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ARTICLE OPEN Deficiency in intestinal epithelial Reg4 ameliorates intestinal inflammation and alters the colonic bacterial composition

Yongtao Xiao1,2,3, Ying Lu2,3, Ying Wang1,3, Weihui Yan1,3 and Wei Cai1,2,3

The regenerating islet-derived family member 4 (Reg4) in the gastrointestinal tract is up-regulated during intestinal inflammation. However, the physiological function of Reg4 in the inflammation is largely unknown. In the current study, the functional roles and involved mechanisms of intestinal epithelial Reg4 in intestinal inflammation were studied in healthy and inflamed states using human intestinal specimens, an intestinal conditional Reg4 knockout mouse (Reg4ΔIEC) model and dextran sulfate sodium (DSS)-induced colitis model. We showed that the elevated serum Reg4 in pediatric intestinal failure (IF) patients were positively correlated with the serum concentrations of proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). In inflamed intestine of IF patients, the crypt base Reg4 protein was increased and highly expressed towards the luminal face. The Reg4 was indicated as a novel target of activating transcription factor 2 (ATF2) that enhanced Reg4 expression during the intestinal inflammation. In vivo, the DSS- induced colitis was significantly ameliorated in Reg4ΔIEC mice. Reg4ΔIEC mice altered the colonic bacterial composition and reduced the bacteria adhere to the colonic epithelium. In vitro, Reg4 was showed to promote the growth of colonic organoids, and that this occurs through a mechanism involving activation of signal transducer and activator of transcription 3 (STAT3). In conclusion, our findings demonstrated intestinal-epithelial Reg4 deficiency protects against experimental colitis and mucosal injury via a mechanism involving alteration of bacterial homeostasis and STAT3 activation. Mucosal Immunology (2019) 12:919–929; https://doi.org/10.1038/s41385-019-0161-5ARTICLE

INTRODUCTION interacted relationships between microbiota and the intestinal The regenerating islet-derived family member 4 (Reg4) is the most surface. recently discovered member of the Reg gene family.1–3 In human, In the present study, to unravel the roles and mechanisms of the Reg gene family members Reg1A, Reg1B, and Reg3A are found Reg4 in the intestinal inflammation, we initially performed encoded in tandem on chromosome 2p12, whereas Reg4 is population based cross-sectional study on Reg4 in relation to located on chromosome 1p12.4,5 Unlike Reg1A, Reg1B, and Reg3A inflammatory response in pediatric intestinal failure (IF) patients, that are derived from Paneth cells,6–9 the Reg4 is physiologically who usually have severe intestinal inflammation.27 In addition, we expressed in enteroendocrine cells and expands to epithelial cells generated mice lacking Reg4 specifically in intestinal epithelial during intestinal inflammation.10–14 Recent studies reported that cells (Reg4ΔIEC) to study the exact roles of Reg4 in intestinal Reg4 is overexpressed in several types of gastrointestinal tract (GI inflammation. tract) malignancies, indicating Reg4 might have a prognostic or a predictive value in cancers of the GI tract.15–20 Although the biological function of Reg4 in cancer of GI tract is still unclear, Reg4 RESULTS protein seems to act as growth factor in malignant cells.21,22 Reg4 is selectively expressed in the mucosa of uninflammed Additionally, Reg4 is also strongly up-regulated during intestinal intestine inflammation,23,24 but the functional roles of Reg4 as well as the Under normal states, Reg4 expressed at the mucosa of mouse regulation of Reg4 expression in intestinal inflammation remained proximal (pro), middle (mid), distal (dis) small bowel and colon elusive. In intestine, the accumulation of several Paneth and (Fig. 1a–c). The expression of Reg4 mRNA was higher in the colon epithelial cell-derived antimicrobial peptides and proteins (AMP) is then in the small intestine (Fig. 1a). The colorimetric in situ vital to maintain the immune homeostasis via avoiding coloniza- hybridization (CISH) assay and immunofluorescence (IF) staining tion of the epithelial cell surface and invasion by opportunistic showed that Reg4 protein exclusively expressed in intestinal pathogens.25 One key human antimicrobial protein is Reg3α, mucosa under normal condition, mainly presenting at the base- which has been reported to be bactericidal via binding to ment of crypts (Fig. 1b, c). Consistent with this finding, the real-time peptidoglycan of Gram-positive bacteria.6,26 It is reminded that PCR (RT-PCR) showed that Reg4 mRNA was also strongly expressed Reg4 mayRETRACTED be involved in intestinal inflammation via alteration of in mucosa of human jejunum, Ileum, and colon (Fig. 1d, e).

