Podosomes of dendritic cells facilitate antigen sampling

Abstract

Dendritic cells sample the environment for antigens and play an important role in establishing the link between innate and acquired immunity. Dendritic cells contain mechanosensitive adhesive structures called podosomes that consist of an actin-rich core surrounded by integrins, adaptor proteins and actin network filaments. They facilitate cell migration via localized degradation of extracellular matrix. Here, we show that podosomes of human dendritic cells locate to spots of low physical resistance in the substrate (soft spots) where they can evolve into protrusive structures. Pathogen recognition receptors locate to these protrusive structures where they can trigger localized antigen uptake, processing and presentation to activate T-cells. Our data demonstrate a novel role in antigen sampling for the podosomes of dendritic cells.

Keywords: Antigen presentation; Dendritic cell; Podosome; Receptor-mediated endocytosis.

Figure 1. Podosomes locate to spots of low physical resistance of the substrate

(A) Human monocyte derived dendritic cells cultured on glass substrate and stained with primary antibodies directed against vinculin, talin and paxillin and secondary antibodies conjugated to Alexa fluor 488 (AB; green). Actin was stained with phalloidin-Alexa fluor 564 (Phal; magenta). Shown are confocal images immediately at the glass surface of representative cells with podosomes. Yellow arrow heads indicate randomly chosen podosomes. Bar graphs show quantification of AB positive actin cores (± SD). (B) Same as panel A, but now for cells on polycarbonate filters with pore sizes of 400 nm. The filters were soaked in gelatin conjugated to Alexa fluor 633 (Filter; grey). Yellow lines indicate the positions of the orthogonal views. The red arrow head indicates the approximate filter surface. (C) Quantification of the alignment of the podosomes to the filter pores from panel B (± SD). (D–E) Schemes of dendritic cells with non-protrusive podosome on glass (D) and filters with pore sizes of 400 nm (E) (actin: magenta; vinculin: green). Scale bars, 5 μm.

Figure 2. Podosome-like protrusive structures of dendritic cells

(A) Dendritic cells cultured on polycarbonate membrane filters impregnated with Alexa fluor 633-labeled gelatin (grey) with pore sizes of 1 μm. Cells were immunostained for vinculin (green) and actin was visualized by phalloidin (Phal; magenta) similar to figure 1. Shown are confocal sections just above the surface of the filters. The yellow line indicates the position of the orthogonal views showing the protrusion of actin in the lumen of the pores. Yellow arrow heads depict randomly selected actin-rich cores. The red arrow head indicates the approximate filter surface. The right hand panel shows a schematic diagram of the protrusions (actin: magenta; vinculin: green). (B) Same as panel A, but now for filters with pore sizes of 2 μm and showing the protrusion of both the actin core and vinculin in the pores as indicated by the white arrows. The cells also formed podosomes that did not overlap with the pores as indicated by the yellow arrow heads. (C) Same as panel B, but now with pore sizes of 3 μm. (D) Quantification of the fraction of actin cores aligning with the filter pores from panels A–C (± SD). (E) Same as panel C, showing cell migration through the 3 μm pores in the filter (white arrows) and the formation of podosomes on both sides of the filter (yellow arrow heads). Confocal sections above, through and underneath the filter are shown. Scale bars, 5 μm.

Figure 3. Characterization of protrusive podosome-like structures

(A) Confocal images of human dendritic cells cultured on membrane filters with pore sizes of 1 μm. Actin was stained with phalloidin-Alexa fluor 546 (Phal; magenta) and talin, paxillin, ITGAM and ITGB1 were labeled by specific monoclonal antibodies and secondary antibodies conjugated to Alexa fluor 488 (AB; green). Yellow lines indicate the positions of the orthogonal views. Yellow arrow heads indicate randomly chosen actin cores. Red arrow head indicates the approximate surfaces of the filter substrates. Bar-graphs show quantifications of the fractions of AB positive actin cores (± SD). (B) Confocal image of a dendritic cell on gelatin-Alexa fluor 633 impregnated filters with pore sizes of 1 μm (Filter; magenta) and stained with phalloidin-Alexa fluor 488 (green). The insets show magnifications of actin cores marked i–iv. The graphs show fluorescence intensity distributions through the cross-sections marked by the red boxes (y-averaging). The orange arrow heads mark intensity peaks at the edges of ring structures of some of the actin cores. (C) Same as panel A, but instead of antibody staining, PIP₂ was labeled with bacterially expressed pleckstrin homology domain of phospholipase C delta subunit fused to citrine (PLC -PH). (D) Cells precultured for 1 h on glass or gelatin-Alexa fluor 633 impregnated filters with pore sizes of 1 μm (magenta) were treated for 1 hr with 5 μM of the WASP inhibitor wiskostatin or with carrier only (DMSO). Yellow arrow heads mark clusters of actin cores stained by phalloidin-Alexa fluor 488 (green). (E) The fractions of cells containing actin cores from panel D on glass (black) and filter (red) as a function of exposure time to wiskostatin (± SD, 3 independent repeats). Scale bars, 5 μm.

