DHX9 maintains epithelial homeostasis by restraining R-loop-mediated genomic instability in intestinal stem cells.

Epithelial barrier dysfunction and crypt destruction are hallmarks of inflammatory bowel disease (IBD). Intestinal stem cells (ISCs) residing in the crypts play a crucial role in the continuous self-renewal and rapid recovery of intestinal epithelial cells (IECs). However, how ISCs are dysregulated in IBD remains poorly understood. Here, we observe reduced DHX9 protein levels in IBD patients, and mice with conditional DHX9 depletion in the intestinal epithelium (Dhx9ΔIEC) exhibit an increased susceptibility to experimental colitis. Notably, Dhx9ΔIEC mice display a significant reduction in the numbers of ISCs and Paneth cells. Further investigation using ISC-specific or Paneth cell-specific Dhx9-deficient mice demonstrates the involvement of ISC-expressed DHX9 in maintaining epithelial homeostasis. Mechanistically, DHX9 deficiency leads to abnormal R-loop accumulation, resulting in genomic instability and the cGAS-STING-mediated inflammatory response, which together impair ISC function and contribute to the pathogenesis of IBD. Collectively, our findings highlight R-loop-mediated genomic instability in ISCs as a risk factor in IBD.

Mechanisms and regulation of defensins in host defense.

As a family of cationic host defense peptides, defensins are mainly synthesized by Paneth cells, neutrophils, and epithelial cells, contributing to host defense. Their biological functions in innate immunity, as well as their structure and activity relationships, along with their mechanisms of action and therapeutic potential, have been of great interest in recent years. To highlight the key research into the role of defensins in human and animal health, we first describe their research history, structural features, evolution, and antimicrobial mechanisms. Next, we cover the role of defensins in immune homeostasis, chemotaxis, mucosal barrier function, gut microbiota regulation, intestinal development and regulation of cell death. Further, we discuss their clinical relevance and therapeutic potential in various diseases, including infectious disease, inflammatory bowel disease, diabetes and obesity, chronic inflammatory lung disease, periodontitis and cancer. Finally, we summarize the current knowledge regarding the nutrient-dependent regulation of defensins, including fatty acids, amino acids, microelements, plant extracts, and probiotics, while considering the clinical application of such regulation. Together, the review summarizes the various biological functions, mechanism of actions and potential clinical significance of defensins, along with the challenges in developing defensins-based therapy, thus providing crucial insights into their biology and potential clinical utility.

Crohn's disease: Why the ileum?

Crohn's disease (CD) is an inflammatory bowel disease characterized by immune-mediated flares affecting any region of the intestine alternating with remission periods. In CD, the ileum is frequently affected and about one third of patients presents with a pure ileal type. Moreover, the ileal type of CD presents epidemiological specificities like a younger age at onset and often a strong link with smoking and genetic susceptibility genes. Most of these genes are associated with Paneth cell dysfunction, a cell type found in the intestinal crypts of the ileum. Besides, a Western-type diet is associated in epidemiological studies with CD onset and increasing evidence shows that diet can modulate the composition of bile acids and gut microbiota, which in turn modulates the susceptibility of the ileum to inflammation. Thus, the interplay between environmental factors and the histological and anatomical features of the ileum is thought to explain the specific transcriptome profile observed in CD ileitis. Indeed, both immune response and cellular healing processes harbour differences between ileal and non-ileal CD. Taken together, these findings advocate for a dedicated therapeutic approach to managing ileal CD. Currently, interventional pharmacological studies have failed to clearly demonstrate distinct response profiles according to disease site. However, the high rate of stricturing disease in ileal CD requires the identification of new therapeutic targets to significantly change the natural history of this debilitating disease.

LAT1 expression influences Paneth cell number and tumor development in ApcMin/+ mice.

