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All-trans retinoic acid attenuates experimental colitis through inhibition of NF-κB signaling
Immunology Letters 162 (2014) 34–40
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Immunology Letters journal homepage: www.elsevier.com/locate/immlet
All-trans retinoic acid attenuates experimental colitis through inhibition of NF-B signaling Kai Hong a,1 , Yi Zhang a,1 , Yuan Guo a,b,1 , Jun Xie b , Jian Wang a , Xingxing He a , Nonghua Lu a , Aiping Bai a,∗ a b
Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang 330006, China Department of Pharmacy, The First Affiliated Hospital of Nanchang University, Nanchang 330006, China
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Article history: Received 19 March 2014 Received in revised form 13 June 2014 Accepted 24 June 2014 Available online 6 July 2014 Keywords: All-trans retinoic acid RAR Colitis Macrophage NF-B
a b s t r a c t Inflammatory bowel disease (IBD) is characterized by excessive innate immune cell activation, which is responsible for tissue damage and induction of adaptive immune responses. All-trans retinoic acid (ATRA), the ligand of retinoic acid receptors (RAR), has been previously shown to regulate adaptive immune responses and restore Th17/Treg balance, while its role in regulation of innate immune cell function such as macrophages remains to be elucidated. The study was performed to explore the effect of ATRA on regulation of innate immune responses during dextran sulfate sodium (DSS) induced murine colitis. The mice with DSS colitis were administered with vehicle, ATRA, or LE135. Colitis was evaluated by clinical symptoms, tissue myeloperoxidase (MPO) activity, and the expressions of CD68 and nuclear factor (NF) B p65, and tumor necrosis factor (TNF) level in inflamed colon. RAW 264.7 cells were pretreated with vehicle, ATRA, or LE135, followed by LPS challenge in vitro. ATRA administration ameliorates DSS-induced colitis evidenced with decreased TNF level and CD68 expression, while LE135 leads to exacerbation of colitis. ATRA treatment in vitro dampens LPS induced NF-B activation and TNF production of RAW 264.7 cells. Together, our data show a crucial role of ATRA in the progress of acute colitis through inhibiting NF-B activation, and suggest that ATRA represents a novel therapeutic approach for the management of IBD. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Inflammatory bowel disease (IBD), mainly referred to Crohn’s disease and ulcerative colitis, is a serious intestinal immune disorder. The etiology of IBD is not clearly studied so far, but it is strongly suggested that dysregulated immune responses contribute to the progress of IBD [1]. It has been well established that innate immune cells including macrophages determine the course of disease progress [1,2]. After exposure to and activated thereafter by the abundant intestinal bacterial antigens, macrophages become recruited, and induce adaptive immune response via direct contact and release of substantive proinflammatory cytokines such
Abbreviations: ATRA, all-trans retinoic acid; DSS, dextran sulfate sodium; ELISA, enzyme-linked immunosorbent assays; IBD, inflammatory bowel disease; IL, interleukin; LPS, lipopolysaccharides; MPO, myeloperoxidase; NFB, nuclear factor B; RAR, retinoic acid receptors; TNF, tumor necrosis factor. ∗ Corresponding author. Tel.: +86 88692507. E-mail address: [email protected] (A. Bai). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.imlet.2014.06.011 0165-2478/© 2014 Elsevier B.V. All rights reserved.
as tumor necrosis factor (TNF), interleukin (IL)-12, IL-6, and IL23 [2]. Immune cells including macrophages, in combination with proinflammatory cytokines, play critical roles in maintaining the uncontrolled inflammatory response, eventually resulting in the intestinal tissue damage seen in IBD [3,4]. Inhibition of immune cell function has been suggested as one of the crucial targets for the treatment immune diseases including IBD. Nuclear factor (NF) B is one of important transcription factors controlling inflammatory responses of immune diseases [5]. In the presence of the stimulations of lipopolysaccharides (LPS) and other extracellular bacterial antigens, the dimer of NFB composed of the P65 and P50 subunits translocates to nucleus of macrophage, and regulates various target gene expression including a large amount of inflammatory cytokines and chemokines, leading to sustained inflammatory responses and tissue damage [5,6]. Recently, we and others have demonstrated that inhibition of NFB signal can attenuate immune responses, and ameliorate immune diseases in animals [7–9], supporting the beneficial effects of inhibition of NFB signaling. Vitamin A and its derivatives, have been shown to be involved in modulation of immune responses. For example, deficiency of
K. Hong et al. / Immunology Letters 162 (2014) 34–40
vitamin A results in exacerbated experimental colitis [10], and supplementation of vitamin A can decrease prevalence of diarrhea among children [11]. Recently, all-trans retinoic acid (ATRA), a metabolite of vitamin A, has emerged as a novel biological chemical that is critically involved in regulation of adaptive immune responses [12]. Via its nuclear receptor retinoic acid receptors (RAR), ATRA can enhance Treg growth, differentiation, and gut homing, and inhibit Th17 generation and function [13,14]. We and others have shown that ATRA restores the balance between Th17 and Treg cells in the development of IBD, and RAR antagonist LE135 aggravates the aberrant helper T cell responses of experimental colitis [15,16]. However, as the pivotal cells in the pathogenesis of IBD, the impact of ATRA on macrophage activation and the related mechanisms remain to be elucidated. In the study, we demonstrate that ATRA inhibits LPS-induced NF-B activation in macrophage, and protects mice against dextran sulfate sodium (DSS)-induced colitis. 2. Methods 2.1. Cell culture and treatment Murine macrophage cell line RAW 264.7 was cultured in HEPESbuffered Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, penicillin G (100 U/mL), and streptomycin (100 g/mL) at 37 ◦ C. For in vitro study, about 1 × 106 /mL RAW264.7 cells were seeded in 48-well plates. The cells were treated with vehicle, 100 nM ATRA (Sigma–Aldrich) or 1 M LE135 (Santa Cruz Biotechnology) for 1 h, prior to 100 ng/mL or 1 g/mL lipopolysaccharide (LPS, from Escherichia coli 0111:B4; Sigma–Aldrich) challenge. 2.2. Enzyme-linked immunosorbent assays (ELISA) Tumor necrosis factor (TNF) levels in culture supernatants or colon tissues were determined by ELISA, following manufacturer’s instructions (R&D Systems, Inc.). Briefly, polyclonal rat anti-mouse cytokine antibodies were used as capturing antibodies and biotinylated polyclonal rat anti-mouse cytokine antibodies for detection, and the standard curve of TNF was set up meanwhile. Color changes were determined at 450 nm. 2.3. Western blot Proteins were extracted from the cultured cells using a lysis buffer (0.1 M PBS pH7.4 containing 1% deoxycholic acid sodium, 0.2% SDS, and protease inhibitors). After measurement of protein concentration, the proteins of the samples were loaded and separated by SDS-PAGE and then electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were incubated with primary rabbit anti-phospho-NFB P65 (Ser536) or NFB P65 antibody (both from Cell Signaling Technology), or mouse anti--actin antibody (from Santa Cruz Biotechnology). After incubating with secondary antibodies, the immunoreactive bands were visualized using the SuperSignal West Femto Maximum Sensitivity Substrate reagents (Thermo Scientific). -actin was used as an inner control. Relative protein levels were determined by Image J software (NIH). 2.4. Animals Female BALB/c mice were provided by the Experimental Animal Center of Nanchang University, and fed under specific pathogenfree conditions. 7–8 week-old mice were randomly divided into four groups in the study, weighing approximately 22 g: Control, DSS, ATRA, and LE135. All protocols for the projects using mice were
