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BACKGROUND: Environmental enteric dysfunction (EED) is an asymptomatic intestinal disorder associated with growth impairment, delayed neurocognitive development, and impaired oral vaccine responses. OBJECTIVES: We set out to develop and validate a histopathologic scoring system on duodenal biopsies from a cohort study of children with growth failure in Bangladesh, Pakistan, and Zambia ("EED") with reference to biopsies from United States children with no clinically reported histologic pathology (referred to hereafter as "normal") or celiac disease. METHODS: Five gastrointestinal pathologists evaluated 745 hematoxylin and eosin slide images from 291 children with EED (mean age: 1.6 y) and 66 United States children (mean age: 6.8 y). Histomorphologic features (i.e., villus/crypt architecture, goblet cells, epithelial and lamina propria acute/chronic inflammation, Brunner's glands, Paneth cells, epithelial detachment, enterocyte injury, and foveolar metaplasia) were used to score each histopathologic slide. Generalized estimating equations were used to determine differences between EED, normal, and celiac disease, and receiver operating characteristic curves were used to assess predictive value. RESULTS: Biopsies from the duodenal bulb showed higher intramucosal Brunner's gland scores and lower intraepithelial lymphocyte scores than from the second or third parts of the duodenum (D2/3), so only D2/3 were included in the final analysis. Although 7 parameters differed significantly between EED and normal biopsies in regression models, only 5 (blunted villus architecture, increased intraepithelial lymphocytosis, goblet cell depletion, Paneth cell depletion, and reduced intramucosal Brunner's glands) were required to create a total score percentage (TSP-5) that correctly identified EED against normal biopsies (AUC: 0.992; 95% CI: 0.983, 0.998). Geographic comparisons showed more severe goblet cell depletion in Bangladesh and more marked intraepithelial lymphocytosis in Pakistan. CONCLUSIONS: This scoring system involving 5 histologic parameters demonstrates very high discrimination between EED and normal biopsies, indicating that this scoring system can be applied with confidence to studies of intestinal biopsies in EED.

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The small intestinal crypts harbor secretory Paneth cells (PCs) which express bactericidal peptides that are crucial for maintaining intestinal homeostasis. Considering the diverse environmental conditions throughout the course of the small intestine, multiple subtypes of PCs are expected to exist. We applied single-cell RNA-sequencing of PCs combined with deep bulk RNA-sequencing on PC populations of different small intestinal locations and discovered several expression-based PC clusters. Some of these are discrete and resemble tuft cell-like PCs, goblet cell (GC)-like PCs, PCs expressing stem cell markers, and atypical PCs. Other clusters are less discrete but appear to be derived from different locations along the intestinal tract and have environment-dictated functions such as food digestion and antimicrobial peptide production. A comprehensive spatial analysis using Resolve Bioscience was conducted, leading to the identification of different PC's transcriptomic identities along the different compartments of the intestine, but not between PCs in the crypts themselves.

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The cytokine tumor necrosis factor (TNF) plays important roles in limiting infection but is also linked to sepsis. The mechanisms underlying these paradoxical roles are unclear. Here, we show that TNF limits the antimicrobial activity of Paneth cells (PCs), causing bacterial translocation from the gut to various organs. This TNF-induced lethality does not occur in mice with a PC-specific deletion in the TNF receptor, P55. In PCs, TNF stimulates the IFN pathway and ablates the steady-state unfolded protein response (UPR), effects not observed in mice lacking P55 or IFNAR1. TNF triggers the transcriptional downregulation of IRE1 key genes Ern1 and Ern2, which are key mediators of the UPR. This UPR deficiency causes a significant reduction in antimicrobial peptide production and PC antimicrobial activity, causing bacterial translocation to organs and subsequent polymicrobial sepsis, organ failure, and death. This study highlights the roles of PCs in bacterial control and therapeutic targets for sepsis.

