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

Paneth cells are antimicrobial peptide-secreting cells located at the base of the crypts of the small intestine. The proteome of Paneth cells is not well defined because of their coexistence with stem cells, making it difficult to culture Paneth cells aloneiin vitro/i. Using a simplified toluidine blue O method for staining mouse intestinal tissue, laser capture microdissection (LCM) to isolate cells from the crypt region, and surfactant-assisted one-pot protein digestion, we identified more than 1300 proteins from crypts equivalent to 18,000 cells. Compared with the proteomes of villi and smooth muscle regions, the crypt proteome is highly enriched in defensins, lysozymes, and other antimicrobial peptides that are characteristic of Paneth cells. The sensitivity of the LCM-based proteomics approach was also assessed using a smaller number of cell equivalent tissues: a comparable proteomic coverage can be achieved with 3600 cells. This work is the first proteomics study of intestinal tissue enriched with Paneth cells. The simplified workflow enables profiling of Paneth cell-associated pathological changes at the proteome level directly from frozen intestinal tissue. It may also be useful for proteomics studies of other spatially resolved cell types from other tissues.

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

Opposing roles have been proposed for IL-22 in intestinal pathophysiology. We have optimized human small intestinal organoid (hSIO) culturing, constitutively generating all differentiated cell types while maintaining an active stem cell compartment. IL-22 does not promote the expansion of stem cells but rather slows the growth of hSIOs. In hSIOs, IL-22 is required for formation of Paneth cells, the prime producers of intestinal antimicrobial peptides (AMPs). Introduction of inflammatory bowel disease (IBD)-associated loss-of-function mutations in the IL-22 co-receptor gene IL10RB resulted in abolishment of Paneth cells in hSIOs. Moreover, IL-22 induced expression of host defense genes (such as REG1A, REG1B, and DMBT1) in enterocytes, goblet cells, Paneth cells, Tuft cells, and even stem cells. Thus, IL-22 does not directly control the regenerative capacity of crypt stem cells but rather boosts Paneth cell numbers, as well as the expression of AMPs in all cell types.

Background & aimsLoss-of-function variants in the PTPN2 gene are associated with increased risk of inflammatory bowel disease. We recently showed that Ptpn2 is critical for intestinal epithelial cell (IEC) barrier maintenance, IEC-macrophage communication, and modulation of the gut microbiome in mice, restricting expansion of a small intestinal pathobiont associated with inflammatory bowel disease. Here, we aimed to identify how Ptpn2 loss affects ileal IEC subtypes and their function invivo.MethodsConstitutive Ptpn2 wild-type, heterozygous, and knockout (KO) mice, as well as mice with inducible deletion of Ptpn2 in IECs, were used in the study. Investigation was performed using imaging techniques, flow cytometry, enteroid culture, and analysis of gene and protein levels of IEC markers.ResultsPartial transcriptome analysis showed that expression of Paneth cell-associated antimicrobial peptides Lyz1, Pla2g2a, and Defa6 was down-regulated markedly in Ptpn2-KO mice compared with wild-type and heterozygous. In parallel, Paneth cell numbers were reduced, their endoplasmic reticulum architecture was disrupted, and the endoplasmic reticulum stress protein, C/EBP-homologous protein (CHOP), was increased in Ptpn2-KO mice. Despite reduced Paneth cell number, flow cytometry showed increased expression of the Paneth cell-stimulatory cytokines interleukin 22 and interferon γ+ in CD4+ T cells isolated from Ptpn2-KO ileum. Key findings in constitutive Ptpn2-KO mice were confirmed in epithelium-specific Ptpn2ΔIEC mice, which also showed impaired lysozyme protein levels in Paneth cells compared with Ptpn2fl/fl control mice.ConclusionsConstitutive Ptpn2 deficiency affects Paneth cell viability and compromises Paneth cell-specific antimicrobial peptide production. The observed effects may contribute to the increased susceptibility to intestinal infection and dysbiosis in these mice.

Antimicrobial proteins possess a broad spectrum of bactericidal activity and play an important role in shaping the composition of gut microbiota, which is related to multiple diseases such as metabolic syndrome. However, it is incompletely known for the regulation of defensin expression in the gut Paneth cells. Here, we found that FABP4 in the Paneth cells of gut epithelial cells and organoids can downregulate the expression of defensins. FABP4

A marked elevation of TLR4 was observed in various organs of septic mice. The mechanism of TLR4 in intestinal epithelial cell damage in sepsis remains unclear. CLP mice models were used to assess the role of TLR4 in intestinal Paneth cell damage by histological, polymerase chain reaction, western-blot analyses. The ileal expression of TLR4 was increased by more than five-fold after CLP. CLP significantly increased 7-day mortality and was associated with a higher murine sepsis score (MSS), closely related with increased TLR4 expression. Histological staining revealed that a reduced number of Paneth cells, accompanied by reduced lysozyme and defensin alpha 5(DEF-5) expression as detected by PCR. Of note, the expression levels of ATF6, XBP1 and CHOP increased in the ileal of the sepsis group. Meanwhile, the uncleaved p90 ATF6 was markedly reduced and cleaved p50 ATF6 was increased in the sepsis group. Intriguingly, The TAK-242 had improved intestinal mucosal injury, reduced the expression of ATF6, XBP1 and CHOP and relieved the cleavage of ATF6. We found that increased the expression level of TLR4 in the ileal of CLP mice promoted the depletion of Paneth cell and reduced LYZ and DEF-5 expression. Furthermore, our findings suggested that TLR4-mediated the hyperactivation of ER stress, via activating the ATF6/CHOP pathway, might be one of the mechanisms associated with Paneth cells loss and dysfunction during intestinal barrier impairment of sepsis.

Cre-lox system is a powerful and widely used research tool developed to understand the specific role of genes. It allows to control the spatial and temporal expression of genes in experimental models. Several limitations including toxicity of Cre recombinase or incomplete excision of floxed loci have been reported in the past. To date, this is the first research study reporting that gut microbes also influence the expected phenotype of Paneth cell depletion in the genetically modified Math1lox/loxVilCreERT2 mouse model.

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-gamma (IFN-gamma), 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-gamma 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-gamma 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-gamma. 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-gamma, further contribute to understanding the mechanism for maintaining intestinal homeostasis by stem cell niche. (C) 2021 The Authors. Published by Elsevier Inc.

