Intestinal Stem Cell Expansion Research Articles

The gut microbial metabolite indole-3-aldehyde alleviates impaired intestinal development by promoting intestinal stem cell expansion in weaned piglets

BackgroundWeaning stress-induced diarrhea is widely recognized as being associated with gut microbiota dysbiosis. However, it has been challenging to clarify which specific intestinal microbiota and their metabolites play a crucial role in the antidiarrhea process of weaned piglets.ResultsIn this study, we first observed that piglets with diarrhea exhibited a lower average daily gain and higher diarrhea score, and elevated levels of lipopolysaccharide (LPS) and D-lactate (D-LA) compared to healthy piglets. Subsequently, we analyzed the differences in intestinal microbial composition and metabolite levels between healthy and diarrheal weaned piglets. Diarrheal piglets demonstrated intestinal microbiota dysbiosis, characterized primarily by a higher Firmicutes to Bacteroidota ratio, a deficiency of Lactobacillus amylovorus and Lactobacillus reuteri, and an increased abundance of Bacteroides sp.HF-5287 and Bacteroides thetaiotaomicron. Functional profiling of the gut microbiota based on Kyoto Encyclopedia of Genes and Genomes (KEGG) data was performed, and the results showed that tryptophan metabolism was the most significantly inhibited pathway in piglets with diarrhea. Most tryptophan metabolites were detected at lower concentrations in diarrheal piglets than in healthy piglets. Furthermore, we explored the effects of dietary indole-3-aldehyde (IAld), a key tryptophan metabolite, on intestinal development and gut barrier function in weaned piglets. Supplementation with 100 mg/kg IAld in the diet increased the small intestine index and improved intestinal barrier function by promoting intestinal stem cell (ISC) expansion in piglets. The promotion of ISC expansion by IAld was also confirmed in porcine intestinal organoids.ConclusionsThese findings revealed that intestinal microbial tryptophan metabolite IAld alleviates impaired intestinal development by promoting ISC expansion in weaned piglets.

Early Weaning Inhibits Intestinal Stem Cell Expansion to Disrupt the Intestinal Integrity of Duroc Piglets via Regulating the Keap1/Nrf2 Signaling.

There are different stress resistance among different breeds of pigs. Changes in intestinal stem cells (ISCs) are still unclear among various breeds of piglets after early weaning. In the current study, Taoyuan Black and Duroc piglets were slaughtered at 21 days of age (early weaning day) and 24 days of age (3 days after early weaning) for 10 piglets in each group. The results showed that the rate of ISC-driven epithelial renewal in local Taoyuan Black pigs hardly changed after weaning for 3 days. However, weaning stress significantly reduced the weight of the duodenum and jejunum in Duroc piglets. Meanwhile, the jejunal villus height, tight junction-related proteins (ZO-1, Occludin, and Claudin1), as well as the trans-epithelial electrical resistance (TEER) values, were down-regulated after weaning for 3 days in Duroc piglets. Moreover, compared with Unweaned Duroc piglets, the numbers of Olfm4+ ISC cells, PCNA+ mitotic cells, SOX9+ secretory progenitor cells, and Villin+ absorptive cells in the jejunum were reduced significantly 3 days after weaning. And ex vivo jejunal crypt-derived organoids exhibited growth disadvantages in weaned Duroc piglets. Notably, the Keap1/Nrf2 signaling activities and the expression of HO-1 were significantly depressed in weaned Duroc piglets compared to Unweaned Duroc piglets. Thus, we can conclude that ISCs of Duroc piglets were more sensitive to weaning stress injury than Taoyuan Black piglets, and Keap1/Nrf2 signaling is involved in this process.

Indole-3-aldehyde Alleviates High-Fat Diet-Induced Gut Barrier Disruption by Increasing Intestinal Stem Cell Expansion.

