These viral synapse dynamics were recorded by live imaging for the first time

During sexual intercourse, HIV-1 crosses epithelial barriers composing the genital mucosa, a poorly understood feature that requires an HIV-1-infected cell vectoring efficient mucosal HIV-1 entry. Therefore, urethral mucosa comprising a polarized epithelium and a stroma composed of fibroblasts and macrophages were reconstructed in vitro. Using this system, we demonstrate by live imaging that efficient HIV-1 transmission to stromal macrophages depends on cell-mediated transfer of the virus through virological synapses formed between HIV-1-infected CD4 + T cells and the epithelial cell mucosal surface. We visualized HIV-1 translocation through mucosal epithelial cells via transcytosis in regions where virological synapses occurred. In turn, interleukin-13 is secreted and HIV-1 targets macrophages, which develop a latent state of infection reversed by lipopolysaccharide (LPS) activation. The live observation of virological synapse formation reported herein is key in the design of vaccines and antiretroviral therapies aimed at blocking HIV-1 access to cellular reservoirs in genital mucosa.

Here, using urethral mucosa reconstructed in vitro, we describe by quantitative 3D live microscopy the transfer of HIV-1 across T cell virological synapses toward the epithelial cell surface and subsequent mucosal penetration, and we demonstrate that efficient HIV-1 transmission to mucosal tissue macrophages is mediated by virological synapses formed between infected CD4 + T cells and genital mucosa epithelial cells.

Virological synapses are transient and polarized zones of contact between HIV-1-infected and non-infected cells in which viral particles are formed extemporaneously, concentrate, and are transferred from cell to cell. At least in vitro, HIV-1 cell-to-cell transmission between immune cells occurs by virological synapses and polysynapses between donor, here HIV-1-infected, cells and one or several target immune cells. This dynamic phenomenon in its essence has been successfully assessed by live-imaging microscopy ().

HIV-1 gains access to the body by crossing epithelial barriers that compose the ano-rectal and female/male genital mucosa during sexual intercourse. One pathway of HIV-1 entry at mucosal sites is via transcytosis through epithelial cells after the formation of cell-cell contacts between epithelial cells and HIV-1-infected cells () present in all secretions that act as vector for HIV-1 infection (). The contact between HIV-1-infected cells and monostratified epithelial target cells triggers the polarized budding of cell-free HIV-1 in a cleft at the site of cell-to-cell contact. Viral particles are then rapidly internalized and transcytosed by the epithelial cells toward the basal pole without infecting epithelial cells, culminating in the release of infectious HIV-1 into the basal environment (). Next, viruses reach immune cell targets in the stroma and establish infection. In contrast, cell-free HIV-1 inoculated on the epithelial surface transcytoses poorly (). Thus, transcytosis and establishment of HIV-1 initial foci at mucosal sites depend on HIV-1 cell-mediated infection, and they occur after the formation of virus-containing clefts, a feature of virological synapses. However, this sequence of events was mainly inferred from biochemical studies or microscopy performed on fixed cells. Furthermore, the dynamic evidence resulting from observations in real time and at a single-cell level that are required for demonstrating virological synapse formation with non-immune target cells, such as epithelial cells, in the context of viral transmission to mucosa, has never been obtained.

The adult penile urethra is a novel entry site for HIV-1 that preferentially targets resident urethral macrophages.

Within 1 h, HIV-1 uses viral synapses to enter efficiently the inner, but not outer, foreskin mucosa and engages Langerhans-T cell conjugates.

Caveats associated with the use of human cervical tissue for HIV and microbicide research.

How HIV-1 reaches these safe niches, established in immune cells such as T cells or Langerhans cells or macrophages depending on the mucosa (), in the midst of stromal mucosal cells, sheltered from foreign aggressions by the efficient barrier of epithelial cells, remains an open question.

Macrophages in vaginal but not intestinal mucosa are monocyte-like and permissive to human immunodeficiency virus type 1 infection.

The adult penile urethra is a novel entry site for HIV-1 that preferentially targets resident urethral macrophages.