Interleukin-22 (IL-22) signaling in the intestines is critical for promoting tissue-protective functions. However, since a diverse array of cell types (absorptive and secretory epithelium as well as stem cells) express IL-22Ra1, a receptor for IL-22, it has been difficult to determine what cell type(s) specifically respond to IL-22 to mediate intestinal mucosal host defense. Here, we report that IL-22 signaling in the small intestine is positively correlated with Paneth cell differentiation programs. Our Il22Ra1fl/fl;Lgr5-EGFP-creERT2-specific knockout mice and, independently, our lineage-tracing findings rule out the involvement of Lgr5+ intestinal stem cell (ISC)-dependent IL-22Ra1 signaling in regulating the lineage commitment of epithelial cells, including Paneth cells. Using novel Paneth cell-specific IL-22Ra1 knockout mice (Il22Ra1fl/fl;Defa6-cre), we show that IL-22 signaling in Paneth cells is required for small intestinal host defense. We show that Paneth cell maturation, antimicrobial effector function, expression of specific WNTs, and organoid morphogenesis are dependent on cell-intrinsic IL-22Ra1 signaling. Furthermore, IL-22 signaling in Paneth cells regulates the intestinal commensal bacteria and microbiota-dependent IL-17A immune responses. Finally, we show ISC and, independently, Paneth cell-specific IL-22Ra1 signaling are critical for providing immunity against Salmonella enterica serovar Typhimurium. Collectively, our findings illustrate a previously unknown role of IL-22 in Paneth cell-mediated small intestinal host defense.

A detailed understanding of intestinal stem cell (ISC) self-renewal and differentiation is required to treat chronic intestinal diseases. However, the different models of ISC lineage hierarchy1–6 and segregation7–12 are subject to debate. Here, we have discovered non-canonical Wnt/planar cell polarity (PCP)-activated ISCs that are primed towards the enteroendocrine or Paneth cell lineage. Strikingly, integration of time-resolved lineage labelling with single-cell gene expression analysis revealed that both lineages are directly recruited from ISCs via unipotent transition states, challenging the existence of formerly predicted bi- or multipotent secretory progenitors7–12. Transitory cells that mature into Paneth cells are quiescent and express both stem cell and secretory lineage genes, indicating that these cells are the previously described Lgr5+ label-retaining cells7. Finally, Wnt/PCP-activated Lgr5+ ISCs are molecularly indistinguishable from Wnt/β-catenin-activated Lgr5+ ISCs, suggesting that lineage priming and cell-cycle exit is triggered at the post-transcriptional level by polarity cues and a switch from canonical to non-canonical Wnt/PCP signalling. Taken together, we redefine the mechanisms underlying ISC lineage hierarchy and identify the Wnt/PCP pathway as a new niche signal preceding lateral inhibition in ISC lineage priming and segregation. Polarity cues regulate intestinal stem cell fate. Bottcher et al. demonstrate that mouse intestinal stem cells, which express the Wnt/planar cell polarity reporter Flattop, are primed either towards the enteroendocrine or Paneth cell lineage.

Paneth cells are antimicrobial peptide-secreting epithelial cells located at the bottom of the intestinal crypts of Lieberkühn. The crypts begin to form around postnatal day 7 (P7) mice, and Paneth cells usually appear within the first 2 weeks. Paneth cell dysfunction has been reported to correlate with Crohn's disease-like inflammation, showing narrow crypts or loss of crypt architecture in mice. The morphology of dysfunctional Paneth cells is similar to that of Paneth/goblet intermediate cells. However, it remains unclear whether the formation of the crypt is related to the maturation of Paneth cells. In this study, we investigated the histological changes including epigenetic modification in the mouse ileum postnatally and assessed the effect of the methyltransferase inhibitor on epithelium development using an organoid culture. The morphological and functional maturation of Paneth cells occurred in the first 2 weeks and was accompanied by histone H3 lysine 27 (H3K27) trimethylation, although significant differences in DNA methylation or other histone H3 trimethylation were not observed. Inhibition of H3K27 trimethylation in mouse ileal organoids suppressed crypt formation and Paneth cell maturation, until around P10. Overall, our findings show that post-transcriptional modification of histones, particularly H3K27 trimethylation, leads to the structural and functional maturation of Paneth cells during postnatal development.

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 Math1Lox/LoxVilcreERT2 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.

ObjectiveAlthough perturbations in mitochondrial function and structure have been described in the intestinal epithelium of Crohn’s disease and ulcerative colitis patients, the role of epithelial mitochondrial stress in the pathophysiology of inflammatory bowel diseases (IBD) is not well elucidated. Prohibitin 1 (PHB1), a major component protein of the inner mitochondrial membrane crucial for optimal respiratory chain assembly and function, is decreased during IBD.DesignMale and female mice with inducible intestinal epithelial cell deletion of Phb1 (Phb1iΔIEC) or Paneth cell-specific deletion of Phb1 (Phb1ΔPC) and Phb1fl/fl control mice were housed up to 20 weeks to characterise the impact of PHB1 deletion on intestinal homeostasis. To suppress mitochondrial reactive oxygen species, a mitochondrial-targeted antioxidant, Mito-Tempo, was administered. To examine epithelial cell-intrinsic responses, intestinal enteroids were generated from crypts of Phb1iΔIEC or Phb1ΔPC mice.ResultsPhb1iΔIEC mice exhibited spontaneous ileal inflammation that was preceded by mitochondrial dysfunction in all IECs and early abnormalities in Paneth cells. Mito-Tempo ameliorated mitochondrial dysfunction, Paneth cell abnormalities and ileitis in Phb1iΔIEC ileum. Deletion of Phb1 specifically in Paneth cells (Phb1ΔPC) was sufficient to cause ileitis. Intestinal enteroids generated from crypts of Phb1iΔIEC or Phb1ΔPC mice exhibited decreased viability and Paneth cell defects that were improved by Mito-Tempo.ConclusionOur results identify Paneth cells as highly susceptible to mitochondrial dysfunction and central to the pathogenesis of ileitis, with translational implications for the subset of Crohn’s disease patients exhibiting Paneth cell defects.

