We isolated Lactobacillus brevis G-101 from kimchi lactic acid bacteria (LAB) strains, which induced IL-10 expression in lipopolysaccharide (LPS)-stimulated peritoneal macrophages. To evaluate the inflammatory effect of G-101, we examined its inhibitory effect in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitic mice.
Materials and Results
The colitic mice were prepared by intrarectal injection of TNBS. We measured intestinal mucosal cytokines by enzyme-linked immunosorbent assay; activation of transcription factors, by immunoblotting; and macrophage polarization markers, by real-time polymerase chain reaction. Of 200 LAB strains tested, Lact. brevis G-101 showed most potent activity for induction of IL-10 expression in LPS-stimulated peritoneal macrophages. However, it significantly inhibited the expression of TNF-α, IL-1β and IL-6 and the phosphorylation of IRAK1 and AKT, and activated NF-κB and MAPKs. Treatment with TNBS caused colon shortening; increased myeloperoxidase activity; and increased IL-1β, IL-6 and TNF-α expression in mice. Oral administration of Lact. brevis G-101 significantly inhibited these activities. Lactobacillus brevis G-101 inhibited TNBS-induced IRAK-1 phosphorylation and NF-κB activation, as well as the expression of COX-2 and iNOS. Lactobacillus brevis G-101 inhibited the expression of M1 macrophage markers, but increased the expression of M2 macrophages in the colons of TNBS-treated mice.
Lactobacillus brevis G-101 may improve colitis by inhibiting the IRAK1/NF-κB, MAPK and AKT pathways and by polarizing M1 macrophages to M2-like macrophages.
Significance and Impact of the Study
These results suggest that IL-10 expression-inducing LAB can ameliorate colitis by inhibiting NF-κB activation and macrophage polarization.
Acute and chronic inflammations are highly regulated immune processes characterized by the release of cytokines, chemokines and growth factors and by the transmigration of inflammatory cells, such as neutrophils, monocytes and lymphocytes, from the blood to the affected tissue (Collins et al. 1995; Johnson and Koval 2009). Acute inflammation is a normal and helpful response to injury. However, chronic inflammation is persistent and excessive. This inflammatory response causes progressive damages to the body, leading to a variety of diseases, such as colitis, rheumatoid arthritis and even cancer. Of these inflammatory mediators, pro-inflammatory cytokines such as tumour necrosis factor (TNF)-α and interleukin (IL)-1β are activated through nuclear factor-kappaB (NF-κB), and they also activate NF-κB (Collins et al., 1995; Baldwin 1996). However, anti-inflammatory cytokine IL-10 downregulates NF-κB-activated inflammatory pathway. These inflammatory reactions mainly proceed via signalling pathways through toll-like receptors (TLRs) and/or cytokine receptors (David et al. 2010). Among this family of receptors, TLR4, which is associated with the activation of transcription factor NF-κB via IL-1 receptor-associated kinases (IRAKs), may serve as the primary mediator of LPS signalling (Chow et al. 1999; Cario and Podolsky 2000). All IRAK members form multimeric receptor complexes (Takeda and Akira 2004). Phosphorylated IRAK-1 activates a multimeric protein complex composed of TRAF6, TAK1, TAB1 and TAB2, leading to activation of NF-κB and mitogen-associated MAPK pathways, as well as induction of pro-inflammatory cytokines. Regulating expression of these inflammatory mediators can therefore be beneficial in decreasing inflammatory diseases, such as colitis and arthritis (Campieri and Gionchetti 1999; Kawaguchi et al. 2011; Paradkar et al. 2004).
