Cystic Fibrosis Intestine Research Articles

Intestinal Inflammation and Alterations in the Gut Microbiota in Cystic Fibrosis: A Review of the Current Evidence, Pathophysiology and Future Directions.

Cystic fibrosis (CF) is a life-limiting autosomal recessive multisystem disease. While its burden of morbidity and mortality is classically associated with pulmonary disease, CF also profoundly affects the gastrointestinal (GI) tract. Chronic low-grade inflammation and alterations to the gut microbiota are hallmarks of the CF intestine. The etiology of these manifestations is likely multifactorial, resulting from cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction, a high-fat CF diet, and the use of antibiotics. There may also be a bidirectional pathophysiological link between intestinal inflammation and changes to the gut microbiome. Additionally, a growing body of evidence suggests that these GI manifestations may have significant clinical associations with growth and nutrition, quality of life, and respiratory function in CF. As such, the potential utility of GI therapies and long-term GI outcomes are areas of interest in CF. Further research involving microbial modulation and multi-omics techniques may reveal novel insights. This article provides an overview of the current evidence, pathophysiology, and future research and therapeutic considerations pertaining to intestinal inflammation and alterations in the gut microbiota in CF.

Goblet cell hyperplasia is not epithelial-autonomous in the Cftr knockout intestine.

Goblet cell hyperplasia is an important manifestation of cystic fibrosis (CF) disease in epithelial-lined organs. Explants of CF airway epithelium show normalization of goblet cell numbers; therefore, we hypothesized that small intestinal enteroids from Cftr knockout (KO) mice would not exhibit goblet cell hyperplasia. Toll-like receptors 2 and 4 (Tlr2 and Tlr4) were investigated as markers of inflammation and influence on goblet cell differentiation. Ex vivo studies found goblet cell hyperplasia in Cftr KO jejunum compared with wild-type (WT) mice. IL-13, SAM pointed domain-containing ETS transcription factor (Spdef), Tlr2, and Tlr4 protein expression were increased in Cftr KO intestine relative to WT. In contrast, WT and Cftr KO enteroids did not exhibit differences in basal or IL-13-stimulated goblet cell numbers, or differences in expression of Tlr2, Tlr4, and Spdef. Ileal goblet cell numbers in Cftr KO/Tlr4 KO and Cftr KO/Tlr2 KO mice were not different from Cftr KO mice, but enumeration was confounded by altered mucosal morphology. Treatment with Tlr4 agonist LPS did not affect goblet cell numbers in WT or Cftr KO enteroids, whereas the Tlr2 agonist Pam3Csk4 stimulated goblet cell hyperplasia in both genotypes. Pam3Csk4 stimulation of goblet cell numbers was associated with suppression of Notch1 and Neurog3 expression and upregulated determinants of goblet cell differentiation. We conclude that goblet cell hyperplasia and inflammation of the Cftr KO small intestine are not exhibited by enteroids, indicating that this manifestation of CF intestinal disease is not epithelial-automatous but secondary to the altered CF intestinal environment.NEW & NOTEWORTHY Studies of small intestinal organoids from cystic fibrosis (CF) mice show that goblet cell hyperplasia and increased Toll-like receptor 2/4 expression are not primary manifestations of the CF intestine. Intestinal goblet cell hyperplasia in the CF mice was not strongly altered by genetic ablation of Tlr2 and Tlr 4, but could be induced in both wild-type and CF intestinal organoids by a Tlr2-dependent suppression of Notch signaling.

EVALUATING THE ROLE OF GOBLET CELL ASSOCIATED ANTIGEN PASSAGES (GAPS) IN THE DEVELOPMENT OF MUCOSAL IMMUNE TOLERANCE IN THE CFTR KO INTESTINE

