ORIGINAL RESEARCH published: 30 April 2019 doi: 10.3389/fimmu.2019.00825 Dendritic Cells From the Cervical Mucosa Capture and Transfer HIV-1 via Siglec-1 Daniel Perez-Zsolt 1,2† , Jon Cantero-Pérez 3,4† , Itziar Erkizia 1 , Susana Benet 1,2 , Maria Pino 1 , Carla Serra-Peinado 3 , Alba Hernández-Gallego 4,5 , Josep Castellví 6,7 , Gustavo Tapia 4,5,7 , Vicent Arnau-Saz 2,3 , Julio Garrido 8 , Antoni Tarrats 9 , Maria J. Buzón 3 , Javier Martinez-Picado 1,10,11 , Nuria Izquierdo-Useros 1,4*‡ and Meritxell Genescà 3,4*‡ 1 IrsiCaixa AIDS Research Institute, Badalona, Spain, 2 Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, Barcelona, Spain, 3 Department of Infectious Diseases, Vall d’Hebron Institut de Recerca, Barcelona, Spain, 4 Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, Badalona, Spain, 5 Pathology Department, Hospital Universitari Germans Trias i Pujol, Badalona, Spain, 6 Pathology Department, Hospital Edited by: Universitari Vall d’Hebron, Barcelona, Spain, 7 Department of Morphological Sciences, Universitat Autònoma de Barcelona, Paul Urquhart Cameron, Barcelona, Spain, 8 Hospital Municipal de Badalona, Badalona, Spain, 9 Department of Obstetrics and Gynecology, Hospital The University of Melbourne, Australia Universitari Germans Trias i Pujol, Badalona, Spain, 10 University of Vic-Central University of Catalonia (UVic-UCC), Vic, Spain, Reviewed by: 11 Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain Christiane Moog, Institut National de la Santé et de la Recherche Médicale (INSERM), Antigen presenting cells from the cervical mucosa are thought to amplify incoming HIV-1 France and spread infection systemically without being productively infected. Yet, the molecular Philippe Benaroch, Centre National de la Recherche mechanism at the cervical mucosa underlying this viral transmission pathway remains Scientifique (CNRS), France unknown. Here we identified a subset of HLA-DR+ CD14+ CD11c+ cervical DCs at the *Correspondence: lamina propria of the ectocervix and the endocervix that expressed the type-I interferon Nuria Izquierdo-Useros nizquierdo@irsicaixa.es inducible lectin Siglec-1 (CD169), which promoted viral uptake. In the cervical biopsy Meritxell Genescà of a viremic HIV-1+ patient, Siglec-1+ cells harbored HIV-1-containing compartments, meritxell.genesca@vhir.org demonstrating that in vivo, these cells trap viruses. Ex vivo, a type-I interferon antiviral † These authors have contributed environment enhanced viral capture and trans-infection via Siglec-1. Nonetheless, HIV-1 equally to this work transfer via cervical DCs was effectively prevented with antibodies against Siglec-1. ‡ These authors share last authorship Our findings contribute to decipher how cervical DCs may boost HIV-1 replication and promote systemic viral spread from the cervical mucosa, and highlight the importance of Specialty section: This article was submitted to including inhibitors against Siglec-1 in microbicidal strategies. Viral Immunology, a section of the journal Keywords: cervix, Siglec-1, HIV-1, trans-infection, myeloid cells Frontiers in Immunology Received: 10 January 2019 Accepted: 28 March 2019 INTRODUCTION Published: 30 April 2019 Women account for 51% of people living with HIV worldwide, and in 2017 this represented Citation: 18.8 million of females (1). Since HIV-1 infection is mostly acquired by sexual transmission Perez-Zsolt D, Cantero-Pérez J, (2), understanding the female genital tract immunobiology is imperative not only to halt novel Erkizia I, Benet S, Pino M, Serra-Peinado C, infections, but also to design strategies that will limit HIV-1 spread within the mucosa and contain Hernández-Gallego A, Castellví J, the virus during the early stages of infection. HIV-1 acquisition requires a series of orchestrated Tapia G, Arnau-Saz V, Garrido J, events that lead to systemic infection, beginning with viral entry through the genital epithelium Tarrats A, Buzón MJ, and followed by the productive infection of distinct CD4+ target cells that reside within the Martinez-Picado J, mucosa. Local infection is early disseminated to draining lymph nodes, aiding to spread HIV-1 Izquierdo-Useros N and Genescà M systemically and boost viral replication (3). While current antiviral agents are potent inhibitors of (2019) Dendritic Cells From the Cervical Mucosa Capture and Transfer viral infection in the mucosa, efficacy of preventive methods is also critically dependent on effective HIV-1 via Siglec-1. blockade of all potential receptors involved in HIV-1 dissemination from the genital mucosa to Front. Immunol. 10:825. the lymphoid tissues (4–6). Yet, the precise molecular mechanisms underlying viral dissemination doi: 10.3389/fimmu.2019.00825 routes from the genital mucosa that boost systemic HIV-1 infection remain unknown. Frontiers in Immunology | www.frontiersin.org 1 April 2019 | Volume 10 | Article 825 Perez-Zsolt et al. Cervical Siglec-1+ DCs trans-infect HIV-1 Although productive infection during the first days after RESULTS vaginal SIV exposure is low and mainly restricted to the cervicovaginal tissues (3), evidence for rapid dissemination to Myeloid Cells From Human Cervical the draining lymph nodes has been demonstrated as soon as Submucosa Express Siglec-1 24 h post-infection (7–9). These studies suggest a critical role We first analyzed Siglec-1 expression by flow cytometry on for cervical myeloid cells and, particularly, dendritic cells (DCs) human cervical mononuclear cells obtained from tissues from in the early dissemination of mucosal viruses to lymphoid benign hysterectomies processed immediately after prescribed tissues. Importantly, viral spread does not only rely on de surgery. Gating on hematopoietic CD45+ , single and viable cells novo productive infection of myeloid cells (10–12), but can be allowed identifying myeloid CD3− HLA-DR+ cells (Figure 1A), triggered upon activation of mucosal myeloid cells via the capture which represented a mean of 6 and 7.5 % of cells at the and storage of large amounts of HIV-1 particles that are later ectocervix and endocervix, respectively (red gate and bar graph, transferred to target cells, as previously reported for monocyte- Figure 1B). Of note, HLA-DR− cells did not express Siglec- derived DCs (13–15). Once mucosal myeloid cells migrate to 1 (gray and brown gates, Figure 1C). However, among the secondary lymphoid tissues for induction of antiviral immune myeloid HLA-DR+ fraction, a mean of 24.5 and 11.5 % of cells responses, trapped viruses can be efficiently transferred to CD4+ expressed Siglec-1 in the ectocervix and endocervix, respectively T cells (10, 11), which become productively infected and fuel (blue gate and bar graph, Figure 1D). Moreover, these cells systemic viral dispersion. This highly