Figure 4. Podosome-like protrusive structures contain MMP14 and tubulin

(A) Confocal images of dendritic cells cultured on filters with 1 μm pore sizes that were impregnated with double-quenched FITC-labeled collagen in gelatin (DQ collagen; green). Actin was stained with phalloidin-Alexa fluor 633 (Phal; magenta). Collagen degradation results in loss of self-quenching of the FITC fluorophore and an increase in fluorescence. (B) Fluorescence intensity profiles from panel A marked by the dashed lines. (C) Confocal images (left) and quantification (right) of dendritic cells cultured on glass and membrane filters with pore sizes of 1 μm. Actin was stained with phalloidin-Alexa fluor 546 (magenta) and the metalloprotease MMP-14 was labeled by specific monoclonal antibodies and secondary antibodies conjugated to Alexa fluor 488 (AB; green). The yellow line indicates the positions of the orthogonal view. The yellow arrow heads indicate randomly chosen actin cores. The red arrow head indicates the approximate surface of the filter. (D) Same as panel C, but now for tubulin. Error bars show the spread of data for multiple cells from at least two independent experiments. Scale bars, 5 μm.

Figure 5. Protrusive podosome-like structures contain pattern recognition receptors

(A–B) Confocal images of dendritic cells cultured on glass (A) and on filters with 1 μm pore size (B). Actin was labeled with phalloidin-Alexa fluor 546 (Phal; magenta) and clathrin was visualized by immunostaining (AB; green). The filters were impregnated with Alexa fluor 633-labeled gelatin (Filter; grey). The yellow line indicates the position of the orthogonal view. Yellow arrow heads indicate randomly selected actin-rich cores. The red arrow head indicates the approximate filter surface. (C) Quantification of the fraction of clathrin positive actin cores from panels A–B. (D–F) Same as panels A–C, but now with immunostaining for DC-SIGN, DCIR, Dectin-1, CD206 and CD71. Error bars show the spread of data for multiple cells from at least two independent experiments. Scale bars, 2 μm.

Figure 6. Transmission electron microscopy of protrusive podosome-like structures

(A) Electron micrographs of a human dendritic cell cultured on gelatin impregnated filters with 1 μm pore size and immuno-gold labeled for DC-SIGN (inset, overview). (B–C) Details of the ventral membrane (B, marked i in panel A) and of a protrusive structure (C, ii) from panel A. Yellow arrow heads mark positions of clusters of gold beads. (D) Quantification of the gold beads per μm imaged membrane for 10 different cells at the ventral membrane (black) and the protrusions (red). Data points for individual cells and the average are shown (± SEM). Bead density was 3-fold increased at the protrusions relative to ventral membrane (P = 0.0005, 2-tailed paired t-test). (E) Same as panel A, but now with immuno-gold labeling for CD206. (F) Magnifications of panel E as indicated (i, ii). (G) Same as panel D, but now for CD206. Bead density was 9-fold increased at the protrusions relative to ventral membrane (P = 0.002, 2-tailed paired t-test). Scale bars, 1 μm.

Figure 7. Antigen uptake by protrusive podosome-like structures

(A) Schematics of the control experiments for passive leakage of quantum dots or OVA-Alexa fluor 647 (OVA-647) through the filters. (B–C) Leakage assay of quantum dots (B) or OVA-647 (C) through filters with different pore sizes and with or without gelatin impregnation. (D) Scheme of the antigen uptake experiments. (E) Confocal images of dendritic cells cultured on gelatin-coated filters with 1 μm pore sizes. A suspension of quantum dots linked to gp120 (Qdot; left; magenta) or a solution of OVA-647 (right; magenta) was applied to the other side of the filter. The cells were stained with phalloidin-Alexa fluor 488 (Phal; green) and imaged after 1 hr incubation (see Fig. 8A–B for quantification). (F) Live cell imaging of dendritic cells tranfected with LifeAct-GFP and cultured on filter. At time t = 0, OVA-647 was applied to the other side of the filter. The inset shows the increase of OVA-647 fluorescence in time at the position of 3 actin cores marked with orange arrow heads (i – iii). Full dataset is in Supplementary Movie 3. (G) Time course of OVA-647 uptake for dendritic cells on filter treated with 20 μM Pitstop 2 (red) or Pitstop 2-negative control (black) (± SEM of three independent repeats). Scale bars: E, 10 μm; F: 2 μm.

Figure 8. Antigen uptake and processing by protrusive podosome-like structures

(A) Uptake assay as described in figure 7 for human dendritic cells and CHO cells stably expressing DC-SIGN cultured on gelatin-coated filters with 1 μm or 400 nm pore sizes and in the presence or absence of 25 μg ml⁻¹ mannan. After 1 h uptake, the fraction of cells that took up gp120 or biotin labeled quantum dots (gp120-Qdot; Biotin-Qdot) was determined by analysis of confocal images by 2 or 3 independent experts (> 3 independent repeats; ± SEM; *, P < 0.02; **, P << 0.01). (B) Same as panel A, but now for OVA-647. (C) Time course of OVA-647 uptake for dendritic cells on filter treated with 5 μM wiskostatin (black) or carrier only (DMSO; red) (± SEM of three independent repeats). (D) Confocal images of dendritic cells cultured on gelatin-coated filters with 1 μm pore sizes and with uptake of double quenched OVA (OVA-DQ; green) and OVA-Alexa fluor 647 (OVA-647; magenta) through the filter. Actin was stained with phalloidin-Alexa fluor 564 (Phal; grey). OVA-DQ was dequenched as apparent from the increased fluorescence compared to cells treated with bafilomycin A1 (control). (E) Distribution of the OVA-DQ fluorescence of OVA-647 positive compartments for the bafilomycin treated and control cells from panel A (at least 5 cells each). (F) Uptake of OVA-647 (magenta) by dendritic cells on filter and immunostained for MHC class II (green). Actin was stained with phalloidin-Alexa fluor 546 (Phal; grey). Yellow arrow heads indicate MHC class II compartments. Bar-graphs show quantifications of MHC class II positive OVA-647 compartments (± SD). Scale bars, 10 μm.