Greater than 70,000 individuals worldwide are living with the monogenetic disease cystic fibrosis (CF). The development of chronic intestinal inflammation, with clinical signs resembling inflammatory bowel disease-like conditions, is a common yet poorly understood occurrence in CF patients. This inflammation is typically neutrophilic in human and animal models with a heightened basal pro-inflammatory cytokine release. Prior research utilizing intestinal organoids (enteroids) cultured from Cftr knockout mice has shown that goblet cells in the CF mouse intestine demonstrate defective clearance of mucin granules and abnormal mucus retention. Goblet cell-associated antigen passages (GAPs), located in the small intestine and colon, deliver intraluminal antigens to antigen-presenting dendritic cells in the submucosa. This mechanism serves as an important step in the development and maintenance of tolerogenic dendritic cell populations expressing receptors to luminal antigens with involvement of regulatory T cell activation and release of IL-10. We hypothesized that mucus plugging of goblet cells in the CF intestine leads to defective GAP formation and a consequent decrease in the expansion of tolerogenic dendritic cells. To test this hypothesis, Cftrtm1Unc (Cftr KO) and wild type (WT) sex-matched littermate pairs (n=2) maintained on a commercially available liquid diet (Peptamen®) were anesthetized with ketamine/xylazine for a laparotomy to inject a luminal fluorescent 10kD dextran dye into the mid-jejunum. After 30 min, the mice were euthanized with CO2, and the intestine was collected for immunofluorescent staining to evaluate GAP formation. In the WT intestine, the dextran dye was observed within goblet cells outlined by CK18 immunofluorescence, a goblet cell marker. Punctate dextran dye was observed in the submucosa, suggestive of dendritic cell uptake. In contrast, the Cftr KO mice demonstrated defective GAP formation, i.e., without dye penetration of goblet cells, and the lack of punctate dextran fluorescence in the submucosa. To evaluate the population of tolerogenic dendritic cells, small intestinal segments from Cftr KO-WT sex-matched littermate pairs (3-female and 2-male pairs) were collected for FACS sorting of submucosal CD103+ (tolerogenic) and CD103- (pro-inflammatory) dendritic cells. The WT mice had a significantly higher population of CD103+ tolerogenic dendritic cells compared to the CF mice (WT: 20.5+/-2, CF: 9.2+/-3, P < 0.006). A trend towards an increase in CD103- dendritic cells was seen in the CF intestine. In summary, the CF mice were found to have defective intraluminal antigen transfer through the GAP pathway and a significant decrease in tolerogenic dendritic cells in the intestine.

EVALUATING THE ROLE OF GOBLET CELL ASSOCIATED ANTIGEN PASSAGES (GAPS) IN THE DEVELOPMENT OF MUCOSAL IMMUNE TOLERANCE IN THE CFTR KO INTESTINE

Abstract Greater than 70,000 individuals worldwide are living with the monogenetic disease cystic fibrosis (CF). The development of chronic intestinal inflammation, with clinical signs resembling inflammatory bowel disease-like conditions, is a common yet poorly understood occurrence in CF patients. This inflammation is typically neutrophilic in human and animal models with a heightened basal pro-inflammatory cytokine release. Prior research utilizing intestinal organoids (enteroids) cultured from Cftr knockout mice has shown that goblet cells in the CF mouse intestine demonstrate defective clearance of mucin granules and abnormal mucus retention. Goblet cell-associated antigen passages (GAPs), located in the small intestine and colon, deliver intraluminal antigens to antigen-presenting dendritic cells in the submucosa. This mechanism serves as an important step in the development and maintenance of tolerogenic dendritic cell populations expressing receptors to luminal antigens with involvement of regulatory T cell activation and release of IL-10. We hypothesized that mucus plugging of goblet cells in the CF intestine leads to defective GAP formation and a consequent decrease in the expansion of tolerogenic dendritic cells. To test this hypothesis, Cftrtm1Unc (Cftr KO) and wild type (WT) sex-matched littermate pairs (n=2) maintained on a commercially available liquid diet (Peptamen®) were anesthetized with ketamine/xylazine for a laparotomy to inject a luminal fluorescent 10kD dextran dye into the mid-jejunum. After 30 min, the mice were euthanized with CO2, and the intestine was collected for immunofluorescent staining to evaluate GAP formation. In the WT intestine, the dextran dye was observed within goblet cells outlined by CK18 immunofluorescence, a goblet cell marker. Punctate dextran dye was observed in the submucosa, suggestive of dendritic cell uptake. In contrast, the Cftr KO mice demonstrated defective GAP formation, i.e., without dye penetration of goblet cells, and the lack of punctate dextran fluorescence in the submucosa. To evaluate the population of tolerogenic dendritic cells, small intestinal segments from Cftr KO-WT sex-matched littermate pairs (3-female and 2-male pairs) were collected for FACS sorting of submucosal CD103+ (tolerogenic) and CD103- (pro-inflammatory) dendritic cells. The WT mice had a significantly higher population of CD103+ tolerogenic dendritic cells compared to the CF mice (WT: 20.5+/-2, CF: 9.2+/-3, P &amp;lt; 0.006). A trend towards an increase in CD103- dendritic cells was seen in the CF intestine. In summary, the CF mice were found to have defective intraluminal antigen transfer through the GAP pathway and a significant decrease in tolerogenic dendritic cells in the intestine.

