Intestinal Epithelial Cell Turnover Research Articles

Intestinal-specific Hdac3 deletion increases susceptibility to colitis and small intestinal tumour development in mice fed a high fat diet.

High fat diets, and inflammation are risk factors for colon cancer, however the underlying mechanisms remain to be fully elucidated. The transcriptional co-repressor HDAC3 has recently emerged as a key regulator of intestinal epithelial responses to diet and inflammation with intestinal-specific Hdac3 deletion (Hdac3IKO) in mice increasing lipid oxidation genes and the rate of lipid oxidation in enterocytes. Hdac3IKO mice are also predisposed to experimentally induced colitis, however whether this is driven by the intestinal metabolic reprogramming, and whether this predisposes these mice to intestinal tumourigenesis is unknown. Herein, we examined the effects of intestinal-specific Hdac3 deletion on colitis-associated intestinal tumourigenesis in mice fed a standard (STD) or HF diet (HFD). Hdac3IKO mice were highly prone to experimentally induced colitis, which was further enhanced by a HFD. Hdac3deletion also accelerated intestinal tumour development, specifically when fed a HFD and most notably in the small intestine where lipid absorption is maximal. Expression of proteins involved in fatty acid metabolism and oxidation (SCD1, EHHADH) were elevated in the small intestine of Hdac3IKO mice fed a HFD, and these mice displayed increased levels of lipid peroxidation, DNA damage, and apoptosis in their villi, as well as extensive expansion of the stem cell and progenitor cell compartment. These findings reveal a novel role for Hdac3 in suppressing colitis and intestinal tumorigenesis, particularly in the context of consumption of a HFD, and reveal a potential mechanism by which HFDs may increase intestinal tumorigenesis by increasing lipid oxidation, DNA damage and intestinal epithelial cell turnover.

Cyberlindnera jadinii yeast as a functional protein source for Atlantic salmon (Salmo salar L.): Early response of intestinal mucosal compartments in the distal intestine.

The interplay between nutrition and the immune system is well recognized, and several studies show that experimental diets elicit local morphological changes and alteration of gene and protein expression in the intestinal mucosa of Atlantic salmon. In this study the pathophysiological effects of experimental diets on mucosal responses in the distal intestine of Atlantic salmon were investigated. Atlantic salmon were fed diets with inclusion of soybean meal (SBM) and Cyberlindnera jadinii (CJ) yeast for 7 days. A standard fish meal (FM) diet was used as a control. Morphological, immunohistochemical and gene expression analyses were used to evaluate the presence of immune cells, proliferating cells, and stem cell populations in mucosal compartments of the simple folds in the distal intestine. Fish fed SBM developed morphological changes consistent with SBM induced enteritis. Immunohistochemistry showed an increased presence of apoptotic cells, CD3ϵ and CD8α labelled cells in the simple fold epithelium of SBM group compared with the CJ group. For the investigated genes, expression levels in all three groups were mostly higher in the epithelial compartment of the simple fold than in the compartment beneath the folds. Most changes within the epithelial compartment were observed in fish fed SBM, where expression of CD3ζ, CD8α, MHC I and MHC II were lower than the FM control group. The CJ group had an increased expression of the stem cell marker Lgr5 in the epithelial compartment compared with SBM group. The division of the simple fold into an apical and basal compartment showed that the increase in Lgr5 was evident along the whole length of the simple folds and not confined to the base of the folds. Similarly, proliferation (PCNA, MCM2) and apoptosis (Caspase-3) gene expression was present in the entire length of the simple folds, suggesting that intestinal epithelial cell turnover is not confined to the basal or apical part of the fold. This study shows that the epithelial compartment is active in the early immunoregulatory response towards dietary stimuli and that the level of an intestinal stem cell marker in salmon was influenced by a diet containing CJ yeast.

