Impaired Epithelial Barrier Function Research Articles

Increasing Evidence That Irritable Bowel Syndrome and Functional Gastrointestinal Disorders Have a Microbial Pathogenesis.

The human gastrointestinal tract harbors most of the microbial cells inhabiting the body, collectively known as the microbiota. These microbes have several implications for the maintenance of structural integrity of the gastrointestinal mucosal barrier, immunomodulation, metabolism of nutrients, and protection against pathogens. Dysfunctions in these mechanisms are linked to a range of conditions in the gastrointestinal tract, including functional gastrointestinal disorders, ranging from irritable bowel syndrome, to functional constipation and functional diarrhea. Irritable bowel syndrome is characterized by chronic abdominal pain with changes in bowel habit in the absence of morphological changes. Despite the high prevalence of irritable bowel syndrome in the global population, the mechanisms responsible for this condition are poorly understood. Although alterations in the gastrointestinal microbiota, low-grade inflammation and immune activation have been implicated in the pathophysiology of functional gastrointestinal disorders, there is inconsistency between studies and a lack of consensus on what the exact role of the microbiota is, and how changes to it relate to these conditions. The complex interplay between host factors, such as microbial dysbiosis, immune activation, impaired epithelial barrier function and motility, and environmental factors, including diet, will be considered in this narrative review of the pathophysiology of functional gastrointestinal disorders.

A Novel Cecropin-LL37 Hybrid Peptide Protects Mice Against EHEC Infection-Mediated Changes in Gut Microbiota, Intestinal Inflammation, and Impairment of Mucosal Barrier Functions.

Intestinal inflammation can cause impaired epithelial barrier function and disrupt immune homeostasis, which increases the risks of developing many highly fatal diseases. Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes intestinal infections worldwide and is a major pathogen that induces intestinal inflammation. Various antibacterial peptides have been described as having the potential to suppress and treat pathogen-induced intestinal inflammation. Cecropin A (1–8)-LL37 (17–30) (C-L), a novel hybrid peptide designed in our laboratory that combines the active center of C with the core functional region of L, shows superior antibacterial properties and minimized cytotoxicity compared to its parental peptides. Herein, to examine whether C-L could inhibit pathogen-induced intestinal inflammation, we investigated the anti-inflammatory effects of C-L in EHEC O157:H7-infected mice. C-L treatment improved the microbiota composition and microbial community balance in mouse intestines. The hybrid peptide exhibited improved anti-inflammatory effects than did the antibiotic, enrofloxacin. Hybrid peptide treated infected mice demonstrated reduced clinical signs of inflammation, reduced weight loss, reduced expression of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interferon-gamma (IFN-γ)], reduced apoptosis, and reduced markers of jejunal epithelial barrier function. The peptide also affected the MyD88–nuclear factor κB signaling pathway, thereby modulating inflammatory responses upon EHEC stimulation. Collectively, these findings suggest that the novel hybrid peptide C-L could be developed into a new anti-inflammatory agent for use in animals or humans.

Granzyme B Contributes to Barrier Dysfunction in Oxazolone-Induced Skin Inflammation through E-Cadherin and FLG Cleavage

Atopic dermatitis (AD) is the most common inflammatory skin condition. Skin barrier dysfunction is of major importance in AD because it facilitates allergen sensitization and systemic allergic responses. Long regarded as a pro-apoptotic protease, emerging studies indicate granzyme B (GzmB) to have extracellular roles involving the proteolytic cleavage of extracellular matrix, cell adhesion proteins, and basement membrane proteins. Minimally expressed in normal skin, GzmB is elevated in AD and is positively correlated with disease severity and pruritus. We hypothesized that GzmB contributes to AD through extracellular protein cleavage. A causative role for GzmB was assessed in an oxazolone-induced murine model of dermatitis, comparing GzmB-/- mice with wild-type mice, showing significant reductions in inflammation, epidermal thickness, and lesion formation in GzmB-/- mice. Topical administration of a small-molecule GzmB inhibitor reduced disease severity compared with vehicle-treated controls. Mechanistically, GzmB impaired epithelial barrier function through E-cadherin and FLG cleavage. GzmB proteolytic activity contributes to impaired epidermal barrier function and represents a valid therapeutic target for AD.

Targeting desmosomal adhesion and signalling for intestinal barrier stabilization in inflammatory bowel diseases-Lessons from experimental models and patients.

