Paneth Cells Research Articles

Inhibition of Pck1 in intestinal epithelial cells alleviates acute pancreatitis via modulating intestinal homeostasis.

Intestinal barrier dysfunction usually occurred in acute pancreatitis (AP) but the mechanism remains unclear. In this study, RNA sequencing of ileum in L-arginine-induced AP mice demonstrated that phosphoenolpyruvate kinase 1 (Pck1) was significantly up-regulated. Increased Pck1 expression in intestinal epithelial cells (IECs) was further validated in ileum of AP mice and duodenum of AP patients. In AP mice, level of Pck1 was positively correlated with pancreatic and ileal histopathological scores, serum amylase activity, and intestinal permeability (serum diamine oxidase (DAO), D-lactate, and endotoxin). In AP patients, level of Pck1 had a positive correlation with Ranson scores, white blood cell count and C-reactive protein. Inhibition of Pck1 by 3-Mercaptopicolinic acid hydrochloride (3-MPA) alleviated pancreatic and ileal injuries in AP mice. AP + 3-MPA mice showed improved intestinal permeability, including less epithelial apoptosis, increased tight junction proteins (TJPs) expression, decreased serum DAO, D-lactate, endotoxin, and FITC-Dextran levels, and reduced bacteria translocation. Lysozyme secreted by Paneth cells and mucin2 (MUC2) secretion in goblet cells were also partly restored in AP + 3-MPA mice. Meanwhile, inhibition of Pck1 improved intestinal immune response during AP, including elevation of M2/M1 macrophages ratio and secretory immunoglobulin A (sIgA) and reduction in neutrophils infiltration. Invitro, administration of 3-MPA dramatically ameliorated inflammation and injuries of epithelial cells in enteroids treated by LPS. In conclusion, inhibition of Pck1 in IECs might alleviate AP via modulating intestinal homeostasis.

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.

LPA5-Dependent Signaling Regulates Regeneration of the Intestinal Epithelium Following Irradiation.

Lysophosphatidic acid (LPA) is a bioactive lipid molecule that regulates a wide array of cellular functions, including proliferation, differentiation, and survival, via activation of cognate receptors. The LPA5 receptor is highly expressed in the intestinal epithelium, but its function in restoring intestinal epithelial integrity following injury has not been examined. Here, we use a radiation-induced injury model to study the role of LPA5 in regulating intestinal epithelial regeneration. Control mice (Lpar5f/f) and mice with an inducible, epithelial cell-specific deletion of Lpar5 in the small intestine (Lpar5IECKO) were subjected to 10 Gy total body X-ray irradiation and analyzed during recovery. Repair of the intestinal mucosa was delayed in Lpar5IECKO mice, with reduced epithelial proliferation and increased crypt cell apoptosis. These effects were accompanied by reduced numbers of OLFM4+ intestinal stem cells (ISCs). The effects of LPA5 on ISCs were corroborated by studies using organoids derived from Lgr5-lineage tracking reporter mice with deletion of Lpar5 in Lgr5+-stem cells (Lgr5Cont or Lgr5ΔLpar5). Irradiation of organoids resulted in fewer numbers of Lgr5ΔLpar5 organoids retaining Lgr5+-derived progenitor cells compared to Lgr5Cont organoids. Finally, we observed that impaired regeneration in Lpar5IECKO mice was associated with reduced numbers of Paneth cells and decreased expression of YAP, a critical factor for intestinal epithelial repair. Our study highlights a novel role for LPA5 in regeneration of the intestinal epithelium following irradiation and its effect on the maintenance of Paneth cells that support the stem cell niche.

The role of goblet cells in Crohn' s disease.

