Paneth Cells Research Articles

Chromatin remodelling in damaged intestinal crypts orchestrates redundant TGFβ and Hippo signalling to drive regeneration.

Cell state dynamics underlying successful tissue regeneration are undercharacterized. In the intestine, damage prompts epithelial reprogramming into revival stem cells (revSCs) that reconstitute Lgr5+ intestinal stem cells (ISCs). Here single-nuclear multi-omics of mouse crypts regenerating from irradiation shows revSC chromatin accessibility overlaps with ISCs and differentiated lineages. While revSC genes themselves are accessible throughout homeostatic epithelia, damage-induced remodelling of chromatin in the crypt converges on Hippo and the transforming growth factor-beta (TGFβ) signalling pathway, which we show is transiently activated and directly induces functional revSCs. Combinatorial gene expression analysis further suggests multiple sources of revSCs, and we demonstrate TGFβ can reprogramme enterocytes, goblet and paneth cells into revSCs and show individual revSCs form organoids. Despite this, loss of TGFβ signalling yields mild regenerative defects, whereas interference in both Hippo and TGFβ leads to profound defects and death. Intestinal regeneration is thus poised for activation by a compensatory system of crypt-localized, transient morphogen cues that support epithelial reprogramming and robust intestinal repair.

Metastasis of colon cancer requires Dickkopf-2 to generate cancer cells with Paneth cell properties.

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.

Metastasis of colon cancer requires Dickkopf-2 to generate cancer cells with Paneth cell properties

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.

Adaptation of the small intestine mucosa after gastric bypass surgery with a single anastomosis

Objective. To compare the morphological and morphometric changes of the small intestine mucosa in its common and biliopancreatic loops at different times after gastric bypass surgery with a single anastomosis. Materials and methods. The study included 36 patients who received surgical treatment at the Department of Thoracoabdominal Surgery of the Shalimov National Research Center for Surgery and Transplantation of the National Academy of Medical Sciences of Ukraine for morbid obesity, which consisted of gastric bypass surgery with a single anastomosis in various variants (long–loop, distal, mini–gastric bypass) in the period from 2016 to 2022. Further outpatient follow–up included scheduled endoscopic examinations at 3, 12, and 24 months after surgery. Results. At 3 months after the operation, no morphological and morphometric changes were detected in the studied biopsies of the mucous membrane of the biliopancreatic and common loops of the small intestine. The first morphological and morphometric changes in the small intestinal mucosa were observed 12 months after surgery. There was a statistically significant (p < 0.05) difference in the length of intestinal villi in the common and biliopancreatic loops of the small intestine – (0.390 ± 0.199) and (0.377 ± 0.184) mm, respectively. These changes indicate hypertrophy of villi in the colon to increase the absorption area. The thickness of the basal layer was greater in the biliopancreatic loop than in the total loop and amounted to (0.196 ± 0.068) and (0.167 ± 0.043) mm, respectively (p > 0.05). There was no statistically significant difference between the number of crypts containing Paneth cells in the biliopancreatic and common loops, so the regulatory function of these cells was preserved in any variant of gastric bypass with a single anastomosis. Conclusions. Morphological and morphometric changes in the small intestinal mucosa after gastric bypass with a single anastomosis may be associated with various physiological conditions and have clinical significance for understanding the mechanisms of absorption and intestinal protection. Additional studies with a larger number of samples and the use of modern methods of morphological analysis are needed to deepen the understanding of these processes.

The cellular-differential composition of the intestinal epithelium in various phases of inflammatory bowel diseases