Cftr Modulates Wnt/β-Catenin Signaling and Stem Cell Proliferation in Murine Intestine.

Background & AimsCystic fibrosis (CF) patients and CF mouse models have increased risk for gastrointestinal tumors. CF mice show augmented intestinal proliferation of unknown etiology and an altered intestinal environment. We examined the role of the cystic fibrosis transmembrane conductance regulator (Cftr) in Wnt/β-catenin signaling, stem cell proliferation, and its functional expression in the active intestinal stem cell (ISC) population. Dysregulation of intracellular pH (pHi) in CF ISCs was investigated for facilitation of Wnt/β-catenin signaling.MethodsCrypt epithelia from wild-type (WT) and CF mice were compared ex vivo and in intestinal organoids (enteroids) for proliferation and Wnt/β-catenin signaling by standard assays. Cftr in ISCs was assessed by immunoblot of sorted Sox9enhanced green fluorescent protein(EGFP) intestinal epithelia and pHi regulation by confocal microfluorimetry of leucine-rich G-protein–coupled receptor 5 ISCs. Plasma membrane association of the Wnt transducer Dishevelled 2 (Dvl2) was assessed by fluorescence imaging of live enteroids from WT and CF mice crossed with Dvl2-EGFP/ACTB-tdTomato,-EGFP)Luo/J (RosamT/mG) mice.ResultsRelative to WT, CF intestinal crypts showed an ∼30% increase in epithelial and Lgr5+ ISC proliferation and increased Wnt/β-catenin signaling. Cftr was expressed in Sox9EGFPLo ISCs and loss of Cftr induced an alkaline pHi in ISCs. CF crypt-base columnar cells showed a generalized increase in plasma membrane Dvl2-EGFP association as compared with WT. Dvl2-EGFP membrane association was charge- and pH-dependent and increased in WT crypt-base columnar cells by Cftr inhibition.ConclusionsCF intestine shows increased ISC proliferation and Wnt/β-catenin signaling. Loss of Cftr increases pHi in ISCs, which stabilizes the plasma membrane association of the Wnt transducer Dvl, likely facilitating Wnt/β-catenin signaling. Absence of Cftr-dependent suppression of ISC proliferation in the CF intestine may contribute to increased risk for intestinal tumors.

Bacterial overgrowth, dysbiosis, inflammation, and dysmotility in the Cystic Fibrosis intestine

Gastrointestinal disease in Cystic Fibrosis (CF) is caused by defective chloride and bicarbonate transport in intestinal cells leading to reduced intraluminal fluidity, increased mucous viscosity and consequently development of intestinal inflammation, dysbiosis and often times dysmotility. This triad is also referred to as the “CF gut”. A diagnosis is mainly based on clinical observation and treatment is often times decided empirically. This review of the literature should provide CF caregivers with some tools to identify intestinal inflammation, dysbiosis and dysmotility as possible cause for their patient's gastrointestinal complaints and provide an overview of our current approach to its management.