1Department of Pediatric Surgery, Xin Hua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; 2Shanghai Institute of Pediatric Research, Shanghai, China and 3Shanghai Key Laboratory of Pediatric Gastroenterology and Nutrition, Shanghai, China Correspondence: Yongtao Xiao ([email protected]) or Wei Cai ([email protected]) Received: 9 October 2018 Revised: 26 February 2019 Accepted: 11 March 2019 Published online: 5 April 2019

© The Author(s) 2019 Deficiency in intestinal epithelial Reg4 ameliorates intestinal. . . Y Xiao et al. 920 1234567890();,: ARTICLE

Fig. 1 Reg4 predominantly expressed in intestine of mice and human. a Quantification of Reg4 mRNA in the mouse liver, stomach, pancreas, proximal (pro), middle (mid), distal (dis) small bowel and colon. b Colorimetric in situ hybridization (CISH) analysis for Reg4 in mouse proximal (pro), middle (mid), distal (dis) small bowel and colon. c Immunofluorescence staining for Reg4 in mouse proximal (pro), middle (mid), distal (dis) small bowel and colon. d Real time-PCR (RT-PCR) analysis for Reg4 in jejunum, ileum and colon of intestinal failure (IF) patients. e Representative images of the DNA agarose gels of panel d

Reg4 is increasingly expressed in intestinal epithelial cells during Reg4 protein expression markedly increased in epithelial surface of inflammation inflamed mucosa (Fig. 3a). Furthermore, Reg4 co-localized with the When mice challenged with 2% dextran sulfate sodium (DSS), the goblet cell marker MUC2 and the phosphorylated STAT3 (p-STAT3, Reg4 mRNA increased significantly in the mucosa of colon (Fig. 2a). Tyr705) in the inflamed mucosa of patients (Fig. 3b, c). In mice given DSS, Reg4 expanded from crypt base to the epithelial cells (Fig. 2b). Consistent with the findings in Reg4 Activating transcription factor 2 (ATF2) enhances Reg4 expression mRNA, the Reg4 protein was evidently increased in the mucosa of during intestinal inflammation mice with DSS treatment (Fig. 2c). In pediatric IF patients, serum During the DSS induced intestinal inflammation, Western blot Reg4 levels were significantly higher [n = 40, 5.441 ng/ml analysis showed that the levels of activated ATF2 (phosphory- (1.626–24.06), p < 0.001] than in age-matched healthy controls lated-ATF2, p-ATF2, Thr71) increased evidently in colonic [n = 16,RETRACTED 0.284 ng/ml (0.082–.910)] using ELISA analysis (Fig. 2d). mucosa (Fig. 4a). Consistently, immunohistochemistry (IHC) The proinflammatory cytokines interleukin-6 (IL-6) and tumor staining indicated that the p-ATF2-positive cells were markedly necrosis factor-α (TNF-α) were also increased significantly in serum increased after DSS administration (Fig. 4b). Using bioinforma- of pediatric IF patients when compared to controls (Fig. 2e, f). In tive software, it predicted that there was a binding site for ATF2 addition, serum Reg4 protein levels were positively correlated with (CTCTGAGGAAACTC) located at 1 kb region upstream of Reg4 the concentrations of serum IL-6 (r = 0.48, p < 0.001) and serum transcription start site (TSS) (Fig. 4c). Intestinal cells Caco2 TNF-α (r = 0.63, p < 0.001) (Fig. 2g, f). In uninflammed areas of transfected with Reg4 promoter-luciferase vectors and patients’ intestine, the Reg4 protein was selectively expressed at ATF2 siRNA for 72 h were treated with lipopolysaccharide (LPS, crypts or villus of jejunum, ileum, and colon (Fig. 3a). In contrast, 100 μg/mL) for 30 min. As shown Fig. 4d, Caco2 cells treatment