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BACKGROUND: Paneth cells play multiple roles in maintaining intestinal homeostasis. However, the clinical role of Paneth cell metaplasia (PCM) in ulcerative colitis (UC) remains unclear. We aimed to investigate the relationship between PCM and relapse in patients with UC and compare the usefulness of PCM with other histological indexes, including mucin depletion (MD) and basal plasmacytosis (BP). METHODS: Patients with UC in clinical remission (CR) who underwent colonoscopy to confirm a Mayo endoscopic subscore (MES) ≦1 with biopsies from the distal colon were enrolled into this retrospective cohort study. Biopsy samples were evaluated for histological findings of PCM, MD, and BP. Clinical relapse was defined as partial Mayo score ≧3 or medication escalation. Multivariate analysis was performed to determine independent predictors of relapse among the three histological findings, MES, and patient background, and relapse prediction models were generated. RESULTS: Eighty-three patients were enrolled in this study (MES 0, n = 47; MES 1, n = 36). The number of PCM cases was significantly higher in patients with prolonged CR than that in those with relapse (p = 0.01). Multivariate analysis showed that the absence of PCM and MD were related to relapse in all the patients. In patients with MES 1, the absence of PCM was the only risk factor significantly and independently associated with relapse (hazard ratio, 4.51 [1.15-17.7]; p = 0.03). CONCLUSION: The absence of PCM was a histological risk factor for relapse in patients with MES 1, implying a protective role for PCM in remission and a new index for mucosal healing.

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OBJECTIVES: Paneth cells and Paneth cell metaplasia are well-known in pathology as foundational components in the gastrointestinal system. When within malignant cells (Paneth cell differentiation [PCD]), however, the function and significance of these cells is less well understood. Here, we present findings from the first study focused on PCD in postneoadjuvant esophageal adenocarcinoma (EAC) resection specimens. METHODS: Patients with EAC treated with neoadjuvant chemoradioation and followed by esophagectomy between 2012 and 2018 in our institution were retrospectively evaluated. A tissue microarray was constructed, and special and immunohistochemical stains were performed. RESULTS: A total of 64 cases were collected, of which 8 had PCD, as highlighted by periodic acid-Schiff with diastase staining. Adenocarcinomas with PCD were more commonly seen in patients 60 to 70 years of age and typically had a poorly differentiated morphology, observationally fewer stromal mucinous changes, and less lymph node metastasis. ß-catenin activation induced by neoadjuvant therapy was more frequent in the PCD-positive cases. Patients with PCD-positive disease had low programmed cell death 1 ligand 1 levels, no positive or equivocal ERBB2 (HER2) expression, and low CD8-positive T-cell infiltration; they were also mismatch repair proficient. Patients with PCD-positive disease showed a survival pattern inferior to that of patients with PCD-negative disease. CONCLUSIONS: When induced by neoadjuvant therapy in EAC, PCD is associated with high ß-catenin activation, less expression of targetable biomarkers, and a potentially worse clinical prognosis.

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The study of intestinal stem cells is a prerequisite for the development of therapies aimed at regenerating the gut. To enable investigation of adult slow-cycling H2B-GFP-retaining putative small intestinal (SI) stem cells in vitro, we have developed a three-dimensional (3D) SI organoid culture model based on the Tet-Op histone 2 B (H2B)-green fluorescent protein (GFP) fusion protein (Tet-Op-H2B-GFP) transgenic mouse. SI crypts were isolated from 6- to 12-week-old Tet-Op-H2B-GFP transgenic mice and cultured with appropriate growth factors and an animal-derived matrix (Matrigel). For in vitro transgene expression, doxycycline was added to the culture medium for 24 h. By pulse-chase experiments, H2B-GFP expression and retention were assessed through direct GFP fluorescence observations, both by confocal and fluorescence microscopy and by immunohistochemistry. The percentages of H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells persisting in organoids were determined by scoring relevant GFP-positive cells. Our results indicate that 24 h exposure to doxycycline (pulse) induced ubiquitous expression of H2B-GFP in the SI organoids. During subsequent culture, in the absence of doxycycline (chase), there was a gradual loss (due to cell division) of H2B-GFP. At 6-day chase, slow-cycling H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells were detected in the SI organoids. The developed culture model allows detection of slow-cycling H2B-GFP-retaining putative SI stem cells and will enable the study of self-renewal and regeneration for further characterization of these cells.

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Ciguateric syndrome is a food poisoning associated with the consumption of some species of fish that have accumulated ciguatoxins (CTXs) in their tissues. The effects of the syndrome occur with nervous imbalances which have been described for quite some time, and mentioned in sailing literature for centuries. In the last decade, research has been focused on the implementation of analytical methods for toxin identification and the study of action modes of CTXs to design effective treatments. However, an important aspect is to determine the damage that CTXs caused in the organs of affected individuals. In this work, the damages observed in tissues of mice, mainly in the small intestine, were analyzed. The animals were fed with CTX-contaminated fish muscle at concentrations 10-times below the median lethal dose (LD50) for 10 weeks. The analysis of tissues derived from the oral treatment resulted in an increased occurrence of Paneth cells, presence of lymphoid tissue infiltrating the mucosa and fibrous lesions in the mucosal layer of the small intestine. A decreasing weight in animals fed with toxic muscle was observed.

