Increased levels of mucins in the cystic fibrosis mouse small intestine, and modulator effects of the Muc1 mucin expression

The mouse model (Cftrtm1UNC/Cftrtm1UNC) for cystic fibrosis (CF) shows mucus accumulation and increased Muc1 mucin mRNA levels due to altered splicing (Hinojosa-Kurtzberg AM, Johansson MEV, Madsen CS, Hansson GC, and Gendler SJ. Am J Physiol Gastrointest Liver Physiol 284: G853–G862, 2003). However, it is not known whether Muc1 is a major mucin contributing to the increased mucus and why CF/Muc1−/− mice show lower mucus accumulation. To address this, we have purified mucins from the small intestine of CF mice using guanidinium chloride extraction, ultracentrifugation, and gel filtration and analyzed them by slot blot, gel electrophoresis, proteomics, and immunoblotting. Normal and CF mice with wild-type (WT) Muc1 or Muc1−/− or that are transgenic for human MUC1 (MUC1.Tg, on a Muc1−/− background) were analyzed. The total amount of mucins, both soluble and insoluble in guanidinium chloride, increased up to 10-fold in the CF mice compared with non-CF animals, whereas the CF mice lacking Muc1 showed intermediate levels between the CF and non-CF mice. However, the levels of Muc3 (orthologue of human MUC17) were increased in the CF/Muc1−/− mice compared with the CF/MUC1.Tg animals. The amount of MUC1 mucin was increased several magnitudes in the CF mice, but MUC1 did still not appear to be a major mucin. The amount of insoluble mucus of the large intestine was also increased in the CF mice, an effect that was partially restored in the CF/Muc1−/− mice. The thickness of the firmly adherent mucus layer of colon in the Muc1−/− mice was significantly lower than that of WT mice. The results suggest that MUC1 is not a major component in the accumulated mucus of CF mice and that MUC1 can influence the amount of other mucins in a still unknown way.

Download Full-text

Bacterial Overgrowth in the Cystic Fibrosis Transmembrane Conductance Regulator Null Mouse Small Intestine

Infection and Immunity ◽

10.1128/iai.72.10.6040-6049.2004 ◽

2004 ◽

Vol 72 (10) ◽

pp. 6040-6049 ◽

Cited By ~ 116

Author(s):

Oxana Norkina ◽

Tim G. Burnett ◽

Robert C. De Lisle

Keyword(s):

Cystic Fibrosis ◽

Small Intestine ◽

Intestinal Inflammation ◽

Bacterial Load ◽

Paneth Cell ◽

Bacterial Overgrowth ◽

Intestinal Lumen ◽

Cytokine Signaling ◽

Null Mouse ◽

Mouse Small Intestine

ABSTRACT We recently reported the inflammation of the cystic fibrosis (CF) mouse small intestine, and we hypothesized bacterial overgrowth as a possible cause. Quantitative PCR of bacterial 16S genomic DNA in the CF mouse small intestine revealed an increase of greater than 40-fold compared to controls. Sequencing of 16S PCR products and Gram staining showed that the majority of bacteria in the CF mouse intestine were gram negative. Bacteria were observed to colonize the mucus that accumulates in the intestinal lumen of mice with CF. Impaired Paneth cell defenses were suggested by observation of partially dispersed Paneth granules in the mucus plugs of CF mouse intestinal crypts, and this mucus was strongly immunoreactive for Paneth cell bactericidal products. The role of bacterial overgrowth in intestinal inflammation in CF was tested by treating mice with oral antibiotics (ciprofloxacin and metronidazole) for 3 weeks, which reduced bacterial load in the CF mouse small intestine over 400-fold. Antibiotic treatment decreased the expression of the inflammation-related genes mast cell protease 2, leucine-rich α2 glycoprotein/leucine-rich high endothelial venule glycoprotein, suppressor of cytokine signaling 3, hematopoietic cell transcript 1, and resistin-like molecule β/found in inflammatory zone 2, all of which were no longer expressed at levels significantly different from control levels. The reduction of intestinal bacteria also significantly improved the growth of CF mice but had no effect on the growth of wild-type mice. These data suggest that bacterial overgrowth in the CF mouse small intestine has a role in inflammation and contributes to the failure to thrive in this mouse model of CF.