Once initial infection foci are established, HIV-1 rapidly forms cell reservoirs that cannot be eradicated despite combined antiretroviral treatment. Apart from the best described CD4T cell reservoir, HIV-1 establishes myeloid cell reservoirs in tissues constituting an additional obstacle to virus eradication (). In addition, the vast majority of sexually transmitted HIV-1 uses the CCR5 co-receptor, being thus R5 tropic or macrophage tropic, the variants that predominate in newly transmitted HIV-1 infection (). Therefore, CCR5-expressing cells like macrophages would represent a very likely first HIV-1 target during sexual transmission, gaining increasing importance as actors of HIV-1 persistence independently of CD4T cells (). At least in vitro, macrophages have a self-limiting production of new viral particles throughout time post-infection, interrupting or keeping a very low-level production after weeks and limiting viral lytic egress and cell-cell spread (), indicating that macrophages may be important long-lasting reservoirs for the virus.

Urethral mucosa displays CD4T cells and macrophages as main HIV-1 cell targets, covered by a single thin layer of epithelial cells. Additionally, the expression of mRNA for HIV-1 cellular co-receptors CXCR4 and CCR5 was detected in urethral swabs (), thus characterizing the male urethra as a potential entry and establishment site for HIV-1. Accordingly, we have previously shown that HIV-1 efficiently penetrates the urethral mucosa reaching urethral macrophages that in turn traffic from the epithelial to the dermal compartment, in a mechanism controlled by HIV-1-induced cytokines (). In contrast with foreskin where HIV-1 targets Langerhans cells (), in the urethral epithelia—in which Langerhans cells are absent—HIV-1 targets macrophages, sparing urethral T cells from infection (). Therefore, the urethra represents an excellent model of genital mucosa for the study of HIV-1 transmission specific to tissue macrophages, from which productive infection and further dynamics have not been assessed yet.

The adult penile urethra is a novel entry site for HIV-1 that preferentially targets resident urethral macrophages.

Within 1 h, HIV-1 uses viral synapses to enter efficiently the inner, but not outer, foreskin mucosa and engages Langerhans-T cell conjugates.

The adult penile urethra is a novel entry site for HIV-1 that preferentially targets resident urethral macrophages.

The early steps of HIV-1 acquisition during sexual intercourse in men and the establishment of HIV-1 cell reservoirs at the male genital tract are poorly understood features of HIV-1 pathogenesis. The apparent protection against HIV-1 conferred by circumcision (around 50% of reduction) () is far from being complete, pointing to the existence of other penile tissues serving as HIV-1 entry sites. The penile urethra is the initial site of infection by many bacterial/viral pathogens that commonly infect the male genital tract, such as N. gonorrhea, C. trachomatis, E. coli, cytomegalovirus, human papilloma virus, and herpes simplex virus-2, demonstrating that the urethral mucosa is accessible to pathogen entry and invasion during sexual intercourse (). Experiments with the simian immunodeficiency virus (SIV) revealed that infusion of this virus into the urethra of male macaques resulted in the development of acquired immunodeficiency syndrome-like disease, suggesting that the urethral mucosa is also an efficient entry site for HIV-1 ().

Direct cell-mediated HIV-1 infection of target macrophages embedded in the collagen matrix did not occur when tissue reconstructions devoid of epithelial cells interacted with infected CD4T cells ( Figure 4 G), indicating that the epithelial barrier is needed as an active intermediate between HIV-1 cell-mediated inoculation and macrophage targeting. Such an active role of the epithelial barrier resulted from cytokine/chemokine secretion induced by the virological synapse formation. Hence, the interaction between HIV-1-infected CD4T cells and epithelium and the formation of virological synapses increased the secretion in the reconstruction culture medium of anti-inflammatory interleukin-13 (IL-13) in the following 3 days p.i. ( Figure 4 H). Furthermore, the macrophage chemoattractant cytokine MCP-1/CCL2 secretion was increased upon epithelium interaction with CD4T cell, although regardless of infection. Other tested cytokines, such as the anti-inflammatory IL-4 (<500 pg/mL; Figure 4 H) but also pro-inflammatory ones, were also constitutively secreted by tissue reconstructions at low levels, not modified by viral synapses (MCP-4/CCL13, <100 pg/mL; MIP-3α/CCL20, <40 pg/mL; MIP-1α/CCL3, <500 pg/mL; and RANTES/CCL5 and TRAIL/TNFSF10, <100 pg/mL).