High-fat diet (HFD) feeding is known to cause intestinal barrier disruption, thereby triggering severe intestinal inflammatory disease. Indole-3-aldehyde (IAld) has emerged as a potential candidate for mitigating inflammatory responses and maintaining intestinal homeostasis. However, the role of IAld in the HFD-related intestinal disruption remains unclear. In this study, 48 7 week-old male C57BL/6J mice were assigned to four groups: the normal chow diet (NCD) group received a NCD; the HFD group was fed an HFD; the HFD + IAld200 group was supplemented with 200 mg/kg IAld in the HFD; and the HFD + IAld600 group was supplemented with 600 mg/kg IAld in the HFD. The results showed that dietary IAld supplementation ameliorated fat accumulation and metabolic disorders, which are associated with reduced intestinal permeability. This reduction potentially led to decreased systemic inflammation and enhanced intestinal barrier function in HFD-fed mice. Furthermore, we found that IAld promoted intestinal stem cell (ISC) proliferation by activating aryl hydrocarbon receptors (AHRs) in vivo and ex vivo. These findings suggest that IAld restores the HFD-induced intestinal barrier disruption by promoting AHR-mediated ISC proliferation.

Mulberry Leaf-Derived Morin Activates β-Catenin by Binding to Frizzled7 to Promote Intestinal Stem Cell Expansion upon Heat-Stable Enterotoxin b Injury.

Intestinal stem cells (ISCs) sustain epithelial renewal by dynamically altering behaviors of proliferation and differentiation in response to various nutrition and stress inputs. However, how ISCs integrate bioactive substance morin cues to protect against heat-stable enterotoxin b (STb) produced by Escherichia coli remains an uncertain question with implications for treating bacterial diarrhea. Our recent work showed that oral mulberry leaf-derived morin improved the growth performance in STb-challenged mice. Furthermore, morin supplementation reinstated the impaired small-intestinal epithelial structure and barrier function by stimulating ISC proliferation and differentiation as well as supporting intestinal organoid expansion ex vivo. Importantly, the Wnt/β-catenin pathway, an ISC fate commitment signal, was reactivated by morin to restore the jejunal crypt-villus architecture in response to STb stimulation. Mechanically, the extracellular morin-initiated β-catenin axis is dependent or partially dependent on the Wnt membrane receptor Frizzled7 (FZD7). Our data reveal an unexpected role of leaf-derived morin, which represents molecular signaling targeting the FZD7 platform instrumental for controlling ISC regeneration upon STb injury.

Corticosteroids impair epithelial regeneration in immune-mediated intestinal damage.

Corticosteroid treatment (CST) failure is associated with poor outcomes for patients with gastrointestinal (GI) graft-versus-host disease (GVHD). CST is intended to target the immune system, but the glucocorticoid receptor (GR) is widely expressed, including within the intestines, where its effects are poorly understood. Here, we report that corticosteroids (CS) directly targeted intestinal epithelium, potentially worsening immune-mediated GI damage. CS administered to mice in vivo and intestinal organoid cultures ex vivo reduced epithelial proliferation. Following irradiation, immediate CST mitigated GI damage but delayed treatment attenuated regeneration and exacerbated damage. In a murine steroid-refractory (SR) GVHD model, CST impaired epithelial regeneration, worsened crypt loss, and reduced intestinal stem cell (ISC) frequencies. CST also exacerbated immune-mediated damage in organoid cultures with SR, GR-deficient T cells or IFN-γ. These findings correlated with CS-dependent changes in apoptosis-related gene expression and STAT3-related epithelial proliferation. Conversely, IL-22 administration enhanced STAT3 activity and overcame CS-mediated attenuation of regeneration, reducing crypt loss and promoting ISC expansion in steroid-treated mice with GVHD. Therefore, CST has the potential to exacerbate GI damage if it fails to control the damage-inducing immune response, but this risk may be countered by strategies augmenting epithelial regeneration, thus providing a rationale for clinical approaches combining such tissue-targeted therapies with immunosuppression.

Gut-liver axis calibrates intestinal stem cell fitness

The gut and liver are recognized to mutually communicate through the biliary tract, portal vein, and systemic circulation. However, it remains unclear how this gut-liver axis regulates intestinal physiology. Through hepatectomy and transcriptomic and proteomic profiling, we identified pigment epithelium-derived factor (PEDF), a liver-derived soluble Wnt inhibitor, which restrains intestinal stem cell (ISC) hyperproliferation to maintain gut homeostasis by suppressing the Wnt/β-catenin signaling pathway. Furthermore, we found that microbial danger signals resulting from intestinal inflammation can be sensed by the liver, leading to the repression of PEDF production through peroxisome proliferator-activated receptor-α (PPARα). This repression liberates ISC proliferation to accelerate tissue repair in the gut. Additionally, treating mice with fenofibrate, a clinical PPARα agonist used for hypolipidemia, enhances colitis susceptibility due to PEDF activity. Therefore, we have identified a distinct role for PEDF in calibrating ISC expansion for intestinal homeostasis through reciprocal interactions between the gut and liver.

