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The activation of the vitamin D receptor (VDR) in the ileum has been shown to regulate Paneth cell-specific defensins, a large family of antimicrobial peptides; hence, this may serve as a potential mechanism to maintain intestinal homeostasis. Previously, we have demonstrated that a combination of vitamin D 3 (VD) and fructooligosaccharides (FOSs) upregulates colonic Vdr in mice. Here, we aim to examine the effect of VD, alone or in combination with FOSs, on intestinal barrier integrity and the secretion of antimicrobial peptides, as well as the gut microbial community. Male and female C57BL/6J mice at 6 weeks old were randomized into three groups to receive the following dietary regimens (n = 10/sex/group) for 8 weeks: (1) standard AIN-93G control diet (CTR), (2) CTR + 5000 IU vitamin D 3 (VD), and (3) VD + 5% fructooligosaccharides (VF). VD and VF differentially regulated the mRNA expressions of tight junction proteins in the colon and ileum. VF suppressed the upregulation of colonic ZO-1 and occludin , which was induced by VD supplementation alone. In the ileum, occludin but not ZO-1 was upregulated 20-fold in the VF-treated mice. While VD did not alter the mRNA expressions of Vdr and defensins in the ileum, these targets were downregulated by VF. Microbial analysis further reveals a shift of microbial beta diversity and a reduction in Romboutsia ilealis , a pathobiont, in VF-treated mice. Though the implications of these phenotypical and microbial changes remain to be determined, the administration of FOSs in the presence of VD may serve as an effective dietary intervention for maintaining intestinal homeostasis.

Paneth cells (PCs), a subset of intestinal epithelial cells (IECs) found at the base of small intestinal crypts, play an essential role in maintaining intestinal homeostasis. Altered PCs function is associated with diverse intestinal pathologies, including ileal Crohn's disease (CD). CD patients with ileal involvement have been previously demonstrated to display impairment in PCs and decreased levels of anti-microbial peptides. Although the immunosuppressive drug Azathioprine (AZA) is widely used in CD therapy, the impact of AZA on IEC differentiation remains largely elusive. In the present study, we hypothesized that the orally administered drug AZA also exerts its effect through modulation of the intestinal epithelium and specifically via modulation of PC function. AZA-treated CD patients exhibited an ileal upregulation of AMPs on both mRNA and protein levels compared to non-AZA treated patients. Upon in vitro AZA stimulation, intestinal epithelial cell line MODE-K exhibited heightened expression levels of PC marker in concert with diminished cell proliferation but boosted mitochondrial OXPHOS activity. Moreover, differentiation of IECs, including PCs differentiation, was boosted in AZA-treated murine small intestinal organoids and was associated with decreased D-glucose consumption and decreased growth rates. Of note, AZA treatment strongly decreased Lgr5 mRNA expression as well as Ki67 positive cells. Further, AZA restored dysregulated PCs associated with mitochondrial dysfunction. AZA-dependent inhibition of IEC proliferation is accompanied by boosted mitochondria function and IEC differentiation into PC., (© 2024. The Author(s).)

Research has shown that maternal sucralose (MS) exposure alters the gut microbiota of offspring at weaning and predisposes the offspring to developing obesity, non-alcoholic fatty liver disease and metabolic syndrome later in life. However, the underlying mechanism remains unclear. Paneth cells are thought to critically influence the gut microbiota. This study aimed to investigate whether MS exposure induced Paneth cell defects and exacerbated gut dysbiosis of offspring. Female C57BL/6 mice were divided into the MS and control (water) groups during pregnancy and lactation. Progeny mice were fed a normal sucralose-free diet after weaning until adulthood. MS inhibited intestinal development and increased the expression of proinflammatory cytokines in the small intestines of 3-week-old progeny mice. MS increased the proportions of abnormal granule secretion by Paneth cells. The number of Paneth cells and mRNA expression of AMPs such as cryptdins and lysozyme were reduced in the MS group. MS disturbed the gut microbiota composition and diversity in the 3-week-old offspring mice. The relative abundances of pro-inflammatory bacteria, such as Desulfovibrionales , Helicobacter , Pasteurellales and Campylobacterales were significantly increased in the MS group, while anti-inflammatory bacteria, including Clostridium XI , were decreased. This dysbiosis continued into adulthood. These findings showed that MS exposure induced Paneth cell defects and exacerbated gut dysbiosis in offspring mice. Sucralose should be consumed with caution, especially during pregnancy and in early life.

Arsenic is a global health concern that causes toxicity through ingestion of contaminated water and food. In vitro studies suggest that arsenic reduces stem and progenitor cell differentiation. Thus, this study determined if arsenic disrupted intestinal stem cell (ISC) differentiation, thereby altering the number, location, and/or function of intestinal epithelial cells. Adult male C57BL/6 mice were exposed to 0 or 100 ppb sodium arsenite (As III ) through drinking water for 5 weeks. Duodenal sections were collected to assess changes in morphology, proliferation, and cell types. qPCR analysis revealed a 40% reduction in Lgr5 transcripts, an ISC marker, in the arsenic-exposed mice, although there were no changes in the protein expression of Olfm4. Secretory cell-specific transcript markers of Paneth (Defa1), Goblet (Tff3), and secretory transit amplifying (Math1) cells were reduced by 51%, 44%, and 30% respectively, in the arsenic-exposed mice, indicating significant impacts on the Wnt-dependent differentiation pathway. Further, protein levels of phosphorylated β-catenin were reduced in the arsenic-exposed mice, which increased the expression of Wnt-dependent transcripts CD44 and c-myc. PCA analysis, followed by MANOVA and regression analyses, revealed significant changes and correlations between Lgr5 and the transit amplifying (TA) cell markers Math1 and Hes1, which are in the secretory cell pathway. Similar comparisons between Math1 and Defa1 show that terminal differentiation into Paneth cells is also reduced in the arsenic-exposed mice. The data suggests that ISCs are not lost following arsenic exposure, but rather, specific Wnt-dependent progenitor cell formation and terminal differentiation in the small intestine is reduced., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Intestinal Paneth cells modulate innate immunity and infection. In Crohn's disease, genetic mutations together with environmental triggers can disable Paneth cell function. Here, we find that a western diet (WD) similarly leads to Paneth cell dysfunction through mechanisms dependent on the microbiome and farnesoid X receptor (FXR) and type I interferon (IFN) signaling. Analysis of multiple human cohorts suggests that obesity is associated with Paneth cell dysfunction. In mouse models, consumption of a WD for as little as 4 weeks led to Paneth cell dysfunction. WD consumption in conjunction with Clostridium spp. increased the secondary bile acid deoxycholic acid levels in the ileum, which in turn inhibited Paneth cell function. The process required excess signaling of both FXR and IFN within intestinal epithelial cells. Our findings provide a mechanistic link between poor diet and inhibition of gut innate immunity and uncover an effect of FXR activation in gut inflammation., Competing Interests: Declaration of interests T.-C.L. received research funding from Pfizer on Paneth cell phenotype in IBD, and advises Interline. R.D.H. and C.S. may receive royalty income based on the CompBio technology developed by R.D.H. and licensed by Washington University to PercayAI. T. Stappenbeck advises Janssen, Boehringer Ingelheim (Ingelheim, Germany), Kallyope, Takada, and Roche. All other authors declare no relevant competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Non-muscle myosin IIA plays an important role in cell adhesion, cell migration, and tissue architecture. We previously showed that low activity of the heavy chain of non-muscle myosin II Myh9 is beneficial to LGR5 + intestinal stem cell maintenance. However, the function of Myh9 in adult mouse intestinal epithelium is largely unclear. In this study, we used the inducible Villin-creERT2 knockout approach to delete Myh9 in adult mouse intestinal epithelium and observed that homozygous deletion of Myh9 causes colitis-like morphologic changes in intestine, leads to a high sensitivity to dextran sulfate sodium and promotes colitis-related adenoma formation in the colon. Myh9 deletion disturbs cell junctions and impairs intestinal lumen barrier integrity, promoting the necroptosis of epithelial cells. Consistently, these changes can be partially rescued by Ripk3 knockout. Our results indicate that Myh9 is required for the maintenance of intestinal epithelium integrity and the prevention of cell necroptosis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Organoids allow the recapitulation of intestinal homeostasis and cancerogenesis in vitro; however, RNA sequencing (RNA-seq)-based methods for drug screens are missing. We develop targeted organoid sequencing (TORNADO-seq), a high-throughput, high-content drug discovery platform that uses targeted RNA-seq to monitor the expression of large gene signatures for the detailed evaluation of cellular phenotypes in organoids. TORNADO-seq is a fast, highly reproducible time- and cost-effective ($5 per sample) method that can probe cell mixtures and their differentiation state in the intestinal system. We apply this method to isolate drugs that enrich for differentiated cell phenotypes and show that these drugs are highly efficacious against cancer compared to wild-type organoids. Furthermore, TORNADO-seq facilitates in-depth insight into the mode of action of these drugs. Our technology can easily be adapted to many other systems and will allow for more systematic, large-scale, and quantitative approaches to study the biology of complex cellular systems., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Paneth cells and Lgr5+ intestinal stem cells (Lgr5+ ISCs) constitute the stem cell niche and maintain small intestinal epithelial integrity by recognizing various niche factors derived from subepithelial cells and external antigens. Although it has been known that interferon-γ (IFN-γ), a Th1 cytokine, is associated with intestinal epithelial disruption during inflammation as a niche factor, dynamics of Paneth cells and Lgr5+ ISCs in response to IFN-γ remain to be understood. Here we show that CAG-tdTomato;Lgr5-EGFP (CT-LE) mice generated in this study enable to identify Paneth cells and Lgr5+ ISCs separately by fluorescence signals. Lgr5+ ISCs underwent cell death a little earlier than Paneth cells in response to IFN-γ by simultaneous tracking using CT-LE mice. In addition, the timing of cell death in most Paneth cells overlapped with Lgr5+ ISCs, suggesting that Paneth cell depletion is induced directly by IFN-γ. Taken together, we established a novel simultaneous stem cell niche tracking method and clarified the involvement of both Paneth cells and Lgr5+ ISCs in stem cell niche damage induced by IFN-γ, further contribute to understanding the mechanism for maintaining intestinal homeostasis by stem cell niche., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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

