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Autophagy induction by rapamycin ameliorates experimental colitis and improves intestinal epithelial barrier function in IL-10 knockout mice
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Autophagy induction by rapamycin ameliorates experimental colitis and improves intestinal epithelial barrier function in IL-10 knockout mice Jie Zhaoa,b,1, Honggang Wangc,1, Haojun Yanga, Yan Zhoua, Liming Tanga,
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a
Department of Gastrointestinal Surgery, The Affiliated Changzhou No. 2 People’s Hospital of Nanjing Medical University, PR China Department of General Surgery, The First Affiliated Hospital of Soochow University, PR China c Department of General Surgery, Taizhou People’s Hospital, Taizhou People’s Hospital, Medical School of Nantong University, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Crohn’s disease IL-10 knockout mice Autophagy Intestinal barrier function
Background: An impairment of the intestinal barrier function is one of the major characteristics of Crohn’s disease (CD). This study aimed to evaluate the impact of autophagy induction by rapamycin on the intestinal epithelial barrier function in CD model mice. Methods: IL-10 knockout (IL-10 KO) mice were used as the human CD models in this study. All the mice were randomly assigned into four groups, (a) wild-type (WT) group; (b) IL-10 KO group; (c) IL-10 KO + rapamycin group and (d) IL-10 KO + 3-methyladenine (3-MA), containing 6 mice in each group. The disease activity index (DAI), histology, pro-inflammatory cytokines and chemotactic factors in colon tissues, intestinal and colonic permeability, distributions and expressions of tight junction (TJ) proteins, epithelial apoptosis of mice in four groups were evaluated and compared. Results: Autophagy induction by rapamycin treatment ameliorated DAI and histological colitis, decreased proinflammatory cytokines (TNF-α, IFN-γ and IL-17) and chemotactic factors (CXCL-1 and CXCL-2), decreased intestinal and colonic permeability, improved the distribution and expression of TJ proteins in IL-10 KO mice. Conclusion: Autophagy induction by rapamycin significantly improved intestinal barrier function and protected IL-10 KO mice from the experimental chronic colitis.
1. Introduction Inflammatory bowel disease (IBD), which consists of ulcerative colitis (UC) and Crohn’s disease (CD), is widely accepted as an autoimmune disorder characterized by chronic inflammation with unknown etiology [1]. Much of our current understanding of the possible molecular mechanisms and potential therapeutic targets in IBD mainly comes from animal models of IBD [2,3]. Interleukin-10-knockout (IL-10 KO) mice have been widely reported to display similar characteristics to that of human CD [4]. Functional gene polymorphism studies have clearly revealed the close association between CD and autophagy-related proteins including nucleotide-binding oligomerization domaincontaining protein (NOD)2, IRGM and ATG16L1, which strongly suggests the pathological role of defective autophagy in CD [5,6]. Increasing evidence has shown that autophagy probably involves in the pathogenesis of CD through variable mechanisms including the modulation of T-cell development, activation and differentiation [7],
clearance of pathogens [8], activation of nuclear factor-kappa B (NFκB) [9]. Furthermore, autophagy has also been suggested as a new therapeutic target for CD [10]. The underlying pathogenesis of CD remains unclear until now, an impairment of the intestinal barrier function is one of the major characteristics of CD [11]. Furthermore, some studies have revealed that the dysfunction of intestinal barrier function involves in the pathogenesis of CD [12]. The disturbance of the intestinal barrier leads to an increased intestinal permeability, an enhanced bacterial translocation and an over-activated mucosal immune response, thus aggravating the intestinal chronic inflammation in CD [13]. Therefore, the intestinal barrier plays an imperative role in the maintenance of intestinal mucosal homeostasis. Recently, the close association between autophagy and intestinal barrier function has drawn a lot of attention. It has been widely proved that goblet cells can defend against intestinal microbe and regulate the microecology by secreting mucins [14,15]. Furthermore, the
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Corresponding author at: Department of Gastrointestinal Surgery, The Affiliated Changzhou No. 2 People's Hospital of Nanjing Medical University, 68 Gehu Road, Changzhou, Jiangsu 213003, PR China. E-mail address: [email protected] (L. Tang). 1 Jie Zhao and Honggang Wang contributed equally to this article. https://doi.org/10.1016/j.intimp.2019.105977 Received 21 September 2019; Received in revised form 11 October 2019; Accepted 13 October 2019 1567-5769/ © 2019 Published by Elsevier B.V.
Please cite this article as: Jie Zhao, et al., International Immunopharmacology, https://doi.org/10.1016/j.intimp.2019.105977
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2.6. Ussing chamber studies
development and function of goblet cells are controlled by cell autophagy [14]. Autophagy plays important roles in the clearance of polyubiquitinated protein aggregates, degradation of invading pathogens, modulation of pathogen induced pro-inflammatory cytokines release, lymphocyte development and antigen presentation [16,17]. Moreover, the process mentioned above is critically important in the mucosal immune response, anti-microbial defense and intestinal barrier integrity maintaining [18]. However, the relationship between epithelial barrier function and autophagy modulation remains unclear. In this study, we aimed to evaluate the impact of autophagy on the intestinal epithelial barrier function in CD model mice.
Segments of proximal colon were obtained for the assessment of the colon permeability according to the descriptions by Wang et al. [19]. In brief, Lucite chambers were used for mounting the mucosa, exposing serosal and mucosal surfaces to 10 ml of Ringer’s buffer (1.25 mM CaCl2, 8 mM-KCl, 115 mM NaCl, 1.2 mM MgCl2, 2.0 mM KHPO4, 25 mM NaCO3, pH 7.33–7.37). The buffers were maintained by a heated water jacket and circulated by CO2 at a temperature of 37 °C. To measure the basal mannitol fluxes, 1 mM of mannitol with 370 KBOr (H3-mannitol) was then added to the mucosal side. The determination of spontaneous transepithelial potential difference (mV) was performed, and the tissue was clamped by introducing an appropriate short circuit current (Isc, mA/cm2) with an automatic voltage clamp (DVC 1000; World Precision Instruments, Sarasota, FL). Following the descriptions of Ohm’s law, tissue ion resistance was calculated with the potential difference and Isc.