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The gut microbiota is responsible for several metabolic functions, producing various metabolites with numerous roles for the host. The gut microbiota plays a key role in constructing the microvascular network in the intestinal villus, depending on the Paneth cells, strategically positioned to coordinate the development of both the microbiota and the microvasculature. The gut microbiota secretes several molecules and chemokines involved in the induction of the secretion of pro-angiogenic factors.

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Symbiosis between the host and intestinal microbial communities is essential for human health. Disruption in this symbiosis is linked to gastrointestinal diseases, including inflammatory bowel diseases, as well as extra-gastrointestinal diseases. Unbalanced gut microbiome or gut dysbiosis contributes in multiple ways to disease frequency, severity and progression. Microbiome taxonomic profiling and metabolomics approaches greatly improved our understanding of gut dysbiosis features; however, the precise mechanisms involved in gut dysbiosis establishment still need to be clarified. The aim of this review is to present new actors and mechanisms underlying gut dysbiosis formation following parasitic infection or in a context of altered Paneth cells, revealing the existence of a critical crosstalk between Paneth and tuft cells to control microbiome composition.

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Intestinal homeostasis is maintained by specialized host cells and the gut microbiota. Wnt/ß-catenin signaling is essential for gastrointestinal development and homeostasis, and its dysregulation has been implicated in inflammation and colorectal cancer. Axin1 negatively regulates activated Wnt/ß-catenin signaling, but little is known regarding its role in regulating host-microbial interactions in health and disease. Here, we aim to demonstrate that intestinal Axin1 determines gut homeostasis and host response to inflammation. Axin1 expression was analyzed in human inflammatory bowel disease datasets. To explore the effects and mechanism of intestinal Axin1 in regulating intestinal homeostasis and colitis, we generated new mouse models with Axin1 conditional knockout in intestinal epithelial cell (IEC; Axin1 ΔIEC) and Paneth cell (PC; Axin1 ΔPC) to compare with control (Axin1 LoxP; LoxP: locus of X-over, P1) mice. We found increased Axin1 expression in the colonic epithelium of human inflammatory bowel disease (IBD). Axin1 ΔIEC mice exhibited altered goblet cell spatial distribution, PC morphology, reduced lysozyme expression, and enriched Akkermansia muciniphila (A. muciniphila). The absence of intestinal epithelial and PC Axin1 decreased susceptibility to dextran sulfate sodium (DSS)-induced colitis in vivo. Axin1 ΔIEC and Axin1 ΔPC mice became more susceptible to DSS-colitis after cohousing with control mice. Treatment with A. muciniphila reduced DSS-colitis severity. Antibiotic treatment did not change the IEC proliferation in the Axin1 Loxp mice. However, the intestinal proliferative cells in Axin1 ΔIEC mice with antibiotic treatment were reduced compared with those in Axin1 ΔIEC mice without treatment. These data suggest non-colitogenic effects driven by the gut microbiome. In conclusion, we found that the loss of intestinal Axin1 protects against colitis, likely driven by epithelial Axin1 and Axin1-associated A. muciniphila. Our study demonstrates a novel role of Axin1 in mediating intestinal homeostasis and the microbiota. Further mechanistic studies using specific Axin1 mutations elucidating how Axin1 modulates the microbiome and host inflammatory response will provide new therapeutic strategies for human IBD.

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Immune mediators affect multiple biological functions of intestinal epithelial cells (IECs) and, like Paneth and Paneth-like cells, play an important role in intestinal epithelial homeostasis. IFN-γ a prototypical proinflammatory cytokine disrupts intestinal epithelial homeostasis. However, the mechanism underlying the process remains unknown. In this study, using in vivo and in vitro models we demonstrate that IFN-γ is spontaneously secreted in the small intestine. Furthermore, we observed that this cytokine stimulates mitochondrial activity, ROS production, and Paneth and Paneth-like cell secretion. Paneth and Paneth-like secretion downstream of IFN-γ, as identified here, is mTORC1 and necroptosis-dependent. Thus, our findings revealed that the pleiotropic function of IFN-γ also includes the regulation of Paneth cell function in the homeostatic gut.

