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Laquinimod ameliorates spontaneous colitis in interleukin-10-gene-deficient mice with improved barrier function
International Immunopharmacology 29 (2015) 423–432
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Laquinimod ameliorates spontaneous colitis in interleukin-10-gene-deficient mice with improved barrier function Jing Sun 1, Xiao Shen 1, Jianning Dong, Jie Zhao, Lugen Zuo, Honggang Wang, Yi Li, Weiming Zhu ⁎, Jianfeng Gong, Jieshou Li Department of General Surgery, Jinling Hospital, Medical School of Nanjing University, 305 East Zhongshan Road, Nanjing, Jiangsu Province 210002, China
a r t i c l e
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Article history: Received 19 May 2015 Received in revised form 16 October 2015 Accepted 16 October 2015 Available online 23 October 2015 Keywords: Crohn's disease IL-10 gene-deficient mice Laquinimod Intestinal barrier function NF-κB pathway
a b s t r a c t Background and aims: Crohn's disease is an autoimmune disease associated with imbalanced mucosal immunity, mediated with increased Th1 and Th17 cells. Laquinimod, an immunomodulatory drug, has shown efficacy in regulating the differentiation of T cells. The aim of the study was to investigate the therapeutic effect of laquinimod on spontaneous colitis in interleukin-10-gene-deficient mice, an animal model of Crohn's disease. Methods: Male Il10−/− mice aged 16 weeks in the laquinimod group were treated with laquinimod with distilled water at a dose of 25 mg/kg by oral gavage, 3 times a week. Il10−/− mice in the IL-10-KO group and wild type mice received equal volume of phosphate buffered saline by oral gavage, 3 times a week. After 4 weeks, mice were sacrificed for analysis. Severity of colitis, epithelial expression of T-cell-associated cytokines, expression and distribution of tight junction proteins in the lamina propria and NF-κB signaling pathway associated mRNA expression were measured at the end of the experiment. Results: Laquinimod treatment ameliorated spontaneous colitis in Il10−/− mice, which was associated with decreased T-cell-associated pro-inflammatory cytokines. Increased expression and correct distribution of tight junction proteins (occludin and ZO-1) were found in Il10−/− mice treated with laquinimod. In addition, in mice treated with laquinimod, NF-κB signaling pathway associated mRNA in the colon was also downregulated. Conclusions: Our results indicated that laquinimod treatment ameliorates colitis in Il10−/− mice and improves intestinal barrier function, which may support a new therapeutic approach to Crohn's disease. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Crohn's disease (CD) is a chronic relapsing inflammatory disorder characterized by chronic inflammation and mucosal tissue damage of the gastrointestinal tract. Although the precise etiology of Crohn's disease remains unclear, research has thus far demonstrated that CD is caused by a combination of genetic, environmental and immunoregulatory factors [1–3]. Increasing evidence suggests that an imbalanced mucosal barrier may play an important role in the pathogenesis and disease progression of CD. Massive numbers of microorganisms reside in the gut lumen, and the intestinal mucosa constitutes an immunologic organ, which tolerates and defends against harmful organisms [4]. Epithelial cells and tight junctions (TJ) comprise the largest number of intestinal barriers [5,6]. When the epithelium is intact, intestinal barrier function is largely defined by the construction and expression of TJ as well as intestinal barrier permeability characteristics [7]. In Crohn's disease, the intestinal barrier at the interface between the intestinal microbiome and the lymphoid tissue plays a critical role in ⁎ Corresponding author. E-mail address: [email protected] (W. Zhu). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.intimp.2015.10.019 1567-5769/© 2015 Elsevier B.V. All rights reserved.
shaping the mucosal immune response. In this situation, the intestinal barrier is impaired, and bacterial products and dietary antigens cross the epithelium and enter the lamina propria. Antigen-presenting cells (APCs) take up foreign materials and regulate the differentiation of T cells [8]. For many years, it has been assumed that Crohn's disease is a T helper 1 (Th1)-cell-mediated disease [9], and a novel subset of IL17-producing CD4+ Th cells, Th17 cells, have more recently been implicated in the pathogenesis of CD [10]. In the most appropriate model of human CD, interleukin-10-gene-deficient (Il10−/−) mice [11] exhibit increases in CD4 + Th1 and Th17 cells, which secrete a large number of pro-inflammatory mediators such as tumor necrosis factor-α (TNFα), IL-1β, interferon-γ (IFN-γ), and IL-17A [12,13]. Laquinimod (C19H17ClN2O3, 5-chloro-N-ethyl-4-hydroxy-1-methyl-2-oxo-N-phenyl-1, 2-dihydroquinoline-3-carboxamide) is an immunomodulatory drug that has extensively shown its efficacy in inflammatory and autoimmune disorders, especially multiple sclerosis (MS) [14]. Laquinimod is a small molecule that concentrates in the peripheral immune system as well as in the central nervous system (CNS) [15]. In MS model mice, laquinimod has shown immunomodulatory effects by downregulating Th1 and Th17 cells. Preclinical data suggest that laquinimod has a direct inhibitory effect on antigen-presenting cells and results in anti-inflammatory T cell polarization manifested by a
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Table 1 Primer for polymerase chain reaction. TNF-α IFN-γ IL-1β IL-17A GAPDH
Forward: Reverse: Forward: Reverse: Forward: Reverse: Forward: Reverse: Forward: Reverse:
5′-TGGGAGTAGACAAGGTACAACCC-3′ 5′-CATCTTCTCAAAATTCGAGTGACAA-3′ 5′-AACGCTACACACTGCATCTTGG-3′ 5′-GCCGTGGCAGTAACAGCC-3′ 5′-CAACCAACAAGTGATATTCTCCATG-3′ 5′-GATCCACACTCTCCAGCTGCA-3′ 5′-GCTCCAGAAGGCCCTCAGA-3′ 5′-AGCTTTCCCTCCGCATTGA-3′ 5′-AGGCCGGTGCTGAGTATGTC-3′ 5′-TGCCTGCTTCACCACCTTCT-3′
reduction in the frequencies of pro-inflammatory Th1 (IFN-γ+CD4+) cells and Th17 (IL-17A+CD4+) cells [16]. Thus, the proposed mechanism of laquinimod may make it ideally suited to reduce gastrointestinal inflammation. However, the effects of laquinimod on colitis have been heretofore unstudied. In this investigation, we determined whether laquinimod ameliorates established spontaneous colitis in Il10−/− mice, and we attempted to explain the potential mechanisms that account for these effects. 2. Materials and methods 2.1. Animals Both wild type (WT) and Il10−/− mice on a C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were bred and maintained under specific pathogen-free (SPF) conditions at the Model Animal Research Center of Nanjing University (Nanjing, China). Previous experiment has demonstrated that most Il10−/− mice on the C57BL/6 strain under SPF conditions develop spontaneous colitis at 12 weeks of age [17]. The mice used in our study were 16 weeks of age. The experimental procedures were performed in accordance with the Guidelines for Animal Experiments at Jinling Hospital and were approved by the Ethics Committee of Jinling Hospital (Jiangsu, China). 2.2. Drug treatment of mice Wild-type and Il10−/− mice were divided into wild-type group (WT, wild-type mice), control group (IL-10-KO, Il10−/− mice) and treatment group (laquinimod, Il10−/− mice), and each group contains 6 mice. To investigate the effects of laquinimod on wild type mice, 6 wild type mice (control group) also received laquinimod treatment. Laquinimod (TEVA Pharmaceuticals Industries, Ltd (Israel), purity N 98%) was dissolved in distilled water and administered 25 mg/kg by oral gavage, 32 times a week. As reported in a study by Lourenço EV et al. [16], we found that this dose was well tolerated in mice, and no obvious macroscopic adverse effects were observed. Mice in the IL-10-KO group and