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Fig. 2 Reg4 levels are correlated with intestinal inflammation. a Quantification of Reg4 mRNA in mouse colon with or without DSS treatment using RT-PCR assay. b Colorimetric in situ hybridization (CISH) analysis for Reg4 in mouse colon. c The Reg4 protein levels were determined in mouse colon using western blot. d–f The levels of serum Reg4, IL-6 and TNF-α were determined in healthy controls (HC, n = 16) and children with intestinal failure (IF, n = 40) using the enzyme linked immunosorbent assay (ELISA). (g, h) The serum Reg4 levels were correlated with serum IL-6 and TNF-α.**p < 0.01, ***p <0.001 with LPS resulted in a 25-fold induction of Reg4 promoter During DSS treatment, Reg4ΔIEC mice exhibited less body weight activity (Fig. 4d). The ATF2 knockdown significantly counteracted loss than Reg4fl/fl mice (Fig. 5a). Histologically, Reg4ΔIEC mice had the LPS-induced Reg4 promoter activity (Fig. 4d). Consistently, less colonic mucosal damage and decreased inflammatory Reg4 mRNA and protein were reduced after ATF2 depletion infiltrates compared to DSS-treated Reg4fl/fl mice (Fig. 5b, c). IHC (Fig. 4e–g). analysis indicated Reg4ΔIEC mice exhibited had lower expression of Muc2, Ki67, and p-STAT3 in colonic mucosa (Fig. 5c, d). Indeed, Intestinal epithelial Reg4 deficiency protects against DSS-induced western-blots analysis revealed the levels of PCNA, Lgr5, and p- colitis STAT3 reduced evidently in colon of Reg4ΔIEC mice with DSS To study the potential roles of Reg4 in intestinal inflammation, we treatment (Fig. 5e). In agreement with histological findings, the generated mice lacking Reg4 specifically in intestinal epithelial inflammatory genes, including Il6, Il1b, Infg, Tnfa, Ccl28, Cx3cl1, and cells (Reg4ΔIEC) by crossing mice carrying loxP-flanked (floxed, fl) Il22, were significantly reduced in colonic mucosa of Reg4ΔIEC mice Reg4 alleles (Reg4fl/fl) mice with villin-Cre transgenics (Supplemen- following DSS-treatment compared to DSS-treated Reg4fl/fl mice tary Fig. 1). As shown in Supplementary Fig. 2, the average body (Fig. 6). Moreover, the ISC genes, including Lgr5, Axin2, Olfm4, and weight, villus height and crypt number in Reg4ΔIEC mice did not also proliferative gene Ki67 decreased significantly in colon of obviouslyRETRACTED differ from the Reg4fl/fl littermates (Supplementary DSS-treated Reg4ΔIEC mice (Fig. 6). The DSS-treated Reg4ΔIEC Fig. 2A–C). IHC staining indicated Reg4ΔIEC mice had fewer cells mice also had a reduction in levels of goblet cell maker, that stained positive with Muc2, Ki67 or p-STAT3 than Reg4fl/fl mice Muc2, enteroendocrine gene, Chga and enterocyte marker, Alpi (Supplementary Fig. 2C and 2D). In colonic epithelium, transmis- (Fig. 6). sion electron microscopy (TEM) showed Reg4ΔIEC mice had immature enterocyte cells with having numerous brush borders Intestinal epithelial Reg4 deficiency alters the colonic bacterial (Supplementary Fig. 2E). The RT-PCR analysis showed the mRNA composition levels of Muc2, Ki67, Lgr5 and especially Alpi were decreased in Given that Reg4 is a member of the calcium-dependent (C-type) colon of Reg4ΔIEC mice (Supplementary Fig. 2f). lectin superfamily, we further study its roles in the commensal

