Primary Intestinal Epithelial Cells Research Articles

Bioengineered Systems and Designer Matrices That Recapitulate the Intestinal Stem Cell Niche.

The relationship between intestinal stem cells (ISCs) and the surrounding niche environment is complex and dynamic. Key factors localized at the base of the crypt are necessary to promote ISC self-renewal and proliferation, to ultimately provide a constant stream of differentiated cells to maintain the epithelial barrier. These factors diminish as epithelial cells divide, migrate away from the crypt base, differentiate into the postmitotic lineages, and end their life span in approximately 7 days when they are sloughed into the intestinal lumen. To facilitate the rapid and complex physiology of ISC-driven epithelial renewal, in vivo gradients of growth factors, extracellular matrix, bacterial products, gases, and stiffness are formed along the crypt-villus axis. New bioengineered tools and platforms are available to recapitulate various gradients and support the stereotypical cellular responses associated with these gradients. Many of these technologies have been paired with primary small intestinal and colonic epithelial cells to re-create select aspects of normal physiology or disease states. These biomimetic platforms are becoming increasingly sophisticated with the rapid discovery of new niche factors and gradients. These advancements are contributing to the development of high-fidelity tissue constructs for basic science applications, drug screening, and personalized medicine applications. Here, we discuss the direct and indirect evidence for many of the important gradients found in vivo and their successful application to date in bioengineered in vitro models, including organ-on-chip and microfluidic culture devices.

Long-Term Culture of Intestinal Organoids.

The in vitro long-term expansion of primary intestinal epithelial cells has been hampered by the inability to maintain an immature stem cell population. Recent technical advances have led to the development of a novel in vitro culture system that can sustain intestinal stem cells (ISCs) using growth factors that mimic the intestinal microenvironment in combination with a three-dimensional (3D) culture. The resulting intestinal organoids display a crypt-villus architecture that recapitulates the native intestinal epithelium. Here, we describe our method for the long-term culture of intestinal epithelial organoids via consistent passaging using a gentle cell dissociation reagent to easily break the organoid into smaller pieces. The long-term cryopreservation and defining characteristics of these intestinal organoids also make this work relevant for the advancement of epithelial organoid-based therapeutic technologies by allowing the production of large numbers of cells for use in clinical applications.

Nonsteroidal Anti-Inflammatory Drug-Induced Leaky Gut Modeled Using Polarized Monolayers of Primary Human Intestinal Epithelial Cells

The intestinal epithelium provides a critical barrier that separates the gut microbiota from host tissues. Nonsteroidal anti-inflammatory drugs (NSAIDs) are efficacious analgesics and antipyretics and are among the most frequently used drugs worldwide. In addition to gastric damage, NSAIDs are toxic to the intestinal epithelium, causing erosions, perforations, and longitudinal ulcers in the gut. Here, we use a unique in vitro human primary small intestinal cell monolayer system to pinpoint the intestinal consequences of NSAID treatment. We found that physiologically relevant doses of the NSAID diclofenac (DCF) are cytotoxic because they uncouple mitochondrial oxidative phosphorylation and generate reactive oxygen species. We also find that DCF induces intestinal barrier permeability, facilitating the translocation of compounds from the luminal to the basolateral side of the intestinal epithelium. The results we outline here establish the utility of this novel platform, representative of the human small intestinal epithelium, to understand NSAID toxicity, which can be applied to study multiple aspects of gut barrier function including defense against infectious pathogens and host-microbiota interactions.

Development and characterization of 2-dimensional culture for buffalo intestinal cells.

