Modulation of P-glycoprotein 170 function as a therapeutic target in ulcerative colitis

Ulcerative colitis is a common and chronic inflammatory disease affecting the large intestines. The cause of the illness is unknown, although there is strong evidence that both genetic and environmental factors contribute. Current treatment is unsatisfactory, relying on toxic drugs which suppress the immune system. Recently studies from our laboratory suggest that variations in a gene (MDR-1) that codes for a pump that transports foreign particles out of cells might be involved in causing the disease. I plan to study this in detail in this project. I will manipulate the levels of gene and pump levels in mouse models of ulcerative colitis and human gut cells and determine if this influences the level of gut inflammation. I will use the latest gene manipulation technology to change the levels of gene expression and to study gene changes that occurs after PgP manipulation. This will also provide the opportunity to study how PgP interact with other mechanisms of gut defence and the immune system. I will test how PgP respond to fragments of bacteria and chemicals which provoke inflammation. These experiments provide the basis of a possible new mechanism for how UC develops and importantly, the development of a future treatment.

Background: Ulcerative colitis (UC) is a common relapsing inflammatory disease affecting 250/100 000 individuals in UK. The morbidity associated with active UC is high, 15% develop severe disease, of which 30% will require emergency surgery. There is compelling evidence for the multidrug resistance gene 1 (MDR1) in the pathogenesis of UC. MDR1 encodes P-glycoprotein 170 (PgP), a transmembrane ATP-dependent efflux protein which drives the transport of a diverse range of molecules to the outside of cells. The MDR1 gene maps to chromosome 7q22, a susceptibility locus for IBD and germline variations of this gene are strongly associated with UC (haplotypic association, p<0.001). This is further strengthened by significantly lower PgP expression on intestinal epithelium in UC (basal and inflamed states; p<0.001). The MDR1 knock-out murine model develops colitis.
Aims: I will study the contribution of PgP to intestinal inflammation and if modulation of PgP expression can result in improvement. I will question if changes in PgP expression in vivo modifies intestinal inflammation, and if in vitro changes of protein expression on intestinal epithelial cells alter cell response to inflammatory stimuli.
Experimental designs: The effect of modulating PgP expression on intestinal inflammation will be examined by using established inducers and inhibitors in 2 animal models (chronic HLA B27/?2-microglobulin transgenic and acute DSS-colitis models). These represent inflammation induced by bacterial flora and hapten (biochemical inflammation) respectively. I will perform comparative analyses of histological and disease parameters of inflammation; and determine the cytokine profile and degree of bacterial translocation in PgP high and low states. I hypothesise that the upregulation of PgP will lead to the improvement of colitis, which will worsen in specific PgP inhibition. To provide relevance to human disease and to further elucidate the underlying mechanism of PgP-mediated protection, I will investigate the effects of PgP modulation on cell response after exposure to pro-inflammatory cytokines and pathogen associated molecular patterns stimulation in human intestinal epithelial cell line (LS180). This will be done using whole genome expressions approach in PgPhigh and PgPnull state following stable knock-down of the MDR1 gene. Confirmatory work will be performed on short-term culture of intestinal epithelial cells of controls and newly-diagnosed (treatment naïve) UC patients.
Potential outcomes: The proposed study will be the first in-depth study in the role of PgP in intestinal inflammation and a direct translation from genetic to molecular function. Since PgP function can be manipulated, there is distinct scope for translation work.