Liver fibrosis a consequence of chronic liver disease is a major cause of mortality and morbidity. Studies in mouse models have demonstrated an integral role for interleukin-33 (IL-33) driven group 2 innate lymphocyte cell (IL2C) expansion and activation in hepatic fibrosis. In humans, the role of ILCs is not well defined and human hepatic ILC2 populations have not previously been investigated. We provide preliminary data that establishes the presence of IL-13 expressing ILC2 populations in normal and cirrhotic human liver and to suggest that natural killer T (NKT) cells are involved in the regulation of ILC2s at this site. We hypothesize that: IL-33 produced by activated NKT cells in the inflamed liver drives expansion and activation of hepatic ILC2s which promote fibrosis through IL-13-dependent effects on hepatic stellate cells (HSCs) and macrophages.
Three specific aims are directed at testing this hypothesis:
In Specific Aim 1 we will examine the levels, phenotype and functional characteristics of ILC2s in healthy and cirrhotic human liver. In addition to direct ex vivo flow cytometric analysis, ILC2s will be isolated and expanded from tissue using an established protocol. The effects of IL-33 on migration, cytokine/chemokine production, gene regulation, and IL-33 (ST2) receptor expression will be measured with chemotaxis, multiplex-ELISA, flow cytometry and RT-PCR.
Specific Aim 2 is designed to explore the direct effects of ILC2s on the activation of HSCs and monocyte maturation. Immunohistochemistry, flow cytometry and co-culture experiments will be used to correlate ILC2 levels with fibrosis markers/stage and accumulation/ activation of pro-fibrogenic kupffer cells and/or alternatively activated macrophages.
In Specific Aim 3 we will explore the regulation of fibrosis by IL-33, NKTs and ILC2s in vivo. We will use the AFC8-hu HSC/Hep model which contains human liver cells and a human immune system. Fibrosis is inducible in this model and we propose to examine ILC2s, NKTs, HSC activation and expression of human fibrogenic genes at various stages of fibrosis. As this model requires co-transplantation of cells of haematopoietic origin to develop fibrosis, we will selectively transfer ILC2s/NKTs in the presence and absence of sST2 to dissect the contribution of each of these components to fibrosis in the liver. In depth characterization of human ILC2s in diseased compared to healthy liver will delineate their role in pathology and determine if targeting this population represents an effective novel strategy to prevent/treat hepatic fibrosis.
Increasingly, murine group-2 innate lymphocyte cells (ILC2s) are being implicated in promoting fibrosis in various organs including the liver. Their contributio to hepatic fibrosis in humans is unknown. This study is designed to determine the role played by human ILC2s in hepatic fibrosis induced by chronic liver diseases of different etiologies. The identification of a novel target population, namely ILC2s, which could ultimately be manipulated, would represent a paradigm-shift in how we treat fibrosis.
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