Small intestinal epithelial cells (IEC) play a major role in the absorption of nutrients and toxins. Due to the similarity of genome-wide single copy protein orthologues between cattle and human, establishment of ruminant's primary small IEC culture could be a valuable tool for toxicity studies. Therefore, the current study focused on the development and characterization of buffalo IEC culture, as cattle slaughter is banned in India. The buffalo jejunum fragments were washed consecutively several times in saline, warm phosphate buffered saline (PBS), PBS with 5mM dithiothreitol, digesting solution and 2% sorbitol in PBS. The cells were cultured on 17µg/cm2 collagen coated plates and transwell plates with serum (2% Fetal bovine serum (FBS) and 10% FBS) and serum-free culture conditions. The cells were differentiated into typical epithelial cobblestone morphology from day 5 onwards in 50% successful cultures. The cultured IEC were characterized by gene expression of epithelial cell markers, cytokeratin and vimentin, and enterocyte markers like villin, zonula occluden (ZO1), fatty acid binding protein 2 (FABP2) and small intestinal peptidase (IP). Based on the morphology and gene expression profile, 10% FBS has been recommended for culturing primary buffalo IEC on collagen coated plates for 10days. However, 50% of the successful cultures could not show epithelial phenotype on 10% FBS culture conditions even on collagen coated plates. Interestingly, undifferentiated IEC showed an increasing expression of FABP2, IP and ZO1 transcripts compared to differentiated intestinal cells with 10% FBS on collagen plates. Therefore, future studies are needed to understand the role of FABP2, IP and ZO1 in differentiation of buffalo IEC.

DNA Methylation and Transcription Patterns in Intestinal Epithelial Cells From Pediatric Patients With Inflammatory Bowel Diseases Differentiate Disease Subtypes and Associate With Outcome

Background & AimsWe analyzed DNA methylation patterns and transcriptomes of primary intestinal epithelial cells (IEC) of children newly diagnosed with inflammatory bowel diseases (IBD) to learn more about pathogenesis.MethodsWe obtained mucosal biopsies (N = 236) collected from terminal ileum and ascending and sigmoid colons of children (median age 13 years) newly diagnosed with IBD (43 with Crohn’s disease [CD], 23 with ulcerative colitis [UC]), and 30 children without IBD (controls). Patients were recruited and managed at a hospital in the United Kingdom from 2013 through 2016. We also obtained biopsies collected at later stages from a subset of patients. IECs were purified and analyzed for genome-wide DNA methylation patterns and gene expression profiles. Adjacent microbiota were isolated from biopsies and analyzed by 16S gene sequencing. We generated intestinal organoid cultures from a subset of samples and genome-wide DNA methylation analysis was performed.ResultsWe found gut segment-specific differences in DNA methylation and transcription profiles of IECs from children with IBD vs controls; some were independent of mucosal inflammation. Changes in gut microbiota between IBD and control groups were not as large and were difficult to assess because of large amounts of intra-individual variation. Only IECs from patients with CD had changes in DNA methylation and transcription patterns in terminal ileum epithelium, compared with controls. Colon epithelium from patients with CD and from patients with ulcerative colitis had distinct changes in DNA methylation and transcription patterns, compared with controls. In IECs from patients with IBD, changes in DNA methylation, compared with controls, were stable over time and were partially retained in ex-vivo organoid cultures. Statistical analyses of epithelial cell profiles allowed us to distinguish children with CD or UC from controls; profiles correlated with disease outcome parameters, such as the requirement for treatment with biologic agents.ConclusionsWe identified specific changes in DNA methylation and transcriptome patterns in IECs from pediatric patients with IBD compared with controls. These data indicate that IECs undergo changes during IBD development and could be involved in pathogenesis. Further analyses of primary IECs from patients with IBD could improve our understanding of the large variations in disease progression and outcomes.

Deletions at SLC18A1 increased the risk of CRC and lower SLC18A1 expression associated with poor CRC outcome.

Copy number variations (CNVs) contribute to the development of colorectal cancer (CRC). We conducted a two-stage association study to identify CNV risk loci for CRC. We performed a gene-based rare CNV study on 694 sporadic CRC and 1641 controls using Illumina Human-OmniExpress-12v1.0 BeadChips, and further replicated in 934 CRC cases and 2680 controls for risk CNVs by using TaqMan Copy Number Assay. Tumor buddings, cancer cells in the center of primary tumor and normal intestinal epithelial cells were captured using laser capture microdissection (LCM) and were assayed using AffymetrixGeneChip® Human Genome U133 Plus 2.0 Array. In addition, The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus data were assessed for the effects of risk CNVs. We found that germline deletions affecting the last six exons of SLC18A1 significantly associated with CRC with a combined P value of 6.4 × 10-5 by a two-stage analysis. Both in TCGA CRC RNA seq dataset and GDS4382, SLC18A1 was significantly down regulated in CRC tissues than in paired normal tissues (N = 32 and 17 pairs, P = 0.004 and 0.009, respectively). In LCM samples, similar observations were obtained that the expression levels of SLC18A1 in the tumor buddings, cancer cells in the center of primary tumor, and stroma of both tumor budding and cancer cells were lower than normal intestinal epithelial and stromal cells (fold change = 0.17-0.62, 0.12-0.57 and 0.37-0.68, respectively). In summary, the germline deletions at SLC18A1 contributed to the development of CRC. The role of SLC18A1 required further exploration.

