HIV-1 immune activation induces Siglec-1 expression and enhances viral trans-infection in blood and tissue myeloid cells
- Maria Pino1,
- Itziar Erkizia1,
- Susana Benet1,
- Elina Erikson2, 8,
- Maria Teresa Fernández-Figueras3,
- Dolores Guerrero4,
- Judith Dalmau1,
- Dan Ouchi1,
- Antonio Rausell5, 6,
- Angela Ciuffi5,
- Oliver T Keppler2,
- Amalio Telenti5, 7,
- Hans-Georg Kräusslich8,
- Javier Martinez-Picado1, 9, 10Email author and
- Nuria Izquierdo-Useros1Email author
© Pino et al.; licensee BioMed Central. 2015
Received: 15 January 2015
Accepted: 24 March 2015
Published: 7 May 2015
Myeloid cells are key players in the recognition and response of the host against invading viruses. Paradoxically, upon HIV-1 infection, myeloid cells might also promote viral pathogenesis through trans-infection, a mechanism that promotes HIV-1 transmission to target cells via viral capture and storage. The receptor Siglec-1 (CD169) potently enhances HIV-1 trans-infection and is regulated by immune activating signals present throughout the course of HIV-1 infection, such as interferon α (IFNα).
Here we show that IFNα-activated dendritic cells, monocytes and macrophages have an enhanced ability to capture and trans-infect HIV-1 via Siglec-1 recognition of viral membrane gangliosides. Monocytes from untreated HIV-1-infected individuals trans-infect HIV-1 via Siglec-1, but this capacity diminishes after effective antiretroviral treatment. Furthermore, Siglec-1 is expressed on myeloid cells residing in lymphoid tissues, where it can mediate viral trans-infection.
Siglec-1 on myeloid cells could fuel novel CD4+ T-cell infections and contribute to HIV-1 dissemination in vivo.
KeywordsDendritic cells HIV-1 Trans-infection Siglec-1
Antigen presenting cells of the myeloid lineage, such as dendritic cells (DCs), monocytes and macrophages, initiate antiviral immune responses and are crucial to control invading viruses. On the other hand, myeloid cells may also be productively infected with HIV-1 and thus promote pathogenesis. Compared to activated CD4+ T cells, the permissivity of myeloid cells for HIV-1 is limited [1-3]. This is largely due to restriction by the cellular factor SAMHD1 in these cells [4,5]. Nevertheless, myeloid cells can contribute to HIV-1 dissemination through the alternative pathway of trans-infection of CD4+ T cells [6,7]. This mechanism involves HIV-1 capture and uptake by myeloid cells and the subsequent release of trapped viruses at a cell-to-cell contact zone, the infectious synapse, that facilitates infection of CD4+ T cells .
We and others have previously shown that HIV-1 trans-infection requires the sialic acid binding Ig-like lectin 1 (Siglec-1, Sialoadhesin or CD169) [9,10]. Siglec-1 is expressed on the surface of DCs and other myeloid cells [11,12] and recognizes sialyllactose molecules exposed on HIV-1 membrane gangliosides [13,14]. Siglec-1 expression on myeloid cells is induced by activating signals that are present upon acute and chronic immune activation, which is observed in HIV-1-infected patients [15,16]. Various pro-inflammatory factors associated with HIV-1 disease progression stimulate Siglec-1 expression on myeloid cells. Lipopolysaccharide (LPS) is a component of the bacterial cell wall that is significantly increased in chronically HIV-1-infected individuals owing to the depletion of gastrointestinal CD4+ T cells, which causes systemic translocation of bacteria from the intestinal lumen . In vitro, LPS is able to induce Siglec-1 expression on DCs, potently enhancing their capacity to trans-infect HIV-1 . Another factor is interferon alpha (IFNα) that exerts antiviral effects on HIV-1 replication [10,18], but also serves as a marker of poor clinical prognosis during late-stage disease [19,20]. Although IFNα is produced in large quantities by plasmacytoid DCs after HIV-1 challenge [21,22] the key cells responsible for sustained IFNα production in vivo are currently under intensive reexamination [23,24]. Regardless of its source, IFNα potently induces the in vitro expression of Siglec-1 on myeloid cells [10,18].