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Alcohol consumption perturbs the gut immune barrier and ultimately results in alcoholic liver diseases, but little is known about how immune-related cells in the gut are perturbed in this process. In this study, we employed laser capture microdissection and a label-free proteomics approach to investigate the consequences of alcohol exposure to the proteomes of crypts and villi in the proximal small intestine. Intestinal tissues from alcohol-fed and pair-fed mice were microdissected to selectively capture cells in the crypts and villi regions, followed by one-pot protein digestion and data-independent LC-MS/MS analysis. We successfully identified over 3000 proteins from each of the crypt or villi regions equivalent to ∼3000 cells. Analysis of alcohol-treated tissues indicated an enhanced alcohol metabolism and reduced levels of α-defensins in crypts, alongside increased lipid metabolism and apoptosis in villi. Immunofluorescence imaging further corroborated the proteomic findings. Our work provides a detailed profiling of the proteomic changes in the compartments of the mouse small intestine and aids in molecular-level understanding of alcohol-induced tissue damage.

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Metastasis is the leading cause of cancer-related mortality. Paneth cells provide stem cell niche factors in homeostatic conditions, but the underlying mechanisms of cancer stem cell niche development are unclear. Here we report that Dickkopf-2 (DKK2) is essential for the generation of cancer cells with Paneth cell properties during colon cancer metastasis. Splenic injection of Dkk2-knockout (KO) cancer organoids into C57BL/6 mice resulted in a significant reduction of liver metastases. Transcriptome analysis showed reduction of Paneth cell markers such as lysozymes in KO organoids. Single cell RNA sequencing analyses of murine metastasized colon cancer cells and patient samples identified the presence of lysozyme positive cells with Paneth cell properties including enhanced glycolysis. Further analyses of transcriptome and chromatin accessibility suggested Hepatocyte nuclear factor 4-alpha (HNF4A) as a downstream target of DKK2. Chromatin immunoprecipitation followed by sequencing analysis revealed that HNF4A binds to the promoter region of Sox9, a well-known transcription factor for Paneth cell differentiation. In the liver metastatic foci, DKK2 knockout rescued HNF4A protein levels followed by reduction of lysozyme positive cancer cells. Taken together, DKK2-mediated reduction of HNF4A protein promotes the generation of lysozyme positive cancer cells with Paneth cell properties in the metastasized colon cancers.

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The intricate architecture of the intestinal epithelium, crucial for nutrient absorption, is constantly threatened by environmental factors. The epithelium undergoes rapid turnover, which is essential for maintaining homeostasis, under the control of intestinal stem cells (ISCs). The central regulator, Wnt/ß-catenin signaling plays a key role in intestinal integrity and turnover. Despite its significance, the impact of environmental factors on this pathway has been largely overlooked. This study, for the first time, investigates the influence of Cd on the intestinal Wnt signaling pathway using a mouse model. In this study, male BALB/c mice were administered an environmentally relevant Cd dose (0.98 mg/kg) through oral gavage to investigate the intestinal disruption and Wnt signaling pathway. Various studies, including histopathology, immunohistochemistry, RT-PCR, western blotting, ELISA, intestinal permeability assay, and flow cytometry, were conducted to study Cd-induced changes in the intestine. The canonical Wnt signaling pathway experienced significant downregulation as a result of sub-chronic Cd exposure, which caused extensive damage throughout the small intestine. Increased intestinal permeability and a skewed immune response were also observed. To confirm that Wnt signaling downregulation is the key driver of Cd-induced gastrointestinal toxicity, mice were co-exposed to LiCl (a recognized Wnt activator) and Cd. The results clearly showed that the harmful effects of Cd could be reversed, which is strong evidence that Cd mostly damages the intestine through the Wnt/ß-catenin signalling axis. In conclusion, this research advances the current understanding of the role of Wnt/ß catenin signaling in gastrointestinal toxicity caused by diverse environmental pollutants.

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Introduction: Paneth cells are critically important to intestinal health, including protecting intestinal stem cells, shaping the intestinal microbiome, and regulating host immunity. Understanding Paneth cell biology in the immature intestine is often modeled in rodents with little information in larger mammals such as sheep. Previous studies have only established the distribution pattern of Paneth cells in healthy adult sheep. Our study aimed to examine the ontogeny, quantification, and localization of Paneth cells in fetal and newborn lambs at different gestational ages and with perinatal transient asphyxia. We hypothesized that ovine Paneth cell distribution at birth resembles the pattern seen in humans (highest concentrations in the ileum) and that ovine Paneth cell density is gestation-dependent. Methods: Intestinal samples were obtained from 126-127 (preterm, with and without perinatal transient asphyxia) and 140-141 (term) days gestation sheep. Samples were quantified per crypt in at least 100 crypts per animal and confirmed as Paneth cells through in immunohistochemistry. Results: Paneth cells had significantly higher density in the ileum compared to the jejunum and were absent in the colon. Discussion: Exposure to perinatal transient asphyxia acutely decreased Paneth cell numbers. These novel data support the possibility of utilizing ovine models for understanding Paneth cell biology in the fetus and neonate.