Regulation of epithelial cell expressed C3 in the intestine – Relevance for the pathophysiology of inflammatory bowel disease?

The complement system not only plays a critical role in efficient detection and clearance of bacteria, but also in intestinal immune homeostasis as mice deficient for key complement components display enhanced intestinal inflammation upon experimental colitis. Because underlying molecular mechanisms for this observation are unclear, we investigated the crosstalk between intestinal epithelial cells (IEC), bacteria and the complement system in the course of chronic colitis.Surprisingly, mouse intestinal epithelial cell lines constitutively express high mRNA levels of complement component 3 (C3), Toll-like receptor 2 (Tlr2) and Tlr4. Stimulation of these cells with lipopolysaccharide (LPS), but not with flagellin, LD-muramyldipeptide or peptidoglycan, triggered increased C3 expression, secretion and activation. Stimulation of the C3aR on these cell lines with C3a resulted in an increase of LPS-triggered pro-inflammatory response. Tissue biopsies from C57BL/6J mice revealed higher expression of C3, Tlr1, Tlr2 and Tlr4 in colonic primary IECs (pIECs) compared to ileal pIECs, while in germ-free mice no differences in C3 protein expression was observed. In DSS-induced chronic colitis mouse models, C3 mRNA expression was upregulated in colonic biopsies and ileal pIECs with elevated C3 protein in the lamina propria, IECs and the mucus. Notably, increased C3b opsonization of mucosa-attached bacteria and decreased fecal full-length C3 protein was observed in DSS-treated compared to untreated mice. Of significant interest, non-inflamed and inflamed colonic biopsy samples from CD but not UC patients displayed exacerbated C3 expression compared to controls.These findings suggest that a novel TLR4-C3 axis could control the intestinal immune response during chronic colitis.

A simple, cost-effective method for generating murine colonic 3D enteroids and 2D monolayers for studies of primary epithelial cell function.

Cancer cell lines have been the mainstay of intestinal epithelial experimentation for decades, due primarily to their immortality and ease of culture. However, because of the inherent biological abnormalities of cancer cell lines, many cellular biologists are currently transitioning away from these models and toward more representative primary cells. This has been particularly challenging, but recent advances in the generation of intestinal organoids have brought the routine use of primary cells within reach of most epithelial biologists. Nevertheless, even with the proliferation of publications that use primary intestinal epithelial cells, there is still a considerable amount of trial and error required for laboratories to establish a consistent and reliable method to culture three-dimensional (3D) intestinal organoids and primary epithelial monolayers. We aim to minimize the time other laboratories spend troubleshooting the technique and present a standard method for culturing primary epithelial cells. Therefore, we have described our optimized, high-yield, cost-effective protocol to grow 3D murine colonoids for more than 20 passages and our detailed methods to culture these cells as confluent monolayers for at least 14 days, enabling a wide variety of potential future experiments. By supporting and expanding on the current literature of primary epithelial culture optimization and detailed use in experiments, we hope to help enable the widespread adoption of these innovative methods and allow consistency of results obtained across laboratories and institutions.NEW & NOTEWORTHY Primary intestinal epithelial monolayers are notoriously difficult to maintain culture, even with the recent advances in the field. We describe, in detail, the protocols required to maintain three-dimensional cultures of murine colonoids and passage these primary epithelial cells to confluent monolayers in a standardized, high-yield and cost-effective manner.