Macrophages were retained in urethral mucosal reconstructions after long-term cell-mediated HIV-1 infection, although their location remained restricted to stromal regions at later time points p.i. ( Figure 4 D). In situ hybridization performed using the recently developed DNA/RNA scope technology () revealed that tissular macrophages displayed integrated proviral DNA as early as 3 days p.i. Furthermore, HIV-1 DNA was detected exclusively in macrophages colabeled with CD68, not epithelial cells ( Figure 4 E, arrowheads). At long-term infection, when macrophages were latently infected, e.g., 25 days p.i., HIV-1 capsid proteins were detected in intracellular clusters within CD68macrophage, as shown by immunofluorescent labeling observed by confocal microscopy ( Figure 4 F), suggestive of viral-containing compartments typical of HIV-1 infection in myeloid cells () (unpublished data).

At later time points (up to day 25), viral production became undetectable, suggesting that HIV-1 infection of macrophages had become latent. Latent HIV-1 infection in macrophages was ascertained using lipopolysaccharide (LPS), a molecule known to re-activate viral production from latency specifically in infected macrophages (). Hence, treatment with LPS at day 25 p.i. of reconstruction otherwise arrested in viral production induced a viral rebound, as compared with non-treated reconstructions ( Figure 4 C, graph on the right).

When cell-free viruses were added to urethral reconstructions and interacted with the mucosal pole of the epithelium, no viral production was detected in the serosal medium up to 26 days following inoculation (p.i.) ( Figure 4 C, open circles). Doubling the number of macrophages inserted into the reconstruction had no effect on mucosal infection. This indicates that cell-free viruses were inefficient in targeting macrophages, in agreement with the low efficiency of cell-free HIV-1 to penetrate mucosal epithelia (). In contrast, when R5-tropic gag-iGFP HIV-1-infected T cells where inoculated into the mucosal reconstruction in a cell-mediated fashion, viral production was detected up to 18 days p.i. in the serosal medium ( Figure 4 C, closed circles). Viral production was proportional to the number of macrophages inserted in the reconstruction ( Figure 4 C, blue and magenta circles), establishing that the detected virus was indeed produced by macrophages inserted in the reconstructed mucosa.

The adult penile urethra is a novel entry site for HIV-1 that preferentially targets resident urethral macrophages.

The in vitro urethral tissue reconstruction successfully mimicked the physical barrier provided by mucosal epithelia to block direct contact between HIV-1-infected CD4T cells outside the mucosa and stromal macrophages (Figures 1 D and 4 B). Infected CD4T cells did not cross the epithelial barrier, remaining localized above the epithelial layer, as recorded by live imaging ( Figure S2 A; Video S7 ). Furthermore, after being washed out from tissue reconstructions at the end of the interaction period, HIV-1-infected CD4T cells were not detected in the reconstructed mucosa, as in tissue cell suspensions prepared up to 13 days post-infection lacked infected T cells, as demonstrated by flow cytometry ( Figure S2 B).

We first confirmed the infectiousness of the R5-tropic gag-iGFP HIV-1 to macrophages after co-culture with infected CD4T cells ( Figure S1 C) or in macrophage single cultures infected with cell-free gag-iGFP virus that has been previously produced by HIV-1-infected CD4T cells ( Figure S1 D). Resulting infected macrophages displayed the characteristic arrest in viral production around 15 days post-infection (p.i.) with cell-free virus ( Figure S1 D).

Next, having demonstrated that virological synapses formed with epithelial cell surface lead in turn to extemporaneous HIV-1 production and viral translocation resembling transcytosis across epithelial cells as shown above, we assessed whether transcytosed viruses could then productively infect macrophages in the following days after interaction between urethral tissues within the reconstructed mucosa and HIV-1-infected CD4T cells. In parallel experiments, cell-free virus was instead inoculated selectively on the tissue mucosal pole. Viral production was assessed by weekly collection of 24-hr serosal tissue culture medium ( Figure 4 A).