The prevalence of Crohn's disease (CD), a subtype of inflammatory bowel disease (IBD), is increasing worldwide. The pathogenesis of CD is hypothesized to be related to environmental, genetic, immunological, and bacterial factors. Current studies have indicated that intestinal epithelial cells, including columnar, Paneth, M, tuft, and goblet cells dysfunctions, are strongly associated with these pathogenic factors. In particular, goblet cells dysfunctions have been shown to be related to CD pathogenesis by direct or indirect ways, according to the emerging studies. The mucus barrier was established with the help of mucins secreted by goblet cells. Not only do the mucins mediate the mucus barrier permeability and bacterium selection, but also, they are closely linked with the endothelial reticulum stress during the synthesis process. Goblet cells also play a vital role in immune response. It was indicated that goblet cells take part in the antigen presentation and cytokines secretion process. Disrupted goblet cells related immune process were widely discovered in CD patients. Meanwhile, dysbiosis of commensal and pathogenic microbiota can induce myriad immune responses through mucus and goblet cell-associated antigen passage. Microbiome dysbiosis lead to inflammatory reaction against pathogenic bacteria and abnormal tolerogenic response. All these three pathways, including the loss of mucus barrier function, abnormal immune reaction, and microbiome dysbiosis, may have independent or cooperative effect on the CD pathogenesis. However, many of the specific mechanisms underlying these pathways remain unclear. Based on the current understandings of goblet cell's role in CD pathogenesis, substances including butyrate, PPARγagonist, Farnesoid X receptor agonist, nuclear factor-Kappa B, nitrate, cytokines mediators, dietary and nutrient therapies were all found to have potential therapeutic effects on CD by regulating the goblet cells mediated pathways. Several monoclonal antibodies already in use for the treatment of CD in the clinical settings were also found to have some goblet cells related therapeutic targets. In this review, we introduce the disease-related functions of goblet cells, their relationship with CD, their possible mechanisms, and current CD treatments targeting goblet cells.

Abstract 5636: CUX1 transcriptionally regulates intestinal epithelial homeostasis

Abstract The small intestine digests and absorbs nutrients, a role made possible by specialized secretory and absorptive cells that constantly replenish from the intestinal stem cell niche. Disruption in intestinal epithelium homeostasis has been linked to pathologies including inflammatory bowel disease (IBD) and cancer. However, the processes regulating differentiation in healthy and disease setting are incompletely understood. CUX1 is a highly conserved homeodomain-containing transcription factor that is expressed in small intestinal crypts and has been previously shown to be essential in mice and Drosophila. CUX1 is upregulated upon inflammatory stress conditions, including intestinal damage and in IBD. In addition, CUX1 is predicted to be protective against IBD from genome-wide association studies. The aim of the current work is to determine the mechanistic role of CUX1 in secretory intestinal cells differentiation and epithelial homeostasis. To this end, we have generated transgenic mouse models that modulate CUX1 levels in an inducible manner. Mice with near-null CUX1 levels exhibit rapid weight loss requiring euthanasia while mice with ~50% residual CUX1 halt normal weight gain. Histologic examination of tissues from mice with CUX1 knock-down reveals a lack of mature secretory cells, including goblet and Paneth cells, the latter playing a supportive role to intestinal stem cells. Moreover, mouse intestinal tissues from our knock-down models demonstrated decreased proliferation with increased apoptosis. In transcriptome profiling, we observed downregulation of proliferation, intestinal stem cell, and digestive system developmental gene signatures and decreased levels of key secretory cell regulators, pointing towards a role for CUX1 in stem cell maintenance and differentiation. Indeed, reducing CUX1 levels decreased β-catenin protein levels, while Wnt/β-catenin signaling is required for the organization of the crypt-villus axis. Finally, CUX1 knock-down compromises the ability to form budding organoids, demonstrating the cell intrinsic role for CUX1 in stem cell differentiation. In ongoing studies, we are identifying CUX1 genome-wide binding targets and the epigenetic impact of CUX1 in intestinal epithelium cells. Our findings indicate that CUX1 is a critical regulator of intestinal epithelium homeostasis that has a role in differentiation and potential role in injury-induced de-differentiation in healthy as well as IBD patients. We propose that CUX1 regulates stem cell maintenance and crypt composition by regulating Wnt/β-catenin signaling and inducing fetal-like transcriptomic repair program shown to be coopted by colon cancers. Resolving an intricate network of transcriptional regulation of differentiation in the intestinal crypt holds the potential to successfully target inflammation-related damage in human intestines. Citation Format: Katarzyna Zawieracz, Ningfei An, Megan McNerney. CUX1 transcriptionally regulates intestinal epithelial homeostasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5636.