Primary culture of cat intestinal epithelial cell and construction of its cDNA library

Objective To establish the primary cat intestinal epithelial cells (IECs) culture methods and construct the cDNA library for the following yeast two-hybrid experiment, so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection, by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMART™ technology, and then the double-strand cDNAs were acquired by LD-PCR, which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recombination. Matchmaker™ Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calculation of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of collagenase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1×106 independent clones. The titer was 2.8×109 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research, and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

Use of a Collagen Membrane to Enhance the Survival of Primary Intestinal Epithelial Cells

Intestinal epithelial cell culture is important for biological, functional, and immunological studies. Since enterocytes have a short in vivo life span due to anoikis, we aimed to establish a novel and reproducible method to prolong the survival of mouse and human cells. Cells were isolated following a standard procedure, and cultured on ordered-cow's collagen membranes. A prolonged cell life span was achieved; cells covered the complete surface of bio-membranes and showed a classical enterocyte morphology with high expression of enzymes supporting the possibility of cryopreservation. Apoptosis was dramatically reduced and cultured enterocytes expressed cytokeratin and LGR5 (low frequency). Cells exposed to LPS or flagellin showed the induction of TLR4 and TLR5 expression and a functional phenotype upon exposure to the probiotic Bifidobacterium bifidum or the pathogenic Clostridium difficile. The secretion of the homeostatic (IL-25 and TSLP), inhibitory (IL-10 and TGF-β), or pro-inflammatory mediators (IL-1β and TNF) were induced. In conclusion, this novel protocol using cow's collagen-ordered membrane provides a simple and reproducible method to maintain intestinal epithelial cells functional for cell-microorganism interaction studies and stem cell expansion. J. Cell. Physiol. 232: 2489-2496, 2017. © 2016 Wiley Periodicals, Inc.

Development of an improved in vitro culture system for Cryptosporidium parvum

The protozoan parasite Cryptosporidium parvum is a leading cause of pediatric diarrhea in developing countries, yet effective treatments for the disease are lacking due to the difficulty of studying Cryptosporidium in the laboratory. Specifically, current culturing methods rely on adenocarcinoma cell lines that do not support continual growth of C. parvum in vitro, hindering the development of reagents such as stage‐specific antibodies and genetic tools for this parasite. To better mimic the native intracellular niche of C. parvum, we have utilized primary intestinal epithelial cell (IEC) monolayers as an improved system for the study of C. parvum. We have found that human and mouse IECs support enhanced growth of C. parvum compared to adenocarcinoma cell lines. Importantly, parasite growth in mouse IECs, the first mouse cell line to support robust asexual C. parvum growth, enabled us to generate a unique panel of monoclonal antibodies against the intracellular life cycle stages of the parasite. We have also developed quantitative PCR assays to measure expression of genes associated with sexual‐stage development of the parasite independent of absolute parasite replication. With the development of these new tools and the enhanced culture system, we are now able to screen culture conditions mimicking the hypoxic, reducing environment of the small intestine to identify conditions that further improve C. parvum growth and/or complete development of the life cycle in vitro. Additionally, we are also screening a panel of gut microbial metabolites for their effects on C. parvum growth to gain insight into the complex community interactions that may contribute to C. parvum infection in vivo. In all, these new tools and improved culture system will facilitate the study of Cryptosporidium in vitro, allowing us to gain a better understanding of the parasite's biology as a means to more effective therapeutics for the disease in the future.Support or Funding InformationThis work is supported by the Bill and Melinda Gates Foundation.

Establishment of immortalized mouse intestinal epithelial cells line and study of effects of Arg-Arg on inflammatory response.

Primary mouse intestinal epithelial cells (MIEs) are not ideal models for long-term culture in vitro and a limited amount of approximate three generations. In addition, the mechanism that arginine-arginine dipeptide (Arg-Arg) regulates mouse intestinal inflammatory response remains unknown. Therefore, the aim of this study was to establish immortal MIEs and study the effects of Arg-Arg on inflammatory response after challenging the MIEs with lipopolysaccharide (LPS) or staphylococcal enterotoxin C (rSEC). Our data showed that immortalized MIEs could be cultured over 100 generations. The immortalized MIEs showed positive reaction against cytokeratine 18 antigen, E-cadherin, and peptide transporters (Pept1) using indirect immunofluorescence. Cytokeratine 18 and Pept1 can be expressed in immortalized MIEs by immunoblotting. Fatty acid-binding proteins (FABPs) and villin known as intestinal epithelial cell functional protein were constitutively expressed in immortalized MIEs. For inflammatory response, these results showed that Arg-Arg can decrease the LPS-induced expression of IL-1β and the rSEC-induced expression of TNF-α; however, it can upregulate the LPS-induced expression of IL-6 and TNF-α and the rSEC-induced expression level of IL-1β. In addition, in the MAPK signaling pathway, pSAPK/JNK and p-Erk1/2 in LPS with Arg-Arg treatment were upregulated than that in LPS treatment. p-p38 in LPS with Arg-Arg treatment was attenuated than that in LPS treatment. pSAPK/JNK and p-p38 in rSEC with Arg-Arg treatment were enhanced than that in rSEC treatment. Conversely, p-Erk1/2 in rSEC with Arg-Arg treatment was attenuated than that in rSEC treatment. These novel findings suggest that Arg-Arg dipeptide plays an important role for regulation of the immunologic balance in mouse intestinal inflammatory response.