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Fig. 3 Intestinal Reg4 increased in inflamed mucosa of IF patients. a Immunofluorescence staining for Reg4 in the uninflammed and inflamed intestine of IF patients (n = 8 each group) (b, c). Reg4 and Muc2 co-staining, and Reg4 and p-STAT3 co-staining were analyzed in mucosa of patients (n = 6 each group)

microbiota homeostasis. We initially homogenized colonic tissues treatment ameliorated the colitis in DSS-treated Reg4ΔIEC mice and feces from Reg4fl/fl and Reg4ΔIEC mice, and cultured on agar (Supplementary Figs. 4 and 5). Mice treated with vancomycin plates for 16 h. As shown in Supplementary Fig. 3A, the number of (VCM), which is active against Gram-positive bacteria, resulted in colony-forming units (CFU) in colon tissues and in feces of Reg4ΔIEC increased numbers of fecal Lactobacillus and Escherichia (Fig. 7a). mice did not evidently differ from Reg4fl/fl mice (Supplementary VCM treatment did not ameliorated DSS-induced colitis in Reg4ΔIEC Fig. 3A). The fluorescence in situ hybridization (FISH) using a mice (Supplementary Figs. 4 and 5). universal bacterial 16S ribosomal RNA (rRNA) probe and co- stained with Reg4 showed that presence of intestinal bacteria Reg4 knockdown reduces bacterial adhering to the colonic overlapped or adjacent to Reg4 protein in inflamed intestine of epithelial cells Reg4fl/fl mice (Supplementary Fig. 3B). In Reg4ΔIEC mice, bacteria In this study, we further address the mechanisms by which Reg4 were not observed adjacent to epithelial cell layers (Supplemen- affected bacterial homeostasis. The fecal suspensions from tary Fig.RETRACTED 3B). We further isolated DNA from the colonic tissues and Reg4ΔIEC mice or Reg4fl/fl mice were incubated with or without fecal content and analyzed for the presence of bacteria by PCR recombinant Reg4 protein. After removing larger particles, the using bacterial genus-specific primers. It showed that the numbers bacterial suspensions were added to cultures of polarized Caco2 of several bacterial genera were decreased in colonic tissues of cells with or without Reg4 siRNA transfection, and after 8 h the Reg4ΔIEC mice, particularly the Prevotella, Escherichia and Lactoba- bacteria that had attached to Caco2 cells were analyzed (Fig. 7b). cillus (Supplementary Fig. 3C). Mice were then orally treated with As shown in Fig. 7c, the fecal suspensions from Reg4ΔIEC mice had GM, which is active against Gram-negative bacteria. Reg4ΔIEC mice more Proteus, Lactobacillus, Bifidobacterium, Staphylococcus, and treated with GM had decreased numbers of Prevotella, Escherichia, Bacteroides adherence to Caco2 cells (Fig. 7c). Reg4 knockdown in Helicobacter, and Proteus in feces (Fig. 7a). In addition, GM Caco2 cells or pretreatment with recombinant Reg4 protein

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Fig. 4 ATF2 promotes Reg4 transcription in during the intestinal inflammation. a Western blot analysis for p-ATF2 and ATF2 in the colonic mucosa with or without DSS treatment. b Representative images of the immunohistochemistry (IHC) staining of p-ATF2. c The binding motif of ATF2 on the promoter of Reg4. a The binding motif of ATF2 on the promoter of Reg4. c–f The promoter activity, mRNA and protein of Reg4 were reduced by ATF2 knockdown. C, The promoter activity was analyzed by luciferase reporter system. D, The mRNA levels were determined by RT-PCR. E, Western blot analysis for Reg4 and ATF2. F, Immunofluorescence staining for Reg4 and ZO1. *p < 0.05, **p < 0.01 reduced the Proteus, Lactobacillus, Bifidobacterium, Staphylococcus, DISCUSSION and Bacteroides bound to Caco2 cells (Fig. 7c). Reg4, a newly discovered member of the Reg gene family, was firstly isolated from a cDNA library of ulcerative colitis (UC) Reg4 promotes growth of colonic organoids via activation of tissues,3 implying that the Reg4 may play some roles in the STAT3 intestinal inflammation. In this study, we demonstrated that in the Since the above data show the Reg4 is critical to colitis, we next normal crypt base Reg4 protein expands to epithelial cells may investigate its roles and mechanisms in colonic regeneration. due to ATF2 activation during intestinal inflammation. The Consistent with findings in vivo (Fig. 5), we found that the number population-based cross-sectional study indicated that the levels of colonic organoids from Reg4ΔIEC mice evidently less than the of serum Reg4 reflect the presence and the degrees of intestinal ones from Reg4fl/fl mice (Fig. 8a). After 3 days of recombinant Reg4 inflammation in pediatrics with IF. In DSS-induced murine colitis, protein treatment, the colonic organoids increased significantly intestinal-specific Reg4 deficiency protects intestinal inflamma- compared to untreated ones (Fig. 8a). In line with increased tion and alters the bacterial composition. number ofRETRACTED organoids, Reg4 treatment enhanced EdU incorpora- In normal intestine, other investigators and we showed that tion in colonic organoids (Fig. 8b). Given found that Reg4 Reg4 protein was selectively expressed at crypts or villus at low increased phosphorylation of STAT3 (Y705), we further evaluated levels, which was consistent with it’s a marker of enteroendocrine STAT3 in Reg4-induced growth of colonic organoids. As shown cells.14,23 In inflamed mucosa of intestine, Reg4 protein markedly in Fig. 8a, b, the treatment with the STAT3 inhibitor Stattic increased in outer parts of crypts and in a surface pattern. In successfully abrogated Reg4-mediated the growth of colonic addition, serum Reg4 protein levels were significantly higher in IF organoids (Fig. 8a, b). We also showed that recombinant Reg4 patients and positively correlated with cytokines IL-6 and TNF-α, protein increased the expression of Lgr5 and Ki67, and this effect indicating that serum Reg4 could reflect the degrees of intestinal was blocked by Stattic treatment (Fig. 8c, d). inflammation. Up to date, the mechanisms involved in regulating

