Innate control of actin nucleation determines two distinct migration behaviours in dendritic cells

Abstract

Dendritic cell (DC) migration in peripheral tissues serves two main functions: antigen sampling by immature DCs, and chemokine-guided migration towards lymphatic vessels (LVs) on maturation. These migratory events determine the efficiency of the adaptive immune response. Their regulation by the core cell locomotion machinery has not been determined. Here, we show that the migration of immature DCs depends on two main actin pools: a RhoA-mDia1-dependent actin pool located at their rear, which facilitates forward locomotion; and a Cdc42-Arp2/3-dependent actin pool present at their front, which limits migration but promotes antigen capture. Following TLR4-MyD88-induced maturation, Arp2/3-dependent actin enrichment at the cell front is markedly reduced. Consequently, mature DCs switch to a faster and more persistent mDia1-dependent locomotion mode that facilitates chemotactic migration to LVs and lymph nodes. Thus, the differential use of actin-nucleating machineries optimizes the migration of immature and mature DCs according to their specific function.

Figure 1

LPS activation induces fast and persistent DC migration. (a–c) Analysis of BMDCs migrating in 4 μm × 5 μm fibronectin-coated micro-channels. Cells were imaged between 6 and 16 h after LPS treatment (100 ng ml⁻¹ for 30 min). (a) Mean instantaneous cell speed. Each dot represents the mean of one experiment (n = 29 experiments, >40 cells in each experiment). (b) Kymograph representative of an iDC and an LPS-DC migrating in micro-channels. (c) Analysis of CCR7KO DC migration in micro-channels (n =150, 99, 145 and 151 cells for iDC, LPS-DC, iCCR7KO and LPS CCR7KO respectively). One representative experiment out of two is shown. (d–g) Analysis of DC migration under agarose. (d) Cell tracks of DCs migrating under agarose. Cells were imaged for 200 min. The starting point of each trajectory was translated to the origin of the plot. One representative experiment out of three is shown. (e,f) Mean instantaneous speed and path persistence of data depicted in d (n =63 and 76 cells for iDC and LPS-DC respectively). (g) Mean square displacement (MSD) obtained from the data depicted in d. The Mann–Whitney test was applied for statistical analysis. In the box plots of c,e,f the bars include 90% of the points, the centre corresponds to the median and the box contains 75% of the data.

Figure 2

LPS activation of DCs modifies the dynamics of their actin cytoskeleton. (a–d) LifeAct–GFP imaging of BMDCs migrating in 8 μm × 5 μm fibronectin-coated micro-channels. (a) Sequential images of LifeAct–GFP DCs acquired on an epifluorescence microscope every 1 min with a ×20 objective. (b) LifeAct–GFP density maps. Scale bars, 2.5 μm. The signal recorded at each time point was integrated into a single image for single migrating cells (see Supplementary Fig. 2). The mean intensity obtained for each cell was then averaged into a single density map (n = 31 and 27 cells for iDC and LPS-DC respectively). One representative experiment out of four is shown. (c) Correlation between the LifeAct–GFP front/back ratio and instantaneous speed values from DCs migrating in micro-channels. Values were obtained from data shown in b. The inset shows the mean fraction of time spent by DCs with LifeAct–GFP concentrated at their front (first third of the cell). The Mann–Whitney test was applied for statistical analysis. Graphic shows mean and error bars correspond to s.e.m. (d) LifeAct–GFP DCs migrating in micro-channels and time lapsed on a spinning-disc microscope (×100). Middle (M) and cortical (C) planes were imaged. The red arrows on the zoomed image show actin cables formed at the rear of LPS-DC.