Decreased Expression of Enterocyte Nutrient Assimilation Genes and Proteins in the Small Intestine of Cystic Fibrosis Mouse

Cystic fibrosis (CF) has major effects on the intestinal tract with potential consequences on nutrition, but these are not fully understood. I investigated the possibility of altered enterocyte maturation in CF, as suggested by decreased enterocyte nutrient assimilation gene expression in published transcriptome analysis of the small intestine of CF mouse. In CF and wild-type (WT) mice, enterocyte gene/protein expression was analyzed by quantitative realtime polymerase chain reaction (qRT-PCR), enzyme histochemistry, immunohistochemistry, and Western blot. One group of mice was maintained on a control liquid diet; to manipulate the gut microbiota, a second group was treated with oral antibiotics; to improve hydration of the gut lumen, a third group was given a laxative drinking solution. On the control diet in the CF intestine, there were decreased levels (67%-85% reduction of WT levels) of enterocyte genes/proteins. Antibiotics did not normalize the expression of enterocyte markers in the CF mouse. In contrast, the laxative treatment of CF mice significantly increased expression to near WT levels. These studies suggest that the environment of the CF intestinal lumen plays a role in reduced maturation of enterocytes. Because changing the gut lumen environment can affect enterocyte maturation, this is not a cell-autonomous effect of loss of CF transmembrane conductance regulator.

Vitamin D attenuates inflammation in CFTR knockdown intestinal epithelial cells but has no effect in cells with intact CFTR.

The cystic fibrosis (CF) intestine is characterized by chronic inflammation. CF patients are instructed to ingest supplemental vitamin D on a daily basis thereby exposing their intestinal tract to pharmacological amounts of this vitamin. It has been shown that vitamin D exerts intestinal anti-inflammatory properties. We therefore postulate that vitamin D may be beneficial in the management of CF intestinal inflammation by attenuating cellular inflammatory responses. In this study, we investigated the anti-inflammatory effects of the oral form of vitamin D3 (cholecalciferol) and its metabolites, 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3, on cytokine-induced inflammatory responses in intestinal epithelial Caco-2/15 cells with intact expression of CF transmembrane conductance regulator (CFTR) and knockdown for CFTR. We show that 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 inhibited p38MAPK phosphorylation and that these effects were not mediated by changes in the expression of MAPK phosphatase-1 (MKP-1). However, 1,25-dihydroxyvitamin D3 exhibited superior anti-inflammatory effects as it furthermore reduced cytokine-induced NF-κB nuclear translocation and interleukin-8 mRNA stability and secretion. Intriguingly, the anti-inflammatory effects of vitamin D metabolites were only observed in CFTR knockdown cells, which may be explained by alterations in its catabolism associated with changes in CYP24A1 expression. These observations were supported in vivo whereby Cftr(-/-) mice fed large amounts of vitamin D3 for 2 mo led to a reduction in the number of eosinophils and apoptotic cells in the duodenal mucosa of females but not males. Altogether, these findings suggest that vitamin D exerts intestinal anti-inflammatory actions under specific circumstances and may thus prove beneficial in CF.

Su1368 Undernutrition and Altered Gut Secretory IgA Synergistically Increase Bacterial Burdens in the Mesenteric Lymph Nodes

Background Cystic fibrosis (CF) is a life-limiting disease. Microbial infections, colonization and airway inflammation are the hallmarks of CF lung disease. Similarly, small bowel bacterial overgrowth has been long recognized as a complication of CF. With the recent availability of non-culture-based microbiological techniques, the presence of intestinal dysbiosis in CF has been confirmed. Defects in bacterial host defenses in the CF airways have been speculated and extensively investigated. However, the role of the innate immune system in the intestines of patients with CF remain poorly studied. Human β-defensin 2 (HBD-2) is an antimicrobial protein produced by epithelial surfaces, and upregulated by inflammation. Its expression in the CF intestine is unknown. Methods Faeces from children (aged 0-18 years) with CF and age-matched healthy controls (HC) were collected for faecal HBD-2 and calprotectin. Patients with CF with a pulmonary exacerbation requiring intravenous antibiotics were excluded. Any CF and HC subjects with gastroenteritis, on oral corticosteroids, probiotics and/or non-steroidal anti-inflammatory drugs in the preceding two weeks were also excluded. Results Thirty-three CF patients with mean (SD) age of 7.3 (3.8) years were recruited. Nineteen subjects (58%) were males. Twenty-nine (88%) were PI and 4 (12%) were PS. As a comparison, 33 age-matched HC were enrolled; 14 (42%) were males. There were no significant differences between CF and HC subjects for age (mean (SD) of 7.3 (3.8) vs. 7.0 (4.6) years respectively; P = 0.78) and gender (P = 0.33). All CF patients had detectable faecal HBD-2. There were no difference between faecal HBD-2 in CF and HC (median (IQR): 49.1 (19.7-77.2) vs. 43.4 (26.5-71.9)ng/g; P=0.7), and between PI and PS CF subjects (median (IQR): 49.1 (19.3 77.2) and 46.9 (32.0 104.4) ng/g; P = 0.6). CF subjects with faecal calprotectin ≥50mg/kg and <50mg/kg were also not different (50.5 (19.6-80.2) vs. 43.0 (19.0-70.4); P=0.7). There was no correlation between faecal HBD-2 and calprotectin in CF (r = 0.14; P = 0.4). Conclusions Faecal HBD-2 levels were not increased in children with CF, in inflamed or non-inflamed states. The lack of HBD-2 induction and upregulation under inflammatory conditions may suggest a diminished intestinal innate immune response in CF.

