Figure 1.
HIV-1-infected cells affect secretion of chemokines by the inner foreskin.
Inner foreskin explants were inoculated in a polarized manner for 4 h with either non-infected or HIV-1-infected PBMCs, washed, and further incubated in medium in a non-polarized manner. The levels of twelve chemokines and cytokines were measured the next day in the culture supernatants, using a custom multiplex bead immunoassay and quantified by flow cytometry or specific ELISA. Shown are mean folds±SEM secretion of each analyte (derived from two independent explants), calculated as [secretion after exposure to HIV-1-infected PBMCs/secretion after exposure to non-infected PBMCs], for either chemokines/cytokines not altered (A) or altered (B) following exposure to HIV-1-infected cells. In (B), p = 0.0105, 0.0308, 0.0166, 0.0045 for INF gamma, CCL3/MIP-1 alpha, CCL20/MIP-3 alpha and CCL5/RANTES, respectively, Student's t-test, n = 2.
Figure 2.
HIV-1-infected cells induce recruitment of T-cells into the epidermis.
Parallel inner (A) and outer (B) foreskin explants were exposed for 4 h to either HIV-1-infected (solid lines) or non-infected (broken lines) PBMCs. Explants were then stained with anti-CD3 Ab and visualized with DAB peroxidase substrate. Shown are means±SEM CD3+ cell densities (cells/mm2) in either epidermis or dermis derived from three independent explants. Cells were counted in a minimum of 10 fields/section in each experiment. In (A): p = 0.0423 and 0.0175, 4 h vs. 0 h for infected epidermis and dermis, respectively; p = 0.0316 and 0.0269, HIV-1-infected vs. non-infected at 4 h for epidermis and dermis, respectively; Student's t-test, n = 3. (C) Electron micrograph of inner foreskin explant exposed for 4 h to HIV-1-infected PBMCs. Shown is a T-cell, identified by its typical morphology and size/shape of nucleus, migrating into the epidermis across a disrupted basement membrane (non-continuous black dotted line). The rear of the cell is still localized in the dermis. Scale bar = 0.5 µm.
Figure 3.
CCL5/RANTES mediates T-cell recruitment into the epidermis.
(A) Representative FACS profiles out of two independent experiments, showing epidermal single-cell suspensions derived from inner foreskin explants exposed for 4 h to either medium alone (left), non-infected PBMCs (middle), or HIV-1-infected PBMCs (right). Cells were stained with PE-conjugated anti-human CD3 mAb and numbers represent the percentages of CD3+ cells in the framed windows (determined based on the non-specific staining of a matched isotype control Ab). (B) Inner foreskin explants were exposed for 4 h to either non-infected PBMCs (white bar) or HIV-1-infected PBMCs: alone (black bar); in the presence of 40 µg/ml control goat IgG Ab (dark grey bar); in the presence of 20 or 80 µg/ml neutralizing goat Ab to CCL5/RANTES (light grey bars). Following infection, epidermal single-cell suspensions were prepared and stained for CD3 expression as described above. Shown are mean folds increase±SEM of the percentages of CD3+ cells, normalized against that following exposure to non-infected PBMCs and derived from two independent explants. p = 0.0129 infected vs. non-infected and p = 0.0128 infected+CCL5 Ab 80 µg/ml vs. infected; Student's t-test.
Figure 4.
(A, B) HIV-1-infected cells induce retention of LCs within the epidermis.
Parallel inner (A) and outer (B) foreskin explants were exposed for 4 h to either HIV-1-infected (solid lines) or non-infected (broken lines) PBMCs. Explants were then stained with anti-langerin Ab and visualized with DAB peroxidase substrate. Shown are means±SEM langerin+ cell densities (cells/mm2) in epidermis derived from three independent explants. Cells were counted in a minimum of 10 fields/section in each experiment. In (A): p = 0.0322 non-infected epidermis 4 h vs. 0 h and p = 0.0415 HIV-1-infected vs. non-infected at 4 h; Student's t-test. (C, D) HIV-1 entry and capture by LCs in inner, but not outer, foreskin explants. Single sections observed by confocal microscopy of parallel inner (C) and outer (D) foreskin explants, following 4 h exposure to HIV-1-infected PBMCs. Horizontal and vertical lines represent the localization of the sectioned stacks along the z axis (25 sections, 0.2 µm apart) that are shown below and to the right of each image. Sections were double-stained with goat-anti-human langerin Ab followed by TRITC-conjugated anti-goat IgG Ab and a mixture of several human/mouse anti-HIV-1 mAbs followed by FITC-conjugated anti-human/mouse IgG Abs. In (C), langerin is detected around cell bodies and dendrites reaching the apical surface (red arrowheads); HIV-1 virions are detected as dots in the epidermis associated with epithelial cells or in dermis (green arrowheads), as well as co-localized with LCs (green arrows). Higher magnification image with the xyz planes rotated shows viral particles internalized into LCs. Cell nuclei were counterstained with DAPI. White dotted lines denote the epidermis/dermis interface. Scale bars = 10 µm. Representative images of n = 3 experiments.
