End-stage liver disease (ESLD) is among the top 10 causes of death in the US, and more alarmingly, the 4th leading cause of death among the 45-64 year old age group. Orthotopic liver transplantation (LTx) is the most important therapeutic option for these ESLD patients; however, at present, too many donor livers are considered to be marginal due to steatosis or other causes and thus not acceptable for transplantation because they present a high likelihood of triggering severe ischemia reperfusion injury (IRI), an inflammatory response which contributes significantly to graft dysfunction and multiorgan failure after LTx. Building on the well-established roles of neutrophils and macrophages, the proposed work seeks to better understand additional immunological mechanisms behind IRI in normal and marginal livers, both those that trigger IRI and those that can protect from it. In this context, our prior work has shown that certain immune cells such as innate lymphoid cells (ILCs) and T cells are functionally heterogeneous effector cell populations in hepatic IRI based on their regulation by key transcription factors such as retinoic acid receptor-related orphan nuclear receptor ? t (ROR?t). Importantly, ROR?t-expressing type 3 ILCs protect from hepatic IRI in an IL-22- dependent manner, whereas ROR?t-expressing ?? T cells exacerbate hepatic IRI via release of proinflammatory IL-17. Building on this, a hypothesis has emerged that these effector cells also have counterparts, and that the interplay of all of these cells in concert with neutrophils and macrophages can determine IRI. Through mechanistic studies in genetically modified mice, using mouse models and methods that have already been established and that mimic conditions of typical warm IRI in human LTx (i.e., length of ischemia times, livers that are marginal/fatty), the project has two aims: ? Aim 1 will demonstrate the existence and effect of protective T cells in IRI by testing the hypothesis that IL-22 producing Th22 cells are protective effector cells through their regulation by the Tbox transcription factor Tbet in a model of partial warm hepatic IRI. Hepatoprotective Th22 cells will be studied for their IRI-related location, phenotype, transcription profile, effector functions, recruitment, and activation in regular and fatty livers, in wild type mice and in mice genetically deficient for the transcription factors Tbet or ROR?t via flow cytometry, IHC, qRT-PCR, intracellular cytokine staining, and ELISAs. ? Aim 2 will demonstrate the existence and effect of proinflammatory ILCs in IRI by testing the hypothesis that IFN-? and TNF-? producing ILC subsets, such as type 1 ILCs, are proinflammatory effector cells through their regulation by Tbet in a model of partial warm hepatic IRI. Proinflammatory ILC1 cells will be studied for their IRI-related location, phenotype, transcription profile, effector functions, recruitment, and activation in regular and fatty wild type and T and B cell deficient Rag knockout mice, and in Tbet or ROR?t deficient mice on a Rag knockout background, with the aforementioned methods.
For patients with end-stage liver disease ? the 4th leading cause of death in the US among 45-64 year olds ? orthotopic liver transplantation is the most important therapeutic option. Unfortunately, at present too many donor livers are fatty and thus marginal ? a phenomenon that will keep increasing in line with the obesity crisis in the US ? and thus not acceptable for transplantation because they present a high likelihood of triggering severe ischemia reperfusion injury, an inflammatory response which contributes significantly to graft dysfunction and multiorgan failure after liver transplantation. The proposed work seeks to better understand innate and adaptive immunological mechanisms behind liver IRI, both those that trigger IRI and those that can protect from it, to lay early groundwork for future cellular immunotherapy approaches that limit hepatic IRI of marginal livers, and thereby enable more end-stage liver disease patients to benefit from successful life-saving orthotopic liver transplantation.
