ABSTRACT
The colonic epithelium can undergo multiple rounds of damage and repair, often in response to excessive inflammation. The responsive stem cell that mediates this process is unclear, in part because of a lack of in vitro models that recapitulate key epithelial changes that occur in vivo during damage and repair. Here, we identify a Hopx+ colitis-associated regenerative stem cell (CARSC) population that functionally contributes to mucosal repair in mouse models of colitis. Hopx+ CARSCs, enriched for fetal-like markers, transiently arose from hypertrophic crypts known to facilitate regeneration. Importantly, we established a long-term, self-organizing two-dimensional (2D) epithelial monolayer system to model the regenerative properties and responses of Hopx+ CARSCs. This system can reenact the "homeostasis-injury-regeneration" cycles of epithelial alterations that occur in vivo. Using this system, we found that hypoxia and endoplasmic reticulum stress, insults commonly present in inflammatory bowel diseases, mediated the cyclic switch of cellular status in this process.
Subject(s)
Cell Culture Techniques/methods , Colon/pathology , Stem Cells/pathology , 3T3 Cells , Animals , Colitis/pathology , Epithelial Cells/drug effects , Epithelial Cells/pathology , Homeodomain Proteins/metabolism , Mice , Models, Biological , Oxygen/pharmacology , Regeneration/drug effects , Stem Cells/drug effects , Stress, Physiological/drug effectsABSTRACT
Paneth cells (PCs), a specialized secretory cell type in the small intestine, are increasingly recognized as having an essential role in host responses to microbiome and environmental stresses. Whether and how commensal and pathogenic microbes modify PC composition to modulate inflammation remain unclear. Using newly developed PC-reporter mice under conventional and gnotobiotic conditions, we determined PC transcriptomic heterogeneity in response to commensal and invasive microbes at single cell level. Infection expands the pool of CD74+ PCs, whose number correlates with auto or allogeneic inflammatory disease progressions in mice. Similar correlation was found in human inflammatory disease tissues. Infection-stimulated cytokines increase production of reactive oxygen species (ROS) and expression of a PC-specific mucosal pentraxin (Mptx2) in activated PCs. A PC-specific ablation of MyD88 reduced CD74+ PC population, thus ameliorating pathogen-induced systemic disease. A similar phenotype was also observed in mice lacking Mptx2. Thus, infection stimulates expansion of a PC subset that influences disease progression.
Subject(s)
Microbiota , Paneth Cells , Humans , Animals , Mice , Paneth Cells/metabolism , Paneth Cells/pathology , Intestine, Small , Inflammation/pathology , Cytokines/metabolismABSTRACT
Heme-oxidized IRP2 ubiquitin ligase-1 (HOIL1)-deficient patients experience chronic intestinal inflammation and diarrhea as well as increased susceptibility to bacterial infections. HOIL1 is a component of the linear ubiquitin chain assembly complex that regulates immune signaling pathways, including NF-κB-activating pathways. We have shown previously that HOIL1 is essential for survival following Citrobacter rodentium gastrointestinal infection of mice, but the mechanism of protection by HOIL1 was not examined. C. rodentium is an important murine model for human attaching and effacing pathogens, enteropathogenic and enterohemorrhagic Escherichia coli that cause diarrhea and foodborne illnesses and lead to severe disease in children and immunocompromised individuals. In this study, we found that C. rodentium infection resulted in severe colitis and dissemination of C. rodentium to systemic organs in HOIL1-deficient mice. HOIL1 was important in the innate immune response to limit early replication and dissemination of C. rodentium. Using bone marrow chimeras and cell type-specific knockout mice, we found that HOIL1 functioned in radiation-resistant cells and partly in radiation-sensitive cells and in myeloid cells to limit disease, but it was dispensable in intestinal epithelial cells. HOIL1 deficiency significantly impaired the expansion of group 3 innate lymphoid cells and their production of IL-22 during C. rodentium infection. Understanding the role HOIL1 plays in type 3 inflammation and in limiting the pathogenesis of attaching and effacing lesion-forming bacteria will provide further insight into the innate immune response to gastrointestinal pathogens and inflammatory disorders.
