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Aryl hydrocarbon receptor activation alleviates dextran sodium sulfate-induced colitis through enhancing the differentiation of goblet cells
Biochemical and Biophysical Research Communications 514 (2019) 180e186
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Aryl hydrocarbon receptor activation alleviates dextran sodium sulfate-induced colitis through enhancing the differentiation of goblet cells Jiuheng Yin a, 1, Kunqiu Yang b, 1, Chao Zhou a, Pengyuan Xu c, Weidong Xiao a, *, Hua Yang a, ** a b c
Department of General Surgery, Xinqiao Hospital, Army Military Medical University, Chongqing, China Department of General Surgery, Sixth Medical Center of PLA General Hospital, Beijing, China Department of Gastrointestinal Surgery, Second Affiliated Hospital of Kunming Medical University, Kunming, China
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Article history: Received 20 March 2019 Accepted 18 April 2019 Available online 24 April 2019
Background: The intestinal inflammation induces disruption of the intestinal barrier function and leads to bacteria invasion. Accumulating evidences revealed that the aryl hydrocarbon receptor (AhR) plays a vital role in maintaining the intestinal barrier function. However, the precise mechanism remains to be unclear. Methods: Adult C57BL/6J mice were randomly divided into three groups: Sham, DSS and DSS þ 6formylindolo (3, 2-b) carbazole (FICZ)group. The colons and epithelial cell were harvested for histological examination, pro-inflammatory cytokines detection, bacterial load analysis, immunohistochemistry and Muc2 protein analysis. Under physiological condition, AhRKO model and FICZ treatment were used to evaluate the roles of AhR in the differentiation of goblet cells and the expression of Muc2 in mice. In vitro, we used HT29 mol to research the signaling pathway. Results: AhR activation by FICZ could increase the Muc2 expression and the number of goblet cells and reduce bacterial infiltration to ameliorate DSS-induced Colitis. Under physiological conditions, the treatment of FICZ promote the differentiation of goblet cell and the expression of Muc2 and inhibit the notch-signaling. Genetic deletion of AhR led to the loss of goblet cells and the decrease of Muc2 expression and enhance the notch-signaling. In HT29 cells, the differentiation of goblet cell meditated by AhR can be abolished by the inhibitor of AhR, pErk1/2 and knocking-down AhR. Conclusion: FICZ promoted the differentiation of goblet cell through AhR-pErk1/2 signaling pathway and ameliorate DSS-induced Colitis. © 2019 Elsevier Inc. All rights reserved.
Keywords: Aryl hydrocarbon receptor Dextran sodium sulfate FICZ Goblet cell differentiation Muc2
1. Introduction The pathogenesis of inflammatory bowel disease (IBD) is not fully understood and is thought to be caused by a variety of factors, such as genetic predisposition, environmental factors, immune disorders, and microbiota [1].
* Corresponding author. Department of General Surgery, Xinqiao Hospital, 183 Xinqiao Main Street, Shapingba, Chongqing, 400037, PR China. ** Corresponding author. Department of General Surgery, Xinqiao Hospital, 183 Xinqiao Main Street, Shapingba, Chongqing, 400037, PR China. E-mail addresses: [email protected] (W. Xiao), [email protected] (H. Yang). 1 Co-first authors. https://doi.org/10.1016/j.bbrc.2019.04.136 0006-291X/© 2019 Elsevier Inc. All rights reserved.
The mucin produced by the goblet cells of the gut have an important role in preventing attachment and removing the adhered load from the mucosal surface, defensing against multiple enteric pathogens [2,3]. There are increasing evidences showing that Aryl hydrocarbon receptor (AhR) could regulate the expression of mucin. For example, the activation of AhR by benzopyrene (B[a] P) in airway epithelial cells could induce the expression of mucin 5AC via producing oxygen species (ROS) [4]. 2,3,7,8Tetrachlorodibenzo-p- dioxin (TCDD), a ligand for AhR, increased mucin 5AC production in NCI-H441 cells [5]. But little is known about the role of AhR activation in the mucin expression in gut. AhR is a conservative nuclear transcription factor which belongs to the basic region-helix-loop-Helix (bHLH) superfamily [6]. AhR forms a stable complex with HSP90 which is present in the
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cytoplasm. Many genes of xenobiotic metabolizing enzymes, such as the genes of CYP1A1 and CYP1B1 are regarded as classic target genes of AhR that could be used to reflect its activation level of AhR. Previous studies have reported that the AhR could suppress the inflammatory response and attenuate experimental colitis [7]. FICZ, an ultraviolet photoproduct of L-tryptophan, is a high-affinity endogenous ligand for AhR and it was used in our study because it might activate AhR and significantly inhibit the inflammatory response [8]. In the present study, we focused on the effect of AhR activation on the expression of mucin and the differentiation of goblet cells. We found that AhR activation by FICZ could promote the differentiation of goblet cell through AhR-pErk1/2 signaling pathway to ameliorate DSS-induced Colitis.
2. Materials and methods 2.1. Animals Experiments were performed using male C57BL/6 mice which were purchased from the Experimental Animal Center at Xinqiao hospital of the Army Military Medical University (AMMU) (Chongqing, P.R. China). Male C57BL/6J AhRKO mice were obtained from the Experimental Animal Center at Daping Hospital of the AMMU (Chongqing, China). C57 L/6 mice were housed in a pathogen-free facility, which is accredited by the American Association for Accreditation of Laboratory Animal Care. All animal experiments and protocols were approved by the Animal Care and Use Committee of the AMMU. Colitis was induced by administration of 3% DSS (Sigma-Aldrich, Inc.; Merck KGaA) which was dissolved in distilled water for 7 days. FICZ (1 mg/mouse, Enzo Life Science; NY, USA) was administered 2 days after the starting of DSS administration daily by intraperitoneal injection.