Siglec-1 up-regulation by immune activating signals during HIV-1 infection could play an important role by allowing myeloid trans-infection of multiple target cells. This function could be particularly relevant in lymphoid tissues, the major sites of HIV-1 replication, where myeloid cells migrate and repeatedly establish interactions with CD4+ T cells [25-27], the primary targets of productive HIV-1 infection. In the study described here, we investigated whether Siglec-1 expression on myeloid cells can be induced during chronic HIV-1 infection and thus contribute to systemic viral dissemination. We found that Siglec-1 on myeloid cells is up-regulated in vitro by IFNα, allowing for HIV-1 capture and transmission. In vivo, HIV-1 infection enhanced Siglec-1 expression on peripheral blood monocytes, but diminished after effective antiretroviral treatment, reducing the trans-infection capacity of monocytes. Moreover, Siglec-1 is present on myeloid cells from lymphoid tissues, where it can mediate HIV-1 trans-infection.
Siglec-1 mediates HIV-1 capture by IFNα-treated myeloid cells
We have previously reported that Siglec-1 expression levels determine the capacity of DCs to capture HIV-1 . To test whether this also holds true for monocytes and macrophages, we first compared the density of Siglec-1 surface expression applying a quantitative FACS assay that determines the absolute number of Siglec-1 antibody binding sites. This number was highest in IFNα-activated monocyte-derived DCs, followed by macrophages and monocytes (Figure 1B). Next, we analyzed binding of infectious HIV-1 to IFNα-activated myeloid cells. HIV-1 was incubated with the cells for 4 h at 4°C to avoid viral internalization and cell-associated p24Gag was quantified by ELISA after extensive washing. Consistent with their respective Siglec-1 expression levels (Figure 1B), IFNα-activated monocyte-derived DCs showed a higher HIV-1 binding capacity than monocytes (Figure 1C). To investigate whether this binding was specific for Siglec-1, cells were pre-treated with a monoclonal antibody (mAb) against Siglec-1. This treatment led to a reduction of HIV-1 binding by 83% in all cases, while isotype control treatment had no inhibitory effect (Figure 1D). Similar results were obtained with fluorescent HIV-1 Virus-Like Particles (VLPs), which lack the viral envelope glycoprotein but carry sialyllactose-containing gangliosides recognized by Siglec-1 (Additional file 1: Figure S1A-B). These data indicate that Siglec-1 is the main molecule responsible for HIV-1 capture by IFNα-activated myeloid cells, and that its expression correlates with the viral binding capacity of the respective cell type.
Siglec-1 mediates HIV-1 uptake into a storage compartment and enhances HIV-1 trans-infection specially in IFNα-treated monocytes and DCs
To elucidate the HIV-1 trafficking differences in macrophages compared to other myeloid cells, we investigated viral uptake by confocal microscopy. IFNα-treated myeloid cells were pulsed with fluorescent HIV-1Cherry for 4 h at 37°C and subsequently stained with mAbs against Siglec-1 and against HLA-DR to reveal cellular membranes (Figure 2D, top images). While most of the monocyte-derived DCs and monocytes accumulated HIV-1Cherry within a sac-like compartment enriched in Siglec-1, macrophages exhibited a more scattered pattern for cell-associated HIV-1Cherry (Figure 2D). Thus, the complementary approaches of confocal microscopy analysis and viral uptake experiments indicate that Siglec-1 is essential for HIV-1 capture in all myeloid cells, while it is not sufficient for further downstream uptake and trafficking involved in the formation of a condensed viral compartment.
We next assessed the capacity for HIV-1 trans-infection by the different myeloid cell types. Cells were pulsed with equal amounts of the X4-tropic HIV-1NL4–3 followed by extensive washing and were subsequently co-cultured with a CD4+ reporter cell line for two days. Monocyte-derived DCs had the highest capacity for trans-infection followed by monocytes, while macrophages showed only weak trans-infection capacity (Figure 2E), consistent with their faster viral degradation kinetics (Figure 2B). However, trans-infection depended on Siglec-1 in all cases (Figure 2F).
