Researcher: Dr Andrew Brain, Weatherall Institute of Molecular Medicine, Oxford University.

Fellowship: Three Year Research Fellowship with partial funding from the National Association of Colitis and Crohn’s

Grant Total: £186,246

 
The pathogenesis of Crohn’s disease (CD) is determined by genetic factors together with mucosal immune dysregulation involving an association between exposure to luminal bacterial products and inappropriate activation of the innate immune system. Nucleotide oligomerization domain NOD2 is an intracellular pattern recognition receptor (PRR) that is mutated in a variable but significant percentage of CD patients whose function in the immunopathology of CD remains unclear. NOD2 senses the bacterial component muramyl dipeptide (MDP) via its leucine–rich repeats (LRRs), an effect that is lost in cells expressing mutant NOD2 associated with CD (3020insC). CD-associated NOD2 mutants display inadequate activation NF-?B in response to MDP. Paradoxically this leads to an uncontrolled inflammatory response to non-pathogenic gut flora, normally recognised as self. The molecular basis of this phenomenon remains unclear. In this work I will examine the function of wild type (WT) and CD 3020insC NOD2 using two approaches. Firstly I will characterise the transcriptional regulators of NOD2 signalling. Primary human antigen presenting cells (APCs) will be stimulated with MDP and large scale gene expression profiling performed. Early expressed genes will be clustered and groups of co-regulated genes will be compared for transcription factor target motifs in their promoter/enhancer regions. Candidate transcriptional regulators will be screened for biological function following NOD2 signalling. Those found to be biologically active will be examined further by Cytoscape and LC-MS/MS analysis to define interacting proteins. Once a full transcription complex governing WT NOD2 signalling has been defined, the results will be compared to those occurring following MDP stimulation of 3020insC NOD2 expressing APC. Secondly, the link between NOD2 and antigen presentation will be examined. Here magnetic microbeads conjugated to MDP and antigen will be internalised by WT and 3020insC NOD2 APC. APC will be exposed to antigen specific T cells and the adaptive immune response characterised to determine characteristic features defective in the context of 3020insC NOD2 expression. In addition a proteomic analysis of the NOD2 antigen presentation compartment will be undertaken. Here internalised MDP and antigen conjugated microbead associating proteins will be retrieved from APC and subject to LC-MS/MS analysis to define a list of proteins associated with a NOD2 antigen presentation compartment. The role of individual proteins in WT and 3020insC NOD2 mediated antigen presentation will be characterised following siRNA knockdown.