Identification of novel innate immune mechanisms regulating inflammation in the gastrointestinal tract

The Gastro-Intestinal (GI) tract is a unique region in the body. Our innate immune system retains a fine homeostatic balance between avoiding inappropriate inflammatory responses against the myriad commensal microbes residing in the gut while also remaining active enough to prevent invasive pathogenic attack. The intestinal epithelium represents the frontline of this interface. It has long been known to act as a physical barrier preventing the lumenal bacteria of the gastro-intestinal tract from activating an inflammatory immune response in the immune cells of the underlying mucosa. However, in recent years, an appreciation has grown surrounding the role played by the intestinal epithelium in regulating innate immune responses, both in the prevention of infection and in maintaining a homeostatic environment through modulation of innate immune signalling systems. The aim of this thesis was to identify novel innate immune mechanisms regulating inflammation in the GI tract. To achieve this aim, we chose several aspects of regulatory mechanisms utilised in this region by the innate immune system. We identified several commensal strains of bacteria expressing proteins containing signalling domains used by Pattern Recognition Receptors (PRRs) of the innate immune system. Three such bacterial proteins were studied for their potentially subversive roles in host innate immune signalling as a means of regulating homeostasis in the GI tract. We also examined differential responses to PRR activation depending on their sub-cellular localisation. This was investigated based on reports that apical Toll-Like Receptor (TLR) 9 activation resulted in abrogation of inflammatory responses mediated by other TLRs in Intestinal Epithelial Cells (IECs) such as basolateral TLR4 activation. Using the well-studied invasive intra-cellular pathogen Listeria monocytogenes as a model for infection, we also used a PRR siRNA library screening technique to identify novel PRRs used by IECs in both inhibition and activation of inflammatory responses. Many of the PRRs identified in this screen were previously believed not to be expressed in IECs. Furthermore, the same study has led to the identification of the previously uncharacterised TLR10 as a functional inflammatory receptor of IECs. Further analysis revealed a similar role in macrophages where it was shown to respond to intracellular and motile pathogens such as Gram-positive L.monocytogenes and Gram negative Salmonella typhimurium. TLR10 expression in IECs was predominantly intracellular. This is likely in order to avoid inappropriate inflammatory activation through the recognition of commensal microbial antigens on the apical cell surface of IECs. Moreover, these results have revealed a more complex network of innate immune signalling mechanisms involved in both activating and inhibiting inflammatory responses in IECs than was previously believed. This contribution to our understanding of innate immune regulation in this region has several direct and indirect benefits. The identification of several novel PRRs involved in activating and inhibiting inflammation in the GI tract may be used as novel therapeutic targets in the treatment of disease; both for inducing tolerance and reducing inflammation, or indeed, as targets for adjuvant activation in the development of oral vaccines against pathogenic attack.

Novel insights into the inflammatory basis of gastrointestinal disease

It has become clear that inflammation is beneficial to man, there are situations though that the inflammatory response causes damage to the host that is harmful to health. When the inflammatory response fails or is too strong, the health of the host is damaged and disease can occur. The implication of intestinal disease caused by an ineffective immune response is of great social and economic burden to society. The overarching purpose of this thesis is to assess inflammatory signalling targets associated with immune mediated disorders such as IBD, IBS and inflammatory liver disease. By assessing these targets and modifying their function I hope to contribute and expand further the pre-existing information on these disorders and improve the therapeutic interventions available in these debilitating conditions. I will assess the role of inflammation in disorders of the GI tract and liver IBD, IBS, hepatic inflammatory injury and furthermore, I will use pharmaceutical agents to activate and suppress components of the immune system. I will examine the inflammatory response in experimental models of disease for IBD and liver injury, I will attempt to alter these pathways using pharmaceutical intervention to delineate the disease causing mechanism that may lead to clinically relevant therapeutic interventions. In regards to IBS, I will attempt to improve the existing knowledge that exists in relation to the pathogenesis of this functional bowel disorder. I will attempt to define a mechanism by which the low grade mucosal inflammation that has been demonstrated by others arises and what this inflammation is induced by. The overall aim of this thesis is to attempt to further understand the mechanisms behind GI and liver disease. Looking at the inflammatory response in these specific conditions and how they can be altered may lead to exciting new therapies for inflammatory conditions in the gastrointestinal tract.