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reviewed and approved by the Institutional Animal Care Committee of Nanchang University. 2.5. Induction of dextran sulphate sodium (DSS) colitis Acute DSS colitis was induced in BALB/c mice according to the previously published method with minor modification [17]. The mice of DSS, ATRA, and LE135 groups were fed 3% (w/v) DSS (molecular mass, 36–50 kDa; MP Biomedicals) dissolved in the drinking water on day one. Fresh DSS solution was provided every second day. Control mice drank only distilled water. ATRA (0.5 mg) or LE135 (0.1 mg) was administered intraperitoneally (i.p.) since day three and repeated daily until the mice were killed on day eight. Disease symptoms of colitis were assessed daily by measurement of body weight, evaluation of stool consistency and detection of bloody stools. Disease severity was scored using a clinical disease activity index (DAI) ranging from 0 to 4, calculated as described previously [17] using the following parameters: stool consistency, presence or absence of fecal blood and weight loss. Mice were killed on day eight, and the middle section of colon was fixed in 10% formaldehyde–saline. Hematoxylin and eosin stain (HE)-stained sections were graded based on a scoring system modified from a previous study [18]. Histology scoring was performed, and a combined score of inflammatory cell infiltration and tissue damage was determined as follows: cell infiltration: score 0, occasional inflammatory cells in the lamina propria (LP); 1, increased infiltrate in the LP predominantly at the base of crypts; 2, confluence of inflammatory infiltrate extending into the mucosa; and 3, transmural extension of infiltrate. Tissue damage: score 0, no mucosal damage; 1, partial (up to 50%) loss of crypts in large areas; 2, partial to total 50–100% loss of crypts in large areas, epithelium intact; and 3, total loss of crypts in large areas and epithelium lost. 2.6. Immunohistochemistry Colon tissues of mice were taken and fixed immediately in 10% buffered formalin, embedded in paraffin, and cut into 4 m sections. After blockade of inner peroxidase, sections were incubated sequentially with the first antibody solution including rabbit anti-CD68 (Abcam), or anti-NF-B p65 (C20, from Santa Cruz Biotechnology) antibody. After three washes in PBS (pH 7.4), the sections were then incubated in secondary goat anti-rabbit immunoglobulin (Ig)G conjugated with peroxidase labeled polymer, prior to colorization using diaminobenzidine reaction and counterstained with hematoxylin. Negative controls were established using rabbit IgG instead of the first antibodies. The sections were evaluated using light microscopy, and 100 cells in lamina propria per high power field were calculated for statistical analysis. The cells stained with anti-NF-B (P65) antibody in both cytoplasm and the nucleus were counted as NF-B positive cells, whereas those without nucleus stain were regarded as NF-B negative cells [19]. Negative controls were established by using rabbit IgG. 2.7. Measurement of Myeloperoxidase (MPO) activity All experiments were performed within 1 week of tissue collection. MPO activity was measured according to the method previously described with minor modification [15,17]. In short, tissues were homogenized in hexadecyltrimethylammonium bromide in 50 mM potassium phosphate buffer. Aliquots were then added to O-dianisidine hydrochloride solution. Absorbance was read at 450 nm using a microplate reader. MPO was expressed in units/gram tissue, where 1 unit corresponds to the activity required to degrade 1 mmol hydrogen peroxide in 1 min at 24 ◦ C.
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Fig. 1. ATRA blocks LPS induced NFB activation in macrophage. RAW 264.7 cells were pretreated with vehicle, ATRA (100 nM), LE135 (1 M) for 1 h, prior to LPS challenge. (A) 1 h after LPS challenge (1 g/mL), the expression of pNFB p65 was determined by western blot. (B) 24 h after stimulation of 100 ng/mL LPS, TNF levels in supernatants were measured by ELISA (n = 4). **P < 0.01. (C) Western blotting of phosphorylation of pNFB p65 induced by LPS (1 g/mL) in the absence or presence of ATRA for different times.
2.8. Statistical analysis All data in the text and figures are expressed as mean ± standard deviation. Comparisons of more than two groups were made with a one-way analysis of variance using Tukey’s post hoc test. When appropriate, comparison with two groups was made using Student’s t-test for unpaired data. Differences were considered statistically significant if P < 0.05. 3. Results 3.1. ATRA inhibits LPS induced NFB activation in macrophages NFB signal is essential for LPS-induced macrophage activation, and responsible for gene expression of proinflammatory cytokines such as TNF, the key cytokine involving in IBD development [20,21]. In the study, we employed RAW 264.7 cells, the murine macrophage cell line, and investigated NFB activation induced by LPS. As shown in Fig. 1A, LPS stimulation on RAW 264.7 cells was characterized by phosphorylation of NFB p65. Meanwhile, treatment of ATRA rather than LE135 abrogated LPS-induced phosphor-NFB p65 expression (Fig. 1A). The results above were concurrent with TNF production of RAW 264.7 cells (Fig. 1B). Meanwhile, we noted that, upon LPS stimulation, phosphorylation of NFB p65 was gradually elevated in RAW 264.7 cells,
in a time-dependent manner (Fig. 1C). However, phosphor-NFB p65 expression induced by LPS was substantively blocked by ATRA administration (Fig. 1C). The data above indicate that inhibitory effect of ATRA on LPS-induced NFB signal activation, and TNF production. 3.2. ATRA treatment ameliorates DSS-induced colitis To investigate the effect of RAR signaling on the progress of DSS-induced colitis, we induced colitis by administering 3% DSS in drinking water and treated the mice with ATRA or LE135, the agonist or antagonist of RAR. DSS administration was associated with significant clinical changes, including body weight loss (starting on day 4), fecal blood and diarrhea (on day 6). Treatment with ATRA resulted in significant amelioration of colitis, as shown by a decrease in DAI, improvement in stool consistency, and reduced rectal bleeding (Fig. 2). Conversely, treatment with LE135 had no significant effect on changes of body weight and disease activity, in comparison with DSS group (Fig. 2). The mice with DSS-induced colitis exhibit shorten colon length (Fig. 3A), and histological changes evidenced with mucosal congestion, erosion, loss of goblet cells, thickening of the colon wall and high level of leukocyte infiltration (Fig. 3B and C). The average histological score of DSS colitis amounted to 9.0 ± 1.3. Treatment of mice with ATRA attenuated the severity of the colon injury, with 4.6 ± 1.1 of histological score, lower than DSS group mice (Fig. 3B and
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3.3. ATRA treatment inhibits immune cell infiltration into colon tissue during DSS colitis As a model of IBD, DSS colitis is characterized by substantial immune cell infiltration into colon tissue. As a specific marker of neutrophil, MPO activity has emerged as an index of polymorphonuclear infiltration [22]. In the study, we measured MPO activity in colon tissues of mice with DSS colitis. As shown in Fig. 4, DSS colitis exhibited high MPO activity, inferring excessive infiltration of neutrophil cells into tissues. ATRA treatment resulted in reduction of MPO activity compared with those DSS colitic mice. However, mice that received LE135 administration showed the highest MPO activity (Fig. 4). Next, we determined CD68 expression, the specific marker of monocyte/macrophage [23], in colon tissues by immunohistochemistry. DSS administration resulted in high CD68 expression in lamina propria cells, while treatment of ATRA reduced CD68 expression in colon tissues (Fig. 5A and B). Meanwhile, injection of LE135 further induced CD68 expression, and exhibited higher CD68 positive cell number in lamina propria than DSS and ATRA groups (Fig. 5A and B). The data show that ATRA inhibits but LE135 enhances immune cell infiltration in colon tissues during the progress of colitis.
3.4. ATRA treatment inhibits NF-B signaling Fig. 2. ATRA treatment ameliorates disease symptoms of DSS colitis. Colitis was induced in mice by administration of 3% DSS dissolved in drinking water. ATRA (0.5 mg) or LE135 (0.1 mg) was administered intraperitoneally 2 days after induction of colitis, and repeated daily until the mice were killed on day 8. Control mice drank only distilled water. (A) Body weight changes in the mice in each group (n = 8–10 per group). *P < 0.05, compared with DSS group or DSS + LE135 group mice. (B) Disease activity index (DAI) was scored on day 7 using the following parameters: stool consistency, presence or absence of fecal blood and weight loss. *P < 0.05.
