Fig. 4

Classical IPAH monocytes are apoptotic. PBMC were isolated from whole blood of Controls and IPAH patients and assessed and/or sorted via FACS. (A) Total monocytes per 10,000 PBMC for Control or IPAH (n = 7 each). (B) Proportion of monocyte subpopulations of the total monocytes. n = 10 Controls and n = 8 IPAH. (C) UMAP of monocyte clusters showing CD14⁻/CD16⁻ cells, and CD14⁺/CD16⁺ cells for Control and IPAH. (D) Schematic outlining the approach to assess the fate of IPAH classical monocytes in vitro over 24 h. Whole blood from Controls (n = 3) and IPAH (n = 3) was FACS sorted to collect CD14⁺/CD16⁻ classical monocytes at T = 0. Classical monocytes were cultured for 24 h with or without stimulation by mCSF, GM-CSF or LPS, and viability was assessed via FACS with a Zombie NIR live/dead stain. (E). Dead classical monocytes following 24 h of culture with or without stimulating factors. n = 3 biological replicates. Bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by Mann-Whitney non-parametric t-test (A, B & E). For panel E, n = 3 biological replicates were used. The ‘+’ symbol indicates that the minimum achievable P-value was reached using a non-parametric t-test with n = 3