Chemotaxis of mature DCs is impaired after HSV-1 infection and CYTIP knockdown. (A) Transwell migration assays on fibronectin-coated transwells of mock-infected (white columns), HSV-1 UV-infected (gray columns), and HSV-1-infected (black columns) DCs harvested at the indicated time points after infection show that the infection with HSV-1 dramatically reduces DC chemotaxis toward CCL19. (B) The chemotaxis of HSV-1–infected DCs toward CCL19 in a 3D-collagen matrix or in collagen matrices containing 20 μg/mL fibronectin or 75 μg/mL ICAM-1 Fc-coated beads is decreased compared with mock-infected cells in all conditions. The presence of immobilized integrin ligands in the gels further reduces migration of HSV-1–infected DCs but has no influence on mock-infected DCs. (C) A total of 4 × 106 immature DCs were electroporated with 10 μg of a nontargeting control siRNA or with 10 μg siRNA targeting CYTIP. Four hours later, maturation cocktail was added and DCs were matured for 48 hours. Where indicated, mature CYTIP-knockdown DCs were additionally infected with HSV-1. Subsequently, DCs were used in 3D migration assays in pure collagen gels or in gels containing 20 μg/mL fibronectin. Silencing of CYTIP strongly impairs migration of DCs in collagen gels containing fibronectin. The infection of CYTIP-silenced DCs further reduces chemotaxis in both conditions but has a more prominent effect in fibronectin-coated gels. DC migration toward a CCL19 gradient was monitored by bright-field time-lapse videomicroscopy beginning 90 minutes after infection. Cells were imaged at a frame rate of 2 minutes. The average speed of a cell was calculated as the step length per minute, including nonmoving periods and displayed in micrometers per minute. Average speed was quantified from 30 cells for each condition over 180 frames (B-C). Error bars represent ± SD. ***P < .001. **P < .01. ns indicates not significant. One representative experiment of 3 is shown.