Lactic acid bacteria (LAB) are safe micro-organisms that improve disturbances of the indigenous microbiota (Campieri and Gionchetti 1999; Perdigon et al. 1991; Collins and Gibson 1999), possess antidiabetic effects (Tabuchi et al. 2003), inhibit carcinogenesis (Perdigon et al. 1991), have anticolitic effects (Campieri and Gionchetti 1999; Daniel et al. 2006; Peran et al. 2007a) and induce nonspecific activation of the host immune system (Perdigon et al. 1991). Lactobacillus casei inhibits the expression of inflammatory cytokines in dextran sulfate sodium (DSS)-induced colitic mice (Chung et al. 2007). Lactobacillus casei, Lact. acidophilus, Lact. sontoryeus, Bifidobacterium lactis and Bif. longum show intestinal anti-inflammatory activity in TNBS-induced colitic animals (Peran et al. 2007b; Lee et al. 2009, 2010). Nevertheless, the anticolitic effects of LAB inducing IL-10 expression in macrophages have not been thoroughly examined.
We isolated Lact. brevis G-101, a strain that induced IL-10 expression most potently, from 200 kimchi LAB strains. To evaluate the inflammatory effect of G-101, we examined its inhibitory effects in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitic mice.
Materials and methods
Dulbecco's modified Eagle's medium (DMEM), sodium thioglycollate, tetramethyl benzidine, TNBS, hexadecyl trimethyl ammonium bromide and radio-immunoprecipitation assay (RIPA) lysis buffer were purchased from Sigma Co. (St Louis, MO, USA). The protease inhibitor cocktail was purchased from Roche Applied Science (Mannheim, Germany). Enzyme-linked immunosorbent assay (ELISA) kits were from Pierce Biotechnology, Inc., (Rockford, IL, USA). Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The enhanced chemiluminescence (ECL) immunoblot system was from Pierce Co. (Rockford, IL, USA).
Bacterial strains and growth conditions
Two hundred strains of LAB were isolated from kimchi using MRS agar, this included 85 Lactobacillus sakei strains, 37 Leuconostoc mesenteroides strains, 45 Lactobacillus plantarum strains, 21 Lactobacillus curvatus strains, nine Lactobacillus brevis strains and three Lactobacillus pentosus strains. Isolated LAB strains were indentified using the Gram stain kit (BioMerieux, Grenoble, France) following the protocol developed by Jung et al. (2012). Additional enzyme activities and biochemical characteristics were determined using the API 20E test strips (BioMerieux, Seoul, Korea). Lactobacillus brevis G-101 was acid tolerant (can survive for 12 h at pH 4). The 16S ribosomal DNA was amplified by PCR using 27F, 1492R primer, followed by purification using the purification kit (Bionics Inc., Seoul, Korea), and the purified 16S ribosomal DNA was sequenced using ABI 3730XL DNA analysis.
For macrophage experiment, Lact. brevis G-101 was anaerobically grown at 37°C in MRS broth without shaking, collected by centrifugation (10 000 g for 30 min) and washed twice with saline. The resulting pellet was suspended in phosphate-buffered saline. The cell suspension (5 ml) was placed in a 50-ml centrifuge tube, heated in boiling water bath for 10 min and used for experiments on its effect in LPS-stimulated peritoneal macrophages.
For mouse experiment, Lact. brevis G-101 was grown to an optical density between 3 and 4 at 600 nm (early stationary phase), harvested by centrifugation (10 000 g for 30 min) and washed with phosphate-buffered saline (PBS). The collected cells (1 × 108 and 1 × 109 CFU) suspended in 50 mmol l−1 NaHCO3 buffer containing 1% glucose were orally administered to mice (Lee et al. 2009).
Male ICR mice (5 weeks old, 20–25 g) were supplied from Orient Experimental Animal Breeding Center (Seoul, Korea). The mice were housed six per cage, allowed access to water and standard laboratory chow (Orient Experimental Animal Breeding Center) ad libitum and maintained at 20–22°C, 50 ± 10% humidity and a 12-h diurnal light cycle (lights on 07:00–19:00 h) prior to testing. All experiments were performed in accordance with the NIH and Kyung Hee University guidelines for Laboratory Animals Care and Use and approved by the Committee for the Care and Use of Laboratory Animals in the College of Pharmacy, Kyung Hee University.
