Intestinal Tissue Regeneration Research Articles

IL-22 promotes mucin-type O-glycosylation and MATH1+ cell-mediated amelioration of intestinal inflammation

The interleukin (IL)-22 cytokine can be protective or inflammatory in the intestine. It is unclear if IL-22 receptor (IL-22Ra1)-mediated protection involves a specific type of intestinal epithelial cell (IEC). By using a range of IEC type-specific Il22Ra1 conditional knockout mice and a dextran sulfate sodium (DSS) colitis model, we demonstrate that IL-22Ra1 signaling in MATH1+ cells (goblet and progenitor cells) is essential for maintaining the mucosal barrier and intestinal tissue regeneration. The IL-22Ra1 signaling in IECs promotes mucin core-2 O-glycan extension and induces beta-1,3-galactosyltransferase 5 (B3GALT5) expression in the colon. Adenovirus-mediated expression of B3galt5 is sufficient to rescue Il22Ra1IEC mice from DSS colitis. Additionally, we observe a reduction in the expression of B3GALT5 and the Tn antigen, which indicates defective mucin O-glycan, in the colon tissue of patients with ulcerative colitis. Lastly, IL-22Ra1 signaling in MATH1+ progenitor cells promotes organoid regeneration after DSS injury. Our findings suggest that IL-22-dependent protective responses involve O-glycan modification, proliferation, and differentiation in MATH1+ progenitor cells.

Myeloid Cell-Derived IL-1 Signaling Damps Neuregulin-1 from Fibroblasts to Suppress Colitis-Induced Early Repair of the Intestinal Epithelium.

Neuregulin-1 (Nrg1, gene symbol: Nrg1), a ligand of the ErbB receptor family, promotes intestinal epithelial cell proliferation and repair. However, the dynamics and accurate derivation of Nrg1 expression during colitis remain unclear. By analyzing the public single-cell RNA-sequencing datasets and employing a dextran sulfate sodium (DSS)-induced colitis model, we investigated the cell source of Nrg1 expression and its potential regulator in the process of epithelial healing. Nrg1 was majorly expressed in stem-like fibroblasts arising early in mouse colon after DSS administration, and Nrg1-Erbb3 signaling was identified as a potential mediator of interaction between stem-like fibroblasts and colonic epithelial cells. During the ongoing colitis phase, a significant infiltration of macrophages and neutrophils secreting IL-1β emerged, accompanied by the rise in stem-like fibroblasts that co-expressed Nrg1 and IL-1 receptor 1. By stimulating intestinal or lung fibroblasts with IL-1β in the context of inflammation, we observed a downregulation of Nrg1 expression. Patients with inflammatory bowel disease also exhibited an increase in NRG1+IL1R1+ fibroblasts and an interaction of NRG1-ERBB between IL1R1+ fibroblasts and colonic epithelial cells. This study reveals a novel potential mechanism for mucosal healing after inflammation-induced epithelial injury, in which inflammatory myeloid cell-derived IL-1β suppresses the early regeneration of intestinal tissue by interfering with the secretion of reparative neuregulin-1 by stem-like fibroblasts.

Spatiotemporal Optimization of Pelvic Radiation Therapy

Conformal and adaptive Radiation Therapy (RT) remains critical in the treatment of locally advanced pelvic cancers despite acute and late bowel complications. This work introduces spatiotemporal optimization of pelvic RT including mathematical modelling of intestinal radiobiology to evaluate a novel treatment planning approach for fractionated pelvic RT. A single-plan fractionated RT delivery has n-fractions at dose/fraction = d and has biologically effective dose BED = nd ( 1 + d / [α/β] ) - ln(2) ([T - Tk] / [αTp]), where Tk and Tp are the kickoff time and doubling time of repopulation, and T is the time to deliver all fractions (including off-days). We present a parameterization of the repopulation BED-term to estimate a range of daily recovered BED including uncertainties in α and Tp. Recovery of BED as currently formulated is independent of the delivered dose; however, the assumption of spatiotemporal optimization is that a regional reduction of dose during RT by mixing treatment plans is critical to allow for normal tissue healing. We evaluate an achievable spatiotemporal optimization treatment planning strategy for an advanced stage prostate and a cervical cancer patient using 25-fraction delivery over 5-weeks and 4-weekends (T = 33 days) and propose 3 treatment plans: (1) a conformal "clinical standard" plan, (2) a right-bowel sparing plan, and (3) a left-bowel sparing plan. The plans are optimized to ensure no increased dose in opposing bowel or normal organs at risk (OARs). For radiobiological parameters ranging from α = 0.2-0.35/Gy, α/β = 2-3 Gy, Tk = 5-10 days, and Tp = 3-7 days, parameterization shows daily BED recovery of 0.44±0.08 Gy (range = 0.28-0.69 Gy), or an average of 4Gy BED recovery for 5-days treatment surrounded by 2 weekends. Conservatively evaluating the 3.6Gy BED distribution in treatment planning may identify regions for complete recovery (after Tk days). In a prostate 3-plan set, the right- and left- bowel sparing plans deliver equivalent mean dose to targets and OARs to within 5cGy/fx. The clinical plan includes 298cc of bowel under 14Gy, but this same bowel will be spared throughout treatment. Delivery of the clinical plan in week 1 to achieve Tk days, followed by alternating the left and right plan each week has the potential to reduce BED to zero for an additional 116cc of bowel using the left-sparing plan in weeks 2 and 4, and 91cc using the right-bowel sparing plan in weeks 3 and 5. The cervical cancer plan-set was more challenging due to right-sided nodal volumes and higher clinical bowel dose; OAR mean dose variations were >8cGy/fx in a few organs, but the left- and right- sided sparing plans results in 394 cc and 139cc, respectively of unique spared bowel at the 3.6Gy/week threshold to potentially reach zero BED during treatment. A novel spatiotemporal optimization of pelvic RT has the potential to preserve bowel, allow for increased intra-treatment intestinal tissue regeneration, and reduce radiation-induced complications.