Subject(s)
Enterobacteriaceae Infections , Immunity, Innate , Child , Humans , Animals , Mice , Citrobacter rodentium/physiology , Ligases , Lymphocytes/pathology , Colon/pathology , Inflammation/pathology , Diarrhea/pathology , Ubiquitins , Mice, Inbred C57BLABSTRACT
It is unclear why disease occurs in only a small proportion of persons carrying common risk alleles of disease susceptibility genes. Here we demonstrate that an interaction between a specific virus infection and a mutation in the Crohn's disease susceptibility gene Atg16L1 induces intestinal pathologies in mice. This virus-plus-susceptibility gene interaction generated abnormalities in granule packaging and unique patterns of gene expression in Paneth cells. Further, the response to injury induced by the toxic substance dextran sodium sulfate was fundamentally altered to include pathologies resembling aspects of Crohn's disease. These pathologies triggered by virus-plus-susceptibility gene interaction were dependent on TNFalpha and IFNgamma and were prevented by treatment with broad spectrum antibiotics. Thus, we provide a specific example of how a virus-plus-susceptibility gene interaction can, in combination with additional environmental factors and commensal bacteria, determine the phenotype of hosts carrying common risk alleles for inflammatory disease.
Subject(s)
Carrier Proteins/genetics , Crohn Disease/genetics , Crohn Disease/virology , Genetic Predisposition to Disease , Ileum/pathology , Norovirus , Animals , Autophagy-Related Proteins , Crohn Disease/pathology , Gene Expression Profiling , Humans , Interferon-gamma/metabolism , Mice , Paneth Cells/metabolism , Paneth Cells/virology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
OBJECTIVE: Fibrosis is a common feature of Crohn's disease (CD) which can involve the mesenteric fat. However, the molecular signature of this process remains unclear. Our goal was to define the transcriptional signature of mesenteric fibrosis in CD subjects and to model mesenteric fibrosis in mice to improve our understanding of CD pathogenesis. DESIGN: We performed histological and transcriptional analysis of fibrosis in CD samples. We modelled a CD-like fibrosis phenotype by performing repeated colonic biopsies in mice and analysed the model by histology, type I collagen-targeted positron emission tomography (PET) and global gene expression. We generated a gene set list of essential features of mesenteric fibrosis and compared it to mucosal biopsy datasets from inflammatory bowel disease patients to identify a refined gene set that correlated with clinical outcomes. RESULTS: Mesenteric fibrosis in CD was interconnected to areas of fibrosis in all layers of the intestine, defined as penetrating fibrosis. We found a transcriptional signature of differentially expressed genes enriched in areas of the mesenteric fat of CD subjects with high levels of fibrosis. Mice subjected to repeated colonic biopsies showed penetrating fibrosis as shown by histology, PET imaging and transcriptional analysis. Finally, we composed a composite 24-gene set list that was linked to inflammatory fibroblasts and correlated with treatment response. CONCLUSION: We linked histopathological and molecular features of CD penetrating fibrosis to a mouse model of repeated biopsy injuries. This experimental system provides an innovative approach for functional investigations of underlying profibrotic mechanisms and therapeutic concepts in CD.
Subject(s)
Crohn Disease , Animals , Crohn Disease/complications , Crohn Disease/drug therapy , Crohn Disease/genetics , Fibrosis , Humans , Intestines/pathology , Mesentery/pathology , Mice , Tumor Necrosis Factor InhibitorsABSTRACT
OBJECTIVE: Recent studies have found aristaless-related homeobox gene (ARX)/pancreatic and duodenal homeobox 1 (PDX1), alpha-thalassemia/mental retardation X-linked (ATRX)/death domain-associated protein (DAXX) and alternative lengthening of telomeres (ALT) to be promising prognostic biomarkers for non-functional pancreatic neuroendocrine tumours (NF-PanNETs). However, they have not been comprehensively evaluated, especially among small NF-PanNETs (≤2.0 cm). Moreover, their status in neuroendocrine tumours (NETs) from other sites remains unknown. DESIGN: An international cohort of 1322 NETs was evaluated by immunolabelling for ARX/PDX1 and ATRX/DAXX, and telomere-specific fluorescence in situ hybridisation for ALT. This cohort included 561 primary NF-PanNETs, 107 NF-PanNET metastases and 654 primary, non-pancreatic non-functional NETs and NET metastases. The results were correlated with numerous clinicopathological features including relapse-free survival (RFS). RESULTS: ATRX/DAXX loss and ALT were associated with several adverse prognostic findings and distant metastasis/recurrence (p<0.001). The 5-year RFS rates for patients with ATRX/DAXX-negative and ALT-positive NF-PanNETs were 40% and 42% as compared with 85% and 86% for wild-type NF-PanNETs (p<0.001 and p<0.001). Shorter 5-year RFS rates for ≤2.0 cm NF-PanNETs patients were also seen with ATRX/DAXX loss (65% vs 92%, p=0.003) and ALT (60% vs 93%, p<0.001). By multivariate analysis, ATRX/DAXX and ALT status were independent prognostic factors for RFS. Conversely, classifying NF-PanNETs by ARX/PDX1 expression did not independently correlate with RFS. Except for 4% of pulmonary carcinoids, ATRX/DAXX loss and ALT were only identified in primary (25% and 29%) and NF-PanNET metastases (62% and 71%). CONCLUSIONS: ATRX/DAXX and ALT should be considered in the prognostic evaluation of NF-PanNETs including ≤2.0 cm tumours, and are highly specific for pancreatic origin among NET metastases of unknown primary.
