EMBO/soda-nel huggingface dataset:
Vaginal bacterium Prevotella timonensis turns protective Langerhans cells into HIV-1 reservoirs for virus dissemination. (2022)
Figure 1

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

(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 LC 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 five days, HIV-1 p24 in LCs was determined by an intracellular staining for CD1a and p24 by flow cytometry.

(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.

(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.
Vaginal bacterium Prevotella timonensis turns protective Langerhans cells into HIV-1 reservoirs for virus dissemination. (2022)
Figure 2

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

(A-D) Isolated epidermal LCs (A, n = 2), isolated vaginal LCs (B-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 five days in presence or absence of HIV-1 replication inhibitors (Inh) zidovudine (AZT), tenofovir (TFV), iamivudine (3TC) or indinavir (IDV). HIV-1 levels were determined by intracellular staining for HIV-1 p24 using flow cytometry.

(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.

(G) Isolated epidermal LCs (n = 3) were treated with T20, Maraviroc (MAR), neutralizing antibodies against CD4 (αCD4) and isotype control (Iso), followed by O/N stimulation with P. timonensis (PT). Next, LCs were exposed to HIV-1 for five 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.
Vaginal bacterium Prevotella timonensis turns protective Langerhans cells into HIV-1 reservoirs for virus dissemination. (2022)
Figure 3

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.).

(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, washed and activation phenotype was determined by flow cytometry. Cells were analysed for CD1a expression and the absolute number of CD1a-positive cells, i.e. 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, i.e. LCs, were analysed 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.
Vaginal bacterium Prevotella timonensis turns protective Langerhans cells into HIV-1 reservoirs for virus dissemination. (2022)
Figure 4

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.

(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).

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

(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. Figure shows representative plots (D) and pooled data (E, n = 3) of viral fusion as determined by β-lactamase-Vpr (BlaM-Vpr) activity.

(F) Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by HIV-1 exposure (SF162; MOI 0.5) in 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).

(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.
Vaginal bacterium Prevotella timonensis turns protective Langerhans cells into HIV-1 reservoirs for virus dissemination. (2022)
Figure 5

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

(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).

(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 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 analysed 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).

(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, i.e. U87.CD4.CCR5 cells, were determined using flow cytometry (n = 5).

(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 analysed 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.
Vaginal bacterium Prevotella timonensis turns protective Langerhans cells into HIV-1 reservoirs for virus dissemination. (2022)
Figure 6

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

(A, B) Isolated epidermal LCs were stimulated by different Prevotella spp. and Bacteriodus spp. followed by T/F variant (CH058; MOI 0.25) exposure in 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.

(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 analysed 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.

Expanded View Figure legends
Enterovirus-A71 exploits peripherin and Rac1 to invade the central nervous system (2021)
Figure 1

Figure 1. PRPH plays a pro-viral role during EV-A71 infection in NSC-34 and SH-SY5Y cells.

(A) NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 strain at MOI 10 and 0.01, respectively. At the indicated time points, the culture supernatants were removed and the cell lysates were prepared for Western blot analysis. Relative protein levels were determined by normalizing against β-actin and compared to mock-infected cells (ImageJ). Values represent relative intensity expressed as fold change.

(B) NSC-34 and SH-SY5Y cells were transfected with various concentrations of siPRPH or siNTC. The efficiency of siRNA knockdown at 48h.p.t. was validated by Western blot while cytotoxicity was determined by AlamarBlue assay. siPRPH- or siNTC-transfected cells were infected with EV-A71 S41, and the viral titers were determined at 48h.p.i. Results are expressed as mean ± SD of technical triplicates. Statistical analysis was performed using two-way ANOVA with Dunnett's post-test (*p<0.05, **p<0.005) against cells treated with siNTC.

