′) [40]. The fold change of IL-8 gene expression relative to GAPDH gene expression was calculated according to the 2 DDCp method [41,42].
Examination of Cell Signaling Pathways by Quantitative Western Blot Lactobacillus-conditioned media-induced changes in the signaling pathway of gastric epithelial cells were analyzed by Western blot as previously described, with
© 2014 John Wiley & Sons Ltd, Helicobacter 19: 144–155
Thiraworawong et al.
minor modifications [24]. AGS cells (2 9 106cells/well) were subjected to co-culture with H. pylori 43504 (6 9 108cells/well) with or without LCM for 0.5, 1, 2, or 4 hours. Culture supernatants were removed by centrifugation, and protein was extracted from whole cell lysates using Mammalian Protein Extraction Reagent (M-PER, Pierce Biotechnology, Rockford, IL, USA) supplemented with Halt protease and phosphatase inhibitors (Pierce Biotechnology) according to the manufacturer’s instructions. Protein concentrations were determined with the Pierceâ BCA Protein Assay Kit (Pierce Biotechnology) according to the manufacturer’s instructions. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Philadelphia, PA, USA) and blocked with 3% bovine serum albumin in TBST (50 mmol/L Tris, pH 7.5, 0.15 mol/L NaCl, 0.05% Tween 20). Blocked membranes were incubated with mouse monoclonal antibodies against NF-kB p65, phospho-NF-kB p65 (Ser 536), c-Jun, phospho-c-Jun and b-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), washed with TBST, and treated with horseradish peroxidaselabeled goat anti-mouse secondary antibodies for 1 hour. Peroxidase signals were detected and imaged by ChemiDocTM XRS (Bio-Rad), and densitometric measurements were completed by ImageJ software (version 1.45s) analysis.
Administration of H. pylori and Lactobacillus plantarum XB7 to Rats The effect of gastric-derived Lactobacillus plantarum XB7 on H. pylori infection and disease pathology was tested in a Sprague–Dawley rat model. The experimental procedures in this study were approved by the Ethics Committee for Animal Care and Use of Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (certificate number: 03/54, research project number: 13/53). Five-week-old male Sprague–Dawley rats (150–200 g) were received from National Laboratory Animal Center, Mahidol University (Bangkok, Thailand) and divided into control, H. pylori-infected, and Lactobacillus-treated groups (eight rats per group). H. pylori infection, treatment with probiotic and assessment of disease pathology was performed according to established protocols with minor modifications [43]. In brief, H. pyloriinfected and Lactobacillus-treated groups were pre-treated with streptomycin (5 mg/mL) for 3 days, then treated with H. pylori ATCC 43504 (108–1010 CFU/mL) by orogastric gavage twice daily for 3 consecutive days. After H. pylori inoculation, the rats were given access to food and water ad libitum for 1 week. Next, the Lactoba-
© 2014 John Wiley & Sons Ltd, Helicobacter 19: 144–155
Gastric Inflammation Suppressed by L. plantarum
cillus-treated groups were gavaged with L. plantarum XB7 (106 CFU/mL) once daily for 7 days and sacrificed. Rats were food-restricted overnight prior to sacrifice.
Macroscopic Assessment of H. pylori-Infected Gastric Tissue Stomachs were collected at time of sacrifice, opened at the side of greater curvature, and washed with PBS. Antral sections (2 mm2) were isolated and used for 1) assessment of H. pylori colonization and 2) cytokine production from tissue. The presence of H. pylori in an antral section was determined by the urease test at room temperature (25 °C) and a change to pink color was observed within 48 hours. Gastric inflammation and H. pylori colonization were determined in a blinded fashion by standard methods for histopathology on fixed stomach tissue (10% formaldehyde, 0.2 mol/L sodium phosphate buffer, pH 7.4). Briefly, fixed tissue was embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) to detect inflammation, and H. pylori colonization. Giemsa staining was performed to detect H. pylori colonization when H&E results were unclear. Gastric inflammation was scored using the updated Sydney System [44]. Mononuclear and polymorphonuclear leukocyte infiltration in the gastric mucosa was scored: Score 0, normal; Score 1, mild; Score 2, moderate; Score 3, marked inflammatory changes. The H. pylori colonization grading scale was: Score 0, no bacteria detected; Score 1, mild colonization in some gastric crypts; Score 2, mild colonization in most gastric crypts; Score 3, moderate colonization in all gastric crypts.
Cytokine Measurements in Serum and Tissue Blood was collected from each rat via cardiac puncture, left at room temperature for 2 hours, and centrifuged (1000 9 g, 4 °C for 20 minutes) to isolate serum. Serum was stored at 20 °C for cytokine analysis. Antral tissue (2 mm2) was homogenized with a micropestle in 500 lL of PBS, supernatant collected by centrifugation (1800 9 g, 4 °C for 10 minutes) and stored at 20 °C until cytokine analysis. Specifically, growth-regulated oncogene/cytokine-induced neutrophil chemo-attractant-1 (GRO/CINC-1), IL-1b, and TNF-a were measured from serum and gastric tissue samples by Quantikine ELISA (R&D Systems) according to manufacturer’s instructions.