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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.

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Objective: The current study sought to clarify the role of lysozyme-regulated gut microbiota and explored the potential therapeutic effects of lysozyme on ileum injury induced by severe traumatic brain injury (sTBI) and bacterial pneumonia in vivo and in vitro experiments. Methods: Male 6-8-week-old specific pathogen-free (SPF) C57BL/6 mice were randomly divided into Normal group (N), Sham group (S), sTBI group (T), sTBI + or Lysozyme-treated group (L), Normal + Lysozyme group (NL) and Sham group + Lysozyme group (SL). At the day 7 after establishment of the model, mice were anesthetized and the samples were collected. The microbiota in lungs and fresh contents of the ileocecum were analyzed. Lungs and distal ileum were used to detect the degree of injury. The number of Paneth cells and the expression level of lysozyme were assessed. The bacterial translocation was determined. Intestinal organoids culture and co-coculture system was used to test whether lysozyme remodels the intestinal barrier through the gut microbiota. Results: After oral administration of lysozyme, the intestinal microbiota is rebalanced, the composition of lung microbiota is restored, and translocation of intestinal bacteria is mitigated. Lysozyme administration reinstates lysozyme expression in Paneth cells, thereby reducing intestinal permeability, pathological score, apoptosis rate, and inflammation levels. The gut microbiota, including Oscillospira, Ruminococcus, Alistipes, Butyricicoccus, and Lactobacillus, play a crucial role in regulating and improving intestinal barrier damage and modulating Paneth cells in lysozyme-treated mice. A co-culture system comprising intestinal organoids and brain-derived proteins (BP), which demonstrated that the BP effectively downregulated the expression of lysozyme in intestinal organoids. However, supplementation of lysozyme to this co-culture system failed to restore its expression in intestinal organoids. Conclusion: The present study unveiled a virtuous cycle whereby oral administration of lysozyme restores Paneth cell's function, mitigates intestinal injury and bacterial translocation through the remodeling of gut microbiota.

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Graft-versus-host disease (GVHD) is one of the most important cause of death in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). The gastrointestinal tract is one of the most common sites affected by GVHD. However, there is no gold standard clinical practice for diagnosing gastrointestinal GVHD (GI-GVHD), and it is mainly diagnosed by the patient's clinical symptoms and related histological changes. Additionally, GI-GVHD causes intestinal immune system disorders, damages intestinal epithelial tissue such as intestinal epithelial cells((IEC), goblet, Paneth, and intestinal stem cells, and disrupts the intestinal epithelium's physical and chemical mucosal barriers. The use of antibiotics and diet alterations significantly reduces intestinal microbial diversity, further reducing bacterial metabolites such as short-chain fatty acids and indole, aggravating infection, and GI-GVHD. gut microbe diversity can be restored by fecal microbiota transplantation (FMT) to treat refractory GI-GVHD. This review article focuses on the clinical diagnosis of GI-GVHD and the effect of GVHD on intestinal flora and its metabolites.

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Most gastric cancers arise in the setting of chronic inflammation which alters gland organization, such that acid-pumping parietal cells are lost, and remaining cells undergo metaplastic change in differentiation patterns. From a basic science perspective, recent progress has been made in understanding how atrophy and initial pyloric metaplasia occur. However, pathologists and cancer biologists have long been focused on the development of intestinal metaplasia patterns in this setting. Arguably, much less progress has been made in understanding the mechanisms that lead to the intestinalization seen in chronic atrophic gastritis and pyloric metaplasia. One plausible explanation for this disparity lies in the notable absence of reliable and reproducible small animal models within the field, which would facilitate the investigation of the mechanisms underlying the development of gastric intestinal metaplasia (GIM). This review offers an in-depth exploration of the current state of research in GIM, shedding light on its pivotal role in tumorigenesis. We delve into the histological subtypes of GIM and explore their respective associations with tumor formation. We present the current repertoire of biomarkers utilized to delineate the origins and progression of GIM and provide a comprehensive survey of the available, albeit limited, mouse lines employed for modeling GIM and engage in a discussion regarding potential cell lineages that serve as the origins of GIM. Finally, we expound upon the myriad signaling pathways recognized for their activity in GIM and posit on their potential overlap and interactions that contribute to the ultimate manifestation of the disease phenotype. Through our exhaustive review of the progression from gastric disease to GIM, we aim to establish the groundwork for future research endeavors dedicated to elucidating the etiology of GIM and developing strategies for its prevention and treatment, considering its potential precancerous nature.

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