Isolation and culture of peritoneal macrophages
Mice were intraperitoneally injected with 2 ml of 4% thioglycolate solution and sacrificed 4 days after injection, and the peritoneal cavities were swilled with 10 ml of RPMI 1640 (Joh et al. 2011). The peritoneal lavage fluids were centrifuged at 300 g for 10 min, and the cells (2 × 107 cells) were resuspended in RPMI 1640 (5 ml) and plated. After incubation at 37 °C for 2 h, the cells were washed three times and nonadherent cells were removed by aspiration. The cells were cultured in 12-well plates at 37°C in RPMI 1640 with 10% FBS (1% antibiotic-antimycotic; Life Technologies, Grand Island, NY, USA). The attached cells (1 × 106 cells per well) were used as peritoneal macrophages. To examine the anti-inflammatory effects of Lact. brevis G-101 (1 × 103, 1 × 104 and 1 × 105 CFU per well), peritoneal macrophages were incubated in the absence or presence of Lact. brevis G-101 with LPS for 24 h.
Preparation of experimental colitic mice
Male ICR mice were randomly divided into five groups: normal and TNBS-induced colitic groups treated with or without Lact. brevis G-101 or mesalazine (10 mg kg−1). Each group is consisted of 6 mice. TNBS-induced colitis was induced by the administration of 2·5% (w/v) TNBS solution (100 μl) in 50% ethanol into the colon of lightly anesthetized mice via a thin round-tip needle equipped with a 1-ml syringe (Joh et al. 2011). The normal group was treated with vehicle alone. The needle was inserted so that the tip was 3·5–4 cm proximal to the anal verge. To distribute the agents within the entire colon and caecum, mice were held in a vertical position for 30 s after the injection. If an animal quickly excreted the TNBS–ethanol solution, it was excluded from the remainder of the study. Lactobacillus brevis G-101 [(1 × 108 or 1 × 109 CFU) per mouse] was orally administered once a day for 3 days after TNBS treatment. The mice were anaesthetized with aether and sacrificed 20 h after the final administration of LAB. The colon was quickly removed, opened longitudinally and gently cleared of stool by PBS. Macroscopic assessment of the disease grade was scored according to a previously reported scoring system (0, no ulcer and no inflammation; 1, no ulceration and local hyperaemia; 2, ulceration without hyperaemia; 3, ulceration and inflammation at one site only; 4, two or more sites of ulceration and inflammation; 5, ulceration extending more than 2 cm) (Lee et al. 2011), and the colon tissue was then used for immunoblot and enzyme-linked immunosorbent assay (ELISA) analysis.
Assay of myeloperoxidase activity in colon
The colons were homogenized in 10 mmol l−1 potassium phosphate buffer (pH 7·0) containing 0·5% hexadecyl trimethyl ammonium bromide, and then centrifuged for 30 min (20 000 g; 4°C). The supernatant was used as a crude enzyme solution. An aliquot (50 μl) of the supernatant was added to a reaction mixture of 1·6 mmol l−1 tetramethyl benzidine and 0·1 mmol l−1 H2O2 and incubated at 37°C. The absorbance of the reaction mixture was obtained at 650 nm over time. Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 μmol ml−1 of peroxide at 37°C and expressed in unit per mg protein (Mullane et al. 1987). The protein content was assayed by the Bradford method (Bradford 1976).
ELISA and immunoblot analysis in peritoneal macrophages
For the assay of cytokines, the peritoneal macrophages (5 × 105 cells) were stimulated with LPS (50 μg ml−1; Invitrogen, CA, USA) for 30 min and 20 h in the presence or absence of Lact. brevis G-101 (1 × 103, 1 × 104 and 1 × 105 CFU per well), lysed ice-cold RIPA lysis buffer containing 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail and centrifuged at 2000 g for 10 min. In addition, colons were homogenized and lysed with ice-cold RIPA lysis buffer. Then the lysates were centrifuged (15 000 g, 4°C) for 15 min, and the supernatant was transferred to 96-well ELISA plates, and the cytokines (IL-10, TNF-α, IL-1β, IL-6) were measured by ELISA kits according to the manufacture's protocol.