Subject(s)
Intellectual Disability , Neuroendocrine Tumors , Pancreatic Neoplasms , alpha-Thalassemia , Co-Repressor Proteins/genetics , Genes, Homeobox , Homeodomain Proteins , Humans , Intellectual Disability/genetics , Molecular Chaperones/genetics , Neoplasm Recurrence, Local/genetics , Neuroendocrine Tumors/genetics , Nuclear Proteins/genetics , Pancreatic Neoplasms/pathology , Telomere/genetics , Telomere/pathology , Transcription Factors/genetics , X-linked Nuclear Protein/genetics , alpha-Thalassemia/geneticsABSTRACT
BACKGROUND: Intestinal disorders such as environmental enteric dysfunction (EED) are prevalent in low- and middle-income countries (LMICs) and important contributors to childhood undernutrition and mortality. Autopsies are rarely performed in LMICs but minimally invasive tissue sampling is increasingly deployed as a more feasible and acceptable procedure, although protocols have been devoid of intestinal sampling to date. We sought to determine (1) the feasibility of postmortem intestinal sampling, (2) whether autolysis precludes enteric biopsies' utility, and (3) histopathologic features among children who died during hospitalization with acute illness or undernutrition. METHODS: Transabdominal needle and endoscopic forceps upper and lower intestinal sampling were conducted among children aged 1 week to 59 months who died while hospitalized in Blantyre, Malawi. Autolysis ratings were determined for each hematoxylin and eosin slide, and upper and lower intestinal scoring systems were adapted to assess histopathologic features and their severity. RESULTS: Endoscopic and transabdominal sampling procedures were attempted in 28 and 14 cases, respectively, with >90% success obtaining targeted tissue. Varying degrees of autolysis were present in all samples and precluded histopathologic scoring of 6% of 122 biopsies. Greater autolysis in duodenal samples was seen with longer postmortem interval (Beta = 0.06, 95% confidence interval, 0.02-0.11). Histopathologic features identified included duodenal Paneth and goblet cell depletion. Acute inflammation was absent but chronic inflammation was prevalent in both upper and lower enteric samples. Severe chronic rectal inflammation was identified in children as young as 5.5 weeks. CONCLUSIONS: Minimally invasive postmortem intestinal sampling is feasible and identifies histopathology that can inform mortality contributors.
Subject(s)
Malnutrition , Autopsy/methods , Biopsy , Child , Humans , Infant , Poverty , Specimen HandlingABSTRACT
Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for in vivo characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule in vivo.
Subject(s)
Gene Deletion , Animals , Autoantigens/analysis , Bacterial Infections/genetics , Bacterial Infections/immunology , CRISPR-Cas Systems , Female , Immunity, Innate , Inflammation/genetics , Inflammation/immunology , Male , Membrane Proteins/analysis , Mice , Mycoses/genetics , Mycoses/immunology , Phylogeny , Virus Diseases/genetics , Virus Diseases/immunologyABSTRACT
Epithelial cells that line lung airways produce and secrete proteins with important roles in barrier function and host defense. Secretion of airway goblet cells is controlled by autophagy proteins during inflammatory conditions, resulting in accumulation of mucin proteins. We hypothesized that autophagy proteins would also be important in the function of club cells, dominant secretory airway epithelial cells that are dysregulated in chronic lung disease. We found that in the absence of an inflammatory stimulus, mice with club cells deficient for the autophagy protein Atg5 had a markedly diminished expression of secreted host defense proteins secretoglobulin family 1A, member 1 (Scgb1a1) and surfactant proteins A1 and D (Sftpa1 and Sftpd), as well as abnormal club cell morphology. Adult mice with targeted loss of Atg5 also showed diminished levels of host defense proteins in regenerating cells following ablation with naphthalene. A mouse strain with global deficiency of Atg16-like 1 (Atg16l1), an Atg5 binding partner, had a similar loss of host defense proteins and abnormal club cell morphology. Cigarette smoke exposure reduced levels of Scgb1a1 in wild-type mice as expected. Smoke exposure was not required to trigger club cell abnormalities in mice bearing the human ATG16 variant Atg16l1T300A/T300A, which had low Scgb1a1 levels independent of this environmental stress. Evaluation of lung tissues from former smokers with severe chronic obstructive pulmonary disease showed evidence of reduced autophagy and SCGB1A1 expression in club cells. Thus, autophagy proteins are required for the function of club cells, independent of the cellular stress of cigarette smoke, with roles that appear to be distinct from those of other secretory cell types.