Figure 3

Arp2/3-dependent actin at the front of iDCs limits migration but promotes antigen uptake. (a) Mean LifeAct–GFP distribution in iDCs migrating in micro-channels and treated with the Arp2/3 inhibitor CK666 (25 μM) or silenced for Arpc4 (n = 27, 42, 28 and 26 cells for iDC, iDC CK666, iDC Ctrl and iArpc4KD respectively). Scale bars, 2.5 μm. One representative experiment out of three is shown. Internal controls are systematically used. (b) Dynamic analysis of the fraction of time spent by cells with LifeAct–GFP at their front obtained from data in a. Graphic shows mean and error bars correspond to s.e.m. (c) Correlation between the LifeAct–GFP front/back ratio and instantaneous speed values from DCs migrating in micro-channels obtained from data shown in a. (d) Mean instantaneous speed of WT or tamoxifen-induced Arpc2KO DCs migrating in micro-channels (n =308, 255, 284 and 209 cells for iWT, LPS WT, iArpc2KO and LPS Arpc2KO respectively). One representative experiment out of three is shown. (e) Spinning-disc images (×100) of Arpc2 WT (TomatoFP⁺) or KO (GFP⁺) iDCs migrating in micro-channels. (f) Cortical LifeAct–GFP signal of control or CK666-treated iDCs migrating in micro-channels. (g) Quantification of fluorescent ovalbumin uptake in iDCs derived from WT and tamoxifen-induced Arpc2KO DCs migrating in micro-channels (n = 36 and 37 cells for Arpc2WT and Arpc2KO respectively). One representative experiment out of two is shown. (h) Immunofluorescence analysis of Arp2 in iDCs migrating in micro-channels analysed using a spinning-disc microscope (×100). The overlay shows LifeAct–GFP (green), Arp2 immunoreactivity (red) and DAPI staining (blue).The Mann–Whitney test was applied for all statistical analyses. In the box plots of d and g the bars include 90% of the points, the centre corresponds to the median and the box contains 75% of the data.

Figure 4

mDia1 is required for fast DC migration. (a,b) Analysis of DC migration in micro-channels. (a) Mean LifeAct–GFP distribution obtained from LPS-DCs migrating in micro-channels and treated with the formin inhibitor Smifh2 (25 μM) or silenced for mDia1. One representative experiment out of three is shown. Scale bars, 2.5 μm. (b) Dynamic analysis of the fraction of time spent by cells with LifeAct–GFP at their front obtained from data shown in a (n = 21, 22 and 31 for iCtrl, LPS Ctrl and LPS mDia1KD respectively). Graphic shows mean and error bars correspond to s.e.m. (c) Quantification of fluorescent ovalbumin uptake in DCs derived from WT and mDia1KO mice or treated with Smifh2 (25 μM) while migrating in micro-channels (n = 33, 36, 38, 45, 23 and 28 cells for WT, LPS DC, imDia1KO, LPS mDia1KO, LPS Ctrl and LPS Smifh2 respectively). One representative experiment out of two is shown. NS, not significant. (d) Mean instantaneous speed of control and mDia1KO DCs migrating in micro-channels (n = 272, 218, 192 and 310 cells for iWT, LPS WT, imDia1KO and LPS mDia1KO respectively). One representative experiment out of three is shown. (e–g) Analysis of DC migration under agarose. (e) Cell tracks of WT and mDia1KO LPS-DCs. Cells were imaged for 200 min. The starting point of each trajectory was translated to the origin of the plot. n = 129 and 69 cells for LPS mDia1WT and LPS mDia1KO respectively. One representative experiment out of three is shown. (f) Mean instantaneous speed obtained from data shown in d. (g) Mean square displacement (MSD) quantified from the data depicted in e. The Mann–Whitney test was applied for all statistical analyses. In the box plots of c,d and f the bars include 90% of the points, the centre corresponds to the median and the box contains 75% of the data.