Abstract 770: The innate immune system response to malignancy- under the radar

Abstract It has become clear that many cancers evade detection by, for example, silencing of immune checkpoints. Re-activation of the immune armamentarium by checkpoint inhibitors has improved survival in many types of cancer. However, we still are unable to gauge the response of the innate immune system (InImS) to incipient malignancy. The InImS is likely both a sentient early warning system and an immediately deployed cancer combatant. We describe a potential marker that quantifies the innate immune system response to multiple deadly tumors. Methods: We obtained a database of patients at high risk of colorectal neoplasia and prospectively noted those who contracted cancer on follow up. We contrasted the historic, premorbid biomarker response from the time of enrolment, primarily using the FERAD ratio. This was obtained by dividing the ferritin blood concentration by the denominator of fecal shedding of a Paneth cell marker, p87. We also compared the FERAD response to the absolute neutrophil:lymphocyte ratio (NLR), a known prognostic biomarker, and also the available, known PDL-1 response for each group of cancers. Results: We found that certain groups of cancers historically had very low FERAD ratios while others were high. Using FERAD ratios for lung, gastric, non-Hodgkin’s lymphoma, melanoma, thyroid, renal, and head and neck cancers, there is a significant inverse correlation between survival and FERAD (r=-0.8;p<0.025) which is similar to that of NLR (r=-0.7;p<0.035). For prostate cancer, breast, colorectal cancer (CRC), leukemia/MDS, there is a direct correlation between survival and FERAD (r=0.99;p<0.002). There is also an inverse relationship between NLR and PDL-1 response rates (r=-0.8;p<0.04). An observed mild positive trend for FERAD PDL-1 response rates may bode well for melanoma, lung, head and neck and renal cancers. Conclusions: Since p87 could be manipulated by dietary means to affect the FERAD ratio favorably, this could be incorporated into more effective chemotherapy regimens and result in better cancer treatment outcomes. Hepatocellular cancer (HCC) and CRC-liver metastasis appear to be immunological outliers. This will need an additional unique approach such as TIGIT intervention therapy (Zheng et al.. Immune checkpoint targeting TIGIT in HCC. Am J Transl Res. 2020). This may allow modulation of the immune response based on FERAD, NLR ratios, and other considerations of mutational burden and genetic mutations, in patients with selected cancers. Citation Format: Martin Tobi, Harvinder Talwar, Benita McVicker. The innate immune system response to malignancy- under the radar [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 770.

Postbiotics from Lactobacillus delbrueckii Alleviate Intestinal Inflammation by Promoting the Expansion of Intestinal Stem Cells in S. Typhimurium-Induced Mice.

Previous studies have demonstrated that L. delbrueckii plays beneficial roles in modulating the gut microbiota, enhancing the intestinal barrier, and promoting animal growth. Postbiotics have a similar or even superior effect in protecting intestinal health compared to probiotics due to their excellent stability, extended shelf life, and safety. However, the protective effects and underlying mechanism of postbiotics from L. delbrueckii in intestinal inflammation remain unclear. In this study, we demonstrated the beneficial impact of postbiotics from L. delbrueckii on intestinal health by establishing a S. Typhimurium-induced intestinal inflammation model in mice, which included inactivated bacteria and supernatant. The results revealed that the probiotics and postbiotics from L. delbrueckii increased the survival rate and body weight of S. Typhimurium-induced mice, increased the level of IL-10, and decreased the levels of TNF-α and IL-6, thereby alleviating intestinal inflammation. Meanwhile, treatment with postbiotics decreased the levels of D-LA, DAO, and LPS and promoted the expression of Occludin, ZO-1, and Claudin-1 in the serum and jejunum, suggesting an improvement in intestinal barrier function by postbiotics. Additionally, the postbiotics modulated gut microbial diversity, increased the ratio of Firmicutes and Bacteroidetes, and restored the abundance of Muribaculaceae, Lachnospiraceae_NK4a136_groups, and Alloprevotella in S. Typhimurium-infected mice. Moreover, postbiotics from L. delbrueckii promoted the expansion of intestinal stem cells (ISCs) and increased the numbers of Paneth and Goblet cells. Taken together, these data revealed the beneficial role of postbiotics from L. delbrueckii in protecting against intestinal inflammation by promoting the expansion of ISCs.

Endoplasmic reticulum stress: A possible connection between intestinal inflammation and neurodegenerative disorders.

Different studies have shown the key role of endoplasmic reticulum (ER) stress in autoimmune and chronic inflammatory disorders, as well as in neurodegenerative diseases. ER stress leads to the formation of misfolded proteins which affect the secretion of different cell types that are crucial for the intestinal homeostasis. In this review, we discuss the role of ER stress and its involvement in the development of inflammatory bowel diseases, chronic conditions that can cause severe damage of the gastrointestinal tract, focusing on the alteration of Paneth cells and goblet cells (the principal secretory phenotypes of the intestinal epithelial cells). ER stress is also discussed in the context of neurodegenerative diseases, in which protein misfolding represents the signature mechanism. ER stress in the bowel and consequent accumulation of misfolded proteins might represent a bridge between bowel inflammation and neurodegeneration along the gut-to-brain axis, affecting intestinal epithelial homeostasis and the equilibrium of the commensal microbiota. Targeting intestinal ER stress could foster future studies for designing new biomarkers and new therapeutic approaches for neurodegenerative disorders.