Subject(s)
Autophagy/physiology , Epithelial Cells/metabolism , Goblet Cells/metabolism , Lung/metabolism , Animals , Bronchioles/metabolism , Female , Humans , Male , Mice, Transgenic , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Surfactant-Associated Protein A/metabolism , Respiratory Mucosa/metabolismABSTRACT
BACKGROUND & AIMS: Crohn disease (CD) presents as chronic and often progressive intestinal inflammation, but the contributing pathogenic mechanisms are unclear. We aimed to identify alterations in intestinal cells that could contribute to the chronic and progressive course of CD. METHODS: We took an unbiased system-wide approach by performing sequence analysis of RNA extracted from formalin-fixed paraffin-embedded ileal tissue sections from patients with CD (n = 36) and without CD (controls; n = 32). We selected relatively uninflamed samples, based on histology, before gene expression profiling; validation studies were performed using adjacent serial tissue sections. A separate set of samples (3 control and 4 CD samples) was analyzed by transmission electron microscopy. We developed methods to visualize an overlapping modular network of genes dysregulated in the CD samples. We validated our findings using biopsy samples (110 CD samples for gene expression analysis and 54 for histologic analysis) from the UNITI-2 phase 3 trial of ustekinumab for patients with CD and healthy individuals (26 samples used in gene expression analysis). RESULTS: We identified gene clusters that were altered in nearly all CD samples. One cluster encoded genes associated with the enterocyte brush border, leading us to investigate microvilli. In ileal tissues from patients with CD, the microvilli were of decreased length and had ultrastructural defects compared with tissues from controls. Microvilli length correlated with expression of genes that regulate microvilli structure and function. Network analysis linked the microvilli cluster to several other down-regulated clusters associated with altered intracellular trafficking and cellular metabolism. Enrichment of a core microvilli gene set also was lower in the UNITI-2 trial CD samples compared with controls; expression of microvilli genes was correlated with microvilli length and endoscopy score and was associated with response to treatment. CONCLUSIONS: In a transcriptome analysis of formalin-fixed and paraffin-embedded ileal tissues from patients with CD and controls, we associated transcriptional alterations with histologic alterations, such as differences in microvilli length. Decreased microvilli length and decreased expression of the microvilli gene set might contribute to epithelial malfunction and the chronic and progressive disease course in patients with CD.
Subject(s)
Crohn Disease/pathology , Ileum/pathology , Intestinal Mucosa/pathology , Intestine, Small/pathology , Microvilli/pathology , Chronic Disease , Crohn Disease/genetics , Disease Progression , Gene Expression Profiling , Humans , Microvilli/genetics , TranscriptomeABSTRACT
Despite prognostic grading and staging systems, it is a challenge to predict outcomes for patients with pancreatic neuroendocrine tumors (PanNETs). Sequencing studies of PanNETs have identified alterations in death domain-associated protein (DAXX) and alpha-thalassemia/mental retardation X-linked chromatin remodeler (ATRX). In tumors, mutations in DAXX or ATRX and corresponding loss of protein expression correlate with shorter times of disease-free survival and disease-specific survival of patients. However, DAXX or ATRX proteins were lost in only 50% of distant metastases analyzed. We performed whole-exome sequencing analyses of 20 distant metastases from 20 patients with a single nonsyndrome, nonfunctional PanNET. We found distant metastases contained alterations in multiple endocrine neoplasia type 1 (MEN1) (n = 8), ATRX (n = 5), DAXX (n = 5), TSC2 (n = 3), and DEP domain containing 5 (DEPDC5) (n = 3). We found copy number loss of cyclin dependent kinase inhibitor 2A (CDKN2A) in 15 metastases (75%) and alterations in genes that regulate chromatin remodeling, including set domain containing 2 (SETD2) (n = 4), AT-rich interaction domain 1A (ARID1A) (n = 2), chromodomain helicase DNA binding protein 8 (CHD8) (n = 2), and DNA methyl transferase 1 (DNMT1) (n = 2). In a separate analysis of 347 primary PanNETs, we found loss or deletion of DAXX and ATRX, disruption of SETD2 function (based on loss of H3 lysine 36 trimethylation), loss of ARID1A expression or deletions in CDKN2A in 81% of primary PanNETs with distant metastases. Among patients with loss or deletion of at least 1 of these proteins or genes, 39% survived disease-free for 5 years and 44% had disease-specific survival times of 10 years. Among patients without any of these alterations, 98% survived disease-free for 5 years and 95% had disease-specific survival times of 10 years. Therefore, primary PanNETs with loss of DAXX, ATRX, H3 lysine 36 trimethylation, ARID1A, and/or CDKN2A associate with shorter survival times of patients. Our findings indicate that alterations in chromatin-remodeling genes and CDKN2A contribute to metastasis of PanNETs.