Figure 5.
HIV-1-infected cells modify the spatial distribution of LCs in inner foreskin.
(A) Representative images of normal inner foreskin (top), and inner foreskin explants exposed to either non-infected (left) or HIV-1-infected (right) PBMCs for 1 h (middle) or 4 h (bottom). Explants were then stained with anti-langerin Ab and visualized with AEC (normal foreskin) or DAB (foreskin explants) peroxidase substrates. The black double-headed arrows denote individual distances of LCs from the apical surface for each condition and are marked as [ai]-[ei] respectively. Images are representative of three independent experiments. Scale bars = 20 µm. (B) Calculated mean±SEM distances covered by LCs, following exposure of inner foreskin explants to either non-infected (white bars) or HIV-1-infected (grey bars) PBMCs. For exposure to non-infected PBMCs, the distances after 1 h and 4 h were calculated as [b]-[a] and [c]-[b], respectively, where a, b, c are means of the individual [ai], [bi], [ci] distances (measured for 106, 193 and 148 different cells in three independent explants). For exposure to HIV-1-infected PBMCs, the distances after 1 h and 4 h were calculated as [a]-[d] and [e]-[d], respectively, where d and e are mean of the individual [di] and [ei] distances (measured for 186 and 108 different cells in three independent explants). *p<0.0001 infected vs. non-infected at 4 h; Student's t-test. (C) shows the actual distances from the apical surface measured for each experimental condition. *p<0.0001 vs. normal foreskin; Student's t-test.
Figure 6.
HIV-1-infected cells increase the formation of epidermal LC-T-cell conjugates.
(A) Inner foreskin explants were exposed for 1 h or 4 h to HIV-1-infected PBMCs. Explants were then double stained with anti-langerin and anti-CD3 Abs, and visualized with DAB and HistoGreen peroxidase substrates, respectively. Shown are calculated means±SEM % LC-T-cell conjugates of the total LCs from three independent explants. Cells were counted in a minimum of 10 fields/section for each experiment. *p = 0.0431 4 h vs. 1 h, Student's t-test. (B–D) Representative images of inner foreskin explants exposed to either non-infected (B) or HIV-1-infected (C and D) PBMCs for 4 h, and double stained for langerin (DAB brown in B and C; Alexa488 green in D) and CD3 (Histogreen blue-green in B and C; Cy5 red in D). Single isolated non-conjugated LCs and T-cells are shown in the framed window in (B), while the framed window in (C) shows LC-T-cell conjugate at the epidermal-dermal interface above the basement membrane (black dotted lines). The confocal microscopy image in (D) shows the close contact between one LC and one T-cell, as well as the LC dendrite in proximity to a second T-cell. Scale bars = 20 µm (B, C) and 8 µm (D). (E) Electron micrograph of LC-T-cell conjugate in inner foreskin explant exposed for 4 h to HIV-1-infected PBMCs. The conjugate is positioned within the epidermis at the epidermal-dermal interface above the basement membrane (black dotted line); scale bar = 1 µm. Higher magnification of the top framed window (right side) shows two rod-shaped Birbeck granule in the LC cytoplasm (black arrows); scale bar = 200 nm. Higher magnification of the bottom framed window (right corner) shows an HIV-1 particle of 90 nm with a central dark core characteristic of mature virions associated with LC; scale bar = 50 nm. “(F) Inner foreskin explants were exposed for 4 h to either non-infected PBMCs (top right) or HIV-1-infected PBMCs: alone (bottom left); in the presence of 40 µg/ml control goat IgG Ab (bottom middle); in the presence of 80 µg/ml neutralizing goat Ab to CCL5/RANTES (bottom right). Following infection, epidermal single-cell suspensions were prepared and double stained with APC-conjugated anti-human langerin and PE-conjugated anti-human CD3 mAbs. Cells were first gated on langerin+ cells (R1 gate, top left profile, insert shows staining with matched isotype control). Numbers represent the percentages of cells in R1 that are both high forward scatter and CD3+. Images are representative of two independent experiments.
Figure 7.
Schematized ‘chain-of-events’ of HIV-1 early entry in the inner foreskin.
At 1 h, HIV-1-infected cells form apical viral synapses, which results in local HIV-1 budding, LCs attraction and HIV-1 capture (1). At 4 h, LCs migrate back towards the basement membrane deeper into the tissue (2), in correlation with reduced CCL20/MIP-3 alpha secretion. At this time point, T-cells are recruited from the dermis into the epidermis (3), as a result of increased CCL5/RANTES secretion, and fuel the formation of LC-T-cell conjugates (4). Blocking CCR5 on T-cells by clinically available CCR5 inhibitors may serve to limit T-cell recruitment and local viral spread (5).