2. Materials and methods 2.1. Animals IL-10 KO mice used in this study were all obtained from the Jackson Laboratory (Bar Harbor, Maine) on the background of C57BL/6. IL-10 KO mice and wild-type (WT) mice included were all housed and maintained under specific pathogen free (SPF) conditions at the Model Animal Research Center of Nanjing University (Nanjing, China). All experimental procedures involving the use of animals were reviewed and approved by the Animal Care and Use Committee of the Model Animal Research Center, Nanjing University (Nanjing, China).
2.7. Intestinal permeability assay Fluorescein isothiocyanate (FITC)–dextran (Sigma-Aldrich; 150 ml) analysis was performed for the intestinal permeability assessment according to our previous published descriptions by Zhao et al. [21]. In brief, a solution containing 25 mg of 4 kDa FITC-dextran, which was diluted in 0.1 ml PBS, was injected into the intestinal lumen. Thirty minutes later, the blood sample via cardiac puncture was obtained and immediately centrifuged (10000g, 10 min) in ice-cold tubes containing heparin. The concentration of FITC-dextran was determined using a fluorescence spectrophotometer (F7000; Hitachi) with the excitation wavelength of 495 nm and emission wavelength of 520 nm.
2.2. Drug administration protocol All the mice included in our study were randomly assigned into four groups, (a) WT group; (b) IL-10 KO group; (c) IL-10 KO + Rapamycin group (2 mg/kg, intragastric administration) and (d) IL-10 KO + 3methyladenine (3-MA, 30 mg/kg, intraperitoneal injection), containing 6 mice in each group. After the final drug administration, all mice were executed, colon tissues were obtained to assess the therapeutic effects of autophagy modulation on the colitis status.
2.8. Epithelial apoptosis assessment Terminal deoxynucleotidyl transferase dUTP nick end labelling assay (TUNEL) In Situ Cell Death Detection Kit (Roche, Basel, Switzerland) was performed for the assessment of epithelial apoptosis. The procedures were performed according to the manufacturer’s instructions. In brief, the obtained sections were permeabilized with 1% Triton X-100, 0.1% sodium citrate, washed and stained. After that, sections were counterstained with 40,6-diamidino-2-phenylindole (DAPI) and then mounted in 50% glycerol after washing with PBS. The confocal microscopy (Olympus, Tokyo, Japan) were utilized for photographing the sections. Six random and non-overlapping pictures of (×200 magnified) optical fields of two different colon layers were chosen. A same laboratory technician who was blinded to this study was invited to count the number of TUNEL-positive cells per field.
2.3. Disease activity index (DAI) assessment As described by previous reports [19], one point was scored for each of the following: occult fecal blood, ruffled fur, soft stool and rectal prolapse < 1 mm. An additional point was scored for severe rectal prolapse > 1 mm or diarrhea. The DAI was calculated by the sum of scores. 2.4. Histology After the mice were euthanized under anesthesia, the entire colons were carefully collected and rinsed with phosphate buffer saline (PBS). The length of colon collected were measured to evaluate the changes in morphology. Thereafter, the proximal colon tissues were obtained and fixed in 10% buffer neutral formalin and embedded in paraffin. 6 umthick sections from the proximal colons were stained with haematoxylin and eosin (H&E). Taking the number of lesions and severity of the disease into consideration, the inflammation score were given to samples assessed by two independent pathologists who were blinded to the study design following well establish criteria by Singh et al. [20].
2.9. Immunofluorescence analysis Immunofluorescence analysis was performed to assess the distribution and integrity of tight junction (TJ) proteins including occludin and zona occludens protein 1 (ZO-1) as described by previous reports [22]. In brief, 6 μm-thick frozen proximal colon sections were transferred to coated slides, fixed in 1% paraformaldehyde, and washed with PBS. 5% normal goat serum diluted in PBS was used to block nonspecific binding. Thereafter, sections were incubated with monoclonal antibodies against occludin (Abcam, UK) and ZO-1 (Abcam, UK) in PBS with 1% goat serum overnight at 4℃. After that, sections were washed with PBS and incubated with Alexa 488-conjugated secondary antibodies for 1 h. A confocal microscopy (Olympus, Tokyo, Japan) was utilized for images visualization.
2.5. Pro-inflammatory cytokines and chemotactic factors in colon tissues by enzyme-linked immunosorbent assay (ELISA) Pro-inflammatory cytokines including tumor necrosis factor-α (TNFα), interferon-γ (IFN-γ) and interleukin-17 (IL-17), chemotactic factors including CXCL-1 and CXCL-2 were measured by ELISA using DuoSet ELISA development kits (R&D systems, Minneapolis, MN). We obtain the protein extracts from the colonic segments in homogenization buffer with a protease inhibitor and performed the measurements of cytokines according to the manufacturer’s instructions.