2.2. Cell culture HT29 cells were purchased from ATCC and were cultured in Dulbecco's Modified Eagle Medium (Rockford, IL, USA) containing 10% fetal calf serum and 1% penicillin/streptomycin (Gibco, Rockville, MD, USA). Cells were cultured at 37 Cin 20% O2 and 5% CO2 conditions (Thermo Fisher, Rockford, IL, USA).
2.3. RNA isolation and real-time PCR Briefly, 1 ml Trizol reagent (TaKaRa Bio Inc) was utilized to isolate total RNA from colon epithelial cells or colon tissues according to the manufacturer's recommendations, and the total RNA was quantified using NanoDrop 2000 (Thermo). Total RNA (1 mg) was reverseetranscribed to cDNA using PrimeScriptR RT reagent kit (TaKaRa Bio Inc). Real-time PCR was performed using Rotor-Gene Q (Qiagen) and the SYBR_PremixR Ex TaqTM II (TaKaRa Bio, Inc.). The primers used in our study were listed as follows: TNF-a(forward 50 -CCACCACGCTCTTCTGTCTACTG-30 and reverse 50 -GGGCTACGGGCTTGTCACTC-30 ) IFN-g(forward 50 -CCACGGCACAGTCATTGA-30 and reverse 50 TGCTGATGGCCTGATTGTCTT-30 ) Muc2 (forward 50 -GGCAACCAGACCACATGTGA-30 and reverse 50 GGCACATGGGTACAGGAGAT-30 ) b-actin(forward 50 -CTTCTTTGCAGCTCCTTCGTT-30 and reverse 50 -AGGAGTCCTTCTGACCCATTC-30 )
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2.4. Quantification of colonic bacteria The colons were collected in sterile tubes on ice and stored at 80 C until being processed. Total genomic bacterial DNA was extracted using the TIANAMP Bacterial DNA Kit (TIANGEN, Beijing, China) as instructed. The 16S ribosomal RNA (rRNA) encoding gene was amplified with barcoded universal bacterial primers (16S (F) 50 -AGAGTTTGATCCTGGCTCAG-30 16S (R) 50 -GGTTACCTTGTTACGACTT-30 ). Lastly, the amplified DNA was used to do AGAR gel electrophoresis. The result was analyzed with molecular imaging system (KODAK Gel Logic 100). 2.5. Intestinal epithelial cell isolation Intestinal epithelial cell isolation was used for Western blot. Briefly, the colon tissue was cut into 5-mm pieces and then placed into tissue culture medium (RPMI 1640, with 10% fetal calf serum). After being washed with the isolation buffer (190 mg ethylenediaminetetraacetic acid, 80 mg DDT and 500 ml phosphatebuffered saline (PBS)), the pieces were then incubated with continuous brisk stirring at 37 C for 30 min. Then a glass capillary column was used to get the supernatant. After being centrifuged, the supernatant was purified with 40% isotonic percoll (GE Healthcare Biosciences). Finally, the cells recovered in the suspension were collected for RNA or protein extraction. 2.6. Western blot analysis Intestinal epithelial cells were collected, sonicated and centrifuged. The BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) were used to measure the protein concentration. The proteins ran on an SDS-PAGE gel and were then electrophoretically transferred to a polyvinylidene difluoride membrane (Millipore) and immunoblotted with appropriate antibodies. The antibodies used for immunoblotting are listed as follows: anti-Muc2 (1:1000) ((Abcam (Cambridge, MA, USA)), anti -phospho-Erk1/2 (1:1000) and anti -Erk1/2(1:1000) (Proteintech Group, Inc. (Wuhan, China)), anti-AhR (1:500)(Abcam (Cambridge, MA, USA)), Anti-NICD1 antibody (1:500) (Cell Signaling Technology, MA, USA). 2.7. Immunohistochemical (IHC) staining The colons of mice were removed and immediately submerged in Methanol-Carnoy's fixative at 4 C for 3 h. Then the tissues were treated with 100% methanol for 30 min, 100% ethanol for 20 min, and xylene for 15 min in turn. Fixed tissues were embedded in paraffin and cut into 4 mm sections. Subsequently, the sections were soaked in citrate buffer (pH 6.0) and heated by microwave for 20 min for antigen retrieval. The sections were blocked with a 5% BSA solution for 60 min and incubated with anti-Muc2 antibody (1:200; Abcam, Cambridge, UK) at 4 Covernight. These sections were washed three times with PBS and incubated with biotinylated goat anti-rabbit or anti-mouse secondary antibody (both from Wuhan Boster Biological Technology Ltd.) at 37 C for 20 min. Sections were washed three times again and incubated with 100 ml of substrate at room temperature for 10 min. The number of Muc2 positive cells was counted under the microscope (400 ). 2.8. Knockdown of AhR by small interfering RNA (siRNA) HT29 cells were cultured to 30e50% confluence in 6-well plates, and then transfected with siRNA (NM 001621, RiboBio, Guangzhou, China) as described in the previous study [9]. The sequences of the
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siRNAs targeting AhR were as follows: sense 50 -GGAACACCUACAUCUAGAAdTdT-30 and antisense 30 -dGdT CCUUGUGGAUGUAGAUCUU-5’.
significantly reduced in mice treated with FICZ. These data indicated that AhR activated by FICZ ameliorated the DSS-induced disruption of intestinal barrier function.
