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Spermatozoa capture HIV-1 through heparan sulfate and efficiently transmit the virus to dendritic cells.

Ceballos A, Remes Lenicov F, Sabatté J, Rodríguez Rodrígues C, Cabrini M, Jancic C, Raiden S, Donaldson M, Agustín Pasqualini R, Marin-Briggiler C, Vazquez-Levin M, Capani F, Amigorena S, Geffner J - J. Exp. Med. (2009)

Bottom Line: Interaction of spermatozoa with DCs not only leads to the transmission of HIV-1 and the internalization of the spermatozoa but also results in the phenotypic maturation of DCs and the production of IL-10 but not IL-12p70.At low values of extracellular pH (approximately 6.5 pH units), similar to those found in the vaginal mucosa after sexual intercourse, the binding of HIV-1 to the spermatozoa and the consequent transmission of HIV-1 to DCs were strongly enhanced.Our observations support the notion that far from being a passive carrier, spermatozoa acting in concert with DCs might affect the early course of sexual transmission of HIV-1 infection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro Nacional de Referencia para SIDA, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires C1121ABG, Argentina.

ABSTRACT
Semen is the main vector for HIV-1 dissemination worldwide. It contains three major sources of infectious virus: free virions, infected leukocytes, and spermatozoa-associated virions. We focused on the interaction of HIV-1 with human spermatozoa and dendritic cells (DCs). We report that heparan sulfate is expressed in spermatozoa and plays an important role in the capture of HIV-1. Spermatozoa-attached virus is efficiently transmitted to DCs, macrophages, and T cells. Interaction of spermatozoa with DCs not only leads to the transmission of HIV-1 and the internalization of the spermatozoa but also results in the phenotypic maturation of DCs and the production of IL-10 but not IL-12p70. At low values of extracellular pH (approximately 6.5 pH units), similar to those found in the vaginal mucosa after sexual intercourse, the binding of HIV-1 to the spermatozoa and the consequent transmission of HIV-1 to DCs were strongly enhanced. Our observations support the notion that far from being a passive carrier, spermatozoa acting in concert with DCs might affect the early course of sexual transmission of HIV-1 infection.