Inflammatory bowel diseases (IBD) such as Crohn's disease (CD) and Ulcerative colitis (UC) have a complex and multifactorial pathogenesis which is incompletely understood. A typical feature closely associated with clinical symptoms is impaired intestinal epithelial barrier function. Mounting evidence suggests that desmosomes, which together with tight junctions (TJ) and adherens junctions (AJ) form the intestinal epithelial barrier, play a distinct role in IBD pathogenesis. This is based on the finding that desmoglein (Dsg) 2, a cadherin-type adhesion molecule of desmosomes, is required for maintenance of intestinal barrier properties both in vitro and in vivo, presumably via Dsg2-mediated regulation of TJ. Mice deficient for intestinal Dsg2 show increased basal permeability and are highly susceptible to experimental colitis. In several cohorts of IBD patients, intestinal protein levels of Dsg2 are reduced and desmosome ultrastructure is altered suggesting that Dsg2 is involved in IBD pathogenesis. In addition to its adhesive function, Dsg2 contributes to enterocyte cohesion and intestinal barrier function. Dsg2 is also involved in enterocyte proliferation, barrier differentiation and induction of apoptosis, in part by regulation of p38MAPK and EGFR signalling. In IBD, the function of Dsg2 appears to be compromised via p38MAPK activation, which is a critical pathway for regulation of desmosomes and is associated with keratin phosphorylation in IBD patients. In this review, the current findings on the role of Dsg2 as a novel promising target to prevent loss of intestinal barrier function in IBD patients are discussed.

Elucidating the Role of the Skin Barrier Protein Filaggrin in the Adaptive Immune Response to Commensals

Atopic dermatitis (AD), commonly known as eczema, is a chronic inflammatory skin disease affecting millions of Americans. Disruption of skin barrier physiology is central to its pathogenesis. Mutations in the gene encoding filaggrin are present in up to 40% of patients and predispose patients to severe and early‐onset AD as well as systemic atopy (asthma and allergic rhinitis). Filaggrin, expressed in the upper layers of the skin epithelium, is a protein critical to the structural integrity of the epidermis and its key physiological functions of maintaining proper skin moisturization and pH. The epidermal barrier also plays a central role in the skin’s response to bacteria. In AD, disease flares are accompanied by skin microbial dysbiosis, specifically increased abundance of Staphylococcus epidermidis (S. epi), a common skin commensal, and Staphylococcus aureus, a pathobiont. Whereas the role of S. aureus in AD is relatively well‐studied, comparatively little is understood about the impact of impaired skin epithelial barrier function (e.g. flg mutations) on the immune response to commensal bacteria and how this might contribute to skin and systemic inflammation in atopy. I hypothesize that early life deficiency of skin barrier function alters the quality of the commensal‐specific T cell response, which contributes to later pro‐inflammatory responses to these bacteria. Using a novel system allowing me to track CD4+ T cells specific for S. epi I have found that mice lacking filaggrin (flg−/−) differ from wildtype (wt) littermates in their immune response to this commensal. Our lab has previously shown that wt mice colonized early in life develop immune tolerance to S. epi via enrichment of antigen‐specific regulatory T cells (Tregs), an immune cell population that controls inflammation. In contrast, S. epi colonization of flg−/− mice elicits reduced antigen‐specific Tregs and increased numbers of antigen‐specific CD4+ effector T cells (Teffs). Ongoing studies are focused on elucidating the mechanistic basis of this key finding and determining its contribution towards propensity for skin and systemic type‐2 inflammation. In summary, our work suggests that skin barrier physiology significantly impacts the quality of the commensal‐specific CD4+ response early in life, which may have enduring consequences for skin immune homeostasis.Support or Funding InformationAmerican Physiological Society: Porter Physiology Development Fellowship

Products of Mature Adipocytes in Visceral Fat of Obese Patients Impair Esophageal Epithelial Barrier Formation and Function