Statistical Analysis Data were presented as mean standard error of mean. Statistical significance of in vitro experiments was
147
Gastric Inflammation Suppressed by L. plantarum
determined by Student’s t-test, while one-way ANOVA and Bonferroni’s post hoc test were used for in vivo experiments, and indicated in both cases by a p < .05.
Results Gastric-derived Lactobacillus spp. Suppressed H. pylori-induced IL-8 Production in Gastric Epithelial Cells Lactobacillus conditioned media (LCM) from eleven gastric-derived Lactobacillus spp. (Table 1) were analyzed for the ability to suppress pro-inflammatory cytokine production from H. pylori-stimulated gastric epithelial cells. LCM from eight of the eleven Lactobacillus isolates significantly (p < .05) inhibited H. pylori-induced IL-8 production in AGS cells when compared with MRS control (Fig. 1). LCM from these 8 lactobacilli did not stimulate IL-8 production when they were co-cultured with AGS cells in the absence of H. pylori (data not shown). The addition of LCM from L. plantarum B6, L. murinus B57, and L. plantarum B70 did not affect IL8 production from H. pylori-stimulated AGS cells when compared to control (Fig. 1). Trypan blue dye exclusion and MTT assays indicated that AGS cell viability (>90%) was not compromised by the presence of any LCM tested (data not shown), and disc diffusion methods confirmed LCM did not inhibit proliferation of H. pylori (data not shown).
Figure 1 Gastric-derived Lactobacillus species secrete factors that suppress IL-8 production by Helicobacter pylori-induced AGS cells. LCM from eleven gastric-derived Lactobacillus isolates were tested for the ability to inhibit IL-8 production from AGS cells stimulated with H. pylori. AGS cells were stimulated with H. pylori in the presence of LCM for 24 hours and IL-8 production was monitored by ELISA. The experiments were performed three times in triplicate. The results are expressed as the mean SEM, *p-value < .05, **p-value < .01, and ***p-value < .001 as compared to media control.
148
Thiraworawong et al.
Lactobacillus spp. Suppressed IL-8 Gene Expression Quantitative RT-PCR was performed to determine the effect of LCM from eight Lactobacillus isolates, shown to suppress IL-8 production, on IL-8 transcription. H. pylori-stimulated AGS cells were treated with LCM for 4 hours prior to total RNA isolation. IL-8 gene expression, relative to gapdh, was significantly downregulated approximately 0.5 fold (p < .05) in the presence of LCM from three isolates: L. salivarius B101 (LS-B101), L. rhamnosus B103 (LR-B103), and L. plantarum XB7 (LP-XB7) (Fig. 2). LCM from the remaining 5 isolates did not affect IL-8 gene expression in this assay and were not studied further (Fig. 2).
NF-kB-directed Signaling is Influenced by Lactobacillus Activation of NF-jB and AP-1 by phosphorylation results in an increase in IL-8 gene expression [22–25]. Western blot analysis of phosphorylated NF-kB and c-Jun (AP-1 subunit) from whole cell lysates were conducted to determine Lactobacillus-mediated effects on the activation of NF-jB and AP-1. H. pylori-stimulated AGS cells were treated with LCM from LS-B101, LRB103, and LP-XB7 for 0.5, 1, 2, and 4 hours. The effects of LCM treatment at each time point on concentrations
Figure 2 Anti-inflammatory Lactobacillus secreted factors suppress IL-8 transcription. IL-8 gene expression was determined in AGS cells stimulated with Helicobacter pylori plus medium control, or LCM from eight IL-8-suppressing Lactobacillus after 4 hours. Quantitative realtime PCR was conducted with primers specific to IL-8 and GAPDH transcripts. Gene expression data were normalized to housekeeping gene, GAPDH. The experiments were performed three times in triplicate. Gene expression ratios of IL-8 (LCM/medium control) were calculated, and results represent the mean SEM, *p-value < .05 and **p-value < .01 as compared to the theoretical mean of 1.0.
© 2014 John Wiley & Sons Ltd, Helicobacter 19: 144–155
Thiraworawong et al.
of phospho NF-kB (p-NF-kB) and phospho c-Jun (p-cJun) were directly assessed using monoclonal antibodies specific for each transcription factor. Two-hour treatments with LCM from LS-B101, LR-B103, and LPXB7 resulted in the most significant decrease in p-NFkB as compared to media control (>0.5 fold change; p < .05) (Fig. 3A). Following treatment with LP-XB7 LCM, significantly decreased concentrations of p-c-Jun were observed after 4 hours (0.585 fold change; p < .05) (Fig. 3B). In contrast, this decrease was not A
B
Figure 3 Gastric-derived Lactobacillus suppress activation of Helicobacter pylori-induced transcription factors. Concentrations of activated NF-kB and c-Jun were determined by Western blot on whole cell lysates of AGS cells stimulated with H. pylori plus medium control, or LCM from LS-B101, LR-B103, or LP-XB7. Concentrations were measured at 0.5, 1, 2, or 4 hours using antibodies corresponding to (A) p-NF-jB p65 and NF-jB p65, or (B) p-c-Jun and c-Jun. Relative protein concentrations were determined by densitometry, and activated transcriptions factors were normalized to their nonactivated counterparts (p-NF-jB p65 (Ser 536) to NF-jB p65 levels; p-c-Jun to c-Jun levels). The experiments were performed three times in triplicate. The results are expressed as the mean SEM, *p-value < .05, or **p-value < .01 as compared to media control.
