Paneth Cell Function Research Articles

Secretory Sorcery: Paneth Cell Control of Intestinal Repair and Homeostasis.

Paneth cells are professional secretory cells that classically play a role in the innate immune system by secreting antimicrobial factors into the lumen to control enteric bacteria. In this role, Paneth cells are able to sense cues from luminal bacteria and respond by changing production of these factors to protect the epithelial barrier. Paneth cells rely on autophagy to regulate their secretory capability and capacity. Disruption of this pathway through mutation of genes, such as Atg16L1, results in decreased Paneth cell function, dysregulated enteric microbiota, decreased barrier integrity, and increased risk of diseases such as Crohn's disease in humans. Upon differentiation Paneth cells migrate downward and intercalate among active intestinal stem cells at the base of small intestinal crypts. This localization puts them in a unique position to interact with active intestinal stem cells, and recent work shows that Paneth cells play a critical role in influencing the intestinal stem cell niche. This review discusses the numerous ways Paneth cells can influence intestinal stem cells and their niche. We also highlight the ways in which Paneth cells can alter cells and other organ systems.

Liver Injury Impaired 25-Hydroxylation of Vitamin D Suppresses Intestinal Paneth Cell defensins, leading to Gut Dysbiosis and Liver Fibrogenesis.

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.

The roles and functions of Paneth cells in Crohn's disease: A critical review.

Paneth cells (PCs) are located at the base of small intestinal crypts and secrete the α‐defensins, human α‐defensin 5 (HD‐5) and human α‐defensin 6 (HD‐6) in response to bacterial, cholinergic and other stimuli. The α‐defensins are broad‐spectrum microbicides that play critical roles in controlling gut microbiota and maintaining intestinal homeostasis. Inflammatory bowel disease, including ulcerative colitis and Crohn's disease (CD), is a complicated autoimmune disorder. The pathogenesis of CD involves genetic factors, environmental factors and microflora. Surprisingly, with regard to genetic factors, many susceptible genes and pathogenic pathways of CD, including nucleotide‐binding oligomerization domain 2 (NOD2), autophagy‐related 16‐like 1 (ATG16L1), immunity‐related guanosine triphosphatase family M (IRGM), wingless‐related integration site (Wnt), leucine‐rich repeat kinase 2 (LRRK2), histone deacetylases (HDACs), caspase‐8 (Casp8) and X‐box‐binding protein‐1 (XBP1), are relevant to PCs. As the underlying mechanisms are being unravelled, PCs are identified as the central element of CD pathogenesis, integrating factors among microbiota, intestinal epithelial barrier dysfunction and the immune system. In the present review, we demonstrate how these genes and pathways regulate CD pathogenesis via their action on PCs and what treatment modalities can be applied to deal with these PC‐mediated pathogenic processes.

Dietary effects on the intestinal wnt signaling molecules in C57bl/6j mice

Rationale: Obesity and metabolic comorbidities are increasingly associated with defects in the intestinal barrier. In previous work we have shown that a high-fat and high-sugar diet resulted in a deterioration of the intestinal integrity, increased bacterial translocation and reduced Paneth cell cryptdins in ileum of C57BL/6J mice. The supplementation with the prebiotic inulin or the short-chain fatty acid (SCFA) butyrate increased Paneth cell cryptdins. The Wnt signaling pathway affects the biological function of Paneth cells by regulating the expression of Paneth cell antimicrobials.

Aberrant TNF Signaling Links RIPK3-Dependent Cell Death and Loss of TLR5 with Intestinal Inflammation and Dysbiosis

Intestinal homeostasis depends on the symbiotic nature of host-microbiota interactions. Whereas elevated TNF and dysbiosis are known hallmarks of inflammatory bowel disease (IBD), the mechanistic link between TNF, its cognate receptors, and the microbiome remains only partially understood. Using XIAP-deficiency as a murine model for a mendelian form of IBD, we show that the host-encoded TNF response cooperated with environmental cues to induce dysbiosis. Specifically, aberrant TNF signaling resulted in Paneth cell dysfunction, deregulated inflammatory pathways, and dysbiosis, which was characterized by loss of beneficial commensals. Mucosal inflammation was dependent on the individual action of TNF receptor (TNFR)1 and TNFR2. Mechanistically, aberrant TNFR2 signaling individually targeted IRF8+ lamina-propria dendritic cells to undergo MLKL-independent but RIPK3-driven cell death, whereas aberrant TNFR1 signaling also compromised Paneth cell function. Genetic loss of either receptor was sufficient to restore Clostridia and prevent intestinal inflammation. Primary patient data support the murine genetic evidence that TNFR2 promotes IBD in human patients. Our data show that the impairment of Paneth cells together with the inflammatory demise of dendritic cells is central for driving IBD and dysbiosis.