Intelectins (intestinal lectins) are highly conserved across chordate evolution and have been implicated in various human diseases, including Crohn's disease (CD). The human genome encodes two intelectin genes, intelectin-1 (ITLN1) and intelectin-2 (ITLN2). Other than its high sequence similarity with ITLN1, little is known about ITLN2. To address this void in knowledge, we report that ITLN2 exhibits discrete, yet notable differences from ITLN1 in primary structure, including a unique amino terminus, as well as changes in amino acid residues associated with the glycan-binding activity of ITLN1. We identified that ITLN2 is a highly abundant Paneth cell-specific product, which localizes to secretory granules, and is expressed as a multimeric protein in the small intestine. In surgical specimens of ileal CD, ITLN2mRNA levels were reduced approximately five-fold compared to control specimens. The ileal expression of ITLN2 was unaffected by previously reported disease-associated variants in ITLN2 and CD-associated variants in neighboring ITLN1 as well as NOD2 and ATG16L1. ITLN2mRNA expression was undetectable in control colon tissue; however, in both ulcerative colitis (UC) and colonic CD, metaplastic Paneth cells were found to express ITLN2. Together, the data reported establish the groundwork for understanding ITLN2 function(s) in the intestine, including its possible role in CD.

Toll-like receptor 4 (TLR4) has been identified as a potentially promising therapeutic target in acute pancreatitis (AP). However, the role of intestinal TLR4 in AP and AP-associated gut injury remains unclear. This study aimed to explore the relationship between intestinal TLR4 and gut microbiota during AP. A mouse AP model was establish by intraperitoneal injection of L-arginine. Pancreatic injury and intestinal barrier function were evaluated in wild-type and intestinal epithelial TLR4 knockout (TLR4ΔIEC) mice. Gut microbiota was analyzed by 16S rRNA sequencing. Quadruple antibiotics were applied to induce microbiota-depleted mice. Differentially expressed genes in gut were detected by RNA sequencing.

Acute pancreatitis (AP) is usually accompanied by intestinal failure, but its mechanism is still unclear. In AP patients, the functions of Paneth cells (lysozyme, HD5, Reg3γ, and Wnt3a) decreased. Compared with AP mice, injuries and inflammation of the pancreas and ileum were aggravated in AP mice treated with dithizone (Dith) (Dith+AP mice). Intestinal permeability and bacterial translocation were also increased. 16S rRNA sequencing showed that the gut microbiota of Dith mice and Dith+AP mice exhibited a marked increase in the pathogenic bacterium

Vitamin D deficiency is co-prevalent with various liver diseases including cirrhosis, while the underlying mechanism remains elusive. Vitamin D receptor (VDR) is abundantly expressed in the distal region of small intestine, where the Paneth cells are enriched, suggesting that vitamin D signaling may modulates the intestinal Paneth cells and their production of defensins to restrain microbiome growth in the small intestine. In this study we found that in carbon tetrachloride-induced liver injury, hepatic 25-hydroxylation of vitamin D was impaired, leading to down regulated expression of Paneth cell fensins in the small intestine, gut dysbiosis, and endotoxinemia. While intraperitoneal injection of endotoxin (lipopolysaccharides) alone did not elicit liver fibrosis, it exacerbated the carbon tetrachloride initiated liver fibrogenesis. Oral gavage of synthetic Paneth cell alpha-defensin 5 (DEFA5) restored the homeostasis of gut microbiota, reduced endotoxemia, relieved liver inflammation, and ameliorated liver fibrosis. Likewise, Cholestyramine, cationic resin that can sequestrate endotoxin in the intestine, attenuated the liver fibrosis as well. Fecal transplant of the microbes derived from the DEFA5-treated donors improved liver fibrosis in the recipient mice. The intestinal Vdrconditional knockout mice exhibited reduction of Paneth cell defensins and lysozyme production, and worsened liver injury and fibrogenesis. Thus, liver injury impairs synthesis of 25(OH)VD3, which consequently impedes the Paneth cells functions in the small intestine, leading to gut dysbiosis for liver fibrogenesis.

Small intestinal Paneth cells play a critical role in acute kidney injury (AKI) and remote organ dysfunction by synthesizing and releasing IL-17A. In addition, intestine-derived norepinephrine is a major mediator of hepatic injury and systemic inflammation in sepsis. We tested the hypothesis that small intestinal Paneth cells synthesize and release norepinephrine to exacerbate ischemic AKI. After ischemic AKI, we demonstrated larger increases in portal venous norepinephrine levels compared with plasma norepinephrine in mice, consistent with an intestinal source of norepinephrine release after renal ischemia and reperfusion. We demonstrated that murine small intestinal Paneth cells express tyrosine hydroxylase mRNA and protein, a critical rate-limiting enzyme for the synthesis of norepinephrine. We also demonstrated mRNA expression for tyrosine hydroxylase in human small intestinal Paneth cells. Moreover, freshly isolated small intestinal crypts expressed significantly higher norepinephrine levels after ischemic AKI compared with sham-operated mice. Suggesting a critical role of IL-17A in Paneth cell-mediated release of norepinephrine, recombinant IL-17A induced norepinephrine release in the small intestine of mice. Furthermore, mice deficient in Paneth cells (SOX9 villin Cre mice) have reduced plasma norepinephrine levels after ischemic AKI. Finally, supporting a critical role for norepinephrine in generating ischemic AKI, treatment with the selective α-adrenergic antagonists yohimbine and phentolamine protected against murine ischemic AKI with significantly reduced renal tubular necrosis, inflammation, and apoptosis and less hepatic dysfunction. Taken together, we identify Paneth cells as a critical source of norepinephrine release that may lead to intestinal and liver injury and systemic inflammation after AKI.