2.10. Western blotting Western blotting analysis of occluding, ZO-1, p62 and LC3B proteins was performed as described previously [23] using the primary 2
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3.2. Autophagy induction decreased pro-inflammatory cytokines and chemotactic factors in IL-10 KO mice As shown in Fig. 4, pro-inflammatory cytokines (TNF-α, IFN-γ and IL-17) are significantly elevated in IL-10 KO mice, indicating the pathogenic role of Th1/Th17 cells differentiation in chronic inflammation development in CD [24]. The results have also indicated that IL-10 KO mice with rapamycin treatment had a significant decrease when comparing with IL-10 KO mice with 3-MA treatment or without any treatment (P < 0.01). Moreover, chemotactic factors (CXCL-1 and CXCL-2) in mice of the four groups showed the same trends (see Fig. 5). 3.3. Autophagy induction decreased intestinal and colonic permeability To study the impact of autophagy modulation in intestinal permeability, we first performed Ussing chamber studies using obtained colon tissues. As shown in Fig. 6, IL-10 KO mice showed a significantly increased colonic permeability to mannitol (Fig. 6a) with a corresponding decrease in electrical resistance (Fig. 6b). Autophagy induction largely improved the increased colonic permeability in IL-10 KO mice. In addition, we also designed the FITC–dextran analysis to assess the intestinal permeability. IL-10 KO mice were associated with greatly increased concentrations of FITC-dextran compared with WT mice and rapamycin-treated IL-10 KO mice (see Fig. 6c). The described results indicated that Rapamycin, as an autophagy inducer, could significantly improve the increased intestinal and colonic permeability in IL-10 KO mice.
Fig. 1. Levels of disease activity index in mice of four groups. Values are presented as means and standard error (SD). ** P < 0.01 (n = 6 in each group).
occluding (Abcam, UK), ZO-1 (Abcam, UK), p62 (Abcam, UK), and LC3B (Novus, Littleton CO, USA) antibodies. ImageJ software was utilized for densitometric analysis of western blots normalized to GAPDH.
3.4. Autophagy induction improved the distribution and expression of TJ proteins TJ proteins are the major determinants for the intestinal barrier permeability maintenance. We performed the immunofluorescence and western blotting analyses in order to investigate the impact of autophagy on TJ proteins (occluding and ZO-1). As exhibited in Fig. 7, occluding and ZO-1 expressions in IL-10 KO mice were decreased when comparing with WT mice. In contrast, the changes in expressions of TJ proteins observed in the IL-10 KO mice were significantly improved by rapamycin treatment (Fig. 7a). Furthermore, occluding and ZO-1 were differently localized in IL-10 KO mice with a lower TJ density, which was also improved by rapamycin treatment (Fig. 7b and c).
2.11. Statistical analysis Statistical analysis was performed using one-way ANOVA test with SPSS (Version 19.0, SPSS Inc., Chicago, IL, USA) and GraphPad Prism (Version 5.0, GraphPad Software Inc., San Diego, CA, USA). P value < 0.05 was considered statistically significant. To ensure the reproducibility, independent experiments were repeated for three times.
3.5. Autophagy induction did not decrease the epithelial apoptosis
3. Result
To further investigate the effect of autophagy on epithelial apoptosis, the apoptotic cells in the proximal colon tissues were identified via TUNEL staining. The results are shown in Fig. 8, indicating that IL10 KO mice exhibited a remarkable increase in apoptosis in comparison with WT mice. However, rapamycin treatment in IL-10 KO mice did not improve the status of epithelial cell apoptosis as we expected.
3.1. Autophagy induction ameliorated DAI and histological colitis in IL-10 KO mice As described in the area of “Methods”, DAI was utilized for evaluating disease progression in IL-10 KO mice. As illustrated in Fig. 1, IL-10 KO mice showed a significantly higher DAI valuein comparison with WT mice, while rapamycin significantly reduced DAI level via autophagy modulation (P < 0.01). Colitis severity was then evaluated in mice of four groups by H&E staining and results are shown in Fig. 2. More infiltration of inflammatory cells in colonic mucosa were obviously observed in IL-10 KO mice when comparing with WT mice. Compared with IL-10 KO mice, the inflammatory cell infiltration and inflammation scores were significantly decreased by rapamycin treatment and aggravated in 3-MA treatment (P < 0.01). The expressions of autophagy-associated proteins (including p62 and LC3B) in proximal colon tissues of four groups were evaluated by western blotting. IL-10 KO mice showed a lower expression of p62 protein and a lower LC3B II/ I ratio (see Fig. 3), indicating the autophagy deficiency in proximal colon tissues when comparing with WT mice. In the meantime, the autophagy levels were significantly induced by rapamycin and inhibited by 3-MA.
4. Discussion Animal models of experimental colitis are frequently used for pathogenesis and therapies for IBD due to the same pathologic processes [25,26]. Of all the animal models, IL-10 KO mice are a very popular model for CD with the pathology mediated by T lymphocytes polarization [27]. IL-10 KO mice develop colitis spontaneously under SPF conditions and infections or drugs can accelerate the colitis [27]. Our present study used IL-10 KO mice as the human CD model and the results indicated that rapamycin, as a widely used autophagy inducer, significantly ameliorated experimental colitis and improved the intestinal barrier function in IL-10 KO mice. More specifically, rapamycin treatment ameliorated DAI and histological colitis, decreased pro-inflammatory cytokines and chemotactic factors, decreased intestinal and colonic permeability, improved the distribution and expression of TJ 3
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Fig. 2. Histological sections of the proximal colon by H&E staining in mice of four groups. (a) WT group; (b) IL-10 KO group; (c) IL-10 KO + Rapamycin group; (d) IL10 KO + 3-MA group; (e) inflammation scores in mice of four groups. ** P < 0.01 (n = 6 in each group).