Abstract 6016: A comprehensive analysis of gut microbiome mediated epigenetic regulation of intestinal stem cells

Abstract Background: Gut microbial colonization, represents a critical time period for establishing the overall health of an individual. The intestine is a highly dynamic tissue with high turnover of intestinal epithelial cells throughout the life span of an individual making intestinal stem cells (ISCs) critical for normal gut homeostasis. Currently, there exists a lack of knowledge in how the gut microbiota regulates DNA methylation to influence the behavior of these ISCs. Aberrant DNA methylation is now documented to be critical in the disease pathogenesis of intestinal disorders such as colorectal cancer, thus understanding how the gut microbiome influences DNA methylation and ISCs is fundamental for improving human health. Methods: We utilized the Lgr5-GFP mouse model in which endogenous GFP expression is driven by the bonafide intestinal stem cell marker Lgr5. These mice were maintained under two different housing conditions, conventional and germ free. We collected mice under the two conditions and performed dissociation of the colon and small intestine epithelial cells using EDTA dissociation. We then analyzed cell populations using flow cytometry, and collected two cell populations: the differentiated cell population (EpiCam +, GFP -) and intestinal stem cell population (EpiCam +, GFP +). To perform a genome wide comprehensive DNA methylation analysis we performed Whole Genome Bisulfite Sequencing (WGBS) which included both stem cell and differentiated cell populations of females and males at the two age groups points (postnatal day 21 (p21) and p100). Results: There was no significant difference in CpG methylation at the global level between the two housing conditions. Next, we analyzed age matched samples from germ free and conventional mice to determine if there are differentially methylated regions (DMRs) associated with the presence of intestinal microbiota. We have identified DMRs that are maintained in germ free stem cells from p21 to p100 compared to conventional mice, as well as DMRs in the differentiated cell population at both ages. Interestingly in our analysis several genes displayed robust hypomethylation of control mice versus germ free mice at the p100 time point in both the stem cell and differentiated cell populations. One such gene, Ifitm3, has been heavily associated with chronic inflammation in the colonic epithelium and a poor risk factor for colonic cancer (Li D et. Al, Clin Cancer Res. 2011). We validated Ifitm3 methylation and expression changes between cohorts using pyrosequencing and RT-PCR respectively. Conclusions: This comprehensive analysis has yielded top candidate genes for studying functional consequences of DNA methylation changes that have been previously linked to severe patient diseases including cancer. Citation Format: Robert Craig Peery, Jiejun Shi, Leah Farmer, Li Yang, Emily Lawrence, Yumei Li, Wei Li, Lanlan Shen. A comprehensive analysis of gut microbiome mediated epigenetic regulation of intestinal stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6016.

Diminished noncanonical NF-κB signaling induces colitis-associated colorectal cancer susceptibility upon de-differentiation of epithelial cells

Abstract Patients with a medical history of Inflammatory Bowel Disease are predisposed to developing colitis-associated colorectal cancer (CAC), with greater than 20% developing CAC within 30 years of disease onset. CAC is a form of inflammation-induced tumorigenesis, where unregulated inflammation causes cells in the colon to accumulate mutations poising them for neoplasia. We demonstrate that NF-κB inducing kinase (NIK) is a critical regulator of intestinal epithelial cell (IEC) regeneration. We hypothesize that IECs exposed to chronic inflammatory conditions mitigated by dysregulated noncanonical NF-κB signaling are most prone to mutagenesis and cancer-initiating events termed Top-down tumorigenesis. In CAC, NIK depletion results in the accumulation of mature IECs that are increasingly susceptible to mutation and neoplastic transformation. This suggests that terminally differentiated IECs dedifferentiate into a stem-like phenotype. Our work has clinical relevancy, where human CAC biopsy samples have diminished noncanonical NF-κB signaling and NIK (MAP3K14) gene expression. Murine models with NIK deletion in IECs have increased susceptibility to inflammation-induced tumorigenesis following the chemical induction of AOM/DSS to induce colitis. Our mechanistic studies evaluating crypts demonstrate accumulation of mature, non-dividing IECs, which are more susceptible to mutations and malignant transformation. Here, we present a novel role for noncanonical NF-κB signaling in regulating intestinal epithelial cell turnover in protecting against CAC development. Focusing on the key niche signaling interplay following noncanonical NF-κB dysregulation gives greater insight into the CAC tumor microenvironment. Supported by grants from the iTHRIVE Pilot Translational and Clinical Studies Program, VCOM Center for One Health Research, Virginia Tech Carilion Research Acceleration Program, and the Virginia-Maryland College of Veterinary Medicine.