Iterative reconstruction as a novel method of radiation dose reduction at computed tomography in patients with Crohn's disease

Prior work of our research group, that quantified the alarming levels of radiation dose to patients with Crohn’s disease from medical imaging and the notable shift towards CT imaging making these patients an at risk group, provided context for this work. CT delivers some of the highest doses of ionising radiation in diagnostic radiology. Once a medical imaging examination is deemed justified, there is an onus on the imaging team to endeavour to produce diagnostic quality CT images at the lowest possible radiation dose to that patient. The fundamental limitation with conventional CT raw data reconstruction was the inherent coupling of administered radiation dose with observed image noise – the lower the radiation dose, the noisier the image. The renaissance, rediscovery and refinement of iterative reconstruction removes this limitation allowing either an improvement in image quality without increasing radiation dose or maintenance of image quality at a lower radiation dose compared with traditional image reconstruction. This thesis is fundamentally an exercise in optimisation in clinical CT practice with the objectives of assessment of iterative reconstruction as a method for improvement of image quality in CT, exploration of the associated potential for radiation dose reduction, and development of a new split dose CT protocol with the aim of achieving and validating diagnostic quality submillisiever t CT imaging in patients with Crohn’s disease. In this study, we investigated the interplay of user-selected parameters on radiation dose and image quality in phantoms and cadavers, comparing traditional filtered back projection (FBP) with iterative reconstruction algorithms. This resulted in the development of an optimised, refined and appropriate split dose protocol for CT of the abdomen and pelvis in clinical patients with Crohn’s disease allowing contemporaneous acquisition of both modified and conventional dose CT studies. This novel algorithm was then applied to 50 patients with a suspected acute complication of known Crohn’s disease and the raw data reconstructed with FBP, adaptive statistical iterative reconstruction (ASiR) and model based iterative reconstruction (MBIR). Conventional dose CT images with FBP reconstruction were used as the reference standard with which the modified dose CT images were compared in terms of radiation dose, diagnostic findings and image quality indices. As there are multiple possible user-selected strengths of ASiR available, these were compared in terms of image quality to determine the optimal strength for this modified dose CT protocol. Modified dose CT images with MBIR were also compared with contemporaneous abdominal radiograph, where performed, in terms of diagnostic yield and radiation dose. Finally, attenuation measurements in organs, tissues, etc. with each reconstruction algorithm were compared to assess for preservation of tissue characterisation capabilities. In the phantom and cadaveric models, both forms of iterative reconstruction examined (ASiR and MBIR) were superior to FBP across a wide variety of imaging protocols, with MBIR superior to ASiR in all areas other than reconstruction speed. We established that ASiR appears to work to a target percentage noise reduction whilst MBIR works to a target residual level of absolute noise in the image. Modified dose CT images reconstructed with both ASiR and MBIR were non-inferior to conventional dose CT with FBP in terms of diagnostic findings, despite reduced subjective and objective indices of image quality. Mean dose reductions of 72.9-73.5% were achieved with the modified dose protocol with a mean effective dose of 1.26mSv. MBIR was again demonstrated superior to ASiR in terms of image quality. The overall optimal ASiR strength for the modified dose protocol used in this work is ASiR 80%, as this provides the most favourable balance of peak subjective image quality indices with less objective image noise than the corresponding conventional dose CT images reconstructed with FBP. Despite guidelines to the contrary, abdominal radiographs are still often used in the initial imaging of patients with a suspected complication of Crohn’s disease. We confirmed the superiority of modified dose CT with MBIR over abdominal radiographs at comparable doses in detection of Crohn’s disease and non-Crohn’s disease related findings. Finally, we demonstrated (in phantoms, cadavers and in vivo) that attenuation values do not change significantly across reconstruction algorithms meaning preserved tissue characterisation capabilities with iterative reconstruction. Both adaptive statistical and model based iterative reconstruction algorithms represent feasible methods of facilitating acquisition diagnostic quality CT images of the abdomen and pelvis in patients with Crohn’s disease at markedly reduced radiation doses. Our modified dose CT protocol allows dose savings of up to 73.5% compared with conventional dose CT, meaning submillisievert imaging is possible in many of these patients.