maintaining of intestinal homeostasis [30]. As reported by previous studies, defective TJ barrier contributes to the access of luminal microbes or antigens to the host immune system, which results in the development of intestinal inflammation [31,32]. The inflammatory status is closely associated with increased paracellular permeability, which is induced by the structural and functional alterations in TJ [17,33]. Autophagy, a physiological process, plays a critical role in the balance maintaining via modulating the functions of cells in the mucosal layer [34]. Autophagy is crucial in mucosal inflammation controlment and intestinal physiology maintaining [35]. Autophagy alterations will lead to the disruption of mucosal immunity in both cell-autonomous and cell non-autonomous manners, such as antigen processing and presentation, the clearance of intracellular bacteria [36]. Furthermore,
proteins via autophagy induction. The intestinal epithelium, the barrier between the intestinal mucosa and lumen, is frequently exposed to microbes or food and it serves as the first-line defense against the various invading gut microbiota [18]. TJs, as multi-protein complexes, regulate paracellular trafficking of macromolecules via selective permeability [28] and provide the foundation to the actin cytoskeleton by anchoring the transmembrane proteins [29]. Functional perturbations or structural abnormalities of TJ can impair the intestinal barrier function and homeostasis, resulting in the intestinal disorders [28]. The increased intestinal permeability leads to internalization of TJ proteins and further contributes to the inflammatory status [28]. The sensitive balance between defense and tolerance is required to maintain homeostasis in the intestine. Intestinal epithelial TJ barrier is widely accepted as a crucial determinant for the
Fig. 3. Expressions of autophagy-associated proteins (p62 and LC3B) in mice of four groups. The ratio of LC3B II/I is presented as means and standard error (SD). ** P < 0.01 (n = 6 in each group). 4
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Fig. 4. Expressions of pro-inflammatory cytokines in proximal colon tissues of mice in four groups. Values are presented as means and standard error (SD). ** P < 0.01 (n = 6 in each group).
Fig. 5. Expressions of chemotactic factors in proximal colon tissues of mice in four groups. Values are presented as means and standard error (SD). ** P < 0.01 (n = 6 in each group).
resistance and disruption of occludin and ZO-1 [45]. Further research is required to determine the exact impact of autophagy alternations on the TJ proteins in various models and cell types. This study has some limitations. First, the unknown target cell type in rapamycin treatment is a major limitation. Second, the involved mechanisms why rapamycin treatment can significantly improve the intestinal barrier function and experimental colitis remain unclear.
autophagy plays an important role in the limitation of proinflammatory TH2 cell expansion and promotion of survival of regulatory T cells [37]. Recently, a critical relationship between autophagy, immune activation, and the metabolic transition of activated T cells has been elucidated [38]. Increasing evidence has indicated that deficient autophagy involves in the complex pathogenesis of intestinal disorders through several cellular processes, including impaired Paneth and Goble cells function [39], microbial sensing, and inadequate clearance of microbes [40]. Furthermore, several studies have indicated that the integrity of intestinal epithelial barrier is also regulated by autophagy [41,42]. Autophagy improves TJ barrier function in Caco-2 intestinal epithelial cells through the enhancement of the lysosomal breakdown of pore forming TJ protein claudin-2 [43]. Similarly, autophagy induction by rapamycin in intestinal epithelial cells partially rescues the intestinal barrier dysfunction by amino acid deprivation [44]. Our results indicated that autophagy induction by rapamycin significantly improved the experimental colitis and intestinal barrier function, which was quite in accordance with their conclusions. On the contrary, another study by Feng et al. has revealed that upregulated autophagy is associated with enhanced paracellular permeability, reduced transepithelial electrical
5. Conclusions In summary, this current study provided evidence that autophagy induction by rapamycin significantly improved intestinal barrier function and protected IL-10 KO mice from the experimental chronic colitis. Given the important role of autophagy in intestinal homeostasis and the pathogenic role of autophagy deficiency in IBD, investigating molecules to activate selective autophagy has attracted much therapeutic interest. Further understanding of the role of autophagy-dependent pathways will provide potential therapeutic targets for CD.
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Fig. 6. The colonic permeability by Ussing chamber and intestinal permeability by fluorescein isothiocyanate (FITC)–dextran of mice in four groups. (a) Mannitol flux; (b) electrical resistance; (c) FITC–dextran. Values are presented as means and standard error (SD). ** P < 0.01 (n = 6 in each group).
Fig. 7. Distribution and expression of occludin and ZO-1 in proximal colon tissues of mice in four groups. (a) expressions by western blotting normalized to GAPDH; resentative immunofluorescence (green) images of occludin (b) and ZO-1 (c) and nuclei (blue) of proximal colon tissues (200x magnification). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 6
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Fig. 8. Representative images of epithelial apoptosis by TUNEL assay (200x magnification). (a) WT group; (b) IL-10 KO group; (c) IL-10 KO + Rapamycin group; (d) IL-10 KO + 3-MA group; (e) numbers of TUNEL-positive cells/field. Values are presented as means and standard error (SD). ** P < 0.01 (n = 6 in each group).
Declarations
References
Author contributions HG W, HJ Y and Y Z: Study design and data analysis. J Z: Patient recruitment, data collection and writing up of the first draft of the paper. LM T: scientific advice, supervision and drafting of the manuscript. Ethics approval and consent to participate Consent to participate is not applicable in this study. All the experimental protocols were approved by the Ethics Committee of the Affiliated Changzhou No. 2 People's Hospital of Nanjing Medical University according to the Chinese Council on Animal Care guidelines. Consent for publication Not applicable. Availability of data and material Please contact the corresponding author (Liming Tang, [email protected]) on reasonable request.
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Funding This work was supported in part by funding from the National Natural Science Foundation of China (Grant 81600434), Natural Science Foundation of Jiangsu Province (Grants BK 20160572 and BK 20170358), Jiangsu Provincial Medical Youth Talent (Grant QNRC2016514), China Postdoctoral Science Foundation (Grant 2018M630581), “333” Level II Talent Project of Jiangsu Province (No. QT201705), and Natural Science Foundation of Jiangsu Province, General Program (No. BK20181115).