© 2014 John Wiley & Sons Ltd, Helicobacter 19: 144–155
Gastric Inflammation Suppressed by L. plantarum
observed following treatment with either LS-B101 LCM or LR-B103 LCM (Fig. 3B). Because LCM produced by L. plantarum XB7 contributed to a decrease in the activation of both p-NF-kB and p-c-Jun, this isolate was selected for further characterization in H. pylori-infected Sprague–Dawley rats.
L. plantarum XB7 Attenuated Urease Production, Gastric Inflammation, and H. pylori Colonization in a Rat Infection Model An H. pylori-infected Sprague–Dawley rat model was previously developed [43] to study the inflammatory effects of H. pylori infection in the gastric mucosa. We adapted this model to study the potential antiinflammatory effects of L. plantarum XB7 in the context of H. pylori infection. Three groups of eight rats were either 1) infected with H. pylori, 2) infected with H. pylori and treated with LP-XB7, 3) or not infected with H. pylori and not treated with LP-XB7 (control group). Urease production by H. pylori can be detected in tissue by a urease test. H. pylori colonization was initially evaluated in the stomach of sacrificed rats by the presence of urease production and histopathologically. Inflammation was assessed by H&E staining. Stomach tissues from sacrificed H. pylori-infected rats were positive for urease production within 24 hours (mean = 21 hours 29 minutes, range 20 hours 3 minutes–22 hours 26 minutes), while tissues from LP-XB7treated rats were urease-positive within 30 hours (mean = 27 hours 46 minutes, range 24 hours 55 minutes–29 hours 11 minutes) (Table 2). This six-hour delay in urease-positive results between LP-XB7untreated and -treated rats was indicative of fewer H. pylori organisms in the LP-XB7-treated group. Stomach tissues from control rats remained negative for urease after 48 hours (Table 2). The indication that fewer H. pylori organisms colonized the LP-XB7-treated group was confirmed histopathologically. H. pylori colonization scores of 1 (mild, 7 rats) and 2 (moderate, 1 rat) were seen in the H. pylori-infected group, while five of eight rats from the LP-XB7-treated group showed a score of 0 (no H. pylori detected) just as in the control group (Table 2). Within the LP-XB7-treated group, the same five rats with no detectable H. pylori also showed no inflammation by H&E stain (score of 0, Table 2). H&E staining showed that seven of eight rats from the H. pylori-infected group demonstrated a score of 1 (mild gastric inflammation) and the control group showed no histopathologic change (Table 2). Images representative of the H&E staining results are shown in Figure 4. The urease test results, H. pylori colonization and gastric inflammation scores are summarized in Table 2.
149
Gastric Inflammation Suppressed by L. plantarum
Thiraworawong et al.
Table 2 Summary of urease test results, gastric inflammation, and Helicobacter pylori colonization scores H. pylori colonizationc
Gastric inflammationb
Urease test Groupa
+
Anti-inflammatory Properties of Gastric-derived Lactobacillus plantarum XB7 in the Context of Helicobacter pylori Infection Thien Thiraworawong,* Jennifer K. Spinler,†,‡ Duangporn Werawatganon,§ Naruemon Klaikeaw,¶ Susan F. Venable,†,‡ James Versalovic†,‡ and Somying Tumwasorn** *Interdisciplinary Program of Medical Microbiology, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand, †Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX 77030, USA, ‡Department of Pathology, Texas Children’s Hospital, Houston, TX 77030, USA, §Department of Physiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand, ¶Department of Pathology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand, **Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
Keywords Lactobacillus plantarum, Helicobacter pylori, interleukin-8, stomach, Sprague–Dawley rat, probiotic. Reprint requests to: Somying Tumwasorn, Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand. E-mail: [email protected]
Abstract Background: Helicobacter pylori colonization of the gastric epithelium induces interleukin-8 (IL-8) production and inflammation leading to host cell damage. We searched for gastric-derived Lactobacillus with the ability to suppress H. pylori-induced inflammation. Materials and methods: Conditioned media from gastric-derived Lactobacillus spp. were tested for the ability to suppress H. pylori-induced IL-8 production in AGS gastric epithelial cells. IL-8 protein and mRNA levels were measured by ELISA and qPCR, respectively. The changes on host cell signaling pathway were analyzed by Western blotting and the anti-inflammatory effect was tested in a Sprague–Dawley rat model. Results: Conditioned media from L. salivarius B101, L. rhamnosus B103, and L. plantarum XB7 suppressed IL-8 production and IL-8 mRNA expression in H. pylori-induced AGS cells without inhibiting H. pylori growth. Conditioned media from LS-B101, LR-B103, and LP-XB7 suppressed the activation of NF-jB in AGS cells, while strain LP-XB7 also suppressed c-Jun activation. The antiinflammatory effect of LP-XB7 was further assessed in vivo using a H. pyloriinfected Sprague–Dawley rat model. Strain LP-XB7 contributed to a delay in the detection and colonization of H. pylori in rat stomachs, attenuated gastric inflammation, and ameliorated gastric histopathology. Additionally, the administration of LP-XB7 correlated with the suppression of TNF-a and CINC-1 in sera, and suppression of CINC-1 in the gastric mucosa of H. pylori-infected rats. Conclusions: These results suggest that L. plantarum XB7 produces secreted factors capable of modulating inflammation during H. pylori infection, and this probiotic Lactobacillus strain shows promise as an adjunctive therapy for treating H. pylori-associated disease.
