Abnormal Paneth Cells Research Articles

Leaky intestine and impaired microbiome in an amyotrophic lateral sclerosis mouse model.

Emerging evidence has demonstrated that intestinal homeostasis and the microbiome play essential roles in neurological diseases, such as Parkinson's disease. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons and muscle atrophy. Currently, there is no effective treatment. Most patients die within 3–5 years due to respiratory paralysis. Although the death of motor neurons is a hallmark of ALS, other organs may also contribute to the disease progression. We examined the gut of an ALS mouse model, G93A, which expresses mutant superoxide dismutase (SOD1G93A), and discovered a damaged tight junction structure and increased permeability with a significant reduction in the expression levels of tight junction protein ZO-1 and the adherens junction protein E-cadherin. Furthermore, our data demonstrated increased numbers of abnormal Paneth cells in the intestine of G93A mice. Paneth cells are specialized intestinal epithelial cells that can sense microbes and secrete antimicrobial peptides, thus playing key roles in host innate immune responses and shaping the gut microbiome. A decreased level of the antimicrobial peptides defensin 5 alpha was indeed found in the ALS intestine. These changes were associated with a shifted profile of the intestinal microbiome, including reduced levels of Butyrivibrio Fibrisolvens, Escherichia coli, and Fermicus, in G93A mice. The relative abundance of bacteria was shifted in G93A mice compared to wild-type mice. Principal coordinate analysis indicated a difference in fecal microbial communities between ALS and wild-type mice. Taken together, our study suggests a potential novel role of the intestinal epithelium and microbiome in the progression of ALS.

Paneth cells

Paneth cells

Critical design aspects involved in the study of Paneth cells and the intestinal microbiota

Paneth cells are long-lived secretory cells that reside in the base of the crypts of Lieberkühn of the small intestine. They produce an arsenal of molecules that are involved in numerous biological processes, ranging from the control of gut microbial populations to supporting the intestinal stem cell niche. Because of these important functions, Paneth cell abnormalities are becoming implicated in a variety of disease processes. As such, it is necessary to establish parameters that will allow for the comprehensive study of Paneth cells in health and disease. In this addendum, we highlight critical design aspects involved in the study of Paneth cells and their downstream effects on the intestinal microbiota. The importance of this approach is demonstrated by our recent findings that Nod2 does not regulate mouse Paneth cell antimicrobial function, in contrast to previous reports. This work defines key issues to consider when studying Paneth cells in mouse systems.

O-025 Paneth Cell Phenotype Correlates with Genetics, Transcriptome Profile, Pathologic Hallmark and Predicts Clinical Outcome in Patients with Crohnʼs Disease

The identification of genetic susceptibility loci in Crohn's Disease (CD) represents an opportunity to define disease subtypes that could be utilized in the development of novel personalized therapies. The major challenges are that the CD susceptibility loci are numerous, complex and likely interact with undefined components of the environment. Paneth cells in the small intestine form part of the innate immunity and play crucial roles in CD pathogenesis. We have previously shown that the morphology of Paneth cell cytoplasmic antimicrobial granules can be used as a readout for host genetics and environmental exposure. We hypothesized that Paneth cell phenotypes could act as an integrative readout to define specific CD subtypes. We retrospectively analyzed 178 genotyped CD patients with small intestinal resections at 2 institutions. Uninvolved ileal sections were analyzed for transcriptional profiles by microarray and Paneth cell phenotype by lysozyme immunofluorescence (up to 250 crypts quantified/sample). The morphology of each individual Paneth cell was categorized. The sum of Paneth cells with abnormal lysozyme distribution was used to determine the patients' phenotype, with “bad” Paneth cell phenotype defined as ≥20% abnormal Paneth cells, and <20% defined as “good” Paneth cell phenotype. The patients' phenotypes were then correlated with genotype, pathologic features, transcriptional profiles, and time to recurrence after surgery. Among the 178 CD patients, 27 (15%) had bad Paneth cell phenotype. Patients with one or more CD-associated NOD2 susceptibility alleles had increased proportions of abnormal Paneth cells compared to those without NOD2 susceptibility alleles. Furthermore, there is an additive effect of CD-associated NOD2 susceptibility alleles and ATG16L1 T300A on Paneth cell abnormalities. We found that the cases with bad Paneth cell phenotype were more likely to be granuloma-poor (P = 0.0160). Bad Paneth cell phenotype was also associated with an altered transcriptional signature most strongly associated with immune activation pathways. Clinically, among the 121 patients who received adjuvant prophylactic therapy, those with bad Paneth cell phenotype showed shorter time to disease recurrence after surgery (median 10 months) compared to patients with good Paneth cell phenotype (median 14 months; P = 0.0198). The difference was not seen in patients who did not receive adjuvant prophylactic therapy. We also found that in the same patients, the Paneth cell phenotype in involved area was consistent with that in the uninvolved area (P = 0.0001). Finally, bioequivalence analysis showed that a minimum of 40 crypts in biopsy was sufficient for Paneth cell morphology analysis. Paneth cell phenotype is associated with patients' genetics, pathologic hallmark, and predicts clinical outcome. It can be used as a “hard” endpoint that can subdivide CD patients into clinically relevant groups. Defining the minimum crypt number required for Paneth cell phenotype analysis enables the use of routine biopsy material for further clinical studies.