Declaration of Competing Interest The authors have no conflicts of interest to declare.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Autophagy induction by rapamycin ameliorates experimental colitis and improves intestinal epithelial barrier function in IL-10 knockout mice Jie Zhaoa,b,1, Honggang Wangc,1, Haojun Yanga, Yan Zhoua, Liming Tanga,
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a
Department of Gastrointestinal Surgery, The Affiliated Changzhou No. 2 People’s Hospital of Nanjing Medical University, PR China Department of General Surgery, The First Affiliated Hospital of Soochow University, PR China c Department of General Surgery, Taizhou People’s Hospital, Taizhou People’s Hospital, Medical School of Nantong University, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Crohn’s disease IL-10 knockout mice Autophagy Intestinal barrier function
Background: An impairment of the intestinal barrier function is one of the major characteristics of Crohn’s disease (CD). This study aimed to evaluate the impact of autophagy induction by rapamycin on the intestinal epithelial barrier function in CD model mice. Methods: IL-10 knockout (IL-10 KO) mice were used as the human CD models in this study. All the mice were randomly assigned into four groups, (a) wild-type (WT) group; (b) IL-10 KO group; (c) IL-10 KO + rapamycin group and (d) IL-10 KO + 3-methyladenine (3-MA), containing 6 mice in each group. The disease activity index (DAI), histology, pro-inflammatory cytokines and chemotactic factors in colon tissues, intestinal and colonic permeability, distributions and expressions of tight junction (TJ) proteins, epithelial apoptosis of mice in four groups were evaluated and compared. Results: Autophagy induction by rapamycin treatment ameliorated DAI and histological colitis, decreased proinflammatory cytokines (TNF-α, IFN-γ and IL-17) and chemotactic factors (CXCL-1 and CXCL-2), decreased intestinal and colonic permeability, improved the distribution and expression of TJ proteins in IL-10 KO mice. Conclusion: Autophagy induction by rapamycin significantly improved intestinal barrier function and protected IL-10 KO mice from the experimental chronic colitis.
1. Introduction Inflammatory bowel disease (IBD), which consists of ulcerative colitis (UC) and Crohn’s disease (CD), is widely accepted as an autoimmune disorder characterized by chronic inflammation with unknown etiology [1]. Much of our current understanding of the possible molecular mechanisms and potential therapeutic targets in IBD mainly comes from animal models of IBD [2,3]. Interleukin-10-knockout (IL-10 KO) mice have been widely reported to display similar characteristics to that of human CD [4]. Functional gene polymorphism studies have clearly revealed the close association between CD and autophagy-related proteins including nucleotide-binding oligomerization domaincontaining protein (NOD)2, IRGM and ATG16L1, which strongly suggests the pathological role of defective autophagy in CD [5,6]. Increasing evidence has shown that autophagy probably involves in the pathogenesis of CD through variable mechanisms including the modulation of T-cell development, activation and differentiation [7],
clearance of pathogens [8], activation of nuclear factor-kappa B (NFκB) [9]. Furthermore, autophagy has also been suggested as a new therapeutic target for CD [10]. The underlying pathogenesis of CD remains unclear until now, an impairment of the intestinal barrier function is one of the major characteristics of CD [11]. Furthermore, some studies have revealed that the dysfunction of intestinal barrier function involves in the pathogenesis of CD [12]. The disturbance of the intestinal barrier leads to an increased intestinal permeability, an enhanced bacterial translocation and an over-activated mucosal immune response, thus aggravating the intestinal chronic inflammation in CD [13]. Therefore, the intestinal barrier plays an imperative role in the maintenance of intestinal mucosal homeostasis. Recently, the close association between autophagy and intestinal barrier function has drawn a lot of attention. It has been widely proved that goblet cells can defend against intestinal microbe and regulate the microecology by secreting mucins [14,15]. Furthermore, the
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Corresponding author at: Department of Gastrointestinal Surgery, The Affiliated Changzhou No. 2 People's Hospital of Nanjing Medical University, 68 Gehu Road, Changzhou, Jiangsu 213003, PR China. E-mail address: [email protected] (L. Tang). 1 Jie Zhao and Honggang Wang contributed equally to this article. https://doi.org/10.1016/j.intimp.2019.105977 Received 21 September 2019; Received in revised form 11 October 2019; Accepted 13 October 2019 1567-5769/ © 2019 Published by Elsevier B.V.
Please cite this article as: Jie Zhao, et al., International Immunopharmacology, https://doi.org/10.1016/j.intimp.2019.105977
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2.6. Ussing chamber studies
development and function of goblet cells are controlled by cell autophagy [14]. Autophagy plays important roles in the clearance of polyubiquitinated protein aggregates, degradation of invading pathogens, modulation of pathogen induced pro-inflammatory cytokines release, lymphocyte development and antigen presentation [16,17]. Moreover, the process mentioned above is critically important in the mucosal immune response, anti-microbial defense and intestinal barrier integrity maintaining [18]. However, the relationship between epithelial barrier function and autophagy modulation remains unclear. In this study, we aimed to evaluate the impact of autophagy on the intestinal epithelial barrier function in CD model mice.
Segments of proximal colon were obtained for the assessment of the colon permeability according to the descriptions by Wang et al. [19]. In brief, Lucite chambers were used for mounting the mucosa, exposing serosal and mucosal surfaces to 10 ml of Ringer’s buffer (1.25 mM CaCl2, 8 mM-KCl, 115 mM NaCl, 1.2 mM MgCl2, 2.0 mM KHPO4, 25 mM NaCO3, pH 7.33–7.37). The buffers were maintained by a heated water jacket and circulated by CO2 at a temperature of 37 °C. To measure the basal mannitol fluxes, 1 mM of mannitol with 370 KBOr (H3-mannitol) was then added to the mucosal side. The determination of spontaneous transepithelial potential difference (mV) was performed, and the tissue was clamped by introducing an appropriate short circuit current (Isc, mA/cm2) with an automatic voltage clamp (DVC 1000; World Precision Instruments, Sarasota, FL). Following the descriptions of Ohm’s law, tissue ion resistance was calculated with the potential difference and Isc.