BACKGROUND: Amino acid transporters play an important role in supplying nutrition to cells and are associated with cell proliferation. L-type amino acid transporter 1 (LAT1) is highly expressed in many types of cancers and promotes tumor growth; however, how LAT1 affects tumor development is not fully understood. METHODS: To investigate the role of LAT1 in intestinal tumorigenesis, mice carrying LAT1 floxed alleles that also expressed Cre recombinase from the promoter of gene encoding Villin were crossed to an ApcMin/+ background (LAT1fl/fl; vil-cre; ApcMin/+), which were subject to analysis; organoids derived from those mice were also analyzed. RESULTS: This study showed that LAT1 was constitutively expressed in normal crypt base cells, and its conditional deletion in the intestinal epithelium resulted in fewer Paneth cells. LAT1 deletion reduced tumor size and number in the small intestine of ApcMin/+ mice. Organoids derived from LAT1-deleted ApcMin/+ intestinal crypts displayed fewer spherical organoids with reduced Wnt/ß-catenin target gene expression, suggesting a low tumor-initiation capacity. Wnt3 expression was decreased in the absence of LAT1 in the intestinal epithelium, suggesting that loss of Paneth cells due to LAT1 deficiency reduced the risk of tumor initiation by decreasing Wnt3 production. CONCLUSIONS: LAT1 affects intestinal tumor development in a cell-extrinsic manner through reduced Wnt3 expression in Paneth cells. Our findings may partly explain how nutrient availability can affect the risk of tumor development in the intestines.

Chronic GPER activation prompted the proliferation of ileal stem cell in ovariectomized mice depending on Paneth cell-derived Wnt3.

Menopausal women often face long-term estrogen treatment. G protein-coupled estrogen receptor (GPER) expressed in intestinal crypt was activated by estrogen therapy, but it was unclear whether chronic GPER activation during menopause had an effect on intestinal stem cells (ISCs). We tested the effect of chronic GPER activation on ISCs of ovariectomized (OVX) mice by injection of the selective GPER agonist G-1 for 28 days, or G-1 stimulation of organoids derived from crypts of OVX mice. G-1 up-regulated crypt depth, the number of Ki67+, bromodeoxyuridine+ cells and Olfm4+ ISCs, and the expression of ISCs marker genes (Lgr5, Olfm4 and Axin2). G-1 administration promoted organoid growth, increased the number of EdU+ cells per organoid and protein expression of Cyclin D1 and cyclin B1 in organoids. After G-1 treatment in vivo or in vitro, Paneth cell-derived Wnt3, Wnt3 effector ß-catenin and Wnt target genes c-Myc and Cyclin D1 increased in ileum or organoids. Once blocking the secretion of Wnt3 from Paneth cells, the effects of G-1 on organoids growth, ISCs marker genes and Wnt/ß-catenin signaling were abolished. G-1 did not affect the number of Paneth cells in ex vivo organoids, while activated Mmp7/cryptdin program in Paneth cells, promoted their maturation, and increased the expression of lysozyme protein. G-1 pretreatment in OVX mice inhibited radiation-induced ISCs proliferation injury and enhanced the resistance of mice to intestinal injury. In conclusion, chronic GPER activation prompted the Wnt3 synthesis in Paneth cells, thus increased the proliferation of ISCs via activation of Wnt3/ß-catenin signaling in OVX mice.

Non-enzymatic role of SOD1 in intestinal stem cell growth.

Superoxide dismutase 1 (SOD1) modulates intestinal barrier integrity and intestinal homeostasis as an antioxidant enzyme. Intestinal homeostasis is maintained by the intestinal stem cells (ISCs). However, whether and how SOD1 regulates ISCs is unknown. In this study, we established intestinal organoids from tamoxifen-inducible intestinal epithelial cell-specific Sod1 knockout (Sod1f/f; Vil-creERT2) mice. We found that loss of Sod1 in organoids suppressed the proliferation and survival of cells and Lgr5 gene expression. SOD1 is known for nearly half a century for its canonical role as an antioxidant enzyme. We identified its enzyme-independent function in ISC: inhibition of SOD1 enzymatic activity had no impact on organoid growth, and enzymatically inactive Sod1 mutants could completely rescue the growth defects of Sod1 deficient organoids, suggesting that SOD1-mediated ISC growth is independent of its enzymatic activity. Moreover, Sod1 deficiency did not affect the ROS levels of the organoid, but induced the elevated WNT signaling and excessive Paneth cell differentiation, which mediates the occurrence of growth defects in Sod1 deficient organoids. In vivo, epithelial Sod1 loss induced a higher incidence of apoptosis in the stem cell regions and increased Paneth cell numbers, accompanied by enhanced expression of EGFR ligand Epiregulin (EREG) in the stromal tissue, which may compensate for Sod1 loss and maintain intestinal structure in vivo. Totally, our results show a novel enzyme-independent function of SOD1 in ISC growth under homeostasis.