Download Full-text

Immunoreactive pancreatic Reg protein in sera from cystic fibrosis patients with and without pancreatic insufficiency

Gut ◽

10.1136/gut.44.4.545 ◽

1999 ◽

Vol 44 (4) ◽

pp. 545-551 ◽

Cited By ~ 11

Author(s):

J Carrère ◽

O Guy-Crotte ◽

E Gaia ◽

C Figarella

Keyword(s):

Gene Expression ◽

Cystic Fibrosis ◽

Molecular Form ◽

Pancreatic Insufficiency ◽

Gel Filtration ◽

Normal Serum ◽

Pancreatic Acinar Cells ◽

Secretory Proteins ◽

Mrna Levels ◽

Dot Blot

BACKGROUNDThe biological function of the Reg protein, a non-enzymic protein produced in fairly large amounts by pancreatic acinar cells, remains elusive. Its susceptibility to proteolysis leading to precipitation of the proteolysis product at neutral pH suggests that it could contribute to the protein plugging observed in cystic fibrosis (CF).AIMSTo study its behaviour in the serum of CF patients with or without pancreatic insufficiency and to compare it with that of other pancreatic secretory proteins.PATIENTS170 patients (93 with CF, 55 controls, and 22 with chronic pancreatitis) were studied.METHODSReg protein was measured using a specific enzyme immunoassay and its molecular form in CF sera was characterised by gel filtration. Molecular gene expression was investigated by dot-blot hybridisation.RESULTSReg protein was present in all CF sera studied from patients with or without pancreatic insufficiency, and in all cases the level was significantly higher than in controls. Its chromatographic behaviour in CF sera was identical with that of the protein present in normal serum. No correlation was found between the levels of Reg protein and trypsin(ogen) (or lipase) in CF, nor in control sera or normal pancreatic juice. Molecular gene expression of the corresponding proteins investigated in pancreatic tissues showed an absence of correlation between the mRNA levels.CONCLUSIONSReg protein may not be a secretory exocrine protein like the digestive enzymes but rather a hormone-like secretory substance with an endocrine or paracrine function.

Download Full-text

Enteric circular muscle dysfunction in the cystic fibrosis mouse small intestine

Neurogastroenterology & Motility ◽

10.1111/j.1365-2982.2009.01418.x ◽

2010 ◽

Vol 22 (3) ◽

pp. 341-e87 ◽

Cited By ~ 28

Author(s):

r. c. de lisle ◽

r. sewell ◽

l. meldi

Keyword(s):

Cystic Fibrosis ◽

Small Intestine ◽

Circular Muscle ◽

Muscle Dysfunction ◽

Cystic Fibrosis Mouse ◽

Mouse Small Intestine

Download Full-text

Inflammation of the cystic fibrosis mouse small intestine

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00473.2003 ◽

2004 ◽

Vol 286 (6) ◽

pp. G1032-G1041 ◽

Cited By ~ 97

Author(s):

Oxana Norkina ◽

Simran Kaur ◽

Donna Ziemer ◽

Robert C. De Lisle

Keyword(s):

Gene Expression ◽

Cystic Fibrosis ◽

Small Intestine ◽

Immune System ◽

Mast Cells ◽

Innate Immune System ◽

Innate Immune ◽

Serum Amyloid ◽

Mouse Small Intestine ◽

Cytochrome P 450

The CFTR null mouse [cystic fibrosis (CF) mouse] has a severe intestinal phenotype that serves as a model for CF-related growth deficiency, meconium ileus, and distal intestinal obstructive syndrome. DNA microarray analysis was used to investigate gene expression in the CF mouse small intestine. Sixty-one genes exhibited a statistically significant twofold or greater increase in expression, and 98 genes were downregulated twofold or greater. Of the upregulated genes, most were associated with inflammation and included markers for cells of the innate immune system (mast cells and neutrophils) and for acute-phase genes (serum amyloid A and complement factors). The downregulated genes include 10 cytochrome P-450 genes; several are involved in lipid metabolism, and several are involved in various transport processes. Confirmation by quantitative RT-PCR showed gene expression was significantly increased for mast cell protease 2 (27-fold), hematopoietic cell transcript 1 (17-fold), serum amyloid A3 (2.9-fold), suppressor of cytokine signaling 3 (2.0-fold), leucine-rich α2-glycoprotein (21-fold), resistin-like molecule-β (49-fold), and Muclin (2.5-fold) and was significantly decreased for cytochrome P-450 4a10 (28-fold) and cubilin (114-fold). Immune cell infiltration was confirmed histologically by staining for mast cells and neutrophils. These data demonstrate that the CF intestine exhibits an inflammatory state with upregulation of components of the innate immune system.

Download Full-text