HIV-1 immune activation induces Siglec-1 expression and enhances viral trans-infection in blood and tissue myeloid cells

Abstract

Background: 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).

Results: 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.

Conclusions: Siglec-1 on myeloid cells could fuel novel CD4(+) T-cell infections and contribute to HIV-1 dissemination in vivo.

Figure 1

Siglec-1 mediates HIV-1 capture by IFNα-treated myeloid cells. A. Representative profiles of Siglec-1 staining in distinct myeloid cells cultured with or without 1000 U/ml of IFNα and assessed by FACS. Staining of matched-isotype control is also shown. The mean fold increase in fluorescence after IFNα treatment of cells derived from three donors is shown in red numbers. B. Mean number of Siglec-1 antibody binding sites per cell displayed by different myeloid cells exposed to 1000 U/ml of IFNα for 48 h and assessed by quantitative FACS analysis. Data show mean values and SEM from four experiments including cells from 12 donors. C. Comparative binding of HIV-1_NL4–3 to different myeloid cells previously exposed to 1000 U/ml of IFNα for 48 h. Cells were cultured with HIV-1_NL4–3 for 4 h at 4°C, washed and lysed to measure p24^Gag by ELISA. Data show mean values and SEM from two experiments including cells from six donors. D. Relative binding of HIV-1_NL4–3 by different IFNα-treated myeloid cells that had been pre-incubated with 10 μg/ml of the indicated mAbs before HIV-1 exposure for 4 h at 4°C as described in C. To compare the effect of the mAbs in different myeloid cells, values were normalized to the level of HIV-1 binding by mock-treated cells (set to 100%). Data show mean values and SEM from two experiments including cells from six donors. Statistical differences were assessed with a paired t test in B and C, and with a one sample t-test in D.

Figure 2

Siglec-1 mediates HIV-1 uptake into a storage compartment and enhances HIV-1 trans-infection specially in IFNα-treated monocytes and DCs. A. Uptake of HIV-1_NL4–3 by different myeloid cells exposed to IFNα. Cells were cultured with HIV-1 to measure p24^Gag by ELISA. Mean values and SEM from four experiments include cells from 12 donors. B. Fold change in HIV-1_NL4–3 uptake of cells treated with bafilomycin A1 compared to untreated cells. Mean values and SEM include cells from three donors. C. Relative uptake of HIV-1_NL4–3 by IFNα-treated myeloid cells pre-incubated with the indicated mAbs. Values are normalized to the level of HIV-1 uptake by mock-treated cells (set at 100%). Mean values and SEM from two experiments include cells from six donors. D. Confocal microscopy analysis of different IFNα-treated myeloid cells pulsed with HIV-1_Cherry and stained for Siglec-1 (Alexa 488), HLA-DR (Alexa 647) and DAPI. (Top) Representative viral pattern for each kind of myeloid cell analyzed, showing maximum fluorescence intensity of four channels. (Bottom) Percentage of myeloid cells with distinct viral patterns: random distribution, polarized accumulation, and sac-like compartment formation, as illustrated in the left drawing. Mean values of 50 cells from two different donors are shown. E. HIV-1 transmission from IFNα-treated myeloid cells to a luciferase reporter CD4⁺ cell line. HIV-1 infection was determined by induced luciferase activity in relative light units (RLUs). Mean values and SEM from four experiments include cells from 12 donors. F. Relative HIV-1 transmission from IFNα-treated myeloid cells pre-incubated with the indicated mAbs. Values are normalized to the level of HIV-1 trans-infected by mock-treated cells. Mean values and SEM from two experiments include cells from six donors. Statistical differences were assessed with a paired t test in A and E, and with a one sample t-test in B, C and F.

