Fig. 4: Hpa2-KO macrophages are shifted towards M2 phenotype.

A M1 and M2 markers. WT and Hpa2-KO mice (n = 12) were administrated with thioglycolate and peritoneal fluid was collected three days later. Cells were subjected to FACS analyses, employing cell surface markers typical of M1 (CD11c + ) and M2 (CD206 + ) macrophages (left panels). Total RNA was extracted from corresponding cells and subjected to qPCR analysis applying primers specific for arginase, typical for M2 macrophages (second right). Purified recombinant Hpa2 protein (1 ug/ml) was added exogenously to Hpa2-KO macrophages, total RNA was extracted after 24 h and was subjected to qPCR applying primers specific for iNOS, typical of M1 macrophages (right panel). B M1 markers. Total RNA was extracted from WT and Hpa2-KO macrophages and was subjected to qPCR analyses applying primers specific for IL-12a, CD40, CD86, and MHC-II, typical for M1 macrophages. C Chemotherapy. WT and Hpa2-KO macrophages were left untreated (Con) or were treated with cisplatin (Cis; 10 μg/ml) or paclitaxel (PCT; 15 μg/ml). After 24 h cells were collected and subjected to FACS analyses employing M2 (CD206⁺/ CD11C^-) and M1 (CD11C⁺) markers. D Phagocytic assay. Macrophages were collected from thioglycolate-treated WT and Hpa2-KO mice (n = 5), plated on fibronectin-coated 96-well dish (2 × 10⁴) for 24 h and, following washes, Zymosan-coated fluorogenic bioparticles (5 µl) were added. Macrophage phagocytosis capacity was quantified over time using IncuCyte methodology. Shown are color intensity over time (upper panel) and representative images of phagocytotic macrophages (lower panels, red). Note the increased phagocytic capacity of Hpa2-KO macrophages, typical for M2.