C). However, the mice with LE135 administration showed severe tissue damage in colon, with 9.3 ± 1.5 of histological score (Fig. 3B and C). The data indicate that RAR agonist ameliorates colon tissue damage induced by DSS colitis.
An immunohistochemical analysis was conducted to identify the positive staining with anti-NF-kB (P65) antibody in both cytoplasm and nucleus of biopsies in colon tissues. As shown in Fig. 6A and B, there were substantial NF-B positive cells in lamina propria of the mice with DSS colitis, while ATRA treatment decreased NF-B positive cell number in colon tissues. Next, TNF levels in colon tissues were determined by ELISA. The mice treated with LE135 injection exhibited the highest TNF level in colonic homogenate, in comparison with that of DSS mice and those mice with ATRA treatment. Concurrent with CD68 expression and NF-B activation, the mice treated with ATRA also showed lower TNF level in colon than those DSS mice, as shown in Fig. 6C. The data
Fig. 3. ATRA treatment attenuates colon tissue damage of DSS colitis. Colitis was induced in mice by administration of 3% DSS dissolved in drinking water on day 1, and were sacrificed on day 8. (A) Colon length of each group mice was measured (n = 8–10 per group), *P < 0.05, compared with DSS group or DSS + LE135 group mice. (B) Colonic inflammation was scored by histological analysis at the end of the experiment. **P < 0.01, in comparison with DSS group or DSS + LE135 group mice. (C) Hematoxylin and eosin staining of colonic tissues of four groups of mice. All images are at shown the same magnification (×40).
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Fig. 4. ATRA treatment decreases myeloperoxidase (MPO) activities in colonic tissues of DSS-induced colitis. Myeloperoxidase (MPO) activities in colonic tissues of each group of mice were determined (n = 8). *P < 0.05.
above infer that ATRA treatment inhibits both NF-B activation and TNF production of immune cells in colon tissues of colitic mice. 4. Discussion Vitamin A and its metabolites are biologically active agents capable to be involved in immune regulation including CD4+ T cell differentiation and maintenance of CD4+ T cell homeostasis. For example, deficiency of vitamin A has been shown to link with exacerbation of experimental colitis [10], and supplementation of vitamin A reduces prevalence of diarrhea in children [11]. Meanwhile, ATRA and other metabolites of vitamin A exhibit to regulate immune responses of various animal models of autoimmune diseases, e.g. rheumatoid arthritis [24], type 1 diabetes [25], and experimental encephalomyelitis [26]. Currently, ATRA has been reported to substantially contribute to helper T cell development, especially Th17 and Treg responses. Via its receptor RAR, ATRA
can modulate the expression of FOXP3, IL-17, and/or ROR␥t, and sequentially regulate Treg and Th17 cell polarizations [13,14]. We and others previously showed that ATRA enhanced Treg response, inhibited Th17 generation and function, and attenuated experimental colitis [14,15]. Here, we further report that ATRA regulates macrophage activation and innate responses, and protects mice against DSS-induced colitis. Macrophages, together with other innate immune cells, play an important role in defending the host against infection by bacteria and viruses in a non-specific manner. However, once stimulated by pathogenic antigens, macrophages become activated, and produce substantial proinflammatory cytokines, and initiate adaptive immune responses thereafter [27]. The intracellular signals involved in macrophage activation have been intensively studied. NFB, one of the key transcription factors, is responsible for macrophage activation and inflammation process in mammals and human beings [5]. During the progression of IBD, macrophages in lamina propria and submucosal region are the main source of proinflammatory cytokines such as TNF [28], and have been recognized as the principal inflammatory cells in the mucosal microenvironment with significant contribution to the development of IBD [28]. Here, we set up in vitro and in vivo systems to study the inhibitory effect of ATRA on functional change of macrophages. With in vitro culture setting and murine colitis model, we found that ATRA administration inhibited NFB activation, which subsequently resulted in inhibition of immune cell infiltration and TNF production, a key proinflammatory cytokine for the pathogenesis of IBD [28]. Overall, our study showed the beneficial effect of ATRA administration on experimental colitis, indicating the mechanism of the anti-inflammatory effect of ATRA mediated by inhibition of macrophage-driven proinflammatory processes of colitis. IBD is a clinical challenge with increasing morbidity and poor prognosis. Recently, a variety of animal models have been established to elucidate the pathogenesis of IBD. For example, severe colonic inflammation is developed by intrarectal administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) in mice [29]. TNBS colitis displays clinical and histopathological changes that resemble those
Fig. 5. ATRA treatment reduces CD68 positive cell numbers in lamina propria of mice with DSS-induced colitis. (A) Immunohistochemical staining was performed in sections from colonic tissues of four group mice. Anti CD68 antibody was used as the primary antibody. All images are at shown the same magnification (×200). (B) CD68 positive cell numbers were counted and summarized in per 100 cells in lamina propria of four groups (n = 8). *P < 0.05.
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Fig. 6. ATRA treatment decreases NF-B positive cell numbers and TNF levels in colon of mice with DSS-induced colitis. (A and B) Immunohistochemistry was performed, and anti-NF-B p65 antibody was used to detect p65 in the nucleus of cells in biopsies (A), all images are at shown the same magnification (×200). The cells with positive staining in the cytoplasm and the nucleus were recognized as NF-B positive cells. NF-kB positive cell numbers in lamina propria of four groups were analyzed (B). n = 8, *P < 0.05, compared with DSS group or DSS + LE135 group mice. (C) TNF levels in colonic homogenates of each group of mice were determined by ELISA (n = 6–8). *P < 0.05.
seen in IBD, and exhibits aberrant adaptive immune responses with dominant Th1 and Th17 responses but deficient Treg function [29]. We have previously shown that ATRA down-regulates inflammatory responses of TNBS colitis through restoration of the balance of Treg/Th17 responses [15]. Nevertheless, intestinal macrophages act as the first line of immunity, and provide immediate immune responses when encountering pathogenic antigens. Once activated, macrophages trigger inflammation process, and also induce helper T cell responses [27]. Macrophages rather than helper T cells play the pivotal role in induction of tissue damage and contribution to the progress of IBD [27,30]. To study innate immune responses in experimental colitis, we established DSS colitis which is characterized by excessive activation of macrophages [18]. In the study, we note that ATRA as the RAR agonist inhibits infiltration of macrophages and neutrophils into colon tissues, and decreases production of TNF, one of the main proinflammatory cytokines released by macrophages [18]. Meanwhile, RAR antagonist exhibits some proinflammatory function, such as promoting infiltration of leukocytes into colon tissues. The data indicate the role of RAR signaling in regulation of macrophage dysfunction during the progress of colitis. ATRA, the major biologically active form of vitamin A, has been clinically used for disease treatment. Since the study in 1925 provided by Wolbach and Howe who were the first to show retinoids as anti-cancer agents, epidemiological, preclinical and clinical studies have established a causative relationship between vitamin A deficiency and risk of cancer [31]. To date, ATRA and other retinoids have been recognized as chemopreventive and chemotherapeutic agents in cancer prevention and treatment, such as acute promyelocytic leukemia and squamous cell carcinoma [32,33]. ATRA has also been reported as an anti-inflammation agent in clinical trial for
human inflammatory diseases such as emphysema [34,35]. In the study, ATRA presents the inhibitory effect on immune responses, and attenuates the experimental colitis, indicating that ATRA might be an alternative to our current clinical approaches to suppress intestinal inflammation in IBD. In summary, ATRA inhibits LPS-induced NF-B activation in macrophage cell line, and protects mice against DSS-induced colitis. RAR-related signaling may provide new alternatives to the current clinical approaches to suppress macrophage responsiveness, and help design novel strategies for treatment of inflammatory diseases. Conflicts of interest The authors declare that they have no conflicts of interest. Acknowledgements Supported by National Natural Science Foundation of China, No. 81270472 and No. 81070310, Science and Technology Program of Education Department of Jiangxi Province, No. GJJ13138, Natural Science Foundation of Jiangxi Province, No. 20142BAB205048, National Science and Technology Major Projects for “Major New Drugs Innovation and Development” of China (No. 2011ZX09302007-03). References [1] Arseneau KO, Tamagawa H, Pizarro TT, Cominelli F. Innate and adaptive immune responses related to IBD pathogenesis. Curr Gastroenterol Rep 2007;9:508–12.