infectious process is known were predominantly CD11c+ CD14+ (pink gate and bar graph, as trans-infection, and pioneering work already identified that Figure 1E) and CD11b+ (orange gate, Figure 1F). In contrast, migratory HLA-DR+ CD3− cervical cells efficiently captured and myeloid HLA-DR+ cells lacking Siglec-1 expression represented transmitted HIV-1 in trans (5). Recent data also demonstrated three distinct subsets: CD11c− CD14+ , CD11c+ CD14− and that among all myeloid cell subsets, vaginal and cervical DCs CD11c+ CD14+ cells (Figure 1G). Of note, Siglec-1+ cells capture and transport transmitted/founder viruses through the displayed a higher activation status than Siglec-1− cells as seen cervicovaginal mucosa and facilitate infection of target cells by HLA-DR expression levels (Supplemental Figure 1). These (16, 17). results identify Siglec-1+ myeloid cells at the ectocervix and Although trans-infection was initially attributed to the endocervix as a subset that displays typical markers of interstitial capacity of C-type lectin receptors such as DC-SIGN to cervical DCs, such as HLA-DR, CD11c, CD14, and CD11b (28). specifically bind to the viral envelope glycoprotein of HIV-1 (11), viral capture in the female genital mucosa is independent of this particular receptor, as the majority of cervical DCs capturing HIV-1 do not express DC-SIGN (18) and neutralizing antibodies Siglec-1+ Cells Accumulate in the against DC-SIGN cannot block viral transmission (5). In addition Submucosa of the Ectocervix to C-type lectin receptors, HIV-1 capture by myeloid cells is and Endocervix mediated by the sialic acid binding immunoglobulin-like lectin- To analyze the localization of Siglec-1+ cells within the cervix, 1 (Siglec-1/CD169) (19–21). This receptor potently enhances we next performed immunofluorescence of mucosal tissues HIV-1 capture and storage in virus containing-compartments from women undergoing benign hysterectomies. Siglec-1+ cells (22), that are later released from DCs to infect target cells via predominantly located in the lamina propria or submucosa virological synapse formation (23). Siglec-1 is present on the and, in agreement with flow cytometry data, were also positive surface of myeloid cells and its expression is potently enhanced for CD14 or CD11c (Figure 2A). In contrast, no Siglec-1+ by type-I interferon signaling (21, 24), which is triggered by cells were found in the lower region of the epithelium, where viral immune sensing or bacterial lipopolysaccharide exposure, CD207/langerin+ Langerhans cells are commonly found (29). both factors associated to HIV-1 infection (25). However, it does Immunohistochemistry staining of cervicovaginal tissues further not bind to the envelope glycoprotein of HIV-1 as C-type lectin identified Siglec-1+ cells displaying myeloid cell morphology receptors do (11), but recognizes sialylated gangliosides that mostly accumulated within the submucosa of the ectocervix, are anchored on the viral membrane of enveloped retroviruses which is lined by a stratified squamous epithelium (Figure 2B, (19, 26). In vivo, Siglec-1 is required for robust infection and left). Siglec-1+ cells were also found in the endocervix, early dissemination of a retrovirus within the lymphoid tissue of a which is covered only by a single-layer columnar epithelium murine model (27), but its potential role during HIV-1 infection (Figure 2B, right), although the frequency was lower than at in the female genital tract remains unexplored. the ectocervix. However, in those endocervical tissues classified Here we sought to clarify if trans-infection mediated by as highly inflamed based on histopathological detection of Siglec-1 could impact the early stages of HIV infection in inflammatory infiltrates, we found a higher number of Siglec- women, and performed a comprehensive analysis of Siglec- 1+ cells (Figure 2C) that were comparable to those observed at 1 expression on human cervical anatomical compartments. the ectocervix (Figure 2B, left graph). Of note, the reasons for a In all samples studied, we identified mucosal DCs expressing higher inflammatory infiltrate could not be attributed to the HIV- this receptor, whose expression was boosted by interferon alfa 1 infection status, the level of viral load, age or any particular (IFNα) antiviral signaling. Moreover, we also found that cervical clinical indication for surgery (Table 1). These results identified DCs enhanced viral capture and trans-infection, and that this the presence of myeloid cells expressing Siglec-1 in the steady mechanism was effectively prevented with antibodies against state just beneath the mucosa of the ectocervix and endocervix, Siglec-1 receptor. where cervical DCs usually accumulate (16). Frontiers in Immunology | www.frontiersin.org 2 April 2019 | Volume 10 | Article 825 Perez-Zsolt et al. Cervical Siglec-1+ DCs trans-infect HIV-1 FIGURE 1 | Myeloid cells from human cervical mucosa express Siglec-1. FACS analysis and representative gating strategy of the cervicovaginal myeloid cell subsets. Colored gates and arrows indicate populations analyzed, and corresponding matching colors are used to identify bar graphs showing frequencies of those (Continued) Frontiers in Immunology | www.frontiersin.org 3 April 2019 | Volume 10 | Article 825 Perez-Zsolt et al. Cervical Siglec-1+ DCs trans-infect HIV-1 FIGURE 1 | populations. (A) Hematopoietic cells were identified by their CD45 expression, and single-live cells were selected by doublet discrimination and live/dead staining. (B) Representative dot plot and frequency of HLA-DR+ and CD3− on hematopoietic cells. (C) Representative dot plot showing that HLA-DR− cells do not express Siglec-1. (D) Representative dot plot and frequency of Siglec-1 expression analyzed in HLA-DR+ myeloid cells compared to matched isotype control. (E) Representative dot plot and frequency of CD11c+ CD14+ cells among myeloid HLA-DR+ cells expressing Siglec-1. (F) Representative dot plot of CD11b expression analyzed in CD11c+ CD14+ Siglec-1+ myeloid cells. (G) Representative dot plot of CD14 and CD11c among the myeloid HLA-DR+ cells that do not express Siglec-1. All bar graphs show mean values and SEM from 14 donors. Statistical differences were assessed with a Mann-Whitney test. FIGURE 2 | Siglec-1+ cells accumulate in the submucosa of the ectocervix and endocervix. (A) Representative immunofluorescent stainings on four ectocervix and endocervix of CD14 (AF488) and Siglec-1 (AF647), or CD11c (AF488) and Siglec-1 (AF647). Scale bars 20 µm. (B) Representative images of Siglec-1 immunostaining (40x) on 10 ectocervix and endocervix. Scale bars 100 µm. Insets show Siglec-1+ cells magnification (100x). Bar graph shows the mean values and SEM of Siglec-1+ cells per field counted in 5 