Siglec-1 is up-regulated on monocytes from HIV-1-infected individuals, and its expression is reduced upon successful antiretroviral treatment
PRE/POST Antiretroviral treatment
Number or Median (IQR)
Number of patients
Antiretroviral treatment regimen
Non-Nucleoside Reverse Transcriptase Inhibitors
Time from diagnosis to antiretroviral treatment (months)
Time between samples (months)
CD4+ T-cell count (cells/μl)
PRE antiretroviral treatment
POST antiretroviral treatment
Plasma Viral Load (Log10 HIV RNA copies/ml)
PRE antiretroviral treatment
POST antiretroviral treatment
The plasma of untreated HIV-1-infected individuals stimulates Siglec-1 expression and signals via the type I IFN receptor
Expression of Siglec-1 on monocytes correlates with clinical parameters
Characteristics of the HIV-1-infected men before initiation of antiretroviral treatment, whose samples were used in Figure 5
PRE Antiretroviral treatment
Number or Median (IQR)
Number of patients
CD4+ T-cell count (cells/μl)
Plasma Viral Load (Log10 HIV RNA copies/ml)
Siglec-1 positive cells accumulate in inflamed lymphoid tissues in areas enriched in CD4+ T cells
We next assessed whether Siglec-1 expressing cells could be detected in lymphoid tissue from an HIV-1-infected individual who had lymph nodes removed before and after initiation of antiretroviral treatment. Of note, this patient also had an untreated HCV infection. Siglec-1 positive cells were detected in perivascular, sub-capsular, and perifollicular areas enriched in CD4+ T cells, but mostly excluded from CD20-positive follicular zones (Figure 6B). A similar pattern was observed for tissue obtained before and after initiation of therapy. Although we cannot rule out the possibility that untreated HCV infection sustained Siglec-1 expression, it is also conceivable that antiretroviral drug concentrations were insufficient to fully suppress HIV-1 replication in the lymphoid tissue of this particular individual [29,30] thus sustaining IFNα production.
Siglec-1 mediates HIV-1 trans-infection by myeloid cells isolated from lymphoid tissue
List of GO biological processes relevant for antigen-presenting cell function found significantly enriched after Bonferroni correction in the 5991 protein coding genes commonly expressed by sorted tonsil-derived Siglec-1 positive cells and the different types of myeloid cells exposed to IFNα
GO biological process
Genes in GO process
Immune system process (GO:0002376)
5,10E + 05
Regulation of immune system process (GO:0002682)
2,96E + 05
Positive regulation of immune system process (GO:0002684)
1,73E + 05
Defense response (GO:0006952)
3,42E + 05
Regulation of defense response (GO:0031347)
1,37E + 05
Immune response (GO:0006955)
3,14E + 05
Regulation of immune response (GO:0050776)
1,94E + 05
Positive regulation of immune response (GO:0050778)
1,16E + 05
Activation of immune response (GO:0002253)
9,35E + 04
Immune response-activating signal transduction (GO:0002757)
8,05E + 04
Immune response-regulating signaling pathway (GO:0002764)
1,10E + 05
Immune effector process (GO:0002252)
1,09E + 05
Response to virus (GO:0009615)
6,80E + 04
Innate immune response (GO:0045087)
2,10E + 05
Regulation of innate immune response (GO:0045088)
6,69E + 04
Positive regulation of innate immune response (GO:0045089)
4,84E + 04
Activation of innate immune response (GO:0002218)
4,02E + 04
Innate immune response-activating signal transduction (GO:0002758)
3,83E + 04
Antigen processing and presentation (GO:0019882)
6,04E + 04
Of peptide antigen (GO:0048002)
5,06E + 04
Of peptide antigen via MHC class I (GO:0002474)
2,75E + 04
Of exogenous antigen (GO:0019884)
4,68E + 04
Of exogenous peptide antigen (GO:0002478)
4,62E + 04