Bifidobacterium longum alleviates irritable bowel syndrome-related visceral hypersensitivity and microbiota dysbiosis via Paneth cell regulation

ABSTRACT Although the oral administration of Bifidobacterium longum (B. longum) relieves the signs of irritable bowel syndrome (IBS) in clinical settings, the mechanisms underlying its effects are unclear. In this study, we evaluated the precise effects of B. longum on IBS via regulation of Paneth cell function. We confirmed the beneficial effects of B. longum on defecation habits and visceral hypersensitivity in WAS rats. Further analysis revealed that B. longum enhanced mucosal repair, promoted lysozyme production, and ameliorated dysbiosis of the microbiota in WAS rats. These processes are closely correlated with Paneth cell functions. In vitro, we incubated primary cultured enteroids with B. longum and found that B. longum promoted the proliferation of these organoids; this may be attributed to the upregulation of the stem niche factors WNT3A and TGF-β, which are secreted by Paneth cells. Based on our findings, we propose that B. longum relieves IBS by restoring the antimicrobial activity and stem niche maintenance function of Paneth cells.

Interleukin-22 receptor signaling in Paneth cells is critical for their maturation and antimicrobial function

Abstract Interleukin-22 (IL-22) acts in the intestine to promote critical tissue protective functions. However, since a diverse array of intestinal cell types (absorptive, secretory, and 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 mucosal host defense. To address this question, we used entire gut epithelium, intestinal stem cell (ISC)-specific, and Paneth cell-specific IL-22Ra1 knockout mice. Entire epithelium-specific IL-22Ra1 knockout (Il22Ra1fl/fl;Villin-cre) mice displayed defects in Paneth cell function. Using ISC-specific IL-22Ra1 knockout mice (Il22Ra1fl/fl;Lgr5-EGFP-creERT2) and lineage tracing mice, we ruled out the involvement of Lgr5+ 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-22Ra1 signaling in Paneth cells is required for small intestinal host defense. We show that Paneth cell maturation, antimicrobial effector functions, gene expression of specific WNTs (Wnt6 and Wnt9b), and enteroid 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 Paneth cell-specific IL-22Ra1 signaling helps provide immunity against Salmonella typhimurium. Collectively, our findings provide a unique and novel role of IL-22 in Paneth cell-mediated small intestinal host defense.

Paneth cells regulate allergic sensitization in the gastrointestinal tract

Abstract The gut immune homeostasis plays a major role in controlling sensitization to food allergens and the development of allergic responses in the gastrointestinal tract, but also to skin or lung. Epithelial cells sensing of commensal microbes is crucial for the maturation of the gut immune system and antibiotic treatment during early life is believed to have contributed to the increased incidence of food allergy. Paneth cells secrete antimicrobial products and cytokines in the crypts of the small intestine, but their role in food allergy remains elusive. We found that SOX9ΔIEC mice, which lack of Paneth cells, display dysbiosis with increased microbial diversity. Interestingly, Paneth cell deficiency have increased numbers of ILC2 in the intestinal lymphoid tissues. Upon oral sensitization with a model food antigen in the presence of cholera toxin, SOX9ΔIEC mice developed significantly higher allergen-specific serum IgE responses, and enhanced signs of allergic responses (i.e., swelling, hypothermia, impairment of lung functions) after nasal allergen challenge. Transplantation of fecal materials from SOX9ΔIEC mice was able to transfer the hyper-allergenic phenotype to control wild-type mice and bacteria-free fecal extract from SOX9ΔIEC mice increased the number of IgE producing B cells than fecal extract from control mice with treatment of anti-CD40 and IL-4 in vitro. Ongoing studies are assessing the metabolites present in the intestinal lumen of SOX9ΔIEC mice and their effect of intestinal epithelial cells and myeloid cells to elucidate the mechanisms by which impaired or defective anti-microbial functions of Paneth cells regulate allergic sensitization of the GI tract.

