Acute stress disrupts intestinal homeostasis via GDNF‐RET

Abstract Objectives Enterochromaffin (EC) cells have been associated with functional gastrointestinal disorders such as IBS. Recently, we found that glial cell‐derived neurotrophic factor (GDNF)‐rearranged during transfection (RET) localized in EC cells in human colonic epithelia. Here, we examine the role of GDNF‐RET in the pathophysiology of diarrhoea‐predominant irritable bowel syndrome (IBS‐D). Materials and Methods GDNF was assessed by ELISA and immunohistochemistry in biopsies from IBS‐D patients and healthy controls. Stress was induced by using a wrap‐restraint stress (WRS) procedure to serve as an acute stress‐induced IBS model. The function of GDNF‐RET axis to intestinal stem cell (ISC) homeostasis, and EC cell numbers were assessed in vivo and in vitro. Results GDNF‐RET was expressed in EC cells in human colon. GDNF was significantly increased in IBS‐D patients. WRS mice showed increased GDNF‐RET levels in colon. WRS induced visceral hypersensitivity by expanding of ISC and differentiation of EC cell via GDNF‐RET. Furthermore, GDNF‐treated mice recapitulated the phenotype of WRS mice. In vitro, GDNF treatment amplified Wnt signal and increased serotonin levels in colonic organoids in a dose‐dependent manner. Conclusions We identified GDNF‐RET was presented in colonic epithelium of patients with IBS‐D. GDNF‐RET played important roles in regulating ISC and EC cell differentiation. Our findings, thus, provide RET inhibitor as new therapeutic targets for treatment of patients with IBS‐D.


| INTRODUC TI ON
Irritable bowel syndrome (IBS) is a common functional bowel disorder characterized by abdominal pain with altered bowel habits. The stress has long been considered to play a role in the aetiology of IBS.
In most patients, IBS is a chronic relapsing disease which significantly reduces health-related quality of life. Although its pathogenesis is not completely understood, the role of visceral hypersensitivity in IBS has recently emerged. 1 4 EC cells are considered to play an important role by acting as chemosensors. It has been shown that EC cells use sensory receptors to detect irritants, metabolites, and catecholamines and convert them into 5-HT release events. 5 Rearranged during transfection (RET) has been reported recently to express predominantly in a subset of enteroendocrine cells in mouse intestine. 6 However, it is still unknown whether intestinal epithelial cells express of RET in human, and the function of RET in EC cells has not yet been identified.
EC cells are terminally differentiated and are unable to replicate themselves. 7 The Lgr5 + stem cells under intestinal crypts have been shown to long-term self-renew and differentiate, giving rise to all cell lineages in the intestinal epithelium. 8 Mouse atonal homolog 1 (Math1), Neurogenin3 (Neurog3) and NeuroD play prominent roles in ISC fate specification. 9 Intestinal stem cells (ISC) and progenitors can switch fate choice to increase the proportion of EC cell progenitors, leading to increased EC cell number. However, the mechanisms may be occurring in patients with IBS-D is unknown.
RET is a transmembrane protein, which binds the members of the glial cell-derived neurotrophic factor (GDNF) family, and is most commonly implicated in Hirschsprung's disease. 10 It is well established that RET activity is required for the development of the enteric nerve system, 11 kidney 12,13 and spermatogenesis. 14 Recent studies demonstrate that RET promotes maturation and activates Wnt signalling in the developing mouse intestinal epithelium. 6 Of interest, in pancreas, GDNF can increase β-cell proliferation by enhancing Pdx1, Ngn3, NeuroD1/2 and Pax4 gene expression. 15 These findings suggest that RET may participate in the development of EC cells.
In IBS-D patients, chronic, low-grade and subclinical inflammation has been implicated in the disease process. 16 GDNF has been shown to prevent colonic epithelial cell apoptosis and ameliorate experimental colitis. 17 In functional dyspepsia, the expression of GDNF is increased in duodenal mucosa. 18 These results suggested that GDNF might be involved in IBS pathophysiology.
In this report, we showed that colon mucosal GDNF expression was increased in IBS-D patients, and GDNF-RET was present in human EC cells. In an acute stress-induced IBS model, stress induced visceral hypersensitivity by expanding of ISC and differentiation of EC cell via GDNF-RET, which was inhibited by treatment with RET inhibitor.
Moreover, GDNF treatment amplified Wnt signal and increased serotonin levels in colonic organoids in a dose-dependent manner.