(H) Measurement of MCP-1/CCL2, IL-13, and IL-4 cytokines constitutively secreted by tissue reconstructions or secreted upon interaction of tissues with cell-free HIV-1 or HIV-1-infected or non-infected CD4 + T cells. Data are represented as boxplots; ∗ p < 0.05 (ANOVA); ns, non-significant. Results are representative of n = 3 independent experiments.

(G) Viral production of 72-hr tissue supernatants (chase period between 3 and 6 days p.i.) after cell-mediated infection of full or incomplete (no THUECs added) reconstructions. Limit of p24 detection is shown by red dotted line. Data are represented as mean ± SEM.

(F) Double indirect immunofluorescence of HIV-1 capsid proteins (p24) observed by confocal microscopy is only detected in CD68 + macrophages, in the stroma of tissue reconstructions infected for 25 days. CD68 staining, red; p24 staining, green; nucleus staining, blue (DAPI). Arrowheads point to clusters of viral proteins, indicating viral-containing compartments. Scale bar, 5 μm.

(E) HIV-1 DNA in situ hybridization (ISH) of infected (cell-mediated) and non-infected (control) tissue reconstructions. Proviral DNA staining is shown in magenta and CD68 co-stainning is shown in cyan (HistoGreen staining), allowing contour of CD68+ macrophages in yellow. Proviral DNA is only detected in CD68 + macrophages early after 3 days of cell-mediated infection (two first images). Arrowhead indicates infected macrophage. Scale bar, 5 μm.

(D) Flow cytometry analysis of the percentage of CD68 + cells in the total population of cells of epithelium and stroma tissue cell suspensions. Macrophages are retained in the stroma after 13 days post-infection, whereas no macrophages were detected in the epithelium at this later time point. Data are represented as mean ± SEM; ∗ p < 0.05 (ANOVA); ns, non-significant.

(C) MΦ viral production after cell-free versus cell-mediated infection of reconstructions containing different amounts (1× or 2×) of inserted MΦ. Treatment of the reconstruction with 1 μg/mL LPS restored viral production during the chase period between 26 and 27 days p.i. The limit of p24 detection is shown by the red dotted line. Results are representative of n = 3 independent experiments. Data are represented as mean ± SEM; ∗ p < 0.05 (ANOVA).

(B) HIV-1-infected CD4 + T cell contacting reconstructed mucosal epithelial cells, but not macrophages (MΦ, identified by electron-dense quantum dots (Qdots) indicated by a red arrowhead), as observed by electron microscopy. Scale bar, 5 μm.

(A) Experimental set-up for cell-mediated or cell-free virus infection of reconstructed mucosa. After 2 hr of interaction with HIV-1-infected CD4 + T cells or cell-free viruses, 24-hr tissue supernatants (chase period) were collected at different indicated time points throughout 28 days post-infection (p.i.), chase periods being separated by a resting period of 1 week of cultivation.

Fluorescent viruses shed following viral synapse formation with the epithelium were then tracked by live imaging in z dimension. Epithelial and HIV-1-infected CD4T cells were also tracked, and their displacements in z coordinate were used as references for fluorescent virus directional migration. We observed that 3 of 9 (≈30%) tracked fluorescent viruses were found to migrate farther from HIV-1-infected CD4T cell and translocate across epithelial cell toward the mucosal stroma, indicating viral transcytosis across the epithelial barrier following viral synapse formation ( Figures 3 D and 3E; Video S5 ).