BACKGROUND: The steady increase in the number of inflammatory bowel diseases and the absence of reliably significant diagnostic markers require the search for new morphological criteria for differential diagnosis. AIM: This study aimed to determine the cellular-differential composition of the intestinal epithelium in the phases of exacerbation and remission in Crohn’s disease and ulcerative colitis. MATERIALS AND METHODS: Tissue samples of 60 patients with inflammatory bowel diseases (Crohn’s disease, n=30; ulcerative colitis, n=30) and with irritable bowel syndrome (control group, n=15) were studied using histological, immunohistochemical, morphometric and statistical methods. RESULTS: Distinct features of the cellular-differential composition of the epithelium in the mucous membrane of the ileum, ascending colon, sigmoid colon, and rectum were identified across these conditions, with differences observed during exacerbation and remission in inflammatory bowel diseases. The proportion of goblet cells in the epithelial lining varied by intestinal region and disease type and phase. Goblet cell differentiation: 25.0% more goblet cells of the superficial epithelium between the crypts of the ileum in acute Crohn’s disease compared with remission (p=0.0002); 42.9% more goblet cells in the superficial epithelium of the ascending colon compared with irritable bowel syndrome (p=0.0001); 23.0% more goblet cells crypt in the sigmoid colon compared with ulcerative colitis in the acute stage (p=0.0024). There were no significant differences in the differentiation of Paneth cells. There was a threefold increase in endocrinocytes in the sigmoid colon in acute Crohn’s disease compared with irritable bowel syndrome (p=0.0238). Nonepithelial differon cells: fewer interepithelial lymphocytes in Crohn’s disease in remission compared with Crohn’s disease in the acute stage by 2.4 times in the sigmoid colon, 4.0 times in the rectum (p 0,0001); compared with irritable bowel syndrome by 4.8 times in the ileum, 2.7 times in the ascending intestine, 4.0 times in the sigmoid colon (p 0,05); 8.0 times in the rectum compared with ulcerative colitis in remission (p=0,0004). Additionally, the study found proliferative activity of crypt cells (mainly goblet-shaped) increased by 4.2 times in the sigmoid colon in acute Crohn’s disease compared with acute ulcerative colitis (p=0,0016). CONCLUSIONS: Morphometric parameters of the cellular-differential composition of the intestinal epithelium can serve as potential differential diagnostic criteria. Acute Crohn’s disease is characterized by a higher proliferation index in the sigmoid colon compared with acute ulcerative colitis, while Crohn’s disease in remission shows a lower number of interepithelial lymphocytes in the rectum compared with ulcerative colitis in remission.

Macrophage LRRK2 hyperactivity impairs autophagy and induces Paneth cell dysfunction.

LRRK2 polymorphisms (G2019S/N2081D) that increase susceptibility to Parkinson's disease and Crohn's disease (CD) lead to LRRK2 kinase hyperactivity and suppress autophagy. This connection suggests that LRRK2 kinase inhibition, a therapeutic strategy being explored for Parkinson's disease, may also benefit patients with CD. Paneth cell homeostasis is tightly regulated by autophagy, and their dysfunction is a precursor to gut inflammation in CD. Here, we found that patients with CD and mice carrying hyperactive LRRK2 polymorphisms developed Paneth cell dysfunction. We also found that LRRK2 kinase can be activated in the context of interactions between genes (genetic autophagy deficiency) and the environment (cigarette smoking). Unexpectedly, lamina propria immune cells were the main intestinal cell types that express LRRK2, instead of Paneth cells as previously suggested. We showed that LRRK2-mediated pro-inflammatory cytokine release from phagocytes impaired Paneth cell function, which was rescued by LRRK2 kinase inhibition through activation of autophagy. Together, these data suggest that LRRK2 kinase inhibitors maintain Paneth cell homeostasis by restoring autophagy and may represent a therapeutic strategy for CD.

Hedgehog Signalling Pathway and Its Role in Shaping the Architecture of Intestinal Epithelium

The hedgehog (Hh) signalling pathway plays a key role in both embryonic and postnatal development of the intestine and is responsible for gut homeostasis. It regulates stem cell renewal, formation of the villous–crypt axis, differentiation of goblet and Paneth cells, the cell cycle, apoptosis, development of gut innervation, and lipid metabolism. Ligands of the Hh pathway, i.e., Indian hedgehog (Ihh) and Sonic hedgehog (Shh), are expressed by superficial enterocytes but act in the mesenchyme, where they are bound by a Patched receptor localised on myofibroblasts and smooth muscle cells. This activates a cascade leading to the transcription of target genes, including those encoding G1/S-specific cyclin-D2 and -E1, B-cell lymphoma 2, fibroblast growth factor 4, and bone morphogenetic protein 4. The Hh pathway is tightly connected to Wnt signalling. Ihh is the major ligand in the Hh pathway. Its activation inhibits proliferation, while its blocking induces hyperproliferation and triggers a wound-healing response. Thus, Ihh is a negative feedback regulator of cell proliferation. There are data indicating that diet composition may affect the expression of the Hh pathway genes and proteins, which in turn, induces changes in mucosal architecture. This was shown for fat, vitamin A, haem, berberine, and ovotransferrin. The Hh signalling is also affected by the intestinal microbiota, which affects the intestinal barrier integrity. This review highlights the critical importance of the Hh pathway in shaping the intestinal mucosa and summarises the results obtained so far in research on the effect of dietary constituents on the activity of this pathway.