Of exogenous peptide antigen via MHC class I (GO:0042590)
2,15E + 04
Of exogenous peptide antigen via MHC class I, TAP-dependent (GO:0002479)
2,04E + 04
Pattern recognition receptor signaling pathway (GO:0002221)
3,75E + 04
Toll-like receptor signaling pathway (GO:0002224)
3,26E + 04
Toll-like receptor 3 signaling pathway (GO:0034138)
2,15E + 04
Toll-like receptor 4 signaling pathway (GO:0034142)
2,58E + 04
Toll-like receptor 9 signaling pathway (GO:0034162)
1,99E + 04
TRIF-dependent toll-like receptor signaling pathway (GO:0035666)
2,07E + 04
Cellular response to cytokine stimulus (GO:0071345)
1,24E + 05
Cytokine-mediated signaling pathway (GO:0019221)
9,22E + 04
Regulation of type I interferon production (GO:0032479)
2,88E + 04
Positive regulation of type I interferon production (GO:0032481)
2,04E + 04
After GO enrichment analysis, 507 GO processes were found significantly enriched, where most of the genes (3564) were related to cellular metabolic processes (GO:0044237). Here we just summarize the enriched GO processes related to immune function, the number of genes categorized in each GO function, the actual number of genes found in the 5991 protein coding genes commonly expressed, the expected P value and the real P value obtained for the genes of interest.
Sorted Siglec-1 positive cells from IFNα-treated tonsils co-stained with several myeloid markers that had been identified in the transcriptomic analysis, including BDCA1, CD11c, HLA-DR, CCR7 and CD86 (Figure 7F, top panels). However, sorted Siglec-1 positive cells could not be employed in functional assays, since mAbs against Siglec-1 block HIV-1 capture (Figure 1D). When we sorted BDCA1-positive cells from IFNα-treated tonsillar cells, they also stained positive for Siglec-1, CD11c, HLA-DR, CCR7 and CD86 (Figure 7F, bottom panels), indicating that this population had a comparable phenotype to that exhibited by Siglec-1 positive cells and could be used for functional assays.
Viral uptake experiments performed with IFNα-treated BDCA1-positive tonsillar cells demonstrated a higher VLP capture capacity when compared to mock-treated BDCA1-positive cells (Figure 7G), and was specifically inhibited by pre-treatment with an anti-Siglec-1 mAb (Figure 7G). Of note, neither the BDCA1-negative cell population nor B cells, which express BDCA1 and could thus be present in the BDCA1-positive cell fraction, were able to up-regulate VLP uptake after IFNα treatment (Additional file 1: Figure S2). In order to investigate HIV-1 trafficking in IFNα-treated BDCA1-positive cells, we added fluorescent HIV-1Cherry for 4 h at 37°C and subsequently stained cells with an anti-Siglec-1 mAb (Figure 7H). Confocal microscopy indicated that most of these BDCA1-positive cells accumulated HIV-1Cherry within a sac-like compartment enriched in Siglec-1, as previously observed for DCs and monocytes (Figure 2C).
Finally, to work with highly purified cell populations, we sorted BDCA1+CD2−CD20−-tonsillar cells cultured in the presence of IFNα and assessed Siglec-1 involvement in HIV-1 trans-infection. IFNα-activated BDCA1-positive cells pre-treated with isotype control or specific mAb were exposed to HIV-1 for 4 h at 37°C, extensively washed and co-cultured with a CD4+ reporter cell line for 2 days (Figure 7I). Trans-infection was readily observed and was specifically inhibited by pre-treatment with a mAb against Siglec-1 (Figure 7I). These results indicated that ex vivo, activation of myeloid cells from tonsils with IFNα leads to Siglec1-dependent enhanced HIV-1 capture and trans-infection, supporting a potential role of Siglec-1 as an important molecule that could contribute to viral capture and trans-infection within lymphoid tissues in HIV-1-infected individuals.