PVA enema ameliorates DSS-induced acute colitis in mice

BackgroundUlcerative colitis (UC) represents a clinically challenging condition characterized by persistent damage to the colonic epithelial mucosa as the principal pathological feature. Polyvinyl alcohol (PVA) solution, primarily composed of glue, is a biodegradable polymer material that has found utility in the medical field. This research endeavors to investigate the therapeutic potential of PVA water solution in ameliorating UC in mice.MethodsUC was induced in 48 C57BL/6 mice by administering 2.5% DSS in their diet for 6 days. Mice were treated with different concentrations of PVA (0.1 mg/ml PVA, 0.3 mg/ml PVA, 1 mg/ml PVA, 3 mg/ml PVA, 10 mg/ml PVA) enemas (n = 6). Disease Activity Index (DAI) and histologic score were evaluated for inflammation degree. Furthermore, mouse colon organoids were cultured, which were used to assess the effects of PVA on expansion in vitro.ResultsPVA aqueous solutions (1 mg/ml and 3 mg/ml) were able to alleviate the DAI in mice. By DAY 6, there was a significant 3/5-fold decrease in DAI within the 1 mg/ml PVA group (p = 0.02). Histopathology scores demonstrated improvements, while the levels of inflammatory factors in the intestinal mucosal tissue were reduced. Additionally, it was confirmed that PVA could promote the expansion of colonic organoids in vitro.ConclusionsIn summary, our investigation has yielded findings indicating that PVA holds the potential to ameliorate symptoms associated with colitis in murine subjects afflicted by DSS-induced colitis, primarily through its facilitation of intestinal stem cell expansion. This study might provide a new candidate for the clinical treatment of ulcerative colitis.

L-glutamate requires β-catenin signalling through Frizzled7 to stimulate porcine intestinal stem cell expansion.

Intestinal stem cells (ISCs) decode and coordinate various types of nutritional information from the diet to support the crypt-villus axis architecture, but how specific dietary molecules affect intestinal epithelial homeostasis remains unclear. In the current study, L-glutamate (Glu) supplementation in either a nitrogen-free diet (NFD) or a corn-soybean meal diet (CSMD) stimulated gut growth and ISC expansion in weaned piglets. Quantitative proteomics screening identified the canonical Wnt signalling pathway as a central regulator of intestinal epithelial development and ISC activity in vivo. Importantly, the Wnt transmembrane receptor Frizzled7 (FZD7) was upregulated in response to dietary Glu patterns, and its perturbations in intestinal organoids (IOs) treated with a specific inhibitor and in FZD7-KO IPEC-J2 cells disrupted the link between Glu inputs and β-catenin signalling and a subsequent reduction in cell viability. Furthermore, co-localization, coimmunoprecipitation (Co-IP), isothermal titration calorimetry (ITC), and microscale thermophoresis (MST) revealed that Glu served as a signalling molecule directly bound to FZD7. We propose that FZD7-mediated integration of the extracellular Glu signal controls ISC proliferation and differentiation, which provides new insights into the crosstalk of nutrients and ISCs.

Recombinant Porcine R-Spondin 1 Facilitates Intestinal Stem Cell Expansion along the Crypt-Villus Axis through Potentiating Wnt/β-Catenin Signaling in Homeostasis and Deoxynivalenol Injury.

R-spondin 1 (RSPO1) is a ligand for the intestinal stem cell (ISC) marker Lgr5 in the crypt, which functions to amplify canonical Wnt signaling to stimulate the division of ISCs. Despite the crucial role of recombinant human RSPO1 (rhRSPO1) in homeostasis and regeneration, little is known about RSPO1 among different species. Here, we cloned the porcine RSPO1 (pRSPO1) gene and obtained rpRSPO1 protein through the expression system of the recombinant Escherichia coli Rosetta (DE3) chemical competent cells. Using the in vitro IPEC-J2 model that combines cell proliferation evaluation approaches, we identified the rpRSPO1 activity in stimulating jejunal epithelial cells. And upon deoxynivalenol challenge in mice, we found that rpRSPO1 ameliorated their growth retardation and jejunal epithelial integrity. Importantly, the ISCs in the jejunum had greater proliferation and differentiation potential that was accompanied by Wnt/β-catenin pathway activation after rpRSPO1 modulation. Subsequently, the jejunal organoids expanded from these ISCs ex vivo presented robust growth advantages. And the rpRSPO1 was able to guide Wnt/β-catenin activity to increase ISC activity. Our work systematically demonstrates that rpRSPO1 facilitates ISC expansion by potentiating Wnt/β-catenin signaling during homeostasis and responding to deoxynivalenol perturbations.