Defective goblet cell exocytosis contributes to murine cystic fibrosis-associated intestinal disease.

Cystic fibrosis (CF) intestinal disease is associated with the pathological manifestation mucoviscidosis, which is the secretion of tenacious, viscid mucus that plugs ducts and glands of epithelial-lined organs. Goblet cells are the principal cell type involved in exocytosis of mucin granules; however, little is known about the exocytotic process of goblet cells in the CF intestine. Using intestinal organoids from a CF mouse model, we determined that CF goblet cells have altered exocytotic dynamics, which involved intrathecal granule swelling that was abruptly followed by incomplete release of partially decondensated mucus. Some CF goblet cells exhibited an ectopic granule location and distorted cellular morphology, a phenotype that is consistent with retrograde intracellular granule movement during exocytosis. Increasing the luminal concentration of bicarbonate, which mimics CF transmembrane conductance regulator-mediated anion secretion, increased spontaneous degranulation in WT goblet cells and improved exocytotic dynamics in CF goblet cells; however, there was still an apparent incoordination between granule decondensation and exocytosis in the CF goblet cells. Compared with those within WT goblet cells, mucin granules within CF goblet cells had an alkaline pH, which may adversely affect the polyionic composition of the mucins. Together, these findings indicate that goblet cell dysfunction is an epithelial-autonomous defect in the CF intestine that likely contributes to the pathology of mucoviscidosis and the intestinal manifestations of obstruction and inflammation.

Loss of Slc26a9 anion transporter alters intestinal electrolyte and HCO3(-) transport and reduces survival in CFTR-deficient mice.

Slc26a9 is an anion transporter that is strongly expressed in the stomach and lung. Slc26a9 variants were recently found associated with a higher incidence of meconium ileus in cystic fibrosis (CF) infants, raising the question whether Slc26a9 is expressed in the intestine and what its functional role is. Slc26a9 messenger RNA (mRNA) was found highly expressed in the mucosae of the murine and human upper gastrointestinal tract, with an abrupt decrease in expression levels beyond the duodenum. Absence of SLC26a9 expression strongly increased the intestinally related mortality in cystic fibrosis transmembrane conductance regulator (CFTR)-deficient mice. Proximal duodenal JHCO3 − and fluid secretion were reduced in the absence of Slc26a9 expression. In the proximal duodenum of young Slc26a9 KO mice, the glands and villi/crypts were elongated and proliferation was enhanced. This difference was lost with ageing, as were the alterations in fluid movement, whereas the reduction in JHCO3 - remained. Laser dissection followed by qPCR suggested Slc26a9 expression to be crypt-predominant in the duodenum. In summary, deletion of Slc26a9 caused bicarbonate secretory and fluid absorptive changes in the proximal duodenal mucosa and increased the postweaning death rates in CFTR-deficient mice. Functional alterations in the duodenum were most prominent at young ages. We assume that the association of meconium ileus and Slc26a9 variants may be related to maldigestion and impaired downstream signaling caused by loss of upper GI tract digestive functions, aggravating the situation of lack of secretion and sticky mucus at the site of obstruction in CF intestine.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-014-1543-x) contains supplementary material, which is available to authorized users.