Gastric-like (pseudopyloric and pseudofoveolar) metaplasia and Paneth cell hyperplasia-neglected histological features of chronic ileal inflammation.

Architectural distortion and basal plasmacytosis are the most widely recognized histologic features of chronic ileal inflammation. However, these features might be difficult to assess in small, poorly oriented, or superficial biopsies. Additional features of chronic mucosal damage, including pseudopyloric or pseudofoveolar metaplasia and Paneth cell hyperplasia, have been less commonly reported, and their broader appreciation could facilitate the diagnosis of chronic ileal inflammatory conditions. The prevalence of gastric-like (pseudopyloric and pseudofoveolar) metaplasia and Paneth cell hyperplasia was evaluated in 102 ileal biopsies obtained from patients with Crohn's disease (n = 47), ulcerative colitis with endoscopically normal ileum (n = 20) or with backwash ileitis (n = 20), and nonsteroidal anti-inflammatory drugs- (NSAIDs-) induced ileitis (n = 15). Gastric-like metaplasia was identified in 23% of CD and 13% of NSAID-induced ileitis cases, whereas it was absent among all ulcerative colitis cases. Pseudopyloric metaplasia, pseudofoveolar metaplasia, or a combination of both was documented in 13%, 2%, and 9% of Crohn's disease cases, respectively. NSAID-associated cases showed only pseudopyloric metaplasia. Paneth cell hyperplasia was detected in 43% of Crohn's disease cases, 13% of NSAID-induced ileitis cases, and 5% of backwash ileitis cases. Accordingly, pseudofoveolar metaplasia, pseudopyloric metaplasia, and Paneth cell hyperplasia are not uncommon in conditions causing chronic ileal inflammation. They are most frequently detected in Crohn's disease, but may also be present in NSAID-induced ileitis, whereas they are significantly less common in backwash ileitis and absent in normal ileum. Given the surface localization of pseudofoveolar metaplasia, its identification can be particularly helpful when dealing with poorly oriented or superficial samples.

The vital role of melatonin in the intestinal mucosal barrier integrity in chick

BackgroundThe intestinal tract, as the main place for nutrient digestion and absorption, is closely related to the health of livestock and poultry. Melatonin secreted by the pineal gland acts as an endocrine transport signaling molecule to regulate intestinal function. However, the effect on intestinal function after pineal removal is unclear. MethodsWe raised 24 chicks under 400-700 nm white light with or without pinealectomy for 21 days. We used electron microscopy, HE staining, PAS staining, immunohistochemistry, immunofluorescence staining and western blot to detect intestinal physical and to explore the effect of melatonin secreted by the pineal gland on the intestinal mucosa barrier function. ResultsThe results showed that after pineal gland removal, the structure of the intestinal villi is severely damaged. Moreover, there was an obviously down-regulation in the villi length, the number of goblet cells and and its secretion of MUC2 protein, the expression level of tight junction proteins (Occludin and ZO-1) and lysozyme secreted by paneth cells, number of PCNA positive cells and macrophage, and an up-regualtion in crypt depth and apoptosis level after pinealectomy, suggesting pinealectomy-mediated melatonin level decrease damaged the intestinal physical, chemical and immune barriers. ConclusionOur findings provide new theoretical support for the future use of melatonin in intestinal development and new ideas about the relationship between endocrine hormone and intestinal physiology.

Establishment of a 3D organoid culture model for the investigation of adult slow-cycling putative intestinal stem cells.

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 24h. 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.

Bitter taste receptors as sensors of gut luminal contents.