Heat-stable enterotoxin inhibits intestinal stem cell expansion to disrupt the intestinal integrity by downregulating the Wnt/β-catenin pathway.

Enterotoxigenic Escherichia coli causes severe infectious diarrhea with high morbidity and mortality in newborn and weanling pigs mainly through the production of heat-stable enterotoxins (STs). However, the precise regulatory mechanisms involved in ST-induced intestinal epithelium injury remain unclear. Consequently, we conducted the experiments in vivo (mice), ex vivo (mouse and porcine enteroids), and in vitro (MODE-K and IPEC-J2 cells) to explore the effect of STp (one type of STa) on the integrity of the intestinal epithelium. The results showed that acute STp exposure led to small intestinal edema, disrupted intestinal integrity, induced crypt cell expansion into spheroids, and downregulated Wnt/β-catenin activity in the mice. Following a similar trend, the enteroid-budding efficiency and the expression of Active β-catenin, β-catenin, Lgr5, PCNA, and KRT20 were significantly decreased after STp treatment, as determined ex vivo. In addition, STp inhibited cell proliferation, induced cell apoptosis, destroyed cell barriers, and reduced Wnt/β-catenin activity by downregulating its membrane receptor Frizzled7 (FZD7). In contrast, Wnt/β-catenin reactivation protected the IPEC-J2 cells from STp-induced injury. Taking these findings together, we conclude that STp inhibits intestinal stem cell expansion to disrupt the integrity of the intestinal mucosa through the downregulation of the Wnt/β-catenin signaling pathway.

PSIX-30 mTORC1 Sensing Glutamate Signal to Promote Porcine Intestinal Development by Accelerating Intestinal Stem Cell Expansion

Abstract Mechanistic target of rapamycin complex 1 (mTORC1) coordinates cell growth and metabolism with environmental cues, such as amino acids, growth factors, and energy. Glutamate (Glu) is a primary metabolic fuel for the intestinal epithelium and extensively involves numerous physiological processes. Crypt intestinal stem cells (ISCs) driven intestinal epithelial renewal that needs a continuous energy supplement. However, the effects of Glu on the expansion of porcine ISCs and intestinal epithelial development remain unclear. Therefore, the objective of this study was to investigate the underlying mechanism that Glu promotes intestinal development. Firstly, a total of 14 weaned piglets (Duroc × Landrace × Large White) with similar body weight (BW) were randomly allocated into the control group, and the 1.0% Glu group with 7 replicates per group and 1 piglet per replicate. The experiment lasts for 21 days. The results showed that dietary Glu increased small intestinal weights, jejunal villus height, and the ratio of the villus height to the crypt depth. Moreover, dietary Glu promoted the proliferation and differentiation of intestinal epithelial cells. Subsequently, iTRAQ proteomics screening indicated that intestinal mTORC1 signaling may participate in Glu-stimulated intestinal epithelial development, which was confirmed by Western blotting. Meanwhile, the IR/IRS/PI3K/Akt pathway and EGFR/ERK pathway are the upstream of Glu-induced mTORC1 signaling activation, which verified in IPEC-J2 cell line and intestinal organoids. Furthermore, the in vivo and ex vivo crypt ISCs experiments showed that Glu accelerated ISC expansion as increased intestinal organoid forming efficiency and budding efficiency. Glu also promoted ISC self-renew and differentiated into various functional cells (enterocytes, goblet cells, enteroendocrine cells, Paneth cells). In summary, mTORC1 integrated Glu signal stimulated ISC expansion and ultimately promoted epithelial development.

Combination treatment of podophyllotoxin and rutin promotes mouse Lgr5+ ve intestinal stem cells survival against lethal radiation injury through Wnt signaling.

