Immune activation of vaginal human Langerhans cells increases susceptibility to HIV-1 infection

Abstract

Vaginal inflammation increases the risk for sexual HIV-1 transmission but underlying mechanisms remain unclear. In this study we assessed the impact of immune activation on HIV-1 susceptibility of primary human vaginal Langerhans cells (LCs). Vaginal LCs isolated from human vaginal tissue expressed a broad range of TLRs and became activated after exposure to both viral and bacterial TLR ligands. HIV-1 replication was restricted in immature vaginal LCs as only low levels of infection could be detected. Notably, activation of immature vaginal LCs by bacterial TLR ligands increased HIV-1 infection, whereas viral TLR ligands were unable to induce HIV-1 replication in vaginal LCs. Furthermore, mature vaginal LCs transmitted HIV-1 to CD4 T cells. This study emphasizes the role for vaginal LCs in protection against mucosal HIV-1 infection, which is abrogated upon activation. Moreover, our data suggest that bacterial STIs can increase the risk of HIV-1 acquisition in women.

Figure 1

Vaginal LC purification. (a) schematic model and pictures of isolation procedures used to obtain immature and mature vaginal LCs from primary human vaginal tissue. Images were adopted from Servier Medical Art by Servier (http://www.servier.com/Powerpoint-image-bank) and modified by the authors under the following terms: CREATIVE COMMONS Attribution 3.0 Unported (CC BY 3.0); (b,c) vaginal LC purity (% CD1a + /Langerin^high cells) after immature ((b), N = 7) and mature ((C), N = 6) isolation procedure. *p < 0,05, **p < 0.01, two-tailed t-test, data are mean ± SD.

Figure 2

Immature and mature vaginal LCs diplay a differential phenotype. (a) representative plots of the expression of LC receptors involved in the interaction with HIV-1 (CD4, CCR5, langerin) and T cells (CD86) on immature and mature vaginal LCs as determined by flow cytometry. ((b), N = 3) pooled data from 3 separate donors of immature and mature vaginal LCs (CD1a +) for the expression of CD4, CCR5, langerin and CD86. **p < 0.01, ***p < 0.001, unpaired t-test, data are mean ± SD.

Figure 3

Vaginal LCs express functional viral and bacterial TLRs. (a) TLR expression levels of immature vaginal LCs (CD1a + , Langerin^high) as determined by RT-PCR (N = 4); (b) relative maturation (MFI CD86 ‘TLR-ligand’/MFI CD86 ‘medium’) of immature LCs exposed to TLR-ligands (N = 4–6); *p < 0,05, **p < 0.01, two-tailed t-test, data are mean ± SD; (c) representative histograms of CD86 upregulation on immature vaginal LCs (CD1a + , Langerin^high) in response to TLR-ligands as determined by flow cytometry (unstimulated—filled grey histogram; TLR-ligand—black line). ((d), N = 5, N = 4 and N = 5 respectively) pooled data of expression of CD80, CD83 and CD86 on seperated donors after stimulation with either LPS or Poly(I:C); ((e), N = 6) pooled data of separate donors for expression of CD4, CCR5 and langerin; *p < 0,05, **p < 0.01, two-tailed t-test, data are mean ± SD.

Figure 4

Immature vaginal LCs efficiently engage HIV-1 through langerin. (a,b) representative flow cytometry plots of HIV-1 gp120 binding to immature (a) and mature (b) vaginal (CD1a + , Langerin^high) left untreated or pre-treated with anti-langerin; (c,d) pooled HIV-1 gp120 binding data (% HIV-1-gp120 +) of immature ((c), N = 3) and mature ((d), N = 3) vaginal LCs pre-treated with medium, anti-langerin, or mannan; *p < 0,05, **p < 0.01, two-tailed t-test; ((e), N = 2) GP120 binding (% HIV-1-gp120 +) on immature vaginal LCs in the presence or absence of anti-langerin (10E2), anti-DC-SIGN (D1, isotype control), anti-CD4 or anti-CCR5 blocking antibody; data are mean ± SD.

Figure 5

Bacterial TLR-ligands increase HIV-1 infection in vaginal LCs and activated vaginal LCs transmit HIV-1 to target cells. (a,b) HIV-1 (NL4.3-Bal, SF162, JR-CSF) infection of immature ((a), N = 3–10) and mature ((b), N = 2–5) vaginal LCs (CD1a + , langerin^high) as measured by double staining for intracellular p24 and CD1a, and analyzed by flow cytometry; (c) HIV-1 (SF162) infection in immature vaginal LCs pre-stimulated with TLR-ligands, N = 4–7 *p < 0,05, two-tailed t-test, (d) HIV-1 (SF162) transmission from immature and mature vaginal LCs to CD4 T cells after co-culture, N = 3–5, **p < 0.01, two-tailed t-test, ((e), N = 4) HIV-1 transmitted/founder virus (CH058) transmission by immature vaginal LCs to CD4/CCR5 expressing U87 cells with or without prior stimulation or LPS for 30 min, data are mean ± SD.