<i>Ex vivo</i> PA GFP HIV-1 interactions with adult human foreskin tissues.

<p>Foreskins obtained from consenting adult donors and inoculated with R5-tropic PA GFP-Vpr HIV-1 for 4 (n = 10) or 24 hours (n = 12) in culture. (A) and (B) Representative images of virion interactions with inner (A) and outer (B) foreskins after 4 hours of HIV exposure <i>ex vivo</i>. When seen, virions (red) were found predominantly on the surface or in the stratum corneum (SC). ES, dotted line, epithelial surface. (C) When co-inoculated with fluorescently labeled bovine serum albumin (BSA, red, right panel), virions (red, top half of inset, pseudo-colored to reveal PA GFP) were seen diffusing to depths that BSA also reached. (D) The majority of penetrating virions (virions seen below the SC) were found interstitially, as determined by tissues stained with fluorescent wheat germ agglutinin (WGA, green, inset). All images: white bar = 10 μm, blue = cell nuclei. (E-G) Estimated means of total virion counts (E), ** = adjusted for virus stock concentrations; proportion of penetrators (F); depths of penetration (G). Dark squares and bars represent inner foreskin; open diamonds and bars represent outer foreskin. *p<0.05, **p<0.01, ***p<0.001</p

HIV-1 and immune cells in cadaveric penile epithelia.

<p>Penile tissues obtained from tissue donation organization banks inoculated with R5-tropic PA GFP-Vpr HIV-1 for 4 hours. (A) Representative image of glans tissue from uncircumcised donor after exposure in culture to HIV-1. Most virions were found on the epithelial surface (ES, white dotted line) in the SC. White bar = 10 μm. Cell nuclei stained with DAPI (blue). (B) Probability density distributions using kernel density estimations of viral penetration depths and tissue resident immune cells in uncircumcised glans (left) and circumcised glans (right). Overlap of 4 hour penetrators (red) and CD4+ cells (blue) appear different between tissues. (C) Interactions of estimated means of virions/image between tissue types and circumcision status, with log ratios presented for ease of reporting. Count ratios with CI >1 are considered statistically significant. (D) Estimated means of proportion of penetrators in tissues from uncircumcised (black circles) and circumcised donors (triangles). (E) Mean depth of virion penetration from uncircumcised (dark bars) and circumcised (gray bars) donors. Uncircumcised glans tissue allows higher proportion of penetrators than foreskin tissues and to greater depths. (F) Analysis of tissue resident immune cell counts shows more LCs found in epithelium than CD4+ cells. (G) Analysis of mean depths of cells shows LCs located more superficially in circumcised glans (white bar) versus shaft (gray dotted bar) and CD4+ cells more superficial in uncircumcised (gray hatched bar) as compared to circumcised shaft (gray dotted bar) tissues and in circumcised glans (white bar) versus shaft (gray dotted bar). *p<0.05, **p<0.01, ***p<0.001.</p

Tissue resident immune cells in foreskin tissues.

<p>Tissue cryosections immunofluorescently stained with OKT6 or α-CD4 antibodies to detect Langerhans cells (LCs) or CD4+ cells, respectively. (A) Representative images of LCs (red, left panel) and CD4+ cells (green, right panel) shown. White bar = 10 μm. Cell nuclei stained with DAPI (blue). Only cells within the epithelium (above the basement membrane, denoted with white solid line and BM) were used in analysis. ES, dotted line, epithelial surface. (B) Probability density distributions using kernel density estimations of viral penetration depths from the epithelial surface after 4 hours (dotted red) and 24 hours (solid red) of exposure in inner (left) and outer (right) foreskins. Overlap of 24 hour penetrators and CD4+ cells (blue) in inner 2X greater than outer foreskin. (C) Cell count analysis shows greater numbers of CD4+ cells in inner (black squares) as compared to outer (white diamonds) foreskin (* p<0.05). (D) Analysis of cell depths show no difference between inner and outer foreskin. (E) Analysis of LCs in foreskin tissue before and after virus exposure in a subset of 4 donor samples. No difference seen in cell counts between inner and outer foreskin, but marginally more cells/image seen in inner foreskin after 24 hours of virus exposure (*p<0.05). (F) No difference in depths of cells before and after virus exposure, but this subset did have differences in LC depths between inner and outer foreskin at both time points. ***p<0.001</p

PA GFP HIV-1 in macaque penile tissues <i>in vivo</i>.

<p>*Adjusted for virus stock concentration</p><p>PA GFP HIV-1 in macaque penile tissues <i>in vivo</i>.</p

Increases in Endogenous or Exogenous Progestins Promote Virus-Target Cell Interactions within the Non-human Primate Female Reproductive Tract

<div><p>Currently, there are mounting data suggesting that HIV-1 acquisition in women can be affected by the use of certain hormonal contraceptives. However, in non-human primate models, endogenous or exogenous progestin-dominant states are shown to increase acquisition. To gain mechanistic insights into this increased acquisition, we studied how mucosal barrier function and CD4+ T-cell and CD68+ macrophage density and localization changed in the presence of natural progestins or after injection with high-dose DMPA. The presence of natural or injected progestins increased virus penetration of the columnar epithelium and the infiltration of susceptible cells into a thinned squamous epithelium of the vaginal vault, increasing the likelihood of potential virus interactions with target cells. These data suggest that increasing either endogenous or exogenous progestin can alter female reproductive tract barrier properties and provide plausible mechanisms for increased HIV-1 acquisition risk in the presence of increased progestin levels.</p></div

Comparison of Target Cell Density in Various Tissue Types of Rhesus and Pigtail Macaques.

