Search

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

The rapid renewal and repair of the intestinal mucosa are based on intestinal stem cells (ISC), which are located at the crypt bottom. Paneth cells are an essential component in the crypt, which served as the niche for ISC development. However, in the chicken, how the function of Paneth cells changes during intestinal inflammation is unclear and is the key to understand the mechanism of mucosal repair. In the present study, 36 HyLine White chickens (7 d of age, n = 6) were randomly divided into 1 control and 5 lipopolysaccharide (LPS) injection groups. The chickens were injected (i.p.) with PBS in the control group, however, were injected (i.p.) with LPS (10 mg/kg BW) in the LPS injection groups, which would be sampled at 5 time points (1 h postinjection [hpi], 2 hpi, 4 hpi, 6 hpi, and 8 hpi). Results showed that tumor necrosis factor-α mRNA transcription in duodenal tissue increased gradually since 1 hpi, peaked at 4 hpi, and then reduced remarkably, indicating that 4 hpi of LPS was the early stage of intestinal inflammation. Meanwhile, the MUC2 expression in duodenal tissue was dramatically reduced since 1 hpi of LPS. The ISC marker, Lgr5 and Bmi1, in the duodenal crypt were reduced from 1 hpi to 4 hpi and elevated later. Accordingly, the hydroethidine staining showed that the reactive oxygen species level, which drives the differentiation of ISC, in the duodenal crypt reduced obviously at 1 hpi and recovered gradually since 4 hpi. The analysis of Paneth cells showed that many swollen mitochondria appeared in Paneth cells at 4 hpi of LPS. Meanwhile, the Lysozyme transcription in the duodenal crypt was substantially decreased since 1 hpi of LPS. However, the Wnt3a and Dll1 in duodenal crypt decreased at 1 hpi of LPS, then increased gradually. In conclusion, Paneth cells were impaired at the early stage of intestinal inflammation, then recovered rapidly. Thus, the ISC activity was reduced at first and recovery soon., (Copyright © 2020. Published by Elsevier Inc.)

HPS1, a BLOC-3 subunit that acts as a guanine nucleotide exchange factor of Rab32/38, may play a role in the removal of VAMP7 during the maturation of large dense core vesicles of Paneth cells. Loss of HPS1 impairs lysozyme secretion and alters the composition of intestinal microbiota, which may explain the susceptibility of HPS-associated inflammatory bowel disease. Hermansky-Pudlak syndrome (HPS) is characterized by oculocutaneous albinism, bleeding tendency, and other chronic organ lesions due to defects in tissue-specific lysosome-related organelles (LROs). For some HPS subtypes, such as HPS-1, it is common to have symptoms of HPS-associated inflammatory bowel disease (IBD). However, its underlying mechanism is largely unknown. HPS1 is a subunit of the BLOC-3 complex which functions in the biogenesis of LROs. Large dense core vesicles (LDCVs) in Paneth cells of the intestine are a type of LROs. We here first report the abnormal LDCV morphology (increased number and enlarged size) in HPS1-deficient pale ear ( ep ) mice. Similar to its role in melanosome maturation, HPS1 plays an important function in the removal of VAMP7 from LDCVs to promote the maturation of LDCVs. The immature LDCVs in ep mice are defective in regulated secretion of lysozyme, a key anti-microbial peptide in the intestine. We observed changes in the composition of intestinal microbiota in both HPS-1 patients and ep mice. These findings provide insights into the underlying mechanism of HPS-associated IBD development, which may be implicated in possible therapeutic intervention of this devastating condition., (Copyright © 2020 Yu, He, Wei, Liu, Zhang, Pan, Hao, Yang, Yuan, Zhang, Zhang, Hao, Liu and Li.)