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Capture of HIV-1 by spermatozoa is mainly mediated through HS. (A) The expression of HS on spermatozoa was analyzed by flow cytometry using anti–HS-specific antibodies (clone 10E4). Isotype control (gray histogram) and 10E4 labeled (open histogram) are depicted. A representative experiment (n = 7) is shown. (B) Spermatozoa (1.5 × 106/200 µl) were incubated with HIV-1 BAL containing 25 ng of p24 for 60 min at 37°C in the absence or presence of 10 and 100 U/ml heparin, washed thoroughly, lysed, and assayed for p24 antigen by ELISA. Results are the mean ± SEM of six experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (C) Spermatozoa were treated with 5 U/ml heparinase II for 60 min at 25°C or 1,000 U/ml trypsin for 15 min at 37°C. Then the expression of HS was analyzed by flow cytometry. The results are expressed as the MFI ± SEM of five experiments performed in duplicate. Asterisks represent statistical significance (P < 0.05 vs. the expression of HS in controls). (D) Spermatozoa were treated with 1 and 5 U/ml heparinase II for 60 min at 25°C. Then their ability to capture HIV-1 was assayed as described for Fig. 2 A. Results are the mean ± SEM of five experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (E) Spermatozoa were treated with 5 U/ml heparinase III for 60 min at 25°C. Then the expression of the neoepitope 3G10 was analyzed by flow cytometry. Isotype (gray histogram) and 3G10-labeled untreated (control) or heparinase III–treated spermatozoa (open histograms) are shown. Isotype controls were similar for untreated and heparinase III–treated spermatozoa. A representative experiment (n = 4) is shown. (F) Spermatozoa (1.5 × 106/200 µl) were incubated with different primary HIV-1 isolates containing 25 ng of p24 for 60 min at 37°C in the absence or presence of 100 U/ml heparin or 5 mg/ml mannan, washed thoroughly, lysed, and assayed for p24 antigen by ELISA. Results are the mean ± SEM of three to five experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (G) HIV-1 pseudotypes were produced as described in Materials and methods. Spermatozoa (1.5 × 106/200 µl) were incubated with HIV-1 env− or HIV-1 env+ pseudotypes containing 40 ng of p24 for 60 min at 37°C in the absence or presence of 5 mg/ml mannan or 100 U/ml heparin, washed thoroughly, lysed, and assayed for p24 antigen by ELISA. Results are the mean ± SEM of three to four experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (H) The expression of syndecans 1–4 was analyzed by flow cytometry. Gray histograms correspond to isotype controls. In each case, a representative experiment (n = 3–6) is shown.
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fig3: Capture of HIV-1 by spermatozoa is mainly mediated through HS. (A) The expression of HS on spermatozoa was analyzed by flow cytometry using anti–HS-specific antibodies (clone 10E4). Isotype control (gray histogram) and 10E4 labeled (open histogram) are depicted. A representative experiment (n = 7) is shown. (B) Spermatozoa (1.5 × 106/200 µl) were incubated with HIV-1 BAL containing 25 ng of p24 for 60 min at 37°C in the absence or presence of 10 and 100 U/ml heparin, washed thoroughly, lysed, and assayed for p24 antigen by ELISA. Results are the mean ± SEM of six experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (C) Spermatozoa were treated with 5 U/ml heparinase II for 60 min at 25°C or 1,000 U/ml trypsin for 15 min at 37°C. Then the expression of HS was analyzed by flow cytometry. The results are expressed as the MFI ± SEM of five experiments performed in duplicate. Asterisks represent statistical significance (P < 0.05 vs. the expression of HS in controls). (D) Spermatozoa were treated with 1 and 5 U/ml heparinase II for 60 min at 25°C. Then their ability to capture HIV-1 was assayed as described for Fig. 2 A. Results are the mean ± SEM of five experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (E) Spermatozoa were treated with 5 U/ml heparinase III for 60 min at 25°C. Then the expression of the neoepitope 3G10 was analyzed by flow cytometry. Isotype (gray histogram) and 3G10-labeled untreated (control) or heparinase III–treated spermatozoa (open histograms) are shown. Isotype controls were similar for untreated and heparinase III–treated spermatozoa. A representative experiment (n = 4) is shown. (F) Spermatozoa (1.5 × 106/200 µl) were incubated with different primary HIV-1 isolates containing 25 ng of p24 for 60 min at 37°C in the absence or presence of 100 U/ml heparin or 5 mg/ml mannan, washed thoroughly, lysed, and assayed for p24 antigen by ELISA. Results are the mean ± SEM of three to five experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (G) HIV-1 pseudotypes were produced as described in Materials and methods. Spermatozoa (1.5 × 106/200 µl) were incubated with HIV-1 env− or HIV-1 env+ pseudotypes containing 40 ng of p24 for 60 min at 37°C in the absence or presence of 5 mg/ml mannan or 100 U/ml heparin, washed thoroughly, lysed, and assayed for p24 antigen by ELISA. Results are the mean ± SEM of three to four experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (H) The expression of syndecans 1–4 was analyzed by flow cytometry. Gray histograms correspond to isotype controls. In each case, a representative experiment (n = 3–6) is shown.

Mentions: Previous studies have shown that HS interacts with gp120 (de Parseval et al., 2005; Crublet et al., 2008), allowing the attachment of HIV-1 to macrophages (Saphire et al., 2001), DCs (de Witte et al., 2007), epithelial cells (Wu et al., 2003), and endothelial cells (Argyris et al., 2003). Studies performed by flow cytometry showed that spermatozoa express HS (Fig. 3 A). Heparin, an agent able to inhibit the attachment of HIV-1 to HS (Saphire et al., 2001; de Parseval et al., 2005; Parish, 2006; Crublet et al., 2008), prevented the capture of HIV-1 by spermatozoa (Fig. 3 B) without affecting the binding of BMA (MFI values of 269 ± 64 and 281 ± 43 for binding assays performed in the absence or presence of 100 U/ml heparin, respectively; mean ± SEM; n = 4). Heparinase II, which removes HS from heparin-like glycosaminoglycans (Parish, 2006; Bishop et al., 2007), reduced the expression of HS and the capture of HIV-1 (Fig. 3, C and D). As expected, treatment with trypsin, which almost completely prevented HIV capture (Fig. 2 A), also resulted in the abrogation of HS expression (Fig. 3 C). Because the expression of HS detected in the spermatozoa surface is relatively low, we then analyzed whether this result could be related to the antibody used to detect HS (mAb 10E4). Ruling out this possibility, Fig. S1 shows high levels of HS revealed by the mAb 10E4 in the human intestinal epithelial cell line HT-29 and a marked reduction in cell staining after heparinase II treatment. Cleavage of HS by heparinase III (heparitinase) results not only in the destruction of the epitope recognized by the anti-HS mAb 10E4 but also in the expression of a neoepitope recognized by the mAb 3G10 (Jones et al., 2005; Kureishy et al., 2006). Consistent with these studies, we found that treatment of spermatozoa with heparinase III resulted in a diminished expression of the HS epitope 10E4 (69 ± 13% decrease in the MFI; mean ± SEM; n = 4; P < 0.05) and the neoexpression of the epitope 3G10 (Fig. 3 E).