ObjectiveVisceral obesity is associated with GERD and its complications of reflux esophagitis, Barrett’s esophagus and esophageal adenocarcinoma. A traditional explanation for these associations is that visceral fat causes pathological acid reflux by increasing intra‐abdominal pressure. However, for unknown reasons, the esophagus of obese patients who have normal esophageal acid exposure can exhibit evidence of impaired epithelial barrier function including dilated intercellular spaces (DIS) and reduced transepithelial resistance (TER). In this study, we explored our hypothesis that the visceral fat of obese individuals secretes substances that impair esophageal barrier function, thus predisposing to injury by GERD.MethodsWe obtained subcutaneous (SQ) and visceral (VIS) fat from 1 non‐obese (BMI 18) and 2 obese (BMIs 51& 42) patients having foregut surgery; VIS included both mesenteric fat and gastroesophageal junction (GEJ) fat pad. Fat contains both mature adipocytes (MAs) and adipocyte derived stem cells (ADSCs), which were isolated from adipocyte tissues by collagenase digestion and differential centrifugation; immunofluorescence (CD90+, CD44+, CD31−, CD45−) was used to confirm ADSC isolation. Telomerase‐immortalized normal, human esophageal squamous cells (NES‐B10T) were seeded into transwells and grown to confluence for 3 days. On day 4, the cells were raised to an air‐liquid interface (ALI) to allow for differentiation, stratification, and formation of an esophageal epithelial barrier. ALI cultures were exposed to conditioned media (CM) from MAs or ADSCs, or to control NES medium beginning at day 4. TER was measured (in Ω·cm2) on days 4, 6, and 8 using the EVOM2 Epithelial Volt/Ohm Meter (World Precision Instruments, Sarasota, FL). On day 10, ALI cultures were stained with H&E and imaged.ResultsCM from ADSCs of SQ and VIS fat from an obese patient did not reduce the TER of NES ALI cultures at any time point; CM from MAs of SQ fat from all 3 patients also did not reduce TER. In contrast, CM from MAs of VIS fat from both obese patients significantly reduced TER on day 6 compared with control medium; TER also remained lower than control on day 8 (Table 1). CM from MAs of VIS fat from the non‐obese patient did not reduce TER of NES ALI cultures at any time point (Table 1). H&E staining demonstrated DIS only in those ALI cultures grown with the CM from MAs of VIS fat from the obese patients.ConclusionsSubstances produced by MAs, but not by ADSCs, in the VIS fat of obese patients impair esophageal epithelial barrier integrity and cause morphologic changes of DIS. No such effects were found for substances produced by MAs in the SQ fat of any patient, or from those produced by MAs in VIS fat of the non‐obese patient. Moreover, VIS fat in the mesentery and at the GEJ exert similar functional and morphologic effects on esophageal barrier formation. We are conducting further investigations to identify the mechanisms underlying the impairment of esophageal barrier formation caused by visceral adiposity, specifically for the GEJ fat pad that surrounds the distal esophagus.Support or Funding InformationBaylor Scott & White Research Institute Transepithelial Resistance (Ω·cm2 ±SEM) of NES on Day 6 and Day 8 of ALI Culture Exposed to Conditioned Media from Mature Adipocytes from Subcutaneous (SQ), Mesenteric, and GEJ Adipocyte Tissues BMI Control Day 6 SQ Day 6 Mesenteric Day 6 GEJ Day 6 Control Day 8 SQ Day 8 Mesenteric Day 8 GEJ Day 8 18 241±2.1 242±2.0 234±2.0 235±2.5 245±3.8 256±2.2 243±4.2 248±7.2 51 212±2.0 204±3.5 194±3.0 * 192±0.5 ** 235±8.5 233±1.0 209±6.5 207±8.0 42 264±3.6 263±5.5 236±2.3 ** 239±3.3 ** 251±2.4 296±45 249±0.9 237±2.6 * *p≤0.05; **p≤0.01 compared with control

Cis 9, trans 11, but not trans 10, cis 12 CLA isomer, impairs intestinal epithelial barrier function in IPEC-J2 cells and mice through activation of GPR120-[Ca2+]i and the MLCK signaling pathway.