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Fig. 5 Intestinal-epithelial Reg4 deficiency ameliorated DSS-mediated histological changes. a The body weight was monitored in DSS-treated Reg4ΔIEC mice and DSS-treated Reg4fl/fl mice. b Quantification of histological scores. c Hematoxylin and eosin (H&E) staining, Alcian blue/ periodic acid Schiff base (AB-PAS) staining and Immunohistochemical analysis with Muc2, Ki67 and p-STAT3 antibodies in Reg4ΔIEC mice and Reg4fl/fl mice (n = 8 per group). (d) Quantification of Immunohistochemical analysis for Ki67 and p-STAT3. (G) The colonic proteins Reg4, p- STAT3, STAT3, Lgr5 and PCNA were analysed in DSS-treated Reg4ΔIEC mice and DSS-treated Reg4fl/fl mice. *p < 0.05, **p < 0.01

Reg4 expression during the intestinal inflammation remains To study the exact roles of Reg4 in intestinal inflammation, we elusive. It reported that other Reg members Reg1 and Reg3 conditionally knockout intestinal Reg4 in mice (Reg4ΔIEC) and expression could be enhanced by several cytokines, such as IL- challenged with a DSS for 10 days. We demonstrated that Reg4fl/fl 6.28–30 In contrast to Reg1 or Reg3 expression, proinflammatory mice exhibited profound weight loss, increased disease severity, cytokines had none stimulatory effect on Reg4 gene expression in increased proinflammatory genes expression, and extensive the human colon cancer cell line.24 It thus suggests that the Reg4 intestinal ulceration, loss of crypt architecture, while Reg4ΔIEC gene expression is cannot be directly stimulated by cytokines. As mice were minimally affected, indicating that Reg4 deficiency is reported previously, Reg4 is a transcriptional target of GATA6 in critical for limiting DSS-induced intestinal damage and inflamma- colon cancer cells.31 It has also been reported that Reg4 is a direct tion. Dysregulation of intestinal homeostasis and susceptibility to target ofRETRACTED the intestinal transcriptional factor CDX2.32 In the present intestinal inflammation are often associated with alterations in study, we identified Reg4 as a novel target gene of ATF2, a commensal bacterial populations.39–41 Recent studies have shown member of the leucine zipper family of DNA-binding proteins,33,34 that other members of the Reg family, such as Reg3, have which has been shown to play an important role in many of the protective effects against experimental colitis via its antimicrobial inflammatory responses.35,36 It reported that GATA6 acts in effects. The murine Reg3γ has antibacterial activities against combination with other transcriptional factors, including TCF437 Gram-positive bacteria by interacting with peptidoglycan carbo- and CDX2,38 to stimulate or repress Reg4 gene expression. We hydrate.6 Reg3β exerts bactericidal activity against Gram-negative thus proposed that the Reg4 might be regulated by ATF2 and/or bacteria by binding to lipopolysaccharide.42,43 Unlike Reg3 showed with other co-factors in the inflamed intestine. an apparently paneth-cell-dependent expansion from the colonic

