NADPH oxidase 1: mutation analysis and function

This thesis investigated the role of NOX1 in the Gastrointestinal (GI) tract using a variety of animal and in vitro models. In the terminal ileum, we show that the microbiome stimulates NOX1 and iNOS expression, leading to superoxide and nitric oxide generation and, subsequently, peroxynitrite formation, a reactive oxygen and nitrogen species (RONS). Sequencing of the bacterial and fungal microbiome did not reveal any substantial changes in Nox1-/y mice, implying that whilst the host responds to the GI microbiome, the RONS generation may not modulate the microbiome populations. However, overproduction of RONS, found in the Nox4-/- mouse, altered the microbiome. The ileal phenotype observed in Nox4 deficiency seems to be primarily mediated by NOX1, as a Nox1-/yNox4-/- mouse did not demonstrate excessive RONS generation or significant microbiome changes. This study also correlated the loss of ileal Nox1 and Nos2 (iNOS) expression in SAMP/YitFc mice, a model of spontaneously developing Crohn’s disease-like ileitis, with decreased RONS generation in the terminal ileum. In vitro experiments indicated that two NOX1 mutations found in IBD patients, N122H and T497A, led to almost total loss of catalytic activity. Moreover, the location of asparagine in position 122 of NOX1 is conserved across the other 6 human NADPH enzymes. Further investigation of this mutation found that it did not affect membrane trafficking but did prevent the catalytic activity of NOX enzymes. Modeling of asparagine 122 in NOX1 suggests that it forms part of the oxygen reducing cavity. In summary, NOX1 functions to generate superoxide, which rapidly reacts to form peroxynitrite in the GI tract. When this is altered, such as in patients with NOX1 mutations, superoxide and, subsequently, peroxynitrite generation will likely be reduced. This may lead to a failure to respond or defective response to the microbiome. Additionally, the microbiome could interact directly with the ileal epithelium, all of which could cause the inflammation seen in IBD patients.