Aberrant miR-29 is a predictive feature of severe phenotypes in pediatric Crohn's disease.

Crohn's disease (CD) is a chronic inflammatory gut disorder. Molecular mechanisms underlying the clinical heterogeneity of CD remain poorly understood. MicroRNAs (miRNAs) are important regulators of gut physiology, and several have been implicated in the pathogenesis of adult CD. However, there is a dearth of large-scale miRNA studies for pediatric CD. We hypothesized that specific miRNAs uniquely mark pediatric CD. We performed small RNA-Seq of patient-matched colon and ileum biopsies from treatment-naive pediatric patients with CD (n = 169) and a control cohort (n = 108). Comprehensive miRNA analysis revealed 58 miRNAs altered in pediatric CD. Notably, multinomial logistic regression analysis revealed that index levels of ileal miR-29 are strongly predictive of severe inflammation and stricturing. Transcriptomic analyses of transgenic mice overexpressing miR-29 show a significant reduction of the tight junction protein gene Pmp22 and classic Paneth cell markers. The dramatic loss of Paneth cells was confirmed by histologic assays. Moreover, we found that pediatric patients with CD with elevated miR-29 exhibit significantly lower Paneth cell counts, increased inflammation scores, and reduced levels of PMP22. These findings strongly indicate that miR-29 upregulation is a distinguishing feature of pediatric CD, highly predictive of severe phenotypes, and associated with inflammation and Paneth cell loss.

Single nuclear RNA sequencing of terminal ileum in patients with cirrhosis demonstrates multi-faceted alterations in the intestinal barrier

Patients with cirrhosis have intestinal barrier dysfunction but the role of the individual cell types in human small intestine is unclear. We performed single-nuclear RNA sequencing (snRNAseq) in the pinch biopsies of terminal ileum of four age-matched men [56 years, healthy control, compensated, early (ascites and lactulose use) and advanced decompensated cirrhosis (ascites and rifaximin use)]. Cell type proportions, differential gene expressions, cell-type specific pathway analysis using IPA, and cellular crosstalk dynamics were compared. Stem cells, enterocytes and Paneth cells were lowest in advanced decompensation. Immune cells like naive CD4 + T cells were lowest while ITGAE + cells were highest in advanced decompensation patients. MECOM had lowest expression in stem cells in advanced decompensation. Defensin and mucin sulfation gene (PAPSS2) which can stabilize the mucus barrier expression were lowest while IL1, IL6 and TNF-related genes were significantly upregulated in the enterocytes, goblet, and Paneth cells in decompensated subjects. IPA analysis showed higher inflammatory pathways in enterocytes, stem, goblet, and Paneth cells in decompensated patients. Cellular crosstalk analysis showed that desmosome, protease-activated receptors, and cadherin-catenin complex interactions were most perturbed in decompensated patients. In summary, the snRNAseq of the human terminal ileum in 4 subjects (1 control and three cirrhosis) identified multidimensional alteration in the intestinal barrier with lower stem cells and altered gene expression focused on inflammation, mucin sulfation and cell–cell interactions with cirrhosis decompensation.

Correlation between intestinal stem cell niche changes and small bowel crypt failure in patients with treatment-naïve celiac disease.

We hypothesized that crypt failure in the small bowel results in villous flattening in patients with celiac disease (CeD). We investigated whether alterations in the stem cell niche (ISC) are responsible for this phenomenon. We included 92 duodenal (D2/3) biopsies from treatment-naive patients of CeD and 37 controls. All underwent screening for serum anti-tissue transglutaminase and endoscopic upper small bowel biopsy. Immunohistochemical markers were used to investigate ISC niche alterations, including LGR5 for crypt basal cells (CBC), Bmi1 for position 4+ cells, β-Defensin for Paneth cells, R-spondin1 as WNT activator, transcription factor-4 as WNT transcription factor, BMP receptor1A as WNT inhibitor, fibronectin-1 as periepithelial stromal cell marker, H2AX as apoptosis marker, and Ki67 as proliferation marker. We also analyzed IgA anti-tTG2 antibody deposits by using dual-color immunofluorescence staining. We found that in biopsies from patients with treatment-naive CeD with modified Marsh grade 3a-3c changes, the epithelial H2AX apoptotic index was upregulated than in controls. LGR5+ crypt basal cells were upregulated in all modified Marsh grades compared to controls. However, the Ki67 proliferation index, expressions of WNT-activator RSPO1, and position-4 cell marker Bmi1 did not significantly alter in patients' biopsies as compared to controls (P = 0.001). We also observed depletion of pericrypt stromal fibronectin-1 in patients with CeD compared to controls. In addition, we identified IgA anti-TG2 antibody deposits in pericrypt stroma. Our data suggests that ISC niche failure is a plausible hypothesis for villous flattening in patients with CeD, resulting from pericrypt IgA anti-TG2 antibody complex-mediated stromal depletion.