Paneth cells secrete bactericidal substances in response to bacterial proliferation on the mucosal surface without directly contacting bacteria. However, the induction mechanism of this transient secretion has not been clarified, although nervous system and/or immunocompetent cells in the lamina propria (LP) might be involved. In this study, we ultrastructurally and immunohistochemically investigated which LP cells are localized beneath Paneth cells and examined the relationship between the Paneth cell-derived cellular processes which extended into the LP and the LP cells. The results showed that various cells-including blood capillary, subepithelial stromal cell, and nerve fiber-were present in the LP beneath Paneth cells. Endothelial cells of blood capillary were the cells most frequently found in this location; they were situated within 1 μm of the Paneth cells and possessed fenestration on the surfaces adjacent to Paneth cells. The Paneth cells rarely extended the cellular processes toward the LP across the basal lamina. Most of the cellular processes of Paneth cells contacted the subepithelial stromal cells. Immunohistochemistry revealed that the CD34 + CD31 - αSMA - stromal cells preferentially localized in the LP beneath the intestinal crypt base, while PDGFRα hi αSMA + stromal cells mainly localized around the lateral portions of the intestinal crypt and PDGFRα hi αSMA - stromal cells localized in the intestinal villus. From these findings, the existence of blood capillaries beneath Paneth cells might reflect the active exocrine function of Paneth cells. Furthermore, subepithelial stromal cells, probably with a CD34 + CD31 - αSMA - PDGFRα -/lo phenotype, beneath the crypt base might affect Paneth cell activity by interacting with their cellular processes. Anat Rec, 301:1074-1085, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Paneth cells are secretory epithelial cells of the innate immune system of the intestine of several mammals, including alpacas. Little is known about the latter; thus, in the present study we described the morphology and histochemical characteristics of Paneth cells in healthy fetuses, and young and adult alpacas. For this purpose, samples of duodenum, jejunum and ileum were taken from 6 fetuses at different days of pregnancy (between days 221-330), 66 offsprings (between 0 and 45-days-old) and 5 adult alpacas (>2-years-old). Samples were fixed in 10% buffered formalin and processed for histological and morphometrical analysis using HE and Masson Trichomićs technique. Immunohistochemistry was used to identify Paneth cells using anti-lysozyme antibody. In addition, the lectinhistochemichal binding-pattern of Paneth celĺs granules was evaluated. Lyzozyme was immunohistochemically detected in the granules of Paneth cells from day 283 of pregnancy in all the small intestinal sections of the studied fetuses. In newborn alpacas Paneth cells were initially found in the duodenum, but the following days (days 18-21 after birth) they were also found in the ileum. Their size gradually increased after birth, but then no significant differences were found. In adult alpacas the number was lower than offsprings. We suggest that Paneth cells early differentiate in the small intestine of alpacas, and the increase in their number during the first two weeks of life strongly support their possible involvement in the intestinal defensive functions against the enteric diseases that occur during the lactancy stage., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The Paneth cells are highly specialized cells in the epithelium of the small intestine of many vertebrate species. These cells reside at the base of crypts of the Lieberkühn and contain abundant secretory granules. Previous studies suggesting the existence of Paneth cells in the chicken (Gallus gallus) remained controversial. Here we seek to identify the Paneth cells in the chicken small intestine through morphological examination and specific gene expression. Histological staining and transmission electron microscope confirmed the presence of granulated secretory cells at the base of the crypts in the chicken small intestine. Western blotting experiment also manifested the expression of lysozyme protein, which is specifically secreted by the Paneth cells in the small intestine. Moreover, lysozyme c and lysozyme g mRNAs were expressed in the small intestine of chickens at different ages. Lysozyme c mRNA, in particular, was located at the base of the small intestinal crypts as displayed by in situ hybridization. Collectively, we provide evidences that the Paneth cells indeed exist in the small intestine of the chicken., (© 2016 Poultry Science Association Inc.)

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

Paneth cells (PCs) contribute to the host defense against indigenous bacteria in the small intestine. We found Paneth cell-like cells (PLCs) in the rat ascending colon, but the nature of PLCs is never clarified. Therefore, the present study aimed to clarify the cytological characteristics of PLCs and discuss their cellular differentiation. PLCs were localized in the bases of intestinal crypts, especially follicle-associated intestinal crypts in proximal colonic lymphoid tissue, but were very seldom found in the ordinary intestinal crypts of the ascending colon. PLCs possessed specific granules with highly electron-dense cores and haloes, as well as PCs in the small intestine. The secretory granules of PLCs were positive for PAS reaction, lysozyme and soluble phospholipase A2, but negative for Alcian blue staining, β-defensin-1 and -2, as well as the ones of PCs. Furthermore, intermediate cells possessing both the PLC-specific granules and the mucus granules similar to those of goblet cells (GCs) were occasionally found in the vicinity of PLCs. Intermediate cells ranged from goblet cell-like cells rich in mucus granules to PLC-like cells with few mucus granules. The cellular condensation and fragmentation were exclusively found in PLCs but never seen in intermediate cells or GCs. The PLCs, which were identified as PC, were suggested to be transformed from GCs through intermediate cells and finally to die by apoptosis in intestinal crypts of proximal colonic lymphoid tissue in the rat ascending colon., (Copyright © 2014 Wiley Periodicals, Inc.)