Considering that cell-to-cell contact, virological synapse formation, and virus transcytosis across epithelium are sequential events in HIV-1 entry into mucosa (), we tracked fluorescent virus shed from virological synapse into epithelial cells ( Figure 3 ). To specifically delineate the epithelial cell plasma membrane, we constructed urethral epithelial cells tagged with red fluorescent ARF6 (ADP-ribosylation factor 6), a protein known to be expressed at the apical plasma membrane of polarized epithelial cells (). Using mucosa reconstructed with fluorescent ARF6-tagged epithelial cells and inoculated with HIV-1-infected CD4T cell in a polarized manner as in Figure 2 , we confirmed the direct transfer of HIV-1 from virological synapse to the epithelium ( Figure 3 A; Video S4 ). Fluorescent viruses are produced extemporaneously by infected CD4T cells upon virological synapse establishment, forming clusters in cell-cell contact zones ( Figure 3 A) corresponding to previous observation with other CD4T cells (). HIV-1 could be detected in intracellular epithelial cell compartments during 3 hr of interaction with HIV-1-infected CD4T cells when observed at the ultrastructural level by electron microscopy ( Figure 3 B). Furthermore, the ultrastructure of particles found in epithelial cells was compared with that of HIV-1 particles produced by gag-iGFP HIV-1-infected CD4T cells, confirming their HIV-1 nature ( Figure 3 C).

(E) Vertical (z coordinate) tracking of fluorescent virus shed from a virological synapse. Displacement in z coordinate of epithelial cells (blue dotted line), infected CD4 + T cell (green dotted line), and transferred gag-iGFP particles (gray, not displaced; orange, displaced in z direction). Displacement was calculated by subtracting the z coordinate at each time point by the initial z coordinate at time point = 0. Negative values represent displacement toward tissue reconstructions, while positive values represent displacement toward the confocal unit objective. From 6 different live recordings analyzed in n = 4 independent experiments, 3 of 9 tracked gag-iGFP particles migrate further in z toward the stroma, thus indicating transcytosis.

(D) Live imaging of an infected CD4+ T cell forming a virological synapse and transferring viruses to epithelium; arrowheads in the sequence indicate examples of particles tracked for vertical (z coordinate) displacement in (E). Time, hours:minutes; scale bar, 10 μm. See also Video S5 . Results are representative of 13 virological synapses recorded in n = 5 independent experiments.

(C) Examples of viral particles shed by an infected CD4 + T cell under electron microscopy. Scale bar, 100 nm.

(B) Contact zone between infected CD4 + T cell and epithelium imaged by electron microscopy. The green dotted square region shown in high magnification (inset) reveals a viral particle within an epithelial cell compartment. Scale bars, 1 μm and 100 nm.

(A) HIV-1-nfected CD4T cell (fluorescent virus in green) interacting with epithelium (THUECs engineered to express a fluorescent ARF6 GTPase at the apical plasma membrane to delineate the cell border in red). Clustering and shedding of fluorescent virus at the zone of contact are indicated by arrowheads. Time of image acquisition is depicted as minutes:seconds. Scale bar, 5 μm. Results are representative of 3 virological synapses with ARF6-tagged THUEC recorded in n = 3 independent experiments. See also Video S4

Viral Particles Formed at the Virological Synapse Transcytose across the Epithelium toward the Mucosal Stroma

Figure 3 Viral Particles Formed at the Virological Synapse Transcytose across the Epithelium toward the Mucosal Stroma

To track macrophages that eventually migrated to upper regions of the epithelium, directly contacting infected CD4T cells, macrophages were loaded with quantum dots before being inserted into tissue reconstruction. In most of regions where viral synapses were recorded, macrophages were absent or not reaching infected CD4T cells. In a few recorded cases (n = 3), macrophages were present in the epithelium close to infected CD4T cell-forming viral synapses, although HIV-1 direct transfers from T cell to these macrophages were not detected, whereas such transfer can be observed in single HIV-1-infected CD4T cell/uninfected macrophage co-cultures ( Figure S1 B; Video S3 ). Therefore, the formation of viral synapses and clear HIV-1 transfers occur exclusively with epithelial cell targets, despite proximity of macrophages to infected CD4T cells ( Figure S1 E; Video S6 ).