Vitamin D receptor (VDR) deficiency is associated with cancer, infection, and chronic inflammation. Prior research has demonstrated VDR regulation of bacteria; however, little is known regarding VDR and viruses. We hypothesize that VDR deficiency impacts on the intestinal virome and viral-bacterial interactions. We specifically deleted VDR from intestinal epithelial cells (VDR ΔIEC ), Paneth cells (VDR ΔPC ), and myeloid cells (VDR ΔLyz ) in mice. Feces were collected for shotgun metagenomic sequencing and metabolite profiling. To test the functional changes, we evaluated pattern recognition receptors (PRRs) and analyzed microbial metabolites. Vibrio phages, Lactobacillus phages, and Escherichia coli typing phages were significantly enriched in all three conditional VDR-knockout mice. In the VDR ΔLyz mice, the levels of eight more virus species (2 enriched, 6 depleted) were significantly changed. Altered virus species were primarily observed in female VDR ΔLyz (2 enriched, 3 depleted) versus male VDR ΔLyz (1 enriched, 1 depleted). Altered alpha and beta diversity (family to species) were found in VDR ΔLyz . In VDR ΔIEC mice, bovine viral diarrhea virus 1 was significantly enriched. A significant correlation between viral and bacterial alterations was found in conditional VDR knockout mice. There was a positive correlation between Vibrio phage JSF5 and Cutibacterium acnes in VDR ΔPC and VDR ΔLyz mice. Also, there were more altered viral species in female conditional VDR knockout mice. Notably, there were significant changes in PRRs: upregulated TLR3, TLR7, and NOD2 in VDR ΔLyz mice and increased CLEC4L expression in VDR ΔIEC and VDR ΔPC mice. Furthermore, we identified metabolites related to virus infection: decreased glucose in VDR ΔIEC mice, increased ribulose/xylulose and xylose in VDR ΔLyz mice, and increased long-chain fatty acids in VDR ΔIEC and VDR ΔLyz female mice. Tissue-specific deletion of VDR changes the virome and functionally changes viral receptors, which leads to dysbiosis, metabolic dysfunction, and infection risk. This study helps to elucidate VDR regulating the virome in a tissue-specific and sex-specific manner.

Omeprazole is a potent inhibitor of gastric acid secretion. It was reported that omeprazole induced dramatic gastric mucosa morphologic changes from the resting state to the stimulated state. However, the effect of omeprazole administration on the ultrastructure and absorptive function of small intestines was largely unknown. Here, male Sprague-Dawley rats were daily treated with a single dose of omeprazole for 12 or 24 weeks. Ultrastructure intestinal mucosal change in duodenum, jejunum, and ileum was observed. We also determined small intestine inflammation, using intraepithelial lymphocytes activation. Finally, magnesium levels were measured in plasma, urine, feces, muscle, and bone to determine systemic magnesium balance. Omeprazole-treated rats had significantly decreased the width of tight junction, villous length, and absorptive area of duodenum, jejunum, and ileum compared to control rats. The small intestine of the omeprazole-treated group showed significantly higher intraepithelial lymphocytes activation levels compared with the control group. Lower secretory granules of Paneth cells at the base of the crypts were showed in omeprazole-treated rats. They also had significantly lower plasma, urinary, bone, and muscle Mg 2+ contents indicating hypomagnesemia with systemic magnesium deficiency. In conclusion, prolonged omeprazole treatment-induced small intestinal inflammation and villous atrophy, which led to decrease small intestinal magnesium absorption in the condition of proton pump inhibitor-induced hypomagnesemia.

Epithelial regeneration is essential for homeostasis and mucosal barrier repair. In this study, we aimed to define the effect of IL-10 on mucosal healing. Intestinal stem cells (ISCs) cultures and mice were treated with recombinant mice IL-10 (rmIL-10). The level of cell proliferation, differentiation, death and related signaling pathways for self-renewal of ISCs were measured in vitro and in vivo. It was uncovered that rmIL-10 increased the size and death, but reduced the total number of organoids. In addition, rmIL-10 depleted Lgr5 + ISCs and reduced epithelial proliferation, but enhanced the differentiation of epithelial cells and expanded numbers of transit-amplifying (TA) cells. These changes are related to the decrease of Wnt and Notch signals in vivo and in vitro. Meanwhile, increased expression of Paneth cells and decreased expression of enteroendocrine cells and goblet cells were induced by rmIL-10. Thus, our data indicate that IL-10 reduces the survival of Lgr5 + ISCs and proliferation of epithelial cells by inhibiting Notch and Wnt signaling, but promotes enhanced the differentiation of epithelial cells and expanded numbers of TA cells. Therefore, IL-10 acts as an anti-inflammatory factor, but may damage intestinal mucosa repair and maybe a potential target for the treatment of intestinal injury., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Acute graft-versus-host disease (GVHD) is a life-threatening complication after allogeneic hematopoietic cell transplantation (allo-HCT). Although currently used GVHD treatment regimens target the donor immune system, we explored here an approach that aims at protecting and regenerating Paneth cells (PCs) and intestinal stem cells (ISCs). Glucagon-like-peptide-2 (GLP-2) is an enteroendocrine tissue hormone produced by intestinal L cells. We observed that acute GVHD reduced intestinal GLP-2 levels in mice and patients developing GVHD. Treatment with the GLP-2 agonist, teduglutide, reduced de novo acute GVHD and steroid-refractory GVHD, without compromising graft-versus-leukemia (GVL) effects in multiple mouse models. Mechanistically GLP-2 substitution promoted regeneration of PCs and ISCs, which enhanced production of antimicrobial peptides and caused microbiome changes. GLP-2 expanded intestinal organoids and reduced expression of apoptosis-related genes. Low numbers of L cells in intestinal biopsies and high serum levels of GLP-2 were associated with a higher incidence of nonrelapse mortality in patients undergoing allo-HCT. Our findings indicate that L cells are a target of GVHD and that GLP-2-based treatment of acute GVHD restores intestinal homeostasis via an increase of ISCs and PCs without impairing GVL effects. Teduglutide could become a novel combination partner for immunosuppressive GVHD therapy to be tested in clinical trials.