2.9. Statistical analysis
3.2. AhR activation by FICZ increased the number of colonic goblet cells and the Muc2 expression
All data were expressed as the mean ± standard deviation(SD). SPSS statistical package (version, 19.0; IBM SPSS, Armonk, NY, USA) was used for data analysis. The statistical significance of differences in mean values was determined by using ANOVA. Statistical significance was defined as P < 0.05. 3. Results 3.1. AhR activation ameliorates DSS-induced Colitis in Mice Firstly, we observed how the weight was influenced by DSS or FICZ. We found that DSS-treated mice exhibited significant weight loss 3e4 days after DSS treatment (Fig. 1A). Conversely, DSS þ FICZtreated mice exhibited less weight loss compared with in the DSStreated mice(Fig. 1A). In addition, colon length is longer in the FICZ treated mice than in the DSS-treated mice (Fig. 1B). To observe the role of FICZ in preventing colon from destruction of enteritis better, we assessed the histological damage degree in various groups by using HE staining. As Fig. 1C shows, mice treated with FICZ developed into less severe colitis. Furthermore, the expression level of proinflammatory cytokines were measured. In Fig. 1D, the mRNA expression levels of IFN-g and TNF-a significantly increased in the DSS-treated group compared with the vehicle-treated group. However, the mRNA expression levels of IFN-g and TNF-a
As the important line of defense, the mucus layer and tight junction protein are critically important in preventing attachment, removing the adhered load from the mucosal surface, and defensing against multiple enteric pathogens into lamina propria [10]. Firstly, we detected bacterial penetration in the colonic tissue sections with semi-quantitative PCR. The expression of 16s rDNA gene showed that bacteria were abundant in colonic tissues of DSS treatment group and less of which were observed in FICZ þ DSS group(Fig. 2A). As described in our previous study, our team have found that FICZ could regulate the expression of tight junction protein [11]. However, the change of mucin is unclear. Goblet cell loss is a hallmark of ulcerative colitis, which can lead to a reduced mucin expression [12]. Among the mucin, Muc2 is the most abundant and is often used as a marker of goblet cell homeostasis and mice with Muc2 knocked out are more susceptible to DSSinduced colitis [13,14]. Therefore, we firstly examine Muc2 expression by Western blot. In our study, DSS treatment significantly decreased the expression of Muc2(Fig. 2B). However, DSS þ FICZ - treated mice exhibited a dramatically increase in the expression of Muc2 compared with the DSS-treated mice(Fig. 2B). We further performed immunohistochemical analysis to detect the number of Muc2 positive cells in the colon of WT, DSS, DSS þ FICZ mice. The results showed a significant increase of yellow-stained
Fig. 1. FICZ ameliorated DSS-induced colitis in Mice. A. Activation of AhR by FICZ reduced weight loss induced by DSS. B. colon length increased in the FICZ treated mice compared with DSS group. C. HE staining showed that the destruction of the colonic structure was relieved by FICZ. D. The mRNA expression levels of IFN-g and TNF-a significantly reduced in mice treated with FICZ. (*P < 0.05).
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Fig. 2. AhR activation by FICZ increased the number of colonic goblet cells and the Muc2 expression. A. Bacterial were abundant in colonic tissues of DSS treatment group, less bacteria were observed in FICZ þ DSS group. B. FICZ-treated mice exhibited a dramatically increase in the expression of Muc2 compared with the DSS-treated mice. CD. AhR activation by FICZ increased the number of colonic goblet cells in the DSS-treated mice. (*P < 0.05).
goblet cells in FICZ þ DSS treated group compared with DSS treated group(Fig. 2.CD). Our results indicated that AhR activation by FICZ increased the number of colonic goblet cells and the Muc2 expression.
3.3. FICZ promoted the differentiation of goblet cells and deletion of AhR led to loss of goblet cells under physiological condition Replacement of mucin-producing goblet cells is vital to prevent dissemination of bacteria. Considering that AhR-activation increased the expression of Muc2 in DSS induced colitis, we investigated whether the AhR activation is connected with the differentiation of mucin-producing goblet cells. To determine the effects of AhR, WT mice were injected with FICZ for 5 days. Firstly, immunohistochemical was used to detected the number of Muc2 positive cells. The result showed a significant increase of yellowstained goblet cells in FICZ treated group (Fig. 3.AB). Meanwhile, the expression of Muc2 is consistent with the differentiation of goblet cells(Fig. 3C). The Notch-signaling pathway is the critical regulator of the intestinal epithelial cell fate determination, especially the goblet cells [15]. when the activation of the Notchsignaling, the Notch intracellular domain (NICD) is released which regulated relative genes expression to arrest the differentiation of goblet cells. Our results shown that FICZ could significantly decrease the expression of NICD(Fig. 3C). These indicated that FICZ could promoted the differentiation of goblet cells. Recently studies showed that endogenous AhR, which didn't bind to any ligand, played very important roles in tissue development and cell maintenance by regulating cell differentiation [16]. Given the profound impact of AhR deficiency on intestinal homeostasis, we set out to define whether AhR deficiency led to reduction of goblet cells. Firstly, we evaluated the effects of AhR knocking out by Western
blot. As Fig. 3D showed, the expression of AhR significantly decreased in epithelial cell of AhR deficient mice compared with the WT mice. We subsequently observed the number of goblet cells and the expression of Muc2. Mice with AhR deficiency exhibited a larger number of goblet cell loss, decreased Muc2 expression and increased the expression of NICD (Fig. 3.EFG) which suggested that AhR was important in maintaining the differentiation of goblet cells.
3.4. FICZ promoted the differentiation of goblet cells though AhRpErk1/2 signaling pathway To determine whether FICZ has a role in goblet cell differentiation, HT29 cells were treated with FICZ for 24 h and the protein and mRNA levels of the goblet cell markers Muc2 was determined. As shown in Fig. 4.AB, stimulation of HT29 cells with FICZ induced Muc2 mRNA and protein expression. Meanwhile, we observed a down-regulation of NICD (Fig. 4B). Furthermore, we next investigated whether these results were dependent on the AhR. The HT29 cells were treated with vehicle, FICZ, 50 mM of CH223191(inhibitor of AhR), or FICZ þ 50 mM of CH223191 for 24 h, after which the proteins were collected and analyzed by Western blotting. The results demonstrated the increase in Muc2 expression and the decrease in NICD expression could be abolished by cotreatment with CH223191 (Fig. 4B). Previous study found that Erk1/2-MAPK signaling pathway controls the differentiation of goblet cell [15]. Noteworthily, our laboratory's preliminary experiment found that FICZ could promote the intraepithelial T cells (IEL) differentiating towards KGFþ IEL subpopulation through AhRpErk1/2 signaling pathway (Data not shown). As shown in Fig. 4B, a high pErk1/2 level is in FICZ treated HT29 and the phospho-Erk1/ 2 level are abolished by co-treatment with CH223191. To
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Fig. 3. FICZ promoted the differentiation of goblet cell and deletion of AhR led to loss of goblet cells under physiological condition AB. Treatment of FICZ increased the number of goblet cell numbers. C. FICZ effectively increased the expression of Muc2 and decreased the expression of NICD. D. The expression of AhR significantly decreased in all gut tissues of AhR deficient mice compared with the WT mice. EF. Mice with AhR deficiency exhibited a larger number of goblet cells loss. G. Deletion of AhR significantly reduced the expression of Muc2 and increased the expression of NICD. (*P < 0.05).