For the immune blotting, the lysates of macrophages and colon tissues were separated by 10% SDS–PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dried-milk proteins in 0·05% PBST, then probed with COX-2, iNOS, p-IRAK-1, IRAK-1, p65, p-p65, EKR, p-ERK, p38, p-p38, JNK, p-JNK, AKT, p-AKT or β-actin antibody. After washing with PBST, proteins were detected with HRP-conjugated secondary antibodies for 50 min. Bands were visualized with enhanced chemiluminescence reagent (Joh et al. 2011).
Real-time polymerase chain reaction (RT-PCR)
Total RNAs were extracted from the colon tissues with the RNeasy Mini kit (Qiagen, Hilden, Germany), and first-strand cDNA synthesis for arginase (ARG) I, II, TNF-α, IL-1β, IL-10, CD206 and β-actin was performed using reverse transcriptase (Takara, Shiga, Japan) according to the manufacturer's protocol. Real-time PCRs were performed on the Rotor-Gene Q® (Qiagen) using DNA polymerase (Takara) and SYBR Green I (Qiagen) in a reaction volume of 20 μl. Primers used for real-time PCR are listed in Table 1. The normalized expression of the target gene, with respect to β-actin, was computed for all samples using Microsoft Excel, as previously reported (Kim et al. 2012),
Table 1. Primers of polarization markers for Real-time polymerase chain reaction
Forward primer sequence
Reverse primer sequence
All data are expressed as the mean ± standard deviation (SD), with statistical significance analysed using one-way anova followed by a Student–Newman–Keuls test (P <0·05).
Lactobacillus brevis G-101 induces IL-10 expression in LPS-stimulated peritoneal macrophages
We measured the ability of 200 LAB strains isolated from kimchi to induce IL-10 expression in LPS-stimulated peritoneal macrophages. LPS treatment significantly reduced IL-10 expression, but induced the expression of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 (Fig. 1). Of these LAB strains (heat-treated), Lact. brevis G-101 induced IL-10 expression reduced by LPS most potently (data not shown). Furthermore, Lact. brevis G-101 also inhibited LPS-induced NF-κB and AP1 activation. Treatment with Lact. brevis G-101 (1 × 105 CFU ml−1) in LPS-stimulated peritoneal macrophages significantly reversed IL-10 expression to 93% of normal control group.
Anti-inflammatory effect of Lactobacillus brevis G-101 in TNBS-induced colitic mice
We examined the ability of Lact. brevis G-101 to inhibit colitis induced by intrarectal injection of TNBS. Oral administration of TNBS induced loss of body weight and severe inflammation, and manifested in the form of shortened, thickened and erythematous colons (Fig. 2). Histological examination of the TNBS-treated colon showed massive bowel oedema, dense infiltration of the superficial layers of the mucosa and epithelial cell disruption due to large ulcerations. Lactobacillus brevis G-101 treatment inhibited body weight reduction, colon shortening and inflammation and thickening on the third day after TNBS treatment. Lactobacillus brevis G-101 treatment inhibited TNBS-induced MPO activity, a representative inflammatory marker. The efficacy of Lact. brevis G-101 was comparable to that of mesalazine, a commercial drug.
TNBS also increased the activation of NF-κB, MAPKs and AKT, as well as the expression of COX-2 and iNOS (Fig. 3). Lactobacillus brevis G-101 blocked the induction of p-IRAK-1, p-p65, p-p38, p-ERK, p-JNK and p-AKT by TNBS. Lactobacillus brevis G-101 also inhibited TNBS-induced iNOS and COX-2 expression. Lactobacillus brevis G-101 (1 × 109 CFU) was comparable to that of mesalazine (10 mg kg−1). We also measured the levels of the pro-inflammatory cytokines, namely TNF-α, IL-1β, IL-6 and IL-10, in the colon of TNBS-induced colitic mice by ELISA (Fig. 4). TNBS increased the protein expression of IL-1β, IL-6 and TNF-α by 5·6-, 4·0- and 10·5-folds, respectively; however, it reduced IL-10 expression. Lactobacillus brevis G-101 treatment increased anti-inflammatory cytokine IL-10 expression and reduced the expression of the pro-inflammatory cytokines, namely IL-1β, IL-6 and TNF-α. Treatment with Lact. brevis G-101 (1 × 109 CFU per mice) inhibited the expression of these cytokines by 89, 69 and 73%, respectively, but reversed IL10 expression to 89% of the normal control group.