Subject(s)
Biomarkers, Tumor/genetics , Cyclin-Dependent Kinase Inhibitor p18/genetics , Neuroendocrine Tumors/genetics , Nuclear Proteins/genetics , Pancreatic Neoplasms/genetics , Adult , Aged , Aged, 80 and over , Chromatin Assembly and Disassembly/genetics , Cyclin-Dependent Kinase Inhibitor p16 , DNA Copy Number Variations , Disease-Free Survival , Female , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Mutation , Neuroendocrine Tumors/mortality , Neuroendocrine Tumors/pathology , Neuroendocrine Tumors/surgery , Pancreatectomy , Pancreatic Neoplasms/mortality , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/surgery , Prognosis , Exome SequencingABSTRACT
BACKGROUND: Environmental Enteropathy (EE), characterized by alterations in intestinal structure, function, and immune activation, is believed to be an important contributor to childhood undernutrition and its associated morbidities, including stunting. Half of all global deaths in children < 5 years are attributable to under-nutrition, making the study of EE an area of critical priority. METHODS: Community based intervention study, divided into two sub-studies, 1) Longitudinal analyses and 2) Biopsy studies for identification of EE features via omics analyses. Birth cohorts in Matiari, Pakistan established: moderately or severely malnourished (weight for height Z score (WHZ) < - 2) children, and well-nourished (WHZ > 0) children. Blood, urine, and fecal samples, for evaluation of potential biomarkers, will be collected at various time points from all participants (longitudinal analyses). Participants will receive appropriate educational and nutritional interventions; non-responders will undergo further evaluation to determine eligibility for further workup, including upper gastrointestinal endoscopy. Histopathological changes in duodenal biopsies will be compared with duodenal biopsies obtained from USA controls who have celiac disease, Crohn's disease, or who were found to have normal histopathology. RNA-Seq will be employed to characterize mucosal gene expression across groups. Duodenal biopsies, luminal aspirates from the duodenum, and fecal samples will be analyzed to define microbial community composition (omic analyses). The relationship between histopathology, mucosal gene expression, and community configuration will be assessed using a variety of bioinformatic tools to gain better understanding of disease pathogenesis and to identify mechanism-based biomarkers. Ethical review committees at all collaborating institutions have approved this study. All results will be made available to the scientific community. DISCUSSION: Operational and ethical constraints for safely obtaining intestinal biopsies from children in resource-poor settings have led to a paucity of human tissue-based investigations to understand and reverse EE in vulnerable populations. Furthermore, EE biomarkers have rarely been correlated with gold standard histopathological confirmation. The Study of Environmental Enteropathy and Malnutrition (SEEM) is designed to better understand the pathophysiology, predictors, biomarkers, and potential management strategies of EE to inform strategies to eradicate this debilitating pathology and accelerate progress towards the 2030 Sustainable Development Goals. TRIAL REGISTRATION: Retrospectively registered; clinicaltrials.gov ID NCT03588013 .