Background & Aims: Paneth cells (PCs) synthesize and secrete antimicrobial peptides that are key mediators of host-microbe interactions, establishing a balance between intestinal microflora and enteric pathogens. We observed that their number increases in experimental portal hypertension and aimed to investigate the mechanisms by which these cells can contribute to the regulation of portal pressure., Methods: We first treated Math1 Lox/Lox VilcreERT2 mice with tamoxifen to induce the complete depletion of intestinal PCs. Subsequently, we performed partial portal vein or bile duct ligation. We then studied the effects of these interventions on hemodynamic parameters, proliferation of blood vessels and the expression of genes regulating angiogenesis. Intestinal organoids were cultured and exposed to different microbial products to study the composition of their secreted products (by proteomics) and their effects on the proliferation and tube formation of endothelial cells (ECs). In vivo confocal laser endomicroscopy was used to confirm the findings on blood vessel proliferation., Results: Portal hypertension was significantly attenuated in PC-depleted mice compared to control mice and was associated with a decrease in portosystemic shunts. Depletion of PCs also resulted in a significantly decreased density of blood vessels in the intestinal wall and mesentery. Furthermore, we observed reduced expression of intestinal genes regulating angiogenesis in Paneth cell depleted mice using arrays and next generation sequencing. Tube formation and wound healing responses were significantly decreased in ECs treated with conditioned media from PC-depleted intestinal organoids exposed to intestinal microbiota-derived products. Proteomic analysis of conditioned media in the presence of PCs revealed an increase in factors regulating angiogenesis and additional metabolic processes. In vivo endomicroscopy showed decreased vascular proliferation in the absence of PCs., Conclusions: These results suggest that in response to intestinal flora and microbiota-derived factors, PCs secrete not only antimicrobial peptides, but also pro-angiogenic signaling molecules, thereby promoting intestinal and mesenteric angiogenesis and regulating portal hypertension., Lay Summary: Paneth cells are present in the lining of the small intestine. They prevent the passage of bacteria from the intestine into the blood circulation by secreting substances to fight bacteria. In this paper, we discovered that these substances not only act against bacteria, but also increase the quantity of blood vessels in the intestine and blood pressure in the portal vein. This is important, because high blood pressure in the portal vein may result in several complications which could be targeted with novel approaches., Competing Interests: Conflict of interest The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Enteroids are cultured primary intestinal epithelial cells that recapitulate epithelial lineage development allowing for a more complex and physiologically relevant model for scientific study. The large presence of intestinal stem cells (ISC) in these enteroids allows for the study of metabolite effects on cellular processes and resulting progeny cells. Short-chain fatty acids (SCFA) such as butyrate (BUT) are bacterial metabolites produced in the gastrointestinal tract that are considered to be beneficial to host cells. Therefore, the objective was to study the effects of SCFAs on biomarkers of ISC activity, differentiation, barrier function and epithelial defense in the intestine using mouse and human enteroid models. Enteroids were treated with two concentrations of acetate (ACET), propionate (PROP), or BUT for 24 h. Enteroids treated with BUT or PROP showed a decrease in proliferation via EdU uptake relative to the controls in both mouse and human models. Gene expression of Lgr5 was shown to decrease with BUT and PROP treatments, but increased with ACET. As a result of BUT and PROP treatments, there was an increase in differentiation markers for enterocyte, Paneth, goblet, and enteroendocrine cells. Gene expression of antimicrobial proteins Reg3β, Reg3γ, and Defb1 were stimulated by BUT and PROP, but not by ACET which had a greater effect on expression of tight junction genes Cldn3 and Ocln in 3D enteroids. Similar results were obtained with human enteroids treated with 10 mM SCFAs and grown in either 3D or Transwell™ model cultures, although tight junctions were influenced by BUT and PROP, but not ACET in monolayer format. Furthermore, BUT and PROP treatments increased transepithelial electrical resistance after 24 h compared to ACET or control. Overall, individual SCFAs are potent stimulators of cellular gene expression, however, PROP and especially BUT show great efficacy for driving cell differentiation and gene expression., Competing Interests: The authors have declared that no competing interests exist.

Increasing pressure of life may bring some disease risks and stress injuries, which may destroy the immune system and result in intestinal mucosal immune disorders. In this study, the effects of different doses of ATX (30 mg per kg b.w., 60 mg per kg b.w. and 120 mg per kg b.w.) on intestinal mucosal functions were explored in cyclophosphamide (Cy)-induced immunodeficient mice. The results showed that continuous intraperitoneal injection of 100 mg per kg b.w. Cy for three days led to a persistent decrease of body weight and a range of abnormalities in the intestine of C57BL/6 mice. However, administration of ATX at 60 and 120 mg per kg b.w. could effectively prevent intestinal mucosa from this damage, including reduced levels of oxidative stress (MDA, GSH and GSH-PX), increased intestinal morphological structural integrity, stimulative growth of goblet cells and mucous secretion, decreased development of Paneth cells and expression levels of antimicrobial peptides (AMPs) (Reg-3γ and lysozyme), increased IgA secretion, ameliorative main gut flora (especially total bacteria, Lactobacillus and Enterobacteriaceae spp. ) and its metabolites (acetic acid, propionic acid and butyric acid). These protective effects of ATX were better than those of control-β-carotene in general. Our results may provide a new protective measure to keep intestinal mucosal barriers, which is of great significance for maintaining immune function in the body.

Necrotizing enterocolitis (NEC) remains a significant cause of morbidity and mortality in preterm infants. Formula feeding is a risk factor for NEC and osmolality, which is increased by the fortification that is required for adequate growth of the infant, has been suggested as a potential cause. Our laboratory has shown that Paneth cell disruption followed by induction of dysbiosis can induce NEC-like pathology in the absence of feeds. We hypothesized adding formula feeds to the model would exacerbate intestinal injury and inflammation in an osmolality-dependent manner. NEC-like injury was induced in 14-16 day-old C57Bl/6J mice by Paneth cell disruption with dithizone or diphtheria toxin, followed by feeding rodent milk substitute with varying osmolality (250-1491 mOsm/kg H 2 O). Animal weight, serum cytokines and osmolality, small intestinal injury, and cecal microbial composition were quantified. Paneth cell-disrupted mice fed formula had significant NEC scores compared to controls and no longer required induction of bacterial dysbiosis. Significant increases in serum inflammatory markers, small intestinal damage, and overall mortality were osmolality-dependent and not related to microbial changes. Overall, formula feeding in combination with Paneth cell disruption induced NEC-like injury in an osmolality-dependent manner, emphasizing the importance of vigilance in designing preterm infant feeds., Competing Interests: The authors declare no conflicts of interest.

Reg3β, a lectin, displays antibacterial activity. This study investigated Reg3β-expressing cells using IL-22-stimulated enteroids. IL-22 stimulation elevated the mRNA and protein levels of Reg3β. IL-22 also increased the mRNA levels of CD133 (a transit-amplifying cell marker) and lysozyme (a Paneth cell marker). Immunohistochemistry showed partial colocalization of Reg3β- and lysozyme-positive cells, suggesting that Paneth cells are one of Reg3β-producing cells.

