Using and in vitro model of HIV-1 male-to-female transmission to identify key aspects of the bottleneck hypothesis

Karl G. Siegert

doi:10.17918/etd-7244

When semen comes in contact with the female reproductive tract (FRT), it elicits a polarized- and time-dependent release of cytokines and other soluble factors from the epithelial barrier. These factors are involved in tissue remodeling, as well as immune cell activation and recruitment. This influx of immune cells potentially susceptible to human immunodeficiency virus type (HIV-1) infection leads us to hypothesize that (i) semen alters the subepithelial environment of the FRT to be more pro-inflammatory, (ii) the pro-inflammatory environment induced by semen will modulate surface expression of antigens on immune cells, specifically the HIV-1 co-receptors on T cells, and (iii) that the combined effect of semen-induced inflammation and immune cell alterations will increase the risk of male-to-female R5-tropic HIV-1 transmission. To test these hypotheses in vitro, three different cell lines derived from the vagina, ectocervix, and endocervix were used to establish a polarized, transwell-based polarized epithelial model. After introduction of activated PBMCs into the basolateral chamber and apical exposure of the epithelial cells to semen (from healthy donors), the basolateral medium was collected and assessed for cytokine production using an ELISArray of twelve pro-inflammatory cytokines, while the PBMCs were harvested and assayed by flow cytometry for cell surface markers indicative of cell status and susceptibility to HIV-1 infection. To model HIV-1 transmission, a pseudovirus containing a GFP reporter was constructed in conjunction with either an X4-tropic envelope (HXB2) or an R5-tropic envelope (a known transmitted/founder virus). Immediately after apical semen exposure to the epithelium, PBMCs were collected and infected with a pseudovirus, and then assayed for infection (as indicated by GFP expression). In co-cultures of epithelial cells and PBMCs in the absence of infection, conditioned media in the subepithelial chamber was pro-inflammatory in nature, a condition which was exacerbated in the presence of semen. In three separate co-cultures incorporating each of the three epithelial cell lines, the innate immune response in the absence or presence of semen was dominated by IL-8. Flow cytometric analyses of the PBMCs showed that CCR5+ T lymphocytes decreased in numbers in the absence of semen only in the ectocervical model. However, CXCR4+ T lymphocyte numbers did not change appreciably in any epithelial model system. When PBMCs were pre-conditioned and then infected with an R5 or X4 pseudovirus, only those conditioned by the ectocervical cells displayed increased levels of infection by the R5 virus, but with no change in X4 virus infection. Ongoing studies are investigating the mechanism by which semen preferentially selects for R5 viruses over X4 viruses.

Using and in vitro model of HIV-1 male-to-female transmission to identify key aspects of the bottleneck hypothesis

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Using and in vitro model of HIV-1 male-to-female transmission to identify key aspects of the bottleneck hypothesis

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