consecutive fields. (C) Representative images of Siglec-1 immunostaining (40x) on 10 endocervix displaying a low and high grade of inflammation. Scale bars 100 µm. Bar graph shows the mean values and SEM of Siglec-1+ cells per field counted in 5 consecutive fields of 10 endocervix with different inflammation grades. Statistical differences were assessed with a Mann-Whitney test. DCs From Cervical Mucosa Mediate Viral with fluorescent viruses. Cells were incubated with HIV-1 Uptake via Siglec-1 and Are Detected Virus-Like Particles (HIV-1Gag−eGFP VLPs), which lack the viral envelope glycoprotein but carry sialyllactose-containing in vivo gangliosides recognized by Siglec-1 receptor and recapitulate To investigate whether Siglec-1+ cervical myeloid cells HIV-1 uptake results obtained with wild type viruses in myeloid could capture and store HIV-1 via the interaction with this cells (30, 31). After VLP exposure, cervical mononuclear cells particular I-type lectin receptor, we next pulsed cervical cell were extensively washed and assessed by flow cytometry. Among suspensions processed immediately after prescribed surgery the single-live CD45+ myeloid HLA-DR+ fraction, a mean of Frontiers in Immunology | www.frontiersin.org 4 April 2019 | Volume 10 | Article 825 Perez-Zsolt et al. Cervical Siglec-1+ DCs trans-infect HIV-1 TABLE 1 | Clinical data associated to cervical tissues where Siglec-1 was quantified by IHQ. Patient Siglec-1+ cells per field Siglec-1+ cells per field Age (yr) Indication for surgery HIV status Viral Load (copies/ml) at the Ectocervix at the Endocervix 1 9,6 2,6 52 Prolapse - NA 2 2,3 3,4 53 Prolapse - NA 3 4,2 7,2* 45 Prolapse - NA 4 13,8 9,2* 52 Prolapse - NA 5 3,6 5,8* 60 Prolapse - NA 6 8,8 4,8 59 Unknown + <50 (ART) 7 6,4 2,4 45 Prolapse + <50 (ART) 8 11,6 3,2 57 Cystocele + 140 (ART) 9 5,4 3 41 Uterine fibroids + 400 Controller (naïve) 10 8 3,2 52 Prolapse + Cystocele + 1.164 (naïve) Higher levels of inflammation were observed in these tissues* 11.8 and 3.6 % of cells expressed Siglec-1 in the ectocervix and and transmission efficiency of DCs (20). Hence, right after endocervix (blue gate and bar graph, Figure 3A), while a 14.1 HIV-1 infection and once the antiviral type-I IFN responses and 7 % of cells captured HIV-1Gag−eGFP VLPs (green gate are triggered, we hypothesized that IFNα could up-regulate and bar graph, Figure 3B). Moreover, cells not capturing VLPs Siglec-1 expression on cervical DCs and enhance their viral lacked Siglec-1 expression (gray gate, Figure 3C), while cells that uptake capacity. In the cervical mucosa, resident myeloid cells captured viruses were predominantly Siglec-1+ in most of the quickly induce type-I IFN responses after HIV/SIV infection tissues (purple gate and bar graph, Figure 3D). Viral uptake by (32). Moreover, plasmacytoid dendritic cells (pDCs), the most Siglec-1+ cells was further confirmed by Amnis-imaging FACS potent producers of IFNα, are soon recruited to the site of initial technology, which allowed microscope inspection of acquired retroviral infection (33). Thus, we next assessed if IFNα secreted cells and screening of Siglec-1/VLP co-localization. Amnis viral by HIV-1-exposed pDCs could trigger Siglec-1 expression on capture assays revealed that fluorescent HIV-1Gag−eGFP VLP DCs, mimicking early events of HIV-1 pathogenesis once localized with Siglec-1 within a virus-containing compartment infection has been established and type-I IFN antiviral immunity (Figure 3E), resembling to the sac-like structures previously is mounted. Blood derived pDCs were cultured alone, co- described in monocyte-derived DCs and activated tonsillar cultured with an uninfected T cell line or with an HIV-1-infected myeloid cells (19, 31). As HIV-1Gag−eGFP VLP had no viral T cell line that chronically produces R5-tropic BaL viruses in envelope glycoprotein, observed uptake could only rely on the presence or absence of anti-CD4 or isotype monoclonal Siglec-1 recognition of sialylated gangliosides anchored on viral antibodies (mAb). HIV-1-exposed pDCs released higher IFNα membranes (26, 30). Thus, Siglec-1+ myeloid cells found in the compared to non-exposed pDCs (Figure 4A), while blockage of ectocervix and endocervix capture HIV-1Gag−eGFP VLP in a pDC infection with a mAb against CD4 reduced IFNα secretion virus-containing compartment via Siglec-1 recognition of viral compared to an isotype control (Figure 4A). Accordingly, Siglec- membrane gangliosides. 1 expression was increased on monocyte-derived DCs incubated To further assess if Siglec-1+ cervical cells are capable with supernatants from HIV-1-exposed pDCs as compared to of capturing wild type HIV-1 during the natural course supernatants from uninfected pDCs (Figure 4B), and further of infection, we next investigated the cervical biopsy of inhibited by an anti-CD4 mAb (Figure 4B). Thus, HIV-1 sensing a viremic HIV-1+ patient by immunostaining. Confocal via viral fusion on pDCs induced IFNα secretion that triggered microscopy analysis revealed that distinct Siglec-1+ cells Siglec-1 expression on monocyte-derived DCs as potently as found in the cervical submucosa harbored viral p24 antigens recombinant IFNα (Figure 4C), and was specifically inhibited (Figure 3F). Three-dimensional z-stack reconstructions by the type-I interferon blocking receptor B18R (Figure 4C). of Siglec-1+ cells from distinct tissue areas showed virus- Moreover, HIV-1-exposed pDCs derived from pre-menopause containing compartments that were different from the women released significantly higher amount of IFNα than p24 staining of productively infected cells lacking Siglec- pDCs derived from men (Figure 4D). We also induced Siglec- 1 expression (Figure 3G and Movie 1). Thus, Siglec-1+ 1 expression with equal amounts of recombinant IFNα on cervical cells can trap viruses throughout the course of HIV-1 monocyte-derived DCs from men and women and determined infection in vivo. the absolute number of Siglec-1 antibody binding sites per cell (Figure 4E). Under these experimental conditions, however, pDCs Exposed to HIV-1 Induce Siglec-1 DCs derived from women did not significantly express higher Expression on DCs via IFNα Secretion amounts of Siglec-1 than DCs derived from men (Figure 4E). Although the basal expression of Siglec-1 on cervical DCs All together, these results highlight how upon HIV-1 infection, already allows viral uptake, the level of expression of this type- pDC accumulation in the female genital tract could lead to an I interferon inducible receptor (21) correlates with viral capture increased antiviral IFNα secretion that could limit viral infection Frontiers in Immunology | www.frontiersin.org 5 April 2019 | Volume 10 | Article 825 Perez-Zsolt et al. Cervical Siglec-1+ DCs trans-infect HIV-1 