Lactobacillus plantarum C29 alleviates NF-κB activation and Th17/Treg imbalance in mice with TNBS-induced colitis

ABSTRACT In this study, we examined whether Lactobacillus plantarum C29 could restore 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced T helper 17 (Th17)/regulatory T cells (Tregs) imbalance in mice. Treatment with C29 inhibited the differentiation of splenic T cells into Th17 cells and the expression of retinoic acid receptor-related orphan receptor gamma t (RORγt) and IL-17 in vitro, whereas promoting the differentiation into Tregs. Oral administration of Lactobacillus plantarum C29 in mice attenuated TNBS-induced colon shortening, myeloperoxidase (MPO) activity, inducible Nitric oxide (NO) synthase, and cyclooxygenase-2 expression, and activation of NF-κB in the colon of mice. C29 treatment downregulated TNF-α, IL-17, and IL-1β expression, while increasing IL-10 expression. C29 treatment suppressed TNBS-induced Th17 cell differentiation and reduced IL-17 and RORγt expression, while promoting the TNBS-suppressed Tregs differentiation and IL-10 and forkhead box P3 expression. These findings suggest that C29 can alleviate colitis by modulating NF-κB activation as well as Th17/Treg balance.


Introduction
Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, is a chronically relapsing inflammatory disease of the gastrointestinal (GI) tract (Maloy & Powrie, 2011). The pathogenesis of IBD involves genetic susceptibility and host innate and adaptive immunity (Du et al., 2015;Niess, Leithäuser, Adler, & Reimann, 2008). The stimulation of pathogens in the GI tracts is continuously defended by the gut immune system, which consists of neutrophils, macrophages, dendritic cells (DCs), and T cells involved in innate and adaptive immunity (Du et al., 2015;Niess et al., 2008;Nourshargh & Alon, 2014). Activation of innate immune cells, including DCs and macrophages, by these antigens stimulates the adaptive immune cells such as T cells: the secretion of TNF-α, IL-10, and IL-12 in the immune cells stimulate the differentiation of naïve CD4+ T cells into effector T cells, such as Th1, Th17, and regulatory T cells (Tregs) (Atreya, Atreya, & Neurath, 2008;Owen & Mohamadzadeh, 2013;Rutella & Locatelli, 2011). TNF-α, IL-12, and IL-17 are highly expressed in the inflamed colons of mice and humans with IBD; however, IL-10 expression is down-regulated, leading to colitis (Kaistha & Levine, 2014;Leppkes et al., 2009). Therefore, the down-regulation of IL-12 and TNF-α expression and up-regulation of IL-10 expression may be important for the prevention and treatment of colitis.
Therefore, in the present study, to understand the effect of C29 on the T cell differentiation, we investigated its effect on the differentiation of splenocytes into Th17/Treg cells and TNBS-induced Th17/Treg imbalance in mice.
Briefly, C29 was cultured at 37°C for 24 h in MRS broth (10 mL), successively inoculated in MRS broth (2 L) and cultured at 37°C to an optical density between 1.5 and 2 at a wavelength of 600 nm. Cells were harvested by centrifugation (10,000 g for 20 min), washed with saline, and freeze-dried. Freeze-dried cells were suspended in 1% glucose for the in vivo study (oral gavage in mice), or in 20 mM phosphate buffer (pH7.0) for the in vitro study (experiments in Th cells isolated from spleens).

Animals
Male C57BL/6 mice (20-22 g, 6-week old) were purchased from RaonBio Inc. (Seoul, Korea). Animals were acclimatized for 7 days before experiments. All mice were housed in wire cages at 20-22°C and 50 ± 10% humidity and fed with standard laboratory chow and water ad libitum. All animal experiments were approved by the Committee for the Care and Use of Laboratory Animals in the Kyung Hee University (IRB No., KHUASP(SE)-16-027) and performed in accordance with the Kyung Hee University Guidelines for Laboratory Animal Care and Usage.

Preparation of mice with TNBS-induced colitis and memory impairment
Mice were divided into four groups to measure the effective dose of C29 against TNBSinduced colitis and memory impairment. Each group consisted of 6 mice. Animals were anesthetized with ether and 2.5% (w/v) TNBS solution (100 μL, dissolved in 50% ethanol) was intrarectally administered into the colon 3.5-4 cm proximal to the anus of mice, apart from mice in the first and the second groups (Jang et al., 2014). Mice were held in a vertical position for 30 s after TNBS administration. Mice in the first and second groups were treated with saline instead of TNBS. After 24 h from the administration of the TNBS treatment, test agents (1 × 10 9 CFU/mouse of C29 for the third and 50 mg/kg of sulfasalazine for the fourth group) were orally administered once a day for 3 days. Mice in the first and third groups were vehicle treated (1% dextrose). Their colons removed, longitudinally opened, and macroscopically scored (grade 0, no ulcer and inflammation; grade 1, no ulceration and local hyperaemia; grade 2, ulceration with hyperaemia; grade 3, ulceration and inflammation at one site only; grade 4, two or more sites of ulceration and inflammation; and grade 5, ulceration extending more than 2 cm) . Colon tissues were washed with ice-cold phosphate-buffered saline, divided into eight segments, and used for other experiments. Colon tissues were stored at −80°C for immunoblotting and ELISA analysis. Histological examination of colon tissues (first and fifth segments) was performed according to the method of Jang et al. (2014).