Disrupted tight junctions in the small intestine of cystic fibrosis mice

The tight junction (TJ) is the major determinant of paracellular permeability, which in the gut protects the body from entry of harmful substances such as microbial components. In cystic fibrosis (CF), there is increased permeability of the small intestine both in humans and in CF mice. To gain insight into the mechanisms of increased intestinal permeability in CF, I analyze the composition of the TJ in a cystic fibrosis transmembrane conductance regulator (Cftr) knockout mouse model. Significant changes in TJ gene expression in the CF intestine were found for Cldn1, Cldn7, Cldn8 and Pmp22, which were expressed at lower levels and Cldn2 that was expressed at a higher level. Protein levels of claudin-2 were increased in the CF intestine as compared to wild-type, while other TJ proteins were not significantly different. In the villus epithelium of the CF intestine, all TJ components analyzed were mislocalized to the basal cytoplasm and showed varying degrees of loss from the TJ and apico-lateral surfaces. The pore-forming claudin-2 in the CF intestine showed more intense staining but was correctly localized to the TJ, principally in the crypts that are enlarged in CF. The cytokine TNFα, known to affect TJ, was elevated to 160% of wild-type in the CF intestine. In summary, there is a dramatic redistribution of claudin proteins from the TJ/lateral membrane to the basal cytoplasm of the villus epithelium in the CF intestine. These changes in TJ protein localization in CF are likely to be involved in the increased permeability of the CF small intestine to macromolecules and TNFα may be a causative factor.

The Role of Calprotectin in Pediatric Disease

Calprotectin (CP) is a calcium- and zinc-binding protein of the S100 family expressed mainly by neutrophils with important extracellular activity. The aim of the current review is to summarize the latest findings concerning the role of CP in a diverse range of inflammatory and noninflammatory conditions among children. Increasing evidence suggests the implication of CP in the diagnosis, followup, assessment of relapses, and response to treatment in pediatric pathological conditions, such as inflammatory bowel disease, necrotizing enterocolitis, celiac disease, intestinal cystic fibrosis, acute appendicitis, juvenile idiopathic arthritis, Kawasaki disease, polymyositis-dermatomyositis, glomerulonephritis, IgA nephropathy, malaria, HIV infection, hyperzincemia and hypercalprotectinemia, and cancer. Further studies are required to provide insights into the actual role of CP in these pathological processes in pediatrics.

Intestinal Smooth Muscle Dysfunction Develops Postnatally in Cystic Fibrosis Mice

Intestinal dysmotility is one of the effects of cystic fibrosis (CF), but when and how this develops is not well understood. The goal of the present study was to use the Cftr knockout mouse to determine when in development circular smooth muscle of the small intestine becomes dysfunctional. Wild-type (WT) and CF mice were used at postnatal day 5 (P5) through adult. Pieces of small intestine were used to measure contractile activity of the circular muscle. Bacterial overgrowth was measured by quantitative polymerase chain reaction (PCR) of the bacterial 16S gene. Intestinal gene expression was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). Prostaglandin E2 (PGE2) and its metabolites were measured by enzyme immunoassay. CF circular muscle response to cholinergic stimulation was similar to WT at P5, became somewhat impaired at P7, and was severely impaired by P14. In the CF intestine, bacterial overgrowth occurred by P4 and was maintained into adulthood. Eicosanoid metabolic gene expression in the CF intestine did not differ from WT shortly after birth. The phospholipase A2 genes, Pla2g4c and Pla2g5 exhibited increased expression in CF mice at P24. Prostaglandin degradative genes, Hpgd and Ptgr1, showed lower expression in CF as compared with WT at P16 and P24, respectively. PGE2 levels were significantly greater in CF mice at most ages from P7 through adulthood. The results clearly demonstrate that lack of CFTR itself does not cause smooth muscle dysfunction, because the circular muscle from P5 CF mice had normal activity and dysfunction developed between P7 and P14.