The loss of antioxidant activities impairs intestinal epithelium homeostasis by altering lipid metabolism

Reactive oxygens species (ROS) are common byproducts of metabolic reactions and could be at the origin of many diseases of the elderly. Here we investigated the role of ROS in the renewal of the intestinal epithelium in mice lacking catalase (CAT) and/or nicotinamide nucleotide transhydrogenase (NNT) activities. Cat−/− mice have delayed intestinal epithelium renewal and were prone to develop necrotizing enterocolitis upon starvation. Interestingly, crypts lacking CAT showed fewer intestinal stem cells (ISC) and lower stem cell activity than wild-type. In contrast, crypts lacking NNT showed a similar number of ISCs as wild-type but increased stem cell activity, which was also impaired by the loss of CAT. No alteration in the number of Paneth cells (PCs) was observed in crypts of either Cat−/− or Nnt−/− mice, but they showed an evident decline in the amount of lysozyme. Cat deficiency caused fat accumulation in crypts, and a fall in the remarkable high amount of adipose triglyceride lipase (ATGL) in PCs. Notably, the low levels of ATGL in the intestine of Cat −/− mice increased after a treatment with the antioxidant N-acetyl-L-cysteine. Supporting a role of ATGL in the regulation of ISC activity, its inhibition halt intestinal organoid development. These data suggest that the reduction in the renewal capacity of intestine originates from fatty acid metabolic alterations caused by peroxisomal ROS.

Intestinal lysozyme1 deficiency alters microbiota composition and impacts host metabolism through the emergence of NAD+-secreting ASTB Qing110 bacteria.

The impact of intestinal lumen lysozyme on intestinal health is complex, arising from its multifaceted interactions with the gut microbiota. Lysozyme can both mitigate and worsen certain health conditions, varying with different scenarios. This underscores the necessity of identifying the specific bacterial responses elicited by lysozyme and understanding their molecular foundations. Our research reveals that a deficiency in intestinal lysozyme1 may offer protection against diet-induced obesity by altering bacterial populations. We discovered a strain of bacterium, ASTB Qing110, which secretes NAD+ and is predominantly found in lyz1-deficient mice. Qing110 demonstrates positive effects in both C. elegans and mouse models of ataxia telangiectasia. This study sheds light on the intricate role of lysozyme in influencing intestinal health.

