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. 2022 Aug 16;66(9):e02298-21. doi: 10.1128/aac.02298-21

FIG 4.

FIG 4

Effect of CSY5669 on antimicrobial and inflammatory responses by monocyte-derived macrophages and blood leukocytes. Monocyte-derived macrophages (MDMs, n = 5) or peripheral blood leukocytes (PBL, n = 4) were infected with MRSA and subsequently incubated overnight (MDMs) or 6 h (PBL) in the presence of 1 μM AZM or CSY5669, after which intracellular bacterial numbers were determined (A) and presented, relative to DMSO controls (B). Alternatively, PBLs were pretreated with AZM or CSY5669, washed, and subsequently infected and incubated with MRSA for 6 h (C). MDMs ([D], n = 3) or PBLs (gated on viable CD14+ monocytes [E] or on viable CD66b+ neutrophils [F], n = 4; for the gating strategy used, see Fig. S3 in the supplemental material) were incubated with heat-killed MRSA for 60 min in the presence of ROS-reactive dye, after which cells were measured with flow cytometry. Monocyte-derived macrophages (MDMs) were incubated with heat-killed, FITC-labeled MRSA for 30 min and measured with flow cytometry ([G], n = 4). MDMs were infected with viable MRSA and incubated for 120 min, after which cells were fixed and measured using confocal microscopy to determine the phagosomal abundance (number of lysotracker+ organelles per cell) or the colocalization of phagolysosomes with bacteria ([H], n = 4). Isolated neutrophils were incubated for 60 min with heat-killed MRSA, after which the expression of degranulation markers (CD63: azurophilic/primary granules, CD66b: specific/secondary and tertiary granules, CD11b: secretory vesicles, CD18: general activation marker) was determined ([I], n = 4). Expression levels were normalized to unstimulated DMSO-treated controls. MRSA-induced cytokine release was determined in MDMs ([J], overnight incubation, n = 5) and PBLs ([K], 4 h incubation, n = 6). Data are presented as mean ± standard deviation, with each dot representing (the average of) individual experiments. Statistical significance was tested using a one-way analysis of variance with Tukey’s post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.