Fig. 7
Inhibition of the CXCR3-CXCL10 axis limits migration of cultured mouse and primary human macrophages. A Murine macrophages (J774A.1) were cultured in a serum-rich medium until the cells were 80% confluent, followed by 12 h in serum-reduced medium and then 12 h in serum-rich medium. Afterward, the cells were exposed to normoxia (21% O2, NOX) or hyperoxia (85% O2) for 24 h. Measurement of Cxcl10 mRNA using quantitative RT-PCR; 18 s rRNA served as a housekeeping gene. B, C Experimental setup to determine the functional role of CXCL10 in macrophage differentiation (B). Assessment of mRNA expression of Il6 (interleukin 6), Il1β (interleukin 1beta), Nos2 (nitric oxide synthase), and Ccl2 in macrophages treated with CXCL10 (10 ng/ml) or vehicle for 48 h; 18 s rRNA served as a housekeeping gene. D, E Representative images of migrated J774A.1 macrophages treated with either only CXCL10 (10 ng/ml) or CXCL10 plus CXCR3 antagonist (300 nm/ml) for 24 h; controls were treated with DMSO (D). Quantification of migrated macrophages per field of view (20X) (E). F, G Experimental plan to study the impact of HYX on differentiation of primary human M1-like macrophages: human M0 macrophages were differentiated into M1-like macrophages by treatment with GM-CSF (100 ng/ml) for 7 days, followed by exposure to NOX or HYX (F). Assessment of CXCL10, IL1B, NOS2, and CCL2 expression in human M1-like macrophages; 18 s rRNA served as housekeeping gene (G). H, I Representative images of migrated human M0-like macrophages treated with either only CXCL10 (10 ng/ml) or CXCL10 plus CXCR3 antagonist (300 nm/ml) for 24 h; controls were treated with DMSO (H). Quantification of migrated human M0-like macrophages per field of view (20X) (I). Mean ± SEM; n = 4–9/group; Mann–Whitney test: #p < 0.05; ##p < 0.01; ###p < 0.001, Paired t-test: $ p < 0.05; scale bar 20X