We measured the effect of Lact. brevis G-101 on the expression of TNF-α, IL-1β, IL-10, ARG I, ARG II and CD206, which are the markers of M1/M2 macrophages (Ambarus et al. 2012), in TNBS-induced colitic mice by real-time PCR (Fig. 5). Treatment with TNBS increased the expression of ARG II, IL-1β and TNF-α, which are the markers of M1 macrophages, but reduced the expression of ARG I, CD206 and IL-10 expression. Treatment with Lact. brevis G-101 in TNBS-treated mice blocked the expression of the M1 macrophage markers, namely ARG 1, TNF-α and IL-1β, but increased the expression of the M2 macrophage markers, namely ARG II, IL-10 and CD206.
IBD does not progress significantly in germ-free animals (Chandran et al. 2003), indicating that intestinal microflora, which comprise approx. 1000 different bacterial species (Benno et al. 1993; Chandran et al. 2003), may play an important role in initiating and perpetuating colonic inflammation. Of intestinal microbiota, gram-negative bacteria, including Enterobacteriaceae, are significantly increased in the colitic patients, as well as in mice treated with colitic inducers such as TNBS and dextran sulfate sodium. These gram-negative bacteria produce LPS, a bacterial endotoxin. The LPS activate the biosynthesis of diverse mediators of inflammation, such as TNF-α, IL-1β, and IL-6, via a Toll-like receptor (TLR) 4-linked NF-κB, MAPK, and AKT pathways in macrophages (Aderem and Ulevitch 2000). LPS reduces IL-10 expression. To improve IBD, inclusion of dietary ingredients regulating LPS signalling, such as β-sitosterol and VSL#3, has recently gathered attention (Sartor 2004; Lee et al. 2012). Of them, LAB suppress the growth of pathogens, improve gut microbiota disturbance (Perdigon et al. 1991; Campieri and Gionchetti 1999), and improve IBD (Campieri and Gionchetti 1999; Chung et al. 2007; Peran et al. 2007a; Lee et al. 2009, 2010). These LAB strains inhibit the expression of pro-inflammatory cytokines by regulating NF-kB activation [Lee et al. 2009, 2010]. However, IL-10 expression-inducing LAB was not studied thoroughly.
Therefore, in the present study, we screened for IL-10 expression-inducing LAB among the LAB strains isolated from kimchi. Then, we selected Lact. brevis G-101 (heat-treated), which significantly increased IL-10 expression in LPS-stimulated macrophages. Lactobacillus brevis G-101 inhibited the expression of the pro-inflammatory cytokines, namely, TNF-α, IL-1β, and IL-6, and the activation of their transcription factors NF-κB and AP1. Lactobacillus brevis G-101 ameliorated inflammatory markers (shortening of the colon, increase of MPO activity and pro-inflammatory cytokines, and activation of NF-κB) in TNBS-induced colitic mice, as reported previously (Lee et al. 2009, 2010). Furthermore, Lact. brevis G-101 inhibited the phosphorylation of MAPKs, which is activated by TAK1 in TLR4/NF-κB pathway, and AKT, which is phosphorylated by MyD88-PI3K signalling, in TNBS-induced colitic mice. These results suggest that Lact. brevis G-101 may inhibit the inflammation by regulating the signalling pathway of upstream molecule(s) of IRAK1 such as TLR4. Additionally, G-101 polarized TNBS-induced M1 macrophages, which express TNF-α, IL-1β, and ARG I, to M2 macrophages, which express IL-10, ARG II and CD206. G-101 may be able to reverse a series of molecular, cellular and immunological responses observed during the inflammation process in vivo and then polarize LPS-stimulated macrophages to M2 macrophages.
Based on these findings, Lact. brevis G-101, which induces IL-10 expression in LPS-stimulated macrophages, may be able to improve colitis by inhibiting TLR-4-linked NF-κB, MAPK and AKT signalling pathways and polarizing M1 macrophages to M2 macrophages,