Executioner caspases 3 and 7 are dispensable for intestinal epithelium turnover and homeostasis at steady state

Apoptosis is widely believed to be crucial for epithelial cell death and shedding in the intestine, thereby shaping the overall architecture of the gastrointestinal tract, but also regulating tolerance induction, pinpointing a role of apoptosis intestinal epithelial cell (IEC) turnover and maintenance of barrier function, and in maintaining immune homeostasis. To experimentally address this concept, we generated IEC-specific knockout mice that lack both executioner caspase-3 and caspase-7 (Casp3/7ΔIEC), which are the converging point of the extrinsic and intrinsic apoptotic pathway. Surprisingly, the overall architecture, cellular landscape, and proliferation rate remained unchanged in these mice. However, nonapoptotic cell extrusion was increased in Casp3/7ΔIEC mice, compensating apoptosis deficiency, maintaining the same physiological level of IEC shedding. Microbiome richness and composition stayed unaffected, bearing no sign of dysbiosis. Transcriptome and single-cell RNA sequencing analyses of IECs and immune cells revealed no differences in signaling pathways of differentiation and inflammation. These findings demonstrate that during homeostasis, apoptosis per se is dispensable for IEC turnover at the top of intestinal villi intestinal tissue dynamics, microbiome, and immune cell composition.

The developmental changes in intestinal epithelial cell proliferation, differentiation, and shedding in weaning piglets.

Intestinal epithelial homeostasis plays an important role in intestinal morphology and function. However, the developmental changes in intestinal epithelial cell turnover in piglets during early weaning are unknown so far. Thus, the aim of this work was to detect changes in piglet gut development from weaning to post-weaning d 14. Accordingly, 40 piglets were used in the present study, and 8 piglets were randomly selected for sampling at d 0, 1, 3, 7 and 14 post-weaning, respectively. The results showed that weaning stress significantly affected small intestinal morphological architecture, and this impact was the worst on d 3, and then returned to normal on d 14. Furthermore, the number of the marker of proliferation Ki-67 (Ki67) positive cells was decreased on d 1 and 3, and then recovered on d 14 (P < 0.001). Also, weaning strikingly increased jejunal epithelial cell shedding on d 1 to 7 compared on d 0 (P < 0.05). Moreover, weaning remarkably affected the number of small intestinal enterocytes, goblets and endocrine cells (P < 0.05), and there were also significant differences in genes expression related to proliferation and differentiation (P < 0.05). Additionally, the mechanistic target of rapamycin (mTOR) phosphorylation level was higher on d 3 (P < 0.05). However, the Wingless/Int1 (WNT)/β-catenin pathway was not influenced by post-weaning days. Taken together, weaning induced noteworthy changes in intestinal epithelial cell proliferation, differentiation and shedding, and the mTOR signaling pathway was involved in this process. Our findings provide a cellular mechanism for intestinal developmental changes during weaning periods. This may provide nutritionists with better insight into designing efficient in-feed alternatives for preventing the unfavorable gut development in weaning piglets.

Mitochondrial transcription factor A plays opposite roles in the initiation and progression of colitis-associated cancer.