FIGURE 3 | DCs from cervical mucosa mediate viral uptake via Siglec-1 and are detected in vivo. (A) Cervical mononuclear cells isolated from the ectocervix and endocervix of benign hysterectomies were pulsed with VLPs for 18 h at 37◦ C, extensively washed, labeled with the indicated mAbs and assessed by FACS. Colored (Continued) Frontiers in Immunology | www.frontiersin.org 6 April 2019 | Volume 10 | Article 825 Perez-Zsolt et al. Cervical Siglec-1+ DCs trans-infect HIV-1 FIGURE 3 | gates and arrows indicate populations analyzed, and corresponding matching colors are used to identify bar graphs showing frequencies of those populations. Representative dot plot and frequency of HLA-DR+ and Siglec-1+ cells on hematopoietic cervical cells. (B) Representative dot plot and frequency of cells capturing HIV-1Gag−eGFP VLPs among the myeloid HLA-DR+ fraction. Smaller dot plot in between depicts the control without VLPs. (C) Representative dot plot showing reduced expression of Siglec-1 in the myeloid HLA-DR+ cells not capturing HIV-1Gag−eGFP VLPs. (D) Representative dot plot of Siglec-1+ cells among the cells capturing HIV-1Gag−eGFP VLPs. Bar graphs show mean values and SEM from the ectocervix and endocervix of 4 to 5 donors. (E) Images of Siglec-1+ cervical cells pulsed and labeled as in (A). Cells were acquired by Amnis-imaging FACS, and showed green fluorescent HIV-1Gag−eGFP VLPs accumulation within a sac-like virus-containing compartment enriched in Siglec-1 (labeled in red). (F) Paraffin-embedded cervical tissue from one viremic HIV-infected woman stained for HIV-1 p24 antigen (labeled in red), Siglec-1 (in green), and nucleus (in blue). Scale bar 50 µm. (Inset panels) zoom in of squared region with distinct fluorescences (scale bar 20 µm). (G) 3D volumetric x-y-z data fields reconstruction of Siglec-1+ cells from four distinct areas of the cervical tissue of the viremic HIV-infected woman. Opacity representation of DAPI stained nuclei and fluorescence of the sac-like virus-containing compartment (VCC; white arrows). Right bottom image displays a characteristic cell pattern with p24+ dots reflecting viral production (Infection). FIGURE 4 | pDCs exposed to HIV-1 induce Siglec-1 expression on DCs via IFNα secretion. (A) IFNα release measured by ELISA on supernatants from blood derived pDCs co-cultured 24 h alone, with an uninfected MOLT CD4+ T cell line or an HIV-1 infected MOLT CD4+ T cell line that chronically produces R5-tropic BaL viruses in the presence or absence of 10 µg/ml of an anti-CD4 or an isotype mAb. Bar graph shows mean values and SEM from at least 6 donors and 3 independent experiments. Statistical differences were assessed with a Wilcoxon matched-pairs signed rank test. (B) Induction of Siglec-1 on monocyte-derived DCs incubated with supernatants isolated from pDCs co-cultured as in (A) and assessed by FACS. (C) Representative histograms of Siglec-1 expression on monocyte-derived DCs exposed to medium, recombinant IFNα, supernatants from uninfected pDCs, supernatants from HIV-1-exposed pDCs on mock treated DCs or on DCs previously incubated with the type-I interferon blocking receptor B18R. (D) IFNα release measured by ELISA on supernatants of pDCs isolated from women or men and co-cultured 24 h with an HIV-1 infected MOLT CD4+ T cell line. Bar graph shows mean values and SEM from 19 donors and 4 independent experiments. Statistical differences were assessed with a Mann-Whitney test. (E) Quantification of Siglec-1 expression levels on monocyte-derived DCs from men and women assessed by FACS. Bar graph shows mean values and SEM from 12 donors and 3 independent experiments. Prentice Rank Sum Test was used to assess statistical differences, which did not reach statistical significance (P = 0.073). initially, but also promote viral capture of cervical myeloid cells capture capacity. Small pieces of ectocervix or endocervix were via Siglec-1 induction. cultured overnight (34), in the presence or absence of increasing concentrations of IFNα and analyzed by flow cytometry after IFNα Enhances Viral Capture and enzymatic digestion. IFNα induced a dose-response increase Trans-infection on Siglec-1+ Cervical DCs in the percentage of Siglec-1+ cells among the myeloid HLA- We next investigated if IFNα could trigger Siglec-1 expression on DR+ CD14+ CD11c+ DC fraction (Figure 5A). Moreover, on cervical myeloid cells and enhance their Siglec-1-mediated viral cellular suspensions obtained from cervical tissues immediately Frontiers in Immunology | www.frontiersin.org 7 April 2019 | Volume 10 | Article 825 Perez-Zsolt et al. Cervical Siglec-1+ DCs trans-infect HIV-1 processed after surgery, IFNα treatment also increased the CD3− HLA-DR+ fraction or the cervicovaginal CD11c+ DCs percentage of Siglec-1+ cells (Figure 5B). This effect was more were found to be the myeloid cell subset that preferentially prominent in the endocervix, where basal expression of Siglec- captured and transported HIV-1 out of the cervicovaginal 1 was lower (Figures 1D, 5B). Accordingly, IFNα treatment mucosa (5, 16, 17). Nevertheless, we now show that on DCs of increased the percentage of cells capturing HIV-1Gag−eGFP VLP the ectocervix and endocervix, alternative receptors beyond DC- among the myeloid HLA-DR+ CD14+ CD11c+ DC population, SIGN operate in HIV-1 viral uptake and transfer, as previously especially at the endocervix (Figure 5C). Thus, endocervical reported for monocyte-derived DCs (13, 15, 38–43). Our results myeloid cells could mediate trans-infection more potently provide a plausible mechanism for previous reports where the once antiviral type I IFN responses are mounted and Siglec-1 predominant cervical myeloid cell population that captured HIV- expression is triggered on DCs. 1 was found to be DC-SIGN negative (18), or where there was Our results suggested that α-Siglec-1 mAbs could offer a lack of inhibition of neutralizing antibodies against DC-SIGN protection against HIV-1 uptake and prevent dissemination on viral transmission mediated by cervical myeloid cells (5). The mediated by cervical DCs. We therefore pre-incubated cells from discovery of Siglec-1 expression on myeloid cervical DCs and the ectocervix and endocervix with an α-Siglec-1 mAb or isotype the capacity of Siglec-1+ cells to capture viruses in vivo help to control before viral exposure (Figure 5D). While isotype control understand how this particular receptor can facilitate boosting of had no inhibitory effect, pre-treatment with α-Siglec-1 mAbs led HIV-1 replication and dissemination from the genital mucosa to to a reduction of HIV-1Gag−eGFP VLP uptake even after IFNα the corresponding draining lymph nodes in the absence of DC treatment (Figure 5D). Moreover, this blocking effect was further productive infection. confirmed