Concerning distinctive features of virological synapses formed between HIV-1-infected T cells and epithelial cells as compared with virological synapses formed with uninfected target CD4T cells ( Figure S1 A) or macrophages ( Figure S1 B; Video S3 ), we observed (1) the formation of membrane extensions in regions of viral clustering at cell-cell contact zones and from which viral particles were transferred to epithelium; and (2) the transfer of virus that could move away microns of distance from viral synapses regions, not being necessarily retained in viral synapse regions as in the case of bounded synaptic clefts. Frequency of virological synapse formation with epithelial cells in tissue reconstructions was similar to that obtained when uninfected macrophages were targeted. Viral synapse formation followed by virus transfer depended on the HIV-1 envelope, as no transfer to epithelium was observed when a virus mutant lacking the HIV-1 envelope, namely ΔENV, gag-iGFP virus was employed ( Video S8 Table 1 ).

Upon HIV-1-infected CD4T cell-epithelial cell contact, fluorescent viruses, defined by groups of singular fluorescent dots of 0.77 (±0.05 SEM) μmof area, clearly distinguishable from the background, formed and clustered in zones of contact between infected CD4cells and epithelial cells. Strikingly, a directional transfer of virus toward epithelial cell surface followed the clustering ( Figures 2 C and 2D, arrowheads; Videos S1 and S2 ). The time-spotting of cluster and transfer events ( Figure 2 E) revealed that transfer of virus starts 37.75 (±14.9 SEM) min after clustering, and transfer events were detected during a mean period of 56.75 min (±12.9 SEM). HIV-1-infected CD4T cells transferred virus to the urethral epithelium through virological synapse formation in 35.1% of the recorded cells ( Table 1 ), and synapses were resolved after transfer started in 72.7% of the recordings where viral transfers occurred ( Figure 2 E).

The efficiency of gag-iGFP HIV-1 (wild-type or a mutant virus lacking HIV-1 envelope [ΔENV]) in forming viral synapses and transfer viral particles to target cells was evaluated using target cells such as uninfected primary CD4 + cells, peripheral blood monocyte-derived macrophages (MD-MΦ), and urethral tissue reconstructions. n = independent experiments.

Summary of Detected Virological Synapses Formed between gag-iGFP HIV-1-Infected CD4 + T Cell Donor and Various Target Cells in Live-Imaging Recordings

Table 1 Summary of Detected Virological Synapses Formed between gag-iGFP HIV-1-Infected CD4 + T Cell Donor and Various Target Cells in Live-Imaging Recordings

To characterize dynamically the formation of virological synapses formed between HIV-1-infected CD4T cells and mucosal epithelial cells, we assessed their interaction by quantitative live imaging from 15 min to 6 hr. Therefore, mucosal tissues were reconstructed on the underside of the membrane filters using the same protocol as above, flipped back to the normal orientation, and inoculated with infected CD4T cells deposited on microscopy imaging dishes coupled to an adaptor suitable for microscopy ( Figures 2 A and 2B ). Virological synapses were characterized using the morphological criteria established previously (), namely, (1) detection of stable (>10-min) organization of fluorescent virus into clusters at zones of cell-cell contact, demonstrated by others to be in fact synaptic clefts (); and (2) detection of subsequent transfer of fluorescent virus from the clusters to the target cell.

(E) Time tracking of virological synapses. Each line in the y axis represents one live-imaging recording, and events of viral clustering (formation, green circle; resolution, red circle) and viral transfers (start, green triangle; end, red triangle) were plotted on the x axis according to the recorded time after interaction with HIV-1-infected CD4 + T cells (hours). Virus is transferred either immediately or within ≈40 min after virological synapse formation; transfer events were observed during ≈1 hr. Results are representative of n = 5 independent experiments.

(C and D) HIV-1-infected CD4T cells interacting with reconstructed urethral epithelium, as observed by live confocal imaging (infected CD4T cell producing gag-iGFP HIV-1 in green; nuclei stained with Hoechst in blue). See also Videos S1 and S2 . In (D), gag-iGFP fluorescence is also shown with hue-saturation-value scale ranging from dark blue (less fluorescence) to white (more fluorescence). Detection of viral cluster formation and resolution and the start and end of viral transfers are depicted as green and red circles and green and red triangles, respectively. Time of image acquisition is expressed as minutes:seconds in (C) and hours:minutes in (D). Scale bar, 10 or 5 μm.