BackgroundMitochondria are key regulators in cell proliferation and apoptosis. Alterations in mitochondrial function are closely associated with inflammation and tumorigenesis. This study aimed to investigate whether mitochondrial transcription factor A (TFAM), a key regulator of mitochondrial DNA transcription and replication, is involved in the initiation and progression of colitis‐associated cancer (CAC).MethodsTFAM expression was examined in tissue samples of inflammatory bowel diseases (IBD) and CAC by immunohistochemistry. Intestinal epithelial cell (IEC)‐specific TFAM‐knockout mice (TFAM △IEC) and colorectal cancer (CRC) cells with TFAM knockdown or overexpression were used to evaluate the role of TFAM in colitis and the initiation and progression of CAC. The underlying mechanisms of TFAM were also explored by analyzing mitochondrial respiration function and biogenesis.ResultsThe expression of TFAM was downregulated in active IBD and negatively associated with the disease activity. The downregulation of TFAM in IECs was induced by interleukin‐6 in a signal transducer and activator of transcription 3 (STAT3)/miR‐23b‐dependent manner. In addition, TFAM knockout impaired IEC turnover to promote dextran sulfate sodium (DSS)‐induced colitis in mice. Of note, TFAM knockout increased the susceptibility of mice to azoxymethane/DSS‐induced CAC and TFAM overexpression protected mice from intestinal inflammation and colitis‐associated tumorigenesis. By contrast, TFAM expression was upregulated in CAC tissues and contributed to cell growth. Furthermore, it was demonstrated that β‐catenin induced the upregulation of TFAM through c‐Myc in CRC cells. Mechanistically, TFAM promoted the proliferation of both IECs and CRC cells by increasing mitochondrial biogenesis and activity.ConclusionsTFAM plays a dual role in the initiation and progression of CAC, providing a novel understanding of CAC pathogenesis.

Roles of Src family kinase, Ras, and mTOR signaling in intestinal epithelial homeostasis and tumorigenesis.

The turnover of intestinal epithelial cells (IECs) is relatively rapid (3‐5 days in mouse and human), and this short existence and other aspects of the homeostasis of IECs are tightly regulated by various signaling pathways including Wnt‐β‐catenin signaling. Dysregulation of IEC homeostasis likely contributes to the development of intestinal inflammation and intestinal cancer. The roles of receptor protein tyrosine kinases and their downstream signaling molecules such as Src family kinases, Ras, and mTOR in homeostatic regulation of IEC turnover have recently been evaluated. These signaling pathways have been found to promote not only the proliferation of IECs but also the differentiation of progenitor cells into secretory cell types such as goblet cells. Of note, signaling by Src family kinases, Ras, and mTOR has been shown to oppose the Wnt‐β‐catenin signaling pathway and thereby to limit the number of Lgr5+ intestinal stem cells or of Paneth cells. Such cross‐talk of signaling pathways is important not only for proper regulation of IEC homeostasis but for the development of intestinal tumors and potentially for anticancer therapy.

Accelerated Intestinal Epithelial Cell Turnover Correlates with Stimulated BMP Signaling Cascade following Intestinal Ischemia-Reperfusion in a Rat.

Bone morphogenetic proteins (BMPs) are a family of proteins that regulate proliferation and differentiation of intestinal epithelial cells. The purpose of this study was to evaluate the role of BMP signaling following intestinal ischemia-reperfusion (IR) in a rat model. Male Sprague-Dawley rats were divided into four experimental groups: Sham-24 and Sham-48 rats underwent laparotomy and were sacrificed 24 or 48 hours later, respectively; IR-24 and IR-48 rats underwent occlusion of superior mesenteric artery and portal vein for 30 minutes followed by 24 or 48 hours of reperfusion, respectively. Enterocyte proliferation and apoptosis were determined at sacrifice. BMP-related genes and protein expression were determined using real-time polymerase chain reaction, Western blot, and immunohistochemistry for 48 hours followed by IR. IR rats demonstrated a significant increase in BMP2 (twofold increase, p < 0.05), BMP4 (sevenfold increase), STAT3 (70% increase), BMPR1 (70% increase) messenger ribonucleic acid levels in jejunum and was accompanied by a significant increase in BMP2 and BMP4 protein levels in jejunum (sixfold increase) (Western blot) and upward increase in the number of BMP-positive cells (by immunohistochemistry) in jejunal (48% increase) and ileal (56% increase) villi compared with Sham-48 animals. Elevation in BMP2 and BMP4 levels was associated with increased rates of cell proliferation and increased cell apoptosis. Forty-eight hours following intestinal IR in rats, BMP signaling pathway was stimulated. The increase in BMP signaling pathway activity correlates with accelerated cell turnover.

