Development and use of a mouse model to investigate β-lactam hypersensitivity reactions

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

Activation of Flucloxacillin-Specific CD8+ T-Cells With the Potential to Promote Hepatocyte Cytotoxicity in a Mouse Model

There are currently no animal models of drug-induced liver injury (DILI) where the adaptive immune system has been shown to damage the liver. Thus, it is difficult to explore the mechanistic basis of the tissue injury. The aim of this study was to use C57BL/6 CD4+-deficient mice with a mutation in the αβ gene encoding for Major histocompatibilty complex (MHC) class II molecules to (1) develop a mouse model of flucloxacillin sensitization, (2) explore whether drug-specific CD8+ kill primary hepatocytes, and (3) analyze perturbations in liver integrity following oral exposure to flucloxacillin. CD8+ T-cells from lymph nodes of flucloxacillin-sensitized mice were stimulated to proliferate, secrete interferon (IFN-γ) and granzyme B, and induce hepatocyte apoptosis in a concentration-dependent manner following ex vivo stimulation. The T-cell response was antigen-specific; T-cells were not activated with other β-lactam antibiotics. Furthermore, T-cell responses only occurred in the presence of flucloxacillin-pulsed antigen presenting cells. In separate experiments, flucloxacillin-specific T-cells were induced to migrate to the mesenteric lymph nodes using retinoic acid, prior to administration of oral flucloxacillin, and analysis of plasma biomarkers of liver injury. Oral exposure to flucloxacillin resulted in mild elevations in alanine aminotransferase, liver, and gall bladder leukocyte infiltration and a marked swelling of the gall bladder. Thus, CD4+-deficient mice represent a promising model to study the role of the adaptive immune system in DIL

Activation of flucloxacillin-specific CD8+ T-cells with the potential to promote hepatocyte cytotoxicity in a mouse model

There are currently no animal models of drug-induced liver injury (DILI) where the adaptive immune system has been shown to damage the liver. Thus, it is difficult to explore the mechanistic basis of the tissue injury. The aim of this study was to use C57BL/6 CD4+-deficient mice with a mutation in the αβ gene encoding for Major histocompatibilty complex (MHC) class II molecules to (1) develop a mouse model of flucloxacillin sensitization, (2) explore whether drug-specific CD8+ kill primary hepatocytes, and (3) analyze perturbations in liver integrity following oral exposure to flucloxacillin. CD8+ T-cells from lymph nodes of flucloxacillin-sensitized mice were stimulated to proliferate, secrete interferon (IFN-γ) and granzyme B, and induce hepatocyte apoptosis in a concentration-dependent manner following ex vivo stimulation. The T-cell response was antigen-specific; T-cells were not activated with other β-lactam antibiotics. Furthermore, T-cell responses only occurred in the presence of flucloxacillin-pulsed antigen presenting cells. In separate experiments, flucloxacillin-specific T-cells were induced to migrate to the mesenteric lymph nodes using retinoic acid, prior to administration of oral flucloxacillin, and analysis of plasma biomarkers of liver injury. Oral exposure to flucloxacillin resulted in mild elevations in alanine aminotransferase, liver, and gall bladder leukocyte infiltration and a marked swelling of the gall bladder. Thus, CD4+-deficient mice represent a promising model to study the role of the adaptive immune system in DIL

Chronic endoplasmic reticulum stress in myotonic dystrophy type 2 promotes autoimmunity via mitochondrial DNA release

Abstract Myotonic dystrophy type 2 (DM2) is a tetranucleotide CCTG repeat expansion disease associated with an increased prevalence of autoimmunity. Here, we identified an elevated type I interferon (IFN) signature in peripheral blood mononuclear cells and primary fibroblasts of DM2 patients as a trigger of chronic immune stimulation. Although RNA-repeat accumulation was prevalent in the cytosol of DM2-patient fibroblasts, type-I IFN release did not depend on innate RNA immune sensors but rather the DNA sensor cGAS and the prevalence of mitochondrial DNA (mtDNA) in the cytoplasm. Sublethal mtDNA release was promoted by a chronic activation of the ATF6 branch of the unfolded protein response (UPR) in reaction to RNA-repeat accumulation and non-AUG translated tetrapeptide expansion proteins. ATF6-dependent mtDNA release and resulting cGAS/STING activation could also be recapitulated in human THP-1 monocytes exposed to chronic endoplasmic reticulum (ER) stress. Altogether, our study demonstrates a novel mechanism by which large repeat expansions cause chronic endoplasmic reticulum stress and associated mtDNA leakage. This mtDNA is, in turn, sensed by the cGAS/STING pathway and induces a type-I IFN response predisposing to autoimmunity. Elucidating this pathway reveals new potential therapeutic targets for autoimmune disorders associated with repeat expansion diseases