A61 ELUCIDATING INTESTINAL EPITHELIAL INFLAMMASOME MECHANISMS THAT REPAIR THE MUCOSAL BARRIER DURING ENTERIC INFECTION

Abstract Background To protect the gastrointestinal tract from infection, intestinal epithelial cells (IECs) lining the mucosa possess cytosolic complexes called inflammasomes which detect pathogenic insult and rapidly initiate inflammation to stop infectious spread. Active inflammasomes mediate crucial effector functions such as epithelial cell extrusion, pyroptotic cell death, and the release of interleukin (IL)-18 and prostaglandin E2 (PGE2). Recent literature has shown that IL-18 and PGE2 mediate intestinal repair following epithelial damage from DSS-induced colitis. Exogenous administration of IL-18 in inflammasome deficient mice improves survival and reduces pathology. PGE2 activates intestinal cellular reprogramming to drive intestinal wound healing. Whether inflammasome derived IL-18 and PGE2 mediate epithelial repair during enteric infection is unclear. Aims Using murine small intestinal organoids (mSIOs), we will investigate how IL-18 and PGE2 production by intestinal inflammasomes directly impacts IECs at both transcriptional and physiological levels. We hypothesize that inflammasome derived IL-18 and PGE2 will act on the intestinal epithelium resulting in transcriptional reprogramming that promotes tissue regeneration and barrier reinforcement. Methods WT and Nlrc4-/- mSIOs were stimulated with flagellin or FlaTox to activate the NAIP-NLRC4 inflammasome. The transcriptomic landscape was assessed with bulk-RNA sequencing and inflammasome activation was assessed by Western blot. IL-18 and PGE2 dependent gene targets were identified using Il-18-/- and indomethacin treated mSIOs by RT-qPCR. Results Bulk-RNA sequencing revealed an inflammasome activation dependent transcriptional signature in murine IECs upon NAIP-NLRC4 inflammasome activation. GO term analysis showed these genes to be involved in actin cytoskeleton rearrangement, epidermal growth factor signaling, and regulation of cell-cell adhesion. The expression of PGE2 gene targets were significantly reduced by indomethacin treatment while IL-18 gene targets remained unaffected in Il-18-/- IECs. FlaTox induced the same transcriptional changes but substantially amplified their expression. FlaTox but not flagellin led to a significant drop in Lgr5 (stem cells) and Lyz1 (Paneth cell) expression and an increase in Ly6a, a marker of fetal-like stem cells. Conclusions This data demonstrates that inflammasome signaling causes transcriptional reprogramming of IECs that activates pathways involved in maintaining epithelial barrier integrity. While IL-18 had no effects on gene expression at 4 hours, PGE2 paracrine signaling contributed to the expression of wound healing genes. Stronger inflammasome mediated damage with FlaTox amplified the repair response at the transcript level and we observed transient reprogramming of IECs into a fetal-like state suggesting active tissue regeneration. Funding Agencies CCC, CIHREPIC: Emerging & Pandemic Infections Consortium

A23 IMPAIRMENT OF INTESTINAL CRYPTAL STEM CELL CYCLE AND PANETH CELL PROLIFERATION BY CHRONIC STRESS

Abstract Background The small intestine’s epithelial barrier is essential for intestinal homeostasis and host defense. It consists of epithelial cells that are constantly renewed by crypt stem cells and their progeny, which have different functions in the gut. Chronic stress can impair the barrier by modulating the gut-brain axis, which involves the hypothalamic-pituitary-adrenal (HPA) axis, the vagus nerve, and the immune system. Chronic stress can also alter the gut microbiota, which can further influence the barrier and immune system. Aims The present study examines how chronic stress affects the crypt stem cell niche and function in the small intestine. Methods Restraint stress was applied to 6-8 weeks old mice in a plastic container for 1 hour daily for 7 days. The dark/light transition box test was used to evaluate the stress-induced behavior change. The Ussing chamber technique was used to assess the paracellular permeability of the small intestine. RNA was extracted from the mucosa and submucosa layers of the small intestine, after removing the muscle layer, and RNA sequencing analysis was performed. Immunofluorescence analysis was also performed on the small intestinal tissues to detect the expression of various markers. To assess the in vivo effect of cortisol, dexamethasone (a synthetic steroid activating same receptors as cortisol), or metyrapone (an 11β-hydroxylation blocker to inhibit cortisol production), was applied to the mice. Results We found that restraint stress induced anxiety-like behavior, increased small intestinal epithelial permeability, and reduced villus and crypt length in mice. RNA sequencing analysis revealed that restraint stress upregulated steroid-driven pathways and downregulated cell cycle and cell mitosis pathways in the small intestinal mucosa, suggesting increased cortisol production and decreased stem cell activity. Immunofluorescence analysis confirmed that restraint stress reduced the expression of stem cell marker Olfm-4 and proliferation markers Ki-67 and p-H3 in the small intestinal crypts. Dexamethasone, a synthetic glucocorticoid, mimicked the effects of restraint stress on crypt stem cell proliferation, while metyrapone, a steroid synthesis inhibitor, attenuated the effects of restraint stress on crypt stem cell proliferation. Restraint stress and dexamethasone also reduced Paneth cell RNA expression and marker in the small intestine. Conclusions Chronic stress induces anxiety, barrier dysfunction, and epithelial shortening. Chronic stress also alters crypt stem cell and Paneth cell expression. These findings show how chronic stress impacts intestinal epithelial renewal and differentiation, which may affect intestinal health and disease. Funding Agencies JSPS

Ion Transport Basis of Diarrhea, Paneth Cell Metaplasia, and Upregulation of Mechanosensory Pathway in Anti-CD40 Colitis Mice.