Spermatozoa capture HIV-1 through heparan sulfate and efficiently transmit the virus to dendritic cells.

Ceballos A, Remes Lenicov F, Sabatté J, Rodríguez Rodrígues C, Cabrini M, Jancic C, Raiden S, Donaldson M, Agustín Pasqualini R, Marin-Briggiler C, Vazquez-Levin M, Capani F, Amigorena S, Geffner J - J. Exp. Med. (2009)

Capture of HIV-1 by spermatozoa is mainly mediated through HS. (A) The expression of HS on spermatozoa was analyzed by flow cytometry using anti–HS-specific antibodies (clone 10E4). Isotype control (gray histogram) and 10E4 labeled (open histogram) are depicted. A representative experiment (n = 7) is shown. (B) Spermatozoa (1.5 × 106/200 µl) were incubated with HIV-1 BAL containing 25 ng of p24 for 60 min at 37°C in the absence or presence of 10 and 100 U/ml heparin, washed thoroughly, lysed, and assayed for p24 antigen by ELISA. Results are the mean ± SEM of six experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (C) Spermatozoa were treated with 5 U/ml heparinase II for 60 min at 25°C or 1,000 U/ml trypsin for 15 min at 37°C. Then the expression of HS was analyzed by flow cytometry. The results are expressed as the MFI ± SEM of five experiments performed in duplicate. Asterisks represent statistical significance (P < 0.05 vs. the expression of HS in controls). (D) Spermatozoa were treated with 1 and 5 U/ml heparinase II for 60 min at 25°C. Then their ability to capture HIV-1 was assayed as described for Fig. 2 A. Results are the mean ± SEM of five experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (E) Spermatozoa were treated with 5 U/ml heparinase III for 60 min at 25°C. Then the expression of the neoepitope 3G10 was analyzed by flow cytometry. Isotype (gray histogram) and 3G10-labeled untreated (control) or heparinase III–treated spermatozoa (open histograms) are shown. Isotype controls were similar for untreated and heparinase III–treated spermatozoa. A representative experiment (n = 4) is shown. (F) Spermatozoa (1.5 × 106/200 µl) were incubated with different primary HIV-1 isolates containing 25 ng of p24 for 60 min at 37°C in the absence or presence of 100 U/ml heparin or 5 mg/ml mannan, washed thoroughly, lysed, and assayed for p24 antigen by ELISA. Results are the mean ± SEM of three to five experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (G) HIV-1 pseudotypes were produced as described in Materials and methods. Spermatozoa (1.5 × 106/200 µl) were incubated with HIV-1 env− or HIV-1 env+ pseudotypes containing 40 ng of p24 for 60 min at 37°C in the absence or presence of 5 mg/ml mannan or 100 U/ml heparin, washed thoroughly, lysed, and assayed for p24 antigen by ELISA. Results are the mean ± SEM of three to four experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (H) The expression of syndecans 1–4 was analyzed by flow cytometry. Gray histograms correspond to isotype controls. In each case, a representative experiment (n = 3–6) is shown.
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fig3: Capture of HIV-1 by spermatozoa is mainly mediated through HS. (A) The expression of HS on spermatozoa was analyzed by flow cytometry using anti–HS-specific antibodies (clone 10E4). Isotype control (gray histogram) and 10E4 labeled (open histogram) are depicted. A representative experiment (n = 7) is shown. (B) Spermatozoa (1.5 × 106/200 µl) were incubated with HIV-1 BAL containing 25 ng of p24 for 60 min at 37°C in the absence or presence of 10 and 100 U/ml heparin, washed thoroughly, lysed, and assayed for p24 antigen by ELISA. Results are the mean ± SEM of six experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (C) Spermatozoa were treated with 5 U/ml heparinase II for 60 min at 25°C or 1,000 U/ml trypsin for 15 min at 37°C. Then the expression of HS was analyzed by flow cytometry. The results are expressed as the MFI ± SEM of five experiments performed in duplicate. Asterisks represent statistical significance (P < 0.05 vs. the expression of HS in controls). (D) Spermatozoa were treated with 1 and 5 U/ml heparinase II for 60 min at 25°C. Then their ability to capture HIV-1 was assayed as described for Fig. 2 A. Results are the mean ± SEM of five experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (E) Spermatozoa were treated with 5 U/ml heparinase III for 60 min at 25°C. Then the expression of the neoepitope 3G10 was analyzed by flow cytometry. Isotype (gray histogram) and 3G10-labeled untreated (control) or heparinase III–treated spermatozoa (open histograms) are shown. Isotype controls were similar for untreated and heparinase III–treated spermatozoa. A representative experiment (n = 4) is shown. (F) Spermatozoa (1.5 × 106/200 µl) were incubated with different primary HIV-1 isolates containing 25 ng of p24 for 60 min at 37°C in the absence or presence of 100 U/ml heparin or 5 mg/ml mannan, washed thoroughly, lysed, and assayed for p24 antigen by ELISA. Results are the mean ± SEM of three to five experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (G) HIV-1 pseudotypes were produced as described in Materials and methods. Spermatozoa (1.5 × 106/200 µl) were incubated with HIV-1 env− or HIV-1 env+ pseudotypes containing 40 ng of p24 for 60 min at 37°C in the absence or presence of 5 mg/ml mannan or 100 U/ml heparin, washed thoroughly, lysed, and assayed for p24 antigen by ELISA. Results are the mean ± SEM of three to four experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (H) The expression of syndecans 1–4 was analyzed by flow cytometry. Gray histograms correspond to isotype controls. In each case, a representative experiment (n = 3–6) is shown.
Mentions: Previous studies have shown that HS interacts with gp120 (de Parseval et al., 2005; Crublet et al., 2008), allowing the attachment of HIV-1 to macrophages (Saphire et al., 2001), DCs (de Witte et al., 2007), epithelial cells (Wu et al., 2003), and endothelial cells (Argyris et al., 2003). Studies performed by flow cytometry showed that spermatozoa express HS (Fig. 3 A). Heparin, an agent able to inhibit the attachment of HIV-1 to HS (Saphire et al., 2001; de Parseval et al., 2005; Parish, 2006; Crublet et al., 2008), prevented the capture of HIV-1 by spermatozoa (Fig. 3 B) without affecting the binding of BMA (MFI values of 269 ± 64 and 281 ± 43 for binding assays performed in the absence or presence of 100 U/ml heparin, respectively; mean ± SEM; n = 4). Heparinase II, which removes HS from heparin-like glycosaminoglycans (Parish, 2006; Bishop et al., 2007), reduced the expression of HS and the capture of HIV-1 (Fig. 3, C and D). As expected, treatment with trypsin, which almost completely prevented HIV capture (Fig. 2 A), also resulted in the abrogation of HS expression (Fig. 3 C). Because the expression of HS detected in the spermatozoa surface is relatively low, we then analyzed whether this result could be related to the antibody used to detect HS (mAb 10E4). Ruling out this possibility, Fig. S1 shows high levels of HS revealed by the mAb 10E4 in the human intestinal epithelial cell line HT-29 and a marked reduction in cell staining after heparinase II treatment. Cleavage of HS by heparinase III (heparitinase) results not only in the destruction of the epitope recognized by the anti-HS mAb 10E4 but also in the expression of a neoepitope recognized by the mAb 3G10 (Jones et al., 2005; Kureishy et al., 2006). Consistent with these studies, we found that treatment of spermatozoa with heparinase III resulted in a diminished expression of the HS epitope 10E4 (69 ± 13% decrease in the MFI; mean ± SEM; n = 4; P < 0.05) and the neoexpression of the epitope 3G10 (Fig. 3 E).