Alternative pathway NF-κB signalling regulates susceptibility towards developing inflammatory bowel disease (IBD), colitis-associated cancer and sepsis-associated intestinal epithelial cell apoptosis and shedding. However, the cell populations responsible for the perturbed alternative pathway NF-κB signalling in intestinal mucosal pathology remain unclear. In order to investigate the contribution of the epithelial compartment, we have tested whether NF-κB2 regulated transcription in intestinal epithelial cells controls the intestinal epithelial response to cytokines that are known to disrupt intestinal barrier permeability. Enteroids were generated from the proximal, middle and distal regions of small intestine (SI) from C57BL/6J wild-type mice and displayed region-specific morphology that was maintained during sub-culture. Enteroids treated with 100 ng/mL TNF were compared with corresponding regions of SI from C57BL/6J mice treated systemically with 0.33 mg/kg TNF for 1.5 h. TNF-induced apoptosis in all regions of the intestine in vitro and in vivo but resulted in Paneth cell degranulation only in proximal tissue-derived SI and enteroids. TNF also resulted in increased enteroid sphericity (quantified as circularity from two-dimensional bright field images). This response was dose and time-dependent and correlated with active caspase-3 immunopositivity. Proximal tissue-derived enteroids generated from Nfκb2 -/- mice showed a significantly blunted circularity response following the addition of TNF, IFNγ, lipopolysaccharide (LPS) activated C57BL/6J-derived bone marrow-derived dendritic cells (BMDC) and secreted factors from LPS-activated BMDCs. However, Nfκb1 -/- mouse-derived enteroids showed no significant changes in response to these stimuli. In conclusion, the selection of SI region is important when designing enteroid studies as region-specific identity and response to stimuli such as TNF are maintained in culture. Intestinal epithelial cells are at least partially responsible for regulating their own fate by modulating NF-κB2 signalling in response to stimuli known to be involved in multiple intestinal and systemic diseases. Future studies are warranted to investigate the therapeutic potential of intestinal epithelial NF-κB2 inhibition.

We previously reported that acute necrotizing pancreatitis (ANP) after normal or high-fat diet is associated with a decreased number of Paneth cells in ileal crypts. Here, we ablated Paneth cells in a rat model of ANP after normal and high-fat diet to investigate the effects on disease symptoms. Adult male Sprague-Dawley rats received standard rat chow or a high-fat diet for 2 weeks, after which they were treated with dithizone to deplete Paneth cells. Six hours later, ANP was established by retrograde injection of sodium taurocholate into the biliopancreatic duct. Rats were sacrificed at 6, 12, and 24 h for assessment. We found dithizone aggravated ANP-associated pathological injuries to the pancreas and ileum in rats on high-fat or standard diets. Lysozyme expression in ileal crypts was decreased, while serum inflammatory cytokines (TNF α , IL-1 β , and IL-17A) and intestinal permeability (serum DAO activity and D-lactate) were increased. Expression of tight junction proteins (claudin-1, zo-1, and occludin) was decreased. Using high-throughput 16S rRNA sequencing, we found dithizone reduced microbiota diversity and altered microbiota composition in rats on high-fat or standard diets. Dithizone decreased fecal short-chain fatty acids (SCFAs) in rats on high-fat or standard diets. Changes in intestinal microbiota correlated significantly with SCFAs, lysozyme, DAO activity, D-lactate, inflammatory cytokines, and pathological injury to the pancreas and ileum in rats on high-fat or standard diets. In conclusion, ablation of Paneth cells exacerbates pancreatic and intestinal injuries in ANP after normal and high-fat diet. These symptoms may be related to changes in the intestinal microbiota., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2019 Yuecheng Guo et al.)

Purpose: Necrotizing enterocolitis is associated with decreased intestinal perfusion and ischemia. Paneth cells, specialized epithelial cells, have been shown to regulate the intestinal vasculature and disruption of these cells has been associated with NEC. We hypothesized that Paneth cell disruption in immature mice intestine would decrease the perfusion of the intestinal microvasculature., Methods: Paneth cells were disrupted in P14-16 mice using chemical (dithizone) and transgenic (diphtheria toxin) methodology. Six hours after Paneth cell disruption, Dylight 488 was injected directly into the left ventricle and allowed to perfuse for 5 minutes prior to intestinal harvesting. Tissue samples were evaluated with confocal fluorescence microscopy to quantify intestinal perfusion and samples were quantified by real time RT-PCR for gene expression., Results: Dithizone treatment significantly decreased intestinal perfusion compared to controls (p < 0.01). However, diphtheria toxin treatment demonstrated no significant difference in perfusion (p > 0.21). Intestines from all treatment groups had similar PECAM staining, but intestines treated with dithizone had significantly decreased nNOS and iNOS gene expression compared to controls (p < 0.007)., Conclusions: Paneth cell disruption significantly decreases the perfusion of the small intestinal microvasculature in a dithizone-specific manner. Dithizone has no effect on the amount of microvasculature, but does impact genes critical to nitric oxide signaling likely contributing to mesenteric vasoconstriction., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Gut microbial translocation contributes to alcoholic hepatitis. Using a mouse model of alcoholic hepatitis, we investigated the effects of chronic alcohol plus binge and found increased abundance of Paneth cells and IL-17A in the proximal small intestine (PSI). Alcohol increased IL-17A production and pro-apoptotic signaling evidenced by Bax, Bim, caspase-3, and caspase-8 increases via endoplasmic reticulum (ER) stress indicated by C/EBP homologous protein (CHOP) upregulation; this was prevented by the ER stress inhibitor, 4-PBA, in isolated crypts in vitro and in vivo. Mechanistically, IL-17 augmented alcohol-induced ER stress in isolated crypts. In vivo IL-17A blocking antibody administration in alcohol-treated mice attenuated ER stress-mediated apoptosis and IL-18 induction and prevented alcohol-induced impairment of tight junctions in the PSI and LPS translocation to the liver. Acute-on-chronic alcohol resulted in inflammasome activation, caspase-1 cleavage, and IL-18 production in the PSI. In vivo treatment with antibiotics or 4-PBA prevented CHOP upregulation and inflammasome activation. Our data suggest that alcohol upregulates innate immune mechanisms by increasing Paneth cell numbers and IL-17A release contributing to apoptosis amplification, inflammasome activation, and gut leakiness in the PSI. Binge alcohol-induced Paneth cell expansion, ER stress, and inflammasome activation in the PSI are modulated by the gut microbiome.

The gut is the biggest immune organ in the body that encloses commensal microbiota which aids in food digestion. Paneth cells, positioned at the frontline of host-microbiota interphase, can modulate the composition of microbiota. Paneth cells achieve this via the delivery of microbicidal substances, among which enteric α-defensins play the primary role. If microbiota is dysregulated, it can impact the function of the local mucosal immune system. Importantly, this system is also exposed to an enormous number of antigens which are derived from the gut-resident microbiota and processed food, and may potentially trigger undesirable local inflammatory responses. To understand the intricate regulations and liaisons between Paneth cells, microbiota and the immune system in this intestinal-specific setting, one must consider their mode of interaction in a wider context of regulatory processes which impose immune tolerance not only to self, but also to microbiota and food-derived antigens. These include, but are not limited to, tolerogenic mechanisms of central tolerance in the thymus and peripheral tolerance in the secondary lymphoid organs, and the intestine itself. Defects in these processes can compromise homeostasis in the intestinal mucosal immunity. In this review, which is focused on tolerance to intestinal antigens and its relevance for the pathogenesis of gut immune diseases, we provide an outline of such multilayered immune control mechanisms and highlight functional links that underpin their cooperative nature., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Butyrate produced by the intestinal microbiota is essential for proper functioning of the intestinal immune system. Total dependence on parenteral nutrition (PN) is associated with numerous adverse effects, including severe microbial dysbiosis and loss of important butyrate producers. We hypothesised that a lack of butyrate produced by the gut microbiota may be compensated by its supplementation in PN mixtures. We tested whether i.v. butyrate administration would (a) positively modulate intestinal defence mechanisms and (b) counteract PN-induced dysbiosis. Male Wistar rats were randomised to chow, PN, and PN supplemented with 9 mM butyrate (PN+But) for 12 days. Antimicrobial peptides, mucins, tight junction proteins, and cytokine expression were assessed by RT-qPCR. T-cell subpopulations in mesenteric lymph nodes (MLN) were analysed by flow cytometry. Microbiota composition was assessed in caecum content. Butyrate supplementation resulted in increased expression of tight junction proteins ( ZO-1, claudin-7, E-cadherin ), antimicrobial peptides ( Defa 8, Rd5, RegIIIγ ), and lysozyme in the ileal mucosa. Butyrate partially alleviated PN-induced intestinal barrier impairment and normalised IL-4, IL-10, and IgA mRNA expression. PN administration was associated with an increase in Tregs in MLN, which was normalised by butyrate. Butyrate increased the total number of CD4+ and decreased a relative amount of CD8+ memory T cells in MLN. Lack of enteral nutrition and PN administration led to a shift in caecal microbiota composition. Butyrate did not reverse the altered expression of most taxa but did influence the abundance of some potentially beneficial/pathogenic genera, which might contribute to its overall beneficial effect.