This study aimed to investigate the effects of conjugated linoleic acid (CLA) on intestinal epithelial barrier function and explore the underlying mechanisms. IPEC-J2 cells and mice were treated with different CLA isomers. The intestinal epithelial barrier function determined by transepithelial electrical resistance (TEER), the expression of tight junction proteins, and the involvement of G-protein coupled receptor 120 (GPR120), intracellular calcium ([Ca2+]i) and myosin light chain kinase (MLCK) were assessed. In vitro, c9, t11-CLA, but not t10, c12-CLA isomer, impaired epithelial barrier function in IPEC-J2 by downregulating the expression of tight junction proteins. Meanwhile, c9, t11-CLA isomer enhanced GPR120 expression, while knockdown of GPR120 eliminated the impaired epithelial barrier function induced by c9, t11-CLA isomer. In addition, c9, t11-CLA isomer increased [Ca2+]i and activated the MLCK signaling pathway in a GPR120-dependent manner. However, chelation of [Ca2+]i reversed c9, t11-CLA isomer-induced MLCK activation and the epithelial barrier function impairment of IPEC-J2. Furthermore, inhibition of MLCK totally abolished the impairment of epithelial barrier function induced by c9, t11-CLA. In vivo, dietary supplementation of c9, t11-CLA rather than t10, c12-CLA isomer decreased the expression of intestinal tight junction proteins and GPR120, increased intestinal permeability, and activated the MLCK signaling pathway in mice. Taken together, our findings showed that c9, t11-CLA, but not t10, c12-CLA isomer, impaired intestinal epithelial barrier function in IPEC-J2 cells and mice through activation of GPR120-[Ca2+]i and the MLCK signaling pathway. These data provided new insight into the regulation of the intestinal epithelial barrier by different CLA isomers and more references for CLA application in humans and animals.

Transcriptomic and barrier responses of human airway epithelial cells exposed to cannabis smoke.

Globally, many jurisdictions are legalizing or decriminalizing cannabis, creating a potential public health issue that would benefit from experimental evidence to inform policy, government regulations, and user practices. Tobacco smoke exposure science has created a body of knowledge that demonstrates the conclusive negative impacts on respiratory health; similar knowledge remains to be established for cannabis. To address this unmet need, we performed in vitro functional and transcriptomic experiments with a human airway epithelial cell line (Calu‐3) exposed to cannabis smoke, with tobacco smoke as a positive control. Demonstrating the validity of our in vitro model, tobacco smoke induced gene expression profiles that were significantly correlated with gene expression profiles from published tobacco exposure datasets from bronchial brushings and primary human airway epithelial cell cultures. Applying our model to cannabis smoke, we demonstrate that cannabis smoke induced functional and transcriptional responses that overlapped with tobacco smoke. Ontology and pathway analysis revealed that cannabis smoke induced DNA replication and oxidative stress responses. Functionally, cannabis smoke impaired epithelial cell barrier function, antiviral responses, and increased inflammatory mediator production. Our study reveals striking similarities between cannabis and tobacco smoke exposure on impairing barrier function, suppressing antiviral pathways, potentiating of pro‐inflammatory mediators, and inducing oncogenic and oxidative stress gene expression signatures. Collectively our data suggest that cannabis smoke exposure is not innocuous and may possess many of the deleterious properties of tobacco smoke, warranting additional studies to support public policy, government regulations, and user practices.

Glutamine Blocks Interleukin-13-Induced Intestinal Epithelial Barrier Dysfunction

Introduction: Impaired intestinal epithelial barrier function is a hallmark of a variety of pathological conditions such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). IBD patients with IBS-like symptoms show higher interleukin-13 (IL-13) serum levels and poor psychological well-being. Supplementary glutamine reduced the daily bowel movement frequency, improved the stool form, and normalized intestinal hyperpermeability. This study was aimed at assessing the effects of IL-13 and supplementary glutamine on human intestinal epithelial function in vitro. Methods: Caco-2 cells were grown on Transwell^TM inserts. ­IL-13 was added to the basolateral compartment, and transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC) labeled-dextran permeability measured. Effects of glutamine or the phosphatidylinositol-3-kinase inhibitor LY294002 were assessed. Involvement of tight junction proteins was assessed using Western blotting and immunofluorescence staining. Results: IL-13 significantly decreased TEER and increased FITC labeled-dextran epithelial permeability. IL-13 stimulation decreased the claudin-1 expression and increased the claudin-2 expression. Glutamine alleviated IL-13-induced decrease of TEER and increase of FITC labeled-dextran permeability. Further, the phosphatidylinositol-3-kinase inhibitor showed this alleviating effect while the signal transducer and activator of transcription 6 inhibitor did not. Conclusions: IL-13 induced barrier integrity impairment by decreasing claudin-1 and increasing claudin-2. Glutamine alleviated IL-13-induced barrier dysfunction by increasing claudin-1 expression, via disruption of the phosphatidylinositol-3-kinase/Akt signaling pathway.

