Podoplanin-rich stromal networks induce dendritic cell motility via activation of the C-type lectin receptor CLEC-2

Abstract

To initiate adaptive immunity, dendritic cells (DCs) move from parenchymal tissues to lymphoid organs by migrating along stromal scaffolds that display the glycoprotein podoplanin (PDPN). PDPN is expressed by lymphatic endothelial and fibroblastic reticular cells and promotes blood-lymph separation during development by activating the C-type lectin receptor, CLEC-2, on platelets. Here, we describe a role for CLEC-2 in the morphodynamic behavior and motility of DCs. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes, thereby reducing T cell priming. CLEC-2 engagement of PDPN was necessary for DCs to spread and migrate along stromal surfaces and sufficient to induce membrane protrusions. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. Thus, activation of CLEC-2 by PDPN rearranges the actin cytoskeleton in DCs to promote efficient motility along stromal surfaces.

Figure 1

Pdpn mRNA and Protein Expression by LECs and FRCs (A) Fluorescence microscopy of tissue whole mounts and schematics of ear skin at resting state. The scale bar represents 100 μm. (B) Representative dot plots showing proportion of stromal cells (CD45⁻) (left panel) and subsets of lymphoid stromal cells (FRCs, CD31⁻PDPN⁺; LECs, CD31⁺PDPN⁺; blood endothelial cells (BECs), CD31⁺PDPN⁻) (right panel) in draining LNs at rest. SSC, side scatter. (C) Graph representing the expression value (EV) of Pdpn mRNA from microarray analysis of sorted BECs, LECs, and FRCs from resting or inflamed LNs. (D) Graph showing mean fluorescence intensity (MFI) of surface PDPN on BECs, LECs, and FRCs isolated from draining LNs 24 hr after intradermal injection of PBS (resting) or LPS (inflamed) into ear skin. Data represent mean values and SD from four independent experiments. (E) Fluorescence microscopy of ear skin whole mounts at 0 (resting) and 24 hr (inflamed) after intradermal LPS injection. Fluorescence images show overlays of PDPN (red) and Lyve-1 (green) antibody staining in the tips and along the lengths of afferent lymphatic vessels. Representative black and white images from three independent experiments show individual PDPN and Lyve-1 stains. The scale bar represents 20 μm.

Figure 2

Clec1b mRNA and Protein Expression by DCs (A) Quantitative PCR analysis of Clec1b mRNA levels in FRCs (negative control), BMDCs, LN DCs, and skin DCs. LN and skin DCs were sorted from primary tissues via flow cytometry. Values above bars depict the mRNA level relative to the negative control. Error bars represent mean and SD for three independent experiments. (B) Flow-cytometry analysis of surface CLEC-2 protein using rPDPN-Fc on WT (solid line) and Clec1b^−/− (dashed line) BMDCs. The gray line (filled histogram) represents the secondary control. (C) FACS analysis of surface CLEC-2 protein using rPDPN-Fc on freshly isolated WT LN DCs (solid line). Gray line (filled histogram), secondary control. (D) Flow cytometry analysis of surface CLEC-2 protein using rPDPN-Fc on BMDCs treated for 12 hr with LPS (solid line) or left untreated (dashed line). The gray line (filled histogram) represents the secondary control. (E) Representative fluorescence microscopic images from three independent experiments of A375 cells transfected with CLEC-2-GFP that were either stimulated with rhodocytin or left untreated. Top panels, overlay of F-actin (red), DAPI (blue), and CLEC-2-GFP (green). Bottom panels, CLEC-2-GFP fluorescence alone. Scale bars represent 50 μm.