Paneth cells at the base of small intestinal crypts secrete granules containing α-defensins in response to bacteria and maintain the intestinal environment by clearing enteric pathogens and regulating the composition of the intestinal microbiota. However, Paneth cell secretory responses remain debatable and the mechanisms that regulate the secretion are not well understood. Although enteroids, three-dimensional cultures of small intestinal epithelial cells, have proven useful for analyzing intestinal epithelial cell functions including ion transport, their closed structures have imposed limitations to investigating interactions between Paneth cells and the intestinal microbiota. Here, we report that microinjection of bacteria or lipopolysaccharide (LPS) into the enteroid lumen provides an ex vivo system for studying Paneth cell secretion in real-time. The results show that Paneth cells released granules immediately when the apical surfaces of enteroid epithelial cells were exposed to LPS or live bacteria by microinjection. However, Paneth cells did not respond to LPS delivered in culture media to enteroid exterior basolateral surface, although they responded to basolateral carbamyl choline. In addition, Paneth cells replenished their granules after secretion, enabling responses to second stimulation. These findings provide new insight for apically-induced Paneth cell secretory responses in regulating the intestinal environment.

Symbionts play an indispensable role in gut homeostasis, but underlying mechanisms remain elusive. To clarify the role of lactic-acid-producing bacteria (LAB) on intestinal stem-cell (ISC)-mediated epithelial development, we fed mice with LAB-type symbionts such as Bifidobacterium and Lactobacillus spp. Here we show that administration of LAB-type symbionts significantly increased expansion of ISCs, Paneth cells, and goblet cells. Lactate stimulated ISC proliferation through Wnt/β-catenin signals of Paneth cells and intestinal stromal cells. Moreover, Lactobacillus plantarum strains lacking lactate dehydrogenase activity, which are deficient in lactate production, elicited less ISC proliferation. Pre-treatment with LAB-type symbionts or lactate protected mice in response to gut injury provoked by combined treatments with radiation and a chemotherapy drug. Impaired ISC-mediated epithelial development was found in mice deficient of the lactate G-protein-coupled receptor, Gpr81. Our results demonstrate that LAB-type symbiont-derived lactate plays a pivotal role in promoting ISC-mediated epithelial development in a Gpr81-dependent manner., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Aim: To explore whether Paneth cells (PCs) and complement system collaborate in the repair of enteric epithelia during acute gastrointestinal injury (AGI)., Methods: Wild-type C57BL/6 mice were employed to induce AGI by performing colon ascendens stent surgery, with sham-operated as control. Exogenous C3 treatment was applied at 6-h postsurgery. After 48 h, overall survival, intestinal damage severity, and C3 intracellular activation were assessed in both epithelial cells and PCs., Results: AGI caused a high mortality, while C3 therapy significantly attenuated epithelial damages and improved survival. Besides, exogenous C3 in vitro enhanced the proliferation and activity of PCs. Importantly, intracellular C3 activation was observed inside of PCs under C3 co-stimulation in vitro., Conclusion: C3 immunotherapy might play a valuable role in turnover of gut epithelia through intracellular activation in PCs.

Necrotizing enterocolitis (NEC) remains the leading cause of gastrointestinal morbidity and mortality in premature infants. Human and animal studies suggest a role for Paneth cells in NEC pathogenesis. Paneth cells play critical roles in host-microbial interactions and epithelial homeostasis. The ramifications of eliminating Paneth cell function on the immature host-microbial axis remains incomplete. Paneth cell function was depleted in the immature murine intestine using chemical and genetic models, which resulted in intestinal injury consistent with NEC. Paneth cell depletion was confirmed using histology, electron microscopy, flow cytometry, and real time RT-PCR. Cecal samples were analyzed at various time points to determine the effects of Paneth cell depletion with and without Klebsiella gavage on the microbiome. Deficient Paneth cell function induced significant compositional changes in the cecal microbiome with a significant increase in Enterobacteriacae species. Further, the bloom of Enterobacteriaceae species that occurs is phenotypically similar to what is seen in human NEC. This further strengthens our understanding of the importance of Paneth cells to intestinal homeostasis in the immature intestine., Competing Interests: The authors have declared that no competing interests exist.

Paneth cells contribute to small intestinal homeostasis by secreting antimicrobial peptides and constituting the intestinal stem cell (ISC) niche. Certain T cell-mediated enteropathies are characterized by extensive Paneth cell depletion coincident with mucosal destruction and dysbiosis. In this study, mechanisms of intestinal crypt injury have been investigated by characterizing responses of mouse intestinal organoids (enteroids) in coculture with mouse T lymphocytes. Activated T cells induced enteroid damage, reduced Paneth cell and Lgr5+ ISC mRNA levels, and induced Paneth cell death through a caspase-3/7-dependent mechanism. IFN-γ mediated these effects, because IFN-γ receptor-null enteroids were unaffected by activated T cells. In mice, administration of IFN-γ induced enteropathy with crypt hyperplasia, villus shortening, Paneth cell depletion, and modified ISC marker expression. IFN-γ exacerbated radiation enteritis, which was ameliorated by treatment with a selective JAK1/2 inhibitor. Thus, IFN-γ induced Paneth cell death and impaired regeneration of small intestinal epithelium in vivo, suggesting that IFN-γ may be a useful target for treating defective mucosal regeneration in enteric inflammation.

Recently, 3D small intestinal organoids (enteroids) have been developed from cultures of intestinal stem cells which differentiate in vitro to generate all the differentiated epithelial cell types associated with the intestine and mimic the structural properties of the intestine observed in vivo. Small-molecule drug treatment can skew organoid epithelial cell differentiation toward particular lineages, and these skewed enteroids may provide useful tools to study specific epithelial cell populations, such as goblet and Paneth cells. However, the extent to which differentiated epithelial cell populations in these skewed enteroids represent their in vivo counterparts is not fully understood. This study utilises label-free quantitative proteomics to determine whether skewing murine enteroid cultures toward the goblet or Paneth cell lineages results in changes in abundance of proteins associated with these cell lineages in vivo. Here, proteomics data confirms that skewed enteroids recapitulate important features of the in vivo gut environment, demonstrating that they can serve as useful models for the investigation of normal and disease processes in the intestine. Furthermore, comparison of mass spectrometry data with histology data contained within the Human Protein Atlas identifies putative novel markers for goblet and Paneth cells., (© 2018 The Authors. Proteomics Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Paneth cells are post-mitotic intestinal epithelial cells supporting the stem cell niche and mucosal immunity. Paneth cell pathologies are observed in various gastrointestinal diseases, but their plasticity and response to genomic and environmental challenges remain unclear. Using a knockin allele engineered at the mouse Lyz1 locus, we performed detailed Paneth cell-lineage tracing. Irradiation induced a subset of Paneth cells to proliferate and differentiate into villus epithelial cells. RNA sequencing (RNA-seq) revealed that Paneth cells sorted from irradiated mice acquired a stem cell-like transcriptome; when cultured in vitro, these individual Paneth cells formed organoids. Irradiation activated Notch signaling, and forced expression of Notch intracellular domain (NICD) in Paneth cells, but not Wnt/β-catenin pathway activation, induced their dedifferentiation. This study documents Paneth cell plasticity, particularly their ability to participate in epithelial replenishment following stem cell loss, adding to a growing body of knowledge detailing the molecular pathways controlling injury-induced regeneration., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Opposing activities of Notch and Wnt signaling regulate mucosal barrier homeostasis and differentiation of intestinal epithelial cells. Specifically, Wnt activity is essential for differentiation of secretory cells including Wnt3-producing Paneth cells, whereas Notch signaling strongly promotes generation of absorptive cells. Loss of caspase-8 in intestinal epithelium (casp8 ∆int ) is associated with fulminant epithelial necroptosis, severe Paneth cell death, secondary intestinal inflammation, and an increase in Notch activity. Here, we found that pharmacological Notch inhibition with dibenzazepine (DBZ) is able to essentially rescue the loss of Paneth cells, deescalate the inflammatory phenotype, and reduce intestinal permeability in casp8 ∆int mice. The secretory cell metaplasia in DBZ-treated casp8 ∆int animals is proliferative, indicating for Notch activities partially insensitive to gamma-secretase inhibition in a casp8 ∆int background. Our data suggest that casp8 acts in the intestinal Notch network.