Subject(s)
Biomarkers/analysis , Celiac Disease/diagnosis , Duodenum/pathology , Infant Nutrition Disorders/diagnosis , Malnutrition/diagnosis , Biopsy , Celiac Disease/pathology , Female , Growth , Growth Disorders/etiology , Humans , Infant , Infant, Newborn , Male , Nutritional Status , Pakistan , Research DesignABSTRACT
A coding polymorphism (Thr300Ala) in the essential autophagy gene, autophagy related 16-like 1 (ATG16L1), confers increased risk for the development of Crohn disease, although the mechanisms by which single disease-associated polymorphisms contribute to pathogenesis have been difficult to dissect given that environmental factors likely influence disease initiation in these patients. Here we introduce a knock-in mouse model expressing the Atg16L1 T300A variant. Consistent with the human polymorphism, T300A knock-in mice do not develop spontaneous intestinal inflammation, but exhibit morphological defects in Paneth and goblet cells. Selective autophagy is reduced in multiple cell types from T300A knock-in mice compared with WT mice. The T300A polymorphism significantly increases caspase 3- and caspase 7-mediated cleavage of Atg16L1, resulting in lower levels of full-length Atg16Ll T300A protein. Moreover, Atg16L1 T300A is associated with decreased antibacterial autophagy and increased IL-1ß production in primary cells and in vivo. Quantitative proteomics for protein interactors of ATG16L1 identified previously unknown nonoverlapping sets of proteins involved in ATG16L1-dependent antibacterial autophagy or IL-1ß production. These findings demonstrate how the T300A polymorphism leads to cell type- and pathway-specific disruptions of selective autophagy and suggest a mechanism by which this polymorphism contributes to disease.
Subject(s)
Carrier Proteins/genetics , Crohn Disease/immunology , Paneth Cells/pathology , Polymorphism, Single Nucleotide/genetics , Salmonella Infections/immunology , Animals , Autophagy/genetics , Autophagy-Related Proteins , Blotting, Western , Chromatography, Liquid , Crohn Disease/genetics , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Gene Knock-In Techniques , Goblet Cells/pathology , Mice , Proteomics , Real-Time Polymerase Chain Reaction , Tandem Mass SpectrometryABSTRACT
BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Histologic analysis of Paneth cell phenotypes can be used to divide patients with CD into subgroups with distinct pathognomonic and clinical features.
Subject(s)
Carrier Proteins/genetics , Crohn Disease/genetics , Nod2 Signaling Adaptor Protein/genetics , Paneth Cells/pathology , Secretory Vesicles/pathology , Alleles , Autophagy-Related Proteins , Cohort Studies , Crohn Disease/pathology , Female , Genetic Predisposition to Disease , Genotype , Granuloma/genetics , Granuloma/pathology , Humans , Male , Phenotype , Polymorphism, Single Nucleotide , Retrospective StudiesABSTRACT
The majority of hepatocellular carcinomas arise in background chronic liver disease, particularly cirrhosis. The pathogenesis of noncirrhotic hepatocellular carcinomas remains unclear. While malignant transformation reportedly occurs in <15% of hepatocellular adenoma, the prevalence of noncirrhotic hepatocellular carcinomas arising from a pre-existing adenoma is a challenge to study. Cirrhotic hepatocellular carcinoma and hepatocellular adenoma may be subclassified by molecular pathways, but little is known in noncirrhotic hepatocellular carcinoma. We aim to delineate clinical, morphologic and immunohistochemical features of noncirrhotic hepatocellular carcinoma to evaluate for possible derivation from hepatocellular adenoma. We evaluated the clinicopathologic features of 74 noncirrhotic hepatocellular carcinomas from 72 patients for underlying clinical conditions and immunohistochemical markers known to be associated with hepatocellular adenoma. Men were more commonly affected (59%); however, in the <50-year-old group, women predominated (8:1). The age range was wide: 18-83 years; median-64 years. Underlying liver diseases were identified in only 7%; however, 25% had diabetes mellitus, 69% were overweight or obese and 58% had metabolic syndrome. Only 50% of the noncirrhotic hepatocellular carcinoma were encapsulated. As published in hepatocellular adenoma, multifocality and larger tumor size were more common in liver fatty acid-binding protein-negative noncirrhotic hepatocellular carcinoma. Beta-catenin nuclear positivity was uncommon (5%), and was restricted to hepatocellular carcinomas in older men. Serum amyloid A positivity was not restricted to any subtype. In summary, we present the largest series to date examining noncirrhotic hepatocellular carcinoma. We evaluated these with current hepatocellular adenoma subclassification markers for possible associations. Thirty percent of the 74 noncirrhotic hepatocellular carcinoma had some clinical, morphological or immunophenotypical associations currently described in hepatocellular adenoma. Our data also confirm the association of noncirrhotic hepatocellular carcinoma in middle-aged to elderly men, an association with metabolic syndrome, and, as with hepatocellular adenoma, that women predominated in the noncirrhotic hepatocellular carcinoma subjects <50 years of age.