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[2] Brown SJ, Mayer L. The immune response in inflammatory bowel disease. Am J Gastroenterol 2007;102:2058–69. [3] Yamamoto-Furusho JK, Podolsky DK. Innate immunity in inflammatory bowel disease. World J Gastroenterol 2007;13:5577–80. [4] Xavier RJ, Podolsky DK. Unravelling the pathogenesis of inflammatory bowel disease. Nature 2007;448:427–34. [5] Gasparini C, Feldmann M. NF-kappaB as a target for modulating inflammatory responses. Curr Pharm Des 2012;18:5735–45. [6] Rakonczay Jr Z, Hegyi P, Takacs T, McCarroll J, Saluja AK. The role of NF-kappaB activation in the pathogenesis of acute pancreatitis. Gut 2008;57:259–67. [7] Ohira H, Fujioka Y, Katagiri C, Mamoto R, Aoyama-Ishikawa M, Amako K, et al. Butyrate attenuates inflammation and lipolysis generated by the interaction of adipocytes and macrophages. J Atheroscler Thromb 2013;20:425–42. [8] Bai A, Ma AG, Yong M, Weiss CR, Ma Y, Guan Q, et al. AMPK agonist downregulates innate and adaptive immune responses in TNBS-induced murine acute and relapsing colitis. Biochem Pharmacol 2010;80:1708–17. [9] Bai A, Guo Y, Lu N. The effect of the cholinergic anti-inflammatory pathway on experimental colitis. Scand J Immunol 2007;66:538–45. [10] Reifen R, Nur T, Ghebermeskel K, Zaiger G, Urizky R, Pines M. Vitamin A deficiency exacerbates inflammation in a rat model of colitis through activation of nuclear factor-kappaB and collagen formation. J Nutr 2002;132:2743–7. [11] Long KZ, Santos JI, Rosado JL, Lopez-Saucedo C, Thompson-Bonilla R, Abonce M, et al. Impact of vitamin A on selected gastrointestinal pathogen infections and associated diarrheal episodes among children in Mexico City, Mexico. J Infect Dis 2006;194:1217–25. [12] Benson MJ, Pino-Lagos K, Rosemblatt M, Noelle RJ. All-trans retinoic acid mediates enhanced T reg cell growth, differentiation, and gut homing in the face of high levels of co-stimulation. J Exp Med 2007;204:1765–74. [13] Elias KM, Laurence A, Davidson TS, Stephens G, Kanno Y, Shevach EM, et al. Retinoic acid inhibits Th17 polarization and enhances FoxP3 expression through a Stat-3/Stat-5 independent signaling pathway. Blood 2008;111:1013–20. [14] Mucida D, Park Y, Kim G, Turovskaya O, Scott I, Kronenberg M, et al. Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science 2007;317:256–60. [15] Bai A, Lu N, Guo Y, Liu Z, Chen J, Peng Z. All-trans retinoic acid down-regulates inflammatory responses by shifting the Treg/Th17 profile in human ulcerative and murine colitis. J Leukoc Biol 2009;86:959–69. [16] Menning A, Loddenkemper C, Westendorf AM, Szilagyi B, Buer J, Siewert C, et al. Retinoic acid-induced gut tropism improves the protective capacity of Treg in acute but not in chronic gut inflammation. Eur J Immunol 2010;40: 2539–48. [17] Bai A, Lu N, Guo Y, Chen J, Liu Z. Modulation of inflammatory response via alpha2-adrenoceptor blockade in acute murine colitis. Clin Exp Immunol 2009;156:353–62. [18] Murano M, Maemura K, Hirata I, Toshina K, Nishikawa T, Hamamoto N, et al. Therapeutic effect of intracolonically administered nuclear factor kappa B (p65)
[19]
[20]
[21] [22]
[23]
[24]
[25]
[26]
[27] [28] [29] [30]
[31] [32] [33] [34]
[35]
antisense oligonucleotide on mouse dextran sulphate sodium (DSS)-induced colitis. Clin Exp Immunol 2000;120:51–8. Bai AP, Ouyang Q, Xiao XR, Li SF. Probiotics modulate inflammatory cytokine secretion from inflamed mucosa in active ulcerative colitis. Int J Clin Pract 2006;60:284–8. Han ES, Oh JY, Park HJ. Cordyceps militaris extract suppresses dextran sodium sulfate-induced acute colitis in mice and production of inflammatory mediators from macrophages and mast cells. J Ethnopharmacol 2011;134:703–10. D’Haens G. Geert Anti-TNF therapy for Crohn’s disease. Curr Pharm Des 2003;9:289–94. Bradley PP, Priebat DA, Christensen RD, Rothstein G. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol 1982;78:206–9. Harris JA, Jain S, Ren Q, Zarineh A, Liu C, Ibrahim S. CD163 versus CD68 in tumor associated macrophages of classical Hodgkin lymphoma. Diagn Pathol 2012;7:12. Miyagawa N, Homma T, Kagechika H, Shudo K, Nagai H. Effect of synthetic retinoid, TAC-101, on experimental autoimmune disease. Pharmacology 2003;67:21–31. Zunino SJ, Storms DH, Stephensen CB. Diets rich in polyphenols and vitamin A inhibit the development of type I autoimmune diabetes in nonobese diabetic mice. J Nutr 2007;137:1216–21. Racke MK, Burnett D, Pak SH, Albert PS, Cannella B, Raine CS, et al. Retinoid treatment of experimental allergic encephalomyelitis. IL-4 production correlates with improved disease course. J Immunol 1995;154:450–8. Sheikh SZ, Plevy SE. The role of the macrophage in sentinel responses in intestinal immunity. Curr Opin Gastroenterol 2010;26:578–82. Mahida YR. The key role of macrophages in the immunopathogenesis of inflammatory bowel disease. Inflamm Bowel Dis 2000;6:21–33. Scheiffele F, Fuss IJ. Induction of TNBS colitis in mice. Curr Protoc Immunol 2002. Chapter 15:Unit 15.19. Uno JK, Rao KN, Matsuoka K, Sheikh SZ, Kobayashi T, Li F, et al. Altered macrophage function contributes to colitis in mice defective in the phosphoinositide-3 kinase subunit p110delta. Gastroenterology 2010;139: 1642–53, 1653 e1-6. Fields AL, Soprano DR, Soprano KJ. Retinoids in biological control and cancer. J Cell Biochem 2007;102:886–98. Theodosiou M, Laudet V, Schubert M. From carrot to clinic: an overview of the retinoic acid signaling pathway. Cell Mol Life Sci 2010;67:1423–45. Rigas JR, Dragnev KH. Emerging role of rexinoids in non-small cell lung cancer: focus on bexarotene. Oncologist 2005;10:22–33. Mao JT, Goldin JG, Dermand J, Ibrahim G, Brown MS, Emerick A, et al. A pilot study of all-trans-retinoic acid for the treatment of human emphysema. Am J Respir Crit Care Med 2002;165:718–23. Frankenberger M, Heimbeck I, Moller W, Mamidi S, Kassner G, Pukelsheim K, et al. Inhaled all-trans retinoic acid in an individual with severe emphysema. Eur Respir J 2009;34:1487–9.