the WT group received an equal volume of phosphate buffered saline (PBS) by oral gavage. Four weeks after administration, the mice were sacrificed by an overdose of anesthesia with pentobarbital sodium, and proximal colons were collected for experiments. 2.3. Weight and disease activity index (DAI) Animals were weighed on a balance, and measurements were recorded to the nearest 0.1 g. The Disease Activity Index (DAI) was recorded as a composite score of stool consistency (0–2) and fecal blood (0–1). Animals were given a score of 0 for hard stools, 1 for soft-formed stools, and 2 for frank diarrhea. The absence (no points) or presence (+ 1 point) of fecal blood was determined using the Hemoccult® Sensa® card (Beckman Coulter, Miami, FL, USA) [18]. 2.4. Histological evaluation After the mice were sacrificed, samples were obtained from the proximal colon. The samples were fixed in 4% paraformaldehyde for 24 h and paraffin-embedded. Fixed tissues were then sectioned at a thickness of 5 μm and stained with hematoxylin and eosin (H&E). A histological score of H&E-stained samples of the proximal colon was determined by two independent pathologists in a blind manner according to the method described by Singh et al. [19]. Briefly, a score (0–4) was given: grade 0 indicated no change from normal tissue; grade 1 indicated one or a few multifocal mononuclear cell infiltrates in the lamina propria, minimal hyperplasia, and no mucus depletion; grade 2 indicated intestinal lesions involving several multifocal, mild, inflammatory cell infiltrates in the lamina propria composed of mononuclear cells with no inflammation in the submucosa; grade 3 indicated lesions involving moderate inflammation and epithelial hyperplasia; grade 4 indicated inflammation involving most of the intestinal sections. Each mouse (6 in every group) got a histological score from 0 to 4, and all scores from the mice were included in the statistics of the corresponding group. Normally, the intestinal epithelial caves in and forms glandular architecture called gland in the lamina propria. The normal architecture of gland is a single straight tubular gland opening to the mucosal surface, and is comprised with columnar cells, goblet cells, endocrine cells and a few of undifferentiated cells. When the score is higher than grade 2, the architecture of gland began destroyed. 2.5. Intestinal permeability in vitro The permeability of mouse intestine was measured with the Ussing Chamber Analyses. Segments of proximal colon were immediately removed for assessment of intestinal permeability. We used the method previously reported by Arrieta et al. [20]. Briefly, the mucosa was mounted in Lucite chambers exposing mucosal and serosal surfaces to 10 ml of oxygenated Krebs buffer (115 mmol/l NaCl, 8 mmol/l KCl, 1.25 mmol/l CaCl2, 1.2 mmol/l MgCl2, 2 mmol/l KH2PO4 and 225
Fig. 1. Clinical manifestation of mice. (A) Body weight weighed every week. (B) Disease Activity Index recorded every day according to stool consistency and fecal blood (0 for absence and 1 for presence of fecal blood). The result of each group was distinguished with different line types. Data are presented as means ± SEM (n = 6 for each group).
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Fig. 2. Colitis activity of mice (H&E, 200× magnification). (A) H&E result of mice in the PBS-treated WT group. (B) H&E result of mice in the PBS-treated IL-10-KO group. (C) H&E result of Il10−/− mice in the laquinimod treatment group. (D) H&E result of wild type mice in the laquinimod-treated control group. (E) The histological inflammation scores of all four groups. Data are presented as means ± SEM (n = 6 for each group, values were significantly different compared with that of the WT group: 0.05, **p b 0.001; values were significantly different compared with that of the IL-10-KO group: #p b 0.05).
mmol/l NaHCO3; pH 7.35), which was maintained at 37 °C by a heated water jacket and gassed with 5% CO2/95% O2. Fructose (10 mmol/l) was added to the serosal and mucosa sides. For the measurement of basal mannitol fluxes, 1 mmol/l of mannitol with 370 KBOr of 3H was added to the mucosal side. The spontaneous transepithelial potential difference was determined, and the tissue was clamped at zero voltage by continuously introducing an appropriate short-circuit current with an automatic voltage clamp (DVC 1000 World Precision Instruments). Tissue ion resistances were calculated from the potential difference and short-circuit current according to Ohm's law and were expressed as ohms times centimeters squared (Ω cm2).
2.6. Western blot Western blotting was performed as previously described by Wang H et al. [21]. Briefly, frozen colon tissues were homogenized in 3 ml of lysis buffer with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Debris was eliminated by centrifugation at 15,000 rpm at 4 °C for 15 min. The primary antibodies were 1:1000 dilutions of rabbit polyclonal antibody against occludin, ZO-1 (Abcam Inc., Cambridge, MA, USA), p65, IκB, phospho-p65, phospho-IκB (Cell Signaling Technology, Inc., Beverly, MA, USA), and GAPDH (Santa Cruz Biotechnology, Santa Cruz, Calif, USA). Quantification was performed by optical density
Fig. 3. Intestinal permeability in vitro: (A) Mannitol flux; (B) electrical resistance. Data are presented as mean ± SEM (n = 6 for each group, values were significantly different compared with that of the WT group: **p b 0.001; values were significantly different compared with that of the IL-10-KO group: #p b 0.05, ##p b 0.001).
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Fig. 4. Immunofluorescence analysis of occludin in proximal colon tissues. Occludin (green), Hoechst 33342 staining (blue), merged occludin and Hoechst 33342, as well as amplified merged occludin and Hoechst 33342 images are presented (200× magnification, n = 3–4), white arrow shows biotin staining ectopic to the lamina propria or deep into the epithelial surface and villi surface lacking focused staining in Il10−/− mice.
methods using ImageJ software. The results are normalized to GAPDH and calculated as target protein expression/GAPDH expression ratios. 2.7. Immunofluorescence Immunofluorescence staining was also performed to determine the localization of the tight junction as well as the expression of p65 and phospho-p65 in each group as described previously [22]. Briefly, 10μm frozen sections of proximal colon samples were collected on coated slides and washed three times with PBS. Nonspecific binding was blocked with 5% normal goat serum in PBS for 30 min at room temperature. After incubation with primary antibodies against occludin, ZO-1 (Abcam Inc., Cambridge, MA, USA), p65 and phospho-p65 (Cell Signaling Technology, Inc., Beverly, MA, USA) in PBS with 1% goat serum overnight at 4 °C, the sections were washed and incubated with an Alexa 488-conjugated secondary antibody for 60 min. Confocal analysis was performed using a confocal microscope (OLYMPUS). 2.8. Cell isolation The colons were opened longitudinally and then were cut into strips 1 cm in length and stirred in Hanks Balanced Salt Solutions (HBSS, Gibco-Invitrogen, Grand Island, NY, USA) containing 2 mM EDTA and 1 mM DTT at 37 °C for 30 min. The cells from intestinal lamina propria were isolated as described previously [23]. In brief, the lamina propria was isolated by digesting intestinal tissue with 40 U/ml collagenase type II (Sigma-Aldrich, St. Louis, MO, USA) 5% fetal bovine serum (FBS, Gibco-Invitrogen Co., Grand Island, NY, USA), and 3 mM Cacl2 in RPMI
1640 (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C with moderate stirring. After each 30 min interval, the released cells were centrifuged, stored in complete medium and mucosal pieces were replaced with fresh collagenase solution at least two times. Lamina propria cells were further purified using a discontinuous Percoll (Sigma-Aldrich, St. Louis, MO, USA) gradient collecting at the 40–70% interface. 2.9. Flow cytometry analysis Cell suspensions from the LP were washed twice in RPMI-1640, and isolated cells were thoroughly suspended in each tube in 100 μl RPMI1640. For cell surface antigen staining, cells were counted and approximately one million cells transferred to each flow test-tube. These cells were stained with FITC-conjugated anti-CD4 (eBiosciences, San Diego, CA, USA), PE-conjugated anti-CD45 (eBiosciences, San Diego, CA, USA) or an appropriate negative control. Then, the stained cells were incubated at room temperature for 30 min in the dark. The cells were washed twice with 2 ml RPMI-1640 at room temperature and suspended in 500ul RPMI-1640, and the cells were evaluated on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). All flow data were analyzed using FlowJo software. 2.10. Quantitative real-time PCR (qRT-PCR) analysis The mRNA levels of TNF-α, IFN-γ, IL-1β, and IL-17A were measured by quantitative real-time PCR analysis. Quantitative RT-PCR analysis of cytokine expression was performed using the method described previously [24,25]. Briefly, total RNA was extracted from frozen colon tissues
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Fig. 5. Immunofluorescence analysis of ZO-1 in proximal colon tissues. ZO-1 (green), Hoechst 33342 staining (blue), merged ZO-1 and Hoechst 33342, as well as amplified merged ZO-1 and Hoechst 33342 images are presented (200× magnification, n = 3–4), white arrow shows villi surface lacking focused staining in Il10−/− mice.
