The role of monocyte subsets in HIV-1 pathogenesis and their contribution to the viral reservoir.

Cells of macrophage lineage have long been known to be a target for HIV-1 infection. Given their potential ability to live for extensive periods of time, to constitutively produce low levels of virus with minimal cytopathic effects and to migrate into tissues and establish local infection, HIV-1 infected macrophages significantly contribute to the viral reservoir and are a major hurdle to clearance of HIV-1 infection. Understanding the origin of infected macrophages and the mechanisms of infection is important in targeting infection in this compartment. Circulating blood monocytes are generally considered to be resistant to HIV-1 infection. However, we have previously shown that a subset of monocytes (CD16+ monocytes which, unless otherwise specified in this thesis, include both CD14++CD16+ and CD14+CD16++ monocytes) are more susceptible to HIV-1 infection than the majority of monocytes (CD14++CD16-). To investigate the role of the CD16+ monocyte subset in HIV-1 pathogenesis and its contribution to the viral reservoir we have investigated HIV-1 infection of CD16+ monocytes, the potential of a monocyte scavenger protein CD163 for clinical application and the cell biology of family of proteins called tetraspanins and their role in HIV-1 infection of monocytes and macrophages. Chapter 2 -HIV-1 infection of CD16+ monocytes Overview: Our laboratory has previously shown the CD16+ monocyte subset is preferentially infected in HIV-1 infected individuals and so may be a major component of the viral reservoir. To investigate this we have measured the contribution of HIV-1 infected CD16+ monocytes to the viral reservoir in comparison to the major monocyte subset and to the known reservoir of resting memory T cells. We have then examined the mechanisms that account for increased susceptibility to HIV-1 infection that is demonstrated by CD16+ monocytes when compared to CD14++CD16-monocytes. Results: This chapter quantifies HIV-1 DNA in monocyte subsets from HIV-1 infected donors compared to resting memory T cells and shows CD16+ monocytes contain more HIV-1 DNA than CD14++CD16-monocytes and comparable levels to resting memory T cells. We have confirmed that CD16+ monocytes can produce infectious virus through experiments using monocyte subsets isolated from the blood of HIV-1 infected donors co-cultured with activated uninfected donor PBMC. Our investigations of the mechanisms which account for increased infection in this subset show that despite increased CCR5 expression, CD16+ monocytes do not bind more HIV-1, nor does HIV-1 entry occur in higher levels than in CD14++CD16-monocytes. Finally, we show that CD16+ monocytes express the HIV-1 resistance host factor APOBEC3G (apolipoprotein B mRNA editing enzyme, catalytic polypeptide like) mainly in an inactive high molecular weight form which may account for their increased susceptibility to infection. Tippett E*, Ellery PJ*, Chui YL, Paukovics G, Cameron PU, Solomon A, Lewin SR, Gorry PR, Jaworowski A, Greene W, Sonza S, Crowe SM. The CD16+ monocyte subset is more permissive to infection and preferentially harbours HIV in vivo. J Immuno/2007; 178(10):6581¬9 (*authors contributed equally) Chapter 3 -Examination of the scavenger receptor CD163 in HIV-1 infection Overview: CD163 has been suggested play an important role in immune resolution, however, relatively little is known about its function. It is expressed on monocytes and shown to be increased in brain tissue from people with HIV-1 associated dementia as well as in simian immunodeficiency virus (SIV) macaque models. We have examined the regulation of CD163 in health and in HIV-1 infection and have investigated the potential of soluble CD163 as a prognostic marker of disease. Results: We show CD163 is differentially expressed on monocyte subsets, with highest levels of expression on CD14++CD16-monocytes. Culture in the presence of M-CSF can stimulate CD163 expression to comparable levels in all subsets. We show CD163 is readily shed from the cell surface during blood processing and that following LPS stimulation shedding predominantly occurs from the CD14++CD16-subset. CD163 expression is increased on CD14++CD16-monocytes in HIV-1 infected patients receiving protease inhibitors as part of their drug regimen compared to uninfected donors. CD163 is also increased on CD14++CD16+ monocytes from HIV-1 positive donors by an undetermined mechanism. Plasma levels of soluble CD163 are not affected by HIV-1 infection in this cohort. Correlation analyses of soluble CD163 with clinical parameters suggests multifactorial regulation of CD163 in the setting of HIV-1 infection. Tippett E, Cheng W, Westhorpe C, Cameron PU, Brew B, Lewin S, Jaworowski A, Crowe S. Differential expression of CD163 on monocyte subsets in healthy and HIV-1 infected individuals PLOS One 2011; 6(5):e19968 Chapter 4 -The role of tetraspanins in HIV-1 infection Overview: There is some evidence that tetraspanins are involved in HIV-1 infection of macrophages, however, their role in the infection of monocytes and the potential that they may contribute to the increased susceptibility to infection shown by CD16+ monocytes has not been investigated. Results: Little is known regarding tetraspanin regulation. Our investigation of tetraspanin expression on monocyte subsets from healthy and HIV-1 infected donors revealed differential expression on monocyte subsets without being significantly altered by HIV-1 infection. We found tetraspanin expression by macrophages was modulated by cytokine exposure and that CD9 is a marker of anti-inflammatory macrophages and CD53 of pro-inflammatory macrophages. Our investigation of the early events in HIV-1 infection has shown that tetraspanins can be found in association with the HIV-1 co-receptor CCR5 in macrophages, and in monocytes tetraspanins are concentrated with virions during HIV-1 entry. However, depletion of tetraspanins with siRNA did not impair HIV-1 entry. Using reporter virus and molecular techniques to investigate HIV-1 reverse transcription and gene expression we demonstrated that CD81, CD63 and to a lesser extent CD53 are required for HIV-1 infection of monocytes and macrophages at a post entry stage; CD81 is required for a stage in the viral replication cycle between early and late reverse transcription and CD63 and CD53 at a point after late reverse transcription but before nuclear importation. Tippett E, Jones KL, Hawkes D, Pereira C, Roche M, Gorry P. Lewin SL, Cameron PU, Marsh M, Crowe SM. Tetraspanins are required for HIV-1 infection of monocytes and macrophaqes Submitted Journal of Immunology.