using a wild type R5-tropic HIV-1NFN−SX to pulse Baseline levels of Siglec-1 on myeloid cells in the lamina mock treated or IFNα-treated CD3− HLA-DR+ cervical DCs, propria of all cervical tissues examined herein already allowed which were sorted and co-cultured with a reporter CD4+ cell viral uptake, demonstrating that Siglec-1 could act as a viral line to measure viral transfer via trans-infection (Figure 5E). Of attachment factor even in the absence of prior viral infection. note, at the steady state, the expression of Siglec-1 on these cells However, as tissues with a high level of inflammatory infiltrate is shown on Figure 1D. In seven independent cervicovaginal showed an increased number in Siglec-1+ cells, ongoing tissues, we consistently observed a significant decrease in the inflammatory events triggered upon infection could magnify levels of HIV-1 transmitted in the α-Siglec-1 mAb treated cells Siglec-1 mediated HIV-1 uptake and trans-infection. Indeed, in a compared to the isotype control, which accounted for blocking cervical biopsy of an HIV-1 viremic woman, we found Siglec-1+ a mean of 70% of the transferred infectivity by myeloid cells. cells with HIV-1-containing compartments, demonstrating that Thus, at the basal state but also upon Siglec-1 induction by IFNα, these cells actually trap viruses and form these compartments the α-Siglec-1 mAb could most likely block viral binding and in vivo. uptake, preventing subsequent viral transmission from myeloid In the female genital tract, type I IFN responses from tissue cells to target cells. These results highlight that specific inhibitors resident cells represent a potent first-line of defense against many against Siglec-1 can halt viral transfer and dispersion mediated by pathogens, including lentiviruses (44–47). Soon after infection, Siglec-1+ cervical DCs. recruited pDCs sensing incoming viruses will strongly contribute to IFNα secretion (33). Here we confirmed that HIV-1 fusion on pDCs induced IFNα secretion (48, 49) and that this mechanism, DISCUSSION which is subjected to hormonal regulation (50), was enhanced in pDCs derived from pre-menopausic women. However, gender A preventative strategy aimed at protecting against HIV did not affect the IFNα response that triggered Siglec-1 induction infection must block several steps during the earliest stages on DCs, as it had been previously reported for other interferon- of infection, avoiding not only the productive infection of stimulated genes during chronic HIV-1 infection (51). Further, primary target cells, but also the viral dissemination toward we demonstrated that CD14+ CD11c+ DCs up-regulate Siglec- distal tissues. In both scenarios, antigen-presenting cells residing 1 expression in a dose response manner after IFNα stimulation in different compartments of the female genital mucosa may of the cervical tissue, especially at the endocervix. In contrast, play a prominent role (7, 9, 24, 35). DC-T cell conjugates higher basal expression of Siglec-1 was found on ectocervical represent an optimal milieu for productive HIV infection, which myeloid cells at the steady state. These data suggest that may boost initial viral replication of CD4+ T cells (10, 36). while pre-existing basal immune activation at the ectocervix Moreover, uninfected DCs could promote HIV dissemination could already favor Siglec-1 capture of early invading viruses, to draining lymph nodes after mucosal challenge (8, 9). Yet, endocervical cells will most likely mediate viral uptake at later the precise molecular mechanism mediating these processes time points, once antiviral type I IFN responses are mounted and remained unexplored in the cervicovaginal tissue. Siglec-1 expression is boosted on DCs. Indeed, this could explain Here we have shown the ability of Siglec-1 to mediate viral why we only observed an increased number of Siglec-1+ cells capture and transmission to target cells on a population of on biopsies from the endocervix that had a high inflammatory cervical myeloid cells expressing HLA-DR, CD11c, CD14, and score. Overall, detection of Siglec-1 on cervical DCs contributes CD11b, all classical markers known to be present on DCs of to understand why the IFN system is not more effective against the submucosa at the lower female genital tract (18, 28, 29, 37). HIV-1 despite its substantial up-regulation early upon infection Our findings concur with prior studies where the migratory (4, 33, 52). Similarly, it may also explain why treatments inducing Frontiers in Immunology | www.frontiersin.org 8 April 2019 | Volume 10 | Article 825 Perez-Zsolt et al. Cervical Siglec-1+ DCs trans-infect HIV-1 FIGURE 5 | IFNα enhances viral capture and trans-infection on Siglec-1+ cervical DCs. (A) Percentage of Siglec-1+ DCs within the myeloid HLA-DR+ CD14+ CD11c+ fraction from small pieces of ectocervix or endocervix cultured in the presence or absence of increasing concentrations of IFNα (1,000; 10,000; and 100,000 IU/ml) and assessed by FACS as in Figure 3. Bar graph shows mean values and SEM from 5 donors and 4 independent experiments. Colors depict each particular donor. Statistical differences were assessed with a one-way repeated measures ANOVA test. (B) Percentage of Siglec-1+ DCs within the myeloid HLA-DR+ CD14+ CD11c+ fraction from cellular suspensions obtained from tissue digestion and cultured in the presence or absence of IFNα. Statistical differences were assessed with a paired t-test. (C) Percentage of cells capturing HIV-1Gag−eGFP VLPs among the myeloid HLA-DR+ CD14+ CD11c+ fraction from cellular suspensions obtained from ectocervix or endocervix digestion and cultured in the presence or absence of IFNα. Statistical differences were assessed with a paired t-test. (D) Percentage of cells capturing HIV-1Gag−eGFP VLPs as in (C) on cells that had been previously pre-incubated with 20 µg/ml of 7D2 anti-Siglec-1 mAb or isotype control. Bar graph shows mean values and SEM from 2-3 donors. Dot plots showing representative inhibition are also depicted. (E) Relative R5 tropic HIV-1NFN−SX transmission to CD4+ target cells from cervical CD45+ CD3− CD19− HLA-DR+ sorted cells pre-incubated with 20 µg/ml of isotype or anti-Siglec-1 mAbs before viral exposure. Values are normalized to isotype-treated cells (set at 100%). Statistical differences were assessed with a one sample t-test. Mean values and SEM from two experiments include cells from 3 or 4 donors. pDC recruitment (53) and a strong type I IFN response (54) infection within the cervical mucosa, but also facilitate early before vaginal challenge in macaques are incapable of limiting dissemination to secondary lymphoid tissues. These findings viral infection beyond the infected mucosa and can even enhance highlight the importance of including