Activation of Epithelial Signal Transducer and Activator of Transcription 1 by Interleukin 28 Controls Mucosal Healing in Mice With Colitis and Is Increased in Mucosa of Patients With Inflammatory Bowel Disease

We investigated the roles of interleukin 28A (also called IL28A or interferon λ2) in intestinal epithelial cell (IEC) activation, studying its effects in mouse models of inflammatory bowel diseases (IBD) and intestinal mucosal healing. Colitis was induced in C57BL/6JCrl mice (controls), mice with IEC-specific disruption of Stat1 (Stat1IEC-KO), mice with disruption of the interferon λ receptor 1 gene (Il28ra-/-), and mice with disruption of the interferon regulatory factor 3 gene (Irf3-/-), with or without disruption of Irf7 (Irf7-/-). We used high-resolution mini-endoscopy and invivo imaging methods to assess colitis progression. We used 3-dimensional small intestine and colon organoids, along with RNA-Seq and gene ontology methods, to characterize the effects of IL28 on primary IECs. We studied the effects of IL28 on the human intestinal cancer cell line Caco-2 in a wound-healing assay, and in mice colon wounds. Colonic biopsies and resected tissue from patients with IBD (n= 62) and patients without colon inflammation (controls, n= 23) were analyzed by quantitative polymerase chain rection to measure expression of IL28A, IL28RA, and other related cytokines; biopsy samples were also analyzed by immunofluorescence to identify sources of IL28 production. IECs were isolated from patient tissues and incubated with IL28; signal transducer and activator of transcription 1 (STAT1) phosphorylation was measured by immunoblots and confocal imaging. Lamina propria cells in colon tissues of patients with IBD, and mice with colitis, had increased expression of IL28 compared with controls; levels of IL28R were increased in the colonic epithelium of patients with IBD and mice with colitis. Administration of IL28 induced phosphorylation of STAT1 inprimary human and mouse IECs, increasing with dose. Il28ra-/-, Irf3-/-, Irf3-/-Irf7-/-, as well as Stat1IEC-KO mice, developed more severe colitis after administration of dextran sulfate sodium than control mice, with reduced epithelial restitution. Il28ra-/- and Stat1IEC-KO mice also developed more severe colitis in response to oxazolone than control mice. We found IL28 to induce phosphorylation (activation) of STAT1 in epithelial cells, leading to their proliferation in organoid culture. Administration of IL28 to mice with induced colonic wounds promoted mucosal healing. IL28 controls proliferation of IECs in mice with colitis and accelerates mucosal healing by activating STAT1. IL28 might be developed as a therapeutic agent for patients with IBD.

Improvement of paracellular transport in the Caco-2 drug screening model using protein-engineered substrates