using TRIzol RNA isolation reagent (Life Technologies Inc., Grand Island, NY, USA), and the oligo (dT)-primed complementary DNA was used for reverse transcription of purified RNA. The amount of transcript of the genes of interest was measured by real-time quantitative RT-PCR assay using SYBR Green detection (Applied Biosystems, Carlsbad, CA, USA). All reactions were independently repeated at least twice to ensure the reproducibility of the results. The primer sets used for quantitative RT-PCR are described in Table 1. Expression levels of each gene were normalized using β-actin gene expression, yielding the relative expression value. 2.11. Statistical analysis The data analysis was performed using Statistical Package for Social Sciences (SPSS Inc., Chicago, IL, USA) software version 19.0. Continuous, normally distributed data were expressed as mean ± standard error of the mean (mean ± SEM). The measurements were subjected to oneway analysis of variance (ANOVA) followed by Tukey's post hoc test. A level of p b 0.05 was considered to be statistically significant. 3. Results 3.1. Laquinimod treatment significantly ameliorated spontaneous colitis in Il10−/− mice Il10−/− mice gained less weight than WT mice, and laquinimod treatment revealed to significantly increase the weight of Il10−/− mice. For wild type mice in the control group, laquinimod treatment did not induce significant weight loss (Fig. 1A). For DAI, the score was
higher in Il10−/− mice compared to WT mice. Laquinimod treatment significantly reduced DAI scores of Il10−/− mice, and did not increase DAI scores of wild type mice (Fig. 1B). After the mice were sacrificed, we first collected the proximal colon tissues to test the effects of laquinimod on colitis through an H&E-stained microscopic study (n = 6 in each group). H&E staining revealed inflammatory cell infiltration in the mucosal of Il10−/− mice (arrow) compared with wild type mice (Fig. 2A–D). The inflammatory score was significantly higher in Il10−/− mice of the IL-10-KO group (p b 0.0001) (Fig. 2E). In the mucosa of Il10−/− mice treated with laquinimod, a significant reduction in colonic inflammation was found with reduced inflammatory cells infiltration and a partially restored glandular architecture (Fig. 2C) compared with the IL-10-KO group. Correspondingly, lower mean inflammation scores were found in Il10−/− mice treated with laquinimod than those treated with PBS (p = 0.0035). In addition, there was no significant inflammatory cells infiltration in wild type mice receiving laquinimod treatment, and the histological scores were similar with mice of the WT group (p = 0.5490) (Fig. 2E). 3.2. Laquinimod treatment protected intestinal barrier function in Il10−/− mice Intestinal permeability of the colon was measured using an Ussing chamber in vitro. Intestinal permeability to mannitol was significantly increased with a corresponding decrease in electrical resistance in Il10−/− mice compared with WT mice. In laquinimod-treated Il10−/− mice, these changes were largely prevented (Fig. 3A, B). To identify whether there were changes in the distribution and expression of apical junctions after laquinimod treatment,
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Fig. 6. Western blot of occludin and ZO-1 expressions in proximal colons. (A) The expressions of occludin and ZO-1 were statistically analyzed relative to GAPDH expression by densitometry. (B), (C) Laquinimod treatment increased the expressions of occludin and ZO-1. Data are presented as mean ± SEM (n = 6 for each group: values were significantly different compared with that of the WT group: **p b 0.001; values were significantly different compared with that of the IL-10-KO group: #p b 0.05, ##p b 0.001).
immunofluorescence and western blotting of occludin and ZO-1 were performed. Immunofluorescence staining revealed that occludin and ZO-1 were continuously distributed with bright spots along the membrane of the colonic mucosa in WT mice. In the Il10−/− group, the fluorescence was faint and discontinuous, especially in regions with infiltrations of inflammatory cells (Figs. 4, 5). Biotin staining ectopic to the lamina propria or deep into the epithelial surface were found, and some of the surface of villi lacked focused staining (arrow). By contrast, the distribution and fluorescence intensity of the staining were significantly improved by laquinimod treatment. Western blotting revealed that occludin and ZO-1 protein levels were reduced in Il10−/− mice when compared to WT mice, and laquinimod treatment partly restored occludin and ZO-1 protein levels (Fig. 6, p b 0.05). 3.3. Laquinimod treatment reduced colonic CD4 + CD45 + lymphocytes and pro-inflammatory cytokines associated with Th1 and Th17 cells It has been shown that Th1/Th17 cells differentiated from CD4 + lymphocytes mainly mediate chronic inflammation in the colon of Il10−/− mice [31]. We first quantified the effect of laquinimod treatment on depleting lamina propria CD4 + CD45 + lymphocytes. Compared with wild-type mice, Il10−/− mice with PBS treatment showed more CD4 + CD45 + lymphocytes in the lamina propria, while laquinimod treatment significantly decreased the percentage of CD4+CD45+ lymphocytes (Fig. 7A) in colonic mucosa of Il10−/− mice, resulting in reduced intestinal inflammation. In the Interleukin-10-gene-deficient spontaneous colitis model, we evaluated the expression of typical inflammatory cytokines in colonic tissues. The pro-inflammatory cytokines secreted by Th1 and Th17 cells, TNF-α, IL-1β, IFN-γ, and IL-17A were significantly increased in
Il10−/− mice when compared with wild type mice. In laquinimod treated mice, the cytokines were significantly lower than Il10−/− controls (Fig. 7B–D). 3.4. Laquinimod treatment reduced the expression of NF-κB pathway proteins To understand the mechanisms underlying the activity of laquinimod, western blotting of the expression of non-phosphorylated and phosphorylated forms of p65 and IκB in mouse colon was performed among the three groups (Fig. 8). The expression levels of p65, phospho-p65, IκB and phospho-IκB were significantly higher in the Il10−/− mice, and treatment with laquinimod significantly reduced the changes of p65, IκB and phospho-IκB. Though the increase of phospho-p65 was not significantly prevented by laquinimod, the level of phospho-p65 was still decreased in the laquinimod group. Immunofluorescence staining of non-phosphorylated and phosphorylated forms of p65 was also undertaken (Fig. 9 and Supplementary Fig. 1). Results revealed that the fluorescence strengthened with some enhanced bright spots (arrow) in the Il10−/− mice compared to control mice. In contrast, the fluorescence was faint and the bright spots were decreased after laquinimod treatment. 4. Discussion To the best of our knowledge, this study is the first to examine the effects of laquinimod in a colitis mouse model, especially a model of Crohn's disease. Our main findings can be summarized as follows: 1) laquinimod treatment ameliorates spontaneous colitis in Il10−/− mice; 2) laquinimod treatment can prevent intestinal barrier
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Fig. 7. Lymphocytes and colonic proinflammatory cytokine. (A) Percentage of CD4+CD45+ lymphocytes in the lamina propria of wild-type, PBS and laquinimod treated Il10−/− mice. (B) TNF-α mRNA. (C) IL-1β mRNA. (D) IFN-γ mRNA. (E) IL-17A mRNA. Data are presented as means ± SEM (n = 6 for each group, values were significantly different compared with that of the WT group: **p b 0.001; values were significantly different compared with that of the IL-10-KO group: ##p b 0.001).