Siglec-1 inhibitors along viral replication. with potent antiretroviral agents in forthcoming microbicidal As other sexually transmitted infections such as herpes virus strategies, to stop not only the productive cellular infection in the or chlamydia infection trigger type I IFN responses in mucosal cervix, but also the systemic viral dissemination from the female tissues via pDC recruitment and/or bacterial lipopolysaccharide genital tract. exposure (55, 56), it would be important to explore the role of Siglec-1 in favoring HIV-1 acquisition, replication and METHODS dissemination in women with pre-existing sexually transmitted infections (2). Moreover, since inflammatory CD14+ CD11c+ Cervical Tissue Digestion DCs are known to induce Th17 T-cell differentiation (57), and and Immunophenotype these are the preferential targets of viral infection in the cervix Human cervical tissue was obtained from women (age range right after retroviral invasion (58), it would also be critical to 39–82 years) undergoing hysterectomy for non-neoplasic evaluate the role of this Siglec-1+ DC subset on susceptibility to indication at either HUGTiP or Hospital Municipal de Badalona. HIV-1 infection. After confirmation of healthy tissue status by the Pathology In summary, myeloid DCs expressing a key molecule involved Service, a piece from ectocervix and endocervix separated in HIV-1 trans-infection, namely Siglec-1, are found in the by anatomical localization was delivered to the laboratory cervical tissues of women in the basal state. IFNα-treated in tissue media [RPMI 1640 supplemented with 15 % FBS, myeloid DCs up-regulate Siglec-1 expression, which increases 500 U/mL penicillin, 500 µg/mL streptomycin, 2.5 µg/mL viral capture and trans-infection, providing an explanation of Fungizone (Life Technologies), 50 µg/mL gentamicin, MEM- how the virus may succeed in an otherwise antiviral environment. non essential aminoacids (Gibco) and 1 mM MEM-sodium We propose that Siglec-1+ cervical DCs may facilitate HIV-1 pyruvate (Gibco)]. Tissue was processed within the next 24 h transfer to bystander CD4+ T cells and favor the nascent after surgery. The mucosal epithelium and the underlying Frontiers in Immunology | www.frontiersin.org 9 April 2019 | Volume 10 | Article 825 Perez-Zsolt et al. Cervical Siglec-1+ DCs trans-infect HIV-1 stroma of both ectocervix and endocervix were separated from PA, USA) attached to a BX41 microscope (Olympus). Siglec- muscular tissue and dissected into approximately 8-mm3 blocks. 1+ cells in five consecutive fields in the subepithelial area Depending on the experiment, tissue blocks were cultured, were counted for ectocervix and endocervix separately. Tissues digested or included in optimal cutting temperature compound where a significant inflammatory infiltrate was detected were for immunofluorescence as previously described (34). considered as highly inflamed. For tissue digestion, five to eight pieces from ectocervix or endocervix were separately placed into 1.5 ml-tubes containing 5 Cell Lines mg/ml collagenase IV (Invitrogen) in RPMI 1640 supplemented HEK-293T cells obtained from ATCC (CRL-11268) and TZM- with 5% fetal bovine serum (FBS, Gibco). Tubes were shaked at bl (obtained through the US National Institutes of Health 400 rpm at 37◦ C for 30 min. After enzymatic digestion, tissue [NIH] AIDS Research and Reference Reagent Program) were blocks were dissociated manually with a disposable pellet pestle maintained in Dulbecco’s Modified Eagle Medium (DMEM). The in two series of 20 rotations while pulling it up and down. The human T cell lines MOLT CD4+ uninfected and MOLT CD4+ suspension was filtered through a 70 µm cell strainer (SPL Life HIV-1BaL infected have been described elsewhere (59) and were Sciences), and cells were collected at 16◦ C after washing with grown in RPMI. All media contained 10% FBS, 100 U/ml of PBS. Cells were then stained with Live/Dead Aqua (Invitrogen) penicillin and 100 µg/ml of streptomycin (all from Invitrogen). for 30 min at RT. Pellet was suspended in staining buffer (1% mouse serum, 1% goat serum in PBS) and stained with HIV-1Gag-eGFP VLP and HIV-1 Generation CD14-V450 (MØP9), CD11c-PE-Cy7 (B-ly6), HLA-DR-PerCP- Fluorescent HIV-1 virus-like particles (HIV-1Gag−eGFP VLP) Cy5.5 (G46-6), (all from BD Biosciences), CD3-eVolve 655 were generated transfecting HEK-293T cells with plasmid (OKT3) (eBiosciences), CD45-Alexa Fluor 700 (Hl30), CD11b- pGag-eGFP obtained from the NIH AIDS Research and FiTC (M1/70), Siglec-1-PE (7-239) (all from BioLegend). Mouse Reference Reagent Program. Replication-competent HIV-1 stock IgG1-PE (BioLegend) was used as isotype control. Cells were was generated by transfecting the proviral construct NFN- acquired using a BD LSRFortessa SORP flow cytometer (Flow SX, an HIV-1NL43 provirus that expresses the HIV-1JRFL Cytometry Platform, IGTP) and analyzed with FlowJo vX.0.7 envelope glycoprotein (kindly provided by W. O’Brien). Thirty software (TreeStar). micrograms of plasmid DNA were added to cells in T75 flasks, and transfection was performed using a calcium phosphate Immunofluorescence kit (CalPhos, Clontech). Forty-eight hours post-transfection, Cervical 5-µm cryosections were dried at RT, fixed in 3.7% supernatants were harvested, filtered (Millex-HV, 0.45 µm; formaldehyde (Sigma Aldrich) diluted in PHEM buffer as Millipore) and frozen at −80◦ C until use. The p24Gag content previously described in (34), permeated with 0.2% Triton X- of the VLP and HIV-1 stock was determined by ELISA 100 (Sigma Aldrich) in PHEM buffer and blocked with 0.2% (PerkinElmer). TCID50 or 50% tissue culture infective doses cold fish gelatin (Sigma Aldrich), 0.1% Triton X-100 and 10% of HIVNFN−SX -infection used for trans-infection assays were normal goat serum (Gibco) diluted in PBS. Sections were first determined by end-point dilution culture on the cell line TZM-bl, incubated with the following primary mAbs: mouse anti-Siglec- which contains an HIV long terminal repeat linked to a luciferase 1 7-239 Ab (Abcam), rabbit anti-CD11c EP1347Y Ab (Abcam) reporter gene. or rabbit anti-CD14 EPR3653 Ab (Abcam). Samples were then washed extensively with PBS and incubated with the secondary VLP Uptake Assays mAbs Alexa 488-conjugated donkey anti-rabbit or Alexa 647- 1 × 106 digested ectocervical and endocervical cells were pre- conjugated donkey anti-mouse IgG (Jackson ImmunoResearch). incubated for 15 min at RT with 20 µg/ml of an anti-Siglec-1 mAb TM Sections were covered with mounting medium (ProLong Gold (7D2) (Abcam), a murine IgG1 κ isotype control (BD Pharmigen) Antifade Mountant with DAPI, Life Technologies, Invitrogen) or left untreated. Cells were pulsed overnight with 2.7- 20 ng and a coverslip. Images were obtained by confocal microscopy p24Gag HIV-1Gag−eGFP VLP at 37◦ C in a 5% CO2 incubator in the using a Zeiss LSM 710 microscope and the Zen Blue Image presence or absence of 1,000 IU/ml of recombinant Interferon- acquisition software. 