Early molecular and functional changes in colonic epithelium that precede increased gut permeability during colitis development in mdr1a(-/-) mice.
BACKGROUND
The early molecular changes preceding the onset of mucosal inflammation in colitis and their temporal relationship with gut permeability remain poorly defined. This study investigated functional and transcriptomic changes in mdr1a(-/-) mice lacking the intestinal transporter P-glycoprotein, which develop colitis spontaneously when exposed to normal enteric flora.


METHODS
Mdr1a(-/-) mice were housed in specific pathogen-free conditions to slow colitis development and compared to congenic controls. Mucosal permeability and cytokine secretion were analyzed in ex vivo colon. Gene expression in colonic mucosal and epithelial preparations was analyzed by microarray and qPCR. Colonocyte responsiveness to bacterial antigens was measured in short-term culture.


RESULTS
Colon from 4-5-week-old, disease-free mdr1a(-/-) mice was histologically normal with no evidence of increased permeability compared to controls. However, these tissues display a distinctive pattern of gene expression involving significant changes in a small number of genes. The majority of upregulated genes were associated with bacterial recognition and the ubiquitin-proteasome system and were gamma-interferon (IFN-gamma) responsive. Expression of the antiinflammatory factor pancreatitis-associated protein (PAP) and the related gene RegIIIgamma were markedly reduced. Colonocytes from 4-5-week mdr1a(-/-) exhibit similar transcriptomic changes, accompanied by higher basal chemokine secretion and increased responsiveness to LPS. Significant increases in colonic permeability were associated with older (12-16-week) mdr1a(-/-) mice displaying molecular and functional evidence of active inflammation.


CONCLUSIONS
These studies show that early epithelial changes associated with altered responsiveness to bacteria precede increased permeability and mucosal inflammation in this model of colitis, highlighting the importance of P-glycoprotein in regulating interactions with the commensal microflora.
Collett
Andrew
A
Infection, Injury and Inflammation Research Group, Division of Medicine and Neurosciences, University of Manchester and Salford Royal Hospitals NHS Trust, Hope Hospital, Salford, UK.
Higgs
Norman B
NB
Gironella
Meritxell
M
Zeef
Leo A H
LA
Hayes
Andy
A
Salmo
Emil
E
Haboubi
Najib
N
Iovanna
Juan L
JL
Carlson
Gordon L
GL
Warhurst
Geoffrey
G
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics*; D000818:Animals; D015153:Blotting, Western; D002463:Cell Membrane Permeability / Q000502:physiology*; D002478:Cells, Cultured; D018925:Chemokines / Q000378:metabolism; D003092:Colitis / Q000378:metabolism* / Q000473:pathology; D003106:Colon / Q000378:metabolism* / Q000473:pathology; D004195:Disease Models, Animal; D018450:Disease Progression; D018432:Drug Resistance, Multiple; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D015870:Gene Expression; D008297:Male; D051379:Mice; D046228:Microarray Analysis; D000073718:Pancreatitis-Associated Proteins; D012313:RNA / Q000096:biosynthesis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018435:ATP Binding Cassette Transporter, Subfamily B; D018925:Chemokines; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D012313:RNA; C095122:multidrug resistance protein 3