Paneth cells regulate diet-induced obesity and trafficking of inflammatory immune cells into adipose tissues

Abstract Paneth cells regulate many key aspects of gastrointestinal health through the antimicrobial products and cytokines they produce. Loss or defective Paneth cell functions leads to dysbiosis and is one of the causes of Inflammatory Bowel Disease (IBD). The prevalence of obesity is growing in the general population and among recently diagnosed IBD patients, leading to the speculation that obesity increases IBD incidence, but the underlying mechanisms remain to be elucidated. We addressed whether Paneth cells of the small intestine play a role in the development of obesity. For this purpose, control wild-type C57BL/6 and Sox9ΔIEC mice, which lack Paneth cells due to Sox9 gene deletion within the intestinal epithelium (Sox9ΔIEC mice), were fed a high fat diet for 13 weeks. Though the consumption of food was similar between the 2 groups, weekly measurement of body weight showed that Sox9ΔIEC mice gained weight much faster and ultimately became more obese than the wild-type mice. The Sox9ΔIEC mice also developed larger abdominal fats, which included increased numbers of inflammatory macrophages (p<0.05) and neutrophils (p<0.05). Interestingly, B cells were the most increased (p<0.01) immune cells population in white fat tissues of Sox9ΔIEC mice. When compared to control mice, the Sox9ΔIEC mice displayed impaired glucose tolerance (p<0.0001) indicating that these mice also developed a metabolic disorder. Finally, the in vivo FITC-dextran permeability assay showed that Sox9ΔIEC mice on high-fat diet have increased intestinal permeability (p<0.05). These results suggest a new role for Paneth cells as regulators of diet-induced obesity via their impact on gut immune homeostasis and recruitment of immune cells into omental fat tissues.

An Update Review on the Paneth Cell as Key to Ileal Crohn's Disease.

The Paneth cells reside in the small intestine at the bottom of the crypts of Lieberkühn, intermingled with stem cells, and provide a niche for their neighbors by secreting growth and Wnt-factors as well as different antimicrobial peptides including defensins, lysozyme and others. The most abundant are the human Paneth cell α-defensin 5 and 6 that keep the crypt sterile and control the local microbiome. In ileal Crohn's disease various mechanisms including established genetic risk factors contribute to defects in the production and ordered secretion of these peptides. In addition, life-style risk factors for Crohn's disease like tobacco smoking also impact on Paneth cell function. Taken together, current evidence suggest that defective Paneth cells may play the key role in initiating inflammation in ileal, and maybe ileocecal, Crohn's disease by allowing bacterial attachment and invasion.

The Paneth Cell: The Curator and Defender of the Immature Small Intestine.

Paneth cells were first described in the late 19th century by Gustav Schwalbe and Josef Paneth as columnar epithelial cells possessing prominent eosinophilic granules in their cytoplasm. Decades later there is continued interest in Paneth cells as they play an integral role in maintaining intestinal homeostasis and modulating the physiology of the small intestine and its associated microbial flora. Paneth cells are highly specialized secretory epithelial cells located in the small intestinal crypts of Lieberkühn. The dense granules produced by Paneth cells contain an abundance of antimicrobial peptides and immunomodulating proteins that function to regulate the composition of the intestinal flora. This in turn plays a significant role in secondary regulation of the host microvasculature, the normal injury and repair mechanisms of the intestinal epithelial layer, and the levels of intestinal inflammation. These critical functions may have even more importance in the immature intestine of premature infants. While Paneth cells begin to develop in the middle of human gestation, they do not become immune competent or reach their adult density until closer to term gestation. This leaves preterm infants deficient in normal Paneth cell biology during the greatest window of susceptibility to develop intestinal pathology such as necrotizing enterocolitis (NEC). As 10% of infants worldwide are currently born prematurely, there is a significant population of infants contending with an inadequate cohort of Paneth cells. Infants who have developed NEC have decreased Paneth cell numbers compared to age-matched controls, and ablation of murine Paneth cells results in a NEC-like phenotype suggesting again that Paneth cell function is critical to homeostasis to the immature intestine. This review will provide an up to date and comprehensive look at Paneth cell ontogeny, the impact Paneth cells have on the host-microbial axis in the immature intestine, and the repercussions of Paneth cell dysfunction or loss on injury and repair mechanisms in the immature gut.