It has been well established that radiation-induced gastrointestinal injury is manifested through loss of intestinal crypt stem cells and disruption of the mucosal layers, resulting in diarrhoea, weight loss, electrolyte imbalance, infection and mortality. Podophyllotoxin and rutin in combination (G-003M) has been reported to regulate endogenous cellular antioxidant defense systems and inflammatory response. However, the mechanism by which G-003M ameliorates radiation-induced intestinal stem cell (ISC) injury remains unclear. Here, we hypothesize the radioprotective potential of G-003M would amplify the intestinal crypt stem cells through upregulation of Wnt/β-catenin signaling and accelerate the reconstitution of the irradiated intestine. Our results showed significant functional and structural intestine regeneration in irradiated animals following G-003M treatment which resulted in improved animal survival. Immunohistochemical examination revealed an enhancement in Lgr5+ ve crypt stem cells. Increased β-catenin nuclear translocation resulted in upregulation of β-catenin target genes that supported ISC renewal and expansion in G-003M-treated mice, as compared to IR-treated mice. However, G-003M could not rescue the Wnt knockdown cohorts (XAV939 treated) which exhibited greater incidence of intestinal apoptosis, DNA damage and crypt depopulation upon radiation exposure. These findings suggest the involvement of Wnt pathway during G-003M mediated amelioration of IR-induced ISC injury. G-003M also minimised acute inflammation by restricting the infiltration of immune cells into the intestinal venules. Furthermore, G-003M treated animals showed improved anti-tumor response compared to FDA approved Amifostine. Taken together, our findings suggest that G-003M may be used as a potential countermeasure for radiation injuries as well as an adjuvant during anti-cancer therapy.

Abstract 4825: Functional role of Ring Finger Protein 43 in intestinal stem cell during colorectal tumorigenesis

Abstract Background: Colorectal cancer is one of the most general causes of cancer related death. Ring Finger Protein 43(RNF43) is an E3 ubiquitin ligase that suppresses the Wint signaling pathway and is known as a tumor suppressor gene. Previous studies reported RNF43 mutation leads to inactivation of RNF43 in pancreatic, ovarian, colorectal cancer and so on. Subsequently, Wnt signaling activation is associtated with tumor progression in these types of cancers. However, functional role of RNF43 for tumor formation is not clear in colorectal cancer. Aim: To investigate the functional role of RNF43 in colorectal cancer by using RNF43 knockout mice. Method: We prepared RNF43 knockout mice by using CRISPR/Cas9 system. To induce colorectal tumor in mice, we used Azoxymethane-Dextran sulfate sodium (AOM-DSS) model. First, we injected AOM 10mg/kg into the peritoneal cavity. After 7days from injection, drunk 2% DSS water to mice for 7days. We sacrificed the mice and evaluated the tumor incidence and tumor size after 8, 10, 12, and 16 weeks from injection. Next, we prepared the small intestine organoid from RNF43 wild and knockout mice. Then we compared the organoid size and formation rate. In vitro analysis, we prepared colon cancer cell lines (COLO205, SW620, HCT116) and evaluated the effect on cell proliferation in suppression of RNF43 and normal condition. Result: There was no significant difference in the tumor incidence and tumor size at 8, 10, and 12weeks after AOM injection. However, at 16 weeks, the tumor size of RNF43 knockout mice was significantly bigger than that of RNF43 wild type mice. (p<0.001) Further examination of the intestinal organoid revealed that the organoids derived from RNF43 knockout mice tended to get bigger than those from wild type mice. In vitro analysis, cell proliferation of RNF43 mutant cell line (HCT116) was significantly increased compared with RNF43 wild type cell lines (COLO205, SW620). RNF43 silencing significantly increased cell proliferation in RNF43 wild type cell lines (p<0.001), whereas did not affected in RNF43 mutant cell line. Conclusion: Our results suggest that RNF43 expression suppresses epithelial cell growth in the intestine, and the dysregulation of Wint signaling via RNF43 disruption promote intestinal stem cell expansion and colorectal tumorigenesis. Citation Format: Tsugio Eto, Takatsugu Ishimoto, Eri Oda, Daisuke Kuroda, Kota Arima, Mayuko Ohuchi, Kenichi Nakamura, Hiroshi Sawayama, Koichi Kinoshita, Masaaki Iwatsuki, Yoshifumi Baba, Yasuo Sakamoto, Naoya Yoshida, Hideo Baba. Functional role of Ring Finger Protein 43 in intestinal stem cell during colorectal tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4825. doi:10.1158/1538-7445.AM2017-4825

Designer matrices for intestinal stem cell and organoid culture.

