Dissecting mechanisms of liver failure using conditional TNF receptor-associated factor 2 (TRAF-2)-deficient mice

Supervisor : Patrick Bertolino (in collaboration with Dr. Robert Brink from the Garvan Institute).

P. Bertolino@centenary.usyd.edu.au, ph : 9565 6186 Fax : 9565 6101

Liver lab (2^nd floor of the Centenary Institute building)

Embryonic and postnatal death due to severe acute hepatitis is a common theme amongst mice lacking genes of the NF-kB signalling pathway. Hepatitis induced death can be prevented or delayed in many of these mice when crossed onto TNF deficient backgrounds. The NF-kB signalling response is thus crucial in protection of hepatocytes from TNF induced death. The signalling response to TNF is largely mediated by TRAF2, a signalling protein that recruits to the receptor complex kinases that initiate NF-kB activation. TRAF-2 deficient mice are embryonic lethal. Thus, our collaborator, Dr. R. Brink (Garvan Institute, Sydney), has developed transgenic mice in which deletion of the TRAF2 gene can be induced. When TRAF2 is deleted, these mice develop severe acute hepatitis and die ~5-9 days after the induction of TRAF2 deficiency. The nature the hepatitis and mechanisms involved remain unknown. This project aims to undertake the following initiatives to further characterise the hepatitis:

1) First, we would like to determine whether hepatitis involves immune cells. This will be performed by characterising the phenotype of lymphocytes present within the liver during the development of hepatitis. The liver contains a population of NK and NKT cells as well as resident B cells, CD4+ and CD8+ T lymphocytes. The total number of lymphocytes and their activation status will be analysed and quantified at different time points.

2) To determine whether the hepatitis is due to an intrinsic sensitivity of liver cells to death pathways, we will analyse the effect of TRAF-2 deficiency in vitro using purified primary hepatocytes. If TRAF-2 deficient hepatocytes die, we will try to analyse the signalling pathways involved in this process and reverse hepatocyte death by activating the NF-kB signalling pathway using cytokines such as IL-6.

3) Given that many of the hepatitis phenotypes seen in mice lacking genes of the NF-kB signalling pathway can be diminished in the absence of TNF, we will investigate the role of TNF in hepatitis. This will be performed by crossing these mice to TNF-deficient mice and by generating bone marrow radiation chimeras expressing TRAF-2 and TNF only on hepatocytes or bone marrow-derived cells.

All transgenic mice are currently breeding in our animal facility. Most techniques are well established in our laboratory. This project will involve mouse work (mouse handling, taking lymph nodes and other organs, tail injection etc..), flow cytometry, immunochemistry, cell culture, Western blot etc…

By using a unique mouse model of fulminant hepatitis, this project will allow us to dissect the critical role of TRAF-2 and TNF in hepatitis. These findings would provide critical information about the role of these molecules in liver damage.