In this report, we show that Siglec-1 on myeloid cells (i) is up-regulated by IFNα; (ii) mediates HIV-1 capture and trans-infection; (iii) correlates in vivo with the levels of plasma viral load and diminishes after effective antiretroviral treatment, and (iv) is expressed in lymphoid tissues in an inflammation-dependent manner where it can mediate HIV-1 trans-infection. Taken together, these findings indicate that inflammatory processes or immune activating signals triggered by HIV-1 replication, such as IFNα release, stimulate Siglec-1 expression on myeloid cells, a process that could enhance viral capture and trans-infection of CD4+ target T cells within lymphoid tissues. Based on our results, this mechanism may be driven by IFNα-activated monocytes and DCs, which exhibited higher Siglec-1 dependent trans-infection than macrophages. Yet, despite the faster viral degradation of captured virions in macrophages, Siglec-1 expression in this cell type may facilitate productive HIV-1 cis-infection .
This model is consistent with our findings that in vivo Siglec-1 expression is up-regulated on monocytes from HIV-1-infected individuals, but diminishes after effective antiretroviral treatment suppresses plasma viral load and virus-induced activating signals [17,28]. Our results are in line with previous reports showing Siglec-1 up-regulation on circulating monocytes of HIV-1-infected individuals with higher plasma viral loads [18,34]. However, assays performed here provide functional evidence that monocytes isolated directly from HIV-1-infected individuals capture HIV-1 and trans-infect CD4+ target cells. We also found that Siglec-1 expression increased with plasma viral load and decreased with CD4+ T-cell counts in HIV-1 infected patients. Furthermore, stimuli present in the plasma of untreated HIV-1-infected individuals induced Siglec-1 expression on myeloid cells via type I IFN receptor signaling. Overall, these data suggest that Siglec-1 could become a useful biomarker to monitor chronic immune activation driven by HIV-1 infection.
Detection of Siglec-1 within lymphoid tissues suggests that this receptor could mediate HIV-1 capture and transmission in these compartments. Lymphoid tissues are the perfect scenarios to fuel novel infections, since they are major sites of HIV-1 replication , where plasmacytoid and myeloid cells accumulate during the course of HIV-1 infection [25,26] and IFNα is detected in lymph nodes of HIV-1-infected individuals . Functional assays performed here with myeloid cells isolated from tonsils and activated with IFNα (to mimic the immune activation state driven by HIV-1 infection in the lymphoid tissues), identified Siglec-1 as a key receptor involved in viral capture and transmission.
We have shown that Siglec-1 expression on distinct primary myeloid cells can be induced during chronic HIV-1 infection in vivo and contribute to systemic viral dissemination. Our results strongly support that Siglec-1 is an important molecule that could accelerate HIV-1 transmission in the crowded cellular environment of lymphatic tissues, where many T-cells can contact myeloid cells. Future studies aimed at blocking Siglec-1 in adequate animal models will be required to shed light on the relative contribution of HIV-1 trans-infection to disease progression and might offer novel therapeutic approaches to halt HIV-1 cell-to-cell transmission.
The institutional review board on biomedical research from Hospital Germans Trias i Pujol (HUGTIP) approved this study. All patients involved in this study gave their written informed consent to participate.
Peripheral blood mononuclear cells (PBMCs) were obtained from HIV-1-seronegative donors by Ficoll-Hypaque density gradient centrifugation and monocyte populations were isolated as described in . Monocytes were differentiated into DCs with 1000 U/ml of granulocyte-macrophage colony-stimulating factor plus 1000 U/ml of Interleukin-4 (both from R&D). In parallel, monocytes from the same donors were differentiated into macrophages with 100 ng/ml of macrophage colony-stimulating factor (Preprotech). Cells were cultured for 7 days, and cytokines and media were replaced every two days. At day five, monocytes, DCs and macrophages were stimulated with 1000 U/ml of Interferon-2α (Sigma-Aldrich) for two days.
HIV-1-infected individuals were selected from a cohort of patients with samples collected before and after antiretroviral treatment. Patient’s characteristics are described in Table 1. HIV-1-negative males matched for age were included as healthy controls. To perform functional assays, PBMCs were thawed and monocytes were isolated with CD14+ magnetic beads (Miltenyi Biotec). Of note, positive isolation did not up-regulate Siglec-1 expression.