Taste is important in the selection of food and is orchestrated by a group of distinct receptors, the taste G protein-coupled receptors (GPCRs). Taste 1 receptors (Tas1rs in mice and TAS1Rs in humans; also known as T1Rs) detect sweet and umami tastes, and taste 2 receptors (Tas2rs in mice and TAS2Rs in humans; also known as T2Rs) detect bitterness. These receptors are also expressed in extraoral sites, including the gastrointestinal mucosa. Tas2rs/TAS2Rs have gained interest as potential targets to prevent or treat metabolic disorders. These bitter taste receptors are expressed in functionally distinct types of gastrointestinal mucosal cells, including enteroendocrine cells, which, upon stimulation, increase intracellular Ca2+ and release signalling molecules that regulate gut chemosensory processes critical for digestion and absorption of nutrients, for neutralization and expulsion of harmful substances, and for metabolic regulation. Expression of Tas2rs/TAS2Rs in gut mucosa is upregulated by high-fat diets, and intraluminal bitter 'tastants' affect gastrointestinal functions and ingestive behaviour through local and gut-brain axis signalling. Tas2rs/TAS2Rs are also found in Paneth and goblet cells, which release antimicrobial peptides and glycoproteins, and in tuft cells, which trigger type 2 immune response against parasites, thus providing a direct line of defence against pathogens. This Review will focus on gut Tas2r/TAS2R distribution, signalling and regulation in enteroendocrine cells, supporting their role as chemosensors of luminal content that serve distinct functions as regulators of body homeostasis and immune response.

Effects of High-Linear-Energy-Transfer Heavy Ion Radiation on Intestinal Stem Cells: Implications for Gut Health and Tumorigenesis.

Heavy ion radiation, prevalent in outer space and relevant for radiotherapy, is densely ionizing and poses a risk to intestinal stem cells (ISCs), which are vital for maintaining intestinal homeostasis. Earlier studies have shown that heavy-ion radiation can cause chronic oxidative stress, persistent DNA damage, cellular senescence, and the development of a senescence-associated secretory phenotype (SASP) in mouse intestinal mucosa. However, the specific impact on different cell types, particularly Lgr5+ intestinal stem cells (ISCs), which are crucial for maintaining cellular homeostasis, GI function, and tumor initiation under genomic stress, remains understudied. Using an ISCs-relevant mouse model (Lgr5+ mice) and its GI tumor surrogate (Lgr5+Apc1638N/+ mice), we investigated ISCs-specific molecular alterations after high-LET radiation exposure. Tissue sections were assessed for senescence and SASP signaling at 2, 5 and 12 months post-exposure. Lgr5+ cells exhibited significantly greater oxidative stress following 28Si irradiation compared to γ-ray or controls. Both Lgr5+ cells and Paneth cells showed signs of senescence and developed a senescence-associated secretory phenotype (SASP) after 28Si exposure. Moreover, gene expression of pro-inflammatory and pro-growth SASP factors remained persistently elevated for up to a year post-28Si irradiation. Additionally, p38 MAPK and NF-κB signaling pathways, which are critical for stress responses and inflammation, were also upregulated after 28Si radiation. Transcripts involved in nutrient absorption and barrier function were also altered following irradiation. In Lgr5+Apc1638N/+ mice, tumor incidence was significantly higher in those exposed to 28Si radiation compared to the spontaneous tumorigenesis observed in control mice. Our results indicate that high-LET 28Si exposure induces persistent DNA damage, oxidative stress, senescence, and SASP in Lgr5+ ISCs, potentially predisposing astronauts to altered nutrient absorption, barrier function, and GI carcinogenesis during and after a long-duration outer space mission.

To sense or not to sense, Paneth cell regulation of mucosal immunity

Paneth cells located within intestinal crypts support epithelial stem cells and immunity through growth factors and antimicrobial peptides. In this issue of Cell Host & Microbe, Wallaeys etal. report that TNF sensing by Paneth cells disrupts the unfolded protein response and decreases antimicrobial peptides, causing bacterial translocation and sepsis.