Since the initial description of granular-rich small-intestinal crypt-based epithelial cells in 1872, today referred to as Paneth cells, a plethora of recent studies underlined their function in intestinal homeostasis. Paneth cells are evolutionary conserved highly secretory cells that produce antimicrobials to control gut microbial communities. Moreover, Paneth cells emerged as stem cell regulators that translate environmental cues into intestinal epithelial responses. Paneth cell disturbances may instigate intestinal inflammation and provide susceptibility to infection. Altered Paneth cell functions have been associated with a variety of inflammatory disease models and were linked to human intestinal disease processes including inflammatory bowel diseases such as Crohn´s disease and ulcerative colitis. This review summarizes our current understanding of Paneth cells and their antimicrobials in health and disease., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The intestinal microbial ecosystem is actively regulated by Paneth cell-derived antimicrobial peptides such as α-defensins. Various disorders, including graft-versus-host disease (GVHD), disrupt Paneth cell functions, resulting in unfavorably altered intestinal microbiota (dysbiosis), which further accelerates the underlying diseases. Current strategies to restore the gut ecosystem are bacteriotherapy such as fecal microbiota transplantation and probiotics, and no physiological approach has been developed so far. In this study, we demonstrate a novel approach to restore gut microbial ecology by Wnt agonist R-Spondin1 (R-Spo1) or recombinant α-defensin in mice. R-Spo1 stimulates intestinal stem cells to differentiate to Paneth cells and enhances luminal secretion of α-defensins. Administration of R-Spo1 or recombinant α-defensin prevents GVHD-mediated dysbiosis, thus representing a novel and physiological approach at modifying the gut ecosystem to restore intestinal homeostasis and host-microbiota cross talk toward therapeutic benefits., (© 2017 Hayase et al.)

Total parenteral nutrition (TPN) is a life-saving therapy for patients with gastrointestinal dysfunction or failure. Long-term TPN impairs gut barrier function and contributes to infections and poor clinical outcomes. However, the underlying mechanisms of TPN-related gut barrier damage have not been fully elucidated, and effective measures are still rare. Here, we compared the effects of a predominantly n-6 polyunsaturated fatty acids emulsion (PUFAs; Intralipid) and a lipid emulsion containing n-3 PUFAs (Intralipid plus Omegaven) on antimicrobial peptides produced by Paneth cells. Our results show for the first time that n-3 PUFAs markedly ameliorated intestine atrophy, and increased protein levels of lysozyme, RegIIIγ, and α-cryptdin 5, and their mRNA expression, compared to the n-6 PUFAs emulsion. Importantly, our study reveals that downregulation of IL-22 and phosphorylated Stat3 (p-Stat3) is associated with Paneth cell dysfunction, which may mediate TPN-related gut barrier damage. Lastly, n-3 PUFAs upregulated levels of IL-22 and increased the p-Stat3/Stat3 ratio in ileal tissue, suggesting that n-3 PUFAs improve Paneth cell function through activation of the IL-22/Stat3 pathway. Therefore, our study provides a cogent explanation for the beneficial effects of n-3 PUFAs, and indicates the IL-22/Stat3 pathway as a promising target in the treatment of TPN-related gut barrier damage., (Copyright © 2017. Published by Elsevier Inc.)

Gut homeostasis is a tightly regulated process requiring finely tuned complex interactions between different cell types, growth factors, or cytokines and their receptors. Previous work has implicated a role for IL-6 and mucosal immune cells in intestinal regeneration following injury and in promoting inflammation and cancer. We hypothesized that IL-6 signaling could also modulate crypt homeostasis. Using mouse in vitro crypt organoid and in vivo models, this study first demonstrated that exogenous IL-6 promoted crypt organoid proliferation and increased stem cell numbers through pSTAT3 activation in Paneth cells. Immunolabeling studies showed that the IL-6 receptor was restricted to the basal membrane of Paneth cells both in vitro and in vivo and that the crypt epithelium also expressed IL-6. Either a blocking Ab to the IL-6 receptor or a neutralizing Ab to IL-6 significantly reduced in vitro basal crypt organoid proliferation and budding, and in vivo significantly reduced the number of nuclei and the number of Lgr5EGFP-positive stem cells per crypt compared with IgG-treated mice, with the number of Paneth cells per crypt also significantly reduced. Functional studies demonstrated that IL-6-induced in vitro crypt organoid proliferation and crypt budding was abrogated by the Wnt inhibitor IWP2. This work demonstrates that autocrine IL-6 signaling in the gut epithelium regulates crypt homeostasis through the Paneth cells and the Wnt signaling pathway., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Necrotizing enterocolitis (NEC) remains a leading cause of morbidity and mortality in premature infants. Both human surgical specimens and animal models suggest a potential involvement of Paneth cells in NEC pathogenesis. Paneth cells play critical roles in epithelial homeostasis, innate immunity and host-microbial interactions. Yet, the complex interplay between Paneth cell disruption, epithelial barrier dysfunction and microbial-driven inflammation remains unclear in the immature intestine. In this study, mucosal intestinal injury consistent with human NEC was induced in postnatal day 14-16 (P14-P16) mice by disrupting Paneth cells, followed by gavage with Klebsiella pneumonia. Mucosal injury was determined by histology, serum cytokine levels and epithelial barrier dysfunction. Toll-like receptor 4 (TLR4) activation was examined using protein expression, gene expression, and TLR4 -/- mice. Finally, the role of bacteria was evaluated using heat-killed bacteria, conditioned media, Bacillus cereus and cecal slurries. We found that live bacteria were required to induce injury; however, TLR4 activation was not required. NEC induced by Paneth cell disruption results in altered localization of tight junction proteins and subsequent loss of barrier function. Prior research has shown a requirement for TLR4 activation to induce NEC-like damage. However, many infants develop NEC in the absence of Gram-negative rod bacteremia, raising the possibility that alternative pathways to intestinal injury exist. In this study, we show a previously unknown mechanism for the development of intestinal injury equivalent to that seen in human NEC and that is not dependent on TLR4 pathways. These data are congruent with the new hypothesis that NEC may be the consequence of several disease processes ending in a final common inflammatory pathway., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Environmental and occupational exposure to aluminum along with ionizing radiation results in serious health problems. This study was planned to investigate the impact of oxidative stress provoked by exposure to ionizing radiation with aluminum administration upon cellular ultra structure and apoptotic changes in Paneth cells of rat small intestine . Animals received daily aluminum chloride by gastric gavage at a dose 0.5 mg/Kg BW for 4 weeks. Whole body gamma irradiation was applied at a dose 2 Gy/week up to 8 Gy. Ileum malondialdehyde, advanced oxidative protein products, protein carbonyl and tumor necrosis factor-alpha were assessed as biomarkers of lipid peroxidation, protein oxidation and inflammation respectively along with superoxide dismutase, catalase, and glutathione peroxidase activities as enzymatic antioxidants. Moreover, analyses of cell cycle division and apoptotic changes were evaluated by flow cytometry. Intestinal cellular ultra structure was investigated using transmission electron microscope.Oxidative and inflammatory stresses assessment in the ileum of rats revealed that aluminum and ionizing radiation exposures exhibited a significant effect upon the increase in oxidative stress biomarkers along with the inflammatory marker tumor necrosis factor-α accompanied by a significant decreases in the antioxidant enzyme activities. Flow cytometric analyses showed significant alterations in the percentage of cells during cell cycle division phases along with significant increase in apoptotic cells. Ultra structurally, intestinal cellular alterations with marked injury in Paneth cells at the sites of bacterial translocation in the crypt of lumens were recorded. The results of this study have clearly showed that aluminum and ionizing radiation exposures induced apoptosis with oxidative and inflammatory disturbance in the Paneth cells of rat intestine, which appeared to play a major role in the pathogenesis of cellular damage. Furthermore, the interaction of these two intestinal toxic routes was found to be synergistic.