Helicobacter pylori is the main cause of gastric-associated disease world-wide. Persistence of H. pylori colonization results in gastric inflammation which can lead to a more severe diseases, such as peptic ulcers and gastric cancer in a minority of infected individuals [1–3]. The controversy surrounding H. pylori, that is, whether it is the direct causative agent or secondary agent after predisposing factors, stems from its specialized coevolution with humans to inhabit the harsh environment of the stomach. While its role in pathogenesis cannot be denied, the possibility that H. pylori could be protective to colonized humans is still debatable [4–6]. Infection
144
with H. pylori strains induces inflammatory cytokine production by host epithelium resulting in host cell damage and inflammation [7–13]. The pro-inflammatory cytokine, interleukin-8 (IL-8), plays a major role in gastric mucosal inflammation [14–16] by recruiting and activating neutrophils [17,18]. Both H. pyloriinfected and gastric cancer patients have been reported to show increased levels of IL-8 in the epithelium of the stomach corpus and antrum [19], and H. pylori can induce IL-8 production in gastric epithelial cell lines [20,21]. Additionally, previous studies demonstrated that major transcription factors responsible for IL-8
© 2014 John Wiley & Sons Ltd, Helicobacter 19: 144–155
Gastric Inflammation Suppressed by L. plantarum
Thiraworawong et al.
activation by H. pylori involved nuclear factor-jB (NFjB) and/or activator protein 1 (AP-1) [22–25]. Triple therapy, consisting of a proton-pump inhibitor (PPI) and two antibiotics, is the standard treatment for H. pylori infection. This drug combination works to decrease the acidity of the stomach, subsequently increasing antibiotic activity, resulting in an eradication rate less than 80% [26–28]. Eradication decreases to below 60% in cases of clarithromycin-resistant H. pylori [29,30]. Probiotics have been proposed as a promising adjunct therapy to triple therapy and have the potential to diminish side effects of antibiotics, increase the H. pylori eradication rate, and decrease the severity of host cell damage [3,31]. L. johnsonii La1 attenuates H. pylori-associated gastritis in mice [32], and contributes to a decrease in MIP-2, a pro-inflammatory chemokine that is a potent murine neutrophil-chemoattractant [33]. L. salivarius WB1004 inhibits the production of H. pylori-stimulated IL-8 by gastric epithelial cells in vitro and prevents H. pylori colonization in the stomach of mice pre-inoculated with L. salivarius [34]. Viable L. gasseri OLL2716 suppressed H. pylori-induced IL-8 production from MKN45 cells in vitro, and in vivo from biopsies of human gastric mucosa [35]. Recently, L. bulgaricus has been reported to specifically inhibit H. pylori SS1-LPS-induced IL-8 production by the inhibition of Toll-like receptor (TLR) 4 pathway in SGC7901 human gastric adenocarcinoma cells [36]. The evolution of gastric-derived Lactobacillus to inhabit the harsh environment of the stomach provides a selective advantage against H. pylori-associated gastric disease. In the current study, we examined the ability of eleven Lactobacillus spp. previously isolated from the human gastric mucosa to suppress H. pylori-induced IL8 production from gastric epithelial cells. In addition, we characterized the signaling pathway affected in
gastric epithelial cells by IL-8 suppressing Lactobacillus strains. Based on the above results, the most promising candidate Lactobacillus plantarum XB7 was chosen and tested for anti-inflammatory effects in a Sprague– Dawley rat model of H. pylori infection. Strain LP-XB7 contributed to a delay in the detection and colonization of H. pylori in rat stomachs, attenuated gastric inflammation, and ameliorated gastric histopathology.