T‐cell Protein Tyrosine Phosphatase is a Key Regulator of Paneth Cell Function

Loss‐of‐function single nucleotide polymorphisms (SNPs) in the Protein Tyrosine Phosphatase non‐receptor Type 2 (PTPN2) gene, are associated with increased susceptibility to Inflammatory Bowel Disease (IBD). PTPN2 encodes the T Cell Protein Tyrosine Phosphatase (TCPTP) which is a negative regulator of intracellular signaling pathways including JAK‐STAT. We previously presented that Paneth and goblet cell numbers are reduced in Ptpn2 knockout (KO) mice, consistent with altered differentiation of these intestinal epithelial cell (IEC) types in IBD. Alterations in IEC populations are detrimental for intestinal homeostasis and increase susceptibility to microbial infection.GoalTo identify whether Ptpn2‐KO mice display altered IEC differentiation and Paneth cell antimicrobial peptide (AMP) production.MethodsIntestinal tissues (ileum, cecum, proximal and distal colon) from 21 day old constitutive Ptpn2‐KO mice and control littermates, and from adult tamoxifen‐inducible intestinal epithelial‐specific Ptpn2ΔIEC mice (Vil‐Cre/Ptpn2), were harvested and processed for imaging, western blot, flow cytometry and qPCR.ResultsConstitutive Ptpn2‐KO mice display reduced numbers of Paneth cells and loss of the AMP lysozyme (p=0.0158; n=5). This was confirmed by Western blots of isolated IECs (~67% reduction; n=4). Expression of Paneth cell markers and differentiation factors (Mmp7, Defa5 and Sox9) were not altered in KO mice (n=10). Interestingly, Reg3‐γ, which like lysozyme also targets gram‐(+) bacteria and is secreted by Paneth cells and enterocytes, was increased in KO mice vs. control littermates (p=0.0066; n=10). Next, we identified by immunofluorescence that loss of Ptpn2 only in intestinal epithelium (Ptpn2ΔIEC) dramatically reduced colocalization of lysozyme and another Paneth cell marker, UEA‐1 (n=6). Decreased lysozyme expression in Ptpn2ΔIEC ileum was confirmed by western blot (p=0.0315; n=6). Since Paneth cells reside in the stem cell compartment, we investigated whether Ptpn2 deficiency affects the intestinal stem cells. Gene expression of Lgr5, an intestinal stem cell marker, Ephb3, a marker of the stem cell compartment, and Ascl2, a gene that controls stem cell renewal in the crypts, was unchanged in Ptpn2‐KO mice (n=10). In addition, expression of early IEC differentiation factors, Hes1 and Math1, were not altered (n=10). Il‐22 and IFN‐γ are known to stimulate secretion of AMPs by Paneth cells. Interestingly, flow cytometry showed increased abundance of Il‐22+ T cells (p=0.0187; n=5) and IFN‐γ+ T cells (p=0.0277; n=5) in the Ileum of constitutive KO mice, indicating that the defect in AMP production is likely not due to a lack of immune cell‐derived stimuli.ConclusionPtpn2‐deficiency decreases Paneth cell number and has opposing effects on their production of specific AMPs. These effects on Paneth cell number and function may contribute to the increased susceptibility to infection and the dysbiotic microbiota in Ptpn2‐deficient mice.Support or Funding InformationSupported by NIH 2R01DK091281 (DFM) and the Crohn’s and Colitis Foundation (DFM)

Impacts of Amino Acids on the Intestinal Defensive System.

The intestine interacts with a diverse community of antigens and bacteria. To keep its homeostasis, the gut has evolved with a complex defense system, including intestinal microbiota, epithelial layer and lamina propria. Various factors (e.g., nutrients) affect the intestinal defensive system and progression of intestinal diseases. This review highlights the current understanding about the role of amino acids (AAs) in protecting the intestine from harm. Amino acids (e.g., arginine, glutamine and tryptophan) are essential for the function of intestinal microbiota, epithelial cells, tight junction, goblet cells, Paneth cells and immune cells (e.g., macrophages, B cells and T cells). Through the modulation of the intestinal defensive system, AAs maintain the integrity and function of the intestinal mucosa and inhibit the progression of various intestinal diseases (e.g., intestinal infection and intestinal colitis). Thus, adequate intake of functional AAs is crucial for intestinal and whole-body health in humansand other animals.

Regulatory network analysis of Paneth cell and goblet cell enriched gut organoids using transcriptomics approaches.