Human tonsils were removed during tonsillectomies of individuals undergoing prescribed surgery at the HUGTIP. After mechanical disruption, mononuclear tonsillar cells were isolated by Ficoll-Hypaque gradient centrifugation. T- and B-lymphocytes were subsequently depleted with magnetic beads against CD3+ and CD19+ (Miltenyi Biotec) prior blocking of the Fc receptor (Miltenyi Biotec). Siglec-1 positive cells were sorted by isolating Siglec-1+/CD20−/CD2−cells with mAbs7–239 α-Siglec-1-PE (AbD Serotec), α-CD2-PerCP and α-CD20-PerCP (both from Becton Dickinson) in a FACSVantage SE after blocking Fc receptors with 1 mg/ml of human IgGs (hIgGs; Privigen, Behring CSL). Myeloid cells were also isolated either by BDCA1 positive selection with magnetic beads (Miltenyi Biotec) or by sorting BDCA1+/CD20−/CD2−cells using mAb α-BDCA1-PE (Miltenyi Biotec), α-CD2-PerCP and α-CD20-PerCP. Cells were stimulated with 1000 U/ml IFNα for 24–48 h. Isolated cells were blocked with 1 mg/ml of hIgGs for 20 min at RT and stained with the following mAbs: α-CD11c-APC-Cy7 (BioLegend), α-CCR7-PerCP (Biolegend), α-Siglec-1-Alexa 488 (AbD Serotec), α-HLA-DR-V450 (BD), α-CD86-FITC (BD), α-BDCA1-PECy7 (Biolegend), α-CD20-PerCPCy5.5 (BD), α-CD3-PerCP (BD) at 4°C for 30 min. Samples were analyzed with LSRII using FlowJo software to evaluate collected data.
All primary cells were cultured in RPMI containing 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 100 μg/ml of streptomycin (all from Invitrogen).
Siglec-1 surface expression analysis by FACS
2x105 myeloid cells were blocked with 1 mg/ml of hIgGs and stained with mAb 7–239 α-Siglec-1-PE or matched isotype-PE control (AbD Serotec) at 4°C for 30 min. The mean number of Siglec-1 mAb binding sites per cell was obtained with a Quantibrite kit (Becton Dickinson) as previously described . Samples were analyzed with FACSCalibur using CellQuest software to evaluate collected data.
Induction of Siglec-1 expression by plasmas of HIV-1-negative individuals and HIV-1-infected individuals before or after successful antiretroviral treatment was assessed on 2x105 DCs derived from HIV-1-negative donors cultured for 24 h in the presence of 2% of each respective plasma. To block Siglec-1 induction by these plasmas, carrier-free recombinant B18R protein (eBioscience) was added at 2 μg/ml. DCs were labeled with mAb 7–239 α-Siglec-1-PE and quantified by Quantibrite. Basal values of Siglec-1 in DCs non-exposed to plasma were subtracted for each sample.
Plasmids, viral stocks and cell lines
HEK-293 T and TZM-bl (obtained through the US National Institutes of Health [NIH] AIDS Research and Reference Reagent Program) were maintained in D-MEM containing 10% FBS, 100 U/ml of penicillin and 100 μg/ml of streptomicin.
HIV-1NL4–3 and VLP (VLPHIV-Gag-eGFP) stocks were generated by transfecting the molecular clones pNL4-3 and pGag-eGFP obtained from the NIH AIDS Research and Reference Reagent Program. HIVNL4–3-Cherry was obtained by cotransfection of pCHIV and pCHIVmCherry  (kindly provided by Dr. B. Muller). HEK-293 T cells were transfected with calcium phosphate (CalPhos, Clontech) in T75 flasks using 30 μg of plasmid DNA. Supernatants containing virus or VLPs were filtered (Millex HV, 0.45 μm; Millipore) and frozen at −80°C until use. The p24Gag content of the infectious viral stocks and VLPs was determined by an ELISA (Perkin-Elmer). HIV-1NL4–3 used in infectious assays was titrated employing the TZM-bl reporter cell line that expresses luciferase under control of the HIV-1 promoter, as described in .