dysfunction caused by IL-10 deficiency; 3) laquinimod maintains the balance of T-cell-associated pro-inflammatory cytokines, and the therapeutic effect of laquinimod may also be associated with the NF-κB signaling pathway. Previous studies have shown that Il10−/− mice spontaneously develop chronic enterocolitis in both a Th1 and Th17 manner with typical release of pro-inflammatory cytokines, which is consistent with the findings in human CD [26]. Among these pro-inflammatory cytokines, TNF-α was reported to cause tight junction barrier dysfunction [27]. TNF-α can impair the epithelial barrier by altering the structure and function of the tight junction in human intestinal epithelial cell line HT29/B6 [28]. IL-1β, IFN-γ and IL-17A, which play an important role in the intestinal inflammatory process, can also decrease the expression of tight junction protein [29–31]. Antigens in the lumen cross the imbalanced mucosal barrier and enter the lamina propria, which promotes APCs to regulate the differentiation of T cells into Th1 and Th17 cells. In a positive feedback loop, ever-increasing pro-inflammatory cytokines are produced and the intestine is in a constant state of inflammation. In our study, laquinimod treatment reduced the increases of proinflammatory cytokines TNF-α, IL-1β, IFN-γ, and IL-17A, and concurrently, barrier function was protected in our colitis model, which may explain the protection of laquinimod in Il10−/− mice. Laquinimod has also been reported to significantly decrease astrocytic NF-κB pathway activation in cuprizone-treated mice in vitro and in vivo [32]. Our results revealed that NF-κB pathway related proteins p65, phospho-p65, IκB and phospho-IκB were increased in the mucosa of Il10−/− mice, which was consistent with previous studies reporting that the NF-κB pathway is activated in the mucosa of Il10−/− mice [33] and IBD patients [34]. Previous studies have reported that NF-κB
pathway plays a role in regulating the differentiation of CD4 + T-cell subsets [35]. A previous study of multiple sclerosis indicated the inhibition of the NF-κB pathway can downregulate the immunogenicity of dendritic cell (DC) responses and thus prevents DC induction of T cell differentiation [36]. In transgenic mice expressing a non-degradable form of IκB specifically in T cells, Th1 response was significantly impaired due to diminished NF-κB pathway activation [37]. More recently, c-Rel- or p65-deficient T cells showed defective IL-17 gene expression and Th17 cell differentiation [38]. Th1 cell related pro-inflammatory cytokines, TNF-α and IL-1β, can further stimulate the NF-κB signaling pathway [39]. In a positive feedback loop, NF-κB signaling pathway is continuously activated. In addition, studies have revealed that upregulation of the NF-κB pathway can reduce the expression tight junction proteins, which may also induce imbalanced mucosal barrier [27,40]. In our study, laquinimod treatment downregulated the expression of the NF-κB pathway, which may explain the protection of mucosal barrier function and the regulation of T cell differentiation. Currently, the most effective therapeutic recommendations for Crohn's disease, the combination of anti-TNF and azathioprine or 6MP, have a relatively inconvenient parenteral route of administration and are costly. Other pharmacological agents, such as azathioprine, were not sufficiently effective and can't control the disease or prevent complications for a long time during the course. Azathioprine is an inexpensive treatment option, which has shown its efficacy in reducing the risk of disease recurrence over a 6 month to 2 year period, and reducing the need for steroid treatment [41]. However, there is still study revealing no difference between azathioprine and mesalazine in the prevention of postoperative clinical recurrence [42], and it was reported that withdrawals due to adverse events were more common in patients
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Fig. 8. Western blot of NF-κB p-65 (A, C), phos-p65 (A, D), IκB (B, E) and phospho-IκB (B, F) expressions in proximal colons. Data are presented as means ± SEM (n = 6 for each group, values were significantly different compared with that of the WT group: **p b 0.001; values were significantly different compared with that of the IL-10-KO group: ##p b 0.001).
treated with azathioprine (Peto OR: 3.74; 95% CI: 1.48 to 9.45, NNH = 20) than with placebo [41]. Therefore, a need exists to find new efficacious and safe agents for the management of CD. Our study demonstrated the therapeutic effect of laquinimod on intestinal inflammation and barrier function in Il10−/− mice. In human monocyte-derived DCs, laquinimod can inhibit CD4 + T cell proliferation and proinflammatory cytokine secretion, and these immunomodulatory effects appeared to correspond to an impaired NF-κB pathway [36,43]. In addition, in Phase III MS clinical trials, laquinimod showed no evident immunosuppression or significant toxicity [14,44]. In addition, our study also found that laquinimod did not induce colitis in wild type mice. Recently, D'Haens et al. conducted a phase II study of laquinimod in Crohn's disease patients, and they found that laquinimod was safe and well tolerated, and the effects on remission and response of the 0.5 mg dose suggest a treatment benefit in patients with CD [45]. Therefore, laquinimod may
be a promising immunomodulatory therapeutic agent for patients with Crohn's disease. In conclusion, the current study, for the first time, has suggested that laquinimod ameliorates spontaneous colitis and improves barrier function in Il10−/− mice, which was associated with the downregulation of the NF-κB pathway. Therefore, laquinimod may represent a novel protective means of restricting inflammation in patients with Crohn's disease, and more clinical studies will be required in the future.
Conflict of interest None. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.intimp.2015.10.019.
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Fig. 9. Immunofluorescence analysis of NF-κB phospho-p-65 in proximal colon tissues. Phospho-p-65 (green), Hoechst 33342 staining (blue), merged phospho-p-65 and Hoechst 33342, as well as amplified merged phospho-p-65 and Hoechst 33342 images are presented (200× magnification, n = 3–4), white arrow shows fluorescence strengthened with some enhanced bright spots in the villi of Il10−/− mice.