Genetic Variants Synthesize to Produce Paneth Cell Phenotypes That Define Subtypes of Crohn's Disease

Genetic susceptibility loci for Crohn's disease (CD) are numerous, complex, and likely interact with undefined components of the environment. It has been a challenge to link the effects of particular loci to phenotypes of cells associated with pathogenesis of CD, such as Paneth cells. We investigated whether specific phenotypes of Paneth cells associated with particular genetic susceptibility loci can be used to define specific subtypes of CD. We performed a retrospective analysis of 119 resection specimens collected from patients with CD at 2 separate medical centers. Paneth cell phenotypes were classified as normal or abnormal (with disordered, diminished, diffuse, or excluded granule phenotypes) based on lysozyme-positive secretory granule morphology. To uncover the molecular basis of the Paneth cell phenotypes, we developed methods to determine transcriptional profiles from whole-thickness and laser-capture microdissected, formalin-fixed, paraffin-embedded tissue sections. The proportion of abnormal Paneth cells was associated with the number of CD-associated NOD2 risk alleles. The cumulative number of NOD2 and ATG16L1 risk alleles had an additive effect on the proportion of abnormal Paneth cells. Unsupervised clustering analysis of demographic and Paneth cell data divided patients into 2 principal subgroups, defined by high and low proportions of abnormal Paneth cells. The disordered and diffuse abnormal Paneth cell phenotypes were associated with an altered transcriptional signature of immune system activation. We observed an inverse correlation between abnormal Paneth cells and presence of granuloma. In addition, high proportions of abnormal Paneth cells were associated with shorter time to disease recurrence after surgery. Histologic analysis of Paneth cell phenotypes can be used to divide patients with CD into subgroups with distinct pathognomonic and clinical features.

Irgm1-deficient mice exhibit Paneth cell abnormalities and increased susceptibility to acute intestinal inflammation

Crohn's disease (CD) is a chronic, immune-mediated, inflammatory disorder of the intestine that has been linked to numerous susceptibility genes, including the immunity-related GTPase (IRG) M (IRGM). IRGs comprise a family of proteins known to confer resistance to intracellular infections through various mechanisms, including regulation of phagosome processing, cell motility, and autophagy. However, despite its association with CD, the role of IRGM and other IRGs in regulating intestinal inflammation is unclear. We investigated the involvement of Irgm1, an ortholog of IRGM, in the genesis of murine intestinal inflammation. After dextran sodium sulfate exposure, Irgm1-deficient [Irgm1 knockout (KO)] mice showed increased acute inflammation in the colon and ileum, with worsened clinical responses. Marked alterations of Paneth cell location and granule morphology were present in Irgm1 KO mice, even without dextran sodium sulfate exposure, and were associated with impaired mitophagy and autophagy in Irgm1 KO intestinal cells (including Paneth cells). This was manifested by frequent tubular and swollen mitochondria and increased LC3-positive autophagic structures. Interestingly, these LC3-positive structures often contained Paneth cell granules. These results suggest that Irgm1 modulates acute inflammatory responses in the mouse intestine, putatively through the regulation of gut autophagic processes, that may be pivotal for proper Paneth cell functioning.

ATG4B/autophagin-1 regulates intestinal homeostasis and protects mice from experimental colitis

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.

A Mouse Model of Acrodermatitis Enteropathica: Loss of Intestine Zinc Transporter ZIP4 (Slc39a4) Disrupts the Stem Cell Niche and Intestine Integrity

Mutations in the human Zip4 gene cause acrodermatitis enteropathica, a rare, pseudo-dominant, lethal genetic disorder. We created a tamoxifen-inducible, enterocyte-specific knockout of this gene in mice which mimics this human disorder. We found that the enterocyte Zip4 gene in mice is essential throughout life, and loss-of-function of this gene rapidly leads to wasting and death unless mice are nursed or provided excess dietary zinc. An initial effect of the knockout was the reprogramming of Paneth cells, which contribute to the intestinal stem cell niche in the crypts. Labile zinc in Paneth cells was lost, followed by diminished Sox9 (sex determining region Y-box 9) and lysozyme expression, and accumulation of mucin, which is normally found in goblet cells. This was accompanied by dysplasia of the intestinal crypts and significantly diminished small intestine cell division, and attenuated mTOR1 activity in villus enterocytes, indicative of increased catabolic metabolism, and diminished protein synthesis. This was followed by disorganization of the absorptive epithelium. Elemental analyses of small intestine, liver, and pancreas from Zip4-intestine knockout mice revealed that total zinc was dramatically and rapidly decreased in these organs whereas iron, manganese, and copper slowly accumulated to high levels in the liver as the disease progressed. These studies strongly suggest that wasting and lethality in acrodermatitis enteropathica patients reflects the loss-of-function of the intestine zinc transporter ZIP4, which leads to abnormal Paneth cell gene expression, disruption of the intestinal stem cell niche, and diminished function of the intestinal mucosa. These changes, in turn, cause a switch from anabolic to catabolic metabolism and altered homeostasis of several essential metals, which, if untreated by excess dietary zinc, leads to dramatic weight loss and death.