VLP and HIV-1 binding and uptake assays
Cells were pre-incubated at 4°C for 30 min with 10 μg/ml of the functional grade mAbα-Siglec-1 7–239, IgG1 isotype control (all from AbD Serotec) or left untreated. Binding was performed at 4°C while uptake was done at 37°C. To assess HIV-1NL4–3 binding and uptake, 4x105 cells were pulsed with 970 ng of p24 for 4 h. After extensive washing, cells were lysed with 0.5% Triton X-100 to measure p24Gag antigen content by an ELISA. To analyze viral degradation, cells were pre-incubated with 250 nM of bafilomycin A1 (Sigma) during 30 min at 37°C and then exposed to HIVNL43 in the presence of the drug or left untreated. To determine VLP binding and uptake, 2x105 myeloid cells were pulsed with 10 ng of VLPs for 3 h and analyzed by FACS.
HIV-1 trans-infection assays
Myeloid cells were treated and pulsed with HIV-1NL4–3 as described above. After extensive washing, cells were co-cultured with the reporter cell line TZM-bl at a ratio 1:1 or 5:1. Cells were assayed for luciferase activity 48 h later (BrightGlo luciferase system; Promega) in a Fluoroskan Ascent FL luminometer (Thermo Labsystems). Background values consisting of non-HIV-1 pulsed co-cultures were measured for each experiment. Of note, we chose the X4-tropic virus NL4-3 and short period co-culture assays to avoid productive cis-infection of myeloid cells and focus on trans-infection.
Confocal microscopy analysis
4x105 myeloid cells were pulsed with HIV-1Gag-Cherry for 4 h at 37°C as previously described . After extensive washing, cells were fixed and permeabilized (Fix & Perm; Invitrogen) and stained with mAbs α-Siglec-1 7–239 Alexa 488 (AbD Serotec), α-HLA-DR-Alexa 647 (Clone L243, Biolegend) and DAPI for 30 min. Cells were cytospun into coverslips and analyzed with an Ultraview ERS Spinning Disk System (Perkin-Elmer) mounted on a Zeiss Axiovert 200 M inverted microscope. Volocity software (Perkin-Elmer) was used to analyze microscopy images as in .
Paraffinized tissues and immunoenzyme staining
Paraffinized tonsils from HIV-1 non-infected individuals were provided by the tissue bank of the National Center for Tumor Diseases (Heidelberg, Germany) and approved by the ethics committee of Heidelberg University (approval No. 206/2005). Immunoenzyme staining of Siglec-1 were performed on 2-μm paraffin sections of formalin-fixed tissues in principle as reported . Antigen retrieval was achieved by steam cooking the slides in 10 mM citrate buffer (pH 6.1, Dako) for 30 min. 10% Earle’s balanced salt solution (EBSS, Sigma Aldrich) supplemented with 1% HEPES, 0.2% bovine serum albumin, and 0.1% saponin (all from Sigma) at pH 7.4 was used as washing and permeabilization buffer. Primary mAb dilutions with α-Siglec-1 7D2 (Novus Biologicals) were also prepared in this buffer and incubated overnight at 4°C. Slides were blocked in 15% sheep serum for 20 min and revealed by biotinylated sheep anti-mouse Ab for 30 min at RT. Immunoenzyme staining was performed with standard avidin-biotin anti-alkaline phosphatase techniques (Vectastain). Naphthol AS-biphosphate (Sigma) with New Fuchsin (Merck) was used as the substrate for alkaline phosphatase. Slides were viewed with an Olympus BX45 microscope. Tonsils were classified by an experienced pathologist as inflamed based on strong tissue infiltration of neutrophil granulocytes.