Understanding & ameliorating enteropathy and malnutrition in impoverished areas.

Understanding & ameliorating enteropathy and malnutrition in impoverished areas.

Long-term effect of parasympathetic or sympathetic denervation on intestinal epithelial cell proliferation and apoptosis

Intestinal epithelial tissue is constantly regenerated as a means to maintain proper tissue function. Previous studies have demonstrated that denervation of the parasympathetic or sympathetic nervous system to the intestine alters this process. However, results are inconsistent between studies, showing both increases and decreases in proliferation after denervation of the parasympathetic or sympathetic. The effect appears to correlate with (1) the timing post-denervation, (2) denervation-induced changes in food intake, (3) the denervation technique used, and (4) which intestinal segment is investigated. Thus, we proposed that parasympathetic or sympathetic denervation does not have an effect on intestinal epithelial regeneration when you (1) evaluate denervation after long-term denervation, (2) control for post-surgical changes in food intake, (3) use minimally invasive surgical techniques and (4) include a segmental analysis. To test this, adult male Sprague Dawley rats underwent parasympathetic denervation via subdiaphragmatic vagotomy, sympathetic denervation via celiacomesenteric ganglionectomy, a parasympathetic denervation sham surgery, or a sympathetic denervation sham surgery. Sham surgery ad libitum-fed groups and sham surgery pair-fed groups were used to control for surgically induced changes in food intake. Three weeks post-surgery, animals were sacrificed and tissue from the duodenum, jejunum, and ileum was excised and immunohistochemically processed to visualize indicators of proliferation (bromodeoxyuridine-positive cells) and apoptosis (caspase-3-positive cells). Results showed no differences between groups in proliferation, apoptosis, or total cell number in any intestinal segment. These results suggest that parasympathetic or sympathetic denervation does not have a significant long-term effect on intestinal epithelial turnover. Thus, intestinal epithelial regeneration is able to recover after autonomic nervous system injury. Impact statement This study investigates the long-term effect of autonomic denervation on intestinal epithelial cell turnover, as measured by proliferation, apoptosis, and total cell number. Although previous research has established that autonomic denervation can alter intestinal epithelial turnover under short-term conditions, here we establish for the first time that these changes do not persist long-term when you control for surgical-induced changes in food intake and use targeted denervation procedures. These findings add to the base of knowledge on autonomic control of tissue turnover, highlight the ability of the intestinal epithelium to recover after autonomic injury and reveal possible implications of the use of ANS denervation for disease treatment in humans.

Signaling and epigenetic mechanisms of intestinal stem cells and progenitors: insight into crypt homeostasis, plasticity, and niches.

The rapid turnover of intestinal epithelial cells is maintained by a small number of stem cells located in pocket-like gland structures called crypts. While our understanding of the identity and function of intestinal stem cells (ISCs) has rapidly progressed, epigenetic and transcriptional regulation in crypt stem cell and progenitor pools remains an active field of investigation. Surrounded by various types of cells in the stroma, crypt progenitors display high levels of plasticity, harboring the ability to interconvert in the face of epithelial damage. Recent studies analyzing epigenetic patterns of intestinal epithelial cells have provided evidence that plasticity is maintained by a broadly permissive epigenomic state, wherein cell-lineage specification is directed through activation of signaling pathways and transcription factor (TF) expression. New studies also have shown that the ISC niche, which is comprised of surrounding epithelial and mesenchymal tissues, plays a crucial role in supporting the maintenance and differentiation of stem cells by providing contextual information in the form of signaling cascades, such as Wnt, Notch, and Hippo. These cascades ultimately govern TF expression to promote early cell-lineage decisions in both crypt stem cells and progenitors. Highlighting recent studies investigating signaling, transcriptional, and epigenetic mechanisms of intestinal epithelial cells, we will discuss the mechanisms underlying crypt homeostasis, plasticity, and niches. WIREs Dev Biol 2017, 6:e281. doi: 10.1002/wdev.281 For further resources related to this article, please visit the WIREs website.

