Human immature Langerhans cells restrict CXCR4-using HIV-1 transmission

Abstract

Background: Sexual transmission is the main route of HIV-1 infection and the CCR5-using (R5) HIV-1 is predominantly transmitted, even though CXCR4-using (X4) HIV-1 is often abundant in chronic HIV-1 patients. The mechanisms underlying this tropism selection are unclear. Mucosal Langerhans cells (LCs) are the first immune cells to encounter HIV-1 and here we investigated the role of LCs in selection of R5 HIV-1 using an ex vivo epidermal and vaginal transmission models.

Results: Immature LCs were productively infected by X4 as well as R5 HIV-1. However, only R5 but not X4 viruses were selectively transmitted by immature LCs to T cells. Transmission of HIV-1 was depended on de novo production of HIV-1 in LCs, since it could be inhibited by CCR5 fusion inhibitors as well as reverse transcription inhibitors. Notably, the activation state of LCs affected the restriction in X4 HIV-1 transmission; immune activation by TNF facilitated transmission of X4 as well as R5 HIV-1.

Conclusions: These data suggest that LCs play a crucial role in R5 selection and that immature LCs effectively restrict X4 at the level of transmission.

Figure 1

Human primary LCs predominantly transmit R5 HIV-1. (A-B) Human epidermal sheets were pulsed with low (4000 IU or 40 ng HIV-1 p24) or high (20000 IU or 400 ng HIV-1 p24) titers of HIV-1 NL4.3 (X4) or HIV-1 NL4.3-Bal (R5) for 5 hours (A) At day 3, emigrated LCs were cocultured with CCR5 vector-transduced Jurkat (CCR5 Jurkat T cells) and infection of Jurkat cells was measured at day 6 by intracellular p24 staining or GFP expression. T cell-marker CD3 and LC-marker CD1a were used to exclude LCs and LCs-T cells conjugates from analysis. Error bars represent the mean ± SEM of at least 3 independent experiments. (B) Emigrated LCs were cocultured with TZM-bl cells for 2 days and infection was determined by measuring luciferase activity (relative luciferase units [RLU]). Error bars represent the mean ± SEM of at least 3 independent experiments. (C-D) CCR5 Jurkat T cells (C) and TZM-bl cells (D) were infected with low or high titers of X4 and R5 virus, and infection was determined by intracellular p24 staining or luciferase activity respectively at day 2. Error bars represent the mean ± SEM of triplicates. (E) Epidermal sheets were infected with different X4 (HIV-1 SF2, LAI and NL4.3) and R5 viruses (SF162 and HIV-1 NL4.3-Bal). Emigrated LCs were cocultured with TZM-bl cells and infection of TZM-bl cells was determined by luciferase activity. Error bars represent the mean ± standard errors of the mean (SEM) of at least 3 independent experiments. (F) Vaginal LCs were exposed to X4 and R5 HIV-1 and after 3 days were co-cultured with CCR5 Jurkat T cells. Transmission was determined by measuring infection of CCR5 Jurkat T cells by intracellular p24 staining after 3 days. Dotplots represents two independent experiments/donors.

Figure 2

Human primary LCs are infected by X4 HIV-1 variants. (A-B) Human epidermal sheets were pulsed with low or high titers of HIV-1 NL4.3 (X4) or HIV-1 NL4.3-Bal (R5) for 5 hours, washed and cultured for 3 days. (A) Infection of emigrant LCs was determined by intracellular p24 staining or GFP expression in combination with LC-marker CD1a by flow cytometric analysis. The percentage of CD1a⁺p24⁺ cells are depicted here as % of infected cells. Error bars represent the mean ± SEM of at least 3 independent experiments. (B) Supernatant of cultured emigrant LCs was collected at day 3, 8 and 12 post-infection and HIV-1 p24 was measured in the supernatant by ELISA. Error bars represent the mean ± SEM of duplicates. (C) Epidermal sheets were pulsed with HIV-1 NL4.3 or HIV-1 NL4.3-Bal in the presence or absence of AZT for 5 hours. At day 3, HIV-1 tat/rev transcription in emigrated LCs was analyzed by real-time qPCR. Error bars represent the mean ± SEM of duplicates. One experiment representative of three is presented. (D) Epidermal sheets were pulsed with different X4 (HIV-1 SF2, LAI and NL4.3) and R5 viruses (SF162 and HIV-1 NL4.3-Bal) and after 3 days infection of LCs was determined. Error bars represent the mean ± SEM of at least 3 independent experiments. (E) Surface expression of HIV-1 coreceptors CCR5 and CXCR4 on immature LCs. Histograms represent at least 3 donors. (F) Epidermal sheets were pulsed with X4 and R5 for 5 hours. After 3 days mRNA expression of CXCR4 was measured in emigrant LCs by real-time qPCR. Error bars represent the mean ± SEM of duplicates. (G) Vaginal LCs were infected with HIV-1 NL4.3 and NL4.3-Bal and infection was measured by intracellular p24 staining. Representative dotplots of one out of two donors are shown.

