Crohn's disease and ulcerative colitis, collectively known as inflammatory bowel diseases (IBD), are caused in part by overly-aggressive, T cell-mediated immune responses to bacterial products in genetically susceptible hosts. Isolates of Escherichia coli (E. coli) have been associated with Crohn's disease and bacterial components, including stress-response proteins, promote intestinal inflammation by stimulating innate and adaptive host immune systems. While the immune response to bacterial products has been extensively studied, little is known about the subsequent effects of host inflammation on bacterial properties. Selective colonization with a commensal murine strain of E. coli (NC101) causes colitis in IL-10-deficient (IL-10-/-), but not wild-type (wt), mice. Intestinal inflammation triggers NC101 to upregulate the bacterial stress-response gene ibpB. IbpB is a small heat shock protein that helps bacteria tolerate heat stress and high levels of reactive oxygen species (ROS) as is found in the inflamed intestine and macrophage phagolysosomes. HYPOTHESIS: The host inflammatory milieu upregulates bacterial ibpB, which in turn allows bacteria to adapt to the environment and perpetuate inflammation by increasing their survival and virulence.
SPECIFIC AIMS : 1) Identify mechanisms by which the host inflammatory milieu stimulates E. coli to upregulate ibpB in vitro. We will study the roles of intracellular and secreted factors in upregulating E. coli ibpB using ibpB-eGFP reporter bacteria and inhibitors of ROS and cytokine production. 2) Ascertain the effects of E. coli ibpB expression on host inflammatory pathways in vitro. We will determine mechanisms by which E. coli IbpB inhibits uptake by macrophages and promotes intracellular survival using genetically altered NC101 and inhibitors of phagocytosis and ROS generation in gentamicin protection assays. 3) Examine the biological relevance of E. coli IbpB in the initiation and perpetuation of chronic experimental colitis in E. coli monoassociated IL-10-/- mice. We will measure histological inflammation, bacterial distribution, innate and adaptive immune responses in wt and IL-10-/- mice monoassociated with NC101 or ibpB-deficient NC101. CAREER DEVELOPMENT: Long-term goals are to lead a research team at an academic institution to: 1) contribute new knowledge to the field of host-microbial interactions, and 2) develop novel diagnostic strategies and therapies for IBD. Short-term goals are to: 1) learn new techniques to complete the proposed and future studies, 2) interact regularly with an exceptional mentoring team, 3) participate in didactic courses and research seminars in microbial genetics and pathogenesis, and 4) differentiate myself from my mentor by publishing manuscripts and submitting applications for independent funding in the area of bacterial stress responses. ENVIRONMENT: The National Gnotobiotic Rodent Resource Center, the Center for Gastrointestinal Biology and Disease, local experts in mucosal immunology and bacteriology, university start-up funds, and an institutional commitment to protect research time will facilitate attainment of these goals.
Inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, are chronic intestinal inflammatory disorders that cause significant morbidity. The studies in this proposal are designed to investigate the mechanisms by which resident intestinal bacteria may cause these diseases and to identify potential targets for novel therapies.