Background: Recent studies link endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) to inflammatory bowel disease. Altered eIF2α phosphorylation (eIF2α-P), a regulatory hub of the UPR, was observed in mucosal tissue of patients with inflammatory bowel disease. In this study, we examined the mechanistic role of eIF2α-P in intestinal epithelial cell (IEC) function and intestinal homeostasis in mice., Methods: We generated mice with villin-Cre-mediated conditional expression of nonphosphorylatable Ser51Ala mutant eIF2α in IECs (AA mice). We analyzed AA mice under normal conditions and on challenge with oral infection of Salmonella Typhimurium or dextran sulfate sodium-induced colitis., Results: Loss of eIF2α-P did not affect the normal proliferation or differentiation of IECs. However, AA mice expressed decreased secretory proteins including lysozyme, suggesting eIF2α-P is required for Paneth cell function. The ultrastructure of AA Paneth cells exhibited a reduced number of secretory granules, a fragmented ER, and distended mitochondria under normal conditions. UPR gene expression was defective in AA IECs. Translation of Paneth cell specific messenger RNAs encoding lysozyme and cryptidins was significantly defective leading to the observed granule-deficient phenotype, which was associated with reduced ribosomal recruitment of these messenger RNAs to the ER membrane. Consequently, AA mice were more susceptible to oral Salmonella infection and dextran sulfate sodium-induced colitis., Conclusions: We conclude eIF2α phosphorylation is required for the normal function of intestinal Paneth cells and mucosal homeostasis by activating UPR signaling and promoting messenger RNA recruitment to the ER membrane for translation.

The identification of inflammatory bowel disease (IBD) susceptibility genes by genome-wide association has linked this pathology to autophagy, a lysosomal degradation pathway that is crucial for cell and tissue homeostasis. Here, we describe autophagy-related 4B, cysteine peptidase/autophagin-1 (ATG4B) as an essential protein in the control of inflammatory response during experimental colitis. In this pathological condition, ATG4B protein levels increase in parallel with the induction of autophagy. Moreover, ATG4B expression is significantly reduced in affected areas of the colon from IBD patients. Consistently, atg4b (-/-) mice present Paneth cell abnormalities, as well as an increased susceptibility to DSS-induced colitis. atg4b-deficient mice exhibit significant alterations in proinflammatory cytokines and mediators of the immune response to bacterial infections, which are reminiscent of those found in patients with Crohn disease or ulcerative colitis. Additionally, antibiotic treatments and bone marrow transplantation from wild-type mice reduced colitis in atg4b (-/-) mice. Taken together, these results provided additional evidence for the importance of autophagy in intestinal pathologies and describe ATG4B as a novel protective protein in inflammatory colitis. Finally, we propose that atg4b-null mice are a suitable model for in vivo studies aimed at testing new therapeutic strategies for intestinal diseases associated with autophagy deficiency.

Using an environmentally sensitized genetic screen we identified mutations that cause inflammatory colitis in mice. The X-linked Klein-Zschocher (KLZ) mutation created a null allele of Yipf6, a member of a gene family believed to regulate vesicular transport in yeast, but without known functions in mammals. Yipf6 is a five transmembrane-spanning protein associated with Golgi compartments. Klein-Zschocher mutants were extremely sensitive to colitis induced by dextran sodium sulfate (DSS) and developed spontaneous ileitis and colitis after 16 mo of age in specific pathogen-free housing conditions. Electron microscopy, gene expression, and immunocytochemistry analyses provided evidence that impaired intestinal homeostasis stemmed from defective formation and secretion of large secretory granules from Paneth and goblet cells. These studies support a tissue- and organ-specific function for Yipf6 in the maintenance of intestinal homeostasis and implicate the orthologous human gene as a disease susceptibility locus.