Methods Bacterial Strains and Culture Conditions Eleven Lactobacillus spp., listed in Table 1, were previously isolated from gastric biopsies of dyspeptic patients and chosen for this study [37]. Conditioned media from these Lactobacillus spp., except L. salivarius B37, have previously been shown to inhibit LPS-induced TNF production in THP-1 monocytoid cells [37]. Five of the eleven isolates were previously reported to suppress H. pylori-stimulated IL-8 production in AGS gastric adenocarcinoma epithelial cells [38]. Lactobacillus spp. were routinely cultured on deMan-Rogosa-Sharpe (MRS) agar (Oxoid, Hampshire, UK) under anaerobic conditions (10% CO2, 10% H2, and 80% N2) at 37 °C for 24 hours. Helicobacter pylori ATCC 43504 (ATCC, Manassas, VA, USA) was cultured on Columbia agar (Oxoid) supplemented with 7% (v/v) horse serum (Gibco New Zealand Ltd, Auckland, New Zealand) and 7% (v/v) sheep blood under microaerophilic conditions (6–12% O2, 5–8% CO2) at 37 °C for 48–72 hours.
Preparation of Lactobacillus-Conditioned Media The Lactobacillus-conditioned media (LCM) were prepared as previously described [39]. Briefly, overnight
Table 1 Gastric-derived Lactobacillus spp. used in this study
Isolate
% Identitya
Patient sex
Patient age (year)
Reference
L. L. L. L. L. L. L. L. L. L. L.
100.0 99.5 96.8 99.0 100.0 99.5 100.0 100.0 99.5 99.0 99.3
Female Male Male Female Male Female Female Female Male Male Male
45 44 53 48 34 59 58 74 67 72 60
Panpetch Panpetch Panpetch Panpetch Panpetch Panpetch Panpetch Panpetch Panpetch Panpetch Panpetch
plantarum B6 salivarius B37 murinus B57 salivarius B60 plantarum B70 salivarius B74 plantarum B90 salivarius B101 rhamnosus B103 casei group B106 plantarum XB7
[37] [37] [37] et al. [37] [37] et al. [37] et al. et al. et al.
2011 [38]
2011 [38] 2011 [38] 2011 [38] 2011 [38]
a
Nucleotide sequences were analyzed using the Ribosomal Database Project II (RDP-II; http://rdp.cme.msu.edu).
© 2014 John Wiley & Sons Ltd, Helicobacter 19: 144–155
145
Gastric Inflammation Suppressed by L. plantarum
cultures of Lactobacillus were adjusted to an OD600 of 0.1 in MRS broth and incubated anaerobically for 48 hours. Cell free supernatants were collected by centrifugation, filter-sterilized using 0.22 lm surfactantfree cellulose acetate membrane filters (Minisart, Sartorius Stedim Biotech GmbH, Goettingen, Germany), and concentrated by speed vacuum drying (Savant instruments, Farmingdale, NY, USA). Pellets were resuspended in an equal volume of Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco-Invitrogen, Carlsbad, CA, USA) and stored at 20 °C until further use.
Effects of LCM on H. pylori Growth by Disc Diffusion A disc diffusion assay was used to ensure that suppression of IL-8 production from AGS cells was not associated with the inhibition of H. pylori growth by LCM. Sterile 6 mm discs (Whatman, Maidstone, UK) were soaked for at least 1 hour at room temperature in LCM (5% v/v) or MRS (media control). A lawn of H. pylori 43504 was cultured as described above in the presence of LCM or MRS saturated discs and incubated microaerophilically at 37 °C for 72 hours. The zone of inhibition around each disc was measured by a vernier caliper (Mitutoyo, Japan).
Effect of LCM on IL-8 Production in Gastric Epithelial Cells Lactobacillus-conditioned media and H. pylori were cocultured with gastric epithelial cells and IL-8 production was monitored as previously described [38]. Briefly, AGS human gastric adenocarcinoma epithelial cells (ATCC CRL-1739) were maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco-Invitrogen) at 37 °C with 5%CO2 for 48 hours. Adherent cells were detached with 0.25% (v/v) Trypsin (Gibco-Invitrogen) in 1 mmol/L EDTA (Gibco-Invitrogen) and resuspended in fresh media. AGS cells (2.0 9 104 cells) were pre-incubated in a 96-well format for 24 hours. LCM (5% v/v) was added to the cells alone or in combination with viable H. pylori 43504 (6.0 9 106 CFU/well), and co-incubated for 24 hours. Cell culture supernatants were collected by centrifugation and stored at 20 °C until further use.
Effects of LCM on Cytokine Production by ELISA and Luminex Assays Supernatants from co-culture assays were tested for the effects of Lactobacillus secreted factors on IL-8
146
Thiraworawong et al.
production and other related cytokines. IL-8 concentrations in treated and untreated H. pylori-stimulated AGS cell supernatants were measured using Human CXCL8/ IL-8 DuoSet (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. To determine whether Lactobacillus secreted factors modulate cytokines other than IL-8 in this assay, supernatants were tested by Human Cytokine/Chemokine-Premixed 14-plex kit (Millipore, Billerica, MA, USA) in a Luminex 100 system (Luminex Corporation, Austin, TX, USA) for quantification of the following analytes: GM-CSF, IFN-c, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, MCP-1, and TNF-a. Analytes at concentrations exceeding the minimum detectable dose were evaluated and raw data were obtained with MasterPlex CT version 1.2.0.7 and analyzed with MasterPlex QT version 5.0.0.73 (Hitachi MiraiBio, San Francisco, CA, USA).