Subject(s)
Adenoma, Liver Cell/pathology , Carcinoma, Hepatocellular/pathology , Cell Transformation, Neoplastic , Liver Neoplasms/pathology , Adenoma, Liver Cell/complications , Adolescent , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/analysis , Carcinoma, Hepatocellular/complications , Comorbidity , Female , Humans , Immunohistochemistry , Liver Neoplasms/complications , Male , Metabolic Syndrome , Middle Aged , Young AdultABSTRACT
INTRODUCTION: Circulating tissue transglutaminase immunoglobulin A concentration is a sensitive and specific indicator of celiac disease, but discrepancies between serologic and histologic findings occur. We hypothesized that fecal markers of inflammation and protein loss would be greater in patients with untreated celiac disease than in healthy controls. Our study aims to evaluate multiple fecal and plasma markers in celiac disease and correlate these findings with serologic and histologic findings as noninvasive means of evaluating disease activity. METHODS: Participants with positive celiac serologies and controls with negative celiac serologies were prospectively enrolled before upper endoscopy. Blood, stool, and duodenal biopsies were collected. Concentrations of fecal lipocalin-2, calprotectin, and alpha-1-antitrypsin and plasma lipocalin-2 were determined. Biopsies underwent modified Marsh scoring. Significance was tested between cases and controls, modified Marsh score and tissue transglutaminase immunoglobulin A concentration. RESULTS: Lipocalin-2 was significantly elevated in the stool ( P = 0.006) but not the plasma of participants with positive celiac serologies. There was no significant difference in fecal calprotectin or alpha-1 antitrypsin between participants with positive celiac serologies and controls. Fecal alpha-1 antitrypsin >100 mg/dL was specific, but not sensitive for biopsy-proven celiac disease. DISCUSSION: Lipocalin-2 is elevated in the stool but not the plasma of patients with celiac disease suggesting a role of local inflammatory response. Calprotectin was not a useful marker in the diagnosis of celiac disease. While random fecal alpha-1 antitrypsin was not significantly elevated in cases compared with controls, an elevation of greater than 100 mg/dL was 90% specific for biopsy-proven celiac disease.
Subject(s)
Biomarkers , Celiac Disease , Duodenum , Feces , GTP-Binding Proteins , Immunoglobulin A , Leukocyte L1 Antigen Complex , Lipocalin-2 , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases , alpha 1-Antitrypsin , Humans , Celiac Disease/diagnosis , Celiac Disease/blood , Celiac Disease/pathology , Female , Biomarkers/blood , Biomarkers/analysis , Male , Child , alpha 1-Antitrypsin/blood , Leukocyte L1 Antigen Complex/analysis , Leukocyte L1 Antigen Complex/blood , Feces/chemistry , Lipocalin-2/blood , Lipocalin-2/analysis , Transglutaminases/immunology , Transglutaminases/blood , Prospective Studies , Child, Preschool , Immunoglobulin A/blood , GTP-Binding Proteins/immunology , GTP-Binding Proteins/blood , Adolescent , Duodenum/pathology , Biopsy , Case-Control Studies , Lipocalins/blood , Acute-Phase Proteins/analysis , Acute-Phase Proteins/metabolism , Inflammation/diagnosis , Inflammation/bloodABSTRACT
The prevalence of obesity in the United States has continued to increase over the past several decades. Understanding how diet-induced obesity modulates mucosal immunity is of clinical relevance. We previously showed that consumption of a high fat, high sugar "Western" diet (WD) reduces the density and function of small intestinal Paneth cells, a small intestinal epithelial cell type with innate immune function. We hypothesized that obesity could also result in repressed gut adaptive immunity. Using small intestinal intraepithelial lymphocytes (IEL) as a readout, we found that in non-inflammatory bowel disease (IBD) subjects, high body mass index correlated with reduced IEL density. We recapitulated this in wild type (WT) mice fed with WD. A 4-week WD consumption was able to reduce IEL but not splenic, blood, or bone marrow lymphocytes, and the effect was reversible after another 2 weeks of standard diet (SD) washout. Importantly, WD-associated IEL reduction was not dependent on the presence of gut microbiota, as WD-fed germ-free mice also showed IEL reduction. We further found that WD-mediated Farnesoid X Receptor (FXR) activation in the gut triggered IEL reduction, and this was partially mediated by intestinal phagocytes. Activated FXR signaling stimulated phagocytes to secrete type I IFN, and inhibition of either FXR or type I IFN signaling within the phagocytes prevented WD-mediated IEL loss. Therefore, WD consumption represses both innate and adaptive immunity in the gut. These findings have significant clinical implications in the understanding of how diet modulates mucosal immunity.