investigate whether Muc2 and NICD regulated by FICZ was dependent on the phosphorylate of Erk1/2. The HT29 cells were treated with vehicle, FICZ, FICZ þ50 mM of U0126 (inhibitor of pErk1/2) for 24 h, after that the proteins were collected and analyzed by Western blotting. The results demonstrated that increased Muc2 expression and decreased NICD expression caused by FICZ could be abolished by co-treatment with U0126 (Fig. 4C). Finally, our study also showed that AhR knocking out decreased the expression of Muc2 and the phosphorylate of Erk1/2 and increased the NICD expression (Fig. 4D). Overall, our research indicated that FICZ promoted the differentiation of goblet cells through AhRpErk1/2 signaling pathway.
4. Discussion The present study showed that AhR activation by FICZ could increase the Muc2 expression, decrease the loss of goblet cells and reduce bacterial infiltration to ameliorate DSS-induced colitis. Under normal physiological conditions, the treatment of FICZ promote the differentiation of goblet cell and the expression of Muc2 and inhibit the notch-signaling. Genetic deletion of AhR led to the loss of goblet cells and the decrease of Muc2 expression and enhance the notch-signaling. Lastly, we demonstrated that FICZ promoted the differentiation of goblet cell by AhR-pErk1/2 signaling pathway in HT29 cells. In recent years, researchers have found that AhR plays a key role in protecting body from disease [17]. In patients with IBD, the expression of AhR is down-regulated [18]. In DSS-induced colitis, we observed the expression of AhR was decreased and activation of
which was inhibited [7]. These observations suggested that AhR might play an important protective role in intestinal inflammation. Now, considerable amount of data has been accumulated to suggest that AhR inhibits inflammation in the gut. For example, Alpinetin exerts anti-colitis efficacy by activating AhR through promoting Treg differentiation [19]. AhR activated by TCDD decreases colonic inflammation in a murine model of colitis by generating regulatory immune cells in part [20]. FICZ is a photoproduct of tryptophan and an endogenous high affinity ligand for AhR [21]. Our previous researches have reported that AhR activated by FICZ maintained the intestinal epithelial barrier function in intestinal ischemia/reperfusion model and intestinal obstruction model [6]. In subsequent studies, we found that administration of FICZ could ameliorate the DSS-induced disruption of intestinal epithelial barrier function. A key component of defensing against the penetration of pathogenic microorganism into the lamina propria is the thick mucus layer [22]. Whether in UC patients or in DSS treated mice, the depletion of recognizable goblet cells, decreased mucus synthesis or secretion are often observed in the colonic epithelium [23,24]. Muc2 is a gelforming mucin and the main component of the intestinal mucus layer and mice with Muc2 knocked out are more susceptible to DSS-induced colitis. Consistently, decreased Muc2 and goblet cell depletion were observed in the diseased colon of mice which were treated with DSS. Treatment of FICZ arrested the depletion of recognizable goblet cells and increased the Muc2 expression to reduce bacterial infiltration and excessive expression of inflammatory cytokines in DSS-induced colitis. In addition, treatment of FICZ promote the differentiation of goblet cell and the expression of the goblet cell marker Muc2 under physiological condition. Genetic
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Fig. 4. FICZ promoted the differentiation of goblet cell though AhR-pErk1/2 signaling pathway A. The mRNA level of Muc2 significantly was increased by FICZ stimulation. B. Treatment of FICZ increased the expression of Muc2, decreased expression of NICD and promoted the phosphorylation of Erk. Those regulation were abolished by CH223191. C. The inhibitor of U0126 inhibited the phosphorylation of Erk, the expression of Muc2 and decreased expression of NICD meditated by FICZ. D. SiAhR significantly decreased the phosphorylation of Erk and the expression of Muc2 and increased expression of NICD.(*P < 0.05).
