title: Development and use of a mouse model to investigate β-lactam hypersensitivity reactions
Creators: name: Nattrass, Ryan
Publisher: name: University of Liverpool Repository
Year: 2015

description: β-lactam hypersensitivity reactions can be severe and are extremely difficult to predict. Drug-specific T-cells have been identified in blood of patients presenting with cutaneous and hepatic hypersensitivity reactions, indicating that they play a role in the disease pathogenesis. Animal models are highly effective tools that have been used extensively to dissect mechanisms of disease and pathways of disease progression; however, animal models of drug hypersensitivity reactions have proven difficult to develop. The aims of this thesis were to utilize 3 β-lactam antibiotics amoxicillin, piperacillin and flucloxacillin to explore antigen-specific T-cell responses in the mouse and to attempt to develop a model of T-cell-mediated drug-induced liver damage. The project utilized the C57/Bl6 CD4+ T-cell deficient mouse with a mutation in the αβ gene encoding for MHC class II molecules, which has previously been used to investigate skin sensitization to drugs. In initial experiments, amoxicillin-specific CD8+ T-cell responses were detected both in vivo and ex vivo. Sensitization was obtained through painting of the drug onto the skin of mice that had been depleted of CD4+ T-cells, which are thought to exert regulatory/suppressor functions. On completion of the sensitization protocol, draining lymph node cells were removed and the drug-specific T-cell response was detected through analysis of proliferation and IFN-γ release. In contrast, proliferative responses and cytokine release were not detected with cells from vehicle control mice. The study was expanded to include 3 β-lactam antibiotics. Activation of CD8+ T-cells was readily detectable following sensitization with flucloxacillin. In contrast, only weak ex-vivo proliferative responses were detected following sensitization with piperacillin, which may relate to the fact that piperacillin preferentially activates CD4+ T-cells in hypertensive human patients. Drug-specific T-cell clones from human patients were generated and tested alongside murine counterparts to provide a detailed assessment of cross-reactivity and variability in the drug-specific T-cell response between species. Amoxicillin and flucloxacillin demonstrated cross-reactivity with both human and murine drug-specific T-cells. Piperacillin cross-reactivity was difficult to assess in mouse. However, human piperacillin-specific T-cells displayed no evident cross-reactivity with amoxicillin or flucloxacillin. The ex vivo activation of flucloxacillin-specific CD8+ T-cells from sensitised mice was discovered to be dependent on the presence of APCs. The concentration of APCs added to cultures of drug-specific draining lymph node cells was directly correlated with the amount of CD8+ T-cell activation. In fact, the removal of APCs ablated the proliferative response and IFNγ secretion when APCs were added to flucloxacillin re-challenged ex vivo cultures of flucloxacillin-specific CD8+ T-cells from the draining lymph nodes of sensitised mice. There are currently no animal models of drug-induced liver injury where the adaptive immune system has been shown to damage hepatocytes. It is therefore difficult to explore the mechanistic basis of the tissue injury. Thus, an aim of the project was to characterize the immunogenicity of flucloxacillin and explore whether flucloxacillin-responsive CD8+ T-cells damage hepatocytes. In initial experiments sensitization was achieved through epicutaneous application. CD8+ T-cells from draining lymph nodes of the flucloxacillin-treated mice proliferated in a concentration-dependent manner following ex vivo secondary stimulation. The proliferative response was associated with IFN-γ and granzyme B release. Flucloxacillin-specific hepatocyte toxicity and apoptosis was observed when CD8+ T-cells were cultured with dendritic cells and flucloxacillin for 24h, washed and transferred to the hepatocyte cultures. In contrast, hepatocyte killing was not detected in with T-cells from vehicle control mice. In separate experiments, flucloxacillin-specific T-cells were forced to migrate to the mesenteric lymph nodes using retinoic acid, prior to administration of oral flucloxacillin for 10 days, followed by analysis of liver histology and plasma biomarkers of liver injury. Oral exposure resulted in gall bladder swelling, hepatic mononuclear cell infiltration (especially around the bile ducts) and mild elevations in plasma ALT. This work has highlighted the usefulness of animal models in studying disease whilst also acting as evidence to the difficulty in developing such models. The experiments show successful sensitization of mice against different β-lactam antibiotics and a promising model to study the role of the adaptive immune system in flucloxacillin-induced cholestatic liver injury.