BACKGROUND: Many hypotheses regarding the pathophysiology of enterocolitis in aganglionic megacolon or Hirschsprung disease (HSCR) has been proposed. Paneth cells are columnar intestinal epithelial cells that have an important role in maintaining of intestinal homeostasis as a bactericide. Since enterocolitis in HSCR may have association with Paneth cells metaplasia and hyperplasia, current study investigated Paneth cells metaplasia and hyperplasia in the sigmoid colon of HSCR rat model and its products, namely a-defensins and IL-1b, in the sigmoid colon tissues.METHODS: Aganglionosis-induced and control Sprague-Dawley rats were euthanized on Day (D)-7, -14, -17, -19, -21, -23, -25, and -28. Sigmoid colon tissue was isolated at each time point, and degree of enterocolitis as well as Paneth cells metaplasia and hyperplasia were analyzed by Hematoxylin-eosin staining, then protein levels of a-defensins and interleukin (IL)-1b were determined by enzyme-linked immunosorbent assay (ELISA).RESULTS: Enterocolitis scores increased with time. The Paneth cells metaplasia and hyperplasia were observed on D14 until D28 (p

The quality of life in patients with inflammatory bowel diseases (IBD) is strongly impaired. Alterations of intestinal epithelial homeostasis contribute to the development and establishment of IBD. Intestinal Paneth and Paneth-like cells produce and secrete luminal proteins sustaining epithelial homeostasis. Here we show that IFN-γ stimulates Paneth and Paneth-like cells degranulation that triggers the proliferation of intestinal epithelial cells (IEC) in a Wnt/β-catenin independent manner. Degranulation in Paneth and Paneth-like cells was mTORC1 and necroptosis dependent. Remarkably, lack of IFN-γ, inhibition of mTORC1, or impeding necroptosis reduces IEC proliferation cytokine-mediated. Our findings identify a new role for IFN-γ in stimulating IEC proliferation through inducing degranulation of Paneth and Paneth-like cells which is mTORC1 and necroptosis- dependent. In a mouse model of colitis, mTORC1 activation and necroptosis regulate Paneth and Paneth-like cell secretion. Furthermore, the colitogenic environment triggers PC metaplasia in the distal region of the large intestine to simulate cell proliferation.Highlights:IFN-γ stimulates proliferation,β-catenin independent.IFN-γ enhances mitochondrial activity and proliferationIFN-γ regulates PC biogenesis.mTORC1-dependent necroptosis mediates secretion in Paneth and Paneth-like cells.

The decline in mucosal immunity during aging increases susceptibility, morbidity and mortality to infections acquired via the gastrointestinal and respiratory tracts in the elderly. We previously showed that this immunosenescence includes a reduction in the functional maturation of M cells in the follicle-associated epithelia (FAE) covering the Peyer’s patches, diminishing the ability to sample of antigens and pathogens from the gut lumen. Here, co-expression analysis of mRNA-seq data sets revealed a general down-regulation of most FAE- and M cell-related genes in Peyer’s patches from aged mice, including key transcription factors known to be essential for M cell differentiation. Conversely, expression of ACE2, the cellular receptor for SARS-Cov-2 virus, was increased in the aged FAE. This raises the possibility that the susceptibility of aged Peyer’s patches to infection with the SARS-Cov-2 virus is increased. Expression of key Paneth cell-related genes was also reduced in the ileum of aged mice, consistent with the adverse effects of aging on their function. However, the increased expression of these genes in the villous epithelium of aged mice suggested a disturbed distribution of Paneth cells in the aged intestine. Aging effects on Paneth cells negatively impact on the regenerative ability of the gut epithelium and could indirectly impede M cell differentiation. Thus, restoring Paneth cell function may represent a novel means to improve M cell differentiation in the aging intestine and increase mucosal vaccination efficacy in the elderly.

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.

The colon embryology in vertebrates recognizes the endoderm layer of the middle and caudal intestine as source of epithelium, and the surrounding lateral splanchnic mesoderm as a source of lamina propria, submucosa, muscular propria and adventitial or serosa tunic. The enterocytes, enteroendocrine cells and goblet cells of distal colon are generated by cranio-caudal expression of homeotic genes Hoxa13 and Hoxd13 both in the mesoderm and endoderm layer reported, and also spatially and temporally, everything under regulation of homeotic genes Cdx2. Concerning the Paneth Cell or PC, the endoderm layer is a source by sponsoring mesodermal layer related with lower expressions to Cdx2, leading to high expressions Hoxa13 and Hoxd13; and PC absence by high Cdx-2 expressions lead to lack of co-expression of same Hoxa genes. This case report contributes to the histology of PC from a domestic feline with a diagnosis of colon adenomatous, a species that does not normally express PC in the colon, reasonably proposing that PC metaplasia in feline adenomatous may imply expressive disabilities of Cdx-2 and / or Hoxa13 and Hoxd13 expressions

Summary The grading and prognosis of prostatic adenocarcinoma with Paneth cell–like differentiation (PanEC) is controversial with limited available data. We identified 80 cases, not previously published, of PanEC first identified on biopsy (n = 69), transurethral resection of the prostate (n = 1), and radical prostatectomy (RP) (n = 10). Of 69 biopsies, 22 did not have a grade assigned. In the remaining 47 biopsies, the Grade Groups (GGs) of the associated usual prostatic adenocarcinoma were GG1–2 (n = 34) and GG3–5 (n = 13). Of 10 RPs, the GGs were as follows: GG1–2 (n = 8), GG4 (n = 1), and no grade due to treatment effect (n = 1); pathological stages were pT2 (n = 8) and pT3a (n = 2), all with negative lymph nodes. We analyzed 19 cases with available follow-up only associated with GG1–2 conventional cancer; 9 underwent RP, and GGs at RP were as follows: GG1–2 (n = 7), no grade due to treatment effect (n = 1), and missing data (n = 1); pathologic stages were pT2 (n = 6) and pT3a (n = 3); there were no positive regional lymph nodes; 3 were managed with active surveillance, without follow-up progression; 5 patients underwent radiation therapy with or without hormone therapy; none showed follow-up progression; 2 (10.5%) patients were recommended to undergo radiotherapy, with no further follow-up. Of the cases with available follow-up, 9 were not associated with conventional adenocarcinoma; the majority of these cases were treated with radiation therapy or active surveillance without evidence of progression. In summary, although a minority of PanECs are associated with conventional higher grade adenocarcinoma and have progression after treatment, the majority have favorable findings, justifying the consideration of them as more indolent tumors despite cases in which PanEC resembles Gleason pattern 5 adenocarcinoma.