Implications of Paneth cell dysfunction on gastrointestinal health and disease.

PURPOSE OF REVIEW: Paneth cells are specialized, secretory epithelial cells located in the small intestine. Although their existence was first described in 1872, their precise role in the gut remained unclear for over a century. Over the past few decades, elegant studies have shown Paneth cells play a key role enhancing gut barrier function, as niche cells for the intestinal stem cell compartment and via secreting antimicrobial peptides to establish an antimicrobial barrier at the epithelial surface. This review describes what is known about Paneth cell biology from human and animal studies with a focus on their putative role in clinical gastrointestinal disease. RECENT FINDINGS: Recent work has demonstrated important associations of dysfunctional Paneth cells with several gastrointestinal disorders. These include Crohn's disease, enteric infections, graft-versus-host disease, necrotizing enterocolitis, and environmental enteric dysfunction. Ongoing studies are examining precisely how Paneth cell biology is altered in these various disease states. SUMMARY: By understanding the mechanisms of Paneth cell regulation - and how these processes go awry in specific gastrointestinal diseases - we set the stage for using Paneth cells as biomarkers for disease progression and developing novel therapeutics that augment Paneth cell function to treat a spectrum of gastrointestinal disorders.

IκBζ controls IL-17-triggered gene expression program in intestinal epithelial cells that restricts colonization of SFB and prevents Th17-associated pathologies.

Control of gut microbes is crucial for not only local defense in the intestine but also proper systemic immune responses. Although intestinal epithelial cells (IECs) play important roles in cytokine-mediated control of enterobacteria, the underlying mechanisms are not fully understood. Here we show that deletion of IκBζ in IECs in mice leads to dysbiosis with marked expansion of segmented filamentous bacteria (SFB), thereby enhancing Th17 cell development and exacerbating inflammatory diseases. Mechanistically, the IκBζ deficiency results in decrease in the number of Paneth cells and impairment in expression of IL-17-inducible genes involved in IgA production. The decrease in Paneth cells is caused by aberrant activation of IFN-γ signaling and a failure of IL-17-dependent recovery from IFN-γ-induced damage. Thus, the IL-17R-IκBζ axis in IECs contributes to the maintenance of intestinal homeostasis by serving as a key component in a regulatory loop between the gut microbiota and immune cells.

Intestinal TLR4 deletion exacerbates acute pancreatitis through gut microbiota dysbiosis and Paneth cells deficiency.

Toll-like receptor 4 (TLR4) has been identified as a potentially promising therapeutic target in acute pancreatitis (AP). However, the role of intestinal TLR4 in AP and AP-associated gut injury remains unclear. This study aimed to explore the relationship between intestinal TLR4 and gut microbiota during AP. A mouse AP model was establish by intraperitoneal injection of L-arginine. Pancreatic injury and intestinal barrier function were evaluated in wild-type and intestinal epithelial TLR4 knockout (TLR4ΔIEC) mice. Gut microbiota was analyzed by 16S rRNA sequencing. Quadruple antibiotics were applied to induce microbiota-depleted mice. Differentially expressed genes in gut were detected by RNA sequencing. L. reuteri treatment was carried out in vivo and vitro study. Compared with wild-type mice, AP and AP-associated gut injury were exacerbated in TLR4ΔIEC mice in a gut microbiota-dependent manner. The relative abundance of Lactobacillus and number of Paneth cells remarkably decreased in TLR4ΔIEC mice. The KEGG pathway analysis derived from RNA sequencing suggested that genes affected by intestinal TLR4 deletion were related to the activation of nod-like receptor pathway. Furthermore, L. reuteri treatment could significantly improve the pancreatic and intestinal injury in TLR4ΔIEC mice through promoting Paneth cells in a NOD2-dependent manner. Loss of intestinal epithelial TLR4 exacerbated pancreatic and intestinal damage during AP, which might be attributed to the gut microbiota dysbiosis especially the exhausted Lactobacillus. L. reuteri might maintain intestinal homeostasis and alleviate AP via Paneth cells modulation.Abbreviations: AP Acute pancreatitis, TLR4 Toll-like receptor 4, IL-1ß Interleukin-1ß, IL-6 Interleukin-6, TNF-α Tumor necrosis factor-α, SIRS Systematic inflammatory response syndrome, LPS Lipopolysaccharides, SPF Specific pathogen-free, ZO-1 Zonula occludens-1, CON Control, H&E Hematoxylin and eosin, FISH Fluorescence in situ hybridization, DAPI 4',6-diamidino-2-phenylindole, PCoA Principal co-ordinates analysis, SCFA Short chain fatty acid, LEfSe Linear discriminant analysis Effect Size, ANOVA Analysis of variance, F/B Firmicutes/Bacteroidetes, PCA Principal component analysis, NOD2 Nod-like receptor 2, ABX antibiotics, PCNA proliferating cell nuclear antigen.

