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Pharmacological and genetic interventions of granuloma neutrophil functions lead to the reduction in mycobacterial burden. (A) Altered neutrophil morphology in explants 5 hours posttreatment, duvelisib versus vehicle. Red arrows: elongated neutrophils, vehicle-treated granuloma; white arrows: rounded neutrophils, duvelisib treated. (B) Mean neutrophil circularity from (A). Point denotes mean circularity from single granuloma. Vehicle: 300 neutrophils; duvelisib: 389 neutrophils (12 granulomas each). (C) Granuloma neutrophils tracked for 230 min posttreatment with vehicle/duvelisib. (D) Mean neutrophil velocities from vehicle and duvelisib treatment. Point denotes mean velocity of five neutrophils tracked per granuloma. Sixty neutrophils (12 granulomas) analyzed for each treatment. (E) M. marinum (red)–infected granulomas 4 days posttreatment. (F) Burden in granulomas 4 days posttreatment as mean arbitrary fluorescence units (AU). Two independent experiments. Point represents bacterial fluorescence from single granuloma, 60 granulomas (vehicle); 62 granulomas (duvelisib), six animals each. No significant differences before treatment. (G) In vitro growth profile of M. marinum with duvelisib/vehicle. (H) Neutrophils recruited to Rac2D57N granuloma and WT sibling. (I) Mean neutrophil number in Rac2D57N granulomas, WT siblings. Point represents neutrophil number from a single granuloma; 31 granulomas (WT); 23 granulomas (Rac2D57N), three animals each; (J) Representative images showing burden in WT and Rac2D57N granulomas. (K) Quantitation for WT and Rac2D57N granuloma burden (AU); 33 granulomas (WT); 35 granulomas (Rac2D57N), three animals each; (L) Mean burden, 14 dpi WT and Rac2D57N animals. Three independent experiments. 27 WT; 30 Rac2D57N fish. [(A), (C), and (H) to (K)] Representative of three independent experiments. [(E) and (G)] Representative of two independent experiments. [(B) and (D)] Data from three independent experiments, nine animals for each group. [(B) and (L)] Two-tailed, unpaired t test. [(D), (F), (I), & (K)] Unpaired t test with Welch’s correction. [(B), (D), and (L)] Error bars: SEM. [(F), (G), (I), and (K)] Error bars: SD. Scale bars, 50 μm.
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