Analysis of IL-8 Gene Expression by qPCR To quantitate the effects of LCM on IL-8 gene expression, AGS cells (5 9 105 cells/well) were pre-incubated in a 24-well format and treated with viable H. pylori 43504 (1.5 9 108 CFU/well) alone or in combination with LCM (5% v/v), and co-incubated for 4 hours. Cell culture supernatants were removed by centrifugation and total RNA of treated AGS cells was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). IL-8 gene-specific cDNA was synthesized using the SuperScriptâ VILO cDNA Synthesis kit (Invitrogen) [40]. Quantitative PCR was carried out using LightCyclerâ FastStart DNA MasterPLUS SYBR Green I (Roche, Germany) in a LightCyclerâ 2.0 instrument (Roche) according to the manufacturer’s instructions under the following settings: 45 cycles of: denaturation at 95 °C for 10 seconds, annealing at 65 °C for 10 seconds, and extension at 72 °C for 25 seconds. Gene targets were amplified using gene specific primers: IL-8 forward (5′ACA CTG CGC CAA CAC AGA AAT TA-3′) and IL-8 reverse (5′-TTT GCT TGA AGT TTC ACT GGC ATC-3′); GAPDH forward primer (5′-GCA CCG TCA AGG CTG AGA AC-3′), GAPDH reverse primer (5′-ATG GTG GTG AAG ACG CCA GT-3′) [40]. The fold change of IL-8 gene expression relative to GAPDH gene expression was calculated according to the 2 DDCp method [41,42].
Examination of Cell Signaling Pathways by Quantitative Western Blot Lactobacillus-conditioned media-induced changes in the signaling pathway of gastric epithelial cells were analyzed by Western blot as previously described, with
© 2014 John Wiley & Sons Ltd, Helicobacter 19: 144–155
Thiraworawong et al.
minor modifications [24]. AGS cells (2 9 106cells/well) were subjected to co-culture with H. pylori 43504 (6 9 108cells/well) with or without LCM for 0.5, 1, 2, or 4 hours. Culture supernatants were removed by centrifugation, and protein was extracted from whole cell lysates using Mammalian Protein Extraction Reagent (M-PER, Pierce Biotechnology, Rockford, IL, USA) supplemented with Halt protease and phosphatase inhibitors (Pierce Biotechnology) according to the manufacturer’s instructions. Protein concentrations were determined with the Pierceâ BCA Protein Assay Kit (Pierce Biotechnology) according to the manufacturer’s instructions. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Philadelphia, PA, USA) and blocked with 3% bovine serum albumin in TBST (50 mmol/L Tris, pH 7.5, 0.15 mol/L NaCl, 0.05% Tween 20). Blocked membranes were incubated with mouse monoclonal antibodies against NF-kB p65, phospho-NF-kB p65 (Ser 536), c-Jun, phospho-c-Jun and b-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), washed with TBST, and treated with horseradish peroxidaselabeled goat anti-mouse secondary antibodies for 1 hour. Peroxidase signals were detected and imaged by ChemiDocTM XRS (Bio-Rad), and densitometric measurements were completed by ImageJ software (version 1.45s) analysis.
Administration of H. pylori and Lactobacillus plantarum XB7 to Rats The effect of gastric-derived Lactobacillus plantarum XB7 on H. pylori infection and disease pathology was tested in a Sprague–Dawley rat model. The experimental procedures in this study were approved by the Ethics Committee for Animal Care and Use of Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (certificate number: 03/54, research project number: 13/53). Five-week-old male Sprague–Dawley rats (150–200 g) were received from National Laboratory Animal Center, Mahidol University (Bangkok, Thailand) and divided into control, H. pylori-infected, and Lactobacillus-treated groups (eight rats per group). H. pylori infection, treatment with probiotic and assessment of disease pathology was performed according to established protocols with minor modifications [43]. In brief, H. pyloriinfected and Lactobacillus-treated groups were pre-treated with streptomycin (5 mg/mL) for 3 days, then treated with H. pylori ATCC 43504 (108–1010 CFU/mL) by orogastric gavage twice daily for 3 consecutive days. After H. pylori inoculation, the rats were given access to food and water ad libitum for 1 week. Next, the Lactoba-
© 2014 John Wiley & Sons Ltd, Helicobacter 19: 144–155
Gastric Inflammation Suppressed by L. plantarum
cillus-treated groups were gavaged with L. plantarum XB7 (106 CFU/mL) once daily for 7 days and sacrificed. Rats were food-restricted overnight prior to sacrifice.