The Caco-2 assay has achieved wide popularity among pharmaceutical companies in the past two decades as an invitro method for estimation of invivo oral bioavailability of pharmaceutical compounds during preclinical characterization. Despite its popularity, this assay suffers from a severe underprediction of the transport of drugs which are absorbed paracellularly, that is, which pass through the cell-cell tight junctions of the absorptive cells of the small intestine. Here, we propose that simply replacing the collagen I matrix employed in the standard Caco-2 assay with an engineered matrix, we can control cell morphology and hence regulate the cell-cell junctions that dictate paracellular transport. Specifically, we use a biomimetic engineered extracellular matrix (eECM) that contains modular protein domains derived from two ECM proteins found in the small intestine, fibronectin and elastin. This eECM allows us to independently tune the density of cell-adhesive RGD ligands presented to Caco-2cells as well as the mechanical stiffness of the eECM. We observe that lower amounts of RGD ligand presentation as well as decreased matrix stiffness results in Caco-2 morphologies that more closely resemble primary small intestinal epithelial cells than Caco-2cells cultured on collagen. Additionally, these matrices result in Caco-2 monolayers with decreased recruitment of actin to the apical junctional complex and increased expression of claudin-2, a tight junction protein associated with higher paracellular permeability that is highly expressed throughout the small intestine. Consistent with these morphological differences, drugs known to be paracellularly transported invivo exhibited significantly improved transport rates in this modified Caco-2 model. As expected, permeability of transcellularly transported drugs remained unaffected. Thus, we have demonstrated a method of improving the physiological accuracy of the Caco-2 assay that could be readily adopted by pharmaceutical companies without major changes to their current testing protocols.

Functional analysis of carboxylesterase in human induced pluripotent stem cell-derived enterocytes

Human carboxylesterase (CES) is a key esterase involved in the metabolism and biotransformation of drugs. Hydrolysis activity in the human small intestine is predominantly mediated by CES2A1 rather than CES1A. In drug development studies, Caco-2 cells are commonly used as a model to predict drug absorption in the human small intestine. However, the expression patterns of CES2A1 and CES1A in Caco-2 cells differ from those in the human small intestine. There are also species-specific differences in CES expression patterns between human and experimental animals. Furthermore, it is difficult to obtain primary human intestinal epithelial cells. Therefore, there is currently no system that can precisely predict features of drug absorption, such as CES-mediated metabolism, in the human intestine. To develop a novel system to evaluate intestinal pharmacokinetics, we analyzed CES expression and function in human induced pluripotent stem (iPS) cell-derived enterocytes. CES2A1 mRNA and protein levels in human iPS cell-derived enterocytes were comparable to Caco-2 cells, whereas CES1A levels were lower in human iPS cell-derived enterocytes compared with Caco-2 cells. p-nitrophenyl acetate hydrolysis in human iPS cell-derived enterocytes was significantly inhibited by the CES2A1-specific inhibitor telmisartan. Hydrolysis levels of the CES2A1-specific substrate aspirin were similar in human iPS cell-derived enterocytes and Caco-2 cells, whereas hydrolysis of the CES1A-specific substrate monoethylglycylxylidine was observed in Caco-2 cells but not in human iPS cell-derived enterocytes. These findings demonstrated that the expression and activity of CES isozymes in human iPS cell-derived enterocytes are more similar to the human small intestine compared with Caco-2 cells.

HADHA, the alpha subunit of the mitochondrial trifunctional protein, is involved in long-chain fatty acid-induced autophagy in intestinal epithelial cells

Genome-wide association studies have identified autophagy-related susceptibility genes for inflammatory bowel disease (IBD); however, whether autophagy regulators can be utilized as therapeutic targets remains unclear. To identify novel microtubule-associated protein 1 light chain 3 (LC3)-interacting proteins in intestinal epithelial cells (IECs), we isolated primary IECs from green fluorescent protein (GFP)-LC3 mice. We performed immunoprecipitation with a GFP antibody and then analyzed co-immunoprecipitates by mass spectrometry. HADHA was identified as an LC3-interacting protein from primary IECs. The HADHA gene encodes the alpha subunit of the mitochondrial trifunctional protein. Given that HADHA catalyzes the last three steps of mitochondrial beta-oxidation of long-chain fatty acids, we investigated whether long-chain fatty acids induce autophagy in IECs. We found that palmitic acid induced autophagy in DLD-1, HT29, and HCT116 cells. HADHA was expressed in not only the mitochondria but also the cytosol. LC3 puncta co-localized with HADHA, which were enhanced by palmitic acid stimulation. However, LC3 puncta did not co-localize with Tom20, suggesting that HADHA was induced to associate with LC3 puncta at sites other than the mitochondria. Thus, HADHA may have extra-mitochondrial functions. Furthermore, we found that palmitic acid induced cell death in IECs, which was accelerated by bafilomycin A and chloroquine. These findings suggested that palmitic acid-induced autophagy supports the survival of IECs. Taken together, these results suggested that HADHA is involved in long-chain fatty acid-induced autophagy in IECs, thus providing new insights into the pathology of IBD and revealing novel therapeutic targets of IBD.

