Caveolin-1 mediated uptake via langerin restricts HIV-1 infection in human Langerhans cells

Abstract

Background: Human Langerhans cells (LCs) reside in foreskin and vaginal mucosa and are the first immune cells to interact with HIV-1 during sexual transmission. LCs capture HIV-1 through the C-type lectin receptor langerin, which routes the virus into Birbeck granules (BGs), thereby preventing HIV-1 infection. BGs are langerin-positive organelles exclusively present in LCs, however, their origin and function are unknown.

Results: Here, we not only show that langerin and caveolin-1 co-localize at the cell membrane and in vesicles but also that BGs are langerin/caveolin-1-positive vesicles are linked to the lysosomal degradation pathway in LCs. Moreover, inhibition of caveolar endocytosis in primary LCs abrogated HIV-1 sequestering into langerin(+) caveolar structures. Notably, both inhibition of caveolar uptake and silencing of caveolar structure protein caveolin-1 resulted in increased HIV-1 integration and subsequent infection. In contrast, inhibition of clathrin-mediated endocytosis did not affect HIV-1 integration, even though HIV-1 uptake was decreased, suggesting that clathrin-mediated endocytosis is not involved in HIV-1 restriction in LCs.

Conclusions: Thus, our data strongly indicate that BGs belong to the caveolar endocytosis pathway and that caveolin-1 mediated HIV-1 uptake is an intrinsic restriction mechanism present in human LCs that prevents HIV-1 infection. Harnessing this particular internalization pathway has the potential to facilitate strategies to combat HIV-1 transmission.

Figure 1

Langerin co-localizes with caveolin-1 in steady state. Confocal scanning laser microscopic analyses for THP-langerin cell line (A), MUTZ3-LC cell line (MUTZ-LCs, B) and human LCs (C) for langerin and caveolin-1 in steady-state condition in permeablized cells; bars represent 10 μm (A, B, C). Langerin and caveolin-1 were immuno-precipitated from LC cell lysates and immuno-blotted with antibodies against caveolin-1 or langerin (D). These data are representative of at least 3 donors or 3 independent experiments.

Figure 2

Langerin, caveolin-1 and HIV-1 partly co-localize in the same organelle. LCs were incubated with HIV-1 for 4 h in the absence (A) or presence of the caveolar inhibitor filipin (1 μg/ml, B) LCs were stained for p24-HIV-1, langerin and caveolin-1; representative for 3 donors; bars represent 10 μm (A, B). LCs were incubated with HIV-1 for 4 h in the absence or presence of filipin (1 μg/ml). Cells were treated with Trypsin-EDTA (0.05%) or left untreated as control. HIV-1 uptake was quantified by permeabilizing cells and measuring subsequent HIV-1-p24 content by flow cytometry. n = 3 paired students t-test; *p < 0.05; SD and mean are depicted (C).

Figure 3

Langerin and caveolin-1 are present in Birbeck Granules. MUTZ-LCs were analyzed by immuno-electron microscopy for the presence of BGs that appear tubular (filled arrows) or tennis racquet shaped (A). Sections were stained for langerin with 10 nm gold particles (B), caveolin-1 with 10 nm gold particles (C), langerin and caveolin with 10 nm and 15 nm gold particles, respectively (D). MUTZ-LCs were incubated for 24 hours with HIV-1 and stained for p24-HIV-1 and CD63 or LAMP2 with 10 nm and 15 nm gold particles (E). Bars represent 200 nm and these data are representative of two independent experiments.

Figure 4

Inhibition of caveolar uptake increased HIV-1 infection. LCs were incubated for 2 hours with HIV-1NL4.3/Vpr-BlaM and viral fusion to the host membrane was measured by BlaM assay as described by [31] (A). MUTZ-LCs were incubated with HIV-1 NL4.3-BaL (MOI = 0.2, B) or with VSV-G pseudotyped HIV-1 (MOI = 0.2, C) in the absence or presence of filipin (1 μg/ml) and integration of HIV-1 DNA was analyzed by Alu-PCR. MUTZ-LCs were treated with small interfering RNA (siRNA) of caveolin-1-specific (Cav-1 siRNA) or with non-target control (Control siRNA). Cells were incubated for 18 hours with HIV-1 NL4.3-BaL (D) or HIV-1 NL4.3 (E). Integration of HIV-1 DNA was determined by Alu-PCR. At day 8 post-infection, HIV-1 infection was measured by intracellular p24 staining. Percentage of CD1a⁺p24⁺ cells are depicted here as % of HIV-1 infection. MUTZ-LCs were incubated with HIV-1 for 4 h in the absence or presence of the clathrin inhibitor monodansylcadaverine (MDC, 10 or 50 μM). Cells were treated with Trypsin-EDTA (0.05%) or left untreated as control. HIV-1 uptake was quantified by permeabilizing cells and measuring subsequent HIV-1-p24 content by flow cytometry (F). MUTZ-LCs were incubated for 18 hours with HIV-1 NL4.3-BaL in the absence or presence of monodansylcadaverine (MDC, 10 or 50 μM) and integration of HIV-1 DNA was analyzed by Alu-PCR (G). n = 3 paired students t-test; *p < 0.05; SD and mean are depicted (A, B, F, G). One representative experiment out of two is shown (C). n = 4 paired students t-test; *p < 0.05; SD and mean are depicted (D, E).