(B) The interaction is allowed in a system composed of the membrane filter coupled to an adaptor placed on a microscope dish containing the infected cells. The adaptor adjusts the optical distance between epithelium and high-magnification objectives of the inverted confocal microscope. The upper photograph depicts how the system is mounted (as indicated by the arrow), and the lower photograph shows the ensemble ready for live imaging.

(A) Reconstruction of mucosal tissue for live-imaging recordings: Tissues were reconstructed on the underside of the chamber membrane filter and incubated at their mucosal pole with HIV-1-infected CD4 + T cells.

Transfer of HIV-1 to the Mucosal Epithelium Is Mediated by Virological Synapses

When inoculated selectively at the mucosal pole of the urethral epithelium for 3 hr prior to fixation and observation by confocal microscopy, the CD4T cells infected with gag-iGFP HIV-1 interacted exclusively with the surface epithelium, without migrating toward the stroma ( Figure 1 E). After 3 hr of interaction between HIV-1-infected CD4T cells and the urethral mucosa, 17.1% of gag-iGFP HIV-1-infected cells (6 of 35 cells observed) shed fluorescent virus at zones of infected T cell-epithelial cell contact, indicating virological synapses ( Figures 1 E and 1F).

To monitor the virological synapse in the context of human mucosa and subsequent HIV-1 translocation across polarized epithelial cells, a polarized urethral epithelium was reconstructed in vitro and inoculated at its mucosal pole with CD4T cells expressing a fluorescent R5-tropic HIV-1 strain (gag-iGFP HIV-1). Human urethral tissue reconstruction consists in the sequential seeding on membrane filters of fibroblasts embedded in collagen matrix together with macrophages, prelabeled with engulfed fluorescent quantum dots, for 2 days forming the stroma. The stroma is overlayed by urethral epithelial cells allowed to grow on the stromal matrix in liquid-liquid and air-liquid environments each for an additional 7 days ( Figure 1 A). The tissue reconstructions mimic structurally and immunologically the human urethral mucosa, displaying a stroma rich in collagen fibrils ( Figure 1 B) and a polarized epithelium presenting active caveolar endocytosis and epithelial cell junctions ( Figure 1 C). The epithelium expresses cytokeratins 10 and 13, allowing for the clear separation of epithelial and stromal regions, the latter containing scattered macrophages vizualized as they were loaded with quantum dots before being inserted in the tissue reconstuction ( Figure 1 D).

(F) Two HIV-1-infected CD4 + T cells interacting with the epithelium (top view) and shedding fluorescent viruses to the epithelium (arrowheads). Scale bar, 10 μm. Results are representative of n = 2 independent experiments.

(E) Side and top views of an HIV-1-infected CD4 + T cell producing fluorescent viruses (green) interacting with reconstructed urethral epithelium (nuclei stained with Hoechst in blue).

(D) Macrophages (arrowheads) distributed in the stroma of reconstructed mucosae (side view). Epithelium and stromal regions were separated by the yellow line, defined after cytokeratin 10/13 expression, in pink. MΦ is shown in green (quantum dot fluorescence), and nuclei are shown in blue (Hoechst). Scale bar, 50 μm.

(C) Electron microscopy images of reconstructed urethra, revealing the presence of caveolae (cyan arrowhead) and desmosomes (red arrowheads) in the epithelium. Scale bar, 100 nm.

(B) Collagen fibrils (red) in reconstructed urethral mucosa, detected by second harmonics generation (cell nuclei stained with Hoechst in blue). Side and top views are shown. Scale bar, 50 μm.

(A) Reconstruction of mucosal tissue. Human foreskin primary fibroblasts and monocyte-derived macrophages (MΦ) tagged with fluorescent cell trackers were embedded in the extracellular matrix and seeded on a two-chamber unit membrane for 2 days, subsequently covered by urethral epithelial cells (THUECs) and cultivated for 2 weeks in liquid-liquid then air-liquid environment in a culture medium favoring epithelial polarization. HIV-1-infected T cells producing fluorescent viruses were added on the mucosal side of reconstructed mucosa to initiate infection.