Myeloperoxidase (MPO) activity assay
Colon tissues (second and sixth segments) were homogenized in 10 mM potassium phosphate buffer (pH 7.0) containing 0.5% hexadecyl trimethyl ammonium bromide, and centrifuged for 10 min (20,000 × g at 4°C) . The supernatant (50 μL) was incubated with 0.75 mL of the pre-incubated reaction mixture containing 0.1 mM hydrogen peroxide and 1.6 mM TMB at 37°C for 5 min and periodically monitored for its absorbance at a wavelength of 650 nm.

Immunoblotting and ELISA
Colon (third and seventh segments) stored at −80°C were homogenized on ice in 1 mL of RIPA lysis buffer containing 1% each of phosphatase and protease inhibitor cocktail and centrifuged at 15,000 × g for 15 min at 4°C (Jang et al., 2014). Immunoblotting analysis and cytokines assays were performed according to the method of Kim, Gu, Lee, Joh, and Kim (2012).

Flow cytometric analysis of Th17 and Tregs in the lamina propria of the colon
Colon tissues (fourth and eighth segments) were cut into small pieces and agitated in 2.5 mM ethylenediaminetetraacetic acid (EDTA) at 37°C for 20 min. Samples treated with EDTA were minced, digested at 37°C for 20 min in RPMI containing 1 mg/mL type VIII collagenase, and lamina propria cells were separated Roy & Kumar, 2015). T cells were purified using the Pan T cell Isolation Kit II. Cells were fixed and stained with anti-Foxp3 or anti-IL-17A antibodies, and the number of Th17 and Treg cells was estimated using a flow cytometer.

Statistical analysis
All experimental data are indicated as the mean ± standard deviation (SD) and statistical significance was analyzed using one-way ANOVA followed by a Student-Newman-Keuls test (P < .05).

C29 suppressed the differentiation of splenic Th cells into Th17 cells and induced the differentiation into Tregs
To investigate whether C29 could modulate the T cell differentiation involved in the adaptive immunity, we purified Th cells from mouse splenocytes, stimulated them with Th17differentiating cytokines (anti-CD3, anti-CD28, rIL-6, and rTGF-β) in the presence or absence of C29, and measured subsets of Th17 cells as well as the expression of the Th17-specific transcription factor RORγt and cytokine IL-17 using a flow cytometer and qPCR (Figure 1). The cytokine treatment significantly induced the differentiation of Th17 cells and increased the expression of RORγt and IL-17. In contrast, C29 treatment (1 × 10 5 CFU/mL) significantly inhibited the differentiation of Th cells into Th17 cells and the expression of RORγt and IL-17 by 52.3%, 60.1%, and 62.0%, respectively. However, C29 treatment (1 × 10 5 CFU/mL) showed a 1.68-fold increase in the differentiation of Th cells into Tregs as well as a 1.21-and a 1.76-fold increase in the expression of Foxp3 and IL-10, respectively.