Single-Cell Analysis Reveals Unexpected Cellular Changes and Transposon Expression Signatures in the Colonic Epithelium of Treatment-Naïve Adult Crohn's Disease Patients.

BACKGROUND & AIMS: The intestinal barrier comprises a monolayer of specialized intestinal epithelial cells (IECs) that are critical in maintaining mucosal homeostasis. Dysfunction within various IEC fractions can alter intestinal permeability in a genetically susceptible host, resulting in a chronic and debilitating condition known as Crohn's disease (CD). Defining the molecular changes in each IEC type in CD will contribute to an improved understanding of the pathogenic processes and the identification of cell type-specific therapeutic targets. We performed, at single-cell resolution, a direct comparison of the colonic epithelial cellular and molecular landscape between treatment-naïve adult CD and non-inflammatory bowel disease control patients. METHODS: Colonic epithelial-enriched, single-cell sequencing from treatment-naïve adult CD and non-inflammatory bowel disease patients was investigated to identify disease-induced differences in IEC types. RESULTS: Our analysis showed that in CD patients there is a significant skew in the colonic epithelial cellular distribution away from canonical LGR5+ stem cells, located at the crypt bottom, and toward one specific subtype of mature colonocytes, located at the crypt top. Further analysis showed unique changes to gene expression programs in every major cell type, including a previously undescribed suppression in CD of most enteroendocrine driver genes as well as L-cell markers including GCG. We also dissect an incompletely understood SPIB+ cell cluster, revealing at least 4 subclusters that likely represent different stages of a maturational trajectory. One of these SPIB+ subclusters expresses crypt-top colonocyte markers and is up-regulated significantly in CD, whereas another subcluster strongly expresses and stains positive for lysozyme (albeit no other canonical Paneth cell marker), which surprisingly is greatly reduced in expression in CD. In addition, we also discovered transposable element markers of colonic epithelial cell types as well as transposable element families that are altered significantly in CD in a cell type-specific manner. Finally, through integration with data from genome-wide association studies, we show that genes implicated in CD risk show heretofore unknown cell type-specific patterns of aberrant expression in CD, providing unprecedented insight into the potential biological functions of these genes. CONCLUSIONS: Single-cell analysis shows a number of unexpected cellular and molecular features, including transposable element expression signatures, in the colonic epithelium of treatment-naïve adult CD.

Human intelectin-2 (ITLN2) is selectively expressed by secretory Paneth cells.

Intelectins (intestinal lectins) are highly conserved across chordate evolution and have been implicated in various human diseases, including Crohn's disease (CD). The human genome encodes two intelectin genes, intelectin-1 (ITLN1) and intelectin-2 (ITLN2). Other than its high sequence similarity with ITLN1, little is known about ITLN2. To address this void in knowledge, we report that ITLN2 exhibits discrete, yet notable differences from ITLN1 in primary structure, including a unique amino terminus, as well as changes in amino acid residues associated with the glycan-binding activity of ITLN1. We identified that ITLN2 is a highly abundant Paneth cell-specific product, which localizes to secretory granules, and is expressed as a multimeric protein in the small intestine. In surgical specimens of ileal CD, ITLN2 mRNA levels were reduced approximately five-fold compared to control specimens. The ileal expression of ITLN2 was unaffected by previously reported disease-associated variants in ITLN2 and CD-associated variants in neighboring ITLN1 as well as NOD2 and ATG16L1. ITLN2 mRNA expression was undetectable in control colon tissue; however, in both ulcerative colitis (UC) and colonic CD, metaplastic Paneth cells were found to express ITLN2. Together, the data reported establish the groundwork for understanding ITLN2 function(s) in the intestine, including its possible role in CD.