Intestinal epithelial suppressor of cytokine signaling 3 (SOCS3) impacts on mucosal homeostasis in a model of chronic inflammation.

IntroductionSuppressor of cytokine signaling 3 (SOCS3) is a tumour suppressor, limiting intestinal epithelial cell (IEC) proliferation in acute inflammation, and tumour growth, but little is known regarding its role in mucosal homeostasis. Resistance to the intestinal helminth Trichuris muris relies on an “epithelial escalator” to expel the parasite. IEC turnover is restricted by parasite‐induced indoleamine 2,3‐dioxygenase (IDO).MethodsMice with or without conditional knockout of SOCS3 were infected with T. muris. Crypt depth, worm burden, and proliferating cells and IDO were quantified. SOCS3 knockdown was also performed in human IEC cell lines.ResultsChronic T. muris infection increased expression of SOCS3 in wild‐type mice. Lack of IEC SOCS3 led to a modest increase in epithelial turnover. This translated to a lower worm burden, but not complete elimination of the parasite suggesting a compensatory mechanism, possibly IDO, as seen in SOCS3 knockdown.ConclusionsWe report that SOCS3 impacts on IEC turnover following T. muris infection, potentially through enhancement of IDO. IDO may dampen the immune response which can drive IEC hyperproliferation in the absence of SOCS3, demonstrating the intricate interplay of immune signals regulating mucosal homeostasis, and suggesting a novel tumour suppressor role of SOCS3.

Emerging role of zinc homeostasis by zinc transporter ZIP7 in intestinal homeostatic self-renewal

There has been a growing interest in the biological significance of zinc and its regulatory mechanism in the development of tissue and in the maintenance of tissue homeostasis. The intestinal epithelium undergoes a continuous self-renewing process to maintain the intestinal homeostasis. Moreover, dysregulation of this process often causes various intestinal disorders including inflammatory bowel disease, ulcer, and cancer. However, the molecular basis of zinc-dependent regulation of intestinal epithelial cell turnover is not fully understood. In this research highlight, we describe that the zinc transporter ZIP7 (highly expressed in undifferentiated epithelial cells) plays a critical role in the intestinal epithelial self-renewal process by alleviating the ER stress during vigorous proliferative response. Our findings also provide evidence showing that the fine-tuning of intracellular zinc homeostasis by ZIP7 is essential to maintain epithelial homeostasis in the intestine.

Review Article. Absent in melanoma 2 (AIM2) in the intestine: diverging actions with converging consequences

AbstractThe intestinal mucosa is a difficult environment to maintain homeostasis as it is constantly challenged by microbial and food antigens. Maintaining an intact epithelial barrier, a continuous turnover of intestinal epithelial cells and normobiosis of the gut microbiota are essential components to prevent intestinal diseases such as inflammatory bowel diseases (IBD) and colorectal cancer (CRC). Inflammasomes are critical immune regulators that are involved in all of these processes. They are multiprotein complexes able to assemble upon interaction with a noxious stimulus that will subsequently lead to caspase-1 activation. Activated caspase-1 will orchestrate the maturation and release of proinflammatory cytokines IL-1β and IL-18, and induce pyroptosis, an inflammatory form of cell death. Both cytokine release and pyroptosis are initiated after detection of molecular patterns by a distinct inflammasome sensor protein. Absent in melanoma 2 (AIM2) is such an inflammasome sensor that specifically responds to the presence of double stranded DNA (dsDNA) in the cytoplasm, leading to the recruitment and activation of caspase-1. Recent studies revealed additional roles of AIM2 in controlling epithelial cell proliferation, tight junction expression and the microbiome. Therefore, AIM2 plays a significant role in maintaining intestinal homeostasis. This review focuses on the multifunctional role of AIM2 in intestinal homeostasis by regulating intestinal immunity and preventing colorectal cancer development.