Infection of human intestinal epithelial cells by invasive bacteria activates NF-κB and increases ICAM-1 expression through NOD1.

Background/AimsNucleotide-binding oligomerization domain 1 (NOD1) is required for primary intestinal epithelial cells (IECs) to respond to natural mucopeptides secreted by gram-negative bacteria. Infection of human IECs with invasive bacteria up-regulates intercellular adhesion molecule-1 (ICAM-1) expression. However, the role of NOD family members in host defense has been largely unknown. The aim of this study was to determine whether there is a functional role for NOD1 in the up-regulation of ICAM-1 expression in invasive bacteria-infected IECs.MethodsICAM-1 mRNA expression was compared between controls, Caco-2 or HT29 cells transfected with an empty vector, and IECs stably transfected with a dominant-negative (DN) NOD1. Expression was compared using qualitative reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and flow cytometry after infection with enteroinvasive Escherichia coli O29:NM or Shigella flexneri. Nuclear factor kB (NF-κB) activation was determined by electrophoretic mobility shift assays.ResultsDN NOD1 significantly inhibited the up-regulation of ICAM-1 expression in response to an enteroinvasive bacterial infection. The Caco-2 cells transfected with DN NOD1 manifested marked inhibition of NF-kB activation in response to E. coli O29:NM infection.ConclusionsSignaling through NOD1 may play an essential role in neutrophil trafficking following infection with enteroinvasive bacteria.

Intestinal Epithelial Cell Regulation of Adaptive Immune Dysfunction in Human Type 1 Diabetes.

Environmental factors contribute to the initiation, progression, and maintenance of type 1 diabetes (T1D), although a single environmental trigger for disease has not been identified. Studies have documented the contribution of immunity within the gastrointestinal tract (GI) to the expression of autoimmunity at distal sites. Intestinal epithelial cells (IECs) regulate local and systemic immunologic homeostasis through physical and biochemical interactions with innate and adaptive immune populations. We hypothesize that a loss in the tolerance-inducing nature of the GI tract occurs within T1D and is due to altered IECs’ innate immune function. As a first step in addressing this hypothesis, we contrasted the global immune microenvironment within the GI tract of individuals with T1D as well as evaluated the IEC-specific effects on adaptive immune cell phenotypes. The soluble and cellular immune microenvironment within the duodenum, the soluble mediator profile of primary IECs derived from the same duodenal tissues, and the effect of the primary IECs’ soluble mediator profile on T-cell expansion and polarization were evaluated. Higher levels of IL-17C and beta-defensin 2 (BD-2) mRNA in the T1D-duodenum were observed. Higher frequencies of type 1 innate lymphoid cells (ILC1) and CD8+CXCR3+ T-cells (Tc1) were also observed in T1D-duodenal tissues, concomitant with lower frequencies of type 3 ILC (ILC3) and CD8+CCR6+ T-cells (Tc17). Higher levels of proinflammatory mediators (IL-17C and BD-2) in the absence of similar changes in mediators associated with homeostasis (interleukin 10 and thymic stromal lymphopoietin) were also observed in T1D-derived primary IEC cultures. T1D-derived IEC culture supernatants induced more robust CD8+ T-cell proliferation along with enhanced polarization of Tc1 populations, at the expense of Tc17 polarization, as well as the expansion of CXCR3+CCR6+/− Tregs, indicative of a Th1-like and less regulatory phenotype. These data demonstrate a proinflammatory microenvironment of the T1D-duodenum, whereby IECs have the potential to contribute to the expansion and polarization of innate and adaptive immune cells. Although these data do not discern whether these observations are not simply a consequence of T1D, the data indicate that the T1D-GI tract has the capacity to foster a permissive environment under which autoreactive T-cells could be expanded and polarized.