Doxorubicin (DOXO) induces significant, but transient, increases in apoptosis in the stem cell zone of the jejunum, followed by mucosal damage involving a decrease in crypt proliferation, crypt number, and villus height. The gastrointestinal tract is home to a vast population of commensal bacteria and numerous studies have demonstrated a symbiotic relationship between intestinal bacteria and intestinal epithelial cells (IEC) in maintaining homeostatic functions of the intestine. However, whether enteric bacteria play a role in DOXO-induced damage is not well understood. We hypothesized that enteric bacteria are necessary for induction of apoptosis and damage associated with DOXO treatment. Conventionally raised (CONV) and germ free (GF) mice were given a single injection of DOXO, and intestinal tissue was collected at 6, 72, and 120 h after treatment and from no treatment (0 h) controls. Histology and morphometric analyses quantified apoptosis, mitosis, crypt depth, villus height, and crypt density. Immunostaining for muc2 and lysozyme evaluated Paneth cells, goblet cells or dual stained intermediate cells. DOXO administration induced significant increases in apoptosis in jejunal epithelium regardless of the presence of enteric bacteria; however, the resulting injury, as demonstrated by statistically significant changes in crypt depth, crypt number, and proliferative cell number, was dependent upon the presence of enteric bacteria. Furthermore, we observed expansion of Paneth and goblet cells and presence of intermediate cells only in CONV and not GF mice. These findings provide evidence that manipulation and/or depletion of the enteric microbiota may have clinical significance in limiting chemotherapy-induced mucositis.

Introduction: Parenteral nutrition (PN) increases the risk of infection in critically ill patients and is associated with defects in gastrointestinal innate immunity. Goblet cells produce mucosal defense compounds, including mucin (principally MUC2), trefoil factor 3 (TFF3), and resistin-like molecule β (RELMβ). Bombesin (BBS), a gastrin-releasing peptide analogue, experimentally reverses PN-induced defects in Paneth cell innate immunity. We hypothesized that PN reduces goblet cell product expression and PN+BBS would reverse these PN-induced defects., Methods: Two days after intravenous cannulation, male Institute of Cancer Research mice were randomized to chow (n = 15), PN (n = 13), or PN+BBS (15 µg tid) (n = 12) diets for 5 days. Defined segments of ileum and luminal fluid were analyzed for MUC2, TFF3, and RELMβ by quantitative reverse transcriptase polymerase chain reaction and Western blot. Th2 cytokines interleukin (IL)-4 and IL-13 were measured by enzyme-linked immunosorbent assay., Results: Compared with chow, PN significantly reduced MUC2 in ileum (P < .01) and luminal fluid (P = .01). BBS supplementation did not improve ileal or luminal MUC2 compared with PN (P > .3). Compared with chow, PN significantly reduced TFF3 in ileum (P < .02) and luminal fluid (P < .01). BBS addition did not improve ileal or luminal TFF3 compared with PN (P > .3). Compared with chow, PN significantly reduced ileal RELMβ (P < .01). BBS supplementation significantly increased ileal RELMβ to levels similar to chow (P < .03 vs PN; P > .6 vs chow). Th2 cytokines were decreased with PN and returned to chow levels with BBS., Conclusion: PN significantly impairs the goblet cell component of innate mucosal immunity. BBS only preserves goblet cell RELMβ during PN but not other goblet cell products measured., (© 2015 American Society for Parenteral and Enteral Nutrition.)

Paneth cells reside at the base of crypts of the small intestine and secrete antimicrobial factors to control gut microbiota. Paneth cell loss is observed in the chronically inflamed intestine, which is often associated with increased reactive oxygen species (ROS). However, the relationship between Paneth cell loss and ROS is not yet clear. Intestinal epithelial-specific deletion of a protein kinase Tak1 depletes Paneth cells and highly upregulates ROS in the mouse model. We found that depletion of gut bacteria or myeloid differentiation factor 88 (Myd88), a mediator of bacteria-derived cell signaling, reduced ROS but did not block Paneth cell loss, suggesting that gut bacteria are the cause of ROS accumulation but bacteria-induced ROS are not the cause of Paneth cell loss. In contrast, deletion of the necroptotic cell death signaling intermediate, receptor-interacting protein kinase 3 (Ripk3), partially blocked Paneth cell loss. Thus, Tak1 deletion causes Paneth cell loss in part through necroptotic cell death. These results suggest that TAK1 participates in intestinal integrity through separately modulating bacteria-derived ROS and RIPK3-dependent Paneth cell loss.

Low expression of vitamin D receptor (VDR) and dysfunction of vitamin D/VDR signaling are reported in patients with inflammatory bowel disease (IBD); therefore, restoration of VDR function to control inflammation in IBD is desirable. Probiotics have been used in the treatment of IBD. However, the role of probiotics in the modulation of VDR signaling to effectively reduce inflammation is unknown. We identified a novel role of probiotics in activating VDR activity, thus inhibiting inflammation, using cell models and VDR knockout mice. We found that the probiotics Lactobacillus rhamnosus strain GG (LGG) and Lactobacillus plantarum (LP) increased VDR protein expression in both mouse and human intestinal epithelial cells. Using the VDR luciferase reporter vector, we detected increased transcriptional activity of VDR after probiotic treatment. Probiotics increased the expression of the VDR target genes, such as antimicrobial peptide cathelicidin, at the transcriptional level. Furthermore, the role of probiotics in regulating VDR signaling was tested in vivo using a Salmonella-colitis model in VDR knockout mice. Probiotic treatment conferred physiological and histologic protection from Salmonella-induced colitis in VDR(+/+) mice, whereas probiotics had no effects in the VDR(-/-) mice. Probiotic treatment also enhanced numbers of Paneth cells, which secrete AMPs for host defense. These data indicate that the VDR pathway is required for probiotic protection in colitis. Understanding how probiotics enhance VDR signaling and inhibit inflammation will allow probiotics to be used effectively, resulting in innovative approaches to the prevention and treatment of chronic inflammation., (Copyright © 2015 the American Physiological Society.)

Tumor necrosis factor (TNF) is a powerful activator of the immune system and a well-validated target for treatment of autoimmune diseases. Injection of TNF induces systemic lethal inflammation characterized by hypothermia, induction of multiple cytokines, and extensive damage to multiple organs. Previously, we reported that TNF-induced lethal inflammation is strictly TNFR1(P55)-dependent. We also uncovered a crucial role for P55 expression levels in intestinal epithelial cells (IECs), in which P55+/+ expression is sufficient to sensitize to TNF lethality in an otherwise fully protected P55+/- background. Here, we investigated the molecular mechanism that drives TNF toxicity in IECs. Unexpectedly, we found that the degree of TNF-induced enterocyte damage and apoptosis in IECs is equally strong in TNF-sensitive P55+/+ mice and TNF-resistant P55+/- mice. Our results suggest that P55+/+-induced signaling causes goblet and Paneth cell dysfunction, leading to severe epithelial barrier dysfunction. As a result, intestinal permeability and systemic bacterial spread are induced, causing lethal systemic inflammation. In conclusion, we identified P55-induced goblet and Paneth cell dysfunction as a crucial mechanism for TNF-induced systemic and lethal inflammation.

Notch signaling largely determines intestinal epithelial cell fate. High Notch activity drives progenitors toward absorptive enterocytes by repressing secretory differentiation programs, whereas low Notch permits secretory cell assignment. Myeloid translocation gene-related 1 (MTGR1) is a transcriptional corepressor in the myeloid translocation gene/Eight-Twenty-One family. Given that Mtgr1(-/-) mice have a dramatic reduction of intestinal epithelial secretory cells, we hypothesized that MTGR1 is a key repressor of Notch signaling. In support of this, transcriptome analysis of laser capture microdissected Mtgr1(-/-) intestinal crypts revealed Notch activation, and secretory markers Mucin2, Chromogranin A, and Growth factor-independent 1 (Gfi1) were down-regulated in Mtgr1(-/-) whole intestines and Mtgr1(-/-) enteroids. We demonstrate that MTGR1 is in a complex with Suppressor of Hairless Homolog, a key Notch effector, and represses Notch-induced Hairy/Enhancer of Split 1 activity. Moreover, pharmacologic Notch inhibition using a γ-secretase inhibitor (GSI) rescued the hyperproliferative baseline phenotype in the Mtgr1(-/-) intestine and increased production of goblet and enteroendocrine lineages in Mtgr1(-/-) mice. GSI increased Paneth cell production in wild-type mice but failed to do so in Mtgr1(-/-) mice. We determined that MTGR1 can interact with GFI1, a transcriptional corepressor required for Paneth cell differentiation, and repress GFI1 targets. Overall, the data suggest that MTGR1, a transcriptional corepressor well characterized in hematopoiesis, plays a critical role in intestinal lineage allocation., (© FASEB.)