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Fig. 6 Reg4 deficiency inhibited DSS-induced colon inflammation. The genes for Il6, Il1b, Infg, Tnfa, Ccl28, Cx3cl1, Cxcl2, Il22, Lgr5, Axin2, Olfm4, Ki67, Muc2, Lysozyme, Chga, and Alpi were determined in colon of Reg4ΔIEC mice and Reg4fl/fl mice with or without DSS-treatment using RT-PCR analysis (n = 8 per group). *p < 0.05, **p < 0.01 crypts during inflammation,44 whereas we showed that Reg4 was In response to inflammation-induced mucosal injury, epithelial expressed in enteroendocrine cells and expanded to epithelial regeneration is critical for barrier maintenance and organ function. cells of the upper colonic crypts during inflammation, suggests The intestinal stem cell (ISC) niche provides Wnt, Notch, and that Reg4 may have different effects on microbiota composi- epidermal growth factor (EGF) signals supporting Lgr5-positive tion. Indeed, we demonstrated that several bacteria, including crypt base columnar ISCs for epithelial maintenance.45,46 Sasaki Prevotella, Escherichia, and Lactobacillus, decreased in colonic et al.47 recently reported that Reg4 could promote organoids tissues of Reg4ΔIEC mice, and Reg4ΔIEC mice had less bacteria formation of single Lgr5-positive colon stem cells, but involved presenting adjacent to the epithelial cell layers. Mice were then mechanisms remained not fully understood. The signal transducer orally treated with gentamicin (GM), which is active against Gram- and activator of transcription 3 (STAT3) has been reported to negative bacteria, decreased numbers of Prevotella, Escherichia, promote ISCs survival and ISCs-mediated epithelial regenera- Helicobacter, and Proteus, and, accordingly, no bacteria were tion.48,49 In vivo, we showed that Reg4 deficiency significantly present just above the intestinal epithelial layers. Additionally, GM inhibited DSS-induced activation of STAT3 in colonic muscoa. In treatment ameliorated the intestinal inflammation in DSS-treated vitro, the recombinant Reg4 protein treatment promoted the Reg4ΔIEC mice,RETRACTED suggests that Reg4 deficiency decreased mucus growth of colonic organoids, but this effect was successfully penetration by Gram-negative bacteria such as Proteus, Escher- abrogated by treatment of STAT3 inhibitor. Two key cytokines are ichia, and Helicobacter and reduced sensitivity to intestinal IL6 and IL22, which activate their respective receptors, followed by inflammation. Furthermore, Reg4 knockdown in Caco2 cells phosphorylation and activation of STAT3 in intestine to promote significantly reduced the bacteria, including Proteus, Bacteroides, their regeneration or growth.50,51 We here demonstrated that Lactobacillus, Bifidobacterium, and Staphylococcus, attaching to the several proinflammatory cytokines, including IL6 and IL22, cells. Given that Reg4 had a carbohydrate recognition domain reduced significantly in colon of DSS-treated Reg4ΔIEC mice. Thus, (CRD),3,23 which suggests that CRD might be essential to Reg4 it suggests that inflammation-induced Reg4 may promote the bounding the bacteria. colon regeneration via IL6/22-STAT3 signaling.

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Fig. 7 Intestinal Reg4 potentially affects the intestinal bacteria homeostasis. a Quantitative PCR of bacterial DNA isolated from 1 mg of feces from untreated, gentamicin (GM)-treated and vancomycin (VCM)-treated mice. Data show the bacterial DNA amounts compared to untreated Reg4fl/fl mice group (n = 8 per group). b Schema of binding assay of fecal bacteria. c Bound analysis for fecal bacteria attached to the Caco2 cells with or without Reg4 expression. *p <0.05, **p < 0.01