The epithelial lining of the small intestine consists of multiple cell types, including Paneth cells and goblet cells, that work in cohort to maintain gut health. 3D in vitro cultures of human primary epithelial cells, called organoids, have become a key model to study the functions of Paneth cells and goblet cells in normal and diseased conditions. Advances in these models include the ability to skew differentiation to particular lineages, providing a useful tool to study cell type specific function/dysfunction in the context of the epithelium. Here, we use comprehensive profiling of mRNA, microRNA and long non-coding RNA expression to confirm that Paneth cell and goblet cell enrichment of murine small intestinal organoids (enteroids) establishes a physiologically accurate model. We employ network analysis to infer the regulatory landscape altered by skewing differentiation, and using knowledge of cell type specific markers, we predict key regulators of cell type specific functions: Cebpa, Jun, Nr1d1 and Rxra specific to Paneth cells, Gfi1b and Myc specific for goblet cells and Ets1, Nr3c1 and Vdr shared between them. Links identified between these regulators and cellular phenotypes of inflammatory bowel disease (IBD) suggest that global regulatory rewiring during or after differentiation of Paneth cells and goblet cells could contribute to IBD aetiology. Future application of cell type enriched enteroids combined with the presented computational workflow can be used to disentangle multifactorial mechanisms of these cell types and propose regulators whose pharmacological targeting could be advantageous in treating IBD patients with Crohn's disease or ulcerative colitis.

Transmissible gastroenteritis virus targets Paneth cells to inhibit the self-renewal and differentiation of Lgr5 intestinal stem cells via Notch signaling

Infection with transmissible gastroenteritis virus (TGEV) has been associated with villous atrophy within 48 h, which seriously disrupts intestinal homeostasis. However, the underlying mechanisms remain elusive. In this study, we found that TGEV infection severely disrupted intestinal homeostasis via inhibition of self-renewal and differentiation in Lgr5 intestinal stem cells (ISCs). Profoundly, TGEV-encoded NSP10/NSP16 protein complex-mediated the inactivation of Notch signaling provided a mechanistic explanation for this phenomenon. Initial invasions by TGEV-targeted Paneth cells through aminopeptidase N (APN) receptor, then inducing mitochondrial damage and ROS generation in them, ultimately causing Paneth cell decrease and loss of Notch factors (DII4 and Hes5), which are essential for Lgr5 ISCs self-renewal and differentiation. Interestingly, loss of Notch signaling induced goblet cells differentiation at the cost of absorptive enterocytes and promoted mucins secretion, which accelerated TGEV replication. Therefore, the more differentiation of goblet cells, the greater TGEV infection in jejunum. These results provide a detailed mechanistic pathway by which villous atrophy sharply occurs in TGEV-infected jejunum within 48 h. Thus, the pathogenesis of TGEV can be described as a “bottom up scenario”, which is contrary to the traditional “top down” hypothesis. Together, our findings provide a potential link between diarrheal virus infection and crypt cells response that regulates Paneth cells function and Lgr5 ISCs fate and could be exploited for therapeutic application.

Long Noncoding RNA H19 Impairs the Intestinal Barrier by Suppressing Autophagy and Lowering Paneth and Goblet Cell Function.

Background & AimsThe protective intestinal mucosal barrier consists of multiple elements including mucus and epithelial layers and immune defense; nonetheless, barrier dysfunction is common in various disorders. The imprinted and developmentally regulated long noncoding RNA H19 is involved in many cell processes and diseases. Here, we investigated the role of H19 in regulating Paneth and goblet cells and autophagy, and its impact on intestinal barrier dysfunction induced by septic stress.MethodsStudies were conducted in H19-deficient (H19-/-) mice, mucosal tissues from patients with sepsis, primary enterocytes, and Caco-2 cells. Septic stress was induced by cecal ligation and puncture (CLP), and gut permeability was detected by tracer fluorescein isothiocyanate–dextran assays. The function of Paneth and goblet cells was examined by immunostaining for lysozyme and mucin 2, respectively, and autophagy was examined by microtubule-associated proteins 1A/1B light chain 3 II immunostaining and Western blot analysis. Intestinal organoids were isolated from H19-/- and control littermate mice and treated with lipopolysaccharide (LPS).ResultsIntestinal mucosal tissues in mice 24 hours after exposure to CLP and in patients with sepsis showed high H19 levels, associated with intestinal barrier dysfunction. Targeted deletion of the H19 gene in mice enhanced the function of Paneth and goblet cells and promoted autophagy in the small intestinal mucosa. Knockout of H19 protected Paneth and goblet cells against septic stress, preserved autophagy activation, and promoted gut barrier function after exposure to CLP. Compared with organoids from control littermate mice, intestinal organoids isolated from H19-/- mice had increased numbers of lysozyme- and mucin 2–positive cells and showed increased tolerance to LPS. Conversely, ectopic overexpression of H19 in cultured intestinal epithelial cells prevented rapamycin-induced autophagy and abolished the rapamycin-induced protection of the epithelial barrier against LPS.ConclusionsIn investigations of mice, human tissues, primary organoids, and intestinal epithelial cells, we found that increased H19 inhibited the function of Paneth and goblet cells and suppressed autophagy, thus potentially contributing to barrier dysfunction in intestinal pathologies.