The term "microbiota" means genetic inheritance associated with microbiota, which is about 100 times larger than the guest. The tolerance of the resident bacterial flora is an important key element of immune cell function. A key role in the interaction between the host and the microbiota is played by Paneth cell, which is able to synthesize and secrete proteins and antimicrobial peptides, such as α/β defensins, cathelicidin, 14 β-glycosidases, C-type lectins, and ribonuclease, in response to various stimuli. Recent studies found probiotics able to preserve intestinal homeostasis by downmodulating the immune response and inducing the development of T regulatory cells. Specific probiotic strain, as well as probiotic-driven metabolic products called "postbiotics," has been recently recognized and it is able to influence innate immunity. New therapeutic approaches based on probiotics are now available, and further treatments based on postbiotics will come in the future.

Ribosome-inactivating lectins (RILs) are A-B type toxins like ricin whose molecular target is the large rRNA of eukaryotic ribosome. Administration of lethal doses of the RIL nigrin b isolated from elderberry (Sambucus nigra L.) bark triggers specific intestinal derangement. The aim of the present research was to explore the early effects of a lethal dose of nigrin b (16 mg/kg body weight) on the small intestine using light and electron microscopy to ascertain intestinal epithelium changes. 6 h after nigrin administration, the small intestine crypts began to show signs of damage with cells appearing at different stages of apoptosis. 16 h after injection crypts appeared more impaired, including the derangement of Paneth cells. The novelty of our results is that the Paneth cells in the small intestine in addition to stem cells are the early cellular targets for nigrin b.

Krüppel-like factor 5 (KLF5) is a pro-proliferative transcriptional regulator primarily expressed in the intestinal crypt epithelial cells. Constitutive intestine-specific deletion of Klf5 is neonatal lethal suggesting a crucial role for KLF5 in intestinal development and homeostasis. We have previously shown Klf5 to play an active role regulating intestinal tumorigenesis. Here we examine the effect of inducible intestine-specific deletion of Klf5 in adult mice. Klf5 is lost from the intestine beginning at day 3 after the start of a 5-day treatment with the inducer tamoxifen. Although the mice have no significant weight loss or lethality, the colonic tissue shows signs of epithelial distress starting at day 3 following induction. Accompanying the morphological changes is a significant loss of proliferative crypt epithelial cells as revealed by BrdU or Ki67 staining at days 3 and 5 after start of tamoxifen. We also observed a loss of goblet cells from the colon and Paneth cells from the small intestine upon induced deletion of Klf5. In addition, loss of Klf5 from the colonic epithelium is accompanied by a regenerative response that coincides with an expansion in the zone of Sox9 expression along the crypt axis. At day 11, both proliferation and Sox9 expression return to baseline levels. Microarray and quantitative PCR analyses reveal an up-regulation of several regeneration-associated genes (Reg1A, Reg3G and Reg3B) and down-regulation of many Klf5 targets (Ki-67, cyclin B, Cdc2 and cyclin D1). Sox9 and Reg1A protein levels are also increased upon Klf5 loss. Lentiviral-mediated knockdown of KLF5 and exogenous expression of KLF5 in colorectal cancer cell lines confirm that Sox9 expression is negatively regulated by KLF5. Furthermore, ChIP assays reveal a direct association of KLF5 with both the Sox9 and Reg1A promoters. We have shown that disruption of epithelial homeostasis due to Klf5 loss from the adult colon is followed by a regenerative response led by Sox9 and the Reg family of proteins. Our study demonstrates that adult mouse colonic tissue undergoes acute physiological changes to accommodate the loss of Klf5 withstanding epithelial damage further signifying importance of Klf5 in colonic homeostasis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The intestinal epithelium continually self-renews and can rapidly regenerate after damage. Lgr5 marks mitotically active intestinal stem cells (ISCs). Importantly, intestinal homeostasis can be maintained after depletion of Lgr5(+) cells due to the activation of Lgr5(-) reserve ISCs. The Lgr5(-) ISC populations are thought to play a similar role during intestinal regeneration following radiation-induced damage. We tested this regeneration hypothesis by combining depletion of Lgr5(+) ISCs with radiation exposure. In contrast to the negligible effect of Lgr5(+) ISC loss during homeostasis, depletion of Lgr5(+) cells during radiation-induced damage and subsequent repair caused catastrophic crypt loss and deterioration of crypt-villus architecture. Interestingly though, we found that crypts deficient for Lgr5(+) cells are competent to undergo hyperplasia upon loss of Apc. These data argue that Lgr5(-) reserve stem cells are radiosensitive and that Lgr5(+) cells are crucial for robust intestinal regeneration following radiation exposure but are dispensable for premalignant hyperproliferation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Paneth cell-derived enteric antimicrobial peptides significantly contribute to antibacterial host defense and host-microbial homeostasis. Regulation occurs by enzymatic processing and release into the small intestinal lumen, but the stimuli involved are incompletely understood. Here, the capacity of various microbial and immune stimuli to induce antimicrobial peptide release from small intestinal tissue was systematically evaluated using antibacterial activity testing, immunostaining for Paneth cell granules and mass spectrometry. We confirmed the stimulatory activity of the muscarinic receptor agonist carbachol and the nucleotide-binding oligomerization domain ligand muramyl dipeptide. In contrast, no release of antibacterial activity was noted after treatment with the Toll-like receptor ligands poly(I:C), lipopolysaccharide or CpG, and the cytokines interleukin (IL)-15, IL-22, IL-28 and interferon-γ. Rapid Paneth cell degranulation and antimicrobial activity release, however, was observed after stimulation with the endogenous mediators IL-4 and IL-13. This process required phosphatidylinositol 3-kinase and was associated with protein kinase B phosphorylation in Paneth cells. Flow cytometric analysis confirmed expression of the IL-13 receptor α1 on isolated Paneth cells. Our findings identify a novel role of IL-13 as inducer of Paneth cell degranulation and enteric antimicrobial peptide release. IL-13 may thus contribute to mucosal antimicrobial host defense and host microbial homeostasis., (© 2014 S. Karger AG, Basel.)

Human digestive system is colonized by a large number of bacteria, estimated to 10(6) - 10(12) per one gram. Those bacteria through a network of interactions and interdependencies, are integrated superorganism. The intestinal flora is a very important element in host's defense against infections of the gastrointestinal tract, caused by for example Salmonella. Therefore, this bacteria have evolved a number of mechanisms, which adapt pathogen to the conditions of the gastrointestinal tract, and on the other hand to the change this environment, for easier colonization and internalization into host cells. One of elements of mentioned above interactions are antimicrobial peptides produced by host's Paneths cells, which have antimicrobial feature. Salmonella mostly are resistant for those peptides, moreover they can stimulate AMPs production for increasing their abilities in competition for ecological niche. In case of Salmonella quorum sensing mechanism was also identified. It allows for recognition of other bacteria presence, which stimulate Salmonella for higher expression of SPI-1, SPI-4 genes. These genes encoded proteins are involved in many host-pathogens interaction, inter alia inflammatory induction. Using of antibiotics in case of Salmonella infections always cause dramatic changes in intestinal flora compositions, which facilitate Salmonella internalizations to host's cells and sometimes could even stimulate to this process. Antibiotic treatment could also cause increase of antimicrobial resistance. Also antibiotics influence on Salmonella carriage was confirmed. Moreover antibiotics could cause super-shedder phenotype, what was detected on streptomycin-treated mice with Salmonella carriage.