Acknowledgments J. S. and X. S. carried out the majority of the biochemical analysis, designed the experiment and contributed to the writing. W. Z., and J. L. contributed to the supervision and drafting of the manuscript. J. D., H. W., L. Z., J. Z., J. L., Y. L., W. Z., and J. G. contributed with technical support, scientific advice and revised the manuscript. The present study was also partly supported by the Model Animal Research Center, Nanjing University (Nanjing, China). The authors would like to acknowledge the expert technical assistance of Professor Xiang Gao and the members of his lab (the Model Animal Research Center of Nanjing University, China). References [1] S.Y. Salim, J.D. Soderholm, Importance of disrupted intestinal barrier in inflammatory bowel diseases, Inflamm. Bowel Dis. 17 (2011) 362–381. [2] D.C. Baumgart, W.J. Sandborn, Crohn's disease, Lancet 380 (2012) 1590–1605. [3] J.F. Colombel, A.J. Watson, M.F. Neurath, The 10 remaining mysteries of inflammatory bowel disease, Gut 57 (2008) 429–433. [4] L. Shen, Tight junctions on the move: molecular mechanisms for epithelial barrier regulation, Ann. N. Y. Acad. Sci. 1258 (2012) 9–18. [5] J.B. Ewaschuk, H. Diaz, L. Meddings, et al., Secreted bioactive factors from Bifidobacterium infantis enhance epithelial cell barrier function, Am. J. Physiol. Gastrointest. Liver Physiol. 295 (2008) G1025–G1034. [6] J.R. Turner, Molecular basis of epithelial barrier regulation: from basic mechanisms to clinical application, Am. J. Pathol. 169 (2006) 1901–1909. [7] J. Madara, Regulation of the movement of solutes across tight junctions, Annu. Rev. Physiol. 60 (1998) 143–159. [8] J.R. Turner, Intestinal mucosal barrier function in health and disease, Nat. Rev. Immunol. 9 (2009) 799–809. [9] S. Brand, Crohn's disease: Th1, Th17 or both? The change of a paradigm: new immunological and genetic insights implicate Th17 cells in the pathogenesis of Crohn's disease, Gut 58 (2009) 1152–1167.
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Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Laquinimod ameliorates spontaneous colitis in interleukin-10-gene-deficient mice with improved barrier function Jing Sun 1, Xiao Shen 1, Jianning Dong, Jie Zhao, Lugen Zuo, Honggang Wang, Yi Li, Weiming Zhu ⁎, Jianfeng Gong, Jieshou Li Department of General Surgery, Jinling Hospital, Medical School of Nanjing University, 305 East Zhongshan Road, Nanjing, Jiangsu Province 210002, China
a r t i c l e
i n f o
Article history: Received 19 May 2015 Received in revised form 16 October 2015 Accepted 16 October 2015 Available online 23 October 2015 Keywords: Crohn's disease IL-10 gene-deficient mice Laquinimod Intestinal barrier function NF-κB pathway
a b s t r a c t Background and aims: Crohn's disease is an autoimmune disease associated with imbalanced mucosal immunity, mediated with increased Th1 and Th17 cells. Laquinimod, an immunomodulatory drug, has shown efficacy in regulating the differentiation of T cells. The aim of the study was to investigate the therapeutic effect of laquinimod on spontaneous colitis in interleukin-10-gene-deficient mice, an animal model of Crohn's disease. Methods: Male Il10−/− mice aged 16 weeks in the laquinimod group were treated with laquinimod with distilled water at a dose of 25 mg/kg by oral gavage, 3 times a week. Il10−/− mice in the IL-10-KO group and wild type mice received equal volume of phosphate buffered saline by oral gavage, 3 times a week. After 4 weeks, mice were sacrificed for analysis. Severity of colitis, epithelial expression of T-cell-associated cytokines, expression and distribution of tight junction proteins in the lamina propria and NF-κB signaling pathway associated mRNA expression were measured at the end of the experiment. Results: Laquinimod treatment ameliorated spontaneous colitis in Il10−/− mice, which was associated with decreased T-cell-associated pro-inflammatory cytokines. Increased expression and correct distribution of tight junction proteins (occludin and ZO-1) were found in Il10−/− mice treated with laquinimod. In addition, in mice treated with laquinimod, NF-κB signaling pathway associated mRNA in the colon was also downregulated. Conclusions: Our results indicated that laquinimod treatment ameliorates colitis in Il10−/− mice and improves intestinal barrier function, which may support a new therapeutic approach to Crohn's disease. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Crohn's disease (CD) is a chronic relapsing inflammatory disorder characterized by chronic inflammation and mucosal tissue damage of the gastrointestinal tract. Although the precise etiology of Crohn's disease remains unclear, research has thus far demonstrated that CD is caused by a combination of genetic, environmental and immunoregulatory factors [1–3]. Increasing evidence suggests that an imbalanced mucosal barrier may play an important role in the pathogenesis and disease progression of CD. Massive numbers of microorganisms reside in the gut lumen, and the intestinal mucosa constitutes an immunologic organ, which tolerates and defends against harmful organisms [4]. Epithelial cells and tight junctions (TJ) comprise the largest number of intestinal barriers [5,6]. When the epithelium is intact, intestinal barrier function is largely defined by the construction and expression of TJ as well as intestinal barrier permeability characteristics [7]. In Crohn's disease, the intestinal barrier at the interface between the intestinal microbiome and the lymphoid tissue plays a critical role in ⁎ Corresponding author. E-mail address: [email protected] (W. Zhu). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.intimp.2015.10.019 1567-5769/© 2015 Elsevier B.V. All rights reserved.
shaping the mucosal immune response. In this situation, the intestinal barrier is impaired, and bacterial products and dietary antigens cross the epithelium and enter the lamina propria. Antigen-presenting cells (APCs) take up foreign materials and regulate the differentiation of T cells [8]. For many years, it has been assumed that Crohn's disease is a T helper 1 (Th1)-cell-mediated disease [9], and a novel subset of IL17-producing CD4+ Th cells, Th17 cells, have more recently been implicated in the pathogenesis of CD [10]. In the most appropriate model of human CD, interleukin-10-gene-deficient (Il10−/−) mice [11] exhibit increases in CD4 + Th1 and Th17 cells, which secrete a large number of pro-inflammatory mediators such as tumor necrosis factor-α (TNFα), IL-1β, interferon-γ (IFN-γ), and IL-17A [12,13]. Laquinimod (C19H17ClN2O3, 5-chloro-N-ethyl-4-hydroxy-1-methyl-2-oxo-N-phenyl-1, 2-dihydroquinoline-3-carboxamide) is an immunomodulatory drug that has extensively shown its efficacy in inflammatory and autoimmune disorders, especially multiple sclerosis (MS) [14]. Laquinimod is a small molecule that concentrates in the peripheral immune system as well as in the central nervous system (CNS) [15]. In MS model mice, laquinimod has shown immunomodulatory effects by downregulating Th1 and Th17 cells. Preclinical data suggest that laquinimod has a direct inhibitory effect on antigen-presenting cells and results in anti-inflammatory T cell polarization manifested by a
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Table 1 Primer for polymerase chain reaction. TNF-α IFN-γ IL-1β IL-17A GAPDH
Forward: Reverse: Forward: Reverse: Forward: Reverse: Forward: Reverse: Forward: Reverse:
5′-TGGGAGTAGACAAGGTACAACCC-3′ 5′-CATCTTCTCAAAATTCGAGTGACAA-3′ 5′-AACGCTACACACTGCATCTTGG-3′ 5′-GCCGTGGCAGTAACAGCC-3′ 5′-CAACCAACAAGTGATATTCTCCATG-3′ 5′-GATCCACACTCTCCAGCTGCA-3′ 5′-GCTCCAGAAGGCCCTCAGA-3′ 5′-AGCTTTCCCTCCGCATTGA-3′ 5′-AGGCCGGTGCTGAGTATGTC-3′ 5′-TGCCTGCTTCACCACCTTCT-3′