Figure 3

DCs Utilize CLEC-2 for Efficient Migration from Skin to Draining LNs (A) RT-PCR analysis of Clec1b mRNA in CD11c⁺ and CD11c⁻ cells that were magnetic-activated cell sorting (MACS)-purified from WT or Clec1b^−/− FLCs. (B) Percentages of migratory (MHCII^hiFITC⁺) DCs among total donor (CD45.2⁺) DCs in draining LNs of WT and Clec1b^−/− FLC mice at 24 and 72 hr post-FITC painting. Error bars represent mean and SD. (C) Representative dot plots (gated on CD45.2⁺CD11c⁺ cells) showing MHCII^hiFITC⁺ DCs in WT and Clec1b^−/− FLCs 24 hr after FITC painting. (D) Total numbers of migratory donor (CD45.2⁺CD11c⁺MHCII^hiFITC⁺) DCs in draining LNs of WT and Clec1b^−/− FLCs at 24 and 72 hr post-FITC painting. Error bars represent mean and SD. (E) Total cellularity in draining LNs collected from WT and Clec1b^−/− FLCs 24 and 72 hr after FITC painting. Error bars represent mean and SD. (B, D, and E) Data represent ten mice per experimental condition from three independent experiments. (F) FACS analysis of popliteal (draining) and axillary (distal) LN 24 hr after injection of WT (CFSE⁺) and Clec1b^−/− (Far red⁺) DCs mixed in equal numbers (2 × 10⁵ of each) prior to injection into the footpad. (G) Quantification of DCs arriving in popliteal LNs 24 hr after footpad injection. Data represent 15 mice per experimental condition from three independent experiments. Error bars represent mean and SD. (H) Histograms showing OT-1 T cell proliferation (CFSE dilution) in popliteal LN following footpad injection of OVA-peptide-loaded WT and Clec1b^−/− DCs. Numbers show the percentage of divided cells among donor OT-1 T cells. (I) Summary of data, as in (H). Data are shown as a division index. Error bars represent mean and SD. (J) Histograms showing proliferation of OT-1 T cells upon coculture of naive, CFSE-labeled OT-1 T cells with OVA-peptide-loaded WT and Clec1b^−/− DCs for 48 hr. Numbers indicate the percentage of divided cells among donor OT-1 T cells. Data are representative of three independent experiments. (K) Percentage of WT or Clec1b^−/− DCs that captured particulate antigen (fluorescently labeled latex beads). Error bars represent mean and SD.

Figure 4

Clec1b^−/− DCs Exhibit Impaired Migration In Vivo (A) Z projection of ear dermis incubated with WT BMDCs (Ai). Lymphatic vessels are shown in green and infiltrating DCs are shown in red. The scale bar indicates 100 μm. Zoomed image showing DCs interacting with lymphatic vessel (Aii). (B) Z projection of ear dermis incubated with Clec1b^−/− BMDCS (Bi). The scale bar indicates 100 μm. Zoomed image showing DCs interacting with lymphatic vessel (Bii). (C) Quantification of localization of WT and Clec1b^−/− DCs within ear sheets. Data are collated from three independent experiments. Error bars represent mean and SD. (D) Z projection of LN from a Ub-GFP BM chimeric mouse (WT BM > Ub-GFP host) injected with WT and Clec1b^−/− DCs 24 hr prior to dissection and fixation (Di). The GFP⁺ stroma is shown in white, WT DCs are shown in red, and Clec1b^−/− DCs are shown in green. The scale bar indicates 100 μm. Location of WT DCs relative to LN capsule (dotted line) (Dii). Location of Clec1b^−/− DCs relative to LN capsule (dotted line) (Diii). (E) Quantification of distance from the LN capsule. Error bars represent mean and SD. (F) Z projection of vibratome-cut GFP-ubiquitin LN slice showing the position of WT (red) and Clec1b^−/− (green) infiltrated DCs (Fi). GFP⁺ lymph node stromal cells are shown in white. The scale bar indicates 100 μm. Overlay showing tracks of WT (red) and Clec1b^−/− (green) DCs on GFP⁺ LN (white) over 2 hr period of time-lapse imaging (Fii). Tracks of WT (red) and Clec1b^−/− (green) DCs within LN slice (Fiii). (G) Representative plots showing directionality of migrating WT and Clec1b^−/− DCs within LN slices of Ub-GFP BM chimeric mouse with GFP⁺ stroma. Tracks were adjusted to begin at origin (0.0) and overlaid. The axes span 150 μm. (H and I) Displacement (H) and velocity (I) of WT and Clec1b^−/− DCs within LN slices of Ub-GFP BM chimeric mouse. Data are collated from eight LN slices from four different donors, from three independent experiments. Error bars represent mean and SD.