deletion of AhR led to the loss of goblet cells and the decrease of Muc2 expression. Importantly, Notch-signaling is one of the master regulators of colonic epithelial differentiation and cell lineage determination of secretory cell lineage, especially goblet Cells [25]. However, how AhR controls the goblet cells differentiation and the Muc2 expression is still unclear. Further mechanism studies are needed in our study. Studies shown that MAPK Signaling pathway controls the differentiation of goblet cell [15]. In an effort to identify, we found that Erk1/2 phosphorylation was stimulated by FICZ. Using a specific inhibitor of Erk phosphorylation(U0126), we went on to show that activation of pErk-MAPK pathway is required for FICZ induced up-regulation of Muc2 and inhibited the NICD expression. This is the firstly demonstration about FICZ promote the differention of goblet cells by AhR-pErk signaling pathway. Our study reveals the importance of AhR in attenuating DSSinduced colitis. AhR plays its protective role by enhancing Muc2 expression and promoting the differentiation of goblet cells. Meanwhile, the effect is depend on AhR-pErk signaling pathway. Conflicts of interest No potential conflict of interest relevant to this article was reported. Acknowledgments This research was supported by grants from the National Natural Science Foundation of China (NSFC 81700454 to JH. Y/NSFC 81330013 to H Y/NSFC81770524 to WD. X), the Program of Changjiang Scholars and Innovative Research (IRT_1716 to HY) and the program of Yunnan academician and expert workstation
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Aryl hydrocarbon receptor activation alleviates dextran sodium sulfate-induced colitis through enhancing the differentiation of goblet cells Jiuheng Yin a, 1, Kunqiu Yang b, 1, Chao Zhou a, Pengyuan Xu c, Weidong Xiao a, *, Hua Yang a, ** a b c
Department of General Surgery, Xinqiao Hospital, Army Military Medical University, Chongqing, China Department of General Surgery, Sixth Medical Center of PLA General Hospital, Beijing, China Department of Gastrointestinal Surgery, Second Affiliated Hospital of Kunming Medical University, Kunming, China
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a b s t r a c t
Article history: Received 20 March 2019 Accepted 18 April 2019 Available online 24 April 2019
Background: The intestinal inflammation induces disruption of the intestinal barrier function and leads to bacteria invasion. Accumulating evidences revealed that the aryl hydrocarbon receptor (AhR) plays a vital role in maintaining the intestinal barrier function. However, the precise mechanism remains to be unclear. Methods: Adult C57BL/6J mice were randomly divided into three groups: Sham, DSS and DSS þ 6formylindolo (3, 2-b) carbazole (FICZ)group. The colons and epithelial cell were harvested for histological examination, pro-inflammatory cytokines detection, bacterial load analysis, immunohistochemistry and Muc2 protein analysis. Under physiological condition, AhRKO model and FICZ treatment were used to evaluate the roles of AhR in the differentiation of goblet cells and the expression of Muc2 in mice. In vitro, we used HT29 mol to research the signaling pathway. Results: AhR activation by FICZ could increase the Muc2 expression and the number of goblet cells and reduce bacterial infiltration to ameliorate DSS-induced Colitis. Under physiological conditions, the treatment of FICZ promote the differentiation of goblet cell and the expression of Muc2 and inhibit the notch-signaling. Genetic deletion of AhR led to the loss of goblet cells and the decrease of Muc2 expression and enhance the notch-signaling. In HT29 cells, the differentiation of goblet cell meditated by AhR can be abolished by the inhibitor of AhR, pErk1/2 and knocking-down AhR. Conclusion: FICZ promoted the differentiation of goblet cell through AhR-pErk1/2 signaling pathway and ameliorate DSS-induced Colitis. © 2019 Elsevier Inc. All rights reserved.
Keywords: Aryl hydrocarbon receptor Dextran sodium sulfate FICZ Goblet cell differentiation Muc2
1. Introduction The pathogenesis of inflammatory bowel disease (IBD) is not fully understood and is thought to be caused by a variety of factors, such as genetic predisposition, environmental factors, immune disorders, and microbiota [1].
* Corresponding author. Department of General Surgery, Xinqiao Hospital, 183 Xinqiao Main Street, Shapingba, Chongqing, 400037, PR China. ** Corresponding author. Department of General Surgery, Xinqiao Hospital, 183 Xinqiao Main Street, Shapingba, Chongqing, 400037, PR China. E-mail addresses: [email protected] (W. Xiao), [email protected] (H. Yang). 1 Co-first authors. https://doi.org/10.1016/j.bbrc.2019.04.136 0006-291X/© 2019 Elsevier Inc. All rights reserved.
The mucin produced by the goblet cells of the gut have an important role in preventing attachment and removing the adhered load from the mucosal surface, defensing against multiple enteric pathogens [2,3]. There are increasing evidences showing that Aryl hydrocarbon receptor (AhR) could regulate the expression of mucin. For example, the activation of AhR by benzopyrene (B[a] P) in airway epithelial cells could induce the expression of mucin 5AC via producing oxygen species (ROS) [4]. 2,3,7,8Tetrachlorodibenzo-p- dioxin (TCDD), a ligand for AhR, increased mucin 5AC production in NCI-H441 cells [5]. But little is known about the role of AhR activation in the mucin expression in gut. AhR is a conservative nuclear transcription factor which belongs to the basic region-helix-loop-Helix (bHLH) superfamily [6]. AhR forms a stable complex with HSP90 which is present in the
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cytoplasm. Many genes of xenobiotic metabolizing enzymes, such as the genes of CYP1A1 and CYP1B1 are regarded as classic target genes of AhR that could be used to reflect its activation level of AhR. Previous studies have reported that the AhR could suppress the inflammatory response and attenuate experimental colitis [7]. FICZ, an ultraviolet photoproduct of L-tryptophan, is a high-affinity endogenous ligand for AhR and it was used in our study because it might activate AhR and significantly inhibit the inflammatory response [8]. In the present study, we focused on the effect of AhR activation on the expression of mucin and the differentiation of goblet cells. We found that AhR activation by FICZ could promote the differentiation of goblet cell through AhR-pErk1/2 signaling pathway to ameliorate DSS-induced Colitis.
2. Materials and methods 2.1. Animals Experiments were performed using male C57BL/6 mice which were purchased from the Experimental Animal Center at Xinqiao hospital of the Army Military Medical University (AMMU) (Chongqing, P.R. China). Male C57BL/6J AhRKO mice were obtained from the Experimental Animal Center at Daping Hospital of the AMMU (Chongqing, China). C57 L/6 mice were housed in a pathogen-free facility, which is accredited by the American Association for Accreditation of Laboratory Animal Care. All animal experiments and protocols were approved by the Animal Care and Use Committee of the AMMU. Colitis was induced by administration of 3% DSS (Sigma-Aldrich, Inc.; Merck KGaA) which was dissolved in distilled water for 7 days. FICZ (1 mg/mouse, Enzo Life Science; NY, USA) was administered 2 days after the starting of DSS administration daily by intraperitoneal injection.
2.2. Cell culture HT29 cells were purchased from ATCC and were cultured in Dulbecco's Modified Eagle Medium (Rockford, IL, USA) containing 10% fetal calf serum and 1% penicillin/streptomycin (Gibco, Rockville, MD, USA). Cells were cultured at 37 Cin 20% O2 and 5% CO2 conditions (Thermo Fisher, Rockford, IL, USA).
