Fig. 5.

Pharmacological break-down of the microtubule cytoskeleton by nocodazole rescues cellular movement but not directionality of NLRP3-deficient neutrophils. (A) Vehicle-treated NLRP3^−/− neutrophils showed a clear impairment of cellular elongation, quantified through the effect ratio of the best fit ellipse. (B) Representative images of cellular elongation in response to LTB4 gradient. Images were taken randomly during the 1-h follow-up (t_n) and 1 min late (t_n+1) to show the dynamic motion of cells in response to external LTB4 gradient of 4,000 pg/mL. While nocodazole promoted NLRP3^−/− cell elongation and migration, it did not improve chemotaxis toward the LTB4 gradient. (C–E) Quantification of cellular movement of neutrophils following real-time tracking of cells in the presence of a 4,000 pg/mL gradient of LTB4. Prior to exposure to LTB4 gradients, neutrophils were pretreated for 1 h with nocodazole or vehicle in order to break down the microtubule cytoskeleton. The mean of NLRP^+/+ neutrophils, taken from (Fig. 2 C and D) are depicted as a dotted line. Total distance traveled of neutrophils over a 1-h time period in an LTB4 gradient. Treatment of NLRP3^−/− neutrophils with nocodazole increased their migration significantly as compared to vehicle-treated NLRP3^−/− neutrophils. (F) Rose plots of cellular movement confirm that NLRP3^−/− neutrophils show increased movement as compared to vehicle-treated NLRP3^−/− cells. However, cellular migration of NLRP3^+/+ cells, treated with nocodazole, was still significantly higher. (G) Directionality plots show that the treatment of NLRP3^−/− neutrophils with nocodazole resulted in a migration without a bias as regard to direction, directionality was also reduced in NLRP3^+/+ neutrophils treated with nocodazole. Kruskal–Wallis test was used for statistical analysis of two groups (P < 0.05 ^*, < 0.01 ^**, < 0.001 ^***, < 0.0001 ^****, n = 6).