Macroscopic Assessment of H. pylori-Infected Gastric Tissue Stomachs were collected at time of sacrifice, opened at the side of greater curvature, and washed with PBS. Antral sections (2 mm2) were isolated and used for 1) assessment of H. pylori colonization and 2) cytokine production from tissue. The presence of H. pylori in an antral section was determined by the urease test at room temperature (25 °C) and a change to pink color was observed within 48 hours. Gastric inflammation and H. pylori colonization were determined in a blinded fashion by standard methods for histopathology on fixed stomach tissue (10% formaldehyde, 0.2 mol/L sodium phosphate buffer, pH 7.4). Briefly, fixed tissue was embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) to detect inflammation, and H. pylori colonization. Giemsa staining was performed to detect H. pylori colonization when H&E results were unclear. Gastric inflammation was scored using the updated Sydney System [44]. Mononuclear and polymorphonuclear leukocyte infiltration in the gastric mucosa was scored: Score 0, normal; Score 1, mild; Score 2, moderate; Score 3, marked inflammatory changes. The H. pylori colonization grading scale was: Score 0, no bacteria detected; Score 1, mild colonization in some gastric crypts; Score 2, mild colonization in most gastric crypts; Score 3, moderate colonization in all gastric crypts.
Cytokine Measurements in Serum and Tissue Blood was collected from each rat via cardiac puncture, left at room temperature for 2 hours, and centrifuged (1000 9 g, 4 °C for 20 minutes) to isolate serum. Serum was stored at 20 °C for cytokine analysis. Antral tissue (2 mm2) was homogenized with a micropestle in 500 lL of PBS, supernatant collected by centrifugation (1800 9 g, 4 °C for 10 minutes) and stored at 20 °C until cytokine analysis. Specifically, growth-regulated oncogene/cytokine-induced neutrophil chemo-attractant-1 (GRO/CINC-1), IL-1b, and TNF-a were measured from serum and gastric tissue samples by Quantikine ELISA (R&D Systems) according to manufacturer’s instructions.
Statistical Analysis Data were presented as mean standard error of mean. Statistical significance of in vitro experiments was
147
Gastric Inflammation Suppressed by L. plantarum
determined by Student’s t-test, while one-way ANOVA and Bonferroni’s post hoc test were used for in vivo experiments, and indicated in both cases by a p < .05.
Results Gastric-derived Lactobacillus spp. Suppressed H. pylori-induced IL-8 Production in Gastric Epithelial Cells Lactobacillus conditioned media (LCM) from eleven gastric-derived Lactobacillus spp. (Table 1) were analyzed for the ability to suppress pro-inflammatory cytokine production from H. pylori-stimulated gastric epithelial cells. LCM from eight of the eleven Lactobacillus isolates significantly (p < .05) inhibited H. pylori-induced IL-8 production in AGS cells when compared with MRS control (Fig. 1). LCM from these 8 lactobacilli did not stimulate IL-8 production when they were co-cultured with AGS cells in the absence of H. pylori (data not shown). The addition of LCM from L. plantarum B6, L. murinus B57, and L. plantarum B70 did not affect IL8 production from H. pylori-stimulated AGS cells when compared to control (Fig. 1). Trypan blue dye exclusion and MTT assays indicated that AGS cell viability (>90%) was not compromised by the presence of any LCM tested (data not shown), and disc diffusion methods confirmed LCM did not inhibit proliferation of H. pylori (data not shown).
Figure 1 Gastric-derived Lactobacillus species secrete factors that suppress IL-8 production by Helicobacter pylori-induced AGS cells. LCM from eleven gastric-derived Lactobacillus isolates were tested for the ability to inhibit IL-8 production from AGS cells stimulated with H. pylori. AGS cells were stimulated with H. pylori in the presence of LCM for 24 hours and IL-8 production was monitored by ELISA. The experiments were performed three times in triplicate. The results are expressed as the mean SEM, *p-value < .05, **p-value < .01, and ***p-value < .001 as compared to media control.
148
Thiraworawong et al.
Lactobacillus spp. Suppressed IL-8 Gene Expression Quantitative RT-PCR was performed to determine the effect of LCM from eight Lactobacillus isolates, shown to suppress IL-8 production, on IL-8 transcription. H. pylori-stimulated AGS cells were treated with LCM for 4 hours prior to total RNA isolation. IL-8 gene expression, relative to gapdh, was significantly downregulated approximately 0.5 fold (p < .05) in the presence of LCM from three isolates: L. salivarius B101 (LS-B101), L. rhamnosus B103 (LR-B103), and L. plantarum XB7 (LP-XB7) (Fig. 2). LCM from the remaining 5 isolates did not affect IL-8 gene expression in this assay and were not studied further (Fig. 2).
NF-kB-directed Signaling is Influenced by Lactobacillus Activation of NF-jB and AP-1 by phosphorylation results in an increase in IL-8 gene expression [22–25]. Western blot analysis of phosphorylated NF-kB and c-Jun (AP-1 subunit) from whole cell lysates were conducted to determine Lactobacillus-mediated effects on the activation of NF-jB and AP-1. H. pylori-stimulated AGS cells were treated with LCM from LS-B101, LRB103, and LP-XB7 for 0.5, 1, 2, and 4 hours. The effects of LCM treatment at each time point on concentrations
Figure 2 Anti-inflammatory Lactobacillus secreted factors suppress IL-8 transcription. IL-8 gene expression was determined in AGS cells stimulated with Helicobacter pylori plus medium control, or LCM from eight IL-8-suppressing Lactobacillus after 4 hours. Quantitative realtime PCR was conducted with primers specific to IL-8 and GAPDH transcripts. Gene expression data were normalized to housekeeping gene, GAPDH. The experiments were performed three times in triplicate. Gene expression ratios of IL-8 (LCM/medium control) were calculated, and results represent the mean SEM, *p-value < .05 and **p-value < .01 as compared to the theoretical mean of 1.0.