Impaired Mucosal Barrier Function in the Small Intestine of the Cystic Fibrosis Mouse

The intestinal mucosal barrier protects the body from the large numbers of microbes that inhabit the intestines and the molecules they release. Intestinal barrier function is impaired in humans with cystic fibrosis (CF), including reduced activity of the lipopolysaccharide-detoxifying enzyme intestinal alkaline phosphatase (IAP) and increased permeability. The objective of this study was to determine the suitability of using the CF mouse to investigate intestinal barrier function, and whether interventions that are beneficial for the CF mouse intestinal phenotype (antibiotics or laxative), would improve barrier function. Also tested were the effects of exogenous IAP administration. The Cftr(tm1UNC) mouse was used. IAP expression (encoded by the murine Akp3 gene) was measured by quantitative reverse transcription-polymerase chain reaction and enzyme activity. Intestinal permeability was assessed by measuring rhodamine-dextran plasma levels following gavage. CF mice had 40% Akp3 mRNA expression and 30% IAP enzyme activity, as compared with wild-type mice. Oral antibiotics and laxative treatments normalized Akp3 expression and IAP enzyme activity in the CF intestine. CF mice had a 5-fold greater transfer of rhodamine-dextran from gut lumen to blood. Antibiotic and laxative treatments reduced intestinal permeability in CF mice. Administration of exogenous purified IAP to CF mice reduced intestinal permeability to wild-type levels and reduced small intestinal bacterial overgrowth by >80%. The CF mouse intestine has impaired mucosal barrier function, similar to human CF. Interventions that improve other aspects of the CF intestinal phenotype (antibiotics and laxative) also increase IAP activity and decrease intestinal permeability in CF mice. Exogenous IAP improve permeability and strongly reduce bacterial overgrowth in CF mice, suggesting this may be a useful therapy for CF.

MicroRNA profiling of cystic fibrosis intestinal disease in mice

Cystic fibrosis (CF) intestinal disease is characterized by alterations in processes such as proliferation and apoptosis which are known to be regulated in part by microRNAs. Herein, we completed microRNA expression profiling of the intestinal tissue from the cystic fibrosis mouse model of cystic fibrosis transmembrane conductance regulator (Cftr) deficient mice (BALBc/J Cftr tm1UNC), relative to that of wildtype littermates, to determine whether changes in microRNA expression level are part of this phenotype. We identified 24 microRNAs to be significantly differentially expressed in tissue from CF mice compared to wildtype, with the higher expression in tissue from CF mice. These data were confirmed with real time PCR measurements. A comparison of the list of genes previously reported to have decreased expression in the BALB × C57BL/6J F2 CF intestine to that of genes putatively targeted by the 24 microRNAs, determined from target prediction software, revealed 155 of the 759 genes of the expression profile (20.4%) to overlap with predicted targets, which is significantly more than the 100 genes expected by chance (p = 1 × 10 − 8 ). Pathway analysis identified these common genes to function in phosphatase and tensin homolog-, protein kinase A-, phosphoinositide-3 kinase/Akt- and peroxisome proliferator-activated receptor alpha/retinoid X receptor alpha signaling pathways, among others, and through real time PCR experiments genes of these pathways were demonstrated to have lower expression in the BALB CF intestine. We conclude that altered microRNA expression is a feature which putatively influences both metabolic abnormalities and the altered tissue homeostasis component of CF intestinal disease.

Lubiprostone ameliorates the cystic fibrosis mouse intestinal phenotype

BackgroundCystic fibrosis (CF) is caused by mutations in the CFTR gene that impair the function of CFTR, a cAMP-regulated anion channel. In the small intestine loss of CFTR function creates a dehydrated, acidic luminal environment which is believed to cause an accumulation of mucus, a phenotype characteristic of CF. CF mice have small intestinal bacterial overgrowth, an altered innate immune response, and impaired intestinal transit. We investigated whether lubiprostone, which can activate the CLC2 Cl- channel, would improve the intestinal phenotype in CF mice.MethodsCftrtm1UNC (CF) and wildtype (WT) littermate mice on the C57BL/6J background were used. Lubiprostone (10 μg/kg-day) was administered by gavage for two weeks. Mucus accumulation was estimated from crypt lumen widths in periodic acid-Schiff base, Alcian blue stained sections. Luminal bacterial load was measured by qPCR for the bacterial 16S gene. Gastric emptying and small intestinal transit in fasted mice were assessed using gavaged rhodamine dextran. Gene expression was evaluated by Affymetrix Mouse430 2.0 microarray and qRT-PCR.ResultsCrypt width in control CF mice was 700% that of WT mice (P < 0.001). Lubiprostone did not affect WT crypt width but, unexpectedly, increased CF crypt width 22% (P = 0.001). Lubiprostone increased bacterial load in WT mice to 490% of WT control levels (P = 0.008). Conversely, lubiprostone decreased bacterial overgrowth in CF mice by 60% (P = 0.005). Lubiprostone increased gastric emptying at 20 min postgavage in both WT (P < 0.001) and CF mice (P < 0.001). Lubiprostone enhanced small intestinal transit in WT mice (P = 0.024) but not in CF mice (P = 0.377). Among other innate immune markers, expression of mast cell genes was elevated 4-to 40-fold in the CF intestine as compared to WT, and lubiprostone treatment of CF mice decreased expression to WT control levels.ConclusionsThese results indicate that lubiprostone has some benefits for the CF intestinal phenotype, especially on bacterial overgrowth and the innate immune response. The unexpected observation of increased mucus accumulation in the crypts of lubiprostone-treated CF mice suggests the possibility that lubiprostone increases mucus secretion.