C29 alleviated TNBS-induced colitis in mice
C29 potently inhibited the Th17 cell differentiation and induced Treg differentiation in vitro. Therefore, to confirm whether C29 could regulate the T cell differentiation, we orally treated C29 at a dose of 1 × 10 9 CFU/mouse in mice with TNBS-induced colitis and investigated its anti-inflammatory effects involved in the innate and adaptive immunities. TNBS treatment significantly caused colon shortening and increased MPO activity in the colon. Histological examination revealed the disruption of epithelial cells in the colon by TNBS treament, attributed to inflammation and ulceration ( Figure 2). However, treatment with C29 significantly inhibited TNBS-induced colon shortening and MPO activity as well as the infiltration of neutrophils in the colon. Treatment with C29 or sulfasalazine increased TNBS-suppressed expression of colonic tight junction proteins, such as claudin-1, occludin, and ZO-1. We also addressed the effect of C29 on the expression of inflammatory markers. TNBS treatment induced the activation of NF-κB and increased the expression of iNOS, and COX-2 ( Figure 3). Conversely, treatment (c) Effect on Foxp3 and IL-10 expression using qPCR. qPCR values (fold changes), which was compared to that of normal control group, were indicated. All data indicate mean ± SD (n = 3); # P < .05 vs. normal control group; *P < .05 vs. group stimulated with anti-CD3/anti-CD28 and IL-6/TGF-β. with C29 had the opposite effect, leading to the inhibition in expression for TNBS-induced markers such as COX-2 and iNOS and to the activation of NF-κB. Furthermore, C29 treatment suppressed TNBS-induced expression of TNF-α, IL-1β, IL-6, and IL-17 and increased IL-10 expression ( Figure 4).
Next, we examined the effect of C29 on the Th17 and Treg cell differentiation in mice with TNBS-induced colitis. Treatment with TNBS increased the differentiation of Th cells into Th17 cells and suppressed their differentiation into Tregs in the colon ( Figure 5). On the contrary, treatment with C29 inhibited TNBS-induced Th17 differentiation and increased TNBS-suppressed Treg differentiation. Furthermore, we confirmed the differentiation of Th17 and Tregs by evaluating the expression levels of Th17 and Treg markers such as IL-10, IL-17, RORγt, and Foxp3 using qPCR. Treatment with C29 significantly inhibited TNBS-induced expression of IL-17 and RORγt in the colon and increased TNBS-suppressed expression of Foxp3 and IL-10. The effect of C29 (1 × 10 9 CFU/ mouse) was similar to that exerted by the treatment with sulfasalazine (50 mg/kg).
The present study showed that oral administration of C29 improved the symptoms of TNBS-induced colitis in mice, suppressed colon shortening, and decreased MPO activity and iNOS and COX-2 expression. C29 also restored TNBS-suppressed tight junction protein ZO-1, occludin, and claudin-1. Moreover, C29 suppressed TNBS-induced differentiation of Th17 cells and t downregulated the expression of IL-17 and that of RORγt, a Th17-specific transcription factor. However, C29 treatment increased TNBS-suppressed Treg cell differentiation and enhanced the expression of IL-10 and of the Treg transcription factor Foxp3. These results suggest that C29 could modulate the innate immune response, as well as the adaptive immune responses including helper T cell differentiation. It was supported by the present study that C29 increased the differentiation of splenic Th cells into Tregs and enhanced the expression of IL-10 and Foxp3 in vitro, while it inhibited the differentiation of Th cells into Th17 cells and the expression of IL-17, TNF-α, and RORγt and the previous study that C29 inhibited LPS-induced NF-κB activation in macrophage cells.
Lactobacillus rhamnosus RC007 and Lactobacillus plantarum 21 improved TNBSinduced colitis in mice and rats, respectively, through the inhibition of TNF-α expression (Dogi, García, De Moreno de LeBlanc, Greco, & Cavaglieri, 2016;Satish Kumar et al., Figure 5. Effects of C29 and sulfasalazine on the differentiation of Th17 and Tregs and expression of their transcription factors and cytokines in mice with TNBS-induced colitis. (A) Effects on Th17 differentiation (a) and RORγt (b) and IL-17 expression (c). (B) Effects on Treg differentiation (a) and Foxp3 (b) and IL-10 expression. RORγt, Foxp3, IL-10, and IL-17 were assessed by qPCR. qPCR values (fold changes), which was compared to that of normal control group, were indicated. All values are mean ± SD (n = 6). # P < .05 vs. normal control group. *P < .05 vs. group treated with TNBS alone. 2015). Studies have reported that Lactobacillus brevis K65 and Lactobacillus paracasei ameliorated DSS-induced ulcerative colitis in mice through the suppression of TNF-α expression (Liu et al., 2016;Pan et al., 2014). Lactobacillus brevis G-101 attenuated colitis by inhibiting TNF-α and IL-1β expression, and by increasing IL-10 expression (Jang et al., 2013). These results suggest that these probiotic organisms may mitigate colitis through the inhibition of innate immune responses such as macrophage activation that stimulates TNF-α and inhibits IL-10 expression. However, Lactobacillus plantarum CLP-0611 attenuated colitis in mice through M1 to M2 macrophage polarization induced via an increased IL-10 expression (Jang et al., 2014). Miyauchi et al. reported that Bifidobacterium longum alleviates inflammatory diseases by suppressing IL-17 expression (Miyauchi et al., 2013). In the previous study, we also reported that C29 inhibited LPS-induced NF-κB activation in macrophages, resulting in the attenuation of colitis in aged mice (Jeong et al., 2015). These results suggest that some lactobacilli can regulate the expression of TNF-α, IL-10, and IL-17, as well as the differentiation of T cells, leading to the attenuation of chronic inflammatory diseases such as colitis. Conclusively, C29 may ameliorate colitis by inhibiting NF-κB activation and restoring the imbalanced Th17/Treg cells.

Disclosure statement
No potential conflict of interest was reported by the authors.