(C) PRPH was overexpressed in NSC-34 cells upon transfection with pCMV6-entry vector containing PRPH gene (Fig. EV1). At 48h.p.t., cells were infected with EV-A71 S41 at MOI 5 and the viral titers were determined at 48h.p.i. Data are expressed as mean of triplicates ± SD. Statistical analysis was performed using t-test (**p<0.005) against vector control.

(D) SH-SY5Y cells were transfected with various concentrations of siPRPH or siNTC. At 48 h.p.t. cells were infected with CA16 or EV-A71 C2 at MOI 0.01. Viral titers were determined 48 h.p.i by plaque assay. Data are expressed as mean of triplicates ± SD. Statistical analysis was performed using two-way ANOVA with Dunnett's post-test ****p<0.0001) against cells treated with siNTC. Data are expressed as mean of triplicates ± SD.

Data information: Data shown in each panel is a representative set obtained from two independent experiments.
Enterovirus-A71 exploits peripherin and Rac1 to invade the central nervous system (2021)
Figure 2

Figure 2. Expression of PRPH during EV-A71 infection in mice.

Two-week old AG129 mice (n=2-3 /group/time point) were infected with EV-A71 S41.

(A) At various time points post-infection, mice were euthanized and the relative expression of PRPH was detected by Western blot in tissue homogenates (brainstem, spinal cord and limb muscle). Actin and GAPDH serve as loading controls, and protein expression was compared to age-matched non-infected mice.

(B) Immunostaining of PRPH and EV-A71 particles on brainstem and spinal cord sections harvested at 6 d.p.i.

(C) Immunostaining of NMJs (nAChR), PRPH, and EV-A71 particles (VP0) in limb muscle sections harvested at 6 d.p.i.

Data information: Data shown in each panel is a representative set obtained from two independent experiments. Scale bars represent 50μm.
Enterovirus-A71 exploits peripherin and Rac1 to invade the central nervous system (2021)
Figure 3

Figure 3. PRPH is involved in EV-A71 entry.

(A) Flow cytometry analysis of surface PRPH expression. NSC-34 cells were infected with EV-A71 S41 at MOI 10 for 1h before staining with Viability Dye and anti-PRPH antibodies. Data are expressed as Mean Fluorescence Intensity.

(B) Antibody blocking assay. NSC-34 and SH-SY5Y cells were pre-treated with anti-PRPH or anti-IgG isotype antibodies, followed by EV-A71 S41 infection at MOI 10 and 0.01, respectively. Culture supernatants were harvested at 24h.p.i. and titrated by plaque assay. Results are expressed as the mean ± SD of technical triplicates. Statistical analysis was performed using one-way ANOVA against IgG control (*, p<0.05, **, p<0.005, ***p<0.0005).

(C) Proximity ligation assay. NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 at MOI 10 and 0.01, respectively. Cells were fixed at 48h.p.i. prior to PLA staining using anti-PRPH and anti-VP0 antibodies. Scale bars represent 10μm.

Data information: Data shown in each panel is a representative set obtained from two independent experiments.
Enterovirus-A71 exploits peripherin and Rac1 to invade the central nervous system (2021)
Figure 4

Figure 4. PRPH plays a role in the replication of EV-A71 genome.

(A) NSC-34 and SH-SY5Y cells were reverse-transfected with siPRPH or siNTC for 48 hrs prior to transfection with EV-A71 RNA. The culture supernatants were harvested for viral titer determination at 24h.p.t. Results are expressed as the mean ± SD of technical triplicates. Statistical analysis was performed using t-test against siNTC treatment (*p<0.05).

(B) Schematic drawing of LucEV-A71 replicon.

(C) NSC-34 and SH-SY5Y cells were reverse-transfected with siPRPH or siNTC for 48 hrs prior to transfection with LucEV-A71 replicon. Luminescence signal was read at 24h.p.t. Results are expressed as the mean ± SD of technical triplicates. Statistical analysis was performed using two-way ANOVA with Dunnett's post-test (*p<0.05, **p<0.005) against siNTC treatment.