ObjectiveReduced Paneth cell (PC) numbers are observed in inflammatory bowel diseases and impaired PC function contributes to the ileal pathogenesis of Crohn’s disease (CD). PCs reside in proximity to Lgr5+ intestinal stem cells (ISC) and mitochondria are critical for ISC-renewal and differentiation. Here, we characterise ISC and PC appearance under inflammatory conditions and describe the role of mitochondrial function for ISC niche-maintenance.DesignIleal tissue samples from patients with CD, mouse models for mitochondrial dysfunction (Hsp60Δ/ΔISC) and CD-like ileitis (TNFΔARE), and intestinal organoids were used to characterise PCs and ISCs in relation to mitochondrial function.ResultsIn patients with CD and TNFΔARE mice, inflammation correlated with reduced numbers of Lysozyme-positive granules in PCs and decreased Lgr5 expression in crypt regions. Disease-associated changes in PC and ISC appearance persisted in non-inflamed tissue regions of patients with CD and predicted the risk of disease recurrence after surgical resection. ISC-specific deletion of Hsp60 and inhibition of mitochondrial respiration linked mitochondrial function to the aberrant PC phenotype. Consistent with reduced stemness in vivo, crypts from inflamed TNFΔARE mice fail to grow into organoids ex vivo. Dichloroacetate-mediated inhibition of glycolysis, forcing cells to shift to mitochondrial respiration, improved ISC niche function and rescued the ability of TNFΔARE mice-derived crypts to form organoids.ConclusionWe provide evidence that inflammation-associated mitochondrial dysfunction in the intestinal epithelium triggers a metabolic imbalance, causing reduced stemness and acquisition of a dysfunctional PC phenotype. Blocking glycolysis might be a novel drug target to antagonise PC dysfunction in the pathogenesis of CD.

The small intestine mucosa is lined by specialized cells that form the crypt-villus axis, which expands its surface. Among the six intestinal epithelial cell types, the Paneth cell is located at the base of the crypt, and it contains numerous granules in its cytoplasm, composed of antimicrobial peptides, such as defensins and lysozyme, and growth factors, such as epidermal growth factor, transforming growth factor-α, and Wnt ligands. Together, these elements act in the defense against microorganisms, regulation of intestinal microbiota, maintenance, and regulation of stem cell identity. Pathologies that target Paneth cells can disturb such defense activity, but they also affect the maintenance of the stem cell niche. In that way, Crohn's disease, necrotizing enterocolitis, and graft-versus-host disease promote a reduction of Paneth cell population, and, consequently, secretion of their products into the lumen of the crypts, making the affected organism predisposed to infections and dysbiosis. Additionally, the emergence of new intestinal cells is also decreased. This review aims to address the main characteristics of Paneth cells, highlighting their multiple functions and the importance of their preservation to ensure bowel homeostasis.

Previous studies suggest that Paneth cells are involved in NEC development. Defensin alpha 6 (DEFA6) and guanylate cyclase activator 2A (GUCA2A) are selective protein markers of Paneth cells. The objective was to explore DEFA6 and GUCA2A expression in intestinal tissue samples from newborn infants with and without NEC. Tissue samples from histologically intact intestine were analyzed from 70 infants: 43 underwent bowel resection due to NEC and 27 controls were operated due to conditions such as intestinal atresia, dysmotility, aganglionosis, pseudo-obstruction or volvulus. Each tissue sample was immunohistochemically stained for DEFA6 and GUCA2A. Semi-automated digital image analysis was performed to determine protein expression. Clinical data and protein expressions were compared between the groups. DEFA6 expression was lower in the NEC group (p = 0.006). Low DEFA6 correlated with risk of developing NEC in a logistic regression analysis, independently of gestational age and birth weight (OR 0.843 [CI 0.732–0.971]; p = 0.018). GUCA2A expression did not differ between the two groups.Conclusion: Lower expression of DEFA6 together with intact GUCA2A expression indicates that NEC patients have well-defined Paneth cells but diminished defensin activity. Our results suggest that DEFA6 could be used as a biomarker for NEC. What is Known:• Previous studies of defensin activity in NEC have been inconsistent, showing that defensin levels may be increased or diminished in NEC. GUCA2A has to our knowledge never been studied in NEC. What is New:• This study benchmarks two specific Paneth cell markers (DEFA6 and GUCA2A) and their activity in individuals with and without NEC.• The key finding is that the NEC group had a lower DEFA6 expression compared to the Controls, while the expression of GUCA2A did not differ between the groups.

New data suggest how epithelial dysfunction and intestinal dysbiosis contribute to inflammatory bowel disease in patients and in models of XIAP deficiency (see the related Research Articles by Strigli et al . and Wahida et al . ).

PurposeNecrotizing 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.MethodsPaneth 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.ResultsDithizone treatment significantly decreased intestinal perfusion compared to controls (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

Background & Aims Paneth cells secrete antimicrobial proteins including lysozyme via secretory autophagy as part of the mucosal protective response. The ELAV like RNA-binding protein 1 (ELAVL1, also called HuR) regulates stability and translation of messenger RNAs (mRNAs) and many aspects of mucosal physiology. We studied the posttranscriptional mechanisms by which HuR regulates Paneth cell function. Methods Intestinal mucosal tissues were collected from mice with intestinal epithelium (IE)-specific disruption of HuR (IE-HuR–/–), HuRfl/fl-Cre– mice (controls), and patients with inflammatory bowel diseases and analyzed by histology and immunohistochemistry. Paneth cell functions were determined by lysozyme-immunostaining assays. We isolated primary enterocytes from IE-HuR–/– and control mice and derived intestinal organoids. HuR and the chaperone CNPY3 were overexpressed from transgenes in intestinal epithelial cells (IECs) or knocked down with small interfering RNAs. We performed RNA pulldown assays to investigate interactions between HuR and its target mRNAs. Results Intestinal tissues from IE-HuR–/– mice had reduced numbers of Paneth cells, and Paneth cells had fewer lysozyme granules per cell, compared with tissues from control mice, but there were no effects on Goblet cells or enterocytes. Intestinal mucosa from patients with inflammatory bowel diseases had reduced levels of HuR and fewer Paneth cells. IE-HuR–/– mice did not have the apical distribution of TLR2 in the intestinal mucosa as observed in control mice. IECs from IE-HuR–/– mice expressed lower levels of CNPY3. Intestinal organoids from IE-HuR–/– mice were smaller and contained fewer buds compared with those generated from controls, and had fewer lysozyme-positive cells. In IECs, knockdown of HuR decreased levels of the autophagy proteins LC3-I and LC3-II, compared with control cells, and prevented rapamycin-induced autophagy. We found HuR to interact directly with the Cnpy3 mRNA coding region and increase levels of CNPY3 by increasing the stability and translation of Cnpy3 mRNA. CNPY3 bound TLR2, and cells with knockdown of CNPY3 or HuR lost membrane localization of TLR2, but increased cytoplasmic levels of TLR2. Conclusions In studies of mice, IECs, and human tissues, we found HuR to increase expression of CNPY3 at the posttranscriptional level. CNPY3 is required for membrane localization of TLR2 and Paneth cell function.