Direct assessment of skin epithelial barrier by electrical impedance spectroscopy.

Many skin and mucosal inflammatory disorders, such as atopic dermatitis, have been associated with an impaired epithelial barrier function, which allows allergens, pollutants, or microbes to enter the tissue and activate the immune response. The aim of this study was to establish a method to directly assess in vivo the epidermal barrier function by electrical impedance (EI) spectroscopy. Mice epidermal barrier was damaged by epicutaneous application of proteases and cholera toxin and by tape stripping. EI and transepidermal water loss (TEWL) were measured before and after the application. Additionally, histological analysis, immunofluorescence staining, and RT-PCR were performed on skin biopsies to evaluate the epithelial barrier. A few hours after papain application, a dose-dependent reduction of EI was detected, reflecting the decreased barrier function. At the same time, an increase of TEWL was observed, with a significant negative correlation with EI, demonstrating that EI changes were directly linked to barrier defects. Twenty-four and 48hours after the treatment, EI starts to increase to background levels, indicating tissue healing and restoration of skin barrier. Barrier disruption was confirmed by histological analysis showing an impaired stratum corneum and higher cellular infiltration after papain application. In addition, immunofluorescence staining and RT-PCR showed downregulation of molecules involved in the barrier function, such as filaggrin, occludin, and claudin-1, and mRNA levels of filaggrin, loricrin, and involucrin. Comparable results were observed after tape stripping and cholera toxin treatment. Electrical impedance spectroscopy is a rapid and reliable diagnostic tool to detect skin barrier defects.

Neurotrophic factor GDNF regulates intestinal barrier function in inflammatory bowel disease.

Impaired intestinal epithelial barrier (IEB) function with loss of desmosomal junctional protein desmoglein 2 (DSG2) is a hallmark in the pathogenesis of inflammatory bowel disease (IBD). While previous studies have reported that glial cell line-derived neurotrophic factor (GDNF) promotes IEB function, the mechanisms are poorly understood. We hypothesized that GDNF is involved in the loss of DSG2, resulting in impaired IEB function as seen in IBD. In the inflamed intestine of patients with IBD, there was a decrease in GDNF concentrations accompanied by a loss of DSG2, changes of the intermediate filament system, and increased phosphorylation of p38 MAPK and cytokeratins. DSG2-deficient and RET-deficient Caco2 cells revealed that GDNF specifically recruits DSG2 to the cell borders, resulting in increased DSG2-mediated intercellular adhesion via the RET receptor. Challenge of Caco2 cells and enteroids with proinflammatory cytokines as well as dextran sulfate sodium-induced (DSS-induced) colitis in C57Bl/6 mice led to impaired IEB function with reduced DSG2 mediated by p38 MAPK-dependent phosphorylation of cytokeratins. GDNF blocked all inflammation-induced changes in the IEB. GDNF attenuates inflammation-induced impairment of IEB function caused by the loss of DSG2 through p38 MAPK-dependent phosphorylation of cytokeratin. The reduced GDNF in patients with IBD indicates a disease-relevant contribution to the development of IEB dysfunction.

Navy bean supplemented high-fat diet improves intestinal health, epithelial barrier integrity and critical aspects of the obese inflammatory phenotype

Obesity is associated with impaired intestinal epithelial barrier function and an altered microbiota community structure, which contribute to host systemic inflammation and metabolic dysfunction. Fiber-rich common beans (Phaseolus vulgaris) promote intestinal health (microbiota and host epithelial barrier integrity) in lean mice. The objective was to assess the intestinal health promoting effects of navy bean supplementation during high-fat (HF)diet-induced obesity. Male C57BL/6 mice were fed either a high-fat (HF) diet (60% of kcal from fat) or an isocaloric HF diet supplemented with 15.7% (by weight) cooked navy bean powder (HF+B) for 12 weeks. Compared to HF, the HF+B diet altered the fecal microbiota community structure (16S rRNA gene sequencing), most notably increasing abundance of Akkermansia muciniphila (+19-fold), whose abundance typically decreases in obese humans and rodents. Additionally, HF+B fecal abundance of carbohydrate fermenting, short chain fatty acid (SCFA) producing Prevotella (+332-fold) and S24–7 (+1.6-fold) and fecal SCFA levels were increased. HF+B improved intestinal health and epithelial barrier integrity versus HF, evidenced by reduced serum fluorescein isothiocyanate (FITC)-dextran concentration in an in vivo gut permeability test, and increased intestinal mRNA expression of tight junction components (ZO-1, occludin), anti-microbial defenses (Reg3γ, IgA, Defα5, Defβ2) and mucins (Muc2). Additionally, HF+B improved the systemic obese phenotype via reduced serum HOMA-IR and leptin:adiponectin ratio, and locally via attenuation of epididymal adipose tissue crown-like structure formation, adipocyte size, and inflammatory transcription factor (NFκBp65 and STAT3) activation. Therefore, navy bean supplementation improved obese intestinal health (microbiota and epithelial barrier integrity) and attenuated the severity of the obese phenotype.