Modeling IBD in the mouse through random germline mutagenesis

Inflammatory bowel diseases (IBD), including both ulcerative colitis and Crohn's disease, have strong genetic components. In mice, several mutations are known to favor IBD or to inhibit intestinal inflammation. But a comprehensive picture of the pathogenesis of IBD cannot be assembled based on the limited information so far available from genetic analyses. To unveil new genes important for the pathogenesis of IBD, we took an unbiased, hypothesis-free view of IBD, using germline mutagenesis to analyze the phenomenon in mice. We screen for mutations that cause susceptibility to IBD on a defined genetic background using the Dextran sodium sulfate (DSS) colitis model, positionally clone these mutations, and form hypotheses concerning the participation of each validated gene in the disease pathogenesis. A total of 5846 mice have been screened by placing them on 1% DSS in drinking water for a period of 7 days. During this time period all animals are weighed daily, and extreme outliers are retrieved for further study. A total of 16 transmissible mutations causing hypersensitivity to DSS have been detected. Of these, 6 have been identified, and fall into 5 genes. Failure of epithelial integrity is sensed by Toll-like receptors (TLR) on epithelial cells. Wound closure also additionally requires water transport via aquaporin 3, signaling via the EGF receptor pathway, and an intact unfolded protein response, which depends on Mbtps1, also known as the site-1 protease. Relatively common mutations affecting the structure of Mucin-2 also enhance susceptibility to colitis, evidently by increasing ER stress. In addition, genes with unknown functions have been identified causing increased susceptibility to DSS colitis. One of the mutant mice identified in this screen was homozygous for a mutation in an uncharacterized gene. The gene is predicted to encode a protein with an unstructured cytoplasmic N-terminal domain and a C-terminal domain containing five transmembrane α helices connected by short loops. The mutation lies in the splice acceptor site of intron 3 of the seven exon gene, and affects a thymine base five nucleotides from the next exon. cDNA sequencing revealed that the mutation results in skipping of exon 4. Splicing of exon 3 to exon 5 creates a frameshift and a premature stop codon. The Klein-Zschocher mouse showed severe weight loss after administration of 1% DSS in the drinking water. Periodic acid-Schiff stain staining of small intestines and colons of Klein-Zschocher mice revealed reduced numbers of Paneth and goblet cells in naïve mutant mice. Furthermore, electron microscopy of the small intestine and colon of naive Klein-Zschocher mice showed abnormal vesicles in Paneth and goblet cells of the mutant mice. Defective Paneth cells of Klein-Zschocher mutant mice show enhanced autophagy as demonstrated by LC3 Western blotting. In addition, the intestinal permeability was increased 85% in Klein-Zschocher mice demonstrating epithelial barrier dysfunction in these mice. Disturbed epithelial integrity together with abnormal Paneth and goblet cells explain the increased susceptibility in the DSS colitis model. Taken together, using random mutagenesis and the DSS mouse model we found new genes and new functions of known genes that might play a role in IBD. A coherent picture of the essential events required for homeostasis has begun to emerge.

Starvation Compromises Paneth Cells

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.

The response of intestinal stem cells and epithelium after alemtuzumab administration

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.

Influence of alemtuzumab on the intestinal Paneth cells and microflora in macaques

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.

Disruption of Paneth and goblet cell homeostasis and increased endoplasmic reticulum stress in Agr2−/− mice

Anterior Gradient 2 (AGR2) is a protein disulfide isomerase that plays important roles in diverse processes in multiple cell lineages as a developmental regulator, survival factor and susceptibility gene for inflammatory bowel disease. Here, we show using germline and inducible Agr2-/- mice that Agr2 plays important roles in intestinal homeostasis. Agr2-/- intestine has decreased goblet cell Mucin 2, dramatic expansion of the Paneth cell compartment, abnormal Paneth cell localization, elevated endoplasmic reticulum (ER) stress, severe terminal ileitis and colitis. Cell culture experiments show that Agr2 expression is induced by ER stress, and that siRNA knockdown of Agr2 increases ER stress response. These studies implicate Agr2 in intestinal homeostasis and ER stress and suggest a role in the etiology of inflammatory bowel disease.

Abnormal Paneth cell granule dissolution and compromised resistance to bacterial colonization in the intestine of CF mice.

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.