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All-trans retinoic acid attenuates experimental colitis through inhibition of NF-B signaling Kai Hong a,1 , Yi Zhang a,1 , Yuan Guo a,b,1 , Jun Xie b , Jian Wang a , Xingxing He a , Nonghua Lu a , Aiping Bai a,∗ a b
Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang 330006, China Department of Pharmacy, The First Affiliated Hospital of Nanchang University, Nanchang 330006, China
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Article history: Received 19 March 2014 Received in revised form 13 June 2014 Accepted 24 June 2014 Available online 6 July 2014 Keywords: All-trans retinoic acid RAR Colitis Macrophage NF-B
a b s t r a c t Inflammatory bowel disease (IBD) is characterized by excessive innate immune cell activation, which is responsible for tissue damage and induction of adaptive immune responses. All-trans retinoic acid (ATRA), the ligand of retinoic acid receptors (RAR), has been previously shown to regulate adaptive immune responses and restore Th17/Treg balance, while its role in regulation of innate immune cell function such as macrophages remains to be elucidated. The study was performed to explore the effect of ATRA on regulation of innate immune responses during dextran sulfate sodium (DSS) induced murine colitis. The mice with DSS colitis were administered with vehicle, ATRA, or LE135. Colitis was evaluated by clinical symptoms, tissue myeloperoxidase (MPO) activity, and the expressions of CD68 and nuclear factor (NF) B p65, and tumor necrosis factor (TNF) level in inflamed colon. RAW 264.7 cells were pretreated with vehicle, ATRA, or LE135, followed by LPS challenge in vitro. ATRA administration ameliorates DSS-induced colitis evidenced with decreased TNF level and CD68 expression, while LE135 leads to exacerbation of colitis. ATRA treatment in vitro dampens LPS induced NF-B activation and TNF production of RAW 264.7 cells. Together, our data show a crucial role of ATRA in the progress of acute colitis through inhibiting NF-B activation, and suggest that ATRA represents a novel therapeutic approach for the management of IBD. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Inflammatory bowel disease (IBD), mainly referred to Crohn’s disease and ulcerative colitis, is a serious intestinal immune disorder. The etiology of IBD is not clearly studied so far, but it is strongly suggested that dysregulated immune responses contribute to the progress of IBD [1]. It has been well established that innate immune cells including macrophages determine the course of disease progress [1,2]. After exposure to and activated thereafter by the abundant intestinal bacterial antigens, macrophages become recruited, and induce adaptive immune response via direct contact and release of substantive proinflammatory cytokines such
Abbreviations: ATRA, all-trans retinoic acid; DSS, dextran sulfate sodium; ELISA, enzyme-linked immunosorbent assays; IBD, inflammatory bowel disease; IL, interleukin; LPS, lipopolysaccharides; MPO, myeloperoxidase; NFB, nuclear factor B; RAR, retinoic acid receptors; TNF, tumor necrosis factor. ∗ Corresponding author. Tel.: +86 88692507. E-mail address: [email protected] (A. Bai). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.imlet.2014.06.011 0165-2478/© 2014 Elsevier B.V. All rights reserved.
as tumor necrosis factor (TNF), interleukin (IL)-12, IL-6, and IL23 [2]. Immune cells including macrophages, in combination with proinflammatory cytokines, play critical roles in maintaining the uncontrolled inflammatory response, eventually resulting in the intestinal tissue damage seen in IBD [3,4]. Inhibition of immune cell function has been suggested as one of the crucial targets for the treatment immune diseases including IBD. Nuclear factor (NF) B is one of important transcription factors controlling inflammatory responses of immune diseases [5]. In the presence of the stimulations of lipopolysaccharides (LPS) and other extracellular bacterial antigens, the dimer of NFB composed of the P65 and P50 subunits translocates to nucleus of macrophage, and regulates various target gene expression including a large amount of inflammatory cytokines and chemokines, leading to sustained inflammatory responses and tissue damage [5,6]. Recently, we and others have demonstrated that inhibition of NFB signal can attenuate immune responses, and ameliorate immune diseases in animals [7–9], supporting the beneficial effects of inhibition of NFB signaling. Vitamin A and its derivatives, have been shown to be involved in modulation of immune responses. For example, deficiency of
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vitamin A results in exacerbated experimental colitis [10], and supplementation of vitamin A can decrease prevalence of diarrhea among children [11]. Recently, all-trans retinoic acid (ATRA), a metabolite of vitamin A, has emerged as a novel biological chemical that is critically involved in regulation of adaptive immune responses [12]. Via its nuclear receptor retinoic acid receptors (RAR), ATRA can enhance Treg growth, differentiation, and gut homing, and inhibit Th17 generation and function [13,14]. We and others have shown that ATRA restores the balance between Th17 and Treg cells in the development of IBD, and RAR antagonist LE135 aggravates the aberrant helper T cell responses of experimental colitis [15,16]. However, as the pivotal cells in the pathogenesis of IBD, the impact of ATRA on macrophage activation and the related mechanisms remain to be elucidated. In the study, we demonstrate that ATRA inhibits LPS-induced NF-B activation in macrophage, and protects mice against dextran sulfate sodium (DSS)-induced colitis. 2. Methods 2.1. Cell culture and treatment Murine macrophage cell line RAW 264.7 was cultured in HEPESbuffered Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, penicillin G (100 U/mL), and streptomycin (100 g/mL) at 37 ◦ C. For in vitro study, about 1 × 106 /mL RAW264.7 cells were seeded in 48-well plates. The cells were treated with vehicle, 100 nM ATRA (Sigma–Aldrich) or 1 M LE135 (Santa Cruz Biotechnology) for 1 h, prior to 100 ng/mL or 1 g/mL lipopolysaccharide (LPS, from Escherichia coli 0111:B4; Sigma–Aldrich) challenge. 2.2. Enzyme-linked immunosorbent assays (ELISA) Tumor necrosis factor (TNF) levels in culture supernatants or colon tissues were determined by ELISA, following manufacturer’s instructions (R&D Systems, Inc.). Briefly, polyclonal rat anti-mouse cytokine antibodies were used as capturing antibodies and biotinylated polyclonal rat anti-mouse cytokine antibodies for detection, and the standard curve of TNF was set up meanwhile. Color changes were determined at 450 nm. 2.3. Western blot Proteins were extracted from the cultured cells using a lysis buffer (0.1 M PBS pH7.4 containing 1% deoxycholic acid sodium, 0.2% SDS, and protease inhibitors). After measurement of protein concentration, the proteins of the samples were loaded and separated by SDS-PAGE and then electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were incubated with primary rabbit anti-phospho-NFB P65 (Ser536) or NFB P65 antibody (both from Cell Signaling Technology), or mouse anti--actin antibody (from Santa Cruz Biotechnology). After incubating with secondary antibodies, the immunoreactive bands were visualized using the SuperSignal West Femto Maximum Sensitivity Substrate reagents (Thermo Scientific). -actin was used as an inner control. Relative protein levels were determined by Image J software (NIH). 2.4. Animals Female BALB/c mice were provided by the Experimental Animal Center of Nanchang University, and fed under specific pathogenfree conditions. 7–8 week-old mice were randomly divided into four groups in the study, weighing approximately 22 g: Control, DSS, ATRA, and LE135. All protocols for the projects using mice were