<p>Menstrual cycle phases were designated as follicular (day 1 of menstruation until day 14), midcycle (days 14–16), or luteal (days 17 to day prior to menstruation). DMPA refers to those animals that were pre-treated with intramuscular injections of 30 mg Depo-provera 4–5 weeks (28–33 days) prior to sacrifice. TCNumber/EpiArea refers to the number of target cells divided by the area of the epithelium analyzed. Each data point represents the mean cell density from a 40x panel image. Each animal had 10 panel images, 1 panel per random section, taken per tissue type from multiple blocks when available. Error bars represent SEM. (a). Analysis of CD4+ T-cell density in untreated (n = 4) and DMPA-treated rhesus macaques (n = 6), comparing vaginal tissue biopsies and terminal tissue collections (Ectocervix and Vagina). (b). Analysis of CD68+ macrophage density in untreated (n = 4) and DMPA-treated rhesus macaques (n = 6), comparing vaginal tissue biopsies and terminal tissue collections (Ectocervix and Vagina). (c). Analysis of CD4+ T-cell and CD68+ macrophage density in the squamous epithelium of infected pigtail macaques by phase of the menstrual cycle, with comparison to 28–33 day post-DMPA treatment in vaginal biopsies of SHIV infected and non-infected animals: (Top left) CD4+ T-cell density in the ectocervix, (top right) CD4+ T-cell density in the vagina, (bottom left) CD68+ macrophage density in the ectocervix, (bottom right) CD68+ macrophage density in the vagina.</p

Average Stratum Corneum (SC) Thickness of the Ectocervix and Vagina of Rhesus and Pigtail Macaques.

<p>Menstrual cycle phases were designated as follicular (day 1 of menstruation until day 14), midcycle (days 14–16), or luteal (days 17 to day prior to menstruation). DMPA refers to those animals that were pre-treated with intramuscular injections of 30 mg Depo-provera 4–5 weeks (28–33 days) prior to sacrifice. Error bars represent SEM. (a). SC thickness of the ectocervix of progesterone-treated and untreated rhesus macaques. Arrows represent those animals whose SC was absent. (b). SC thickness of the vagina of progesterone-treated and untreated rhesus macaques. Arrows represent those animals whose SC was absent. (c). SC thickness of the ectocervix of infected pigtail macaques by phase of the menstrual cycle and post-DMPA treatment. (d). SC thickness of the vagina of infected pigtail macaques by phase of the menstrual cycle and post-DMPA treatment (30mg). (e). Fluorescent deconvolution image (40x) of the SC thickness in a representative infected pigtail macaque at mid-cycle menstrual cycle phase. Adherens junctions (red), DAPI (blue), tissue background (green). Size bar is 40μm. (f). Fluorescent deconvolution image (40x) of the SC thickness in a representative infected pigtail macaque at luteal menstrual cycle phase pigtail macaque. Adherens junctions (red), DAPI (blue), tissue background (green). Size bar is 40μm. (g). Fluorescent deconvolution image (40x) of the SC thickness in a representative infected pigtail macaque after DMPA treatment. Adherens junctions (red), DAPI (blue), tissue background (green). Size bar is 40μm. Tissue layers are described to the right of image.</p

Mechanistic Model of How HIV-1 Interacts with the Female Reproductive Tract during Various Phases of the Menstrual Cycle and/or with the use of DMPA.

<p>(a). The simple columnar epithelium of the endocervix: (Left) as mid-cycle progresses into luteal phase, a small number of virions are able to enter the cervical canal to associate with the simple columnar epithelium of the endocervix. (Middle) During the luteal phase and/or with the use of progesterone contraceptives (e.g., DMPA), virions are more readily able to enter the cervical canal to interact with the simple columnar epithelium of the endocervix. (Right) During the follicular phase, virions rarely enter the cervical canal to interact with the endocervical simple columnar. Tissue layers are labeled as follows: C = simple columnar, LP = lamina propria. (b). The stratified squamous epithelium of the ectocervix and vagina: (Left) At the midcycle phase, the squamous epithelium is thickest and virions may penetrate the non-viable stratum corneum. However, target cells are primarily located below the epidermis, in the lamina propria; therefore, the likelihood of virus interacting with a target cell is minimal. (Middle) Although few virions are visualized penetrating the stratified squamous epithelium due to the lack of stratum corneum during the luteal phase (or with exogenous progesterone treatments; e.g., DMPA), there is a greater propensity for virus to interact with an infiltrating intraepithelial target cell near the luminal surface. (Right) Like the mid-cycle phase, the squamous epithelium during the follicular phase is thick, and virions are associated with the non-viable stratum corneum. However, target cells are primarily located below the epidermis, in the lamina propria; therefore, the likelihood of virus interacting with a target cell is minimal. Tissue layers are labeled as follows: SC = stratum corneum, G = granulosum, S = spinosum, and B = basal layer, LP = lamina propria.</p

HIV-1 and 200nm bead diffusion in pigtail macaque mucus before, during and after administration of DMPA.

<p>Estimated means and standard errors were calculated for (a). Mean-squared displacement (MSD) values for all particle types in each mucus sample collected before (n = 29), during (n = 16) and after (n = 34) DMPA administration. (b). Alpha (α) values for all particle types in each mucus sample collected before (n = 29), during (n = 16) and after (n = 34) DMPA administration. For each treatment condition, generalized linear mixed models were run to compare estimated means for particle MSD and α.</p

Analysis of PA-GFP HIV-1 in rhesus macaques with and without DMPA treatment and pigtail macaques at different menstrual cycle phases.

<p>Analysis of PA-GFP HIV-1 in rhesus macaques with and without DMPA treatment and pigtail macaques at different menstrual cycle phases.</p