T1852 Roles of Cyclic AMP-Dependent HCO 3- Secretion and CA + Mediated Mucus Secretion in Mucus Accumulation in Cystic Fibrosis Intestine

T1852 Roles of Cyclic AMP-Dependent HCO 3- Secretion and CA + Mediated Mucus Secretion in Mucus Accumulation in Cystic Fibrosis Intestine

Defective cAMP‐dependent HCO3‐ Secretion Leads to Abnormal Mucus

Cystic fibrosis (CF) is a hereditary, fatal disease caused by a unique pathology that appears to begin with thick mucus that obstructs the lumina of hollow organs. In the present study, PAS‐AB Staining, morphometry, pH stat and mucus collection assay methods were used to investigate the formation of abnormal mucus in CF mouse small intestine. We found that in the wild type mouse small intestine, carbachol (100 μM, an intracellular Ca2+ mediated activator) induced goblet cell mucus release, but did not stimulate sustained HCO3− secretion. Whereas, forskolin (10 μM) + IBMX (100 μM) applied to activate intracellular cAMP‐mediated pathways did not have any detectable effect on goblet cell mucus release, but did induce sustained HCO3− secretion. With HCO3− in the basolateral solution, PGE2 (1 μM) + 5‐HT (10 μM), cAMP and Ca2+mediated activators, induced more mucus release from goblet cells in the presence than in the absence of HCO3−. In CF mouse small intestine, PGE2 (1 μM) + 5‐HT (10 μM) also stimulated goblet cell mucus release, but there was no significant difference in mucus releasing goblet cells with or without HCO3− in the basolateral solution. cAMP activated HCO3− secretion was missing in CF intestine. These results suggest that a defect in cAMP‐dependent HCO3− secretion, rather than in mucus release, leads to the abnormal mucus observed in CF mouse.Supported by Elizabeth Nash Memorial Fellowship

Acetylcholine induces cytosolic Ca2+ mobilization in isolated distal colonic crypts from normal and cystic fibrosis mice.

In intestinal biopsies from cystic fibrosis (CF) patients acetylcholine fails to elicit a chloride secretion response, and this observation can be explained by a defect in the Ca2+ signalling pathway in CF secretory cells. We tested the hypothesis that in CF intestine, the generation of an intracellular Ca2+ signal upon cholinergic stimulation is absent. A transgenic CF mouse model was used. Electrical measurements on intact jejunum and unstripped colon were performed in Ussing chambers. Intact distal colonic crypts were isolated, and the intracellular Ca2+ concentration was monitored using the Ca2+-sensitive dye fura-2. Acetylcholine increased the short-circuit current generated by wild-type jejunum and colon, but failed to induce a response in CF tissues. Acetylcholine caused a transient elevation in the intracellular Ca2+ concentration in colonic crypts from both wild-type and CF mice; the amplitude and timing of the response in CF crypts was indistinguishable from that in wild-type crypts. The response to acetylcholine was also observed in the absence of extracellular calcium, indicating intracellular stores as the source from which the cytosolic Ca2+ concentration increased. We conclude that the absence of a cholinergically-induced secretory response in CF intestine is not due to a defect in the generation of a Ca2+ signal in intestinal cells upon cholinergic stimulation.