2. Materials and methods 2.1. Animals IL-10 KO mice used in this study were all obtained from the Jackson Laboratory (Bar Harbor, Maine) on the background of C57BL/6. IL-10 KO mice and wild-type (WT) mice included were all housed and maintained under specific pathogen free (SPF) conditions at the Model Animal Research Center of Nanjing University (Nanjing, China). All experimental procedures involving the use of animals were reviewed and approved by the Animal Care and Use Committee of the Model Animal Research Center, Nanjing University (Nanjing, China).
2.7. Intestinal permeability assay Fluorescein isothiocyanate (FITC)–dextran (Sigma-Aldrich; 150 ml) analysis was performed for the intestinal permeability assessment according to our previous published descriptions by Zhao et al. [21]. In brief, a solution containing 25 mg of 4 kDa FITC-dextran, which was diluted in 0.1 ml PBS, was injected into the intestinal lumen. Thirty minutes later, the blood sample via cardiac puncture was obtained and immediately centrifuged (10000g, 10 min) in ice-cold tubes containing heparin. The concentration of FITC-dextran was determined using a fluorescence spectrophotometer (F7000; Hitachi) with the excitation wavelength of 495 nm and emission wavelength of 520 nm.
2.2. Drug administration protocol All the mice included in our study were randomly assigned into four groups, (a) WT group; (b) IL-10 KO group; (c) IL-10 KO + Rapamycin group (2 mg/kg, intragastric administration) and (d) IL-10 KO + 3methyladenine (3-MA, 30 mg/kg, intraperitoneal injection), containing 6 mice in each group. After the final drug administration, all mice were executed, colon tissues were obtained to assess the therapeutic effects of autophagy modulation on the colitis status.
2.8. Epithelial apoptosis assessment Terminal deoxynucleotidyl transferase dUTP nick end labelling assay (TUNEL) In Situ Cell Death Detection Kit (Roche, Basel, Switzerland) was performed for the assessment of epithelial apoptosis. The procedures were performed according to the manufacturer’s instructions. In brief, the obtained sections were permeabilized with 1% Triton X-100, 0.1% sodium citrate, washed and stained. After that, sections were counterstained with 40,6-diamidino-2-phenylindole (DAPI) and then mounted in 50% glycerol after washing with PBS. The confocal microscopy (Olympus, Tokyo, Japan) were utilized for photographing the sections. Six random and non-overlapping pictures of (×200 magnified) optical fields of two different colon layers were chosen. A same laboratory technician who was blinded to this study was invited to count the number of TUNEL-positive cells per field.
2.3. Disease activity index (DAI) assessment As described by previous reports [19], one point was scored for each of the following: occult fecal blood, ruffled fur, soft stool and rectal prolapse < 1 mm. An additional point was scored for severe rectal prolapse > 1 mm or diarrhea. The DAI was calculated by the sum of scores. 2.4. Histology After the mice were euthanized under anesthesia, the entire colons were carefully collected and rinsed with phosphate buffer saline (PBS). The length of colon collected were measured to evaluate the changes in morphology. Thereafter, the proximal colon tissues were obtained and fixed in 10% buffer neutral formalin and embedded in paraffin. 6 umthick sections from the proximal colons were stained with haematoxylin and eosin (H&E). Taking the number of lesions and severity of the disease into consideration, the inflammation score were given to samples assessed by two independent pathologists who were blinded to the study design following well establish criteria by Singh et al. [20].
2.9. Immunofluorescence analysis Immunofluorescence analysis was performed to assess the distribution and integrity of tight junction (TJ) proteins including occludin and zona occludens protein 1 (ZO-1) as described by previous reports [22]. In brief, 6 μm-thick frozen proximal colon sections were transferred to coated slides, fixed in 1% paraformaldehyde, and washed with PBS. 5% normal goat serum diluted in PBS was used to block nonspecific binding. Thereafter, sections were incubated with monoclonal antibodies against occludin (Abcam, UK) and ZO-1 (Abcam, UK) in PBS with 1% goat serum overnight at 4℃. After that, sections were washed with PBS and incubated with Alexa 488-conjugated secondary antibodies for 1 h. A confocal microscopy (Olympus, Tokyo, Japan) was utilized for images visualization.
2.5. Pro-inflammatory cytokines and chemotactic factors in colon tissues by enzyme-linked immunosorbent assay (ELISA) Pro-inflammatory cytokines including tumor necrosis factor-α (TNFα), interferon-γ (IFN-γ) and interleukin-17 (IL-17), chemotactic factors including CXCL-1 and CXCL-2 were measured by ELISA using DuoSet ELISA development kits (R&D systems, Minneapolis, MN). We obtain the protein extracts from the colonic segments in homogenization buffer with a protease inhibitor and performed the measurements of cytokines according to the manufacturer’s instructions.