reduction in the frequencies of pro-inflammatory Th1 (IFN-γ+CD4+) cells and Th17 (IL-17A+CD4+) cells [16]. Thus, the proposed mechanism of laquinimod may make it ideally suited to reduce gastrointestinal inflammation. However, the effects of laquinimod on colitis have been heretofore unstudied. In this investigation, we determined whether laquinimod ameliorates established spontaneous colitis in Il10−/− mice, and we attempted to explain the potential mechanisms that account for these effects. 2. Materials and methods 2.1. Animals Both wild type (WT) and Il10−/− mice on a C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were bred and maintained under specific pathogen-free (SPF) conditions at the Model Animal Research Center of Nanjing University (Nanjing, China). Previous experiment has demonstrated that most Il10−/− mice on the C57BL/6 strain under SPF conditions develop spontaneous colitis at 12 weeks of age [17]. The mice used in our study were 16 weeks of age. The experimental procedures were performed in accordance with the Guidelines for Animal Experiments at Jinling Hospital and were approved by the Ethics Committee of Jinling Hospital (Jiangsu, China). 2.2. Drug treatment of mice Wild-type and Il10−/− mice were divided into wild-type group (WT, wild-type mice), control group (IL-10-KO, Il10−/− mice) and treatment group (laquinimod, Il10−/− mice), and each group contains 6 mice. To investigate the effects of laquinimod on wild type mice, 6 wild type mice (control group) also received laquinimod treatment. Laquinimod (TEVA Pharmaceuticals Industries, Ltd (Israel), purity N 98%) was dissolved in distilled water and administered 25 mg/kg by oral gavage, 32 times a week. As reported in a study by Lourenço EV et al. [16], we found that this dose was well tolerated in mice, and no obvious macroscopic adverse effects were observed. Mice in the IL-10-KO group and
the WT group received an equal volume of phosphate buffered saline (PBS) by oral gavage. Four weeks after administration, the mice were sacrificed by an overdose of anesthesia with pentobarbital sodium, and proximal colons were collected for experiments. 2.3. Weight and disease activity index (DAI) Animals were weighed on a balance, and measurements were recorded to the nearest 0.1 g. The Disease Activity Index (DAI) was recorded as a composite score of stool consistency (0–2) and fecal blood (0–1). Animals were given a score of 0 for hard stools, 1 for soft-formed stools, and 2 for frank diarrhea. The absence (no points) or presence (+ 1 point) of fecal blood was determined using the Hemoccult® Sensa® card (Beckman Coulter, Miami, FL, USA) [18]. 2.4. Histological evaluation After the mice were sacrificed, samples were obtained from the proximal colon. The samples were fixed in 4% paraformaldehyde for 24 h and paraffin-embedded. Fixed tissues were then sectioned at a thickness of 5 μm and stained with hematoxylin and eosin (H&E). A histological score of H&E-stained samples of the proximal colon was determined by two independent pathologists in a blind manner according to the method described by Singh et al. [19]. Briefly, a score (0–4) was given: grade 0 indicated no change from normal tissue; grade 1 indicated one or a few multifocal mononuclear cell infiltrates in the lamina propria, minimal hyperplasia, and no mucus depletion; grade 2 indicated intestinal lesions involving several multifocal, mild, inflammatory cell infiltrates in the lamina propria composed of mononuclear cells with no inflammation in the submucosa; grade 3 indicated lesions involving moderate inflammation and epithelial hyperplasia; grade 4 indicated inflammation involving most of the intestinal sections. Each mouse (6 in every group) got a histological score from 0 to 4, and all scores from the mice were included in the statistics of the corresponding group. Normally, the intestinal epithelial caves in and forms glandular architecture called gland in the lamina propria. The normal architecture of gland is a single straight tubular gland opening to the mucosal surface, and is comprised with columnar cells, goblet cells, endocrine cells and a few of undifferentiated cells. When the score is higher than grade 2, the architecture of gland began destroyed. 2.5. Intestinal permeability in vitro The permeability of mouse intestine was measured with the Ussing Chamber Analyses. Segments of proximal colon were immediately removed for assessment of intestinal permeability. We used the method previously reported by Arrieta et al. [20]. Briefly, the mucosa was mounted in Lucite chambers exposing mucosal and serosal surfaces to 10 ml of oxygenated Krebs buffer (115 mmol/l NaCl, 8 mmol/l KCl, 1.25 mmol/l CaCl2, 1.2 mmol/l MgCl2, 2 mmol/l KH2PO4 and 225
Fig. 1. Clinical manifestation of mice. (A) Body weight weighed every week. (B) Disease Activity Index recorded every day according to stool consistency and fecal blood (0 for absence and 1 for presence of fecal blood). The result of each group was distinguished with different line types. Data are presented as means ± SEM (n = 6 for each group).
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Fig. 2. Colitis activity of mice (H&E, 200× magnification). (A) H&E result of mice in the PBS-treated WT group. (B) H&E result of mice in the PBS-treated IL-10-KO group. (C) H&E result of Il10−/− mice in the laquinimod treatment group. (D) H&E result of wild type mice in the laquinimod-treated control group. (E) The histological inflammation scores of all four groups. Data are presented as means ± SEM (n = 6 for each group, values were significantly different compared with that of the WT group: 0.05, **p b 0.001; values were significantly different compared with that of the IL-10-KO group: #p b 0.05).
mmol/l NaHCO3; pH 7.35), which was maintained at 37 °C by a heated water jacket and gassed with 5% CO2/95% O2. Fructose (10 mmol/l) was added to the serosal and mucosa sides. For the measurement of basal mannitol fluxes, 1 mmol/l of mannitol with 370 KBOr of 3H was added to the mucosal side. The spontaneous transepithelial potential difference was determined, and the tissue was clamped at zero voltage by continuously introducing an appropriate short-circuit current with an automatic voltage clamp (DVC 1000 World Precision Instruments). Tissue ion resistances were calculated from the potential difference and short-circuit current according to Ohm's law and were expressed as ohms times centimeters squared (Ω cm2).
2.6. Western blot Western blotting was performed as previously described by Wang H et al. [21]. Briefly, frozen colon tissues were homogenized in 3 ml of lysis buffer with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Debris was eliminated by centrifugation at 15,000 rpm at 4 °C for 15 min. The primary antibodies were 1:1000 dilutions of rabbit polyclonal antibody against occludin, ZO-1 (Abcam Inc., Cambridge, MA, USA), p65, IκB, phospho-p65, phospho-IκB (Cell Signaling Technology, Inc., Beverly, MA, USA), and GAPDH (Santa Cruz Biotechnology, Santa Cruz, Calif, USA). Quantification was performed by optical density
Fig. 3. Intestinal permeability in vitro: (A) Mannitol flux; (B) electrical resistance. Data are presented as mean ± SEM (n = 6 for each group, values were significantly different compared with that of the WT group: **p b 0.001; values were significantly different compared with that of the IL-10-KO group: #p b 0.05, ##p b 0.001).
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Fig. 4. Immunofluorescence analysis of occludin in proximal colon tissues. Occludin (green), Hoechst 33342 staining (blue), merged occludin and Hoechst 33342, as well as amplified merged occludin and Hoechst 33342 images are presented (200× magnification, n = 3–4), white arrow shows biotin staining ectopic to the lamina propria or deep into the epithelial surface and villi surface lacking focused staining in Il10−/− mice.