| Participants and questionnaires
A total of 11 patients with IBS-D (4 women and 7 men) and 12 control subjects (5 women and 7 men) participated in the study. This study was approved by the Clinical Ethical Committee of the Qilu Hospital of Shandong University, and all participants signed a written informed consent form before participation. The diagnosis of IBS was based on the Rome IV criteria. 19 Controls were selected from patients undergoing colonoscopy for cancer surveillance who received negative results. Exclusion criteria were as follows: patients taking non-steroidal anti-inflammatory drugs or other anti-inflammatory drugs (including mast cell stabilizers, histamine antagonists, probiotics, immunosuppressants and steroids); had undergone major abdominal surgery; or had any organic syndrome, including coeliac disease, allergic diseases and psychiatric disorders as assessed by history taking, appropriate consultations and laboratory tests. All specimens were taken from the rectosigmoid junction to standardize the site of sampling.

| Organoid cultures
The small intestines and colons of male C57BL/6 mice were isolated and cut into 2-4 mm sections after making a longitudinal incision along the entire length of the intestine, then incubated in 2 mmol/L ethylenediaminetetraacetic acid (EDTA) on a rocking platform at 100 rpm for 30 minutes (small intestine) or 60 minutes (colon) at 4°C.
The tissue was then re-suspended in clean PBS and pipetted several times. The supernatant was collected and filtered through a 70 μm filter, then centrifuged at 300 × g for 5 minutes at 4°C. Isolated Organoids were kept in a humidified atmosphere containing 5% CO 2 at 37°C.

| Animal models
Adult male C57BL/10 mice were purchased from the animal centre of Shandong University of Traditional Chinese Medicine. All experiments were approved by the Ethical Committee and Institutional Animal Care and Use Committee of Qilu Hospital. For the acute stress model, stress was induced by using a wrap-restraint stress (WRS) procedure, an acute non-ulcerogenic model of colonic hypersensitivity. All constraint was performed at the same time of the day, between 10 am and 12 am for 7 days, to minimize the influence of circadian rhythms. We forced immobilization of mice by placing them in 50 mL tubes with a hole for air. Mice were then placed back in their home cage.
For oral dosing, GSK3179106 was prepared as a suspension in saline at 1 μg/mL and administered at 10 μg/kg and injected BID at 8 am and 4 pm for 2 days. For intraperitoneal injection, recombinant murine GDNF was administrated at 10 μg/kg and injected BID for 5 days. After 7 days of administration and injection, the mice were evaluated by colorectal distension (CRD) test.

| Visceral hypersensitivity evaluation
The visceral hypersensitivity was evaluated by CRD test, following the previously described protocol. 20 The distension was applied on awake mice by using a 4 mm Fogarty catheter balloon inserted into the descending colon. Graded CRD was performed by rapidly injecting different volumes of normal saline (0, 0.04 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18. 0.2 and 0.22 mL) into the balloon and maintaining the distension for 20 seconds. Distension was given at 4 minutes intervals and repeated 3 times to achieve an accurate result. The abdominal withdrawal reflex (AWR) score was recorded according to a method described before, 21 and the mean scores from three data were used as the final score.

| Immunohistochemistry
Endoscopic biopsy colon tissues and mice colon were fixed overnight at 4°C in 4% paraformaldehyde and was subsequently embedded in paraffin. Fixed and embedded tissues were sectioned (4 μm) into slides.

| Enzyme-linked immunosorbent assays
The total proteins of biopsy colon tissues were extracted and centrifuged, and the supernatants were taken out and quantified. The protein level of GDNF was measured using human GDNF ELISA kit (Abcam) according to the manufacturer's instructions. After organoid treatment, the medium was taken out and centrifuged to furnish the supernatant. The protein level of 5-HT in the medium was measured using Serotonin ELISA kit (Abcam) according to the manufacturer's instructions. Samples and standards were added in duplicate to 96-well ELISA plates.

| Immunofluorescence staining
The slides were incubated overnight at 4°C with the primary antibody, washed in PBS and incubated with secondary antibodies for 1.5 hours at room temperature. This was followed by washing with PBS and mounting with Vectashield-DAPI mounting medium. Primary antibodies used in our experiment were as follows:

| qPCR
Total RNA was extracted from organoid using the RNAprep Pure Cell/Bacteria Kit (TIANGEN, Beijing, China) according to the manufacturer's protocol. Similarly, total RNA of tissue specimens was extracted using RNAprep pure Tissue Kit (TIANGEN) and cDNA was synthesized using ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan).