NF-κB determines Paneth versus goblet cell fate decision in the small intestine.

Although the role of the transcription factor NF-κB in intestinal inflammation and tumor formation has been investigated extensively, a physiological function of NF-κB in sustaining intestinal epithelial homeostasis beyond inflammation has not been demonstrated. Using NF-κB reporter mice, we detected strong NF-κB activity in Paneth cells, in '+4/+5' secretory progenitors and in scattered Lgr5+ crypt base columnar stem cells of small intestinal (SI) crypts. To examine NF-κB functions in SI epithelial self-renewal, mice or SI crypt organoids ('mini-guts') with ubiquitously suppressed NF-κB activity were used. We show that NF-κB activity is dispensable for maintaining SI epithelial proliferation, but is essential for ex vivo organoid growth. Furthermore, we demonstrate a dramatic reduction of Paneth cells in the absence of NF-κB activity, concomitant with a significant increase in goblet cells and immature intermediate cells. This indicates that NF-κB is required for proper Paneth versus goblet cell differentiation and for SI epithelial homeostasis, which occurs via regulation of Wnt signaling and Sox9 expression downstream of NF-κB. The current study thus presents evidence for an important role for NF-κB in intestinal epithelial self-renewal.

The phosphatase PRL-3 affects intestinal homeostasis by altering the crypt cell composition.

Expression of the phosphatase of regenerating liver-3 (PRL-3) is known to promote tumor growth in gastrointestinal adenocarcinomas, and the incidence of tumor formation upon inflammatory events correlates with PRL-3 levels in mouse models. These carcinomas and their onset are associated with the impairment of intestinal cell homeostasis, which is regulated by a balanced number of Paneth cells and Lgr5 expressing intestinal stem cells (Lgr5+ ISCs). Nevertheless, the consequences of PRL-3 overexpression on cellular homeostasis and ISC fitness in vivo are unexplored. Here, we employ a doxycycline-inducible PRL-3 mouse strain to show that aberrant PRL-3 expression within a non-cancerous background leads to the death of Lgr5+ ISCs and to Paneth cell expansion. A higher dose of PRL-3, resulting from homozygous expression, led to mice dying early. A primary 3D intestinal culture model obtained from these mice confirmed the loss of Lgr5+ ISCs upon PRL-3 expression. The impaired intestinal organoid formation was rescued by a PRL inhibitor, providing a functional link to the observed phenotypes. These results demonstrate that elevated PRL-3 phosphatase activity in healthy intestinal epithelium impairs intestinal cell homeostasis, which correlates this cellular mechanism of tumor onset with PRL-3-mediated higher susceptibility to tumor formation upon inflammatory or mutational events.Key messages• Transgenic mice homozygous for PRL-3 overexpression die early.• PRL-3 heterozygous mice display disrupted intestinal self-renewal capacity.• PRL-3 overexpression alone does not induce tumorigenesis in the mouse intestine.• PRL-3 activity leads to the death of Lgr5+ ISCs and Paneth cell expansion.• Impairment of cell homeostasis correlates PRL-3 action with tumor onset mechanisms.

High-content, targeted RNA-seq screening in organoids for drug discovery in colorectal cancer.

Organoids allow the recapitulation of intestinal homeostasis and cancerogenesis in vitro; however, RNA sequencing (RNA-seq)-based methods for drug screens are missing. We develop targeted organoid sequencing (TORNADO-seq), a high-throughput, high-content drug discovery platform that uses targeted RNA-seq to monitor the expression of large gene signatures for the detailed evaluation of cellular phenotypes in organoids. TORNADO-seq is a fast, highly reproducible time- and cost-effective ($5 per sample) method that can probe cell mixtures and their differentiation state in the intestinal system. We apply this method to isolate drugs that enrich for differentiated cell phenotypes and show that these drugs are highly efficacious against cancer compared to wild-type organoids. Furthermore, TORNADO-seq facilitates in-depth insight into the mode of action of these drugs. Our technology can easily be adapted to many other systems and will allow for more systematic, large-scale, and quantitative approaches to study the biology of complex cellular systems.

Non-muscle myosin heavy chain 9 maintains intestinal homeostasis by preventing epithelium necroptosis and colitis adenoma formation.