Selenium nanoparticles affect chicken offspring's intestinal health better than other selenium sources

This study aimed to assess the effects of maternal diets containing various selenium (Se) sources on the intestinal mucosal function in the jejunum of chicken offspring. A total of 630, 18-wk-old Hy-Line Grey hens and 70 18-wk-old Hy-Line Grey breeders were randomly allocated into 7 groups, with 5 replicates in each group (18 hens and tow roosters). After 4 wk of Se depletion, the birds were fed either a nonsupplemented basal diet (control) or the same basal diet supplemented with 0.15 mg/kg selenium nanoparticles (Nano-Se), 0.30 mg/kg Nano-Se, 0.30 mg/kg selenocysteine (Sec), 0.30 mg/kg sodium selenite (SS), 0.30 mg/kg selenomethionine (SeMet), or 0.15 mg/kg Nano-Se + 0.15 mg/kg Sec, for 8 wk. Frtilized eggs were collected and incubated during the final week of the experiment. Jejunal tissues from embryonic d 18 and the hatch day were collected for analysis, and the 7-d survival rate of the offspring was recorded. Compared to the control, the maternal diet of 0.30 mg/kg Nano-Se, 0.30 mg/kg Sec, and 0.30 mg/kg SeMet significantly increased the survival of 7-day-old offspring (P < 0.05). The maternal diet supplemented with 0.30 mg/kg Nano-Se significantly increased intestinal villus height and the villus height/crypt depth ratio in chicks at embryonic d 18 and in 1-day-old (P < 0.05). The maternal diet containing 0.30 mg/kg Nano-Se and Sec increased the mRNA expression levels of tight junction proteins in 1-day-old offspring (P < 0.05). Supplemental 0.30 mg/kg Nano-Se significantly increased the mRNA expression levels of marker genes in intestinal enteroendocrine, stem, and Paneth cells (P < 0.05). In 1-day-old chicks, the number of intestinal goblet cells, as well as the mRNA expression levels of intestinal mucin2 (Muc2) and goblet cell differentiation factors (Spdef and C-myc), were the highest in diets supplemented with 0.30 mg/kg Nano-Se. Moreover, the expression levels of intestinal Muc2 and Spdef in chicks at embryonic d 18 was the highest with 0.30 mg/kg Nano-Se supplementation (P < 0.05). Supplementing with 0.30 mg/kg Nano-Se significantly reduced reactive oxygen species levels and decreased the mRNA expression levels of apoptosis-related genes in 1-day-old chicks (P < 0.05). Additionally, 0.30 mg/kg Nano-Se supplementation significantly down-regulated NLRP3 pathway gene expression in 1-day-old chicks (P < 0.05). In conclusion, maternal dietary supplementation with Nano-Se improved jejunal microarchitecture, antioxidant levels, and the expression of tight-junction protein in chicken offspring along with supporting goblet cell development by inhibiting the NLRP3 signaling pathway.

Metastasis of colon cancer requires Dickkopf-2 to generate cancer cells with Paneth cell properties.

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.

Establishment of an in-vitro inflammatory bowel disease model using immunological differentiation of Caco-2 cells

Studies on intestinal cell differentiation, particularly in dextran sodium sulfate (DSS)-induced inflammatory bowel disease (IBD), have predominantly focused on the disruption of intestinal crypts and suppressive effects on the intestinal microbiota; however, repeated administration of DSS is required to induce inflammatory pathology, and there is a lack of observation of early responses and consideration of differentiation stages. Although colonic adenocarcinoma (Caco-2) cells can be used as intestinal cell models, research on these cells in an immature state is limited. We, therefore, investigated the relationship between Caco-2 cell culture duration and immunological differentiation using α-defensin5 (DEFA5) as an indicator of intestinal immunity and differentiation. Changes in protein and gene expression levels in response to DSS were examined at each differentiation stage. Expression of immune- and differentiation-related proteins, including DEFA5 and lysozyme, was evident from Day 8 of culture. Immune responses to DSS varied with the differentiation stage, affecting cell viability and cytokine expression.•Caco-2 cell culture duration correlates with the differentiation stage of Paneth cells.•DSS exposure elicits different effects depending on the differentiation stage.•Our in-vitro model of IBD facilitates the characterization of the cell differentiation process and provides a methodology to help elucidate the causal mechanisms of IBD.

Paneth-like cells disruption and intestinal dysbiosis in the development of enterocolitis in an iatrogenic rectosigmoid hypoganglionosis rat model.