All reactions were run in triplicate on an Applied Biosystems StepOne
Real-Time PCR System (Thermo, Waltham, MA, USA). The primer sequences used for real-time PCR analysis are listed in Table 1.

| Western blot
Total proteins were extracted and quantified. Protein was separated by 10% SDS-PAGE and transferred onto a PVDF membrane. The membrane was incubated with primary antibody at 4°C overnight, then incubated with the secondary antibody for 1 hour at room temperature. The immunoblots were detected by an enhanced chemiluminescent substrate (Millipore). Antibodies: anti-RET antibody

| Statistical analysis
Branching coefficient was assessed according to previous described Results were analysed using Graphpad prism 5.0c (GraphPad Software, La Jolla, CA, USA). Univariate analysis of the characteristics of the population used the chi-square test. Differences between multiple groups were evaluated using one-way ANOVA. Student's t test or Mann-Whitney non-parametric test was applied to compare two groups. Correlations between the two parameters were assessed by Spearman rank correlation. Results were expressed as means ± SD. Differences were considered significant at P < .05.

| Demographics and clinical characteristics
Demographics and clinical characteristics of control subjects and patients with IBS-D are shown in Table 2. There was no significant difference in age, sex, or the body mass index (BMI) between the control group and IBS-D group. In IBS-D group, the anxiety subscale scores (control vs IBS-D: 2.3 ± 0.8 vs 4.8 ± 1.7, P < .05) and depression subscale scores (control vs IBS-D: 2.6 ± 1.2 vs 4.1 ± 1.8, P < .05) were significantly higher.

| Acute adult stress induces proliferation of EC cells in mice colon
To investigate the potential role of GDNF in acute stress-related disorders in the intestine, we adopted a WRS model which was an adequate model to mimic part of the main symptoms of IBS, such as pain and colonic dysmotility. In WRS mice, a statistically significant increase in the AWR score was shown in volume 0.06 mL and 0.08 mL, P < .001. WRS treatment generated a sharper curve compared to control, and reached score 4 at small balloon volume, indicating a higher colorectal sensitivity ( Figure 3A). WRS significantly increased the expression of GDNF in the colon. The expression of RET in the colon was also upregulated by WRS ( Figure 3E).
To investigate whether GDNF could induce colorectal hypersensitivity, we treated mice with recombinant GDNF intraperitoneally.
Compared with saline controls, recombinant GDNF treatment caused higher visceral sensitivity ( Figure 3B). As hyperplasia of EC cells was found in IBS patients, we examined the density of EC

| GDNF-RET promotes intestinal stem cell proliferation and differentiation during acute adulthood stress
RET has been previously reported to promote maturation and Wnt signalling in developing mouse intestinal epithelium. 6 We speculated Furthermore, GSK3179106 administration reduced the expression levels of Axin2, β-catenin, Sox9 and CD44 ( Figure 3C-E). Put all together, these data indicated that GDNF amplifies Wnt/β-catenin signal via RET in the colon epithelium.
GDNF influences β-cell proliferation by enhancing Ngn3 expression in the pancreas, 15 and Ngn3 is required for EC cell fate specification in intestine. 28 We then investigated whether GDNF/RET axis also regulates EC cell differentiation in the intestine. As shown in Figure 3C-E, the expression of Ngn3 was increased in WRS-and GDNF-treated mice. The Ngn3 expression was inhibited by the treatment with GSK3179106, suggesting that GDNF-induced stem cell differentiation was mainly mediated by RET ( Figure 3C-E).

