| Abstract |
Several clinical and epidemiological studies have provided evidence supporting that hepatic inflammation and tissue damage is the critical cause of liver disease directing the development of liver cirrhosis and hepatocellular carcinoma (HCC). Hence, liver disease corresponds to a major healthcare issue, capable of debilitating many patients worldwide and overall, associated with high morbidity and mortality rate. Improved understanding of immune-mediated liver injury may assist the development of therapeutic approaches to this widespread clinical problem.
Operating at the interface of gastrointestinal and systemic blood circulation, the liver is the recipient of a multitude of metabolic, immune and microbial products which must be appropriately handled to maintain tissue homeostasis. Unlike the majority of secondary immune organs, the liver is typified by an over-representation of cellular components of the innate immunity such as Kupffer, dendritic, natural killer (NK) and NKT cells endowed with antigen recognition and regulation of immunogenic versus tolerogenic functions. Disruption of immunological balance as a result of viral infection, autoimmune reactions, excess alcohol consumption or metabolic disease leads to exaggerated and uncontrolled inflammation, hepatocyte death and, eventually, permanent loss of organ function. Irrespective of the cause, liver injury seems to be facilitated by immune effector mechanisms that are common to various liver diseases.
Emerging evidence underscores an instrumental role for NKT cells in orchestrating immune homeostasis and disease pathogenesis in the liver. NKT cells are a heterogeneous group of nonconventional T lymphocytes which are found with highest frequency in the liver. Studies using a mouse model of fulminant hepatitis induced by concanavalin A (ConA) have revealed a major pathogenic role for iNKT cells in liver injury. Upon activation iNKT cells produce copious amounts of immunoregulatory molecules, including the TH1 cytokine interferon-γ (IFN-γ) and the TH2 cytokine interleukin-4 (IL-4) which endow them with the capacity to bridge innate and adaptive responses, direct the licensing of other immune cell types and thus orchestrate inflammatory immune reactions in the liver. Remarkably, the intracellular signaling pathways that mediate the pathogenic functions of iNKT cells remain poorly defined. In this regard, we reasoned that TPL2 (tumor progression locus 2; also known as COT or MAP3K8), a serine-threonine MAP3 kinase mitogen activated protein kinase, could participate in signaling pathways in iNKT cells. TPL2 plays an obligatory role in signal transduction on the MEK/ERK axis downstream of receptors involved in innate and adaptive immunity, including Toll-like receptors, the tumor necrosis factor (TNF) receptor, interleukin-1beta (IL-1β), CD40 and G-protein–coupled receptors (GPCRs).
The aim of this PhD thesis was to characterize in vivo the functional role of TPL2 kinase in immune-mediated liver injury and hepatocellular carcinoma.
In Chapter I, we present experimental evidence demonstrating that TPL2 is a crucial signaling factor in iNKT cells and powerful mediator of hepatic inflammation. We show that genetic ablation of TPL2 in the mouse ameliorates liver injury induced by Concanavalin A and impinges on hallmarks of iNKT cell activation, including production of the effector cytokines IL-4 and IFN-γ, accumulation of neutrophils and licensing and activation of other immune cell types in the liver. The pivotal role of TPL2 in iNKT cell functions is endorsed by adoptive transfer experiments and studies using the iNKT-specific ligand alpha-galactosylceramide which causes mild hepatitis in the mouse in a TPL2-dependent manner. A TPL2 kinase inhibitor mirrors the effects of genetic ablation of TPL2 in vivo and reveals ERK and Akt as the TPL2-regulated signaling pathways responsible for IL-4 and IFN-γ expression through the activation of the transcription factors JunB and NFAT. Overall, data presented in this thesis uncover a novel function of TPL2 as a key mediator of iNKT signal transduction during immune-mediated liver injury. Congruent with this regulatory link, TPL2 is shown to confer pathogenic effects in mouse models of immune-mediated hepatitis. Further understanding of the cellular and molecular processes involved in the development of autoimmune liver diseases may open new avenues for the development of more effective therapies for patients who suffer from inflammatory liver disease. These findings provide new insight into the intracellular pathways that mediate the pathogenic effects of iNKT cells and highlight TPL2 as a promising target suggesting that its modulation has the potential to minimize the severity of inflammatory liver diseases.
In Chapter II, we study the role of TPL2 kinase in HCC. For this purpose, we have used a model of chemical carcinogen induced-HCC in wild type and in tpl2-/- mice to investigate the impact of TPL2 kinase and its possible contribution in inflammatory mechanisms during development of hepatocellular carcinoma. The results showed that TPL2 does not have a significant role in HCC development.
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