© 2014 John Wiley & Sons Ltd, Helicobacter 19: 144–155
Thiraworawong et al.
of phospho NF-kB (p-NF-kB) and phospho c-Jun (p-cJun) were directly assessed using monoclonal antibodies specific for each transcription factor. Two-hour treatments with LCM from LS-B101, LR-B103, and LPXB7 resulted in the most significant decrease in p-NFkB as compared to media control (>0.5 fold change; p < .05) (Fig. 3A). Following treatment with LP-XB7 LCM, significantly decreased concentrations of p-c-Jun were observed after 4 hours (0.585 fold change; p < .05) (Fig. 3B). In contrast, this decrease was not A
B
Figure 3 Gastric-derived Lactobacillus suppress activation of Helicobacter pylori-induced transcription factors. Concentrations of activated NF-kB and c-Jun were determined by Western blot on whole cell lysates of AGS cells stimulated with H. pylori plus medium control, or LCM from LS-B101, LR-B103, or LP-XB7. Concentrations were measured at 0.5, 1, 2, or 4 hours using antibodies corresponding to (A) p-NF-jB p65 and NF-jB p65, or (B) p-c-Jun and c-Jun. Relative protein concentrations were determined by densitometry, and activated transcriptions factors were normalized to their nonactivated counterparts (p-NF-jB p65 (Ser 536) to NF-jB p65 levels; p-c-Jun to c-Jun levels). The experiments were performed three times in triplicate. The results are expressed as the mean SEM, *p-value < .05, or **p-value < .01 as compared to media control.
© 2014 John Wiley & Sons Ltd, Helicobacter 19: 144–155
Gastric Inflammation Suppressed by L. plantarum
observed following treatment with either LS-B101 LCM or LR-B103 LCM (Fig. 3B). Because LCM produced by L. plantarum XB7 contributed to a decrease in the activation of both p-NF-kB and p-c-Jun, this isolate was selected for further characterization in H. pylori-infected Sprague–Dawley rats.
L. plantarum XB7 Attenuated Urease Production, Gastric Inflammation, and H. pylori Colonization in a Rat Infection Model An H. pylori-infected Sprague–Dawley rat model was previously developed [43] to study the inflammatory effects of H. pylori infection in the gastric mucosa. We adapted this model to study the potential antiinflammatory effects of L. plantarum XB7 in the context of H. pylori infection. Three groups of eight rats were either 1) infected with H. pylori, 2) infected with H. pylori and treated with LP-XB7, 3) or not infected with H. pylori and not treated with LP-XB7 (control group). Urease production by H. pylori can be detected in tissue by a urease test. H. pylori colonization was initially evaluated in the stomach of sacrificed rats by the presence of urease production and histopathologically. Inflammation was assessed by H&E staining. Stomach tissues from sacrificed H. pylori-infected rats were positive for urease production within 24 hours (mean = 21 hours 29 minutes, range 20 hours 3 minutes–22 hours 26 minutes), while tissues from LP-XB7treated rats were urease-positive within 30 hours (mean = 27 hours 46 minutes, range 24 hours 55 minutes–29 hours 11 minutes) (Table 2). This six-hour delay in urease-positive results between LP-XB7untreated and -treated rats was indicative of fewer H. pylori organisms in the LP-XB7-treated group. Stomach tissues from control rats remained negative for urease after 48 hours (Table 2). The indication that fewer H. pylori organisms colonized the LP-XB7-treated group was confirmed histopathologically. H. pylori colonization scores of 1 (mild, 7 rats) and 2 (moderate, 1 rat) were seen in the H. pylori-infected group, while five of eight rats from the LP-XB7-treated group showed a score of 0 (no H. pylori detected) just as in the control group (Table 2). Within the LP-XB7-treated group, the same five rats with no detectable H. pylori also showed no inflammation by H&E stain (score of 0, Table 2). H&E staining showed that seven of eight rats from the H. pylori-infected group demonstrated a score of 1 (mild gastric inflammation) and the control group showed no histopathologic change (Table 2). Images representative of the H&E staining results are shown in Figure 4. The urease test results, H. pylori colonization and gastric inflammation scores are summarized in Table 2.
149
Gastric Inflammation Suppressed by L. plantarum
Thiraworawong et al.
Table 2 Summary of urease test results, gastric inflammation, and Helicobacter pylori colonization scores H. pylori colonizationc
Gastric inflammationb
Urease test Groupa
+