Figure 3

Siglec-1 is up-regulated on monocytes of HIV-1-infected individuals, but its expression is reduced after successful antiretroviral treatment. A. Mean number of Siglec-1 Ab binding sites per cell displayed by monocytes isolated from HIV-1-negative men and from HIV-1-infected men before or after successful antiretroviral treatment (with an initial median of 297 CD4⁺ T-cell count and 5 log₁₀ HIV RNA copies/ml that changed to a median of 505 CD4⁺ T-cell count and 1.7 log₁₀ HIV RNA copies/ml after treatment). Graph shows mean values and SEM from nine HIV-1-negative individuals and 16 HIV-1-infected individuals. Man Whitney t-test was used to compare differences between HIV-1-negative individuals and HIV-1-infected individuals. Paired t-test was used to assess differences between HIV-1-infected men before or after successful antiretroviral treatment. B. Uptake of fluorescent VLPs by monocytes isolated from HIV-1-infected individuals before (red dots) and after (green dots) antiretroviral treatment. Cells were pulsed with VLPs for 3 h at 37°C and assessed by FACS. Graph shows mean values and SEM from 16 HIV-1-infected individuals. C. Uptake of HIV-1_NL4–3 by monocytes isolated from HIV-1-infected individuals before and after antiretroviral treatment. Cells were cultured with HIV-1 for 4 h at 37°C, washed and lysed to measure p24^Gag by an ELISA. Graph shows mean values and SEM of the 13 individuals from which we recovered enough monocytes to perform this assay. D. HIV-1 transmission from monocytes isolated from HIV-1-infected individuals before and after antiretroviral treatment to a reporter CD4⁺ cell line cultured at a ratio 5:1. Cells were pulsed with HIV-1_NL4–3 as in Figure 2D. Graph shows mean values and SEM from the same individuals of panel C.

Figure 4

The plasma of untreated HIV-1-infected individuals stimulates Siglec-1 expression and signals via type I IFN receptor. A. Mean number of Siglec-1 Ab binding sites per cell induced by the plasma of HIV-1-negative individuals and HIV-1-infected individuals before or after successful antiretroviral treatment, respectively. DCs derived from uninfected donors were cultured for 24 h in the presence of plasma and then stained for Siglec-1. Graph shows mean values and SEM of Siglec-1 induction in DCs from two donors that were tested in parallel with the plasmas from five HIV-1-negative individuals and ten HIV-1-infected individuals. B. Relative blockade of Siglec-1 expression by B18R, a soluble recombinant receptor with high affinity for type I IFNs, which inhibits Siglec-1 induction triggered by the plasmas of untreated HIV-1-infected individuals. DCs were cultured for 24 h with the respective plasma in the presence or absence of 2 μg/ml of B18R. Values are normalized to the level of Siglec-1 induction by plasma of mock-treated cells (set at 100%). Mean changes from 100% were assessed with a one sample t-test. Representative histogram also depicts IFNα-treated DCs. C. Mean number of Siglec-1 Ab binding sites per cell induced by the plasma of untreated HIV-1-infected individuals. DCs were cultured for 24 h in the presence of plasma collected from patients displaying the highest levels of Siglec-1 (>5500 Ab binding sites per monocyte), intermediate levels of Siglec-1 (4000–2500 Ab binding sites per monocyte) or the lowest levels of Siglec-1 (<1500 Ab binding sites per monocyte) and then stained for Siglec-1. Graph shows mean values and SEM of Siglec-1 induction in DCs from two donors that were tested in parallel with the plasmas from ten HIV-1-infected individuals. Man Whitney t-test was used to compare the differences between distinct plasmas to induce Siglec-1 expression.