Paraffinized axillary and abdominal lymph nodes from an HIV/HCV co-infected patient were analyzed at the Pathology Department of HUGTIP. For immunohistochemistry, 4-μm paraffin-embedded sections were cut, deparaffinized and rehydrated through xylene and graded alcohols to water. Antigen retrieval was done immersing the slides for 40 minutes in EDTA Buffer in a water bath at 98°C. The staining was performed using as primary mAbs α-Siglec-1 7D2, α-CD4 (Clone SP35, Ventana Medical Systems) and α-CD20cy (Clone L26, DAKO) and the Ventana Discovery XT automated stainer (Ventana Medical Systems,) with ultraView Universal DAB Detection Kit. Of note, patient had been treated with two nucleoside reverse transcriptase inhibitors for 7 years and had stopped treatment for 4 years, when the first biopsy was performed. At that time point, HIV-1 plasma viral load was < 50 HIV-1 RNA copies/ml and CD4+ T cell-count was 485. Patient started antiretroviral treatment again with a protease inhibitor-based regimen and had a second biopsy one year later, when HIV-1 plasma viral load was <25 HIV-1 RNA copies/ml and CD4+ T cell-count was 511. At the second biopsy, HCV viral load was 641.144 UI/ml.
Transcriptome RNA-seq analysis
RNA extraction from IFNα-treated DCs, macrophages and monocytes cells (1–6 x106) was performed using RNeasy Mini kit (Qiagen). RNA extraction from sorted IFNα-treated Siglec-1 tonsillar cells was performed using RNeasy Micro kit (Qiagen). mRNA-Seq library preparation was done with TruSeq RNA sample prep kit, Illumina (starting with capture of polyA-containing transcripts), followed by cluster generation (TruSeq single-end cluster generation kit, Illumina) and high-throughput sequencing on Illumina HiSeq2000 at the Genomics Technology Facility, University of Lausanne. The 100 bp single-end reads obtained were cleaned before alignment as described in . Cleaned reads were aligned to the human reference genome with STAR aligner  using the ensembl gene GRCh37 release 70 annotation file. The number of reads per gene was quantified with HTSeq-count v.0.6.1  with parameters mode = union and type = exon. We obtained an average library size of 45072173 uniquely mapped reads. All downstream analyses were performed taking as gene expression values the log10 of the number of library size-normalized reads per kilobase of exonic sequence. A pseudo-count of 1 was added previous to the log10 transformation to avoid NA’s (impossible log transformation) and obtain a numerical value.
We analyzed mean changes using a paired t-test, which was considered significant at P < 0.05. Mean changes of unpaired observations were assessed using the Man Whitney t-test, which was considered significant at P < 0.05. Significant mean changes from 100% of the data normalized to percentages were assessed with a one sample t-test, considered significant at P < 0.04. Pearson correlation tests were used to determine the level of association between Siglec-1 Ab binding sites per monocyte and VLP capture, HIV-1 capture, HIV-1 trans-infection, plasma viral load or CD4+ T-cell counts from HIV-1-infected individuals. All analyses and figures were generated with the GraphPad Prism v5.0b Software.
For their excellent assistance and advice, we thank Elena Rebollo from the Advanced Fluorescence Microscopy Unit IBMB-PCB, Marco Fernández and Gerard Requena from the Flow Cytometer Unit of IGTP, Angela Fernández Vasalo from the Pathology Department of HUGTIP, the Sample Processing Team of IrsiCaixa, Elisabeth Gómez and Julià Blanco from IrsiCaixa and surgeons from the Otorhinolaryngology Department of HUGTIP. We are grateful to Barbara Mueller for providing key reagents and to Felix Lasitschka for expert histopathological assessment.
Work in JMP group is supported by the Spanish Ministry of Science and Innovation through grant SAF2013-49042-R, SAF2010-21224 and the Spanish AIDS network “Red Temática Cooperativa de Investigación en SIDA” (RD06/0006). NIU is supported by the Mathilde Krim Fellowship in basic biomedical research 108676 founded by “AmfAR” AIDS research Foundation. AC and AT are supported by the Swiss National Science Foundation. HGK is supported by the Deutsche Forschungsgemeinschaft through SFB/TR 83. The funders had no role in the design and preparation of this paper, decision to publish, or preparation of the manuscript.
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