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reviewed and approved by the Institutional Animal Care Committee of Nanchang University. 2.5. Induction of dextran sulphate sodium (DSS) colitis Acute DSS colitis was induced in BALB/c mice according to the previously published method with minor modification [17]. The mice of DSS, ATRA, and LE135 groups were fed 3% (w/v) DSS (molecular mass, 36–50 kDa; MP Biomedicals) dissolved in the drinking water on day one. Fresh DSS solution was provided every second day. Control mice drank only distilled water. ATRA (0.5 mg) or LE135 (0.1 mg) was administered intraperitoneally (i.p.) since day three and repeated daily until the mice were killed on day eight. Disease symptoms of colitis were assessed daily by measurement of body weight, evaluation of stool consistency and detection of bloody stools. Disease severity was scored using a clinical disease activity index (DAI) ranging from 0 to 4, calculated as described previously [17] using the following parameters: stool consistency, presence or absence of fecal blood and weight loss. Mice were killed on day eight, and the middle section of colon was fixed in 10% formaldehyde–saline. Hematoxylin and eosin stain (HE)-stained sections were graded based on a scoring system modified from a previous study [18]. Histology scoring was performed, and a combined score of inflammatory cell infiltration and tissue damage was determined as follows: cell infiltration: score 0, occasional inflammatory cells in the lamina propria (LP); 1, increased infiltrate in the LP predominantly at the base of crypts; 2, confluence of inflammatory infiltrate extending into the mucosa; and 3, transmural extension of infiltrate. Tissue damage: score 0, no mucosal damage; 1, partial (up to 50%) loss of crypts in large areas; 2, partial to total 50–100% loss of crypts in large areas, epithelium intact; and 3, total loss of crypts in large areas and epithelium lost. 2.6. Immunohistochemistry Colon tissues of mice were taken and fixed immediately in 10% buffered formalin, embedded in paraffin, and cut into 4 m sections. After blockade of inner peroxidase, sections were incubated sequentially with the first antibody solution including rabbit anti-CD68 (Abcam), or anti-NF-B p65 (C20, from Santa Cruz Biotechnology) antibody. After three washes in PBS (pH 7.4), the sections were then incubated in secondary goat anti-rabbit immunoglobulin (Ig)G conjugated with peroxidase labeled polymer, prior to colorization using diaminobenzidine reaction and counterstained with hematoxylin. Negative controls were established using rabbit IgG instead of the first antibodies. The sections were evaluated using light microscopy, and 100 cells in lamina propria per high power field were calculated for statistical analysis. The cells stained with anti-NF-B (P65) antibody in both cytoplasm and the nucleus were counted as NF-B positive cells, whereas those without nucleus stain were regarded as NF-B negative cells [19]. Negative controls were established by using rabbit IgG. 2.7. Measurement of Myeloperoxidase (MPO) activity All experiments were performed within 1 week of tissue collection. MPO activity was measured according to the method previously described with minor modification [15,17]. In short, tissues were homogenized in hexadecyltrimethylammonium bromide in 50 mM potassium phosphate buffer. Aliquots were then added to O-dianisidine hydrochloride solution. Absorbance was read at 450 nm using a microplate reader. MPO was expressed in units/gram tissue, where 1 unit corresponds to the activity required to degrade 1 mmol hydrogen peroxide in 1 min at 24 ◦ C.
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Fig. 1. ATRA blocks LPS induced NFB activation in macrophage. RAW 264.7 cells were pretreated with vehicle, ATRA (100 nM), LE135 (1 M) for 1 h, prior to LPS challenge. (A) 1 h after LPS challenge (1 g/mL), the expression of pNFB p65 was determined by western blot. (B) 24 h after stimulation of 100 ng/mL LPS, TNF levels in supernatants were measured by ELISA (n = 4). **P < 0.01. (C) Western blotting of phosphorylation of pNFB p65 induced by LPS (1 g/mL) in the absence or presence of ATRA for different times.
2.8. Statistical analysis All data in the text and figures are expressed as mean ± standard deviation. Comparisons of more than two groups were made with a one-way analysis of variance using Tukey’s post hoc test. When appropriate, comparison with two groups was made using Student’s t-test for unpaired data. Differences were considered statistically significant if P < 0.05. 3. Results 3.1. ATRA inhibits LPS induced NFB activation in macrophages NFB signal is essential for LPS-induced macrophage activation, and responsible for gene expression of proinflammatory cytokines such as TNF, the key cytokine involving in IBD development [20,21]. In the study, we employed RAW 264.7 cells, the murine macrophage cell line, and investigated NFB activation induced by LPS. As shown in Fig. 1A, LPS stimulation on RAW 264.7 cells was characterized by phosphorylation of NFB p65. Meanwhile, treatment of ATRA rather than LE135 abrogated LPS-induced phosphor-NFB p65 expression (Fig. 1A). The results above were concurrent with TNF production of RAW 264.7 cells (Fig. 1B). Meanwhile, we noted that, upon LPS stimulation, phosphorylation of NFB p65 was gradually elevated in RAW 264.7 cells,
in a time-dependent manner (Fig. 1C). However, phosphor-NFB p65 expression induced by LPS was substantively blocked by ATRA administration (Fig. 1C). The data above indicate that inhibitory effect of ATRA on LPS-induced NFB signal activation, and TNF production. 3.2. ATRA treatment ameliorates DSS-induced colitis To investigate the effect of RAR signaling on the progress of DSS-induced colitis, we induced colitis by administering 3% DSS in drinking water and treated the mice with ATRA or LE135, the agonist or antagonist of RAR. DSS administration was associated with significant clinical changes, including body weight loss (starting on day 4), fecal blood and diarrhea (on day 6). Treatment with ATRA resulted in significant amelioration of colitis, as shown by a decrease in DAI, improvement in stool consistency, and reduced rectal bleeding (Fig. 2). Conversely, treatment with LE135 had no significant effect on changes of body weight and disease activity, in comparison with DSS group (Fig. 2). The mice with DSS-induced colitis exhibit shorten colon length (Fig. 3A), and histological changes evidenced with mucosal congestion, erosion, loss of goblet cells, thickening of the colon wall and high level of leukocyte infiltration (Fig. 3B and C). The average histological score of DSS colitis amounted to 9.0 ± 1.3. Treatment of mice with ATRA attenuated the severity of the colon injury, with 4.6 ± 1.1 of histological score, lower than DSS group mice (Fig. 3B and
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3.3. ATRA treatment inhibits immune cell infiltration into colon tissue during DSS colitis As a model of IBD, DSS colitis is characterized by substantial immune cell infiltration into colon tissue. As a specific marker of neutrophil, MPO activity has emerged as an index of polymorphonuclear infiltration [22]. In the study, we measured MPO activity in colon tissues of mice with DSS colitis. As shown in Fig. 4, DSS colitis exhibited high MPO activity, inferring excessive infiltration of neutrophil cells into tissues. ATRA treatment resulted in reduction of MPO activity compared with those DSS colitic mice. However, mice that received LE135 administration showed the highest MPO activity (Fig. 4). Next, we determined CD68 expression, the specific marker of monocyte/macrophage [23], in colon tissues by immunohistochemistry. DSS administration resulted in high CD68 expression in lamina propria cells, while treatment of ATRA reduced CD68 expression in colon tissues (Fig. 5A and B). Meanwhile, injection of LE135 further induced CD68 expression, and exhibited higher CD68 positive cell number in lamina propria than DSS and ATRA groups (Fig. 5A and B). The data show that ATRA inhibits but LE135 enhances immune cell infiltration in colon tissues during the progress of colitis.
3.4. ATRA treatment inhibits NF-B signaling Fig. 2. ATRA treatment ameliorates disease symptoms of DSS colitis. Colitis was induced in mice by administration of 3% DSS dissolved in drinking water. ATRA (0.5 mg) or LE135 (0.1 mg) was administered intraperitoneally 2 days after induction of colitis, and repeated daily until the mice were killed on day 8. Control mice drank only distilled water. (A) Body weight changes in the mice in each group (n = 8–10 per group). *P < 0.05, compared with DSS group or DSS + LE135 group mice. (B) Disease activity index (DAI) was scored on day 7 using the following parameters: stool consistency, presence or absence of fecal blood and weight loss. *P < 0.05.