Data information: Data shown in each panel is a representative set obtained from two independent experiments.
Enterovirus-A71 exploits peripherin and Rac1 to invade the central nervous system (2021)
Figure 5

Figure 5. Intracellular PRPH interacts with capsid and non-structural components of EV-A71.

(A) NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 at MOI 30 and 0.01, respectively. At 48 h.p.i., cells were fixed and permeabilized prior to immunostaining.

(B) Total infected cell lysates were pulled down with anti-PRPH or IgG isotype control antibody prior to immunoblotting. Uninfected cells were included as negative control. IP: immunoprecipitation; IB: immunoblotting.

(C) Proximity ligation assay. NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 at MOI 10 and 0.01, respectively. At 48 h.p.i., the cells were fixed and permeabilized prior to PLA staining using antibodies specific to PPRH, VP0, 3C and 3D.

Data information: Data shown in each panel is a representative set obtained from two independent experiments. Scale bars represent 10μm.
Enterovirus-A71 exploits peripherin and Rac1 to invade the central nervous system (2021)
Figure 7

Figure 7. Rac1 interacts with intracellular EV-A71 in both NSC-34 and SH-SY5Y cells.

(A) NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 at MOI 30 and 0.01, respectively. At 48h.p.i., the cells were fixed and permeabilized prior to immunostaining.

(B) Proximity ligation assay. NSC-34 and SH-SY5Y cells were transfected with LucEV-A71 replicon. Cells were fixed and permeabilized at 24h.p.t. prior to PLA staining using antibodies specific to Rac1, 3C and 3D.

(C) Total cell lysates were pulled down with anti-Rac1 or IgG isotype control antibody prior to immunoblotting. Uninfected cells serve as negative control. IP: immunoprecipitation; IB: immunoblotting.

Data information: Data shown in each panel is a representative set obtained from two independent experiments. Scale bars represent 10μm.

Expanded View Figures Legend.
Lipid droplet breakdown requires dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes. (2013)
Figure 1.

Dyn2 knockdown interferes with starvation-induced LD breakdown in Hep3B hepatocytes. Hep3B hepatocytes were treated with nontargeting control siRNA (siNT) or siRNA targeting Dyn2 (siDyn2), followed by re-expression of either vector alone, or GFP-tagged versions of Dyn2, loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS. LDs were visualized by immunofluorescence using Oil Red O staining. Cell boundaries are outlined and those cells re-expressing GFP, GFP-wtDyn2, or GFP--K44A are denoted with asterisks. Bars, 20 µM. (B) Representative blot showing the efficiency of the Dyn2 knockdown in these cells. (C and D) Quantitation of the average LD number and area (in pixels2) per cell from three independent experiments. The data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01. NS, not significant.
Lipid droplet breakdown requires dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes. (2013)
Figure 2.

Pharmacological and genetic inhibition of Dyn2 function also reduces starvation-induced LD breakdown. HuH-7 (A) and Hep3B (B) hepatocytes were loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS in the presence of Dyn2 inhibitors or DMSO as indicated. Representative images of inhibitor-treated and control cells (stained with Oil Red O) are shown in A and B together with the quantitation of the average LD area per cell from three independent experiments. Pharmacological inhibitors used were: Dynasore (inhibits Dyn2 GTPase activity), MiTMAB (targets PH domain and interferes with membrane binding), Dynole 34-2 (allosteric GTPase inhibitor), and Dynole 31-2 (negative control for Dynole 34-2). Bars, 20 µM. (C) Representative images from control and Dyn2 knockout MEFs after an overnight lipid loading with 400 µM oleate for 17 h. Knockout of Dyn2 was induced by treatment with 2 µM 4-hydroxy-tamoxifen for 7 d and was confirmed by immunostaining of endogenous Dyn2 (top row) and by immunoblot (D). Bars, 20 µM. (E and F) Average LD number (E) and area (F) per cell from five independent experiments. All data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01. (G) Whole-cell lysates and LD fractions isolated from HuH-7 hepatocytes under resting or starved (2 h HBSS starvation) conditions. (H) Primary hepatocyte expressing Dyn2-GFP, showing an absence of colocalization with the LD surface (stained with Oil Red O). Bar, 20 µM. Inset shows magnification of boxed region (bar, 2 µM).
Lipid droplet breakdown requires dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes. (2013)
Figure 3.