Absence of conventional dendritic cells type 1 (cDC1) impairs intestinal homeostasis and permeability and leads to the development of obesity

Abstract The maintenance of intestinal homeostasis depends on a complex interaction between microbiota, intestinal epithelial barrier and immune system. Alteration in of one of these components could lead to the development of chronic inflammatory diseases such as inflammatory bowel diseases (IBD). Recent studies have associated obesity with more severe IBD. In obesity, pro-inflammatory macrophages accumulate in adipose tissue and their numbers correlate with inflammation and insulin resistance. BATF3 is a transcription factor implicated in the development of conventional dendritic cells type 1 (cDC1). Here, we show that aged Batf3−/− mice developed obesity as characterized by increased body weight, fasting glucose levels (Batf3−/−: 213, WT: 115 mg/dl, p < 0.005), size of abdominal white adipocyte, and development of hepatosteatosis. We also observed increased intestinal permeability in Batf3−/− mice (FITC-dextran gavage; p < 0.01) suggesting impaired epithelial barrier function. Feeding high-fat diet for 2 months further enhanced the metabolic phenotype in Batf3−/− mice, leading to increased fasting glucose levels (300 vs. 246 mg/dl, p < 0.05), and increased adipocyte size. Moreover, during high-fat diet Batf3−/− mice were more susceptible to dextran sulfate sodium (DSS) colitis with more severe cecal inflammation, increased gut and mesenteric fat M1 macrophages (p < 0.05). Our data suggest that deficiency of the transcription factor BATF3 and lack of cDC1 alters the intestinal epithelial barrier, promotes the development of obesity, and increases susceptibility to DSS. In conclusion, BATF3-dependent cDC1 play an important role in maintaining the intestinal epithelial barrier and are protective for the development of obesity.

Female mice carrying a defective Alox15 gene are protected from experimental colitis via sustained maintenance of the intestinal epithelial barrier function

Lipoxygenases (ALOXs) are involved in the regulation of cellular redox homeostasis. They also have been implicated in the biosynthesis of pro- and anti-inflammatory lipid mediators and play a role in the pathogenesis of inflammatory diseases, which constitute a major health challenge owing to increasing incidence and prevalence in all industrialized countries around the world. To explore the pathophysiological role of Alox15 (leukocyte-type 12-LOX) in mouse experimental colitis we tested the impact of systemic inactivation of the Alox15 gene on the extent of dextrane sulfate sodium (DSS) colitis. We found that in wildtype mice expression of the Alox15 gene was augmented during DSS-colitis while expression of other Alox genes (Alox5, Alox15b) was hardly altered. Systemic Alox15 (leukocyte-type 12-LOX) deficiency induced less severe colitis symptoms and suppressed in vivo formation of 12-hydroxyeicosatetraenoic acid (12-HETE), the major Alox15 (leukocyte-type 12-LOX) product in mice. These alterations were paralleled by reduced expression of pro-inflammatory gene products, by sustained expression of the zonula occludens protein 1 (ZO-1) and by a less impaired intestinal epithelial barrier function. These results are consistent with in vitro incubations of colon epithelial cells, in which addition of 12S-HETE compromised enantioselectively transepithelial electric resistance. Consistent with these data transgenic overexpression of human ALOX15 intensified the inflammatory symptoms. In summary, our results indicate that systemic Alox15 (leukocyte-type 12-LOX) deficiency protects mice from DSS-colitis. Since exogenous 12-HETE compromises the expression of the tight junction protein ZO-1 the protective effect has been related to a less pronounced impairment of the intestinal epithelial barrier function.