Butyric Acid and Leucine Induce α-Defensin Secretion from Small Intestinal Paneth Cells.

The intestine not only plays a role in fundamental processes in digestion and nutrient absorption, but it also has a role in eliminating ingested pathogenic bacteria and viruses. Paneth cells, which reside at the base of small intestinal crypts, secrete α-defensins and contribute to enteric innate immunity through potent microbicidal activities. However, the relationship between food factors and the innate immune functions of Paneth cells remains unknown. Here, we examined whether short-chain fatty acids and amino acids induce α-defensin secretion from Paneth cells in the isolated crypts of small intestine. Butyric acid and leucine elicit α-defensin secretion by Paneth cells, which kills Salmonella typhimurium. We further measured Paneth cell secretion in response to butyric acid and leucine using enteroids, a three-dimensional ex vivo culture system of small intestinal epithelial cells. Paneth cells expressed short-chain fatty acid receptors, Gpr41, Gpr43, and Gpr109a mRNAs for butyric acid, and amino acid transporter Slc7a8 mRNA for leucine. Antagonists of Gpr41 and Slc7a8 inhibited granule secretion by Paneth cells, indicating that these receptor and transporter on Paneth cells induce granule secretion. Our findings suggest that Paneth cells may contribute to intestinal homeostasis by secreting α-defensins in response to certain nutrients or metabolites.

The assembly of integrated rat intestinal-hepatocyte cultures.

The jejunum is the segment of the small intestine responsible for several metabolism and biotransformation functions. In this report, we have cultured rat jejunum explants in vitro and integrated them with hepatocyte cultures. We have also investigated the changes in jejunum function at different locations since spatial variations in intestinal functions have been reported previously. We divided the length of the rat jejunum into three distinct regions of approximately 9 cm each. We defined the regions as proximal (adjacent to the duodenum), medial, and distal (adjacent to the ileum). Spatiotemporal variations in functions were observed between these regions within the jejunum. Alkaline phosphatase activity (a marker of enterocyte function), decreased twofold between the proximal and distal regions at 4 hr. Lysozyme activity (a marker of Paneth cell function) increased from the proximal to the distal jejunum by 40% at 24 hr. Mucin‐covered areas, a marker of goblet cell function, increased by twofold between the proximal and distal segments of the jejunum at 24 hr. When hepatocytes were integrated with proximal jejunum explants, statistically higher urea (~2.4‐fold) and mucin (57%) production were observed in the jejunum explants. The integrated intestine‐liver cultures can be used as a platform for future investigations.

Functional Changes of Paneth Cells in the Intestinal Epithelium of Mice with Obstructive Jaundice and After Internal and External Biliary Drainage.

To investigate the functional changes of Paneth cells in the intestinal epithelium of mice with obstructive jaundice (OJ) and after internal biliary drainage (ID) and external biliary drainage (ED). The experiment was divided into two stages. First stage: Mice were randomly assigned to two groups: (I) sham operation (SH); (II) OJ. The mice were sacrificed before the operation and on the 1st, 3rd, 5th and 7th day after the operation to collect specimens. Second stage: Mice were randomly assigned to four groups: (I) SH; (II) OJ; (III) OJ and ED; and (IV) OJ and ID. They were reoperated on day 5 for biliary drainage procedure. The specimens were collected on day 10. The expressions of lysozyme and cryptdin-4 increased first and then decreased over time in group OJ, and the number of Paneth cells decreased gradually with the extension of OJ time（p<0.05. After the secondary operation on the mice to relieve OJ, the number of Paneth cells and expressions of lysozyme and cryptdin-4 in group ID increased more significantly than those in group ED（p<0.05）. OJ could cause intestinal Paneth cells to dysfunction in mice. ID was more significant than ED in restoring the function of Paneth cells. It might be one of the mechanisms that make ID superior to ED.

RNA-Binding Protein HuR Regulates Paneth Cell Function by Altering Membrane Localization of TLR2 via Post-transcriptional Control of CNPY3

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. 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. 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. 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.