Epithelial organoids recapitulate multiple aspects of real organs, making them promising models of organ development, function and disease. However, the full potential of organoids in research and therapy has remained unrealized, owing to the poorly defined animal-derived matrices in which they are grown. Here we used modular synthetic hydrogel networks to define the key extracellular matrix (ECM) parameters that govern intestinal stem cell (ISC) expansion and organoid formation, and show that separate stages of the process require different mechanical environments and ECM components. In particular, fibronectin-based adhesion was sufficient for ISC survival and proliferation. High matrix stiffness significantly enhanced ISC expansion through a yes-associated protein 1 (YAP)-dependent mechanism. ISC differentiation and organoid formation, on the other hand, required a soft matrix and laminin-based adhesion. We used these insights to build a fully defined culture system for the expansion of mouse and human ISCs. We also produced mechanically dynamic matrices that were initially optimal for ISC expansion and subsequently permissive to differentiation and intestinal organoid formation, thus creating well-defined alternatives to animal-derived matrices for the culture of mouse and human stem-cell-derived organoids. Our approach overcomes multiple limitations of current organoid cultures and greatly expands their applicability in basic and clinical research. The principles presented here can be extended to identify designer matrices that are optimal for long-term culture of other types of stem cells and organoids.

Innate immunity repairs gut lining

It emerges that innate immune cells called group 3 innate lymphoid cells signal directly to intestinal stem cells to promote the replacement of damaged epithelial cells lining the gut. See Letter p.560 The cellular signals supporting normal epithelial intestine maintenance through regulation of intestinal stem cell (ISC) activity are well characterized, but the signals involved in the regulation of the ISC compartment after damage are still unclear. Alan Hanash and colleagues have found that innate lymphoid cells produce interleukin-22 (IL-22) after injury to increase the growth of mouse intestinal organoids. They further show that recombinant IL-22 promotes ISC expansion in both human and mouse organoids, via a STAT3-dependent pathway and independently of the Paneth cells, which provide for ISC maintenance signals. IL-22 treatment also enhanced the recovery of ISCs from intestinal injury.

Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration.

Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch, and epidermal growth factor (EGF) signals supporting Lgr5+ crypt base columnar ISCs for normal epithelial maintenance1,2. However, little is known about the regulation of the ISC compartment after tissue damage. Utilizing ex vivo organoid cultures, we provide evidence that innate lymphoid cells (ILCs), potent producers of Interleukin-22 (IL-22) after intestinal injury3,4, increased the growth of murine small intestine (SI) organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both murine and human intestinal organoids, increasing proliferation, and promoting ISC expansion. IL-22 induced Stat3 phosphorylation in Lgr5+ ISCs, and Stat3 was critical for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after murine allogeneic bone marrow transplantation (BMT) enhanced recovery of ISCs, increased epithelial regeneration, and reduced intestinal pathology and mortality from graft vs. host disease (GVHD). Atoh1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independent of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support intestinal epithelium, activating ISCs to promote regeneration.

IL-22 Is an Intestinal Stem Cell Growth Factor, and IL-22 Administration in Vivo Reduces Morbidity and Mortality in Murine GvHD

