VZV Research Laboratory

Lab head: Dr Allison Abendroth
Location: Blackburn Building D06 Camperdown

Immune evasion and DCs; Varicella zoster virus evasion of the TLR9 pathway

Primary supervisor: Allison Abendroth

Previously we have shown that VZV infected plasmacytoid dendritic cells (pDCs) downregulate interferon alpha (IFNa) production via the TLR9 pathway. Specifically, in VZV infected cultures, pDCs treated with the TLR9 agonist ODN2216 failed to upregulate IFNa. This was despite only 40-70% of cells becoming VZV infected (Huch et al., 2010). It was shown that a secreted factor was unlikely to be mediating the observed complete inhibition and was suggested that it was contact dependent between infected and uninfected pDCs. The TLR9 pathway can upregulate IFNathrough 3 main mechanisms, via IRF7, the MAPK/AP-1 pathway and via NF-kB signalling. However ODN2216 is known to be a weak stimulator of the NF-kB pathway and therefore the inhibition mediated via VZV is likely to be via the other pathways. Studies have shown that VZV infection upregulates signaling through the MAPK and therefore VZV may be affecting IRF7 function. TLR9 and IRF7 are only expressed in a subset of cells within the body and are normally limited to peripheral lymphocytes, dendritic cells,and cells of the spleen, and lymph nodes. However MUTZ-3 derived DCs (MuDCs) have been shown to express TLR9. Therefore the aims of this project are to determine the suitability of using MuDCs as a model to investigate TLR9/IRF7 signaling, then determine the mechanism behind the VZV-mediated downregulation in IFNaproduction.

MUTZ-3 cells will be differentiated into MuDCs by culturing in GM-CSF, IL-4 and TNF-a for 7 days. Their ability to produce IFNain response to ODN treatment in both VZV and mock-infected cultures will be assessed (ELISA/Flow). Intracellular staining for VZV and IFNa will be performed to confirm if all cells within the infected cultures are unable to produce IFNa. Western blotting will then be performed for IRF7, as well as its phosphorylated and ubiquitinated forms. Other components in the pathway such as MyD88 , IRAK4 and TRAF6 will also be studied.  Components of this work may also be translated into primary human plasmacytoid dendritic cells. The results obtained will provide valuable new information to our understanding of how VZV has evolved strategies to evade immune detection and become such a successful and ubiquitous human pathogen.

This project will involve a molecular and cell biology techniques including specialized primary human dendritic cell culture, mammalian cell culture, virus culture, cell infection, multi-colour flow cytometry, western blotting, immunofluorescent staining and confocal microscopy.

Discipline: Infectious diseases and Immunology
Co-supervisors: Barry Slobedman, Megan Steain
Keywords: Virology, immunobiology, Dendritic cells