Hypoganglionosis resembles Hirschsprung disease (HSCR) which is characterized by severe constipation. Enterocolitis due to hypoganglionosis or Hirschsprung-associated enterocolitis (HAEC) is a life-threatening complication of both diseases. This study investigated the role of Paneth-like cells (PLCs) and gut microbiota in the development of enterocolitis in an iatrogenic rectosigmoid hypoganglionosis rat model. The rectosigmoid serosa of male Sprague-Dawley rats were exposed to 0.1% benzalkonium chloride (BAC). The rats were then sacrificed after 1, 3, 5, 8, and 12 weeks. A sham group was sacrificed on Week 12. With hematoxylin-eosin staining, the ganglionic cells were quantified, the degree of enterocolitis was analyzed, and the PLCs was identified. Intestinal barrier function was assessed for the anti-peripherin, occludin, and acetylcholinesterase (AChE)/butyrylcholinesterase (BChE) ratio. qRT-PCR was used as reference for the evaluation of antimicrobial peptide (AMP) of PLCs using cryptdins, secretory Phospholipase A2, and lysozyme levels. 16S rRNA high-throughput sequencing on fecal samples was performed to analyze the changes in the intestinal microbiota diversity in each group. After 1 week of intervention, the ganglion cells were fewer in all sacrificial 0.1% BAC groups at varying times than those in the sham group. Occludin and peripherin were decreased, while the AChE/BChE ratio was increased. At Week 5 postintervention, the number of α-defensins-positive PLCs increased in the sigmoid colon tissues from BAC-treated rats. Conversely, PLCs-produced AMP decreased from Week 5 to Week 12. The sham group demonstrated increased Lactobacillus and decreased Bacteroides, while the 0.1% BAC group exhibited reciprocal changes, indicating dysbiosis. Enterocolitis occurred from Week 1 postintervention. Application with BAC influences the disruption of PLCs in Week 5 postintervention, and dysbiosis exacerbate the occurrence of enterocolitis. Further research on Paneth cells involvement in HAEC development is warranted.

Paneth cell TNF signaling induces gut bacterial translocation and sepsis

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.

Histopathology underlying environmental enteric dysfunction in a cohort study of undernourished children in Bangladesh, Pakistan, and Zambia compared with United States children

BackgroundEnvironmental enteric dysfunction (EED) is an asymptomatic intestinal disorder associated with growth impairment, delayed neurocognitive development, and impaired oral vaccine responses. ObjectivesWe 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. MethodsFive 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. ResultsBiopsies 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. ConclusionsThis 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.

Protective Effect of Rosavin Against Intestinal Epithelial Injury in Colitis Mice and Intestinal Organoids.

Rhodiola species have been utilized as functional foods in Asia and Europe for promoting health. Research has demonstrated that Rhodiola has the potential to alleviate inflammatory bowel disease (IBD) in animal models. However, the specific active components and the underlying mechanism for ameliorating intestinal damage remain unclear. This study aims to explore the relieving effect of Rosavin (Rov), a known active constituent of Rhodiola, in IBD and the regulatory mechanisms. The therapeutic effect of Rov was evaluated using a murine model of acute colitis induced by dextran sulfate sodium salt (DSS). Inflammatory cytokines and neutrophil activation markers were measured by corresponding kits. Immunohistochemistry, immunofluorescence, TUNEL, and EdU assays were applied to investigate the tight conjunction proteins expression, epithelial marker expression, number of apoptotic cells, and epithelial proliferation, respectively. The protection effect of Rov on gut epithelial injury was assessed using TNF-α-induced intestinal organoids. Additinally, RNA sequencing was applied to observe the genetic alteration profile in these intestinal organoids. Oral administration of Rov significantly attenuated weight loss and restored colon length in mice. Notably, Rov treatment led to decreased levels of pro-inflammatory cytokines and neutrophil activation markers while increasing anti-inflammatory factors. Importantly, Rov restored intestinal despair by increasing the number of Lgr5+ stem cells, Lyz1+ Paneth cells and Muc2+ goblet cells in intestines of colitis mice, displaying reduced epithelial apoptosis and recovered barrier function. In TNF-α-induced intestinal organoids, Rov facilitated epithelial cell differentiation and protected against TNF-α-induced damage. RNA sequencing revealed upregulation in the gene expression associated with epithelial cells (including Lgr5+, Lyz1+ and Muc2+ cells) proliferation and defensin secretion, unveiling the protective mechanisms of Rov on the intestinal epithelial barrier. Rov holds potential as a natural prophylactic agent against IBD, with its protective action on the intestinal epithelium being crucial for its therapeutic efficacy.