2.10. Western blotting Western blotting analysis of occluding, ZO-1, p62 and LC3B proteins was performed as described previously [23] using the primary 2
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3.2. Autophagy induction decreased pro-inflammatory cytokines and chemotactic factors in IL-10 KO mice As shown in Fig. 4, pro-inflammatory cytokines (TNF-α, IFN-γ and IL-17) are significantly elevated in IL-10 KO mice, indicating the pathogenic role of Th1/Th17 cells differentiation in chronic inflammation development in CD [24]. The results have also indicated that IL-10 KO mice with rapamycin treatment had a significant decrease when comparing with IL-10 KO mice with 3-MA treatment or without any treatment (P < 0.01). Moreover, chemotactic factors (CXCL-1 and CXCL-2) in mice of the four groups showed the same trends (see Fig. 5). 3.3. Autophagy induction decreased intestinal and colonic permeability To study the impact of autophagy modulation in intestinal permeability, we first performed Ussing chamber studies using obtained colon tissues. As shown in Fig. 6, IL-10 KO mice showed a significantly increased colonic permeability to mannitol (Fig. 6a) with a corresponding decrease in electrical resistance (Fig. 6b). Autophagy induction largely improved the increased colonic permeability in IL-10 KO mice. In addition, we also designed the FITC–dextran analysis to assess the intestinal permeability. IL-10 KO mice were associated with greatly increased concentrations of FITC-dextran compared with WT mice and rapamycin-treated IL-10 KO mice (see Fig. 6c). The described results indicated that Rapamycin, as an autophagy inducer, could significantly improve the increased intestinal and colonic permeability in IL-10 KO mice.
Fig. 1. Levels of disease activity index in mice of four groups. Values are presented as means and standard error (SD). ** P < 0.01 (n = 6 in each group).
occluding (Abcam, UK), ZO-1 (Abcam, UK), p62 (Abcam, UK), and LC3B (Novus, Littleton CO, USA) antibodies. ImageJ software was utilized for densitometric analysis of western blots normalized to GAPDH.
3.4. Autophagy induction improved the distribution and expression of TJ proteins TJ proteins are the major determinants for the intestinal barrier permeability maintenance. We performed the immunofluorescence and western blotting analyses in order to investigate the impact of autophagy on TJ proteins (occluding and ZO-1). As exhibited in Fig. 7, occluding and ZO-1 expressions in IL-10 KO mice were decreased when comparing with WT mice. In contrast, the changes in expressions of TJ proteins observed in the IL-10 KO mice were significantly improved by rapamycin treatment (Fig. 7a). Furthermore, occluding and ZO-1 were differently localized in IL-10 KO mice with a lower TJ density, which was also improved by rapamycin treatment (Fig. 7b and c).
2.11. Statistical analysis Statistical analysis was performed using one-way ANOVA test with SPSS (Version 19.0, SPSS Inc., Chicago, IL, USA) and GraphPad Prism (Version 5.0, GraphPad Software Inc., San Diego, CA, USA). P value < 0.05 was considered statistically significant. To ensure the reproducibility, independent experiments were repeated for three times.
3.5. Autophagy induction did not decrease the epithelial apoptosis
3. Result
To further investigate the effect of autophagy on epithelial apoptosis, the apoptotic cells in the proximal colon tissues were identified via TUNEL staining. The results are shown in Fig. 8, indicating that IL10 KO mice exhibited a remarkable increase in apoptosis in comparison with WT mice. However, rapamycin treatment in IL-10 KO mice did not improve the status of epithelial cell apoptosis as we expected.
3.1. Autophagy induction ameliorated DAI and histological colitis in IL-10 KO mice As described in the area of “Methods”, DAI was utilized for evaluating disease progression in IL-10 KO mice. As illustrated in Fig. 1, IL-10 KO mice showed a significantly higher DAI valuein comparison with WT mice, while rapamycin significantly reduced DAI level via autophagy modulation (P < 0.01). Colitis severity was then evaluated in mice of four groups by H&E staining and results are shown in Fig. 2. More infiltration of inflammatory cells in colonic mucosa were obviously observed in IL-10 KO mice when comparing with WT mice. Compared with IL-10 KO mice, the inflammatory cell infiltration and inflammation scores were significantly decreased by rapamycin treatment and aggravated in 3-MA treatment (P < 0.01). The expressions of autophagy-associated proteins (including p62 and LC3B) in proximal colon tissues of four groups were evaluated by western blotting. IL-10 KO mice showed a lower expression of p62 protein and a lower LC3B II/ I ratio (see Fig. 3), indicating the autophagy deficiency in proximal colon tissues when comparing with WT mice. In the meantime, the autophagy levels were significantly induced by rapamycin and inhibited by 3-MA.
4. Discussion Animal models of experimental colitis are frequently used for pathogenesis and therapies for IBD due to the same pathologic processes [25,26]. Of all the animal models, IL-10 KO mice are a very popular model for CD with the pathology mediated by T lymphocytes polarization [27]. IL-10 KO mice develop colitis spontaneously under SPF conditions and infections or drugs can accelerate the colitis [27]. Our present study used IL-10 KO mice as the human CD model and the results indicated that rapamycin, as a widely used autophagy inducer, significantly ameliorated experimental colitis and improved the intestinal barrier function in IL-10 KO mice. More specifically, rapamycin treatment ameliorated DAI and histological colitis, decreased pro-inflammatory cytokines and chemotactic factors, decreased intestinal and colonic permeability, improved the distribution and expression of TJ 3
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Fig. 2. Histological sections of the proximal colon by H&E staining in mice of four groups. (a) WT group; (b) IL-10 KO group; (c) IL-10 KO + Rapamycin group; (d) IL10 KO + 3-MA group; (e) inflammation scores in mice of four groups. ** P < 0.01 (n = 6 in each group).
