Effects of Hemozoin and Hemozoin-Generated Molecules on Immune and Non-Immune Host Cells

Blood-stage P. falciparum (Pf) produces and accumulates hemozoin (HZ), a stable crystalline polymer derived from undigested hemoglobin heme. Phagocytes avidly phagocytose large amounts of HZ contained in late parasite forms or in residual bodies. HZ contains large amounts of redox active iron, able to peroxidize HZ-bound or phagocyte-derived polyunsaturated fatty acids (PUFA), to produce an array of bioactive hydroxy-PUFA (HETEs; HODEs) and terminal aldehydes (e.g. 4-hydroxynonenal, HNE). A wide range of inhibitory or stimulatory effects have been described in human (HU) and murine (MU) monocytes/macrophages/phagocytic cell lines (phagocytes) after phagocytosis of Pf-HZ or synthetic HZ (sHZ). 1. Inhibitory effects: 1a) Long term inhibition (but short-term enhancement, see below) of oxidative burst, phagocytosis and killing (after phagocytosis of native PfHZ by HU monocytes); 1b) Expression of MHC class II, accessory cell function and inhibition of antigen presentation (Scorza et al) (after phagocytosis of PfHZ by HU and MU phagocytes); 1c) Inhibition of dendritic cell (DC) differentiation and maturation (after phagocytosis of trophozoites and PfHZ by HU phagocytes, and MU-HZ by MU phagocytes), but 1d) increase in DC maturation after purified HZ phagocytosis (Coban et al). Taken together, effects 1a)-1c) may explain immunodepression typical of falciparum malaria 2. Stimulatory effects: 2a) Short-term enhancement of oxidative burst and MCP-1 secretion (after contact of native PfHZ with HU monocytes); 2b) Short-term and long-term increased production of TFN, IL-1beta, MIP-1alfa; MIP-1beta (same by MU phagocytes); 2c) Induction of gene expression and activity of metalloproteinases (after phagocytosis of native PfHZ by HU monocytes; after phagocytosis of sHZ by MU phagocytes); 2d) Stimulation of DC differentiation by DNA-containing HZ (Parroche et al); 2e) Induction of gene expression and activity of iNOS (after phagocytosis of native PfHZ and sHZ by MU phagocytes). No induction of iNOS was observed in HU monocytes fed with PfHZ. Taken together, effects 2a)-2c) may explain typical cytokine alterations observed in falciparum malaria and likely responsible for fever and pro-inflammatory phenomena. Induction of metalloproteinases is likely to play roles in the lesion of endothelial basal membrane and disruption of the blood-brain barrier typical of cerebral malaria. 3. Mechanistic studies: role of HETE and HNE: Studies have indicated that bioactive products generated by HZ (HETE, HNE) may be responsible for some of the HZ effects listed above. 3a) Inhibition of DC differentiation and maturation was recapitulated by low-micromolar HETE/HODE, ligands of intracellular receptor PPAR-gamma, a transrepressor for the nFκB-induced/controlled CD83 expression. Recapitulation was abrogated by PPAR-gamma inhibitors. Low-micromolar HNE also inhibited DC maturation and CD83 expression, probably by cross-linking critical proteins/receptors, such as CD14 or GMCSF-R. Several HNE-adducted proteins have been identified. 3b) Low-micromolar HETE recapitulated enhancement of gene expression and enzyme activity of metalloproteinase-9 (MMP-9) in HZ-fed HU monocytes. MMP-9 sheds cell-bound TNF and IL-1, and contributes to cytokine increase. Recapitulation was abrogated by PPAR-gamma inhibitors. 4. HZ effects independent of HETE/HNE: Fibrinogen (FI) was found to be firmly and specifically attached to native HZ. In HU monocytes, the complex HZ-FI was found to bind to i) the LPS-receptor, Toll-like receptor 4 (TLR4), and ii) the integrin CD11b/CD18. Binding to both receptors induced a very rapid and intense formation and release of ROS, TNF and MCP-1. FI effect was independent of lipid components and critically dependent on the crystalline poly-heme scaffold. 5. Effects due to transfer of HZ-generated HNE to adherent non-phagocytic cells: HZ-generated HNE may transfer to adherent non-phagocytic cells, such as erythroid cells, non-infected RBCs, endothelia and other cells. The effect of inter-cellular transfer of HNE was studied in developing erythroid cells. Erythropoiesis was inhibited by HNE transferred from HZ or HZ-laden phagocytes, as the consequence of blockage of cell-cycle and down-regulation of protein expression of crucial receptors for erythropoietin, transferrin and stem cell factor. This paradigm may be applied to other non-phagocytic cells (endothelia, lymphocytes) and possibly clarify aspects of malaria pathogenesis.