2.3. RNA isolation and real-time PCR Briefly, 1 ml Trizol reagent (TaKaRa Bio Inc) was utilized to isolate total RNA from colon epithelial cells or colon tissues according to the manufacturer's recommendations, and the total RNA was quantified using NanoDrop 2000 (Thermo). Total RNA (1 mg) was reverseetranscribed to cDNA using PrimeScriptR RT reagent kit (TaKaRa Bio Inc). Real-time PCR was performed using Rotor-Gene Q (Qiagen) and the SYBR_PremixR Ex TaqTM II (TaKaRa Bio, Inc.). The primers used in our study were listed as follows: TNF-a(forward 50 -CCACCACGCTCTTCTGTCTACTG-30 and reverse 50 -GGGCTACGGGCTTGTCACTC-30 ) IFN-g(forward 50 -CCACGGCACAGTCATTGA-30 and reverse 50 TGCTGATGGCCTGATTGTCTT-30 ) Muc2 (forward 50 -GGCAACCAGACCACATGTGA-30 and reverse 50 GGCACATGGGTACAGGAGAT-30 ) b-actin(forward 50 -CTTCTTTGCAGCTCCTTCGTT-30 and reverse 50 -AGGAGTCCTTCTGACCCATTC-30 )
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2.4. Quantification of colonic bacteria The colons were collected in sterile tubes on ice and stored at 80 C until being processed. Total genomic bacterial DNA was extracted using the TIANAMP Bacterial DNA Kit (TIANGEN, Beijing, China) as instructed. The 16S ribosomal RNA (rRNA) encoding gene was amplified with barcoded universal bacterial primers (16S (F) 50 -AGAGTTTGATCCTGGCTCAG-30 16S (R) 50 -GGTTACCTTGTTACGACTT-30 ). Lastly, the amplified DNA was used to do AGAR gel electrophoresis. The result was analyzed with molecular imaging system (KODAK Gel Logic 100). 2.5. Intestinal epithelial cell isolation Intestinal epithelial cell isolation was used for Western blot. Briefly, the colon tissue was cut into 5-mm pieces and then placed into tissue culture medium (RPMI 1640, with 10% fetal calf serum). After being washed with the isolation buffer (190 mg ethylenediaminetetraacetic acid, 80 mg DDT and 500 ml phosphatebuffered saline (PBS)), the pieces were then incubated with continuous brisk stirring at 37 C for 30 min. Then a glass capillary column was used to get the supernatant. After being centrifuged, the supernatant was purified with 40% isotonic percoll (GE Healthcare Biosciences). Finally, the cells recovered in the suspension were collected for RNA or protein extraction. 2.6. Western blot analysis Intestinal epithelial cells were collected, sonicated and centrifuged. The BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) were used to measure the protein concentration. The proteins ran on an SDS-PAGE gel and were then electrophoretically transferred to a polyvinylidene difluoride membrane (Millipore) and immunoblotted with appropriate antibodies. The antibodies used for immunoblotting are listed as follows: anti-Muc2 (1:1000) ((Abcam (Cambridge, MA, USA)), anti -phospho-Erk1/2 (1:1000) and anti -Erk1/2(1:1000) (Proteintech Group, Inc. (Wuhan, China)), anti-AhR (1:500)(Abcam (Cambridge, MA, USA)), Anti-NICD1 antibody (1:500) (Cell Signaling Technology, MA, USA). 2.7. Immunohistochemical (IHC) staining The colons of mice were removed and immediately submerged in Methanol-Carnoy's fixative at 4 C for 3 h. Then the tissues were treated with 100% methanol for 30 min, 100% ethanol for 20 min, and xylene for 15 min in turn. Fixed tissues were embedded in paraffin and cut into 4 mm sections. Subsequently, the sections were soaked in citrate buffer (pH 6.0) and heated by microwave for 20 min for antigen retrieval. The sections were blocked with a 5% BSA solution for 60 min and incubated with anti-Muc2 antibody (1:200; Abcam, Cambridge, UK) at 4 Covernight. These sections were washed three times with PBS and incubated with biotinylated goat anti-rabbit or anti-mouse secondary antibody (both from Wuhan Boster Biological Technology Ltd.) at 37 C for 20 min. Sections were washed three times again and incubated with 100 ml of substrate at room temperature for 10 min. The number of Muc2 positive cells was counted under the microscope (400 ). 2.8. Knockdown of AhR by small interfering RNA (siRNA) HT29 cells were cultured to 30e50% confluence in 6-well plates, and then transfected with siRNA (NM 001621, RiboBio, Guangzhou, China) as described in the previous study [9]. The sequences of the
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siRNAs targeting AhR were as follows: sense 50 -GGAACACCUACAUCUAGAAdTdT-30 and antisense 30 -dGdT CCUUGUGGAUGUAGAUCUU-5’.
significantly reduced in mice treated with FICZ. These data indicated that AhR activated by FICZ ameliorated the DSS-induced disruption of intestinal barrier function.