Pathophysiology of IBD associated diarrhea

ABSTRACTInflammatory bowel diseases broadly categorized into Crohn's disease (CD) and ulcerative colitis (UC), are chronic inflammatory disorders of the gastrointestinal tract with increasing prevalence worldwide. The etiology of the disease is complex and involves a combination of genetic, environmental, immunological and gut microbial factors. Recurring and bloody diarrhea is the most prevalent and debilitating symptom in IBD. The pathogenesis of IBD-associated diarrhea is multifactorial and is essentially an outcome of mucosal damage caused by persistent inflammation resulting in dysregulated intestinal ion transport, impaired epithelial barrier function and increased accessibility of the pathogens to the intestinal mucosa. Altered expression and/or function of epithelial ion transporters and channels is the principle cause of electrolyte retention and water accumulation in the intestinal lumen leading to diarrhea in IBD. Aberrant barrier function further contributes to diarrhea via leak-flux mechanism. Mucosal penetration of enteric pathogens promotes dysbiosis and exacerbates the underlying immune system further perpetuating IBD associated-tissue damage and diarrhea. Here, we review the mechanisms of impaired ion transport and loss of epithelial barrier function contributing to diarrhea associated with IBD.

Cryptosporidium parvumdisrupts intestinal epithelial barrier function via altering expression of key tight junction and adherens junction proteins

Infection with the protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a widespread diarrhoeal disease. Impaired intestinal epithelial barrier function and increased permeability are most commonly associated with diarrhoeal diseases caused by enteric infections. However, studies on barrier disruption and underlying mechanisms in cryptosporidiosis are extremely limited. Epithelial tight junctions (TJs) and adherens junctions (AJs) are important in maintaining barrier integrity. Therefore, we examined the effects of CP infection on paracellular permeability and on the expression of the major TJ and AJ proteins utilising in vitro, ex vivo, and in vivo models. CP infection (0.5×106 oocysts/well in Transwell inserts, 24hr) increased paracellular permeability (FITC-dextran flux) in Caco-2 cell monolayers and substantially decreased the protein levels of occludin, claudin 4, and E-cadherin. Claudin 3, zonula occludens-1 (ZO1) and α-catenin were also significantly decreased, whereas claudins 1 and 2 and β-catenin were not altered. Substantial downregulation of occludin, claudin 4, and E-cadherin was also observed in response to CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mocosa of C57BL/6 mice. The mRNA levels of these proteins were also significantly decreased in CP-infected mouse ileum and jejunum but were unaltered in Caco-2 cells. Further, bafilomycin-A, an inhibitor of lysosomal proton pump, partially abrogated CP effects on occludin expression in Caco-2 cells, suggesting a potential role of posttranslational mechanisms, such as induction of protein degradation pathways, in mediating the effects of the parasite. Our studies suggest that disruption of barrier function via downregulation of specific key components of TJ and AJ could be a major mechanism underlying CP infection-induced diarrhoea.

Blend of organic acids and medium chain fatty acids prevents the inflammatory response and intestinal barrier dysfunction in mice challenged with enterohemorrhagic Escherichia coli O157:H7

Impaired epithelial barrier function disrupts immune homeostasis and increases inflammation in intestines, leading to many intestinal diseases. The blend of organic acids (OAs) and medium chain fatty acids (MCFAs) has been shown to have synergistic bactericidal effect. In this study, we demonstrated that two blends of OAs and MCFAs (OM1 and OM2) could prevent the inflammatory response and intestinal barrier dysfunction in enterohemorrhagic Escherichia coli (EHEC)-infected mice. Treatments of OM1 and OM2 significantly reduced the body weight loss and production of IL-6 and TNF-α induced by EHEC. Mice treated with OM1 and OM2 showed decrease in serum D-lactic concentration, DAO activity and bacterial transfer to liver and spleen. Furthermore, OM1 and OM2 increased the expression of tight junction proteins occludin and ZO-1, mucus protein MUC-2, and host defense peptides mBD1, mBD2 and mBD3. Finally, OM1 and OM2 increased the population of Lactobacillus spp. and Bifidobacterium spp., but decreased that of E. coli in the cecum. These findings indicate that OM1 and OM2 may be used to develop a prophylactic agent for intestinal inflammation and injury in enteric pathogen infection.

Mechanistic Understanding of Herbal Therapy in Inflammatory Bowel Disease.