Figure 5

Cdc42 and RhoA respectively control the migration of iDCs and LPS-DCs. (a) Mean instantaneous speed of Cdc42WT and KO DCs migrating in micro-channels (n = 111, 33, 129 and 110 cells for iWT, LPS WT, iCdc42KO and LPS Cdc42KO respectively). One representative experiment out of two is shown. NS, not significant. (b) Mean LifeAct–GFP distribution in DCs treated with the Cdc42 inhibitor ML141 (50 μM) (n =25, 32, 33 and 33 cells for iDC, iDC ML141, LPS-DC and LPS-DC ML141 respectively). One representative experiment out of two is shown. Scale bars, 2.5 μm. (c) Dynamic analysis of the fraction of time spent by cells with LifeAct–GFP at their front obtained from data in b. Graphic shows mean and error bars correspond to s.e.m. (d) Quantification of fluorescent ovalbumin uptake in iDCs treated with ML141 (50 μM) while migrating in micro-channels (n = 15 and 29 cells for iDC and iDC ML141 respectively). One representative experiment out of two is shown. (e) Mean instantaneous speed of WT and RhoAKO DCs migrating in micro-channels (n=86, 102, 45 and 109 cells for iWT, LPS-DC, iRhoAKO and LPS RhoAKO respectively). One representative experiment out of two is shown. NS, not significant. (f) Mean LifeAct–GFP distribution in DCs migrating in micro-channels and treated with the RhoA inhibitor C3 convertase (1 μg ml⁻¹) (n=42, 97, 34 and 59 cells for iDC, iDC C3, LPS-DC and LPS-DC C3 respectively). One representative experiment out of two is shown. Scale bars, 2.5 μm. (g) Dynamic analysis of the fraction of time spent by cells with LifeAct–GFP at their front obtained from data shown in f. Graphic shows mean and error bars correspond to s.e.m. (h) Quantification of fluorescent ovalbumin uptake in iDCs treated with C3 convertase (1 μg ml⁻¹) while migrating in micro-channels (n = 23 and 28 cells for LPS-DC and LPS-DC C3 respectively). One representative experiment out of two is shown. The Mann–Whitney test was applied for all statistical analyses. In the box plots of a,d,e and h the bars include 90% of the points, the centre corresponds to the median and the box contains 75% of the data.

Figure 6

mDia1 is required for the chemotactic response of mature DCs. (a–g) Chemotactic response of LPS-DCs embedded in a collagen gel containing a CCL21 gradient. Frequency (Freq.) was calculated in 500 random tracks because of oversampling. One representative experiment out of three is shown. (a) Directionality of trajectories during the chemotactic response or in the absence of chemokines (No chem.) of mDia1WT and mDia1KO LPS-DCs. (b) One hundred random tracks of LPS-DCs undergoing chemotaxis. (c) Mean speed of LPS-DCs represented as a function of the distance to the CCL21 source. (d) Frequency of movement of LPS-DCs in the direction of the gradient represented as a function of the distance to the CCL21 source. (e) Speed of LPS-DCs in the absence of CCL21. (f) Directionality of trajectories of LPS-DCs treated or not with CK666 (25 μM) during chemotactic response as in a. (g) Mean speed of CK666-treated LPS-DCs represented as a function of the distance to the CCL21 source. Trend lines with 95% confidence interval are shown in c,d,e and g.

Figure 7

mDia1 is required for migration of mature DCs to LVs and LNs in vivo. (a–f) Migration of LPS-DCs in mouse ear explants (n = 50 and 59 cells for LPS mDia1WT and LPS mDia1KO respectively). Pool of three independent experiments. (a) Cell tracks from mDia1WT (red) or mDia1KO (green) LPS-DCs migrating in the proximity of LVs (stained with LYVE-1; blue) in a mouse epidermal ear sheet. Scale bar, 50 μm. (b) Fifteen randomly selected tracks of mDia1WT and mDia1KO LPS-DCs migrating in an epidermal ear sheet as shown in a. (c) Mean square displacement (MSD) of mDia1WT and mDia1KO LPS-DCs migrating in an epidermal ear sheet. The MSD curve of mDia1KO cells is fitted with a simple linear expression, reflecting the isotropic random walk-like behaviour of this population. Instead, the MSD curve of mDia1WT cells showed a first nonlinear increase followed by a linear dependency, as expected for biased persistent random walk and is fitted with Fürth’s formula. (d) Mean instantaneous speed of LPS-DCs represented as a function of the distance to the closest LV. Error bars correspond to s.d. (e) Frequency of cell movements in the direction of the closest LV. Statistical analysis was performed comparing values with respect to random migration from data shown in d. Student’s t-test and Pearson’s χ² test were applied in d and e, respectively. NS, not significant. (f,g) Fraction of DCs that reach LVs plotted as a function of time. Error bars correspond to s.e.m. (h) In vivo migration of LPS-DCs to LNs. The number of mDia1WT and mDia1KO DCs that arrive at popliteal LNs after injection in the footpad was analysed by fluorescence-activated cell sorting after 16 h (n = 9 mice per condition pooled from three independent experiments). The Mann–Whitney test was applied for statistical analysis in h. Graphic shows mean and error bars correspond to s.e.m.