Anti-Cluster of differentiation (CD)-40-induced colitis, driven by innate inflammatory responses in the intestine, is a potent animal model exhibiting IBD pathophysiology including diarrhea. However, the ion transport basis of diarrhea and some key mucosal pathways (Paneth cells, stem cell niche, and mechanosensory) in this model have not been investigated. Mucosal scrapings and intestinal tissue from control and CD40 antibody (150µg) treated Rag2-/- mice were examined for gut inflammation, Paneth cell numbers, expression of key transporters, tight/adherens junction proteins, stem cell niche, and mechanosensory pathway via hematoxylin and eosin staining, quantitative polymerase chain reaction, and western blotting. Compared with control, anti-CD40 antibody treatment resulted in a significant loss of body weight (P < .05) and diarrhea at day 3 postinjection. Distal colonic tissues of anti-CD40 mice exhibited increased inflammatory infiltrates, higher claudin-2 expression, and appearance of Paneth cell-like structures indicative of Paneth cell metaplasia. Significantly reduced expression (P < .005) of downregulated in adenoma (key Cl- transporter), P-glycoprotein/multidrug resistantance-1 (MDR1, xenobiotic transporter), and adherens junction protein E-cadherin (~2-fold P < .05) was also observed in the colon of anti-CD40 colitis mice. Interestingly, there were also marked alterations in the stem cell markers and upregulation of the mechanosensory YAP-TAZ pathway, suggesting the activation of alternate regeneration pathway post-tissue injury in this model. Our data demonstrate that the anti-CD40 colitis model shows key features of IBD observed in the human disease, hence making it a suitable model to investigate the pathophysiology of ulcerative colitis (UC).

Intestinal epithelial adaptations to vertical sleeve gastrectomy defined at single-cell resolution

The gut plays a key role in regulating metabolic health. Dietary factors disrupt intestinal physiology and contribute to obesity and diabetes, whereas bariatric procedures such as vertical sleeve gastrectomy (VSG) cause gut adaptations that induce robust metabolic improvements. However, our understanding of these adaptations at the cellular and molecular levels remains limited. In a validated murine model, we leverage single-cell transcriptomics to determine how VSG impacts different cell lineages of the small intestinal epithelium. We define cell type-specific genes and pathways that VSG rescues from high-fat diet perturbation and characterize additional rescue-independent changes brought about by VSG. We show that Paneth cells have increased expression of the gut peptide Reg3g after VSG. We also find that VSG restores pathways pertaining to mitochondrial respiration and cellular metabolism, especially within crypt-based cells. Overall, our study provides unprecedented molecular resolution of VSG's therapeutic effects on the gut epithelium.

In-Ovo Glutamine Administration Enhances Intestinal Development and Functions in Broiler Chickens: Insights from Enteroid Models

BackgroundEarly life events play significant roles in tissue development and animal health in their later life. Early nutrition, through in-ovo delivery, has shown beneficial effects on improving intestinal health in broiler chickens. However, the underlying mechanism is not fully investigated. A recently developed enteroid culture technique allows investigations on intestinal epithelial functions that are close to physiologic conditions. ObjectivesIn this study, we evaluated the short- and long-term effects of in-ovo administration of glutamine (Gln) on intestinal epithelial development and functions by using intestinal enteroid culture and tissue electrophysiologic analysis. MethodsA hundred eggs of commercial Cobb500 broilers were in-ovo injected with 0.2 mL of either phosphate-buffered saline (PBS) or 3% Gln at embryonic day 18 (E18). Chicks were killed on the day of hatch, and at 3- and 14-d posthatch. Enteroids were generated from the small intestine. After 4 d of culture, enteroids were harvested for 5-ethynyl-2′-deoxyuridine proliferation, fluorescein isothiocyanate-4 kDa dextran permeability, and glucose absorption assays. At day 3 (d3) and day 14 (d14), intestinal barrier and nutrient transport functions were measured by the Ussing chamber. The gene expression of epithelial cell markers, nutrient transporters, and tight-junction proteins were analyzed in both intestinal tissues and enteroids. ResultsIn comparison with the PBS control group, in-ovo Gln increased intestinal villus morphology, epithelial cell proliferation, and differentiation, and altered epithelial cell population toward increased number of enteroendocrine and goblet cells while decreasing Paneth cells. Enteroids gene expression of nutrient transporters (B0AT1, SGLT1, and EAAT3), tight junction (ZO2), glucose absorption, and barrier functions were enhanced on the day of hatch. Long-term increases of intestinal di-peptide and alanine transport were observed at day 14 posthatch. ConclusionsTogether our results suggested that the in-ovo injection of Gln stimulated intestinal epithelium proliferation and programmed the epithelial cell differentiation toward absorptive cells.

