Differential susceptibility of P‐glycoprotein deficient mice to colitis induction by environmental insults

Inflammatory bowel diseases (IBD) are diseases of unknown etiology characterized by recurrent, spontaneous inflammation of the gastrointestinal tract (GI).(1) IBD consists of two disease phenotypes, ulcerative colitis (UC) and Crohn’s disease (CD). These diseases are proposed to be the result of genetic predispositions for either altered immune function leading to aberrant immune responses to natural stimuli, or overall autoimmune dysregulation.(2) Studies show that 20% of patients with IBD have a relative with either UC or CD, in fact a positive family history is thought to be the single greatest predictive factor associated with the disease.(1, 3) Numerous genes are being examined as carriers of mutations that may result in a predisposition towards IBD, of particular interest is the human multi-drug resistance gene (MDR), an ATP-binding cassette subfamily B member 1A (Abcb1a). In humans the MDR gene is expressed on chromosome 7 (7q21.1) a susceptibility loci for IBD.(4) Studies to date have been inconsistent with regards to the significance of mutations in the MDR gene in relation to the development of IBD. Several recent meta-analysis studies have shown that a significant number of IBD patients carry mutations to this gene, and further studies have demonstrated that MDR mutations are associated with decreased P-gp expression and function.(5–8) Additionally, it was shown that mutations to this gene are strongly associated with early onset UC in German populations.(9) Conversely, it has been reported that subsets of IBD patients in Greek and European populations bear no correlation to MDR mutations.(10, 11) The functional product of the MDR gene is P-glycoprotein (P-gp). P-gp is a glycosylated transmembrane protein, which functions as a drug efflux pump effectively removing hydrophobic and amphipathic substrates from the intracellular environment.(12) There are 2 MDR genes in humans (MDR1 and MDR3) and 3 mdr genes in mice (mdr1a (abcb1a), mdr1b (abcb1b,) and mdr2(abcb4)).(13, 14) The MDR3 (and mdr2) genes are expressed in the liver, and actively pump phosphatydlcholine into bile.(15) The MDR1 genes, those linked to colitis, are found in multiple tissue locations. They are present not only on the apical surface of epithelial cells in the colon and small intestine, but also on epithelial cells of the pancreas, kidney, and adrenal glands, on endothelial cells at the blood brain barrier, and on cells of the hematopoetic lineage.(16–18) Due to the expression of P-gp on the apical epithelium of organs with excretory function, and due to the known role of P-gp in drug extrusion; it has been postulated that P-gp’s physiologic function is to extrude xenobiotic substrates from the intracellular environment.(19) A murine model of this genetic mutation has lent some insight into the potential physiological role of P-gp. FVB/N mice deficient in mdr1a gene expression develop a spontaneous colitis as early as 3–4 months of age.(20, 21) These FVB.mdr1a−/− mice have deficiencies in tight junction proteins in the intestinal epithelium, as well as an increased intestinal permeability prior to disease development.(21, 22) These mice have increased colonic IFNγ and IL8 and treatment of these animals with antibiotics was shown to be prophylactic and therapeutic in mitigating disease progression, confirming the role intestinal microbiota in inducing intestinal inflammation.(20, 23) To expand our knowledge of the mechanisms of the spontaneous colitis in this disease model and to investigate the role of different genetic backgrounds in disease susceptibility, our lab endeavored to backcross the mdr1a genetic deletion on to the C57BL/6J (B6) mouse strain. B6.mdr1a−/− mice were established by 10-generations of backcross breeding to B6 control mice. We initially characterized disease development in these animals; however, the P-gp deficiency on the B6 genetic background did not result in any clinical or histological evidence of spontaneous colitis in animals up to one year of age. Expression of genetic mutations may leave individuals predisposed towards colitis development in response to mitigating factors, such as a bacterial colonization or infection.(1) We therefore hypothesized that the B6.mdr1a−/− mice might be more susceptible to colitis induction after a secondary environmental insult. Environmental factors tested included Helicobacter bilis, and dextran sodium sulfate (DSS), which have both been previously reported to accelerate the development of colitis in the FVB.mdr1a−/− model.(24, 25) DSS has also been shown to alter mdr1a expression prior to clinical colitis symptoms.(26) Additionally, results from our B6.mdr1a−/− animals indicated that prostaglandin (PG) regulation may play a protective role in preventing colitis in this seemingly resistant strain. Therefore, we tested the affects of altering PG synthesis by blocking the key regulatory enzyme of the PG biosynthesis pathway, cyclooxygenase (COX) in our B6.mdr1a−/− animal model. We administered the non-steroidal anti-inflammatory drug (NSAID), Piroxicam, which inhibits both COX isoforms and rapidly induces colitis in IL10 deficient mice.(27)