HIV MOLECULAR PATHOGENESIS LABORATORY

Lab head: Tony Cunningham
Location: Centre for Virus Research, Westmead Millennium Institute, Westmead, NSW, 2145

A fascinating future for HIV research lies ahead as the HIV scientific community focuses on the two major aims of preventing HIV infection through vaccine/microbicide development and curing or eliminating HIV from antiretroviral-controlled chronically infected people.

Lab members: 1 post doc, 1 Research assistant
Funding: NHMRC / ACH2

Characterising ‘Bona-Fide’ Plasmacytoid Dendritic Cell Responses in Initial HIV Infection

Primary supervisor: Najla Nasr

Introduction

More than 30 years after its discovery, human immunodeficiency virus-1 (HIV) still propagates worldwide and as of 2016, 36.7 million individuals are living with HIV. Although the advent of antiretroviral therapy has meant HIV can be managed as a chronic disease, there is currently no effective vaccine or microbicide that can block the sexual transmission of HIV. One of the key events during initial HIV infection is the rapid recruitment of plasmacytoid dendritic cells (pDCs) to mucosal sites of exposure. As HIV blocks IFN responses in its target cells such as myeloid dendritic cells (DCs) and CD4+ T cells, pDCs likely serve as the major source of antiviral interferons (IFNs) during HIV infection. In addition, pDCs have been shown to produce inflammatory cytokines such as TNF-α, IL-6, IL-13 and IL-12 p40 (Saidi et al., 2016) and upregulate CD80, CD83 and CD86 expression when exposed to HIV inocula (Fonteneau et al., 2004, Yonezawa et al., 2003, Schmidt et al., 2005).

However, recent studies utilising high-dimensional single-cell RNA sequencing have redefined the repertoire of DCs in peripheral blood, revealing a novel myeloid DC subset that expresses classical pDC markers, termed AXL+ SIGLEC6+ (AS)DCs (Villani et al., 2017). ‘Bona-fide’ pDCs were redefined as purely interferon-producing cells separate from T cell stimulatory capacity, and this has added to uncertainty over previously reported responses of pDCs to HIV and whether some of these responses might instead be attributed to ASDCs. Indeed, ASDCs were shown to produce IL-12 p40 upon toll-like receptor 7 stimulation whilst ‘bona-fide’ pDCs did not (Villani et al., 2017). As such, it is critical to distinguish the soluble factors produced by these two cell types and their cellular responses to properly understand their roles during initial HIV infection.

Aims and hypothesis of the project:

·      Characterising the gene expression of ‘bona-fide’ pDCs and ASDCs upon exposure to HIV, specifically examining the expression of IFNs, proinflammatory cytokines and chemokines

·      Characterising the cellular infection and maturation status of HIV-exposed ‘bona-fide’ pDCs and ASDCs by flow cytometry

·      Assessing the effects of IFNs, proinflammatory cytokines and chemokines on HIV infection of different T cell subsets and dendritic cells.

We hypothesise that pDCs can produce both IFNs and proinflammatory cytokines and chemokines in response to HIV, but have a distinct signature to ASDCs, which may account for some of the soluble factors traditionally ascribed to pDCs. In addition, we hypothesise that pDCs are resistant to HIV infection due to their production of IFNs whilst ASDCs are more permissive to infection, and that HIV matures both of these cell types.

Techniques used in this project:

•       Primary cell culture including the isolation of peripheral blood mononuclear cells from peripheral blood by Ficoll-Paque density centrifugation, and magnetic sorting of blood DCs

•       Fluorescence activated cell sorting (FACS) for isolation of ‘bona-fide’ pDCs and ASDCs from enriched blood DCs

•       HIV infection of sorted cells in a PC3 laboratory

•       Flow Cytometry sample preparation, acquisition and analysis


Discipline: Applied Medical Sciences, Westmead
Co-supervisors: Orion Tong, Tony Cunningham
Keywords: Microbiology, Molecular biology, HIV infection
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