2.9. Statistical analysis
3.2. AhR activation by FICZ increased the number of colonic goblet cells and the Muc2 expression
All data were expressed as the mean ± standard deviation(SD). SPSS statistical package (version, 19.0; IBM SPSS, Armonk, NY, USA) was used for data analysis. The statistical significance of differences in mean values was determined by using ANOVA. Statistical significance was defined as P < 0.05. 3. Results 3.1. AhR activation ameliorates DSS-induced Colitis in Mice Firstly, we observed how the weight was influenced by DSS or FICZ. We found that DSS-treated mice exhibited significant weight loss 3e4 days after DSS treatment (Fig. 1A). Conversely, DSS þ FICZtreated mice exhibited less weight loss compared with in the DSStreated mice(Fig. 1A). In addition, colon length is longer in the FICZ treated mice than in the DSS-treated mice (Fig. 1B). To observe the role of FICZ in preventing colon from destruction of enteritis better, we assessed the histological damage degree in various groups by using HE staining. As Fig. 1C shows, mice treated with FICZ developed into less severe colitis. Furthermore, the expression level of proinflammatory cytokines were measured. In Fig. 1D, the mRNA expression levels of IFN-g and TNF-a significantly increased in the DSS-treated group compared with the vehicle-treated group. However, the mRNA expression levels of IFN-g and TNF-a
As the important line of defense, the mucus layer and tight junction protein are critically important in preventing attachment, removing the adhered load from the mucosal surface, and defensing against multiple enteric pathogens into lamina propria [10]. Firstly, we detected bacterial penetration in the colonic tissue sections with semi-quantitative PCR. The expression of 16s rDNA gene showed that bacteria were abundant in colonic tissues of DSS treatment group and less of which were observed in FICZ þ DSS group(Fig. 2A). As described in our previous study, our team have found that FICZ could regulate the expression of tight junction protein [11]. However, the change of mucin is unclear. Goblet cell loss is a hallmark of ulcerative colitis, which can lead to a reduced mucin expression [12]. Among the mucin, Muc2 is the most abundant and is often used as a marker of goblet cell homeostasis and mice with Muc2 knocked out are more susceptible to DSSinduced colitis [13,14]. Therefore, we firstly examine Muc2 expression by Western blot. In our study, DSS treatment significantly decreased the expression of Muc2(Fig. 2B). However, DSS þ FICZ - treated mice exhibited a dramatically increase in the expression of Muc2 compared with the DSS-treated mice(Fig. 2B). We further performed immunohistochemical analysis to detect the number of Muc2 positive cells in the colon of WT, DSS, DSS þ FICZ mice. The results showed a significant increase of yellow-stained
Fig. 1. FICZ ameliorated DSS-induced colitis in Mice. A. Activation of AhR by FICZ reduced weight loss induced by DSS. B. colon length increased in the FICZ treated mice compared with DSS group. C. HE staining showed that the destruction of the colonic structure was relieved by FICZ. D. The mRNA expression levels of IFN-g and TNF-a significantly reduced in mice treated with FICZ. (*P < 0.05).
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Fig. 2. AhR activation by FICZ increased the number of colonic goblet cells and the Muc2 expression. A. Bacterial were abundant in colonic tissues of DSS treatment group, less bacteria were observed in FICZ þ DSS group. B. FICZ-treated mice exhibited a dramatically increase in the expression of Muc2 compared with the DSS-treated mice. CD. AhR activation by FICZ increased the number of colonic goblet cells in the DSS-treated mice. (*P < 0.05).
goblet cells in FICZ þ DSS treated group compared with DSS treated group(Fig. 2.CD). Our results indicated that AhR activation by FICZ increased the number of colonic goblet cells and the Muc2 expression.
3.3. FICZ promoted the differentiation of goblet cells and deletion of AhR led to loss of goblet cells under physiological condition Replacement of mucin-producing goblet cells is vital to prevent dissemination of bacteria. Considering that AhR-activation increased the expression of Muc2 in DSS induced colitis, we investigated whether the AhR activation is connected with the differentiation of mucin-producing goblet cells. To determine the effects of AhR, WT mice were injected with FICZ for 5 days. Firstly, immunohistochemical was used to detected the number of Muc2 positive cells. The result showed a significant increase of yellowstained goblet cells in FICZ treated group (Fig. 3.AB). Meanwhile, the expression of Muc2 is consistent with the differentiation of goblet cells(Fig. 3C). The Notch-signaling pathway is the critical regulator of the intestinal epithelial cell fate determination, especially the goblet cells [15]. when the activation of the Notchsignaling, the Notch intracellular domain (NICD) is released which regulated relative genes expression to arrest the differentiation of goblet cells. Our results shown that FICZ could significantly decrease the expression of NICD(Fig. 3C). These indicated that FICZ could promoted the differentiation of goblet cells. Recently studies showed that endogenous AhR, which didn't bind to any ligand, played very important roles in tissue development and cell maintenance by regulating cell differentiation [16]. Given the profound impact of AhR deficiency on intestinal homeostasis, we set out to define whether AhR deficiency led to reduction of goblet cells. Firstly, we evaluated the effects of AhR knocking out by Western
blot. As Fig. 3D showed, the expression of AhR significantly decreased in epithelial cell of AhR deficient mice compared with the WT mice. We subsequently observed the number of goblet cells and the expression of Muc2. Mice with AhR deficiency exhibited a larger number of goblet cell loss, decreased Muc2 expression and increased the expression of NICD (Fig. 3.EFG) which suggested that AhR was important in maintaining the differentiation of goblet cells.
3.4. FICZ promoted the differentiation of goblet cells though AhRpErk1/2 signaling pathway To determine whether FICZ has a role in goblet cell differentiation, HT29 cells were treated with FICZ for 24 h and the protein and mRNA levels of the goblet cell markers Muc2 was determined. As shown in Fig. 4.AB, stimulation of HT29 cells with FICZ induced Muc2 mRNA and protein expression. Meanwhile, we observed a down-regulation of NICD (Fig. 4B). Furthermore, we next investigated whether these results were dependent on the AhR. The HT29 cells were treated with vehicle, FICZ, 50 mM of CH223191(inhibitor of AhR), or FICZ þ 50 mM of CH223191 for 24 h, after which the proteins were collected and analyzed by Western blotting. The results demonstrated the increase in Muc2 expression and the decrease in NICD expression could be abolished by cotreatment with CH223191 (Fig. 4B). Previous study found that Erk1/2-MAPK signaling pathway controls the differentiation of goblet cell [15]. Noteworthily, our laboratory's preliminary experiment found that FICZ could promote the intraepithelial T cells (IEL) differentiating towards KGFþ IEL subpopulation through AhRpErk1/2 signaling pathway (Data not shown). As shown in Fig. 4B, a high pErk1/2 level is in FICZ treated HT29 and the phospho-Erk1/ 2 level are abolished by co-treatment with CH223191. To
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Fig. 3. FICZ promoted the differentiation of goblet cell and deletion of AhR led to loss of goblet cells under physiological condition AB. Treatment of FICZ increased the number of goblet cell numbers. C. FICZ effectively increased the expression of Muc2 and decreased the expression of NICD. D. The expression of AhR significantly decreased in all gut tissues of AhR deficient mice compared with the WT mice. EF. Mice with AhR deficiency exhibited a larger number of goblet cells loss. G. Deletion of AhR significantly reduced the expression of Muc2 and increased the expression of NICD. (*P < 0.05).
