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Visualization of HIV-1 interactions with penile and foreskin epithelia: clues for female-to-male HIV transmission.

Dinh MH, Anderson MR, McRaven MD, Cianci GC, McCoombe SG, Kelley ZL, Gioia CJ, Fought AJ, Rademaker AW, Veazey RS, Hope TJ - PLoS Pathog. (2015)

Bottom Line: Using statistical models to account for repeated measures and zero-inflated datasets, we found no difference in total virions visualized at 4 hours between inner and outer foreskins from local donors.At 24 hours, there were more virions in inner as compared to outer foreskin (0.0495 +/- 0.0154 and 0.0171 +/- 0.0038 virions/image, p = 0.001).In the cadaveric specimens, we observed more virions in inner foreskin (0.0507 +/- 0.0079 virions/image) than glans tissue (0.0167 +/- 0.0033 virions/image, p<0.001), but a greater proportion was seen penetrating uncircumcised glans tissue (0.0458 +/- 0.0188 vs. 0.0151 +/- 0.0100 virions/image, p = 0.099) and to significantly greater mean depths (29.162 +/- 3.908 vs. 12.466 +/- 2.985 μm).

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.

ABSTRACT
To gain insight into female-to-male HIV sexual transmission and how male circumcision protects against this mode of transmission, we visualized HIV-1 interactions with foreskin and penile tissues in ex vivo tissue culture and in vivo rhesus macaque models utilizing epifluorescent microscopy. 12 foreskin and 14 cadaveric penile specimens were cultured with R5-tropic photoactivatable (PA)-GFP HIV-1 for 4 or 24 hours. Tissue cryosections were immunofluorescently imaged for epithelial and immune cell markers. Images were analyzed for total virions, proportion of penetrators, depth of virion penetration, as well as immune cell counts and depths in the tissue. We visualized individual PA virions breaching penile epithelial surfaces in the explant and macaque model. Using kernel density estimated probabilities of localizing a virion or immune cell at certain tissue depths revealed that interactions between virions and cells were more likely to occur in the inner foreskin or glans penis (from local or cadaveric donors, respectively). Using statistical models to account for repeated measures and zero-inflated datasets, we found no difference in total virions visualized at 4 hours between inner and outer foreskins from local donors. At 24 hours, there were more virions in inner as compared to outer foreskin (0.0495 +/- 0.0154 and 0.0171 +/- 0.0038 virions/image, p = 0.001). In the cadaveric specimens, we observed more virions in inner foreskin (0.0507 +/- 0.0079 virions/image) than glans tissue (0.0167 +/- 0.0033 virions/image, p<0.001), but a greater proportion was seen penetrating uncircumcised glans tissue (0.0458 +/- 0.0188 vs. 0.0151 +/- 0.0100 virions/image, p = 0.099) and to significantly greater mean depths (29.162 +/- 3.908 vs. 12.466 +/- 2.985 μm). Our in vivo macaque model confirmed that virions can breach penile squamous epithelia in a living model. In summary, these results suggest that the inner foreskin and glans epithelia may be important sites for HIV transmission in uncircumcised men.

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Ex vivo PA GFP HIV-1 interactions with adult human foreskin tissues.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 ex vivo. 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
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ppat.1004729.g001: Ex vivo PA GFP HIV-1 interactions with adult human foreskin tissues.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 ex vivo. 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

Mentions: Fluorescently labeled CCR5-tropic (R5-tropic) HIV-1 was made by co-transfecting 293T cells with an HIV-1 provirus and photoactivatable GFP-Vpr constructs (PA GFP HIV)[22–24]. Foreskin tissues were obtained from local consenting adult donors and cultured with PA GFP HIV for 4 and 24 hours (n = 10 and 12, respectively). 1612 images of tissue cryosections were obtained using deconvolution epifluorescent microscopy. A subtraction method was used to determine true PA GFP HIV from tissue background autofluorescence, as previously described[24]. Many images captured did not contain virions (∼40%); in those that did, we counted 15626 individual virions, the majority of which were found on the epithelial surface or in the stratum corneum (SC) (Fig. 1A and 1B). Foreskin specimens inoculated with PA GFP HIV and a fluorescent fluid phase marker (bovine serum albumin, BSA) demonstrated that the virus diffused into the SC in a similar manner as BSA (Fig. 1C). That is, there was heterogeneous distribution of both BSA and virions into the SC, with some areas allowing for shallower diffusion and other areas allowing for deeper diffusion. These patterns did not demonstrably differ between the inner and outer foreskin. On average, 1 per 100 virions visualized were seen past the SC, which we termed, “penetrators” (Fig. 1D). The range of penetration depths seen in foreskin tissue was 0–96.69 μm (S1A Fig.). Using wheat germ agglutinin to highlight epithelial cell surfaces, we determined that >80% of penetrators were found between rather than inside a cell (inset, Fig. 1D).


