Malaria pigment hemozoin drives M1 pro-inflammatory macrophage polarization in vitro

Severe malaria, a high burden parasitic disease, is characterized by hyperproduction of proinflammatory cytokines, most likely generated by M1-polarized macrophages. Malaria pigment or hemozoin (HZ), a byproduct of heme detoxification in intra-erythrocytic parasites, is internalized by circulating monocytes and tissue macrophages, modulating their functions. Although the immunomodulatory properties of HZ have been described, its specific role in M1/M2 macrophage polarization remains unclear. This study aims to fill this gap by elucidating whether HZ modulates M1/M2 polarization, contributing to the strong inflammatory response in severe malaria. Primary human monocyte derived macrophages (MDM) and THP-1 cells differentiated into macrophages (dTHP-1) were stimulated with M1 or M2 signals in the presence of native HZ. Gene expression and protein secretion of TNF-α, IL-1β, CXCL8, IL-6, IL-10 and PPARG were evaluated by Real-Time PCR and ELISA, respectively. STAT6 phosphorylation was evaluated by western blot analysis. MDM and dTHP-1 showed a different polarisation response to classical M1/M2 stimuli and to HZ treatment. In both non-polarized (M0) MDM and dTHP-1, HZ induced an M1/pro-inflammatory phenotype, increasing gene expression and protein secretion of CXCL8, TNF-α, and IL-1β. In the presence of M1- or M2-polarizing stimuli, HZ further increased CXCL8 and IL-1β in MDM but not in dTHP-1, where TNF-α secretion was even reduced. HZ did not affect M2 markers (PPARG and IL10 expression, STAT6 phosphorylation) in any condition. This is the first in vitro study investigating the effect of HZ on macrophage polarization, showing its ability to promote M1 pro-inflammatory differentiation. Results vary across experimental models, emphasizing the importance of considering model-specific effects. Clarifying HZ's role remains crucial for understanding malaria pathogenesis and developing new immunomodulatory therapies.