Discussion

We have established a urethral mucosa reconstruction model suitable for live imaging at high-dimensional resolution and appropriate minute-wise temporal resolution, which allowed for the unprecedented demonstration in real time that virological synapses are formed between HIV-1-infected CD4+ T cells and the human genital epithelium, thereby simulating in vitro HIV-1 acquisition as it occurs during sexual intercourses.

Figure 5 Chain of Events of HIV-1 Transmission to Macrophages in Genital Tissue following Virological Synapse Formation Show full caption After contacting the epithelium (1), HIV-1-infected CD4+T cells locally transfer the virus through virological synapses (2) followed by viral transcytosis (3). Macrophages are efficiently targeted in a microenvironment rich in macrophage chemoattractant cytokine MCP-1/CCL2 (induced by contact between the epithelium and CD4+T cells) and IL-13 (secreted specifically upon virological synapse formation). In turn, macrophages display HIV-1 DNA and produce new viral particles for weeks following infected CD4+T cell interaction with the epithelium (4). Macrophage viral production decreases until latency, reversed by LPS administration (5). Altogether, results demonstrate the potential of mucosal stromal macrophages for establishing viral reservoirs early after infected T cell virological synapses formed with epithelial cells. The scenario of cell-mediated targeting of mucosal macrophages via virological synapses, as demonstrated here using urethral tissue reconstructions infected with R5-tropic HIV-1, is schematized in Figure 5 . It comprises the following sequential steps: (1) contact between HIV-1-infected cells vectoring infection and the mucosal epithelial surface; (2) virological synapse formation allowing for extemporaneous virus budding on the epithelial cell layer; (3) HIV-1 translocation in a transcytosis-like process across epithelium toward the mucosal stroma; and, (4) in an anti-inflammatory microenvironment, stromal macrophages being infected, having integrated proviral DNA in their genomes, and producing new viral particles for 3 weeks p.i. prior to (5) the establishment of a reversibly latent macrophage infection.

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Eslahpazir J.

Sekaly R.P.

Bélec L. Differential modulation of CCR5-tropic human immunodeficiency virus-1 transfer from macrophages towards T cells under interleukin-4/interleukin-13 microenvironment. In the present model, virological synapses formed by CD4T cells infected with replication-competent HIV-1 with urethral tissue reconstructions induced an increase in the secretion of the macrophage chemoattractant pro-inflammatory cytokine MCP-1/CCL2 and the anti-inflammatory cytokine IL-13. MCP-1/CCL2 secretion is induced upon the interaction between CD4T cells and tissue reconstructions, but irrespective of T cell infection. MCP-1/CCL2 is associated with monocyte recruitment to sites of inflammation () and could participate to recruit or maintain target macrophages in the submucosa near the basal membrane, ready to capture HIV-1 exiting the epithelium after transcytosis. IL-13 secretion, on the other hand, is increased specifically upon virological synapse formation, as only HIV-1-infected CD4T cells can trigger an upregulated secretion of this cytokine by tissue reconstructions. In this scenario, synapse-dependent IL-13 and MCP-1/CCL2 increased secretion after virological synapse formation promote an anti-inflammatory microenvironment that favors chemoattraction of macrophages to synaptic regions and/or polarizes macrophages toward immunological profiles susceptible for long-lasting viral reservoir formation () (unpublished data).

By simulating the interaction with HIV-1-infected CD4+ T cells with genital mucosal epithelium using tissues reconstructed in vitro, the results altogether demonstrate that the establishment of HIV-1 latent infection in mucosal macrophages in the stroma occurs after virological synapses formed first with the epithelial cell barrier. How HIV-1, produced locally at the virological synapse, gets selective access to immune cell targets in the urethra and other genital mucosa and which mechanisms drive the selectivity of the targeting to macrophages are topics currently under investigation.

Overall, the present study addresses basic physiopathology questions on the early mechanism of HIV-1 penetration in male genital mucosa at the cell-biology level. We have demonstrated that virological synapses are formed between an HIV-1-infected donor immune cell and a target epithelial cell in male urethral mucosa, potentially revealing additional aspects of HIV-1 transmission that may contribute to the design of better-targeted antiviral strategies.