Visualization of HIV-1 interactions with penile and foreskin epithelia: clues for female-to-male HIV transmission.

Dinh MH, Anderson MR, McRaven MD, Cianci GC, McCoombe SG, Kelley ZL, Gioia CJ, Fought AJ, Rademaker AW, Veazey RS, Hope TJ - PLoS Pathog. (2015)

Ex vivo PA GFP HIV-1 interactions with adult human foreskin tissues.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 ex vivo. 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
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352059&req=5

ppat.1004729.g001: Ex vivo PA GFP HIV-1 interactions with adult human foreskin tissues.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 ex vivo. 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
Mentions: Fluorescently labeled CCR5-tropic (R5-tropic) HIV-1 was made by co-transfecting 293T cells with an HIV-1 provirus and photoactivatable GFP-Vpr constructs (PA GFP HIV)[22–24]. Foreskin tissues were obtained from local consenting adult donors and cultured with PA GFP HIV for 4 and 24 hours (n = 10 and 12, respectively). 1612 images of tissue cryosections were obtained using deconvolution epifluorescent microscopy. A subtraction method was used to determine true PA GFP HIV from tissue background autofluorescence, as previously described[24]. Many images captured did not contain virions (∼40%); in those that did, we counted 15626 individual virions, the majority of which were found on the epithelial surface or in the stratum corneum (SC) (Fig. 1A and 1B). Foreskin specimens inoculated with PA GFP HIV and a fluorescent fluid phase marker (bovine serum albumin, BSA) demonstrated that the virus diffused into the SC in a similar manner as BSA (Fig. 1C). That is, there was heterogeneous distribution of both BSA and virions into the SC, with some areas allowing for shallower diffusion and other areas allowing for deeper diffusion. These patterns did not demonstrably differ between the inner and outer foreskin. On average, 1 per 100 virions visualized were seen past the SC, which we termed, “penetrators” (Fig. 1D). The range of penetration depths seen in foreskin tissue was 0–96.69 μm (S1A Fig.). Using wheat germ agglutinin to highlight epithelial cell surfaces, we determined that >80% of penetrators were found between rather than inside a cell (inset, Fig. 1D).

Bottom Line: Using statistical models to account for repeated measures and zero-inflated datasets, we found no difference in total virions visualized at 4 hours between inner and outer foreskins from local donors.At 24 hours, there were more virions in inner as compared to outer foreskin (0.0495 +/- 0.0154 and 0.0171 +/- 0.0038 virions/image, p = 0.001).In the cadaveric specimens, we observed more virions in inner foreskin (0.0507 +/- 0.0079 virions/image) than glans tissue (0.0167 +/- 0.0033 virions/image, p<0.001), but a greater proportion was seen penetrating uncircumcised glans tissue (0.0458 +/- 0.0188 vs. 0.0151 +/- 0.0100 virions/image, p = 0.099) and to significantly greater mean depths (29.162 +/- 3.908 vs. 12.466 +/- 2.985 μm).

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.

ABSTRACT
To gain insight into female-to-male HIV sexual transmission and how male circumcision protects against this mode of transmission, we visualized HIV-1 interactions with foreskin and penile tissues in ex vivo tissue culture and in vivo rhesus macaque models utilizing epifluorescent microscopy. 12 foreskin and 14 cadaveric penile specimens were cultured with R5-tropic photoactivatable (PA)-GFP HIV-1 for 4 or 24 hours. Tissue cryosections were immunofluorescently imaged for epithelial and immune cell markers. Images were analyzed for total virions, proportion of penetrators, depth of virion penetration, as well as immune cell counts and depths in the tissue. We visualized individual PA virions breaching penile epithelial surfaces in the explant and macaque model. Using kernel density estimated probabilities of localizing a virion or immune cell at certain tissue depths revealed that interactions between virions and cells were more likely to occur in the inner foreskin or glans penis (from local or cadaveric donors, respectively). Using statistical models to account for repeated measures and zero-inflated datasets, we found no difference in total virions visualized at 4 hours between inner and outer foreskins from local donors. At 24 hours, there were more virions in inner as compared to outer foreskin (0.0495 +/- 0.0154 and 0.0171 +/- 0.0038 virions/image, p = 0.001). In the cadaveric specimens, we observed more virions in inner foreskin (0.0507 +/- 0.0079 virions/image) than glans tissue (0.0167 +/- 0.0033 virions/image, p<0.001), but a greater proportion was seen penetrating uncircumcised glans tissue (0.0458 +/- 0.0188 vs. 0.0151 +/- 0.0100 virions/image, p = 0.099) and to significantly greater mean depths (29.162 +/- 3.908 vs. 12.466 +/- 2.985 μm). Our in vivo macaque model confirmed that virions can breach penile squamous epithelia in a living model. In summary, these results suggest that the inner foreskin and glans epithelia may be important sites for HIV transmission in uncircumcised men.

Show MeSH
Related in: MedlinePlus