Role of Src Family Kinases in Regulation of Intestinal Epithelial Homeostasis.

Proper regulation of epithelial cell turnover is important for the structural integrity and homeostasis of various tissues, including the intestine. Here we show that ablation of Csk, a negative regulator of Src family kinases (SFKs), specifically in intestinal epithelial cells (IECs) resulted in the development of hyperplasia throughout the intestinal epithelium of mice. Such conditional ablation of Csk also increased the proliferative activity and turnover of IECs, disturbed the differentiation of Paneth and goblet cells, reduced the number of intestinal stem cells, and attenuated the expression of Wnt target genes in the intestine. Moreover, the tyrosine phosphorylation of focal adhesion kinase (FAK) and the activities of both Rac and Yes-associated protein (YAP) were increased in intestinal crypts or organoids of the mutant mice, whereas inhibition of Rac or YAP activity rescued the mutant phenotypes. Our results thus suggest that SFKs promote the proliferation of IECs in intestinal crypts through activation of Rac or YAP and that they thereby contribute to the proper regulation of IEC turnover and intestinal homeostasis.

Protein Malnutrition Impairs Intestinal Epithelial Cell Turnover, a Potential Mechanism of Increased Cryptosporidiosis in a Murine Model.

Malnutrition and cryptosporidiosis form a vicious cycle and lead to acute and long-term growth impairment in children from developing countries. Insights into mechanisms underlying the vicious cycle will help to design rational therapies to mitigate this infection. We tested the effect of short-term protein malnutrition on Cryptosporidium parvum infection in a murine model by examining stool shedding, tissue burden, and histologic change and explored the mechanism underlying the interaction between malnutrition and cryptosporidiosis through immunostaining and immunoblotting. Protein malnutrition increased stool shedding and the number of intestine-associated C. parvum organisms, accompanied by significant suppression of C. parvum-induced caspase 3 activity and expression of PCNA and Ki67, but activation of the Akt survival pathway in intestinal epithelial cells. We find that even very brief periods of protein malnutrition may enhance (or intensify) cryptosporidiosis by suppressing C. parvum-induced cell turnover and caspase-dependent apoptosis of intestinal epithelial cells. This implicates a potential strategy to attenuate C. parvum's effects by modulating apoptosis and promoting regeneration in the intestinal epithelium.

Fenofibrate reduces intestinal damage and improves intestinal recovery following intestinal ischemia-reperfusion injury in a rat.

Fenofibrate (FEN) is known as a nuclear receptor activator which regulates many pathophysiological processes, such as oxidative stress, inflammation, and leukocyte endothelium interactions. Recent studies have demonstrated an anti-oxidant, anti-inflammatory, and anti-ischemic role of FEN in the attenuation of ischemia-reperfusion (IR) injury in the kidney, liver, brain, and heart. The purpose of the present study was to examine the effect of FEN on intestinal recovery and enterocyte turnover after intestinal IR injury in rats. Male Sprague-Dawley rats were divided into four experimental groups: (1) sham rats underwent laparotomy, (2) sham-FEN rats underwent laparotomy and were treated with intraperitoneal (IP) FEN (20mg/kg); (3) IR rats underwent occlusion of both the superior mesenteric artery and the portal vein for 30min followed by 24h of reperfusion, and (4) IR-FEN rats underwent IR and were treated with IP FEN immediately before abdominal closure. Intestinal structural changes, Park's injury score, enterocyte proliferation, and enterocyte apoptosis were determined 24h following IR. The expression of Bax, Bcl-2, p-ERK, and caspase-3 in the intestinal mucosa was determined using real-time PCR, Western blot, and immunohistochemistry. Treatment with FEN resulted in a significant decrease in Park's injury score in jejunum (32%) and ileum (33%) compared to IR animals. IR-FEN rats also demonstrated a significant increase in mucosal weight in jejunum (23%) and ileum (22%), mucosal DNA (38%) and protein (65%) in jejunum, villus height in jejunum (17%) and ileum (21%), and crypt depth in ileum (14%) compared to IR animals. IR-FEN rats also experienced significant proliferation rates as well as lower apoptotic indices in jejunum and ileum which was accompanied with higher Bcl-2 levels compared to IR animals. Treatment with fenofibrate prevents intestinal mucosal damage and stimulates intestinal epithelial cell turnover following intestinal IR in a rat model.