Figure 5

Expression of Siglec-1 on monocytes correlates with clinical parameters. A. Positive correlation between the mean number of Siglec-1 Ab binding sites per cell and VLP uptake of monocytes isolated from antiretroviral treatment-naïve HIV-1-infected men, with a median of 338 CD4⁺ T-cell count and 4.3 log₁₀ HIV RNA copies/ml. Graph shows the linear correlation of 26 individuals. Pearson correlation coefficient of the population is denoted by ρ. B. Positive correlation between the mean number of Siglec-1 Ab binding sites per cell and HIV-1 uptake of monocytes isolated from antiretroviral treatment-naïve HIV-1-infected individuals. Graph shows the linear correlation for the 13 individuals from which we recovered enough monocytes to perform this assay. C. Positive correlation between the mean number of Siglec-1 Ab binding sites per cell and the HIV-1 trans-infection capacity of monocytes isolated from antiretroviral treatment-naïve HIV-1-infected individuals. Graph shows the linear correlation for the same individuals as in panel B. D. Positive correlation between the mean number of Siglec-1 Ab binding sites per cell and the plasma viral load at the time of sample analysis of monocytes isolated from antiretroviral treatment-naïve HIV-1-infected individuals. Graph shows a linear correlation for 26 individuals. E. Negative correlation between the mean number of Siglec-1 Ab binding sites per cell and the CD4⁺ T cell count at the time of sample analysis of monocytes isolated from antiretroviral treatment-naïve HIV-1-infected individuals. Graph shows a linear correlation for 26 individuals.

Figure 6

Siglec-1 positive cells accumulate in inflamed lymphoid tissues in areas enriched in CD4⁺ T cells. A. Siglec-1 expression on sections of tonsils surgically removed either due to acute tonsillitis (n = 3; inflamed) or obstructive hypertrophy (n = 3; non-inflamed). Graph show mean values and SEM. Representative staining images are depicted in the right panels (magnification 40x). B. Staining images of Siglec-1 on lymph nodes surgically removed from an HIV-1-infected individual under antiretroviral treatment. Four continuous sections were cut and labeled independently with the following panel: anti-Siglec-1, anti-CD4, anti-CD20 or hematoxilin/eosin. Top images show perivascular distribution of Siglec-1 positive cells and bottom images display a perifollicular and subcapsular distribution.

Figure 7

Siglec-1 mediates HIV-1 trans-infection in myeloid cells isolated from lymphoid tissue. A. Workflow to enrich or isolate tonsillar myeloid cells. Red numbers on the right are used throughout the figure to identify isolation procedure. B. Hierarchical clustering (by Spearman correlation) of IFNα-treated sorted Siglec-1 positive tonsillar cells and primary cells based on protein-coding gene expression. C. Venn diagram of overlapping protein-coding genes in the indicated IFNα-treated cells. D. Expression heat maps of genes with antigen-presenting cell functions shared between IFNα-treated Siglec-1 sorted tonsillar cells and at least one myeloid cell type. Data in B-D show three donors for each cell type. E. Heat map-like representation of the theoretical presence/absence profile reported for markers proposed for myeloid cell classification [31] in the indicated cellular subsets (dark blue: presence; light blue: absence; blue: heterogeneous expression/unknown/unclear). Observed expression in sorted Siglec-1 tonsillar cells is also depicted (dark blue: >1.5; light blue: <1; blue: ≥1 and ≤1.5 log₁₀). Boxes indicate markers of inflammatory myeloid cells [32]. F. Representative FACS staining of selected myeloid markers in sorted Siglec-1 positive cells (top) and BDCA1-positive cells (bottom). G. Uptake of VLPs by BDCA1-positive tonsillar cells exposed to IFNα or left untreated. Cells were pre-incubated with indicated mAbs. Mean values and SEM from three experiments include cells from eight donors, assessed with a paired t-test. H. Confocal microscopy of IFNα-treated BDCA1-positive tonsillar cells (n = 2) pulsed with HIV-1_Cherry and stained for Siglec-1. Representative viral pattern (maximum fluorescence intensity of three channels and bright field). I. HIV-1 transmission from IFNα-treated sorted BDCA1-positive tonsillar cells to a reporter CD4⁺ cell line. Cells were pre-incubated with indicated mAbs. Values are normalized to the level of HIV-1 trans-infected by isotype-treated cells. Mean values and SEM from two experiments include mixed cells from six donors, assessed with a one sample t-test.