Oral Delivery of Bioactive Glass-Loaded Core-Shell Hydrogel Microspheres for Effective Treatment of Inflammatory Bowel Disease.

Resolving inflammation and promoting intestinal tissue regeneration are critical for inflammatory bowel disease (IBD) treatment. Bioactive glass (BG) is a clinically approved bone graft material and has been shown to modulate inflammatory response, but it is unknown whether BG can be applied to treat IBD. Here, it is reported that BG attenuates pro-inflammatory response of lipopolysaccharide (LPS)-stimulated macrophages and hence reduces inflammatory damage to intestinal organoids in vitro. In addition, zein/sodium alginate-based core-shell microspheres (Zein/SA/BG) are developed for oral delivery of BG, which helps prevent premature dissolution of BG in the stomach. The results show that Zein/SA/BG protects BG from a gastric-simulated environment while dissolved in an intestinal-simulated environment. When administered to acute and chronic colitis mice model, Zein/SA/BG significantly reduces intestinal inflammation, promotes epithelial tissue regeneration, and partially restores microbiota homeostasis. These findings are the first to reveal the therapeutic efficacy of BG against IBD, which may provide a new therapeutic approach at low cost for effective IBD treatment.

Loss of PI3k activity of inositol polyphosphate multikinase impairs PDK1-mediated AKT activation, cell migration, and intestinal homeostasis

Protein kinase B (AKT) is essential for cell survival, proliferation, and migration and has been associated with several diseases. Here, we demonstrate that inositol polyphosphate multikinase (IPMK's) lipid kinase property drives AKT activation via increasing membrane localization and activation of PDK1 (3-Phosphoinositide-dependent kinase 1), largely independent of class I PI3k (cPI3K). Deletion of IPMK impairs cell migration, which is partially associated with the abolition of PDK1-mediated ROCK1 disinhibition and subsequent myosin light chain (MLC) phosphorylation. IPMK is highly expressed in intestinal epithelial cells (IEC). Deleting IPMK in IEC reduced AKT phosphorylation and diminished the number of Panethcells. Ablation of IPMK impaired IEC regeneration both basally and after chemotherapy-induced damage, suggesting a broad role for IPMK in activating AKT and intestinal tissue regeneration. In conclusion, the PI3k activity of IPMK is necessary for PDK1-mediated AKT activation and intestinal homeostasis.