C). However, the mice with LE135 administration showed severe tissue damage in colon, with 9.3 ± 1.5 of histological score (Fig. 3B and C). The data indicate that RAR agonist ameliorates colon tissue damage induced by DSS colitis.
An immunohistochemical analysis was conducted to identify the positive staining with anti-NF-kB (P65) antibody in both cytoplasm and nucleus of biopsies in colon tissues. As shown in Fig. 6A and B, there were substantial NF-B positive cells in lamina propria of the mice with DSS colitis, while ATRA treatment decreased NF-B positive cell number in colon tissues. Next, TNF levels in colon tissues were determined by ELISA. The mice treated with LE135 injection exhibited the highest TNF level in colonic homogenate, in comparison with that of DSS mice and those mice with ATRA treatment. Concurrent with CD68 expression and NF-B activation, the mice treated with ATRA also showed lower TNF level in colon than those DSS mice, as shown in Fig. 6C. The data
Fig. 3. ATRA treatment attenuates colon tissue damage of DSS colitis. Colitis was induced in mice by administration of 3% DSS dissolved in drinking water on day 1, and were sacrificed on day 8. (A) Colon length of each group mice was measured (n = 8–10 per group), *P < 0.05, compared with DSS group or DSS + LE135 group mice. (B) Colonic inflammation was scored by histological analysis at the end of the experiment. **P < 0.01, in comparison with DSS group or DSS + LE135 group mice. (C) Hematoxylin and eosin staining of colonic tissues of four groups of mice. All images are at shown the same magnification (×40).
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Fig. 4. ATRA treatment decreases myeloperoxidase (MPO) activities in colonic tissues of DSS-induced colitis. Myeloperoxidase (MPO) activities in colonic tissues of each group of mice were determined (n = 8). *P < 0.05.
above infer that ATRA treatment inhibits both NF-B activation and TNF production of immune cells in colon tissues of colitic mice. 4. Discussion Vitamin A and its metabolites are biologically active agents capable to be involved in immune regulation including CD4+ T cell differentiation and maintenance of CD4+ T cell homeostasis. For example, deficiency of vitamin A has been shown to link with exacerbation of experimental colitis [10], and supplementation of vitamin A reduces prevalence of diarrhea in children [11]. Meanwhile, ATRA and other metabolites of vitamin A exhibit to regulate immune responses of various animal models of autoimmune diseases, e.g. rheumatoid arthritis [24], type 1 diabetes [25], and experimental encephalomyelitis [26]. Currently, ATRA has been reported to substantially contribute to helper T cell development, especially Th17 and Treg responses. Via its receptor RAR, ATRA
can modulate the expression of FOXP3, IL-17, and/or ROR␥t, and sequentially regulate Treg and Th17 cell polarizations [13,14]. We and others previously showed that ATRA enhanced Treg response, inhibited Th17 generation and function, and attenuated experimental colitis [14,15]. Here, we further report that ATRA regulates macrophage activation and innate responses, and protects mice against DSS-induced colitis. Macrophages, together with other innate immune cells, play an important role in defending the host against infection by bacteria and viruses in a non-specific manner. However, once stimulated by pathogenic antigens, macrophages become activated, and produce substantial proinflammatory cytokines, and initiate adaptive immune responses thereafter [27]. The intracellular signals involved in macrophage activation have been intensively studied. NFB, one of the key transcription factors, is responsible for macrophage activation and inflammation process in mammals and human beings [5]. During the progression of IBD, macrophages in lamina propria and submucosal region are the main source of proinflammatory cytokines such as TNF [28], and have been recognized as the principal inflammatory cells in the mucosal microenvironment with significant contribution to the development of IBD [28]. Here, we set up in vitro and in vivo systems to study the inhibitory effect of ATRA on functional change of macrophages. With in vitro culture setting and murine colitis model, we found that ATRA administration inhibited NFB activation, which subsequently resulted in inhibition of immune cell infiltration and TNF production, a key proinflammatory cytokine for the pathogenesis of IBD [28]. Overall, our study showed the beneficial effect of ATRA administration on experimental colitis, indicating the mechanism of the anti-inflammatory effect of ATRA mediated by inhibition of macrophage-driven proinflammatory processes of colitis. IBD is a clinical challenge with increasing morbidity and poor prognosis. Recently, a variety of animal models have been established to elucidate the pathogenesis of IBD. For example, severe colonic inflammation is developed by intrarectal administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) in mice [29]. TNBS colitis displays clinical and histopathological changes that resemble those
Fig. 5. ATRA treatment reduces CD68 positive cell numbers in lamina propria of mice with DSS-induced colitis. (A) Immunohistochemical staining was performed in sections from colonic tissues of four group mice. Anti CD68 antibody was used as the primary antibody. All images are at shown the same magnification (×200). (B) CD68 positive cell numbers were counted and summarized in per 100 cells in lamina propria of four groups (n = 8). *P < 0.05.
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Fig. 6. ATRA treatment decreases NF-B positive cell numbers and TNF levels in colon of mice with DSS-induced colitis. (A and B) Immunohistochemistry was performed, and anti-NF-B p65 antibody was used to detect p65 in the nucleus of cells in biopsies (A), all images are at shown the same magnification (×200). The cells with positive staining in the cytoplasm and the nucleus were recognized as NF-B positive cells. NF-kB positive cell numbers in lamina propria of four groups were analyzed (B). n = 8, *P < 0.05, compared with DSS group or DSS + LE135 group mice. (C) TNF levels in colonic homogenates of each group of mice were determined by ELISA (n = 6–8). *P < 0.05.
seen in IBD, and exhibits aberrant adaptive immune responses with dominant Th1 and Th17 responses but deficient Treg function [29]. We have previously shown that ATRA down-regulates inflammatory responses of TNBS colitis through restoration of the balance of Treg/Th17 responses [15]. Nevertheless, intestinal macrophages act as the first line of immunity, and provide immediate immune responses when encountering pathogenic antigens. Once activated, macrophages trigger inflammation process, and also induce helper T cell responses [27]. Macrophages rather than helper T cells play the pivotal role in induction of tissue damage and contribution to the progress of IBD [27,30]. To study innate immune responses in experimental colitis, we established DSS colitis which is characterized by excessive activation of macrophages [18]. In the study, we note that ATRA as the RAR agonist inhibits infiltration of macrophages and neutrophils into colon tissues, and decreases production of TNF, one of the main proinflammatory cytokines released by macrophages [18]. Meanwhile, RAR antagonist exhibits some proinflammatory function, such as promoting infiltration of leukocytes into colon tissues. The data indicate the role of RAR signaling in regulation of macrophage dysfunction during the progress of colitis. ATRA, the major biologically active form of vitamin A, has been clinically used for disease treatment. Since the study in 1925 provided by Wolbach and Howe who were the first to show retinoids as anti-cancer agents, epidemiological, preclinical and clinical studies have established a causative relationship between vitamin A deficiency and risk of cancer [31]. To date, ATRA and other retinoids have been recognized as chemopreventive and chemotherapeutic agents in cancer prevention and treatment, such as acute promyelocytic leukemia and squamous cell carcinoma [32,33]. ATRA has also been reported as an anti-inflammation agent in clinical trial for
human inflammatory diseases such as emphysema [34,35]. In the study, ATRA presents the inhibitory effect on immune responses, and attenuates the experimental colitis, indicating that ATRA might be an alternative to our current clinical approaches to suppress intestinal inflammation in IBD. In summary, ATRA inhibits LPS-induced NF-B activation in macrophage cell line, and protects mice against DSS-induced colitis. RAR-related signaling may provide new alternatives to the current clinical approaches to suppress macrophage responsiveness, and help design novel strategies for treatment of inflammatory diseases. Conflicts of interest The authors declare that they have no conflicts of interest. Acknowledgements Supported by National Natural Science Foundation of China, No. 81270472 and No. 81070310, Science and Technology Program of Education Department of Jiangxi Province, No. GJJ13138, Natural Science Foundation of Jiangxi Province, No. 20142BAB205048, National Science and Technology Major Projects for “Major New Drugs Innovation and Development” of China (No. 2011ZX09302007-03). References [1] Arseneau KO, Tamagawa H, Pizarro TT, Cominelli F. Innate and adaptive immune responses related to IBD pathogenesis. Curr Gastroenterol Rep 2007;9:508–12.