Bottom Line: Interaction of spermatozoa with DCs not only leads to the transmission of HIV-1 and the internalization of the spermatozoa but also results in the phenotypic maturation of DCs and the production of IL-10 but not IL-12p70.At low values of extracellular pH (approximately 6.5 pH units), similar to those found in the vaginal mucosa after sexual intercourse, the binding of HIV-1 to the spermatozoa and the consequent transmission of HIV-1 to DCs were strongly enhanced.Our observations support the notion that far from being a passive carrier, spermatozoa acting in concert with DCs might affect the early course of sexual transmission of HIV-1 infection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro Nacional de Referencia para SIDA, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires C1121ABG, Argentina.

ABSTRACT
Semen is the main vector for HIV-1 dissemination worldwide. It contains three major sources of infectious virus: free virions, infected leukocytes, and spermatozoa-associated virions. We focused on the interaction of HIV-1 with human spermatozoa and dendritic cells (DCs). We report that heparan sulfate is expressed in spermatozoa and plays an important role in the capture of HIV-1. Spermatozoa-attached virus is efficiently transmitted to DCs, macrophages, and T cells. Interaction of spermatozoa with DCs not only leads to the transmission of HIV-1 and the internalization of the spermatozoa but also results in the phenotypic maturation of DCs and the production of IL-10 but not IL-12p70. At low values of extracellular pH (approximately 6.5 pH units), similar to those found in the vaginal mucosa after sexual intercourse, the binding of HIV-1 to the spermatozoa and the consequent transmission of HIV-1 to DCs were strongly enhanced. Our observations support the notion that far from being a passive carrier, spermatozoa acting in concert with DCs might affect the early course of sexual transmission of HIV-1 infection.

Show MeSH
Related in: MedlinePlus