Elevated inflammatory cytokines (EMCs) at mucosal surfaces have been associated with HIV susceptibility, but the underlying mechanisms remain unclear. We characterized the soluble mucosal proteome associated with elevated cytokine expression in the female reproductive tract. A scoring system was devised based on the elevation (upper quartile) of at least three of seven inflammatory cytokines in cervicovaginal lavage. Using this score, HIV-uninfected Kenyan women were classified as either having EMC (n=28) or not (n=68). Of 455 proteins quantified in proteomic analyses, 53 were associated with EMC (5% false discovery rate threshold). EMCs were associated with proteases, cell motility, and actin cytoskeletal pathways, whereas protease inhibitor, epidermal cell differentiation, and cornified envelope pathways were decreased. Multivariate analysis identified an optimal signature of 16 proteins that distinguished the EMC group with 88% accuracy. Three proteins in this signature were neutrophil-associated proteases that correlated with many cytokines, especially GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-1β (interleukin-1β), MIP-3α (macrophage inflammatory protein-3α), IL-17, and IL-8. Gene set enrichment analyses implicated activated immune cells; we verified experimentally that EMC women had an increased frequency of endocervical CD4(+) T cells. These data reveal strong linkages between mucosal cytokines, barrier function, proteases, and immune cell movement, and propose these as potential mechanisms that increase risk of HIV acquisition.

The HIV pandemic disproportionately affects women, with most infections acquired through receptive vaginal sex. Although the target cells by which HIV establishes infection in the female genital tract remain poorly defined, it is known that immune activation results in CD4+ T cells with enhanced susceptibility, as does expression of the mucosal integrin α4β7 and the HIV coreceptor CCR5. Blood and cervical cytobrush specimens were collected from female sex workers (FSWs) in Nairobi, Kenya. Genital infection diagnostics were performed, T cell populations were defined by multiparameter flow cytometry based on their expression of surface receptors relevant to mucosal homing and/or HIV acquisition, and cytokine production was assayed by intracellular cytokine staining. The integrin α4β7 was expressed on 26.0% of cervical CD4+ T cells, and these cells were more likely to express both the HIV coreceptor CCR5 (p < 0.0001) and the early activation marker CD69 (p < 0.0001) but not CXCR4 (p = 0.34). Cervical Th17 frequencies were enhanced compared with blood (7.02 versus 1.24%; p < 0.0001), and cervical IL-17A+ CD4+ T cells preferentially coexpressed α4β7 and CCR5. Expression of IFN-γ and IL-22 was greater in cervical Th17 cells than in blood Th17 cells. In keeping with the hypothesis that these cells are preferential HIV targets, gp120 preferentially bound CCR5+ cervical T cells, and cervical Th17 cells were almost completely depleted in HIV+ FSWs compared with HIV− FSWs. In summary, a subset of Th17 CD4+ T cells in the cervical mucosa coexpresses multiple HIV susceptibility markers; their dramatic depletion after HIV infection suggests that these may serve as key target cells during HIV transmission.

A better understanding of the cellular targets of HIV infection in the female genital tract may inform HIV prevention efforts. Proposed correlates of cellular susceptibility include the HIV co-receptor CCR5, peripheral homing integrins, and immune activation. We used a CCR5-tropic pseudovirus to quantify HIV entry into unstimulated endocervical CD4(+) T cells collected by cytobrush. Virus entry was threefold higher into cervix-derived CD4(+) T cells than blood, but was strongly correlated between these two compartments. Cervix-derived CD4(+) T cells expressing CD69, α(4)β(7), or α(4)β(1) were preferential HIV targets; this enhanced susceptibility was strongly correlated with increased CCR5 expression in α(4)β(7)(+) and CD69(+) CD4(+) T cells, and to a lesser extent in α(4)β(1)(+) CD4(+) T cells. Direct binding of gp140 to integrins was not observed, integrin inhibitors had no effect on virus entry, and pseudotypes with an env that preferentially binds α(4)β(7) still demonstrated enhanced entry into α(4)β(1)(+) cells. In summary, a rapid and sensitive HIV entry assay demonstrated enhanced susceptibility of activated endocervical CD4(+) T cells, and those expressing α(4)β(7) or α(4)β(1). This may relate to increased CCR5 expression by these cell subsets, but did not appear to be due to direct interaction of α(4)β(7) or α(4)β(1) with HIV envelope.

BackgroundFunctional analysis of mononuclear leukocytes in the female genital mucosa is essential for understanding the immunologic effects of HIV vaccines and microbicides at the site of HIV exposure. However, the best female genital tract sampling technique is unclear.Methods and FindingsWe enrolled women from four sites in Africa and the US to compare three genital leukocyte sampling methods: cervicovaginal lavages (CVL), endocervical cytobrushes, and ectocervical biopsies. Absolute yields of mononuclear leukocyte subpopulations were determined by flow cytometric bead-based cell counting. Of the non-invasive sampling types, two combined sequential cytobrushes yielded significantly more viable mononuclear leukocytes than a CVL (p<0.0001). In a subsequent comparison, two cytobrushes yielded as many leukocytes (∼10,000) as one biopsy, with macrophages/monocytes being more prominent in cytobrushes and T lymphocytes in biopsies. Sample yields were consistent between sites. In a subgroup analysis, we observed significant reproducibility between replicate same-day biopsies (r = 0.89, p = 0.0123). Visible red blood cells in cytobrushes increased leukocyte yields more than three-fold (p = 0.0078), but did not change their subpopulation profile, indicating that these leukocytes were still largely derived from the mucosa and not peripheral blood. We also confirmed that many CD4+ T cells in the female genital tract express the α4β7 integrin, an HIV envelope-binding mucosal homing receptor.ConclusionsCVL sampling recovered the lowest number of viable mononuclear leukocytes. Two cervical cytobrushes yielded comparable total numbers of viable leukocytes to one biopsy, but cytobrushes and biopsies were biased toward macrophages and T lymphocytes, respectively. Our study also established the feasibility of obtaining consistent flow cytometric analyses of isolated genital cells from four study sites in the US and Africa. These data represent an important step towards implementing mucosal cell sampling in international clinical trials of HIV prevention.

These data support the model of initial CD4 T-cell depletion followed by overall T-cell influx in response to infection and concomitant increases in immune activation, inflammation, and regulatory markers. These data are among the earliest characterization of the cellular milieu in the female genital tract following male-to-female HIV transmission.