Interleukin-7 facilitates HIV-1 transmission to cervico-vaginal tissue ex vivo

Abstract

The majority of HIV-1 infections in women occur through vaginal intercourse, in which virus-containing semen is deposited on the cervico-vaginal mucosa. Semen is more than a mere carrier of HIV-1, since it contains many biological factors, in particular cytokines, that may affect HIV-1 transmission. The concentration of interleukin (IL)-7, one of the most prominent cytokines in semen of healthy individuals, is further increased in semen of HIV-1-infected men. Here, we investigated the potential role of IL-7 in HIV-1 vaginal transmission in an ex vivo system of human cervico-vaginal tissue. We simulated an in vivo situation by depositing HIV-1 on cervico-vaginal tissue in combination with IL-7 at concentrations comparable with those measured in semen of HIV-1-infected individuals. We found that IL-7 significantly enhanced virus replication in ex vivo infected cervico-vaginal tissue. Similarly, we observed an enhancement of HIV-1 replication in lymphoid tissue explants. Analysis of T cells isolated from infected tissues showed that IL-7 reduced CD4⁺ T cell depletion preventing apoptosis, as shown by the decrease in the number of cells expressing the apoptotic marker APO2.7 and the increase in the expression of the anti-apoptotic protein B-cell lymphoma (Bcl)-2. Also, IL-7 increased the fraction of cycling CD4⁺ T cells, as evidenced by staining for the nuclear factor Ki-67. High levels of seminal IL-7 in vivo may be relevant to the survival of the founder pool of HIV-1-infected cells in the cervico-vaginal mucosa at the initial stage of infection, promoting local expansion and dissemination of HIV infection.

Figure 1. IL-7 enhances HIV-1 production in lymphoid tissue explants.

Donor-matched lymphoid tissue blocks were inoculated with an X4 or R5 HIV-1 variant, HIV-1_LAI.04 and HIV-1_BaL respectively, or with the primary isolates HIV-1_96USSN20 (R5/X4 clade A), HIV-1_97USNG30 (R5 clade C), HIV-1_96USNG31 (R5/X4 clade C), and HIV-1_ME1 (R5 clade B) and cultured for 9 or 12 days in the absence or presence of recombinant human IL-7 5 or 25 ng/mL. HIV-1 replication was monitored from measurements of HIV-1 p24_gag accumulated in culture media over 3-days periods. Presented are kinetics of the release of HIV-1 p24_gag in culture media of tissue blocks inoculated with HIV-1_LAI.04 (A), HIV-1_BaL (B), and primary isolates (C–F) from representative donors. Each point represents pooled viral release from 27 tissue blocks over 3-days periods. (G) Presented are the average increases in the cumulative release of HIV-1 p24_gag in culture media of tissue blocks infected with different HIV-1 variants and treated with IL-7 5 ng/mL (n = 3–7) or 25 ng/mL (n = 7–13) compared with infected untreated donor-matched tissue blocks (means ± standard error of the mean (s.e.m.)).

Figure 2. IL-7 increases the number of HIV-1-infected CD4⁺ T cells in lymphoid tissue explants.

(A) Presented are dot plots for CD4⁺ T cells isolated from HIV-1-infected tissue blocks treated or not treated with IL-7 25 ng/mL from a representative donor on day 9 post infection. The amount of HIV-1-infected CD4⁺ (CD8⁻ p24_gag ⁺) T cells is expressed as percentage of CD3⁺ CD8⁻ cells. Upper panel: HIV-1_LAI.04, middle panel: HIV-1_BaL, lower panel: uninfected control. (B) Presented are the average increases in the numbers of HIV-1-infected CD4⁺ T cells isolated from tissue blocks infected with HIV-1_LAI.04 (n = 8) or HIV-1_BaL (n = 6) and treated with IL-7 25 ng/mL compared with infected untreated donor-matched tissue blocks (means ± s.e.m.).

Figure 3. IL-7 increases HIV-1 production and the number of HIV-1-infected CD4⁺ T cells in cervico-vaginal tissue explants.