Autophagy-related 16 like-1 (ATG16L-1), immunity-related GTPase-M (IRGM), and nucleotide-binding oligomerization domain-containing 2 (NOD2) regulate autophagy, and variants in these genes have been associated with predisposition to Crohn's disease (CD). However, little is known about the role of autophagy in CD. Intestinal biopsies from untreated pediatric patients with CD, celiac disease, or ulcerative colitis were analyzed by immunohistochemistry and electron microscopy. We observed that autophagy was specifically activated in Paneth cells from patients with CD, independently of mucosal inflammation or disease-associated variants of ATG16L1 or IRGM. In these cells, activation of autophagy was associated with a significant decrease in number of secretory granules and features of crinophagy. These observations might account for the disorganization of secretory granules previously reported in Paneth cells from patients with CD., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Lack of enteral feeding, with or without parenteral nutritional support, is associated with increased intestinal permeability and translocation of bacteria. Such translocation is thought to be important in the high morbidity and mortality rates of patients who receive nothing by mouth. Recently, Paneth cells, important constituents of innate intestinal immunity, were found to be crucial in host protection against invasion of both commensal and pathogenic bacteria. This study investigates the influence of food deprivation on Paneth cell function in a mouse starvation model. Quantitative PCR showed significant decreases in mRNA expression of typical Paneth cell antimicrobials, lysozyme, cryptdin, and RegIIIγ, in ileal tissue after 48 hours of food deprivation. Protein expression levels of lysozyme and RegIIIγ precursor were also significantly diminished, as shown by Western blot analysis and IHC. Late degenerative autophagolysosomes and aberrant Paneth cell granules in starved mice were evident by electron microscopy, Western blot analysis, and quantitative PCR. Furthermore, increased bacterial translocation to mesenteric lymph nodes coincided with Paneth cell abnormalities. The current study demonstrates the occurrence of Paneth cell abnormalities during enteral starvation. Such changes may contribute to loss of epithelial barrier function, causing the apparent bacterial translocation in enteral starvation., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Intestinal stem cells may have important roles in the maintenance of epithelial integrity during tissue repair. Alemtuzumab is a humanized anti-CD52 lymphocytic antibody that is increasingly being used to induce immunosuppression; intestinal barrier function is impaired during treatment with alemtuzumab. We investigated the response of intestinal stem cells to epithelial damage resulting from alemtuzumab treatment. Intestinal epithelial cell loss and abnormal Paneth cell morphology were found following a single dose of alemtuzumab. The animals receiving alemtuzumab exhibited increased apoptosis in the villi 3 days after alemtuzumab treatment and in the crypt on day 9, but apoptosis was scarce on day 35. We assessed expression of Musashi-1- and Lgr5-positive stem cells following alemtuzumab treatment. Increased numbers of cells staining positive for both Musashi-1 and Lgr5 were found in the stem cell zone after alemtuzumab treatment for 3 and 9 days. These data indicated that the epithelial cells were injured following alemtuzumab treatment, with the associated expansion of intestinal stem cells. After alemtuzumab treatment for 35 days, the numbers of intestinal epithelial cells and intestinal stem cells returned to normal. This study suggests that alemtuzumab treatment induced the increase in stem cells, resulting in the availability of more enterocytes for repair.

Elucidating the mechanisms determining multipotent progenitor cell fate remains a fundamental project of contemporary biology. Various tissues of mice and men with defects in the zinc-finger transcriptional repressor Gfi1 have dramatic perturbations in the proportions of their differentiated cell types. In Gfi1-deficient intestinal epithelium there is a shift from mucous and Paneth towards enteroendocrine cells, leading to the proposal that Gfi1 functions in the allocation of the progeny derived from a hypothetical common granulocytic progenitor. However, studies of clones have yielded no evidence of such a common progenitor prompting us to investigate alternate mechanisms explaining the Gfi1-deficient phenotype. We report that mucous and Paneth but not enteroendocrine lineage cells normally express Gfi1. Sporadic mucous and Paneth lineage cells in the crypts of Gfi1-deficient mice aberrantly express the pro-enteroendocrine transcription factor Neurog3, indicating that stable repression of Neurog3 in these lineages requires Gfi1. Importantly, we also find mucous and Paneth lineage cells in various stages of cellular reprogramming into the enteroendocrine lineage in Gfi1-deficient mice. We propose that mucous and Paneth cell lineage metastability, rather than reallocation at the level of a hypothetical common granulocytic progenitor, is responsible for the shifts in cell type proportions observed in Gfi1-deficient intestinal epithelium., (Crown Copyright 2010. Published by Elsevier Inc. All rights reserved.)