Taken together, our studies demonstrate that Reg4 is highly DSS-induced colitis experiments. Acute colitis was induced by expressed towards the intestinal luminal face during inflamma- administration of 2% DSS (36–50 kDa) in the drinking water for tion, and ATF2 plays transcriptional role in the inflammation- 10 days. The changes in weight were monitored each day. All mediated Reg4 expression. Intestinal-specific depletion of Reg4 animal experiments were performed according to guidelines of ameliorates DSS-induced colonic inflammation and injury the Institutional Animal Care and Use Committee of the Xin hua might through decreasing bacterial aggregating onto the hospital, School of Medicine, Shanghai Jiao Tong University. intestinal epithelial cells, thus reducing the risk of inflamma- tion. During the colonic mucosal regeneration, Reg4 enhances Statistical analysis the growth of organoids via activating the STAT3 signaling The statistics are presented as mean ± SD. In human population, (Fig. 9).RETRACTEDthe Kolmogorov-Smirnov test was used to assess distributions. Abnormally distributed data were logarithmically transformed before analysis. Correlations between serum Reg4 and serum IL6 MATERIALS AND METHODS or TNFα were tested by Spearman rank correlation test. Mann Animal experiments Whitney U-test, Fisher exact test or one-way ANOVA were used to To generate intestinal-specific Reg4 deficiency mice, the Reg4- compare differences between groups. The level of statistical floxed alleles (Reg4fl/fl) were bred with Villin-cre+ mice to significance was set at 0.05. generated Reg4 conditional knockout mice (Reg4ΔIEC). Six-week- Detailed protocols are provided in the Supplementary Materials old Reg4ΔIEC mice and their littermate Reg4fl/fl mice were used for and Methods.

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Fig. 8 Reg4 promotes growth of colonic organoids dependent on STAT3 activation. a The number of colonic organoids was calculated following incubation with or without Reg4 protein for 3 days. b EdU staining and its quantification. c, d Levels of Lgr5 mRNA and Ki67 mRNA were determined in colonic organoids with RT-PCR. *p < 0.05, **p < 0.01

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Fig. 9 Schema of that Reg4 was involved in intestinal inflammation and regeneration. The activated ATF2 enhanced the Reg4 expression during the intestinal inflammation. The intestinal epithelial Reg4 bounded bacteria and induced the colitis. During the inflammation inducing- colonic injuries, Reg4 increased the regeneration via activating the IL6/22-STAT3 signaling

Mucosal Immunology (2019) 12:919 – 929 Deficiency in intestinal epithelial Reg4 ameliorates intestinal. . . Y Xiao et al. 928 ACKNOWLEDGEMENTS 18. Ohara, S. et al. Reg IV is an independent prognostic factor for relapse in We thank Yinuo Xu, Ping Wu, Chuanqi Zhao and Beilin Gu for the administrative, patients with clinically localized prostate cancer. Cancer Sci. 99, 1570–1577 technical, or material support. (2008). 19. Eguchi, H. et al. Serum REG4 level is a predictive biomarker for the response to preoperative chemoradiotherapy in patients with pancreatic cancer. Pancreas 38, AUTHOR CONTRIBUTIONS 791–798 (2009). Y.X. and W.C. accomplished the study concept and design, acquisition of data, 20. Oue, N. et al. Serum concentration of Reg IV in patients with colorectal cancer: analysis and interpretation of data, obtained funding and drafting of the manuscript; overexpression and high serum levels of Reg IV are associated with liver – Y.X., Y.W., and Y.L. performed most of the experiments. Y.L. and W.Y. gave the metastasis. Oncology 72, 371 380 (2007). administrative, technical, or material support. This study was supported by National 21. Bishnupuri, K. S. et al. Reg IV activates the epidermal growth factor receptor/Akt/ Natural Science Foundation of China (81770517 and 81630039), Shanghai Key AP-1 signaling pathway in colon adenocarcinomas. Gastroenterology 130, – Laboratory of Pediatric Gastroenterology and Nutrition (17DZ2272000) and Research 137 149 (2006). Funding of Shanghai Health and Family Planning Commission (201640153). 22. Rafa, L. et al. REG4 acts as a mitogenic, motility and pro-invasive factor for colon cancer cells. Int. J. Oncol. 36, 689–698 (2010). 23. Kamarainen, M. et al. 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