Donor-matched cervico-vaginal tissue blocks were inoculated with HIV-1_BaL and cultured for 12 days in the absence or presence of IL-7 5 or 25 ng/mL. (A) Presented are kinetics of the release of HIV-1 p24_gag in culture media of tissue blocks inoculated with HIV-1_BaL from a representative donor. Each point represents pooled viral release from 24 tissue blocks over 3-days periods. (B) Presented are the average increases in the cumulative release of HIV-1 p24_gag in culture media of tissue blocks infected with HIV-1_BaL and treated with IL-7 5 or 25 ng/mL compared with infected untreated donor-matched tissue blocks (means ± s.e.m., n = 5). (C) Presented are dot plots for CD4⁺ T cells isolated from HIV-1-infected tissue blocks treated or not treated with IL-7 25 ng/mL from a representative donor on day 9 post infection. The amount of HIV-1-infected CD4⁺ (CD8⁻ p24_gag ⁺) T cells is expressed as percentage of total CD3⁺ CD8⁻ cells.

Figure 4. Exposure to IL-7 for a short time is sufficient to enhance HIV-1 production.

Donor-matched lymphoid tissue blocks were treated with IL-7 overnight prior to infection with HIV-1 or until day 3 post infection, and subsequently cultured in the absence of IL-7. Presented are the average increases in the cumulative release of HIV-1 p24_gag in culture media of tissue blocks infected with HIV-1_LAI.04 (n = 4) or HIV-1_BaL (n = 5) and treated with IL-7 25 ng/mL compared with infected untreated donor-matched tissue blocks (means ± s.e.m.).

Figure 5. IL-7 decreases apoptosis of HIV-1-infected CD4⁺ T cells.

Presented are the average fractions of HIV-1-infected CD4⁺ (CD8⁻ p24_gag ⁺) T cells expressing the apoptotic marker APO2.7 isolated from donor-matched lymphoid tissue blocks infected with HIV-1_LAI.04 (n = 8) (A) or HIV-1_BaL (n = 5) (B) treated or not treated with IL-7 25 ng/mL (means ± s.e.m.). The amount of APO2.7⁺ HIV-1-infected CD4⁺ T cells is expressed as a percentage of total CD8⁻ p24_gag ⁺ T cells.

Figure 6. IL-7 up-regulates the expression of Bcl-2.

(A) Presented are the expression levels of the anti-apoptotic protein Bcl-2 in HIV-1-infected CD4⁺ (CD8⁻ p24_gag ⁺) T cells isolated from lymphoid tissue blocks infected with HIV-1_LAI.04 or HIV-1_BaL and treated with IL-7 25 ng/mL (black line) vs. infected untreated tissue blocks (red line) from a representative donor on day 9 post infection. The amount of Bcl-2 is expressed as median fluorescence intensity (MFI) value. (B) Presented are the average increases in Bcl-2 expression in HIV-1-infected CD4⁺ T cells isolated from lymphoid tissue blocks infected with HIV-1_LAI.04 (n = 8) or HIV-1_BaL (n = 6) and treated with IL-7 25 ng/mL compared with infected untreated donor-matched tissue blocks (means ± s.e.m.). (C) Presented are the expression levels of Bcl-2 in HIV-1-infected and -uninfected (CD8⁻ p24_gag ⁻) CD4⁺ T cells isolated from cervico-vaginal tissue blocks infected with HIV-1_BaL and treated with IL-7 (black line) vs. untreated tissue blocks (red line) from a representative donor on day 9 post infection. (D) Presented are the average increases in Bcl-2 expression in HIV-1-infected and -uninfected CD4⁺ T cells isolated from cervico-vaginal tissue blocks infected with HIV-1_BaL and treated with IL-7 25 ng/mL compared with infected untreated donor-matched tissue blocks (means ± s.e.m., n = 5).

Figure 7. IL-7 increases the proliferation of HIV-1-infected CD4⁺ T cells.

(A) Presented are dot plots for HIV-1-infected CD4⁺ (CD8⁻ p24_gag ⁺) T cells isolated from lymphoid tissue blocks infected with HIV-1_LAI.04 or HIV-1_BaL treated or not treated with IL-7 25 ng/mL from a representative donor on day 9 post infection. The amount of Ki-67⁺ HIV-1-infected CD4⁺ T cells is expressed as percentage of total CD8⁻ p24_gag ⁺ T cells. (B) Presented are the average fractions of HIV-1-infected CD4⁺ T cells expressing Ki-67 isolated from donor-matched lymphoid tissue blocks infected with HIV-1_LAI.04 or HIV-1_BaL treated or not treated with IL-7 25 ng/mL (means ± s.e.m., n = 6).