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[2] Brown SJ, Mayer L. The immune response in inflammatory bowel disease. Am J Gastroenterol 2007;102:2058–69. [3] Yamamoto-Furusho JK, Podolsky DK. Innate immunity in inflammatory bowel disease. World J Gastroenterol 2007;13:5577–80. [4] Xavier RJ, Podolsky DK. Unravelling the pathogenesis of inflammatory bowel disease. Nature 2007;448:427–34. [5] Gasparini C, Feldmann M. NF-kappaB as a target for modulating inflammatory responses. Curr Pharm Des 2012;18:5735–45. [6] Rakonczay Jr Z, Hegyi P, Takacs T, McCarroll J, Saluja AK. The role of NF-kappaB activation in the pathogenesis of acute pancreatitis. Gut 2008;57:259–67. [7] Ohira H, Fujioka Y, Katagiri C, Mamoto R, Aoyama-Ishikawa M, Amako K, et al. Butyrate attenuates inflammation and lipolysis generated by the interaction of adipocytes and macrophages. J Atheroscler Thromb 2013;20:425–42. [8] Bai A, Ma AG, Yong M, Weiss CR, Ma Y, Guan Q, et al. AMPK agonist downregulates innate and adaptive immune responses in TNBS-induced murine acute and relapsing colitis. Biochem Pharmacol 2010;80:1708–17. [9] Bai A, Guo Y, Lu N. The effect of the cholinergic anti-inflammatory pathway on experimental colitis. Scand J Immunol 2007;66:538–45. [10] Reifen R, Nur T, Ghebermeskel K, Zaiger G, Urizky R, Pines M. Vitamin A deficiency exacerbates inflammation in a rat model of colitis through activation of nuclear factor-kappaB and collagen formation. J Nutr 2002;132:2743–7. [11] Long KZ, Santos JI, Rosado JL, Lopez-Saucedo C, Thompson-Bonilla R, Abonce M, et al. Impact of vitamin A on selected gastrointestinal pathogen infections and associated diarrheal episodes among children in Mexico City, Mexico. J Infect Dis 2006;194:1217–25. [12] Benson MJ, Pino-Lagos K, Rosemblatt M, Noelle RJ. All-trans retinoic acid mediates enhanced T reg cell growth, differentiation, and gut homing in the face of high levels of co-stimulation. J Exp Med 2007;204:1765–74. [13] Elias KM, Laurence A, Davidson TS, Stephens G, Kanno Y, Shevach EM, et al. Retinoic acid inhibits Th17 polarization and enhances FoxP3 expression through a Stat-3/Stat-5 independent signaling pathway. Blood 2008;111:1013–20. [14] Mucida D, Park Y, Kim G, Turovskaya O, Scott I, Kronenberg M, et al. Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science 2007;317:256–60. [15] Bai A, Lu N, Guo Y, Liu Z, Chen J, Peng Z. All-trans retinoic acid down-regulates inflammatory responses by shifting the Treg/Th17 profile in human ulcerative and murine colitis. J Leukoc Biol 2009;86:959–69. [16] Menning A, Loddenkemper C, Westendorf AM, Szilagyi B, Buer J, Siewert C, et al. Retinoic acid-induced gut tropism improves the protective capacity of Treg in acute but not in chronic gut inflammation. Eur J Immunol 2010;40: 2539–48. [17] Bai A, Lu N, Guo Y, Chen J, Liu Z. Modulation of inflammatory response via alpha2-adrenoceptor blockade in acute murine colitis. Clin Exp Immunol 2009;156:353–62. [18] Murano M, Maemura K, Hirata I, Toshina K, Nishikawa T, Hamamoto N, et al. Therapeutic effect of intracolonically administered nuclear factor kappa B (p65)
[19]
[20]
[21] [22]
[23]
[24]
[25]
[26]
[27] [28] [29] [30]
[31] [32] [33] [34]
[35]
antisense oligonucleotide on mouse dextran sulphate sodium (DSS)-induced colitis. Clin Exp Immunol 2000;120:51–8. Bai AP, Ouyang Q, Xiao XR, Li SF. Probiotics modulate inflammatory cytokine secretion from inflamed mucosa in active ulcerative colitis. Int J Clin Pract 2006;60:284–8. Han ES, Oh JY, Park HJ. Cordyceps militaris extract suppresses dextran sodium sulfate-induced acute colitis in mice and production of inflammatory mediators from macrophages and mast cells. J Ethnopharmacol 2011;134:703–10. D’Haens G. Geert Anti-TNF therapy for Crohn’s disease. Curr Pharm Des 2003;9:289–94. Bradley PP, Priebat DA, Christensen RD, Rothstein G. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol 1982;78:206–9. Harris JA, Jain S, Ren Q, Zarineh A, Liu C, Ibrahim S. CD163 versus CD68 in tumor associated macrophages of classical Hodgkin lymphoma. Diagn Pathol 2012;7:12. Miyagawa N, Homma T, Kagechika H, Shudo K, Nagai H. Effect of synthetic retinoid, TAC-101, on experimental autoimmune disease. Pharmacology 2003;67:21–31. Zunino SJ, Storms DH, Stephensen CB. Diets rich in polyphenols and vitamin A inhibit the development of type I autoimmune diabetes in nonobese diabetic mice. J Nutr 2007;137:1216–21. Racke MK, Burnett D, Pak SH, Albert PS, Cannella B, Raine CS, et al. Retinoid treatment of experimental allergic encephalomyelitis. IL-4 production correlates with improved disease course. J Immunol 1995;154:450–8. Sheikh SZ, Plevy SE. The role of the macrophage in sentinel responses in intestinal immunity. Curr Opin Gastroenterol 2010;26:578–82. Mahida YR. The key role of macrophages in the immunopathogenesis of inflammatory bowel disease. Inflamm Bowel Dis 2000;6:21–33. Scheiffele F, Fuss IJ. Induction of TNBS colitis in mice. Curr Protoc Immunol 2002. Chapter 15:Unit 15.19. Uno JK, Rao KN, Matsuoka K, Sheikh SZ, Kobayashi T, Li F, et al. Altered macrophage function contributes to colitis in mice defective in the phosphoinositide-3 kinase subunit p110delta. Gastroenterology 2010;139: 1642–53, 1653 e1-6. Fields AL, Soprano DR, Soprano KJ. Retinoids in biological control and cancer. J Cell Biochem 2007;102:886–98. Theodosiou M, Laudet V, Schubert M. From carrot to clinic: an overview of the retinoic acid signaling pathway. Cell Mol Life Sci 2010;67:1423–45. Rigas JR, Dragnev KH. Emerging role of rexinoids in non-small cell lung cancer: focus on bexarotene. Oncologist 2005;10:22–33. Mao JT, Goldin JG, Dermand J, Ibrahim G, Brown MS, Emerick A, et al. A pilot study of all-trans-retinoic acid for the treatment of human emphysema. Am J Respir Crit Care Med 2002;165:718–23. Frankenberger M, Heimbeck I, Moller W, Mamidi S, Kassner G, Pukelsheim K, et al. Inhaled all-trans retinoic acid in an individual with severe emphysema. Eur Respir J 2009;34:1487–9.