methods using ImageJ software. The results are normalized to GAPDH and calculated as target protein expression/GAPDH expression ratios. 2.7. Immunofluorescence Immunofluorescence staining was also performed to determine the localization of the tight junction as well as the expression of p65 and phospho-p65 in each group as described previously [22]. Briefly, 10μm frozen sections of proximal colon samples were collected on coated slides and washed three times with PBS. Nonspecific binding was blocked with 5% normal goat serum in PBS for 30 min at room temperature. After incubation with primary antibodies against occludin, ZO-1 (Abcam Inc., Cambridge, MA, USA), p65 and phospho-p65 (Cell Signaling Technology, Inc., Beverly, MA, USA) in PBS with 1% goat serum overnight at 4 °C, the sections were washed and incubated with an Alexa 488-conjugated secondary antibody for 60 min. Confocal analysis was performed using a confocal microscope (OLYMPUS). 2.8. Cell isolation The colons were opened longitudinally and then were cut into strips 1 cm in length and stirred in Hanks Balanced Salt Solutions (HBSS, Gibco-Invitrogen, Grand Island, NY, USA) containing 2 mM EDTA and 1 mM DTT at 37 °C for 30 min. The cells from intestinal lamina propria were isolated as described previously [23]. In brief, the lamina propria was isolated by digesting intestinal tissue with 40 U/ml collagenase type II (Sigma-Aldrich, St. Louis, MO, USA) 5% fetal bovine serum (FBS, Gibco-Invitrogen Co., Grand Island, NY, USA), and 3 mM Cacl2 in RPMI
1640 (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C with moderate stirring. After each 30 min interval, the released cells were centrifuged, stored in complete medium and mucosal pieces were replaced with fresh collagenase solution at least two times. Lamina propria cells were further purified using a discontinuous Percoll (Sigma-Aldrich, St. Louis, MO, USA) gradient collecting at the 40–70% interface. 2.9. Flow cytometry analysis Cell suspensions from the LP were washed twice in RPMI-1640, and isolated cells were thoroughly suspended in each tube in 100 μl RPMI1640. For cell surface antigen staining, cells were counted and approximately one million cells transferred to each flow test-tube. These cells were stained with FITC-conjugated anti-CD4 (eBiosciences, San Diego, CA, USA), PE-conjugated anti-CD45 (eBiosciences, San Diego, CA, USA) or an appropriate negative control. Then, the stained cells were incubated at room temperature for 30 min in the dark. The cells were washed twice with 2 ml RPMI-1640 at room temperature and suspended in 500ul RPMI-1640, and the cells were evaluated on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). All flow data were analyzed using FlowJo software. 2.10. Quantitative real-time PCR (qRT-PCR) analysis The mRNA levels of TNF-α, IFN-γ, IL-1β, and IL-17A were measured by quantitative real-time PCR analysis. Quantitative RT-PCR analysis of cytokine expression was performed using the method described previously [24,25]. Briefly, total RNA was extracted from frozen colon tissues
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Fig. 5. Immunofluorescence analysis of ZO-1 in proximal colon tissues. ZO-1 (green), Hoechst 33342 staining (blue), merged ZO-1 and Hoechst 33342, as well as amplified merged ZO-1 and Hoechst 33342 images are presented (200× magnification, n = 3–4), white arrow shows villi surface lacking focused staining in Il10−/− mice.
using TRIzol RNA isolation reagent (Life Technologies Inc., Grand Island, NY, USA), and the oligo (dT)-primed complementary DNA was used for reverse transcription of purified RNA. The amount of transcript of the genes of interest was measured by real-time quantitative RT-PCR assay using SYBR Green detection (Applied Biosystems, Carlsbad, CA, USA). All reactions were independently repeated at least twice to ensure the reproducibility of the results. The primer sets used for quantitative RT-PCR are described in Table 1. Expression levels of each gene were normalized using β-actin gene expression, yielding the relative expression value. 2.11. Statistical analysis The data analysis was performed using Statistical Package for Social Sciences (SPSS Inc., Chicago, IL, USA) software version 19.0. Continuous, normally distributed data were expressed as mean ± standard error of the mean (mean ± SEM). The measurements were subjected to oneway analysis of variance (ANOVA) followed by Tukey's post hoc test. A level of p b 0.05 was considered to be statistically significant. 3. Results 3.1. Laquinimod treatment significantly ameliorated spontaneous colitis in Il10−/− mice Il10−/− mice gained less weight than WT mice, and laquinimod treatment revealed to significantly increase the weight of Il10−/− mice. For wild type mice in the control group, laquinimod treatment did not induce significant weight loss (Fig. 1A). For DAI, the score was
higher in Il10−/− mice compared to WT mice. Laquinimod treatment significantly reduced DAI scores of Il10−/− mice, and did not increase DAI scores of wild type mice (Fig. 1B). After the mice were sacrificed, we first collected the proximal colon tissues to test the effects of laquinimod on colitis through an H&E-stained microscopic study (n = 6 in each group). H&E staining revealed inflammatory cell infiltration in the mucosal of Il10−/− mice (arrow) compared with wild type mice (Fig. 2A–D). The inflammatory score was significantly higher in Il10−/− mice of the IL-10-KO group (p b 0.0001) (Fig. 2E). In the mucosa of Il10−/− mice treated with laquinimod, a significant reduction in colonic inflammation was found with reduced inflammatory cells infiltration and a partially restored glandular architecture (Fig. 2C) compared with the IL-10-KO group. Correspondingly, lower mean inflammation scores were found in Il10−/− mice treated with laquinimod than those treated with PBS (p = 0.0035). In addition, there was no significant inflammatory cells infiltration in wild type mice receiving laquinimod treatment, and the histological scores were similar with mice of the WT group (p = 0.5490) (Fig. 2E). 3.2. Laquinimod treatment protected intestinal barrier function in Il10−/− mice Intestinal permeability of the colon was measured using an Ussing chamber in vitro. Intestinal permeability to mannitol was significantly increased with a corresponding decrease in electrical resistance in Il10−/− mice compared with WT mice. In laquinimod-treated Il10−/− mice, these changes were largely prevented (Fig. 3A, B). To identify whether there were changes in the distribution and expression of apical junctions after laquinimod treatment,
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Fig. 6. Western blot of occludin and ZO-1 expressions in proximal colons. (A) The expressions of occludin and ZO-1 were statistically analyzed relative to GAPDH expression by densitometry. (B), (C) Laquinimod treatment increased the expressions of occludin and ZO-1. Data are presented as mean ± SEM (n = 6 for each group: values were significantly different compared with that of the WT group: **p b 0.001; values were significantly different compared with that of the IL-10-KO group: #p b 0.05, ##p b 0.001).