Accelerated intestinal epithelial cell turnover after bowel resection in a rat is correlated with inhibited hedgehog signaling cascade.

The hedgehog (Hh) signaling pathway is one of the key regulators of gastrointestinal tract development. Recent studies point to the role of hedgehog signaling in regulating adult stem cells involved in maintenance and regeneration of intestinal stem cells. The purpose of this study was to evaluate the role of Hh signaling during intestinal adaptation in a rat model of short bowel syndrome (SBS). Male rats were divided into two groups: Sham rats underwent bowel transection and SBS rats underwent a 75% bowel resection. Parameters of intestinal adaptation, enterocyte proliferation, and apoptosis were determined 2weeks after operation. Illumina's Digital Gene Expression analysis was used to determine the Hh signaling gene expression profiling. Hh-related genes and protein expression were determined using Real-Time PCR, Western blotting, and immunohistochemistry. Massive small bowel resection resulted in a significant increase in enterocyte proliferation and concomitant increase in cell apoptosis. From the total number of 20,000 probes, 13 genes related to Hh signaling were investigated. In jejunum, eight genes were down-regulated, three genes up-regulated, and two genes remained unchanged. In ileum, five genes were down-regulated and six genes were unchanged in SBS vs sham animals. SBS rats also demonstrated a significant three- to fourfold decrease in SMO, GIL, and PTCH mRNA, and protein levels (determined by Real-Time PCR and Western blot) compared to control animals. Two weeks following massive bowel resection in rats, the accelerated cell turnover was accompanied by an inhibited Hh signaling pathway. Hh signaling may serve as an important mediator of reciprocal interactions between the epithelium and the underlying mesenchymal stroma during intestinal adaptation following massive bowel resection in a rat.

Intestinal Epithelial Cell-Intrinsic Deletion of Setd7 Identifies Role for Developmental Pathways in Immunity to Helminth Infection.

The intestine is a common site for a variety of pathogenic infections. Helminth infections continue to be major causes of disease worldwide, and are a significant burden on health care systems. Lysine methyltransferases are part of a family of novel attractive targets for drug discovery. SETD7 is a member of the Suppressor of variegation 3-9-Enhancer of zeste-Trithorax (SET) domain-containing family of lysine methyltransferases, and has been shown to methylate and alter the function of a wide variety of proteins in vitro. A few of these putative methylation targets have been shown to be important in resistance against pathogens. We therefore sought to study the role of SETD7 during parasitic infections. We find that Setd7 -/- mice display increased resistance to infection with the helminth Trichuris muris but not Heligmosomoides polygyrus bakeri. Resistance to T. muris relies on an appropriate type 2 immune response that in turn prompts intestinal epithelial cells (IECs) to alter differentiation and proliferation kinetics. Here we show that SETD7 does not affect immune cell responses during infection. Instead, we found that IEC-specific deletion of Setd7 renders mice resistant to T. muris by controlling IEC turnover, an important aspect of anti-helminth immune responses. We further show that SETD7 controls IEC turnover by modulating developmental signaling pathways such as Hippo/YAP and Wnt/β-Catenin. We show that the Hippo pathway specifically is relevant during T. muris infection as verteporfin (a YAP inhibitor) treated mice became susceptible to T. muris. We conclude that SETD7 plays an important role in IEC biology during infection.