Figure 3

Activated LCs efficiently transmit X4 HIV-1 variants. (A) Migratory LCs were analysed for expression of CD83 and CD86 by flow cytometry. Error bars represent the mean ± SEM of at least 3 different donors. (B) Surface expression of HIV-1 coreceptors on migratory LCs was determined by CCR5 and CXCR4 staining in combination of LC-markers CD1a and langerin by flow cytometry. (C-D) Immature LCs and migratory LCs were isolated and harvested and mRNA expression of CCR5 (C) and CXCR4 (D) was measured by real-time qPCR. Error bars represent the mean ± SEM of at least 3 donors. (E) Migratory LCs were infected with different titers of NL4.3 (X4) and NL4.3-Bal (R5) HIV-1. After 3 days infection of LCs was determined by intracellular HIV-1 p24 staining or GFP expression in combination with LC-marker CD1a by flow cytometric analysis. Error bars represent the mean ± SEM of at least 3 independent experiments. (F) Migratory LCs were pulsed with X4 or R5 HIV-1 strains and after 3 days, cocultured with CCR5 Jurkat T cells for additional 3 days. LCs mediated HIV-1 transmission was determined via measuring infection of CCR5 Jurkat T cells by intracellular p24 staining or GFP expression in combination with T cell-marker CD3 and LC-marker CD1a following flow cytometry. Error bars represent the mean ± SEM of at least 3 independent experiments.

Figure 4

Virus replication is necessary for HIV transmission. (A) Epidermal sheets were pulsed with low or high titers of HIV-1 NL4.3 (X4) or HIV-1 NL4.3-Bal (R5) in the presence or absence of the reverse transcriptase inhibitor AZT. After 3 days, emigrated LCs were cocultured with TZM-bl cells for 2 days. HIV-1 transmission to TZM-bl cells was determined by measuring luciferase activity (luminescence value or relative luciferase units [RLU]). Error bars represent the mean ± SEM of triplicates. (B) Migratory LCs were infected with X4 or R5 HIV-1 strains in presence or absence of different inhibitors including CCR5 antagonist Maraviroc, AZT and protease inhibitor Indinavir. After 3 days culture, LCs were cocultured with TZM-bl cells. After 2 days, transmission was measured by luciferase assay. Error bars represent the mean ± SEM of triplicates.

Figure 5

TNF-matured LCs transmit both X4 and R5 HIV-1. (A) Epidermal sheets were pretreated with different stimuli including Pam3CSK4 and TNF for 4 hours, pulsed with low and high titers of X4 and R5 HIV-1 for 5 hours (A) At day 3 emigrant LCs were collected, cocultured with CCR5 Jurkat T cells and HIV-1 transmission was determined after 3 days by measuring intracellular HIV-1 p24 or GFP expression by flow cytometry. Using T cell-marker CD3 and LC-marker CD1a, infection of the target cells were exclusively analyzed. Error bars represent the mean ± SEM of at least 3 independent experiments. (B) Epidermal sheets were pretreated with Pam3CSK4 and TNF for 4 hours or left untreated as control, following exposure to low or high titers of X4 and R5 HIV-1 in presence or absence of AZT for 5 hours. After 3 days, emigrated LCs were cocultured with CCR5 Jurkat T cells and transmission rate was determined at day 6 by flow cytometry. Error bars represent the mean ± SEM of at least 3 independent experiments. (C) Treatment of LCs with stimuli highly increased infection. Epidermal sheets were pretreated with Pam3CSK4 and TNF for 4 hours then pulsed with X4 HIV-1 for 5 hours. Infection of emigrant LCs was determined at 6–7 days by intracellular p24 staining in combination with LC-marker CD1a by flow cytometric analysis. Error bars represent the mean ± SEM of 3 independent experiments.