The role of interleukin-22 in mammalian intestinal homeostasis: Friend and foe.

Interleukin-22 (IL-22) is an important cytokine in the intestinal environment. IL-22 is mainly produced by immune cells and targeted at nonimmune cells such as epithelial and stromal cells in a broad array of tissues such as -but not restricted to- the liver and adipose tissue. IL-22 therefore connects immune functions with metabolic functions of the host, and since it is induced by the microbiota, connects host functioning to the outside environment. IL-22 induces epithelial cell proliferation aiding in rapid epithelium regeneration and wound healing. Additionally, IL-22 activates antiapoptotic genes and DNA damage response pathways, enhancing epithelial cell survival. Recently, it has also been shown that IL-22 induces Paneth cell differentiation in humans. However, IL-22 can also contribute to intestinal epithelium damage and reduces microbial diversity in the intestine directly or indirectly by inducing excessive antimicrobial peptide production by epithelial cells. Moreover, IL-22 enhances angiogenesis and may therefore support tumorigenesis in the intestine. In conclusion, it appears that whether IL-22 has a beneficial or harmful effect in the mammalian intestine largely depends on its regulation. This review aims to provide a comprehensive overview of the current literature and emphasizes that IL-22 signaling outcome depends on the timing and duration of IL-22 production, the presence of it regulators such as IL-22BP, and the specific location of the cytokine production in the gastrointestinal tract.

DEVELOPMENT OF A PERSONALIZED HUMAN PANETH CELL MODEL USING INTESTINAL ORGANOIDS

Abstract INTRODUCTION Paneth cells are a specialized intestinal epithelial cell subtype whose major function is to produce antimicrobial peptides (AMPs). Alterations in Paneth cell function are associated with Crohn’s disease (CD) but the confounding influences of environmental factors and/or genetic variations makes it difficult to identify the cause of this. Human intestinal organoid (HIO) technology now allows for the generation of intestinal epithelium from any individual but there are relatively few protocols that allow for the enrichment of Paneth cells within organoids. Our goal was to specifically enrich the Paneth cell population in HIOs derived from induced pluripotent stem cells (iPSCs). METHODS iPSCs from a control individual and 2 CD patients were directed to HIOs and then dissociated to generate purified populations of epithelial only-HIOs (eHIOs). eHIOs were passaged weekly in proliferation medium (EGF, Noggin and CHIR99021) and the γ-secretase inhibitor, DAPT, was added to direct towards a Paneth cell fate. Flow cytometry, qPCR and immunocytochemistry were used to determine Paneth cell numbers, and gene and protein expression of various AMPs respectively. RESULTS iPSC-derived eHIOs from the control individual, which were used to develop this enrichment protocol, could be maintained for at least 4 months in proliferation medium. Flow cytometry analysis of the Paneth marker lysozyme revealed that the population of Paneth cells in eHIOs significantly increased from ~1% in proliferation media to ~28% upon treatment with DAPT. qPCR analysis demonstrated that DAPT treatment significantly increased the expression of the Paneth cell associated genes DEFA5, DEFA6, ITLN2, REG3A and PLA2G2A. Immunocytochemistry revealed that DAPT treated eHIOs were enriched for cells possessing granulated lysozyme, and also for additional AMPs such as DEFA5, ITLN2 and REG3A. The presence of the bacterial sensors TLR2, TLR5 and NOD2 were also detected in our Paneth cells. Finally, to confirm the applicability to CD patients, we applied our protocol and found the population of Paneth cells in CD eHIOs significantly increased from ~1% in proliferation media to ~10% upon treatment with DAPT, had significantly increased expression of the Paneth cell related genes and were also were enriched for cells with granulated AMPs. CONCLUSION We have successfully developed a methodology to enrich Paneth cells in iPSC-derived HIOs from both healthy and CD patients. Given that iPSCs can be generated from donor cells stored in well characterized biorepositories or obtained from a small blood draw from any CD patient, this modeling system now opens up a new avenue of research by allowing an examination of how environmental factors (microbes/cytokines) and/or genetic variations influence human Paneth cell function in a personalized manner. Fluorescent image showing the presence of granulated DEFA5 (green) in E-cadherin+ (red) human iPSC-derived intestinal organoids treated with DAPT (X60). qPCR analysis of Paneth cell related genes in proliferation media compared to DAPT treatment. *P &amp;lt; .05, **P &amp;lt; .01, ***P &amp;lt; .001, and ****P &amp;lt; .0001 as compared to gene expression in proliferation media.