Development of pro-inflammatory phenotype in monocytes after engulfing Hb-activated platelets in hemolytic disorders

Abstract Monocytesand macrophage combat infections and maintain homeostatic balance by engulfing microbes and apoptotic cells, and releasing inflammatory cytokines. Studies have described that these cells develop anti-inflammatory properties upon recycling the free-hemoglobin(Hb) in hemolytic conditions. While investigating the phenotype of monocytesin two hemolytic disorders- paroxysmal nocturnal hemoglobinuria(PNH) and sickle cell disease (SCD), we observed a high number of pro-inflammatory ( CD14+ CD16hi ) monocytesin these patients. We further investigated in vitro the phenotype of these monocytesand found an estimated 55% of CD14+ cells were transformed into the CD14+ CD16hi subset after engulfing Hb-activated platelets. The CD14+ CD16hi monocytes, which were positive for both intracellular Hb and CD42b (platelet marker), secreted significant amounts of TNF-α and IL-1β, unlike monocytestreated with only free Hb, which secreted more IL-10. We have shown recently the presence of a high number of Hb-bound hyperactive platelets in patients with both diseases, and further investigated if the monocytesengulfed these activated platelets in vivo. As expected, we found 95% of CD14+ CD16hi monocyteswith both intracellular Hb and CD42b in both diseases, and they expressed high TNF-α. Furthermore our data showed that these monocyteswhether from patients or developed in vitro after treatment with Hb-activated platelets, secreted significant amounts of tissue factor. Besides, these CD14+ CD16hi monocytesdisplayed significantly decreased phagocytosis of E. coli . Our study therefore suggests that this alteration of monocytephenotype may play a role in the increased propensity to pro-inflammatory/coagulant complications observed in these hemolytic disorders-PNH and SCD.