2α (Sigma-Aldrich) in 10% FBS RPMI with antibiotics. After extensive washing, cells were stained as previously described and Immunohistochemistry acquired using a BD LSRFortessa SORP flow cytometer (Flow Ten cervical formalin-fixed, paraffin-embedded samples from the Cytometry Platform, IGTP) and analyzed with FlowJo v10.3 Pathology Department of HUGTiP were analyzed. Of note, we software (TreeStar). chose samples from five HIV-1 infected and five seronegative For imaging flow cytometer analyses, 1 × 106 digested women, but results were equivalent regardless of the HIV status. ectocervical and endocervical cells were pulsed with HIV- Immunohistochemical stains were performed using a Ventana 1Gag−eGFP VLP as previously described. After extensive washing, Benchmark Ultra (Ventana Medical Systems) in accordance with cells were resuspended in PBS with 1:250 Live/Dead Aqua the manufacturer’s protocol, with standard antigen retrieval (pH (Invitrogen) and incubated for 30 min at RT. Cells were then fixed 9.0; Ventana) and the mAb against α-Siglec-1 (clone SP213, LS and permeabilized (Fix & Perm; Invitrogen), and stained with an Biosciences, dilution 1/100) for 12 min. A pathologist carried anti-Siglec-1-PE mAb (7-239) (BioLegend). Cells were acquired out a blind quantification of Siglec-1+ cells and images were with an Amnis ImageStreamX imaging flow cytometer (Merck), captured using a DP71 digital camera (Olympus, Center Valley, and analyzed using IDEAS v6.1 software. A gradient root mean Frontiers in Immunology | www.frontiersin.org 10 April 2019 | Volume 10 | Article 825 Perez-Zsolt et al. Cervical Siglec-1+ DCs trans-infect HIV-1 square or RMS value > 40 was established as the best focus As a negative control, pDCs were co-cultured with an uninfected threshold, and single cells were selected in the Area vs. Aspect MOLT-4 cell line. After 24 h of co-culture, supernatants were Ratio dot plot of the bright field channel. collected and assessed for IFNα production with VeriKine Human IFN Alpha Elisa Kit (pbl Assay Science). Alternatively, HIV-1 p24 Immunostaining supernatants from these co-cultures were transferred to 0.2 × 106 A cervical formalin-fixed, paraffin-embedded sample from a DCs to assess Siglec-1 induction 24 h later with a FACSCalibur, viremic patient diagnosed with sexually transmitted HIV-1 labeling cells with a mAb anti-Siglec-1-PE or a matched isotype- infection a decade ago was obtained from the Pathology PE control (both from AbD Serotec). Of note, these supernatants Department of the University Hospital Vall d’Hebron. The were also added to DCs that had been previously incubated with sample was collected when the patient had between 5,160 2 µg/ml of carrier-free recombinant B18R protein (eBioscience) and 10,400 HIV-1 RNA copies/ml in blood. Samples were to block type I IFN receptor. DCs were also cultured in de-waxed and placed in decreasing ethanol concentrations. the presence of RPMI media or 1,000 IU/ml of recombinant Heat-induced epitope retrieval was performed in EDTA buffer Interferon-2α. The mean number of Siglec-1 Ab binding sites per pH 9 (Abcam) in a water bath at 100◦ C during 10 min. monocyte-derived DC from men and women was obtained with a Slides were permeabilized with Tris-bufferd saline 1X (TBS) Quantibrite kit (Becton-Dickinson) as previously described (13). (Fisher scientific) with 0.1% Triton X-100 and 1% BSA (Sigma- Aldrich) for 10 min. Subsequently, blocking was performed Cervical Tissue Stimulation With IFNα with TBS 1x supplemented with 10% donkey serum (Jackson After dissection of the tissue as previously described, five pieces Immunoresearch) and 1% BSA for 2h. Mouse anti-p24 mAb (Kal- from ectocervix or endocervix were separately placed into a 12- 1; Dako-Agilent) was incubated overnight at 4◦ C. Later, rabbit well plate containing 1 ml of tissue culture medium. Interferon- anti-Siglec-1 mAb (SP213; Lifespan Bioscience) was incubated 2α was added to the medium at 1,000; 10,000 or 100,000 IU/ml. for 15 min at RT. Samples were then stained with secondary After 24 h at 37◦ C in 5% CO2 , tissue was digested and the antibodies Donkey AlexaFluor 647 anti-mouse (Invitrogen) remaining culture plate was treated with accutase (Thermo Fisher and Donkey AlexaFluor 488 anti-rabbit; counter stained with Scientific) for 30 min at 37◦ C to detach adherent cells. Finally, DAPI (Thermo Fisher) and mounted with Fluoromount G tissue and adherent cells were pooled together and stained for (eBioscience). Samples were imaged on an Olympus Spectral flow cytometry as previously described. Confocal Microscope FV1000 using a 20x and 60x phase objective and sequential mode to separately capture the fluorescence at an image resolution of 800 × 800 pixels. Image Trans-infection Experiments J software was used for image processing. Alternatively, samples Ectocervical and endocervical blocks from HIV-1 non-infected were acquired in z-stacks every 0.2 µm on a Zeiss LSM 780 donors were left untreated or incubated overnight with 10,000 confocal inverted microscope with an apochromatic 63x oil U/ml of IFNα and 100 ng/ml of CCL19 (Mip-3β). Tissue was then (NA = 1.4) and processed with Volocity software using the 3D digested, pooled together and stained with mAbs as previously Opacity module for reconstruction. described to sort single CD45+ CD3− CD19− HLA-DR+ live cells by FACS. Recovered cells were pre-incubated with anti-Siglec- Primary Cell Cultures 1 7D2 or isotype control mAbs for 10 min at RT. Cells were Peripheral blood mononuclear cells (PBMCs) were obtained subsequently incubated with 185 ng of p24/ml of an R5 tropic from buffy coats of the Banc de Sang i Texits of Barcelona HIV-1NFN−SX (with an estimated TCID50 of 116.824) in the by Ficoll-Hypaque density gradient centrifugation (Alere presence of 20 µg/ml of the indicated mAbs for 4 h at 37◦ C. Technologies AS). Plasmacytoid DCs were negatively isolated After extensive washes, myeloid cells were co-cultured with the using magnetic beads from the Plasmacytoid Dendritic Cell reporter TZM-bl cell line at a 1:1 ratio for 48 h. Luciferase activity isolation kit (Miltenyi Biotech) and immediately used for was measured with Britelite plus (Perkin Elmer) in a Synergy MX experiments. Monocytes were isolated using CD14+ selection luminometer (Biotek). magnetic beads (Miltenyi Biotech) and differentiated into monocyte-derived DCs with 1,000 IU/ml of granulocyte- Statistical Analysis macrophage colony-stimulating factor plus 1,000 IU/ml of Data are reported as the mean and the standard error of the Interleukin-4 (both from R&D) during 5 days before supernatant mean (SEM) for each condition. We analyzed mean changes exposure. Cells were maintained in RPMI supplemented with using Mann-Whitney test, Wilcoxon matched-pairs signed rank 10% FBS, 100 U/ml of penicillin and 100 µg/ml of streptomycin. test, paired t-test, and one-way repeated measures ANOVA test as indicated, which were considered significant at P < 0.05. IFNα Release on Supernatants From pDCs Significant mean changes from 100% of the data normalized to and Siglec-1 Induction percentages were assessed with a one sample t-test, considered A total of 0.1 × 106 pDCs were co-cultured with 0.1 × 106 HIV- significant at P < 0.05. Gender main effect inference across 1BaL -infected MOLT-4 cells for 24 h at 37◦ C. Before co-culture, multiple experiments was assessed using the Prentice Rank Sum some pDCs were also pre-treated with 10 µg/ml of anti-CD4 Test, a generalized Friedman rank sum test with replicated mAb (clone RPA T-4) to avoid viral fusion or with an isotype blocked data. All analyses and figures were generated with the mAb control (both from Beckton Dickinson) for 10 min at RT. GraphPad Prism v7 Software and R v3.5. Frontiers in Immunology | www.frontiersin.org 11 April 2019 | Volume 10 | Article 825 Perez-Zsolt et al. Cervical Siglec-1+ DCs trans-infect HIV-1 ETHICS STATEMENT Ministerio de Economía y Competitividad, Instituto de Salud Carlos III (ISCIII, PI14/01235 and PI17/01470) and by the Pla Informed written consent was obtained from all participants estratègic de recerca i innovació en salut (PERIS), from the and the study protocols were approved by the University Catalan government. Hospital Germans Trias i Pujol (HUGTiP, Badalona, Spain) and the Vall d’Hebron University Hospital (HUVH, Barcelona, ACKNOWLEDGMENTS Spain) Clinical Research Ethics Committee (reference numbers PI-14-070 and PR (IR)294/2017). The study was undertaken We would like to thank all the patients who participated in accordance with the Declaration of Helsinki and the in the study. We thank M. A. Fernández and G. Requena requirements of Good Clinical Practice. from the Flow Cytometry Platform at the IGTP for excellent technical assistance, as well as I. Crespo from the Flow AUTHOR CONTRIBUTIONS Cytometry Platform at the IDIBAPS for her excellent technical assistance on Amnis technology. For her excellent assistance, DP-Z, JC-P, NI-U, and MG conceived and designed the advice, and imaging processing we thank Elena Rebollo from experiments. AH-G, JC, GT, JG, and AT obtained, processed, the Advanced Fluorescence Microscopy Unit IBMB-PCB. We and analyzed tissues. DP-Z, JC-P, IE, SB, MP, CS-P, and also would like to thank I. Lorencés and L. Pérez-Roca VA-S performed the experiments. DP-Z, JC-P, IE, SB, MP, from the Tumor Bank of the IGTP-HUGTiP and M. Martín- CS-P, JC, GT, MJB, JM-P, NI-U, and MG analyzed and Céspedes from the Pathology Department of the HUGTiP interpreted the data. DP-Z, JC-P, NI-U, and MG wrote for their outstanding sample management and processing. the paper. We would like to thank V. Urrea from for his excellent statistical assistance. FUNDING SUPPLEMENTARY MATERIAL JM-P and NI-U are supported by the Spanish Secretariat of State of Research, Development and Innovation through grant The Supplementary Material for this article can be found SAF2016-80033-R. DP-Z is supported by the Spanish Ministry of online at: https://www.frontiersin.org/articles/10.3389/fimmu. Science, Innovation and Universities and the European Regional 2019.00825/full#supplementary-material Development Fund under agreement BES-2014-069931. JM-P Movie 1 | 3D volumetric x-y-z data field reconstruction of a Siglec-1+ cell from and MJB are supported by the Spanish AIDS network Red the cervical tissue of a viremic HIV-infected woman. Opacity representation of Temática Cooperativa de Investigación en SIDA RD16/0025/0007. DAPI stained nuclei and fluorescence of the sac-like virus-containing compartment along with Siglec-1 staining. MJB is supported by the Spanish Secretariat of Science and Innovation and FEDER funds (grant SAF2015-67334-R Supplemental Figure 1 | Representative histograms of HLA-DR expression comparing the Siglec-1 positive and negative populations on CD3− CD11c+ [MINECO/FEDER]) and by the Miguel Servet program funded CD14+ cells from the ectocervix and endocervix. Bar graphs show the geometric by the Spanish Health Institute Carlos III (CP17/00179). MG mean fluorescence values and SEM of HLA-DR expression from 14 donors. is supported by a Marie Curie Career Integration Grant (CIG) Statistical differences were assessed with a Wilcoxon matched-pairs signed from the European Commission, grants from the Spanish rank test. REFERENCES and infects intraepithelial dendritic cells. J Virol. (2000) 74:6087–95. doi: 10.1128/JVI.74.13.6087-6095.2000 1. UNAIDS. Global AIDS Update. (2016) Geneva:UNAIDS. 8. Masurier C, Salomon B, Guettari N, Pioche C, Lachapelle F, Guigon M, et al. 2. 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The authors declare that no other competing financial The Toll-like receptor 7 (TLR7) agonist, imiquimod, and the TLR9 interests exist and that the research was conducted in the absence of any agonist, CpG ODN, induce antiviral cytokines and chemokines but do commercial or financial relationships that could be construed as a potential conflict not prevent vaginal transmission of simian immunodeficiency virus when of interest. applied intravaginally to rhesus macaques. J Virol. (2005) 79:14355–70. doi: 10.1128/JVI.79.22.14355-14370.2005 Copyright © 2019 Perez-Zsolt, Cantero-Pérez, Erkizia, Benet, Pino, Serra- 55. Agrawal T, Vats V, Wallace PK, Singh A, Salhan S, Mittal A. Recruitment Peinado, Hernández-Gallego, Castellví, Tapia, Arnau-Saz, Garrido, Tarrats, Buzón, of myeloid and plasmacytoid dendritic cells in cervical mucosa during Martinez-Picado, Izquierdo-Useros and Genescà. This is an open-access article Chlamydia trachomatis infection. Clin Microbiol Infect. (2009) 15:50–9. distributed under the terms of the Creative Commons Attribution License (CC BY). doi: 10.1111/j.1469-0691.2008.02113.x The use, distribution or reproduction in other forums is permitted, provided the 56. Lund JM, Linehan MM, Iijima N, Iwasaki A. Cutting edge: original author(s) and the copyright owner(s) are credited and that the original plasmacytoid dendritic cells provide innate immune protection publication in this journal is cited, in accordance with accepted academic practice. against mucosal viral infection in situ. J Immunol. (2006) 177:7510–4. No use, distribution or reproduction is permitted which does not comply with these doi: 10.4049/jimmunol.177.11.7510 terms. Frontiers in Immunology | www.frontiersin.org 14 April 2019 | Volume 10 | Article 825