Scientific Abstract

Proposal No. IBD-0236
Principal Investigator: Richard Gearry, MBChB, Ph.D.
Applicant Organization: University of Otago (Dunedin, New Zealand)
Project Title: A novel cellular assay system to assess the ptoential role of Mycobacterium avium subsp. paratuberculosis in the development and progression of Crohn's disease
Period of Award: June 1, 2008 – March 31, 2010

Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic intestinal inflammation in ruminants (Johne’s disease, JD) and was first proposed as a cause of CD in 1913. Subsequent studies have demonstrated a significant association of MAP with CD. MAP is able to survive and replicate within phagocytic cells. In many CD patients the phagocytic destruction and clearance of MAP appears to be defective, however the mechanism by which this occurs is poorly understood. MAP also exists in a cell-wall deficient form which has been suggested to be the virulent form of MAP in CD pathogenesis. These spheroplasts may reduce the antigenic signal presented to the host (due to the absence of cell wall antigens) and could be a way in which MAP evades innate host defences. Little is known about how or whether spheroplasts are capable of eliciting an inflammatory response. Interactions of MAP with phagocytic cells (bovine, ovine and murine) have been studied. However, extrapolation of results from animal-based cellular models must be interpreted with care as human phagocytes may respond differently to MAP. The pathobiology of JD differs in a number of ways from CD, further suggesting the existence of species differences with respect to the recognition and processing of MAP. It has been shown that MAP from different hosts varies with respect to entry, survival and persistence in bovine cell phagocytosis assays, and may initiate different host gene responses. There is a fundamental shortage of research investigating the interactions of human MAP isolates with human cells.

In the first year, we will develop a cellular model using monocytes from CD patients. This model will be used to assess how mutations in the CD-associated genes NOD2, ATG16L1, NCF4, and IRGM affect:

1) the uptake, survival and persistence of human isolates of MAP (from CD) in human monocytes
2) the production of host inflammatory mediators in response to MAP infection.

In the second year, we will apply this cellular model to assess the same parameters in relation to other CD-associated genes confirmed from our GWA study. We also intend studying the MAP spheroplast in this cellular model to clarify whether it has a different biological role to the better understood bacillary form. We will also use this model to investigate whether the antimycobacterials clarithromycin, rifabutin, clofazimine and ethambutol are effective against intracellular MAP, and to assess the production of inflammatory mediators in their presence.
This study will provide valuable new insights into the role that CD-associated genes play in the detection and elimination of MAP. It will also clarify the role of the MAP spheroplast in CD. The presence of MAP may also act as a useful prognostic biomarker to indicate whether antibiotic therapy of CD is justified in a subset of CD patients.