Highlights • Colonic Paneth adenocarcinoma is a rare entity with only a few reports in the world literature. • The pathologist must be aware of the existence of this histological subtype. • A specific pathogen pathway is incriminated. • The treatment remains equal to other classic types of colorectal adenocarcinoma., Introduction Amongst the morphotypes of colorectal adenocarcinomas, the rich cell type of Paneth constitutes a rare histopathologic variant of adenocarcinoma, which can be observed all along the digestive tract but also in other organs such as the prostate or the breast. About 24 cases were found in the literature, with only 7 cases within the colon and appendix. Presentation of case We report the case of a 50-year-old man, without past medical history, complaining of abdominal pain and constipation for 3 months. Biological tests were normal. Radiological investigations and endoscopy revealed a sessile polyp in the right colonic angle measuring 4 cm in greatest diameter. Biopsy concluded to a tubular adenoma with low-grade dysplasia. The patient underwent right hemicolectomy. Microscopically, an invasive adenocarcinoma was identified occupying the colonic mucosal with an invasion of the submucosa. The tumor showed a tubulovillous pattern on the surface and was made mostly of jagged crowded glands in the depth. Some areas exhibit Paneth cell differentiation. No metastatic lymph node was found, and the tumor was staged T1N0. The postoperative course was uneventful. The patient remained free of symptoms at the 6-month follow-up and had no evidence of recurrence. Conclusion We reported a Tunisian case of Paneth cell colonic adenocarcinoma. The diagnosis is challenging in biopsies when only well-differentiated areas are sampled. Lysozyme immune-histochemical stain may be helpful when diagnosis difficulty arises. The beta-catenin pathway seems to be activated. More studies are needed for the etiology, pathogenesis, clinical course, prognosis and treatment of Paneth cell carcinoma.

Differentiation and lineage specification are controlled by cooperation of growth factor signalling. The involvement of epigenetic regulators in lineage specification remains largely elusive. Here, we show that the histone methyltransferase Mll1 prevents intestinal progenitor cells from differentiation, whereas it is also involved in secretory lineage specification of Paneth and goblet cells. Using conditional mutagenesis in mice and intestinal organoids, we demonstrate that loss of Mll1 renders intestinal progenitor cells permissive for Wnt-driven secretory differentiation. However, Mll1-deficient crypt cells fail to segregate Paneth and goblet cell fates. Mll1 deficiency causes Paneth cell-determined crypt progenitors to exhibit goblet cell features by unleashing Mapk signalling, resulting in increased numbers of mixed Paneth/goblet cells. We show that loss of Mll1 abolishes the pro-proliferative effect of Mapk signalling in intestinal progenitor cells and promotes Mapk-induced goblet cell differentiation. Our data uncover Mll1 and its downstream targets Gata4/6 as a regulatory hub of Wnt and Mapk signalling in the control of lineage specification of intestinal secretory Paneth and goblet cells.

A decline in stem cell function impairs tissue regeneration during ageing, but the role of the stem-cell-supporting niche in ageing is not well understood. The small intestine is maintained by actively cycling intestinal stem cells that are regulated by the Paneth cell niche(1,2). Here we show that the regenerative potential of human and mouse intestinal epithelium diminishes with age owing to defects in both stem cells and their niche. The functional decline was caused by a decrease in stemness-maintaining Wnt signalling due to production of Notum, an extracellular Wnt inhibitor, in aged Paneth cells. Mechanistically, high activity of mammalian target of rapamycin complex 1 (mTORC1) in aged Paneth cells inhibits activity of peroxisome proliferator activated receptor alpha (PPAR-alpha)(3), and lowered PPAR-a activity increased Notum expression. Genetic targeting of Notum or Wnt supplementation restored function of aged intestinal organoids. Moreover, pharmacological inhibition of Notum in mice enhanced the regenerative capacity of aged stem cells and promoted recovery from chemotherapy-induced damage. Our results reveal a role of the stem cell niche in ageing and demonstrate that targeting of Notum can promote regeneration of aged tissues.

Deficiency in X-linked inhibitor of apoptosis protein (XIAP) is the cause for X-linked lymphoproliferative syndrome 2 (XLP2). About one-third of these patients suffer from severe and therapy-refractory inflammatory bowel disease (IBD), but the exact cause of this pathogenesis remains undefined. Here, we used XIAP-deficient mice to characterize the mechanisms underlying intestinal inflammation. In

Paneth cells (PCs) are intestinal epithelial cells (IECs) that contain eosinophilic granules, which are located in Lieberkühn crypts. An increasing number of animal and human experiments have indicated that PCs are involved in the progression of a variety of intestinal as well as systemic inflammatory responses including necrotizing enterocolitis (NEC). NEC is an enteric acquired disease with high mortality that usually occurs in premature infants and neonates, however the underlying mechanisms remain unclear. In this review, we summarize the features of PCs, including their immune function, association with gut microbiota and intestinal stem cells, and their mechanism of regulating IEC death to explore the possible mechanisms by which PCs affect NEC.