Alemtuzumab has been recently introduced for induction therapy in organ transplantation. However, the pathogenesis and molecular mechanism of the impact of such induction therapy on bacterial infections remain to be clarified. We found the alterations of Paneth cells including abnormal Paneth cell granules and expression of lysozyme and defensin 5 in response to lymphocyte depletion by alemtuzumab. Lymphocyte depletion resulted in decreased expression of TNF-alpha, IFN-gamma, IL-10 and TGF-beta in the intestine. The diversity of gut bacteria varied significantly between different times of alemtuzumab treatment. Abnormal expression of granule peptides might result in impairment of host gut microflora. The alterations in bacterial microflora had almost reversed 56days after alemtuzumab treatment, which was consistent with our results that Paneth cells were recovered to secrete antimicrobial peptides to govern gut microflora. These findings indicated the associations between changes of Paneth cell function and gut microflora and supported the important role of Paneth cells to barrier impairment with the use of alemtuzumab in organ transplantation., (Copyright 2010 Elsevier Inc. All rights reserved.)

Macroautophagy (mAG) is a lysosomal mechanism of degradation of cell self-constituents damaged due to variety of stress factors, including ionizing irradiation. Activation of mAG requires expression of mAG protein Atg8 (LC3) and conversion of its form I (LC3-I) to form II (LC3-II), mediated by redox-sensitive Atg4 protease. We have demonstrated upregulation of this pathway in the innate host defense Paneth cells of the small intestine (SI) due to ionizing irradiation and correlation of this effect with induction of pro-oxidant inducible nitric oxide synthase (iNOS). CD2F1 mice were exposed to 9.25 Gy gamma-ionizing irradiation. Small intestinal specimens were collected during 7 days after ionizing irradiation. Assessment of ionizing irradiation-associated alterations in small intestinal crypt and villus cells and activation of the mAG pathway was conducted using microscopical and biochemical techniques. Analysis of iNOS protein and the associated formation of nitrites and lipid peroxidation products was performed using immunoblotting and biochemical analysis, and revealed increases in iNOS protein, nitrate levels and oxidative stress at day 1 following ionizing irradiation. Increase in immunoreactivity of LC3 protein in the crypt cells was observed at day 7 following ionizing irradiation. This effect predominantly occurred in the CD15-positive Paneth cells and was associated with accumulation of LC3-II isoform. The formation of autophagosomes in Paneth cells was confirmed by transmission electron microscopy (TEM). Up-regulation of LC3 pathway in the irradiated SI was accompanied by a decreased protein-protein interaction between LC3 and chaperone heat shock protein 70. A high-level of LC3-immunoreactivity in vacuole-shaped structures was spatially co-localized with immunoreactivity of 3-nitro-tyrosine. The observed effects were diminished in iNOS knockout B6.129P2-NOS2(tm1Lau)/J mice subjected to the same treatments. We postulate that the observed up-regulation of mAG in the irradiated small intestine is at least in part mediated by the iNOS signalling mechanism., (2009 by John Wiley & Sons, Ltd.)

Background & Aims: Stem cells within the intestinal epithelium generate daughter cells that undergo lineage commitment and maturation through the combined action of the Wnt and Notch signaling cascades. Both pathways, in turn, regulate transcription factor networks that further define differentiation toward either enterocytes or 1 of 3 secretory cell lineages (Paneth, goblet, or enteroendocrine cells). In this study, we investigated the role of the Wnt-responsive, Ets-domain transcription factor Spdef in the differentiation of goblet and Paneth cells., Methods: The in vivo function of Spdef was examined by disrupting the Spdef gene in mice (Spdef(-/-) mice) and analyzing the intestinal phenotype using a range of histologic techniques and DNA microarray profiling., Results: In accordance with expression data, we found that loss of Spdef severely impaired the maturation of goblet and Paneth cells and, conversely, led to an accumulation of immature secretory progenitors. Spdef appears to positively and negatively regulate a specific subset of goblet and Paneth cell genes, including Cryptdins, Mmp7, Ang4, Kallikreins, and Muc2., Conclusions: Spdef acts downstream of Math1 to promote terminal differentiation of a secretory progenitor pool into Paneth and goblet cells.

