Chronic glucocorticoid overexposure predisposes to the development of atherosclerotic cardiovascular disease in humans. Cholestatic liver disease is associated with increased plasma glucocorticoid... Show moreChronic glucocorticoid overexposure predisposes to the development of atherosclerotic cardiovascular disease in humans. Cholestatic liver disease is associated with increased plasma glucocorticoid levels. Here we determined, in a preclinical setting, whether the chronic presence of cholestatic liver disease also induces a concomitant negative impact on atherosclerosis susceptibility. Hereto, regular chow diet-fed atherosclerosis-susceptible hypercholesterolemic apolipoprotein E (APOE) knockout mice were treated with the bile duct toxicant alpha‑naphthylisothiocyanate (ANIT) for 8 weeks. ANIT exposure induced the development of fibrotic cholestatic liver disease as evident from collagen deposits and compensatory bile duct hyperproliferation within the liver and the rise in plasma levels of bilirubin (+60%; P<0.01) and bile acids (10-fold higher; P<0.01). Adrenal weights (+22%; P<0.01) and plasma corticosterone levels (+72%; P<0.01) were increased in ANIT-treated mice. In contrast, atherosclerosis susceptibility was not increased in response to ANIT feeding, despite the concomitant increase in plasma free cholesterol (+30%; P<0.01) and cholesteryl ester (+42%; P<0.001) levels. The ANIT-induced hypercorticosteronemia coincided with marked immunosuppression as judged from the 50% reduction (P<0.001) in circulating lymphocyte numbers. However, hepatic glucocorticoid signaling was not enhanced after ANIT treatment. It thus appears that the immunosuppressive effect of glucocorticoids is uncoupled from their metabolic effect under cholestatic disease conditions. In conclusion, we have shown that cholestatic liver disease-associated endogenous glucocorticoid overexposure does not increase atherosclerosis susceptibility in APOE knockout mice. Our studies provide novel preclinical evidence for the observations that the hypercholesterolemia seen in cholestatic human subjects does not translate into a higher risk for atherosclerotic cardiovascular disease. Show less
This thesis describes the development and use of a novel technology for single-cell fate mapping, called cellular barcoding. With this technology, unique and heritable genetic tags (barcodes) are... Show moreThis thesis describes the development and use of a novel technology for single-cell fate mapping, called cellular barcoding. With this technology, unique and heritable genetic tags (barcodes) are introduced into na_ve T cells. Using cellular barcoding, we investigated I) how different antigen-specific CD8+ T cell clones contribute to the formation of effector and memory T cell subsets II) at what point during in vivo CD8+ T cell differentiation fate decisions take place and III) to what extent the clonal expansion of individual antigen-specific CD8+ T cells shapes the overall response magnitude. Our experiments demonstrate that most if not all effector and memory T cells are progeny of the same na_ve T cells and that the decision to develop into either subset is taken after the first cell division. Furthermore, we show that the overall T cell response magnitude is primarily controlled by the extent of clonal expansion, as the efficiency by which na_ve T cells are recruited into the response is remarkably constants. Quantification of barcode abundances revealed that individual antigen-specific T cells produce highly variable numbers of daughter cells, and that this strong disparity in output is established during the first phase of infection. Show less