AMINO ACID SENSING PATHWAYS IN INFLAMMATORY BOWEL DISEASE

Abstract IBD is a chronic inflammatory disease of the gastrointestinal tract affecting millions of people worldwide. The last few decades have seen rapid increase of IBD cases in the US contributing to exorbitant health-care costs and morbidity rates. IBD comprises of two major subtypes: ulcerative colitis and Crohn’s disease. Although the etiology of IBD is not completely understood, a complex interaction of environmental, genetic, immune, and gut microbial factors contributes to its pathogenesis. These factors ultimately converge to disrupt intestinal epithelial cell homeostasis, compromising mucosal barrier function, leading to unresolved relapsing inflammatory injury. Poor dietary habits such as Western diet are thought to play a pivotal role in the development of colitis. A healthy and balanced diet is important in management of the disease. Epidemiological and experimental studies demonstrate that high-protein diet triggers inflammatory flares and colitis patients are often advised to reduce animal dietary protein. However, colitis patients are also at high risk of a protein malnutrition. Thus, the precise mechanisms by which low dietary protein increases incidence of colitis and/or aggravate already established disease remains unknown. Our preliminary findings suggest that low dietary protein intake aggravated dextran sulfate sodium (DSS)-induced colitis with reduced body weight, colon length and increased histological injury. Dietary habits impact intestinal epithelial cell homeostasis and regeneration process in the event of an injury. The mechanistic target of rapamycin complex 1 (mTORC1), a ‘master’ regulator of cell growth participates in intestinal tissue regeneration. Remarkably, studies inhibiting mTORC1 activity have shown to disrupt the regenerative capacity of intestinal epithelium and increased susceptibility to colitis while activating mTORC1 had a positive effect. As amino acids potently activate mTORC1, we assessed and found that low dietary protein significantly reduce colonic mTORC1 activation. The newly discovered GAP activity towards Rags (GATOR1 & GATOR2) complexes act as amino acid sensing pathways by which mTORC1 activity is modulated. GATOR1 is a negative regulator, whereas GATOR2 is a positive regulator of mTORC1. CRISPR/CAS9 knockout of Wdr24 (GATOR2) led to inactivation of mTORC1 under amino acid culture conditions. Next, we investigated the role of amino acid sensing pathway in colitis models and found that disruption of intestinal epithelial specific GATOR2 complex (Wdr24ΔIE) attenuated mTORC1 activity and increased susceptibility to colitis. These findings suggest that low protein diet impacts colitis by modulating the intestinal epithelial amino acid sensing pathway.

Phenotypic landscape of intestinal organoid regeneration.

The development of intestinal organoids from single adult intestinal stem cells in vitro recapitulates the regenerative capacity of the intestinal epithelium1,2. Here we unravel the mechanisms that orchestrate both organoid formation and the regeneration of intestinal tissue, using an image-based screen to assay an annotated library of compounds. We generate multivariate feature profiles for hundreds of thousands of organoids to quantitatively describe their phenotypic landscape. We then use these phenotypic fingerprints to infer regulatory genetic interactions, establishing a new approach to the mapping of genetic interactions in an emergent system. This allows us to identify genes that regulate cell-fate transitions and maintain the balance between regeneration and homeostasis, unravelling previously unknown roles for several pathways, among them retinoic acid signalling. We then characterize a crucial role for retinoic acid nuclear receptors in controlling exit from the regenerative state and driving enterocyte differentiation. By combining quantitative imaging with RNA sequencing, we show the role of endogenous retinoic acid metabolism in initiating transcriptional programs that guide the cell-fate transitions of intestinal epithelium, and we identify an inhibitor of the retinoid X receptor that improves intestinal regeneration in vivo.

Me6TREN targets β-catenin signaling to stimulate intestinal stem cell regeneration after radiation.

Background: Acute gastrointestinal syndrome (AGS) is one of the most severe clinical manifestations after exposure to high doses of radiation, and is life-threatening in radiological emergency scenarios. However, an unmet challenge is lacking of an FDA-approved drug that can ameliorate the damage of radiation-exposed intestinal tissues and accelerate the regeneration of injured epithelia. In this study, we investigated whether the small molecule Me6TREN (Me6) can regulate intestinal stem cell (ISC) proliferation and promote crypt regeneration after irradiation.Methods: Lethally irradiated mice were administered with Me6 or PBS to study the survival rate, and sections of their small intestine were subjected to immunostaining to evaluate epithelial regeneration. An intestinal organoid culture system was employed to detect the role of Me6 in organoid growth and ISC proliferation. We further investigated the key signaling pathways associated with Me6 using microarray, western blotting, and RNA interference techniques.Results: We identified the small molecule Me6 as a potent intestinal radiation countermeasure. Systemic administration of Me6 significantly improved ISC and crypt cell regeneration and enhanced the survival of mice after high doses of radiation. Using an in vitro intestinal organoid culture system, we found that Me6 not only induced ISC proliferation but also increased the budding rate of intestinal organoids under unirradiated and irradiated conditions. Me6 remarkably activated the expression of ISC-associated and proliferation-promoting genes, such as Ascl2, Lgr5, Myc, and CyclinD1. Mechanistically, Me6 strongly stimulated the phosphorylation of β-catenin at the S552 site and increased the transcriptional activity of β-catenin, a key signaling pathway for ISC self-renewal and proliferation. This is further evidenced by the fact that knockdown of β-catenin abolished the effect of Me6 on intestinal organoid growth in vitro and crypt regeneration in irradiated mice.Conclusion: The small molecule Me6TREN induced ISC proliferation, enhanced intestinal organoid growth in vitro, and promoted intestinal tissue regeneration after radiation injury by activating β-catenin signaling.