The incidence and prevalence of inflammatory bowel diseases (IBD), which comprise ulcerative colitis and Crohn's disease, are increasing dramatically worldwide. Immunomodulators and biological agents can help but cause severe side effects in long-term use. As such, complementary and alternative medicine, in particular herbal remedy, is becoming more and more popular in the treatment of IBD patients. Many natural compounds have been used in clinical trials and some have been proven promising in IBD treatment. To achieve a better understanding of herbal therapy, researchers focus on understanding the underlying mechanisms by using experimental rodent models. The mechanism of the pathogenesis of IBD is complex involving both environmental and genetic factors. IBD is considered as a consequence of impaired epithelial barrier function, gut microbiota dysbiosis, and aberrant immune response. Studies have demonstrated that herbal medicine can improve epithelial proliferation and barrier integrity, restore microbiota homeostasis, and suppress hyper-immune reaction. This review is to summarize current understanding of the molecular basis of herbal treatment of IBD at the levels of epithelial, microbial, and immune regulation.

High mobility group box1 contributes to hypoxia-induced barrier dysfunction of nasal epithelial cells

Objective:To investigated the promotion of high mobility group box1 (HMGB1) under hypoxia, and determined the regulatory role of HMGB1 on the barrier function of nasal epithelial cells.Method:Primary nasal epithelial cells (NECs) collected from patients with septal deviation were cultured at air-liquid interface. The release of HMGB1 under hypoxia was detected by ELISA. The effect of HMGB1 on fluorescein isothiocyanatedextran 4 kDa (FD4) permeability of NECs was measured. Western blot analysis was utilized to examine the level of major junction proteins, namely E-cadherin, ZO-1, Occludin and Claudin-1.Result:The release of HMGB1 was significantly upregulated in NECs under hypoxia. Recombinant human HMGB1 increased FD4 permeability in a dose and time-dependent manner, indicating the impaired epithelial barrier function. HMGB1-mediated barrier hyperpermeability was accompanied by the selective downregulation of ZO-1, occludin and Claudin-1, but not E-cadherin.Conclusion:HMGB1 mediates hypoxia-induce barrier dysfunction of nasal epithelium, which may be a potential target for the treatment of chronic rhinosinusitis.

Abstract 4401: Role of estrogen signaling in colon microenvironment during obesity

Abstract Recent epidemiological studies highlight the strong association between high body mass index (BMI) and enhanced risk of colon cancer. This association appears stronger in men than in women, indicating a possible protective role of estrogens in this context. The biological action of the estrogens is mediated by specific transcription factors, the estrogen receptors (ERs). ERβ is the main ER in the intestine; and it is expressed in both epithelial cells and immune cells. Anti-inflammatory and anti-tumorigenic effects of ERβ in colitis-associated neoplasia have been demonstrated. Several rodent models for obesity show an impaired intestinal epithelial barrier function and an increase of pro-inflammatory markers in the colon. Moreover, in murine colitis models, high fat diet (HFD) exacerbates colonic inflammation. However, the specific molecular mechanisms linking obesity to increased colon cancer development, where estrogen signaling may be involved, is not clear. We hypothesize that ERβ-selective agonists can oppose the HFD-mediated inflammatory pro-tumorigenic colonic signalling in a gender-specific manner during obesity, and thereby protect against colorectal cancer. Our study is focused on the initiation of colon inflammation during obesity and aims to investigate the impact of ERβ activation in this context. To address this question, male and female mice were fed a HFD for 26 weeks and treated with a ERβ-selective agonist for 3 weeks prior sacrifice. Stool pellets were regularly collected. Animals were sacrificed; colon samples and caecal contents were collected for further analysis. We show that the expression pattern of inflammatory genes in colon is modified by the ERβ-selective agonist in HFD fed mice. Moreover the ERβ-selective agonist appears to induce gender-specific changes of the microbiota of HFD-fed mice. Our results show that an ERβ-selective agonist can modulate the colon microenvironment during obesity in mice and will provide a better comprehension of the molecular mechanisms leading to obesity-associated colonic inflammation. Citation Format: Amena Archer, Linnea Pettersson, Marcela González-Granillo, Christina Savva, Marion Korach-André, Cecilia Williams. Role of estrogen signaling in colon microenvironment during obesity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4401. doi:10.1158/1538-7445.AM2017-4401