Non-muscle myosin IIA plays an important role in cell adhesion, cell migration, and tissue architecture. We previously showed that low activity of the heavy chain of non-muscle myosin II Myh9 is beneficial to LGR5+ intestinal stem cell maintenance. However, the function of Myh9 in adult mouse intestinal epithelium is largely unclear. In this study, we used the inducible Villin-creERT2 knockout approach to delete Myh9 in adult mouse intestinal epithelium and observed that homozygous deletion of Myh9 causes colitis-like morphologic changes in intestine, leads to a high sensitivity to dextran sulfate sodium and promotes colitis-related adenoma formation in the colon. Myh9 deletion disturbs cell junctions and impairs intestinal lumen barrier integrity, promoting the necroptosis of epithelial cells. Consistently, these changes can be partially rescued by Ripk3 knockout. Our results indicate that Myh9 is required for the maintenance of intestinal epithelium integrity and the prevention of cell necroptosis.

Gut microbiota shape the inflammatory response in mice with an epithelial defect.

Intestinal epithelial cell endoplasmic reticulum (ER) stress has been implicated in intestinal inflammation. It remains unclear whether ER stress is an initiator of or a response to inflammation. Winnie mice, carrying a Muc2 gene mutation resulting in intestinal goblet cell ER stress, develop spontaneous colitis with a depleted mucus barrier and increased bacterial translocation. This study aims to determine whether the microbiota was required for the development of Winnie colitis, and whether protein misfolding itself can initiate inflammation directly in absence of the microbiota. To assess the role of microbiota in driving Winnie colitis, WT and Winnie mice on the same background were rederived into the germ-free facility and housed in the Trexler-type soft-sided isolators. The colitis phenotype of these mice was assessed and compared to WT and Winnie mice housed within a specific pathogen-free facility. We found that Winnie colitis was substantially reduced but not abolished under germ-free conditions. Expression of inflammatory cytokine genes was reduced but several chemokines remained elevated in absence of microbiota. Concomitantly, ER stress was also diminished, although mucin misfolding persisted. RNA-Seq revealed that Winnie differentiated colon organoids have decreased expression of the negative regulators of the inflammatory response compared to WT. This data along with the increase in Mip2a chemokine expression, suggests that the epithelial cells in the Winnie mice are more responsive to stimuli. Moreover, the data demonstrate that intestinal epithelial intrinsic protein misfolding can prime an inflammatory response without initiating the unfolded protein response in the absence of the microbiota. However, the microbiota is necessary for the amplification of colitis in Winnie mice. Genetic predisposition to mucin misfolding in secretory cells initiates mild inflammatory signals. However, the inflammatory signal sets a forward-feeding cycle establishing progressive inflammation in the presence of microbiota.Abbreviations: Endoplasmic Reticulum: ER; Mucin-2: Muc-2; GF: Germ-Free; Inflammatory Bowel Disease: IBD.

Human placental-derived stem cell therapy ameliorates experimental necrotizing enterocolitis.

Necrotizing enterocolitis (NEC), a life-threatening intestinal disease, is becoming a larger proportionate cause of morbidity and mortality in premature infants. To date, therapeutic options remain elusive. Based on recent cell therapy studies, we investigated the effect of a human placental-derived stem cell (hPSC) therapy on intestinal damage in an experimental NEC rat pup model. NEC was induced in newborn Sprague-Dawley rat pups for 4 days via formula feeding, hypoxia, and LPS. NEC pups received intraperitoneal (ip) injections of either saline or hPSC (NEC-hPSC) at 32 and 56 h into NEC induction. At 4 days, intestinal macroscopic and histological damage, epithelial cell composition, and inflammatory marker expression of the ileum were assessed. Breastfed (BF) littermates were used as controls. NEC pups developed significant bowel dilation and fragility in the ileum. Further, NEC induced loss of normal villi-crypt morphology, disruption of epithelial proliferation and apoptosis, and loss of critical progenitor/stem cell and Paneth cell populations in the crypt. hPSC treatment improved macroscopic intestinal health with reduced ileal dilation and fragility. Histologically, hPSC administration had a significant reparative effect on the villi-crypt morphology and epithelium. In addition to a trend of decreased inflammatory marker expression, hPSC-NEC pups had increased epithelial proliferation and decreased apoptosis when compared with NEC littermates. Further, the intestinal stem cell and crypt niche that include Paneth cells, SOX9+ cells, and LGR5+ stem cells were restored with hPSC therapy. Together, these data demonstrate hPSC can promote epithelial healing of NEC intestinal damage.NEW & NOTEWORTHY These studies demonstrate a human placental-derived stem cell (hPSC) therapeutic strategy for necrotizing enterocolitis (NEC). In an experimental model of NEC, hPSC administration improved macroscopic intestinal health, ameliorated epithelial morphology, and supported the intestinal stem cell niche. Our data suggest that hPSC are a potential therapeutic approach to attenuate established intestinal NEC damage. Further, we show hPSC are a novel research tool that can be utilized to elucidate critical neonatal repair mechanisms to overcome NEC.