Advances in understanding the pathogenesis of graft-versus-host disease.

Acute graft-versus-host disease (GVHD) remains a major complication after allogeneic haematopoietic stem cell transplantation (allo-HSCT). The emergence of different immuno-prophylaxis strategies, such as post-transplant cyclophosphamide or anti-thymocyteglobulin has reduced the incidence of acute GVHD in recent years. The biology of the acute GVHD we observe in the clinic may change due to the use of novel immuno-stimulatory agents, including immune checkpoint inhibitors or anti-neoplastic immune-modifiers, like lenalidomide, given before or after allo-HSCT. Here we discuss the recent advances in our understanding of acute GVHD with a focus on early events of the disease, including tissue damaging factors, innate immune cells, costimulatory pathways, immune cell signalling, immuno-regulatory cell types, biomarkers of GVHD and regenerative approaches. New insight in the pathogenesis of acute GVHD has revealed the role of pro-inflammatory intracellular signalling, defects in intestinal tissue regeneration and anti-bacterial defence, as well as a reduced diversity of the microbiome, which will be the basis for the development of novel therapies.

Ubiquitin-specific protease 22 enhances intestinal cell proliferation and tissue regeneration after intestinal ischemia reperfusion injury.

BACKGROUNDIntestinal ischemia reperfusion (I/R) injury is a serious but common pathophysiological process of many diseases, resulting in a high mortality rate in clinical practice. Ubiquitin-specific protease 22 (USP22) acts as regulator of cell cycle progression, proliferation, and tumor invasion. Depleted USP22 expression has been reported to contribute to arrested cell cycle and disrupted generation of differentiated cell types in crypts and villi. However, the role of USP22 in intestinal damage recovery has not been investigated. Therefore, elucidation of the underlying mechanism of USP22 in intestinal I/R injury may help to improve the tissue repair and patient prognosis in clinical practice.AIMTo investigate the role of USP22 in intestinal cell proliferation and regeneration after intestinal I/R injury.METHODSAn animal model of intestinal I/R injury was generated in male Sprague-Dawley rats by occlusion of the superior mesenteric artery followed by reperfusion. Chiu’s scoring system was used to grade the damage to the intestinal mucosa. An in vitro model was developed by incubating rat intestinal epithelial IEC-6 cells in hypoxia/reoxygenation conditions in order to simulate I/R in vivo. siRNA and overexpression plasmid were used to regulate the expression of USP22. USP22, Cyclin D1, and proliferating cell nuclear antigen (PCNA) expression levels were measured by Western blot analysis and immunohistochemistry staining. Cell survival (viability) and cell cycle were evaluated using the Cell Counting Kit-8 and flow cytometry, respectively.RESULTSUSP22 expression was positively correlated with the expression levels of PCNA and Cyclin D1 both in vivo and in vitro, which confirmed that USP22 was involved in cell proliferation and intestinal regeneration after intestinal I/R injury. Decreased levels of Cyclin D1 and cell cycle arrest were observed in the USP22 knockdown group (P < 0.05), while opposite results were observed in the USP22 overexpression group (P < 0.05). In addition, increased expression of USP22 was related to improved intestinal pathology or IEC-6 cell viability after I/R or hypoxia/reoxygenation. These results suggested that USP22 may exert a protective effect on intestinal I/R injury by regulating cell proliferation and facilitating tissue regeneration.CONCLUSIONUSP22 is correlated with promoting intestinal cell proliferation and accelerating intestinal tissue regeneration after intestinal I/R injury and may serve as a potential target for therapeutic development for tissue repair during intestinal I/R injury.

Substance P accelerates intestinal tissue regeneration after γ‐irradiation–induced damage

Radiation therapy causes varying degrees of damage to biological systems. Many groups are investigating the mechanism underlying radiation-induced cellular damage but there are limited therapeutic solutions for affected patients. Recent studies show that substance P (SP) participates in cell proliferation. In the present study, we characterized the mechanism underlying SP-induced cellular signaling in radiation-induced damage of the intestine. Exposure of Caco-2 cells to SP increases cell proliferation and Erk phosphorylation in a time- and dose-dependent manner. The proliferation of cells exposed to gamma-irradiation is also stimulated by exposure to SP, a phenomenon that may result from inhibition of apoptosis because SP activates Akt and inhibits the cleavage of caspase-3. The effect of SP on cell proliferation and protection was confirmed by investigations in mice. Proliferating cell nuclear antigen staining shows that cell proliferation in radiation-damaged mouse intestine increases significantly upon exposure to SP. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end labeling assay reveals fewer cells stained in SP-treated mice compared with untreated controls. These findings show the potential for SP-induced acceleration of intestinal wound healing and reveal that the mechanism underlying this process involves activation of Erk and Akt and inhibition of caspase-3 cleavage.