ABSTRACT
Environmental enteric dysfunction (EED) is a diffuse small bowel disorder associated with poor growth, inadequate responses to oral vaccines, and nutrient malabsorption in millions of children worldwide. We identify loss of the small intestinal Paneth and goblet cells that are critical for innate immunity, reduced villous height, increased bile acids, and dysregulated nicotinamide adenine dinucleotide (NAD+) synthesis signaling as potential mechanisms underlying EED and which also correlated with diminished length-for-age z score. Isocaloric low-protein diet (LPD) consumption in mice recapitulated EED histopathology and transcriptomic changes in a microbiota-independent manner, as well as increases in serum and fecal bile acids. Children with refractory EED harbor single-nucleotide polymorphisms in key enzymes involved in NAD+ synthesis. In mice, deletion of Nampt, the gene encoding the rate-limiting enzyme in the NAD+ salvage pathway, from intestinal epithelium also reduced Paneth cell function, a deficiency that was further aggravated by LPD. Separate supplementation with NAD+ precursors or bile acid sequestrant partially restored LPD-associated Paneth cell defects and, when combined, fully restored all histopathology defects in LPD-fed mice. Therapeutic regimens that increase protein and NAD+ contents while reducing excessive bile acids may benefit children with refractory EED.
Subject(s)
Bile Acids and Salts , NAD , Humans , Child , Mice , Animals , NAD/genetics , NAD/metabolism , Cytokines/metabolismABSTRACT
Crohn disease (CD) burden has increased with globalization/urbanization, and the rapid rise is attributed to environmental changes rather than genetic drift. The Study Of Urban and Rural CD Evolution (SOURCE, n = 380) has considered diet-omics domains simultaneously to detect complex interactions and identify potential beneficial and pathogenic factors linked with rural-urban transition and CD. We characterize exposures, diet, ileal transcriptomics, metabolomics, and microbiome in newly diagnosed CD patients and controls in rural and urban China and Israel. We show that time spent by rural residents in urban environments is linked with changes in gut microbial composition and metabolomics, which mirror those seen in CD. Ileal transcriptomics highlights personal metabolic and immune gene expression modules, that are directly linked to potential protective dietary exposures (coffee, manganese, vitamin D), fecal metabolites, and the microbiome. Bacteria-associated metabolites are primarily linked with host immune modules, whereas diet-linked metabolites are associated with host epithelial metabolic functions.
Subject(s)
Crohn Disease , Diet , Gastrointestinal Microbiome , Rural Population , Urban Population , Crohn Disease/microbiology , Crohn Disease/genetics , Humans , Male , Female , China/epidemiology , Adult , Israel/epidemiology , Metabolomics , Cohort Studies , Middle Aged , Feces/microbiology , Ileum/microbiology , Ileum/metabolism , Transcriptome , Young AdultABSTRACT
BACKGROUND & AIMS: Biomarkers that integrate genetic and environmental factors and predict outcome in complex immune diseases such as inflammatory bowel disease (IBD; including Crohn's disease [CD] and ulcerative colitis [UC]) are needed. We showed that morphologic patterns of ileal Paneth cells (Paneth cell phenotype [PCP]; a surrogate for PC function) is one such cellular biomarker for CD. Given the shared features between CD and UC, we hypothesized that PCP is also associated with molecular/genetic features and outcome in UC. Because PC density is highest in the ileum, we further hypothesized that PCP predicts outcome in UC subjects who underwent total colectomy and ileal pouch-anal anastomosis (IPAA). METHODS: Uninflamed ileal resection margins from UC subjects with colectomy and IPAA were used for PCP and transcriptomic analyses. PCP was defined using defensin 5 immunofluorescence. Genotyping was performed using Immunochip. UC transcriptomic and genotype associations of PCP were incorporated with data from CD subjects to identify common IBD-related pathways and genes that regulate PCP. RESULTS: The prevalence of abnormal ileal PCP was 27%, comparable to that seen in CD. Combined analysis of UC and CD subjects showed that abnormal PCP was associated with transcriptomic pathways of secretory granule maturation and polymorphisms in innate immunity genes. Abnormal ileal PCP at the time of colectomy was also associated with pouch complications including de novo CD in the pouch and time to first episode of pouchitis. CONCLUSIONS: Ileal PCP is biologically and clinically relevant in UC and can be used as a biomarker in IBD.