Liver injury results from viral hepatitis infection, from other infections such as Dengue Fever, from chemicals such as ethanol, and from biological toxins. Primary hepatocellular injury in complicated by immunopathology mediated by the innate immune system, but the mechanisms are poorly understood. This is partly because experimental models such as ischemia-reperfusion and CCl4 administration impact on many liver cell types simultaneously. To clarify the mechanisms of liver immunopathology we have created a novel model to study the consequences of hepatocyte death. A gene therapy vector is used to express the Diphtheria Toxin Receptor specifically in mouse hepatocytes, then administration of Diphtheria Toxin results in liver injury. There is rapid up-regulation of chemokine genes and a mixed myeloid influx, coinciding with a sharp elevation in serum Alanine Aminotransaminase (ALT). We made two striking observations. First, the pathology is mediated via Toll-Like Receptor-3 (TLR-3), suggesting the hypothesis that RNA is the important molecular trigger. Second, when we dissociate and isolate different subsets of liver cells, the innate immune response is by far most dramatic in hepatocytes. This gives us a second hypothesis that the hepatocytes are the prime movers in sensing hepatocellular injury and orchestrating the innate immune response.
In Specific Aim 1 we directly address the RNA DAMP through experiments to formally prove that RNA is the signal, and to identify what form(s) of RNA are likely to be relevant.
In Specific Aim 2, we dissect the liver injury to determine the role of different forms of hepatocyte death, test whether hepatocytes directly recognize the TLR-3 mediated signal, and then test the hypothesis that the capacity of hepatocytes to take up their dead neighbors through the AsialoGylcoprotein Receptor is critical for such recognition. So far we have focused our preliminary work on the hepatocyte-specific Diphtheria Toxin model, but in Specific Aim 3 we test the hypothesis that RNA- and TLR3-dependent liver injury occurs in a more physiological context, using an experimental model of the immunopathology induced by Hepatitis B Virus. These experiments will give insight into the mechanism of an under-explored mechanism of liver injury, mediated through TLR3, and test its wider applicability. Two aspects of the proposed work enhance its global significance. First, we provoke injury using Diphtheria Toxin, which is a potential biological threat. New ways to ameliorate Diphtheria Toxin-induced cell damage will be of great value in protecting the population in the event of a biological attack Secondly, new experiments in Specific Aims 1 and 3 use a recombinant RNase fusion protein that is genetically engineered for use as a human therapeutic agent, and is already in Phase II clinical trials. This means that if the in vivo degradation of RNA in fact ameliorates liver immunopathology, there is an immediate pathway to translate this discovery to clinical medicine.

Public Health Relevance

Many diseases kill liver cells, but some of the injury is caused by an undesirable immune response. In this project we will understand the immune side effects of dying liver cells, and test methods to prevent them. This might be useful in severe hepatitis from any cause, including viruses, chemical poisons and biological toxins.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI114630-03
Application #
9199394
Study Section
Innate Immunity and Inflammation Study Section (III)
Program Officer
Singleton, Kentner L
Project Start
2015-02-10
Project End
2020-01-31
Budget Start
2017-02-01
Budget End
2018-01-31
Support Year
3
Fiscal Year
2017
Total Cost
$521,263
Indirect Cost
$197,281
Name
University of Washington
Department
Pathology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Brempelis, Katherine J; Yuen, Sebastian Y; Schwarz, Nicole et al. (2017) Central role of the TIR-domain-containing adaptor-inducing interferon-? (TRIF) adaptor protein in murine sterile liver injury. Hepatology 65:1336-1351
Crispe, Ian N (2016) Hepatocytes as Immunological Agents. J Immunol 196:17-21
Grakoui, Arash; Crispe, Ian Nicholas (2016) Presentation of hepatocellular antigens. Cell Mol Immunol 13:293-300