Loss-of-function variants in the Protein Tyrosine Phosphatase non-receptor Type 2 ( PTPN2) gene are associated with increased risk of Inflammatory Bowel Disease (IBD). PTPN2 encodes T Cell Protein Tyrosine Phosphatase (TCPTP), a negative regulator of intracellular signaling pathways including JAK-STAT. PTPN2 is critical for intestinal epithelial cell (IEC) barrier maintenance, IEC-macrophage crosstalk, and modulating the gut microbiome in mice, although the mechanism in which PTPN2 loss affects gut microbiome is unknown. IBD patient biopsies and studies in transgenic mice show that IBD-associated genes can modify IEC subtypes and their functional roles in intestinal homeostasis. Hypothesis: PTPN2 loss compromises modulation of gut microbiome by impairing number and function of IEC subtypes. Methods: To identify how loss of PTPN2 activity in vivo affects ileal IEC subtypes and their function, RNA and protein were isolated from ileal IECs of whole-body Ptpn2-wild-type (WT), heterozygous (Het) and KO mice. RNA was assayed by two separate Nanostring® panels: PanCancer and AutoImmune totaling >1500 targets. Dysregulated targets associated with IEC function were validated by Western blot, immunostaining, and transmission electron microscopy (TEM). Altered genes were then investigated in tamoxifen-inducible intestinal epithelial-specific Ptpn2 knockout mice ( Ptpn2 Δ IEC) and Ptpn2 fl/fl controls. Results: 1398 targets had detectable expression. Notably, Paneth cell (PC)-associated genes Lyz1, Defa6, Pla2g2a, and Fgfr3 were markedly downregulated (pΔ IEC mice showed no change in ileal villus length (n=6), number of PCs (n=7), and TEM showed abundant cytosolic granules compared to Ptpn2 fl/fl (n=4). However, immunostaining (n=6) and Western blotting (pΔ IEC mice (p Supported by NIH 2R01DK091281, 1R01AI153314-01, R21AI152017 and R01AI165490 (DFM) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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

Complement activation is associated with regional inflammation during acute gastrointestinal injury (AGI). This study is designed to explore how intracellular C3 activation in Paneth cells (PCs) affects regeneration of intestinal epithelium during AGI. AGI was induced in wildtype C57BL/6 mice, with sham operation employed as control. Exogenous C3 (1 mg, I.P.) was applied at 6 h post-surgery. Intestinal crypts harvested from ileum were cultured with presence or absence of C3 (20 μg/ml), with small interfering RNA against BST1 and complement activation inhibitor selectively applied in vitro. The intestinal integrity, percentage of PCs and intestinal stem cells (ISCs) were evaluated. Importantly, cADPR, C3 fragments, and S6-related proteins were detected in PCs to inspect the mammalian target of rapamycin complex 1 (mTORC1) signaling. AGI caused breakdown of intestinal mucosa integrity and regional inflammation. Exogenous C3 by itself failed to promote the growth of intestinal epithelium, but distinctly enhanced the activity of PCs via intracellular activation, which subsequently supported the expansion of ISCs inside of intestinal crypts. Inhibition of C3 activation was associated with decreased expressions of S6, S6K1 and cADPR, with blocking BST1 found to depress cADPR only. Collectively, these data confirmed intracellular activation of C3 in PCs enhanced expansion of ISCs in response to acute injury. The mTORC1 signaling pathway in PCs contributed to this crosstalk during exogenous C3 treatment.

Serving as the guardians of small intestine, Paneth cells (PCs) play an important role in intestinal homeostasis maintenance. Although PCs uniquely exist in intestine under homeostasis, the dysfunction of PCs is involved in various diseases not only in intestine but also in extraintestinal organs, suggesting the systemic importance of PCs. The mechanisms under the participation of PCs in these diseases are multiple as well. The involvements of PCs are mostly characterized by limiting intestinal bacterial translocation in necrotizing enterocolitis, liver disease, acute pancreatitis and graft-vs-host disease. Risk genes in PCs render intestine susceptible to Crohn’s disease. In intestinal infection, different pathogens induce varied responses in PCs, and toll-like receptor ligands on bacterial surface trigger the degranulation of PCs. The increased level of bile acid dramatically impairs PCs in obesity. PCs can inhibit virus entry and promote intestinal regeneration to alleviate COVID-19. On the contrary, abundant IL-17A in PCs aggravates multi-organ injury in ischemia/reperfusion. The pro-angiogenic effect of PCs aggravates the severity of portal hypertension. Therapeutic strategies targeting PCs mainly include PC protection, PC-derived inflammatory cytokine elimination, and substituting AMP treatment. In this review, we discuss the influence and importance of Paneth cells in both intestinal and extraintestinal diseases as reported so far, as well as the potential therapeutic strategies targeting PCs.

Paneth cells are a group of unique intestinal epithelial cells, and they play an important role in host-microbiota interactions. At the origin of Paneth cell life, several pathways such as Wnt, Notch, and BMP signaling, affect the differentiation of Paneth cells. After lineage commitment, Paneth cells migrate downward and reside in the base of crypts, and they possess abundant granules in their apical cytoplasm. These granules contain some important substances such as antimicrobial peptides and growth factors. Antimicrobial peptides can regulate the composition of microbiota and defend against mucosal penetration by commensal and pathogenic bacteria to protect the intestinal epithelia. The growth factors derived from Paneth cells contribute to the maintenance of the normal functions of intestinal stem cells. The presence of Paneth cells ensures the sterile environment and clearance of apoptotic cells from crypts to maintain the intestinal homeostasis. At the end of their lives, Paneth cells experience different types of programmed cell death such as apoptosis and necroptosis. During intestinal injury, Paneth cells can acquire stem cell features to restore the intestinal epithelial integrity. In view of the crucial roles of Paneth cells in the intestinal homeostasis, research on Paneth cells has rapidly developed in recent years, and the existing reviews on Paneth cells have mainly focused on their functions of antimicrobial peptide secretion and intestinal stem cell support. This review aims to summarize the approaches to studying Paneth cells and introduce the whole life experience of Paneth cells from birth to death.

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by fibrosis that develops from fatty liver. Disruption of intestinal microbiota homeostasis, dysbiosis, is associated with fibrosis development in NASH. An antimicrobial peptide α-defensin secreted by Paneth cells in the small intestine is known to regulate composition of the intestinal microbiota. However, involvement of α-defensin in NASH remains unknown. Here, we show that in diet-induced NASH model mice, decrease of fecal α-defensin along with dysbiosis occurs before NASH onset. When α-defensin levels in the intestinal lumen are restored by intravenous administration of R-Spondin1 to induce Paneth cell regeneration or by oral administration of α-defensins, liver fibrosis is ameliorated with dissolving dysbiosis. Furthermore, R-Spondin1 and α-defensin improved liver pathologies together with different features in the intestinal microbiota. These results indicate that decreased α-defensin secretion induces liver fibrosis through dysbiosis, further suggesting Paneth cell α-defensin as a potential therapeutic target for NASH.