maintaining of intestinal homeostasis [30]. As reported by previous studies, defective TJ barrier contributes to the access of luminal microbes or antigens to the host immune system, which results in the development of intestinal inflammation [31,32]. The inflammatory status is closely associated with increased paracellular permeability, which is induced by the structural and functional alterations in TJ [17,33]. Autophagy, a physiological process, plays a critical role in the balance maintaining via modulating the functions of cells in the mucosal layer [34]. Autophagy is crucial in mucosal inflammation controlment and intestinal physiology maintaining [35]. Autophagy alterations will lead to the disruption of mucosal immunity in both cell-autonomous and cell non-autonomous manners, such as antigen processing and presentation, the clearance of intracellular bacteria [36]. Furthermore,
proteins via autophagy induction. The intestinal epithelium, the barrier between the intestinal mucosa and lumen, is frequently exposed to microbes or food and it serves as the first-line defense against the various invading gut microbiota [18]. TJs, as multi-protein complexes, regulate paracellular trafficking of macromolecules via selective permeability [28] and provide the foundation to the actin cytoskeleton by anchoring the transmembrane proteins [29]. Functional perturbations or structural abnormalities of TJ can impair the intestinal barrier function and homeostasis, resulting in the intestinal disorders [28]. The increased intestinal permeability leads to internalization of TJ proteins and further contributes to the inflammatory status [28]. The sensitive balance between defense and tolerance is required to maintain homeostasis in the intestine. Intestinal epithelial TJ barrier is widely accepted as a crucial determinant for the
Fig. 3. Expressions of autophagy-associated proteins (p62 and LC3B) in mice of four groups. The ratio of LC3B II/I is presented as means and standard error (SD). ** P < 0.01 (n = 6 in each group). 4
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Fig. 4. Expressions of pro-inflammatory cytokines in proximal colon tissues of mice in four groups. Values are presented as means and standard error (SD). ** P < 0.01 (n = 6 in each group).
Fig. 5. Expressions of chemotactic factors in proximal colon tissues of mice in four groups. Values are presented as means and standard error (SD). ** P < 0.01 (n = 6 in each group).
resistance and disruption of occludin and ZO-1 [45]. Further research is required to determine the exact impact of autophagy alternations on the TJ proteins in various models and cell types. This study has some limitations. First, the unknown target cell type in rapamycin treatment is a major limitation. Second, the involved mechanisms why rapamycin treatment can significantly improve the intestinal barrier function and experimental colitis remain unclear.
autophagy plays an important role in the limitation of proinflammatory TH2 cell expansion and promotion of survival of regulatory T cells [37]. Recently, a critical relationship between autophagy, immune activation, and the metabolic transition of activated T cells has been elucidated [38]. Increasing evidence has indicated that deficient autophagy involves in the complex pathogenesis of intestinal disorders through several cellular processes, including impaired Paneth and Goble cells function [39], microbial sensing, and inadequate clearance of microbes [40]. Furthermore, several studies have indicated that the integrity of intestinal epithelial barrier is also regulated by autophagy [41,42]. Autophagy improves TJ barrier function in Caco-2 intestinal epithelial cells through the enhancement of the lysosomal breakdown of pore forming TJ protein claudin-2 [43]. Similarly, autophagy induction by rapamycin in intestinal epithelial cells partially rescues the intestinal barrier dysfunction by amino acid deprivation [44]. Our results indicated that autophagy induction by rapamycin significantly improved the experimental colitis and intestinal barrier function, which was quite in accordance with their conclusions. On the contrary, another study by Feng et al. has revealed that upregulated autophagy is associated with enhanced paracellular permeability, reduced transepithelial electrical
5. Conclusions In summary, this current study provided evidence that autophagy induction by rapamycin significantly improved intestinal barrier function and protected IL-10 KO mice from the experimental chronic colitis. Given the important role of autophagy in intestinal homeostasis and the pathogenic role of autophagy deficiency in IBD, investigating molecules to activate selective autophagy has attracted much therapeutic interest. Further understanding of the role of autophagy-dependent pathways will provide potential therapeutic targets for CD.
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Fig. 6. The colonic permeability by Ussing chamber and intestinal permeability by fluorescein isothiocyanate (FITC)–dextran of mice in four groups. (a) Mannitol flux; (b) electrical resistance; (c) FITC–dextran. Values are presented as means and standard error (SD). ** P < 0.01 (n = 6 in each group).
Fig. 7. Distribution and expression of occludin and ZO-1 in proximal colon tissues of mice in four groups. (a) expressions by western blotting normalized to GAPDH; resentative immunofluorescence (green) images of occludin (b) and ZO-1 (c) and nuclei (blue) of proximal colon tissues (200x magnification). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 6
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Fig. 8. Representative images of epithelial apoptosis by TUNEL assay (200x magnification). (a) WT group; (b) IL-10 KO group; (c) IL-10 KO + Rapamycin group; (d) IL-10 KO + 3-MA group; (e) numbers of TUNEL-positive cells/field. Values are presented as means and standard error (SD). ** P < 0.01 (n = 6 in each group).
Declarations
References
Author contributions HG W, HJ Y and Y Z: Study design and data analysis. J Z: Patient recruitment, data collection and writing up of the first draft of the paper. LM T: scientific advice, supervision and drafting of the manuscript. Ethics approval and consent to participate Consent to participate is not applicable in this study. All the experimental protocols were approved by the Ethics Committee of the Affiliated Changzhou No. 2 People's Hospital of Nanjing Medical University according to the Chinese Council on Animal Care guidelines. Consent for publication Not applicable. Availability of data and material Please contact the corresponding author (Liming Tang, [email protected]) on reasonable request.
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Funding This work was supported in part by funding from the National Natural Science Foundation of China (Grant 81600434), Natural Science Foundation of Jiangsu Province (Grants BK 20160572 and BK 20170358), Jiangsu Provincial Medical Youth Talent (Grant QNRC2016514), China Postdoctoral Science Foundation (Grant 2018M630581), “333” Level II Talent Project of Jiangsu Province (No. QT201705), and Natural Science Foundation of Jiangsu Province, General Program (No. BK20181115).
Declaration of Competing Interest The authors have no conflicts of interest to declare.
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