The unfolded protein response as a consequence of endoplasmic reticulum (ER) stress has recently been implicated as a novel mechanism that may lead to inflammatory bowel disease (IBD). Impairment of proper ER stress resolution in highly secretory Paneth and, to a lesser extent, goblet cells within the epithelium can primarily lead to intestinal inflammation. An inability to manage ER stress may not only be a primary originator of intestinal inflammation as exemplified by genetic polymorphisms in XBP1 that are associated with IBD but also a perpetuator of inflammation when ER stress is induced secondarily to inflammatory mediators or microbial factors. Furthermore, ER stress pathways may interact with other processes that lead to IBD, notably autophagy.

Lipopolysaccharide (LPS)-binding protein (LBP) is an acute-phase protein involved in the host's response to endotoxin and mainly synthesized and secreted to the blood by the liver. But in addition, LBP is also made by extrahepatic cells, including the enterocyte-like cell line Caco-2. To study in closer detail the synthesis and storage of LBP in the intestinal mucosal epithelium, we performed an immunolocalization of LBP in mouse small intestine. By immunofluorescence microscopy, an antibody recognizing the 58-60 kDa protein of LBP distinctly labeled a small population of cells located deep into the crypts. This cell population was also positive for lysozyme and alpha-defensin 4, identifying Paneth cells as the main intestinal LBP-producing cells. By immunogold electron microscopy, intense labeling was observed in the secretory granules of these cells. We conclude that Paneth cells express LBP together with other proteins acting in the innate immune response of the gut, such as lysozyme, defensins and intelectin.

The accumulation of certain species of bacteria in the intestine is involved in both tissue homeostasis and immune-mediated pathologies. The host mechanisms involved in controlling intestinal colonization with commensal bacteria are poorly understood. We observed that under specific pathogen-free or germ-free conditions, intragastric administration of Pseudomonas aeruginosa, E. coli, Staphylococcus aureus, or Lactobacillus gasseri resulted in increased colonization of the small intestine and bacterial translocation in mice lacking Cd1d, an MHC class I-like molecule, compared with WT mice. In contrast, activation of Cd1d-restricted T cells (NKT cells) with alpha-galactosylceramide caused diminished intestinal colonization with the same bacterial strains. We also found prominent differences in the composition of intestinal microbiota, including increased adherent bacteria, in Cd1d-/- mice in comparison to WT mice under specific pathogen-free conditions. Germ-free Cd1d-/- mice exhibited a defect in Paneth cell granule ultrastructure and ability to degranulate after bacterial colonization. In vitro, NKT cells were shown to induce the release of lysozyme from intestinal crypts. Together, these data support a role for Cd1d in regulating intestinal colonization through mechanisms that include the control of Paneth cell function.

Background & Aims: All 4 differentiated epithelial cell types found in the intestinal epithelium derive from the intestinal epithelial stem cells present in the crypt unit, in a process whose molecular clues are intensely scrutinized. Peroxisome proliferator-activated receptor beta (PPARbeta) is a nuclear hormone receptor activated by fatty acids and is highly expressed in the digestive tract. However, its function in intestinal epithelium homeostasis is understood poorly., Methods: To assess the role of PPARbeta in the small intestinal epithelium, we combined various cellular and molecular approaches in wild-type and PPARbeta-mutant mice., Results: We show that the expression of PPARbeta is particularly remarkable at the bottom of the crypt of the small intestine where Paneth cells reside. These cells, which have an important role in the innate immunity, are strikingly affected in PPARbeta-null mice. We then show that Indian hedgehog (Ihh) is a signal sent by mature Paneth cells to their precursors, negatively regulating their differentiation. Importantly, PPARbeta acts on Paneth cell homeostasis by down-regulating the expression of Ihh, an effect that can be mimicked by cyclopamine, a known inhibitor of the hedgehog signaling pathway., Conclusions: We unraveled the Ihh-dependent regulatory loop that controls mature Paneth cell homeostasis and its modulation by PPARbeta. PPARbeta currently is being assessed as a drug target for metabolic diseases; these results reveal some important clues with respect to the signals controlling epithelial cell fate in the small intestine.