Intestinal homeostasis depends on interactions between the intestinal epithelium, the immune system and the microbiota. Because of these complicated connections, there are many problems that need to be solved. Current research has indicated that genes targeted by Wnt signaling are responsible for controlling intestinal stem cell fate and for modulating intestinal homeostasis. Our data show that loss of frizzled 7 (Fzd7), an important element in Wnt signaling, interrupts the differentiation of mouse intestinal stem cells into absorptive progenitors instead of secretory progenitors (precursors of goblet and Paneth cells). The alteration in canonical Wnt and Notch signaling pathways interrupts epithelial homeostasis, resulting in a decrease in physical protection in the intestine. Several phenotypes in our Fzd7-deleted model were similar to the features of enterocolitis, such as shortened intestines, decreased numbers of goblet cells and Paneth cells, and severe inflammation. Additionally, loss of Fzd7 exacerbated the defects in a chemical-induced colitis model and could initiate tumorigenesis. These findings may provide important information for the discovery of efficient therapeutic methods to treat enterocolitis and related cancers in the intestines.

The mammalian intestine is one of the most rapidly self-renewing tissues, driven by actively cycling stem cells residing at the crypt bottom1,2. Together with stromal cells, Paneth cells form a major element of the niche microenvironment that provides various growth factors to orchestrate intestinal stem cell homeostasis, such as Wnt33. With 19 family members, different Wnt ligands can selectively activate β-catenin dependent (canonical) or independent (non-canonical) signaling4,5. Here, we report that Dishevelled-associated activator of morphogenesis 1 (Daam1) and its paralogue Daam2 asymmetrically regulate canonical and non-canonical Wnt (Wnt/PCP) signaling, and their function is required for Paneth cell progenitor differentiation. We found that Daam1/2 interacts with the Wnt antagonist Rnf43, and Daam1/2 double knockout stimulates canonical Wnt signaling by preventing Rnf43-dependent endo-lysosomal degradation of the ubiquitinated Wnt receptor, Frizzled (Fzd). Moreover, single-cell RNA sequencing analysis revealed that Paneth cell differentiation is impaired by Daam1/2 depletion, as a result of defective Wnt/PCP signaling. Taken together, we identified Daam1/2 as an unexpected hub molecule coordinating both canonical and non-canonical Wnt signaling, the regulation of which is fundamental for specifying an adequate number of Paneth cells while maintaining intestinal stem cell homeostasis.

Paneth cells (PCs), responsible for the secretion of antimicrobial peptides in the small intestine and for niche support to Lgr5+ crypt-base columnar stem cells (CBCs), have been shown to respond to inflammation by dedifferentiating into stem-like cells in order to sustain a regenerative response1,2. Therefore, PCs may represent the cells-of-origin of intestinal cancer in the context of inflammation. To test this hypothesis, we targeted Apc, Kras, and Tp53 mutations in Paneth cells by Cre-Lox technology and modelled inflammation by dextran sodium sulfate (DSS) administration. PC-specific loss of Apc resulted in multiple small intestinal tumors, whereas Kras or Tp53 mutations did not. Compound Apc and Kras mutations in PCs resulted in a striking increase in tumor multiplicity even in the absence of the inflammatory insult. By combining scRNAseq with lineage tracing to capture the conversion of PCs into bona fide tumor cells, we show that they progress through a “revival stem cell” (RSC) state characterized by high Clusterin (Clu) expression and Yap1 signaling, reminiscent of what has been previously observed upon irradiation of the mouse digestive tract3. Accordingly, comparison of PC- and Lgr5-derived murine intestinal tumors revealed differences related to Wnt signaling and inflammatory pathways which match the dichotomy of CBCs and injury-induced RSCs4 between human sporadic colon cancers and those arising in the context of inflammatory bowel diseases. Last, we show that western-style dietary habits, known to trigger a low-grade inflammation throughout the intestinal tract, underlie the analogous de-differentiation of Paneth cells and their acquisition of stem-like features. Taken together, our results show that intestinal cancer arises in the context of inflammation through the dedifferentiation of committed secretory lineages such as Paneth cells and the activation of the revival stem cell state. As such, a true quiescent stem cell identity may be hidden in fully committed and post-mitotic lineages which, upon inflammation, support the regenerative response by re-entering the cell cycle and dedifferentiating into RSCs. The chronic nature of the tissue insult in inflammatory bowel diseases and even in the context of western-style dietary habits is likely to result in the expansion of cell targets for tumor initiation and progression.

Mouse atonal homolog 1 (Math1/Atoh1) is a basic helix-loop-helix transcription factor important for the differentiation of secretory cells within the intestinal epithelium. The analysis of Paneth depletion efficiency upon Math1lox/loxVilCreERT2 (Math1∆IEC) mice treatment with Tamoxifen in the presence or absence of intestinal microbiota, showed a failure on Paneth cell depletion in germ-free mice as compared to SPF mice. However, goblet cells were efficiently depleted in Math1∆IEC germ-free mice. The gene expression of Math1 was significantly reduced in the ileum of germ-free Math1∆IEC mice 5 days post tamoxifen injection as compared to germ-free control, but its protein expression was still detectable in the nuclei of epithelial cells in the crypts. Germ-free mice showed low proliferative ileal crypts as well as apoptotic cells that were mainly detected in the tip of the villus, consistent with a slow turnover rate of epithelial cells. Although Paneth cells were not depleted in germ-free Math1∆IEC mice for the first 7 weeks after the last tamoxifen injection - far already from the 5 days timelaps observed in SPF conditions- but an incomplete depletion of Paneth cells was observed 14 weeks after last tamoxifen injection. Colonization of germ-free mice restored the phenotype observed in SPF mice, highlighting the regulatory role of gut microbes in our model. We conclude that absence of intestinal microbiota in Math1∆IEC mice is associated with reduced epithelial cell renewal and delays the depletion of preexisting Paneth cells.