**Sueli de Oliveira Silva Lautenschlager 1, Tehyung Kim 2, Danielle Lazarim Bidóia 1, Celso Vataru Nakamura 1, Hans-Joachim Anders <sup>2</sup> and Stefanie Steiger 2,\***


Received: 28 November 2019; Accepted: 20 December 2019; Published: 30 December 2019

**Abstract:** Hemozoin is an insoluble crystalline pigment produced by the malaria parasite *Plasmodia* upon digesting host hemoglobin inside red blood cells. Red blood cell rupture releases hemozoin crystals into the circulation from where they are cleared by phagocytes such as neutrophils. We speculated that plasma proteins would affect the ability of neutrophils to clear hemozoin crystals. To test this, we cultured human blood neutrophils with hemozoin ex vivo and found that neutrophils ingested hemozoin (0.1–1 μm crystal size) in a dose-dependent manner into phagosomes and vesicles/vacuoles, resulting in morphological changes including nuclear enlargement, and vesicle formation, but not cell membrane rupture or release of neutrophil extracellular traps. The presence of human plasma significantly inhibited the ability of neutrophils to ingest hemozoin crystals. Platelet-poor plasma further inhibited the uptake of hemozoin by neutrophils. Selective exposure to fibrinogen completely replicated the plasma effect. Taken together, neutrophils cleared hemozoin crystals from the extracellular space via endocytosis into phagosomes and vesicles without inducing the release of neutrophil extracellular traps. However, human plasma components such as fibrinogen limited hemozoin clearance, whereas the presence of platelets augmented this process. These factors may influence the pro-inflammatory potential of hemozoin crystals in malaria.

**Keywords:** hemozoin; neutrophils; plasma; fibrinogen; platelet; malaria
