Vaginal bacterium Prevotella timonensis turns protective Langerhans cells into HIV-1 reservoirs for virus dissemination

Abstract

Dysbiosis of vaginal microbiota is associated with increased HIV-1 acquisition, but the underlying cellular mechanisms remain unclear. Vaginal Langerhans cells (LCs) protect against mucosal HIV-1 infection via autophagy-mediated degradation of HIV-1. As LCs are in continuous contact with bacterial members of the vaginal microbiome, we investigated the impact of commensal and dysbiosis-associated vaginal (an)aerobic bacterial species on the antiviral function of LCs. Most of the tested bacteria did not affect the HIV-1 restrictive function of LCs. However, Prevotella timonensis induced a vast uptake of HIV-1 by vaginal LCs. Internalized virus remained infectious for days and uptake was unaffected by antiretroviral drugs. P. timonensis-exposed LCs efficiently transmitted HIV-1 to target cells both in vitro and ex vivo. Additionally, P. timonensis exposure enhanced uptake and transmission of the HIV-1 variants that establish infection after sexual transmission, the so-called Transmitted Founder variants. Our findings, therefore, suggest that P. timonensis might set the stage for enhanced HIV-1 susceptibility during vaginal dysbiosis and advocate targeted treatment of P. timonensis during bacterial vaginosis to limit HIV-1 infection.

Keywords: HIV-1; Langerhans cells (LCs); Prevotella timonensis; transmission; vaginal microbiome.

Figure 1. P. timonensis increases HIV‐1 content in primary LCs

1. A, B

   Isolated epidermal LCs (A, n = 4) or isolated vaginal LCs (B, n = 5) were stimulated O/N with a variety of vaginal microbiota and subsequently exposed to HIV‐1 (SF162; MOI 0.5). Epidermal LCs were stimulated with L. crispatus (LC), M. elsdenii (ME), and P. timonensis (PT), whereas vaginal LCs were additionally stimulated with L. iners (LI), G. vaginalis (GV), and A. vaginae (AV). After 5 days, HIV‐1 p24 in LCs was determined by an intracellular staining for CD1a and p24 by flow cytometry.

2. C

   Isolated epidermal LCs (n = 3) were stimulated O/N with B. fragilis (BF), B. thetaiotaomicron (BT), P. amnii (PA), P. bivia (PB), P. copri (PC), and P. timonensis (PT), followed by HIV‐1 (SF162; MOI 0.5) infection for 5 days and HIV‐1 p24 determination by flow cytometry.

3. D

   Vaginal epithelium explants (n = 8) were exposed to vaginal microbiota as described for isolated vaginal LCs. HIV‐1 p24 in emigrated LCs was determined by flow cytometry.

Data information: Symbols represent independent donors. Data are mean ± SD. Two‐tailed t‐test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 2. P. timonensis increases HIV‐1 uptake but not productive infection

1. A–D

   Isolated epidermal LCs (A, n = 2), isolated vaginal LCs (B and C, both n = 3), or vaginal explants (D, n = 5) were stimulated O/N with P. timonensis (PT) and exposed to HIV‐1 (SF162; MOI 0.5) for 5 days in the presence or absence of HIV‐1 replication inhibitors (Inh) zidovudine (AZT), tenofovir (TFV), lamivudine (3TC), or indinavir (IDV). HIV‐1 levels were determined by intracellular staining for HIV‐1 p24 using flow cytometry.

2. E, F

   Representative plots (E) and pooled data (F, n = 3) of HIV‐1 (NL4.3eGFP‐Bal) infection of O/N P. timonensis‐stimulated (PT) isolated vaginal LCs as determined by both GFP‐detection (de novo replication) and HIV‐1 p24 (detection of de novo replication and uptake) by flow cytometry.

3. G

   Isolated epidermal LCs (n = 3) were stimulated O/N with P. timonensis (PT). Next, LCs were treated with T20, CCR5 inhibitor Maraviroc (CCR5), neutralizing antibodies against CD4 (CD4) and isotype control (Iso), subsequently followed by HIV‐1 exposure for 5 days (SF162; MOI 0.5). Intracellular HIV‐1 p24 levels were determined using flow cytometry.

Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two‐tailed t‐test, *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3. P. timonensis induces minor maturation in immature and migratory LCs

Immature vaginal (vLC) and epidermal LCs (eLC) were stimulated O/N with L. crispatus (LC), M. elsdenii (ME), or P. timonensis (PT), and activation phenotype was determined by flow cytometry. Same donor emigrated epidermal LCs (mLC) served as a matured and activated control. Same donor data of emigrated vaginal LCs could not be obtained due to limited amount of tissue (N.D.).

1. A–H

   Representative plots of vaginal (A) or epidermal (B) donors and combined experiments (MFI expression relative to untreated condition) of CD80 (C, n = 3), CD86 (D, n = 3), and CCR7 (E, n = 3) surface expression are shown. Ex vivo skin explants were stimulated O/N with L. crispatus (LC), M. elsdenii (ME), P. timonensis (PT) or poly(I:C) and at day 3 post‐inoculation, emigrated LCs were collected and washed and activation phenotype was determined by flow cytometry. Cells were analyzed for CD1a expression, and the absolute number of CD1a‐positive cells, that is LCs, migrated from the epidermis was determined using counting beads; the graph shows the migration relative to the untreated condition (F, n = 5). CD1a‐positive cells, that is LCs, were analyzed using flow cytometry, and the graphs show combined experiments (MFI expression relative to untreated condition) of CD80 (G, n = 5) and CD86 (H, n = 5) surface expression.

Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two‐tailed t‐test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure EV1. Effectiveness of HIV inhibitors

Migratory LCs were treated with replication inhibitors followed by exposure to SF162 (MOI 0.5) for 3 days. Intracellular HIV‐1 p24 levels were measured by flow cytometry. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD.

Figure EV2. Effectiveness of NL4.3eGFP‐Bal

Phytohemagglutinin (PHA)‐activated CD4⁺ T cells were infected with NL4.3eGFP‐Bal for 3 days. GFP expression levels were measured by flow cytometry. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD.

Figure 4. HIV‐1 is internalized and protected from the langerin‐mediated degradation pathway in P. timonensis‐exposed LCs

Isolated vaginal LCs were stimulated O/N with P. timonensis (PT) followed by HIV‐1 (SF162; MOI 0.5) exposure.

1. A

   Intracellular HIV‐1 detection was determined by confocal microscopy (Hoechst in blue, HIV‐1 p24 in green); scale bar represents 5 μm (representative donor).

2. B

   Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by a 4‐h exposure to HIV‐1. HIV‐1 p24 was determined by ELISA after trypsin treatment and lysis of LCs (n = 3).

3. C

   Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by HIV‐1 (SF162; MOI 0.5) exposure for 3 days. After 3 days, LCs were treated with PBS or trypsin and HIV‐1 p24 was determined by flow cytometry (n = 3).

4. D, E

   Isolated vaginal LCs were stimulated O/N with P. timonensis (PT) followed by infection with VSV‐g‐BlaM‐Vpr fusion (positive control) and NL4.3Bal‐BlaM‐Vpr fusion. The figure shows representative plots (D) and pooled data (E, n = 3) of viral fusion as determined by β‐lactamase‐Vpr (BlaM‐Vpr) activity.

5. F

   Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by HIV‐1 exposure (SF162; MOI 0.5) in the presence or absence of anti‐langerin (10E2) or mannan (MAN). HIV‐1 p24 content was determined by ELISA after trypsin treatment and subsequent lysis of LCs (n = 2).

6. G

   Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by treatment with Rapamycin (μM) and exposure to HIV‐1 (SF162). After 3 days, HIV‐1 p24 was measured by ELISA after trypsin treatment and lysis of LCs (n = 2).

Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two‐tailed t‐test, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure EV3. Relative langerin expression

Immature epidermal LCs were stimulated O/N with L. crispatus (LC), M. elsdenii (ME), or P. timonensis (PT), and surface expression of langerin expression was determined by flow cytometry (MFI expression relative to untreated condition; n = 3). Same donor emigrated epidermal LCs (mLC) served as a matured and activated control. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two‐tailed t‐test, **P < 0.01 and ****P < 0.0001.

Figure 5. P. timonensis induces transmission of HIV‐1 by LCs in a replication‐independent manner

1. A

   Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by HIV‐1 (SF162; MOI 0.5) exposure. LCs were subjected to repetitive freeze–thaw cycles, and cell lysates were added to U87.CD4.CCR5 cell line. HIV‐1 infection of U87.CD4.CCR5 was determined by an intracellular staining for p24 by flow cytometry (n = 3).

2. B, C

   Ex vivo skin explants were stimulated O/N with L. crispatus (LC), M. elsdenii (ME), or P. timonensis (PT) and inoculated with HIV‐1 (JRCSF; MOI 0.5) in the presence or absence of tenofovir. At day 2 post‐inoculation, emigrated LCs were collected, washed, and co‐cultured with U87.CD4.CCR5 cells for 3 days. Cells were analyzed for CD1a expression and p24 content by flow cytometry and here presented as representative plots (B) and pooled data of HIV‐1‐positive cells (%) of the CD1a‐negative U87.CD4.CCR5 cells (C, n = 4).

3. D

   Ex vivo skin explants were stimulated with P. timonensis (PT), pre‐treated with T20 or maraviroc (MAR), and exposed to HIV‐1 (JRCSF; MOI 0.5) for 2 days, after which the migratory fraction was co‐cultured with U87.CD4.CCR5 cells. HIV‐1‐positive cells (%) of the CD1a‐negative fraction, that is, U87.CD4.CCR5 cells, were determined using flow cytometry (n = 5).

4. E, F

   Isolated vaginal LCs were stimulated O/N with P. timonensis (PT) and infected with HIV‐1 (SF162; MOI 0.5). After 3 days, LCs were washed and co‐cultured with U87.CD4.CCR5. Infection was analyzed by flow cytometry and here shown as representative plots (E) and pooled data of HIV‐1‐positive cells (%) of the CD1a‐negative U87.CD4.CCR5 cells (F, n = 2).

Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two‐tailed t‐test, *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 6. P. timonensis specifically induces uptake and transmission of Transmitted Founder HIV‐1

1. A, B

   Isolated epidermal LCs were stimulated by different Prevotella spp. and Bacteroides spp. followed by T/F variant (CH058; MOI 0.25) exposure in the presence or absence of AZT (A, n = 3) or tenofovir (B, n = 2). A lower MOI was used due to higher susceptibility of LCs for CH058 than for laboratory strains.

2. C

   HIV‐1 uptake was determined by flow cytometry. Ex vivo skin explants (n = 3) were stimulated O/N with P. timonensis (PT) followed by pre‐treatment with tenofovir (TFV). After pre‐treatment, skin explants were inoculated with lab‐adapted strain (SF162; MOI 0.25) or HIV‐1 T/F strain (CHO58; MOI 0.25). After 2 days, the migratory fraction was collected and co‐cultured with U87.CD4.CCR5 cells. Cells were analyzed for CD1a expression and p24 content in U87.CD4.CCR5 cells by flow cytometry. A lower MOI was used due to higher susceptibility of LCs for CH058 than for laboratory strain SF162.

Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two‐tailed t‐test, *P < 0.05.