Figure 5

CLEC-2 Activation Induces Protrusion Formation in DCs (A and B) Time-lapse imaging of control-, PDPN-Fc-, and rhodocytin-treated WT (A) and Clec1b^−/− (B) BMDCs at 0, 5, and 10 min after stimulation. Scale bars represent 20 μm. Far-right panels, overlays of still traces from each time point. (C–F) Morphology index (C), protrusion length (μm) (D), number of protrusions per cell (E), and protrusion persistence (min) (F) for control, rPDPN-Fc, and rhodocytin-treated DCs from WT and Clec1b^−/− FLCs, as in (A) and (B). Error bars represent mean and SD.

Figure 6

CLEC-2-PDPN Interaction Is Required for DC Migration Along, but Not Attachment to, the FRC Network (A) Z projection of 3D FRC network with DCs. The scale bar represents 100 μm. (B) Representative cross-section of FRC in 3D culture showing PDPN localization at the plasma membrane. The scale bar represents 5 μm. (C) Time-lapse imaging (top) and schematic (bottom) of DC-FRC interaction at 1, 3, 6, and 9 min after contact. (D) Transmitted-light image of WT DCs interacting with WT FRCs in 3D network (Di). The scale bar represents 50 μm. Diagram showing tracks of migrating DCs over 1 hr time course (Dii). Quantification of DC migration along primary FRCs or NIH 3T3 fibroblasts in 3D network (Diii). Each point represents the path of one DC collated from > three independent experiments. (E) Transmitted-light image of Clec1b^−/− DCs interacting with WT FRCs in 3D network (Ei). The scale bar represents 50 μm. Diagram showing tracks of migrating DCs over 1 hr time course (Eii). Quantification of WT and Clec1b^−/− DC migration along WT FRCs in 3D network (Eiii). Each point represents the path of one DC. (F) Transmitted-light image of WT DCs interacting with Pdpn^−/− FRCs in 3D network (Fi). The scale bar represents 50 μm. Diagram showing tracks of migrating DCs over 1 hr time course (Fii). Quantification of WT DCs along FRCs with control siRNA, PDPN-targeted siRNAs, or Pdpn^−/− FRCs in the 3D network (Fiii). Each point represents the path of one DC. (G) Immunofluorescence showing A375 GFP-CLEC-2 transfectants in contact with FRCs in the 3D network. The scale bar represents 20 μm. Error bars represent mean and SD. (H) Quantification of cells with protrusions, as in (G).

Figure 7

CLEC-2 Signaling Coordinately Reduces Actomyosin Contractility and Promotes Actin Polymerization (A) F-actin and DAPI staining of control, rhodocytin, and rPDPN-Fc-treated DCs. The scale bar represents 20 μm. (B) Morphology index of WT and Vav1^−/−Vav2^−/−Vav3^−/− DCs. Data are collated from three individual experiments, and each data point represents the morphology of an individual DC. Error bars represent mean and SD. (C) Immunofluorescence of A375 cells transfected with GFP-CLEC-2 and treated with rhodocytin in 2D culture. DAPI, F-actin, and GFP fluorescence are shown. The scale bar represents 20 μm. (D) Quantification of area comprised by cells plated in 2D following rhodocytin stimulation relative to untransfected A375 cells. Error bars represent mean and SD. (E) Protein blots showing levels of GFP-CLEC-2 and total and activated Rac1 and RhoA in A375 clones expressing either low or high levels of GFP-CLEC-2 or A375 control cells following treatment with rPDPN-Fc or rhodocytin. (F) Quantification of Rac1-GTP, as in (G) (n = 3). (G) Quantification of RhoA-GTP, as in (G) (n = 3). Error bars represent mean and SD. (H) Immunofluorescence of FRCs and DCs in 3D network showing DAPI, CD11c, and pMLC (S19) staining. The scale bar represents 20 μm. (I) Quantification of pMLC fluorescence intensity in 3D network relative to untreated DCs or DCs not in contact with FRCs. Error bars represent mean and SD.