Requirement of Notch activation during regeneration of the intestinal epithelia

Notch signaling regulates cell differentiation and proliferation, contributing to the maintenance of diverse tissues including the intestinal epithelia. However, its role in tissue regeneration is less understood. Here, we show that Notch signaling is activated in a greater number of intestinal epithelial cells in the inflamed mucosa of colitis. Inhibition of Notch activation in vivo using a gamma-secretase inhibitor resulted in a severe exacerbation of the colitis attributable to the loss of the regenerative response within the epithelial layer. Activation of Notch supported epithelial regeneration by suppressing goblet cell differentiation, but it also promoted cell proliferation, as shown in in vivo and in vitro studies. By utilizing tetracycline-dependent gene expression and microarray analysis, we identified a novel group of genes that are regulated downstream of Notch1 within intestinal epithelial cells, including PLA2G2A, an antimicrobial peptide secreted by Paneth cells. Finally, we show that these functions of activated Notch1 are present in the mucosa of ulcerative colitis, mediating cell proliferation, goblet cell depletion, and ectopic expression of PLA2G2A, thereby contributing to the regeneration of the damaged epithelia. This study showed the critical involvement of Notch signaling during intestinal tissue regeneration, regulating differentiation, proliferation, and antimicrobial response of the epithelial cells. Thus Notch signaling is a key intracellular molecular pathway for the proper reconstruction of the intestinal epithelia.

Intestinal regeneration by a novel surgical procedure

Treatment of short bowel syndrome is problematical. Small bowel tissue engineering has achieved modest results in animal studies. The aim of this study was to investigate intestinal regeneration in a novel surgical model. Roux-en-Y bypass procedures were performed on 40 Wistar rats weighing 250-350 g. Animals were killed at 1, 2, 3, 4, 8, 12 and 24 weeks after implantation with a 3-cm silicone tube. The spatio temporal relationship of intestinal regeneration was analysed using three-dimensional multislice computed tomography, and examination of sequential morphological changes on gross or histological findings and measurement of missing intestinal tissue (growth defects). Progressive intestinal regeneration on a silicone tube was identifiable in 35 animals. Most adhesions were initially localized on the tube but spread to a distal site 4 weeks after implantation. Growth defects decreased with time, with a marked reduction in the first 4 weeks and a gradual reduction to week 24 after implantation. Luminal patency shown radiologically as well as sequential histological findings, such as mucosal lining, matrix remodelling and muscular regeneration, suggested that regeneration of intestinal tissue took place, not merely scar contraction. Non-invasive as well as histomorphological assessment followed intestinal regeneration over time in this model, which provides scope for further studies.

Using porcine small intestinal submucosa in intestinal regeneration.

Small intestinal submucosa (SIS) is an unusual tissue that promotes constructive tissue remodeling when applied as a xenogeneic material. The aim of our experimental study was to assess its effectiveness in intestinal regeneration. Twenty white New Zealand rabbits were anesthetized and underwent celiotomy. A 6-cm antimesenteric incision was created at the jejunal segment. An elliptical SIS graft measuring 6 cm long and 2 cm wide was sutured to the jejunal defect as a patch graft. Thirteen living rabbits were divided into groups of three and the grafts were harvested at postoperative weeks 2, 4, and 6. The obtained specimens were evaluated for gross and histologic appearance. In morphometric examination, in the 2, 4, and 6 weeks groups, the diameters of grafted intestines were larger than preoperatively by 50%, 25%, and 25% respectively; also the grafts had contracted to 0%, 25%, and 50% of their original sizes respectively. At the end of 2 weeks, the grafts were intact without evidence of epithelial regeneration. By 4 weeks, intestinal tissue regeneration was started, and epithelial coverage of the grafts was detected. The grafts were covered with a complete intestinal mucosa at 6 weeks. Remarkable regeneration marked fibroplasia, angiogenesis, and mild mononuclear cell infiltration had also occurred throughout the grafts at 6 weeks. Porcine SIS appeared an effective biodegradable scaffold, facilitating regeneration of intestinal tissue. These results suggest that SIS may be useful to increase the mucosal surface of intestine and may provide a new substance for short gut syndrome in the future.