Graft versus host disease (GVHD) remains a major limitation of allogeneic hematopoietic stem cell transplantation (allo-HSCT), and gut GVHD specifically is a major cause of GVHD-related morbidity and mortality. Little is known about regulation of the intestinal stem cell (ISC) compartment in gut GVHD. We have found that Interleukin-22 (IL-22) produced by innate lymphoid cells is important for ISC recovery after transplant. However, the mechanism of action and specific cellular targets of IL-22 leading to ISC recovery are poorly understood.Using clinically modeled LP into C57BL/6 (B6) minor antigen-mismatched HSCT (H-2 into H-2b), we found that daily treatment with recombinant murine (rm)IL-22 (4ug, intraperitoneal injection) starting day seven after transplant led to reduced intestinal pathology from GVHD without altering alloreactive immunity. Both overall GVHD pathology and epithelial apoptosis scores were significantly lower three weeks post-BMT in rmIL-22-treated mice with GVHD compared to PBS-treated controls (p<0.001). We observed that mice treated with rmIL-22 (and no pharmacologic immunosuppression) had increased numbers of Lgr5+ ISCs and significantly greater ISC proliferation (p<0.01). This was not due to IL-22-dependent changes in the ISC niche, as Paneth cell numbers, Paneth cell-derived growth factors (EGF, Wnt3), and stroma-derived growth factors (Rspo3) were all unchanged after IL-22 administration. However, the antimicrobial proteins Reg3β and Reg3γ were both upregulated by qPCR in small intestine (SI) of rmIL-22-treated mice (p<0.01 and p<0.001 respectively), although this did not result in consistent changes in the gut microbial flora.To evaluate direct effects on epithelial regeneration, we performed intestinal organoid culture assays in the presence of IL-22. Organoids generated from SI and large intestine (LI) crypts of wild-type B6 mice demonstrated substantially increased size after seven days of culture with IL-22 (p<0.001, SI, Fig. 1A; p<0.05, LI). Co-culturing crypts with innate lymphoid cells (ILC), potent producers of IL-22 in vivo, led to increased organoid size as well. Furthermore, culture with IL-22 significantly increased organoid budding (new crypt formation), resulting in increased organoid expansion with serial passaging in the presence of IL-22 (1ng/ml) suggesting that IL-22 could directly increase ISC expansion. Indeed, IL-22 culture led to increased organoid EDU incorporation and expansion of Lgr5+ ISCs after culture of SI crypts from Lgr5-GFP reporter mice (p< 0.001, Fig. 1D). Demonstrating a direct effect on ISCs, IL-22 led to STAT3 phosphorylation specifically in Lgr5+ cells and resulted in increased budding of organoids cultured from isolated single SI ISCs after only four days in culture (p<0.01).To investigate the translational potential for use in humans, we tested a human IL-22 dimer/Fc fusion molecule (F-652, Generon Corp., Shanghai) on mouse intestinal crypts and found that F-652 significantly increased the size of SI and LI organoids. Using the LP into B6 allo-HSCT model described above, we found that every other day subcutaneous (SQ) treatment with 100 ug/kg F-652 starting day seven post-BMT led to significant improvement in both clinical GVHD score (P<0.0001) and survival (p<0.05, Fig. 1C).In summary, we found that IL-22 and innate lymphoid cells can bridge immune function and tissue regeneration by acting directly on epithelial stem cells. IL-22 and F-652 therapy may represent a novel approach to promote intestinal recovery in patients with GVHD without increasing post-transplant immunodeficiency. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Intestinal Regeneration: YAP—Tumor Suppressor and Oncoprotein?

The Hippo signaling pathway exerts a growth-suppressive effect by inhibitory phosphorylation of the oncogenic transcription co-activator Yki/YAP. A recent study paradoxically reports that genetic removal of YAP enhances intestinal stem cell expansion and regeneration.

Restriction of intestinal stem cell expansion and the regenerative response by YAP.

A remarkable feature of regenerative processes is their ability to halt proliferation once an organ’s structure has been restored. The Wnt signaling pathway is the major driving force for homeostatic self-renewal and regeneration in the mammalian intestine. The mechanisms that counterbalance Wnt-driven proliferation are poorly understood. We demonstrate here that YAP, a protein known for its powerful growth-inducing and oncogenic properties1-2, has an unexpected growth-suppressive function restricting Wnt signals during intestinal regeneration. Transgenic expression of YAP reduces Wnt target gene expression and results in the rapid loss of intestinal crypts. In addition, loss of YAP results in Wnt hypersensitivity during regeneration, leading to hyperplasia, expansion of intestinal stem cells (ISCs) and niche cells, and formation of ectopic crypts and microadenomas. We find that cytoplasmic YAP restricts elevated Wnt signaling independently of the APC/Axin/GSK3β complex partly by limiting the activity of Dishevelled (DVL). DVL signals in the nucleus of ISCs and its forced expression leads to enhanced Wnt signaling in crypts. YAP dampens Wnt signals by restricting DVL nuclear translocation during regenerative growth. Finally, we provide evidence that YAP is silenced in a subset of highly aggressive and undifferentiated human colorectal carcinomas (CRC) and its expression can restrict the growth of CRC xenografts. Collectively, our work describes a novel mechanistic paradigm for how proliferative signals are counterbalanced in regenerating tissues. Additionally, our findings have important implications for the targeting of YAP in human malignancies.

Restriction of intestinal stem cell expansion and the regenerative response by YAP

Restriction of intestinal stem cell expansion and the regenerative response by YAP