Intelectin is a mammalian Ca2+-dependent, D-galactosyl-specific lectin expressed in Paneth and goblet cells of the small intestine and proposed to serve a protective role in the innate immune response to parasite infection. In addition, it is structurally identical to the intestinal lactoferrin receptor known to reside in the enterocyte brush border. To clarify this apparent discrepancy with regard to localization, the aim of this work was to study the cellular and subcellular distribution of small intestinal intelectin by immunofluorescence and immunogold electron microscopy. Secretory granules of lysozyme-positive Paneth cells in the bottom of the crypts as well as goblet cells along the crypt-villus axis were intensively labeled with intelectin antibodies, but quantitatively, the major site of intelectin deposition was the enterocyte brush border. This membrane is organized in stable glycolipid-based lipid raft microdomains, and like the divalent lectin galectin-4, intelectin was enriched in microvillar "superrafts", i.e., membranes that resist solubilization with Triton X-100 at 37 degrees C. This strategic localization suggests that the trimeric intelectin, like galectin-4, serves as an organizer and stabilizer of the brush border membrane, preventing loss of digestive enzymes to the gut lumen and protecting the glycolipid microdomains from pathogens.

Wnt signalling, which is transduced through beta-catenin/TCF4, maintains the undifferentiated state of intestinal crypt progenitor cells. Mutational activation of the pathway initiates the adenomacarcinoma sequence. Whereas all other differentiated epithelial cells migrate from the crypt onto the villus, Paneth cells home towards the source of Wnt signals--that is, the crypt bottom. Here, we show that expression of a Paneth gene programme is critically dependent on TCF4 in embryonic intestine. Moreover, conditional deletion of the Wnt receptor Frizzled-5 abrogates expression of these genes in Paneth cells in the adult intestine. Conversely, adenomas in Apc-mutant mice and colorectal cancers in humans inappropriately express these Paneth-cell genes. These observations imply that Wnt signals in the crypt can separately drive a stem-cell/progenitor gene programme and a Paneth-cell maturation programme. In intestinal cancer, both gene programmes are activated simultaneously.

To date Paneth cells have not previously been reported to kill Giardia trophozoites and other protozoa in vivo. Here we report the first evidence for in vivo killing of Giardia trophozoites by intestinal Paneth cells. Transmission electron microscopic (TEM) examination of duodenal specimens taken from naturally infected mice revealed that only Giardia trophozoites harbouring peripheral bacterial endosymbionts were destroyed and lysed in the vicinity of the activated Paneth cells. Additionally, intestinal epithelium was more affected by Giardia harbouring bacterial endosymbionts than Giardia with no endosymbionts. Our findings imply that the bacterial endosymbionts within Giardia trophozoites have a role in both host protective and pathological mechanisms, probably through altering the trophozoite antigencity. These observations might shed light on the diversity in infectivity and host specificity of Giardia species.

Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen. Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice. On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., alpha-defensins (cryptdins) and lysozyme, in CF murine intestine. Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion. Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution. Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates. Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine. Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis. Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens.

Ultrastructure, lysozyme and glycoconjugate activity in duodenal Paneth cells were observed concurrently in the horse. Paneth cells were seen to uniformly line the base of the equine intestinal glands. The round secretory granules have centrally located electron densities with peripherally located electron lucent halos. Histochemically, the peripheral halo layer was positively stained for carbohydrates by the periodic acid-thiocarbohydrazide-silver protein-physical development (PA-TCH-SP-PD) method and the entire granules reacted positively to the WGA. The central core area reacted with anti-lysozyme. We identified a young (Type I) and an old (Type II) cell population in the same crypt, but we suggest that the observed populations are variations of the same cell type with the varied appearance due to aging of the secretory granules.

A post-embedding ultrastructural immunogold method was used to detect osteopontin in human intestinal biopsies with special emphasis on secretory and phagocytic organelles. Osteopontin immunoreactivity was localized to phagolysosomes of macrophages, fibroblasts, absorptive epithelial cells of the small intestine and Paneth cells. The mucigen secretory granules and Golgi structures of mucous epithelial cells of the small intestinal epithelium contained osteopontin, but secretory granules of numerous other cells, including Paneth cells, did not. Extracellular and phagocytosed Tropheryma whippelii within macrophage phagolysosomes also bound osteopontin. These localizations are supportive of a role for osteopontin in phagocytic and some secretory cell functions in human intestine.