Dyn2 inhibition leads to enlarged autolysosomal structures and prevents the autophagy of lipid droplets. Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and co-stained with antibodies specific for LAMP1 (red) and LC3 (green). After Dyn2 knockdown, a juxtanuclear aggregation and enlargement of the LAMP1-positive compartment is observed (C and D, arrows). Increased labeling of LC3 is also detectable after knockdown of Dyn2. (E) Western blotting of Hep3B lysates after a 3-d treatment with either the control or Dyn2-targeted siRNA and further treatment with or without 50 µM leupeptin. Densitometry-based analysis of six independent experiments is shown at the bottom of the figure. (F) Western blotting of Hep3B lysates after treatment for 2 h with DMSO or 80 µM Dynasore, in the presence or absence of 50 µM leupeptin. Quantitation of LC3-II levels relative to control are shown below the blots. The data are represented as mean ± SE; *, P ≤ 0.05.
Lipid droplet breakdown requires dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes. (2013)
Figure 4.

Dyn2 knockdown results in the formation of enlarged autophagic structures. (A–D) Transmission electron micrographs (TEMs) of oleate-loaded Hep3B hepatocytes treated with nontargeting control (siNT) or Dyn2 (siDyn2) siRNA for 72 h. Bars: (A and B) 2 µm; (A′ and B′) 1 µm; (C and D) 0.5 µm. Insets in A and B show fluorescent micrographs of LAMP1-stained cells (bars, 10 µM). Control cells (A) contain an abundance of small (<1 µm) electron-dense lysosomes and autolysosomes (arrowheads). Under conditions in which Dyn2 expression is suppressed (B–D), far fewer small lysosomes are observed. Instead, the cells are populated by larger autolysosomes (≥1 µm) with aberrant morphologies and containing putative LDs (*). (E) Quantitative measure of autolysosomal size from cells in which Dyn2 was knocked down as compared with control cells. Values represent the fold-increase in the number of autophagic structures of a given diameter (measured in microns) observed over siNT-treated control cells. Data were obtained from a single experiment with n = 6 cells examined by electron microscopy from each condition.
Lipid droplet breakdown requires dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes. (2013)
Figure 5.

Dyn2 knockdown disrupts autophagic lysosomal reformation (ALR) and lysosomal tubule scission. Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and stained with an antibody specific for LAMP1 after a 24-h starvation period in media containing 0.1% FBS. Control cells displayed normal lysosomal compartments, with LAMP1-stained structures dispersed throughout the cytosol, some containing short reformation tubules (B′). After Dyn2 knockdown, many cells contained enlarged autolysosomes with a noticeable increase in persistent and lengthy reformation tubules emanating from LAMP1-positive compartments (C′ and D′). Bars: (A–D) 20 µM; (A′ and B′) 5 µM; (C′ and D′) 10 µM. (E and F) Measurements of the length (E) and distribution (F) of LAMP1-stained ALR tubules in control (siNT) and siDyn2-treated cells. Data represent the mean derived from measurements of tubules in a minimum of 40 cells for each experimental group over 6 independent experiments. Error bars represent SE; *, P ≤ 0.05; **, P ≤ 0.01. (G) TEM of siDyn2-treated Hep3B hepatocyte exhibiting enlarged autolysosomal structures with extensive tubulation (arrows). Bar, 2 µm. (G′) Inset showing enlargement of autolysosomal tubule (arrows). Bar, 1 µm. (H) Tubulating autolysosome with engulfed lipid droplet. Bar, 0.5 µm.