immunofluorescence and western blotting of occludin and ZO-1 were performed. Immunofluorescence staining revealed that occludin and ZO-1 were continuously distributed with bright spots along the membrane of the colonic mucosa in WT mice. In the Il10−/− group, the fluorescence was faint and discontinuous, especially in regions with infiltrations of inflammatory cells (Figs. 4, 5). Biotin staining ectopic to the lamina propria or deep into the epithelial surface were found, and some of the surface of villi lacked focused staining (arrow). By contrast, the distribution and fluorescence intensity of the staining were significantly improved by laquinimod treatment. Western blotting revealed that occludin and ZO-1 protein levels were reduced in Il10−/− mice when compared to WT mice, and laquinimod treatment partly restored occludin and ZO-1 protein levels (Fig. 6, p b 0.05). 3.3. Laquinimod treatment reduced colonic CD4 + CD45 + lymphocytes and pro-inflammatory cytokines associated with Th1 and Th17 cells It has been shown that Th1/Th17 cells differentiated from CD4 + lymphocytes mainly mediate chronic inflammation in the colon of Il10−/− mice [31]. We first quantified the effect of laquinimod treatment on depleting lamina propria CD4 + CD45 + lymphocytes. Compared with wild-type mice, Il10−/− mice with PBS treatment showed more CD4 + CD45 + lymphocytes in the lamina propria, while laquinimod treatment significantly decreased the percentage of CD4+CD45+ lymphocytes (Fig. 7A) in colonic mucosa of Il10−/− mice, resulting in reduced intestinal inflammation. In the Interleukin-10-gene-deficient spontaneous colitis model, we evaluated the expression of typical inflammatory cytokines in colonic tissues. The pro-inflammatory cytokines secreted by Th1 and Th17 cells, TNF-α, IL-1β, IFN-γ, and IL-17A were significantly increased in
Il10−/− mice when compared with wild type mice. In laquinimod treated mice, the cytokines were significantly lower than Il10−/− controls (Fig. 7B–D). 3.4. Laquinimod treatment reduced the expression of NF-κB pathway proteins To understand the mechanisms underlying the activity of laquinimod, western blotting of the expression of non-phosphorylated and phosphorylated forms of p65 and IκB in mouse colon was performed among the three groups (Fig. 8). The expression levels of p65, phospho-p65, IκB and phospho-IκB were significantly higher in the Il10−/− mice, and treatment with laquinimod significantly reduced the changes of p65, IκB and phospho-IκB. Though the increase of phospho-p65 was not significantly prevented by laquinimod, the level of phospho-p65 was still decreased in the laquinimod group. Immunofluorescence staining of non-phosphorylated and phosphorylated forms of p65 was also undertaken (Fig. 9 and Supplementary Fig. 1). Results revealed that the fluorescence strengthened with some enhanced bright spots (arrow) in the Il10−/− mice compared to control mice. In contrast, the fluorescence was faint and the bright spots were decreased after laquinimod treatment. 4. Discussion To the best of our knowledge, this study is the first to examine the effects of laquinimod in a colitis mouse model, especially a model of Crohn's disease. Our main findings can be summarized as follows: 1) laquinimod treatment ameliorates spontaneous colitis in Il10−/− mice; 2) laquinimod treatment can prevent intestinal barrier
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Fig. 7. Lymphocytes and colonic proinflammatory cytokine. (A) Percentage of CD4+CD45+ lymphocytes in the lamina propria of wild-type, PBS and laquinimod treated Il10−/− mice. (B) TNF-α mRNA. (C) IL-1β mRNA. (D) IFN-γ mRNA. (E) IL-17A mRNA. Data are presented as means ± SEM (n = 6 for each group, values were significantly different compared with that of the WT group: **p b 0.001; values were significantly different compared with that of the IL-10-KO group: ##p b 0.001).
dysfunction caused by IL-10 deficiency; 3) laquinimod maintains the balance of T-cell-associated pro-inflammatory cytokines, and the therapeutic effect of laquinimod may also be associated with the NF-κB signaling pathway. Previous studies have shown that Il10−/− mice spontaneously develop chronic enterocolitis in both a Th1 and Th17 manner with typical release of pro-inflammatory cytokines, which is consistent with the findings in human CD [26]. Among these pro-inflammatory cytokines, TNF-α was reported to cause tight junction barrier dysfunction [27]. TNF-α can impair the epithelial barrier by altering the structure and function of the tight junction in human intestinal epithelial cell line HT29/B6 [28]. IL-1β, IFN-γ and IL-17A, which play an important role in the intestinal inflammatory process, can also decrease the expression of tight junction protein [29–31]. Antigens in the lumen cross the imbalanced mucosal barrier and enter the lamina propria, which promotes APCs to regulate the differentiation of T cells into Th1 and Th17 cells. In a positive feedback loop, ever-increasing pro-inflammatory cytokines are produced and the intestine is in a constant state of inflammation. In our study, laquinimod treatment reduced the increases of proinflammatory cytokines TNF-α, IL-1β, IFN-γ, and IL-17A, and concurrently, barrier function was protected in our colitis model, which may explain the protection of laquinimod in Il10−/− mice. Laquinimod has also been reported to significantly decrease astrocytic NF-κB pathway activation in cuprizone-treated mice in vitro and in vivo [32]. Our results revealed that NF-κB pathway related proteins p65, phospho-p65, IκB and phospho-IκB were increased in the mucosa of Il10−/− mice, which was consistent with previous studies reporting that the NF-κB pathway is activated in the mucosa of Il10−/− mice [33] and IBD patients [34]. Previous studies have reported that NF-κB
pathway plays a role in regulating the differentiation of CD4 + T-cell subsets [35]. A previous study of multiple sclerosis indicated the inhibition of the NF-κB pathway can downregulate the immunogenicity of dendritic cell (DC) responses and thus prevents DC induction of T cell differentiation [36]. In transgenic mice expressing a non-degradable form of IκB specifically in T cells, Th1 response was significantly impaired due to diminished NF-κB pathway activation [37]. More recently, c-Rel- or p65-deficient T cells showed defective IL-17 gene expression and Th17 cell differentiation [38]. Th1 cell related pro-inflammatory cytokines, TNF-α and IL-1β, can further stimulate the NF-κB signaling pathway [39]. In a positive feedback loop, NF-κB signaling pathway is continuously activated. In addition, studies have revealed that upregulation of the NF-κB pathway can reduce the expression tight junction proteins, which may also induce imbalanced mucosal barrier [27,40]. In our study, laquinimod treatment downregulated the expression of the NF-κB pathway, which may explain the protection of mucosal barrier function and the regulation of T cell differentiation. Currently, the most effective therapeutic recommendations for Crohn's disease, the combination of anti-TNF and azathioprine or 6MP, have a relatively inconvenient parenteral route of administration and are costly. Other pharmacological agents, such as azathioprine, were not sufficiently effective and can't control the disease or prevent complications for a long time during the course. Azathioprine is an inexpensive treatment option, which has shown its efficacy in reducing the risk of disease recurrence over a 6 month to 2 year period, and reducing the need for steroid treatment [41]. However, there is still study revealing no difference between azathioprine and mesalazine in the prevention of postoperative clinical recurrence [42], and it was reported that withdrawals due to adverse events were more common in patients
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Fig. 8. Western blot of NF-κB p-65 (A, C), phos-p65 (A, D), IκB (B, E) and phospho-IκB (B, F) expressions in proximal colons. Data are presented as means ± SEM (n = 6 for each group, values were significantly different compared with that of the WT group: **p b 0.001; values were significantly different compared with that of the IL-10-KO group: ##p b 0.001).
treated with azathioprine (Peto OR: 3.74; 95% CI: 1.48 to 9.45, NNH = 20) than with placebo [41]. Therefore, a need exists to find new efficacious and safe agents for the management of CD. Our study demonstrated the therapeutic effect of laquinimod on intestinal inflammation and barrier function in Il10−/− mice. In human monocyte-derived DCs, laquinimod can inhibit CD4 + T cell proliferation and proinflammatory cytokine secretion, and these immunomodulatory effects appeared to correspond to an impaired NF-κB pathway [36,43]. In addition, in Phase III MS clinical trials, laquinimod showed no evident immunosuppression or significant toxicity [14,44]. In addition, our study also found that laquinimod did not induce colitis in wild type mice. Recently, D'Haens et al. conducted a phase II study of laquinimod in Crohn's disease patients, and they found that laquinimod was safe and well tolerated, and the effects on remission and response of the 0.5 mg dose suggest a treatment benefit in patients with CD [45]. Therefore, laquinimod may
be a promising immunomodulatory therapeutic agent for patients with Crohn's disease. In conclusion, the current study, for the first time, has suggested that laquinimod ameliorates spontaneous colitis and improves barrier function in Il10−/− mice, which was associated with the downregulation of the NF-κB pathway. Therefore, laquinimod may represent a novel protective means of restricting inflammation in patients with Crohn's disease, and more clinical studies will be required in the future.
Conflict of interest None. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.intimp.2015.10.019.
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Fig. 9. Immunofluorescence analysis of NF-κB phospho-p-65 in proximal colon tissues. Phospho-p-65 (green), Hoechst 33342 staining (blue), merged phospho-p-65 and Hoechst 33342, as well as amplified merged phospho-p-65 and Hoechst 33342 images are presented (200× magnification, n = 3–4), white arrow shows fluorescence strengthened with some enhanced bright spots in the villi of Il10−/− mice.
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