investigate whether Muc2 and NICD regulated by FICZ was dependent on the phosphorylate of Erk1/2. The HT29 cells were treated with vehicle, FICZ, FICZ þ50 mM of U0126 (inhibitor of pErk1/2) for 24 h, after that the proteins were collected and analyzed by Western blotting. The results demonstrated that increased Muc2 expression and decreased NICD expression caused by FICZ could be abolished by co-treatment with U0126 (Fig. 4C). Finally, our study also showed that AhR knocking out decreased the expression of Muc2 and the phosphorylate of Erk1/2 and increased the NICD expression (Fig. 4D). Overall, our research indicated that FICZ promoted the differentiation of goblet cells through AhRpErk1/2 signaling pathway.
4. Discussion The present study showed that AhR activation by FICZ could increase the Muc2 expression, decrease the loss of goblet cells and reduce bacterial infiltration to ameliorate DSS-induced colitis. Under normal physiological conditions, the treatment of FICZ promote the differentiation of goblet cell and the expression of Muc2 and inhibit the notch-signaling. Genetic deletion of AhR led to the loss of goblet cells and the decrease of Muc2 expression and enhance the notch-signaling. Lastly, we demonstrated that FICZ promoted the differentiation of goblet cell by AhR-pErk1/2 signaling pathway in HT29 cells. In recent years, researchers have found that AhR plays a key role in protecting body from disease [17]. In patients with IBD, the expression of AhR is down-regulated [18]. In DSS-induced colitis, we observed the expression of AhR was decreased and activation of
which was inhibited [7]. These observations suggested that AhR might play an important protective role in intestinal inflammation. Now, considerable amount of data has been accumulated to suggest that AhR inhibits inflammation in the gut. For example, Alpinetin exerts anti-colitis efficacy by activating AhR through promoting Treg differentiation [19]. AhR activated by TCDD decreases colonic inflammation in a murine model of colitis by generating regulatory immune cells in part [20]. FICZ is a photoproduct of tryptophan and an endogenous high affinity ligand for AhR [21]. Our previous researches have reported that AhR activated by FICZ maintained the intestinal epithelial barrier function in intestinal ischemia/reperfusion model and intestinal obstruction model [6]. In subsequent studies, we found that administration of FICZ could ameliorate the DSS-induced disruption of intestinal epithelial barrier function. A key component of defensing against the penetration of pathogenic microorganism into the lamina propria is the thick mucus layer [22]. Whether in UC patients or in DSS treated mice, the depletion of recognizable goblet cells, decreased mucus synthesis or secretion are often observed in the colonic epithelium [23,24]. Muc2 is a gelforming mucin and the main component of the intestinal mucus layer and mice with Muc2 knocked out are more susceptible to DSS-induced colitis. Consistently, decreased Muc2 and goblet cell depletion were observed in the diseased colon of mice which were treated with DSS. Treatment of FICZ arrested the depletion of recognizable goblet cells and increased the Muc2 expression to reduce bacterial infiltration and excessive expression of inflammatory cytokines in DSS-induced colitis. In addition, treatment of FICZ promote the differentiation of goblet cell and the expression of the goblet cell marker Muc2 under physiological condition. Genetic
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Fig. 4. FICZ promoted the differentiation of goblet cell though AhR-pErk1/2 signaling pathway A. The mRNA level of Muc2 significantly was increased by FICZ stimulation. B. Treatment of FICZ increased the expression of Muc2, decreased expression of NICD and promoted the phosphorylation of Erk. Those regulation were abolished by CH223191. C. The inhibitor of U0126 inhibited the phosphorylation of Erk, the expression of Muc2 and decreased expression of NICD meditated by FICZ. D. SiAhR significantly decreased the phosphorylation of Erk and the expression of Muc2 and increased expression of NICD.(*P < 0.05).
deletion of AhR led to the loss of goblet cells and the decrease of Muc2 expression. Importantly, Notch-signaling is one of the master regulators of colonic epithelial differentiation and cell lineage determination of secretory cell lineage, especially goblet Cells [25]. However, how AhR controls the goblet cells differentiation and the Muc2 expression is still unclear. Further mechanism studies are needed in our study. Studies shown that MAPK Signaling pathway controls the differentiation of goblet cell [15]. In an effort to identify, we found that Erk1/2 phosphorylation was stimulated by FICZ. Using a specific inhibitor of Erk phosphorylation(U0126), we went on to show that activation of pErk-MAPK pathway is required for FICZ induced up-regulation of Muc2 and inhibited the NICD expression. This is the firstly demonstration about FICZ promote the differention of goblet cells by AhR-pErk signaling pathway. Our study reveals the importance of AhR in attenuating DSSinduced colitis. AhR plays its protective role by enhancing Muc2 expression and promoting the differentiation of goblet cells. Meanwhile, the effect is depend on AhR-pErk signaling pathway. Conflicts of interest No potential conflict of interest relevant to this article was reported. Acknowledgments This research was supported by grants from the National Natural Science Foundation of China (NSFC 81700454 to JH. Y/NSFC 81330013 to H Y/NSFC81770524 to WD. X), the Program of Changjiang Scholars and Innovative Research (IRT_1716 to HY) and the program of Yunnan academician and expert workstation
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