Cdk5rap3 is essential for intestinal Paneth cell development and maintenance.

Intestinal Paneth cells are professional exocrine cells that play crucial roles in maintenance of homeostatic microbiome, modulation of mucosal immunity, and support for stem cell self-renewal. Dysfunction of these cells may lead to the pathogenesis of human diseases such as inflammatory bowel disease (IBD). Cdk5 activator binding protein Cdk5rap3 (also known as C53 and LZAP) was originally identified as a binding protein of Cdk5 activator p35. Although previous studies have indicated its involvement in a wide range of signaling pathways, the physiological function of Cdk5rap3 remains largely undefined. In this study, we found that Cdk5rap3 deficiency resulted in very early embryonic lethality, indicating its indispensable role in embryogenesis. To further investigate its function in the adult tissues and organs, we generated intestinal epithelial cell (IEC)-specific knockout mouse model to examine its role in intestinal development and tissue homeostasis. IEC-specific deletion of Cdk5rap3 led to nearly complete loss of Paneth cells and increased susceptibility to experimentally induced colitis. Interestingly, Cdk5rap3 deficiency resulted in downregulation of key transcription factors Gfi1 and Sox9, indicating its crucial role in Paneth cell fate specification. Furthermore, Cdk5rap3 is highly expressed in mature Paneth cells. Paneth cell-specific knockout of Cdk5rap3 caused partial loss of Paneth cells, while inducible acute deletion of Cdk5rap3 resulted in disassembly of the rough endoplasmic reticulum (RER) and abnormal zymogen granules in the mature Paneth cells, as well as loss of Paneth cells. Together, our results provide definitive evidence for the essential role of Cdk5rap3 in Paneth cell development and maintenance.

JAK-STAT Pathway Inhibition Partially Restores Intestinal Homeostasis in Hdac1- and Hdac2-Intestinal Epithelial Cell-Deficient Mice.

We have previously reported that histone deacetylase epigenetic regulator Hdac1 and Hdac2 deletion in intestinal epithelial cells (IEC) disrupts mucosal tissue architecture and barrier, causing chronic inflammation. In this study, proteome and transcriptome analysis revealed the importance of signaling pathways induced upon genetic IEC-Hdac1 and Hdac2 deletion. Indeed, Gene Ontology biological process analysis of enriched deficient IEC RNA and proteins identified common pathways, including lipid metabolic and oxidation-reduction process, cell adhesion, and antigen processing and presentation, related to immune responses, correlating with dysregulation of major histocompatibility complex (MHC) class II genes. Top upstream regulators included regulators associated with environmental sensing pathways to xenobiotics, microbial and diet-derived ligands, and endogenous metabolites. Proteome analysis revealed mTOR signaling IEC-specific defects. In addition to mTOR, the STAT and Notch pathways were dysregulated specifically in jejunal IEC. To determine the impact of pathway dysregulation on mutant jejunum alterations, we treated mutant mice with Tofacitinib, a JAK inhibitor. Treatment with the inhibitor partially corrected proliferation and tight junction defects, as well as niche stabilization by increasing Paneth cell numbers. Thus, IEC-specific histone deacetylases 1 (HDAC1) and 2 (HDAC2) support intestinal homeostasis by regulating survival and translation processes, as well as differentiation and metabolic pathways. HDAC1 and HDAC2 may play an important role in the regulation of IEC-specific inflammatory responses by controlling, directly or indirectly, the JAK/STAT pathway. IEC-specific JAK/STAT pathway deregulation may be, at least in part, responsible for intestinal homeostasis disruption in mutant mice.