| GDNF-RET promotes maturation of ISC
In vitro, organoids from small intestine and colon were exposed to GDNF and RET inhibitor. The increased proliferation of ISC induced by GDNF/RET was evidenced by the increased branching efficiency and the size in GDNF-treated organoids, and their reduced branching and size in GSK3179106-treated organoids ( Figure 6A-C). We further examined the distribution of β-catenin by immunofluorescence, GDNF-treated organoids have increased levels of β-catenin, while GSK3179106 prevented the increase in both small intestinal and colonic organoids ( Figure 6D). Intriguingly, treatment with GSK3179106 decreased the numbers of buds and was more likely for organoids to forming a sphere. Since GDNF promotes the specification of EC cell in vivo, we measured the 5-HT in supernatants of organoids stimulated with different concentrations of GDNF.
The increase in 5-HT was observed in 100 nmol/L GDNF-treated colonic organoids and reduced by pre-incubation of GSK3179106.

| D ISCUSS I ON
Epithelial barrier defects are observed in IBS-D patients with associated tight junction alterations. 29 GDNF affects the apical junction complex, resulting in the maturation of epithelial barrier function by autocrine and paracrine regulatory mechanisms. 30 GDNF also has a strong anti-apoptotic effect on colonic epithelial cells and is increased in mucosa of inflammatory bowel disease (IBD) patients. 31 In this study, we demonstrated that GDNF expression was increased in IBS-D patients. Although the enteric glial cells (EGC) are well established as the major resource for GDNF production, 32  Stress has long been considered as an important factor for the onset and exacerbation of IBS, which is manifested as psychiatric disorders such as anxiety and depression and pain. In our study, elevated pre-incubation abrogated such effect, indicating that activation of the 5-HT system was mediated by RET kinase. Interestingly, the GDNF treatment did not affect the activation of 5-HT and TPH1 expression in small intestinal organoids. This regional specificity has also been shown with microbiota-induced 5-HT biosynthesis, which occurs in colonic, but not small intestinal EC cells. 36 As the differential expression of specific nutrient transporters and receptors in small intestinal and colonic EC cells, 37 the ability of EC cells in sensing stimulation may vary along the GI tract.
Wnt signalling has recently been found to be crucial for proliferation, differentiation, and migration of the stem cells 38 and is crucial in intestinal organoids culture. 39,40 In APC min mice where Wnt signalling is constitutively active, the number of EC cells in colon is significantly increased. 41 A recent study proposes that there is positive feedback between RET and Wnt signalling in Drosophila and GDNF upregulated Axin2 expression in mice colonic organoids. 6 F I G U R E 6 GDNF-RET induced ISC self-renew in colonic organoids. A, Representative images of small intestinal and colonic organoids stimulated by GDNF and GDNF with GSK3179106 pre-incubation. B, Surface area of organoids in (A). C, Branching quantifications of organoids in (A). D, Immunofluorescence of β-catenin (green) in small intestinal and colonic organoids stimulated by GDNF and GDNF with GSK3179106 pre-incubation. The data were displayed as mean ± SD; *P < .05, **P < .01 and ***P < .001 Consistently, we showed that GDNF-RET amplified Wnt signalling and increased ISC expansion. Furthermore, GDNF-RET likely promoted ISC differentiation to enteroendocrine cell line by regulating Ngn3 and NeuroD. All together, these data suggest that GDNF-RET may participate in expansion of colonic ISC via Wnt signalling.
In conclusion, our study demonstrated the roles of GDNF-RET axis in regulation of ISC self-renewal state and differentiation, which plays an important role in the pathophysiology of IBS-D. EC cell might be a potential cell for ISC niche. Further investigation into the role of RET in visceral hypersensitivity may provide a novel potential therapeutic target for treatment of patients with IBS.

ACK N OWLED G EM ENT
This study is supported by the National Natural Science Foundation

CO N FLI C T O F I NTE R E S T
All authors declare no conflicts of interests. F I G U R E 7 GDNF-RET regulated epithelial maturation in colonic organoids. A, Effects of GDNF and RET inhibitor GSK3179106 on the 5-HT secretion in small intestinal and colonic organoids. GDNF concentrations were 50, 100 and 200 nmol/L. GSK3179106 were pre-incubated 1 h before GDNF stimulation. The 5-HT levels in intestinal organoids supernatant. Data were normalized to 5-HT levels in Control group. B, The Tph1, Ngn3, NeuroD, Axin2, Lgr5, Sox9 and Cd44 mRNA expression in small intestinal organoids. C, The Tph1, Ngn3, NeuroD, Axin2, Lgr5, Sox9 and Cd44 mRNA expression in colonic organoids. The data were displayed as mean ± SD; *P < .05, **P < .01 and ***P < .001

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.