Biomarkers Induced by the Immunomodulatory Bacterial Extract OM-85: Unique Roles for Peyer’s Patches and Intestinal Epithelial Cells

Objective: The orally administered bacterial lysate OM-85 (Broncho-Vaxom®, Broncho-Munal®, Ommunal®, Paxoral®, Vaxoral®) is known to protect against recurrent respiratory tract infections. Despite mechanistic investigations performed during clinical and pre-clinical studies, little is known regarding the initial immune response to OM-85 following its passage through the stomach. To better understand the primary steps in the OM-85 gut-mediated immune-response cascade, we investigated its effects on two candidate cell types: intestinal epithelial cells (IECs) and Peyer’s patch (PP) leukocytes and confirmed its sustained immune effect in a reconstituted gastric buffer. Methods: In order to confirm OM-85’s continued activity following gastric transit, THP-1 cells were stimulated with OM-85 following its incubation in reconstituted gastric buffer (pH 1.7, 8 pM Pepsin). The ability of OM-85 to stimulate IECs was tested by incubating epithelial-cell lines (Caco-2 and HT-29) and freshly isolated mouse IEC aggregates with OM-85 or standard pattern-recognition receptor (PRR) ligands (Pam3CSK4, LPS, flagellin, or PGN). To test the ability of OM-85 to stimulate mucosal immune cells, PP cells isolated from mouse intestine were incubated with OM-85 or PRR ligands. Results: THP-1 cells released macrophage inflammatory protein-3 alpha (MIP-3α), both when OM-85 was pre-incubated in gastric buffer and when it was left untreated. In the presence of OM-85, functional PP cells freshly isolated from the intestine solely and dose-dependently released MIP-1α, a chemokine produced by myeloid cells which is involved in the recruitment and activation of various immune effector cells. Surprisingly, neither established epithelial-cell lines nor primary IECs of human or mouse origin released any of the tested cytokines in the presence of OM-85 or standard purified toll-like receptor (TLR)/nucleotide-binding oligomerisation domain (NOD)-like receptor ligands. Conclusion: These data suggested that primary mucosal PPs, but not IECs, are activated by ligands from the bacterial lysate OM-85. The secretion of MIP-1α from PPs might be a trigger signal inducing tonic stimulation of mucosal tissues to prepare host immune defense towards invading pathogens.

Crohn's disease-associated mucosal factors regulate the expression of TNF-like cytokine 1A and its receptors in primary subepithelial intestinal myofibroblasts and intestinal epithelial cells

Intestinal subepithelial myofibroblasts (SEMFs) exert a profibrotic role in Crohn's disease (CD). Tumor necrosis factor-like cytokine 1A (TL1A) and its receptors, death-domain receptor 3 (DR3) and decoy receptor 3 (DcR3), are mucosal factors with significant involvement in experimental inflammation and CD. We aimed to determine the regulation of expression of this system of proteins in SEMFs and intestinal epithelial cells. The relative amount of mRNA transcripts for TL1A, DR3, and DcR3 was measured by real-time reverse transcription polymerase chain reaction in cultured primary SEMFs, colonic myofibroblast cell line 18CO, and epithelial cell line HT29. Protein expression was determined by immunofluorescence. The effect of various proinflammatory stimuli in mRNA and protein expression was studied. TL1A mRNA and protein expression in primary SEMFs (and 18CO cells) was significantly upregulated after stimulation with interleukin 1-alpha and/or tumor necrosis factor alpha (TNF-α) (32- to 44-fold increase, P<0.05 vs unstimulated). Following stimulation with interleukin 1-alpha + TNF-α + IFN-γ, HT-29cells highly expressed DR3 (4.1-fold over unstimulated, P=0.008) and DcR3 (56-fold, P=0.009) and secreted soluble factors that led to induction of TL1A mRNA in primary SEMFs (28-fold, P=0.008). Activated epithelial cells significantly upregulated IL-8 expression in response to stimulation with recombinant TL1A. Supernatants from mucosal cultures of patients with CD were able to stimulate theexpression of TL1A in cultured primary SEMFs, in comparison to supernatants from healthy controls (3.8-fold increase, P<0.05) or culture media alone (P<0.05). In conclusion, we found that proinflammatory cytokines are important regulators ofthe expression of TL1A in SEMFs and of its receptors in intestinal epithelial cells. Our results raise the possibility for involvement of TL1A/DR3/DR3-mediated mechanismsin epithelial-mesenchymal interactions and the development of inflammation-induced intestinal fibrosis in CD.