Lipid moiety of malarial pigment promotes NF-kappaB complex nuclear translocation in human monocytes: role of 15-HETE in MMP-9, TNFalpha and IL-1beta regulation

INTRODUCTION. During recent years, increased levels of several matrix metalloproteinases (MMPs) have been related to glial activation in either human or murine models of cerebral malaria (CM), an encephalopathy caused by massive sequestration of parasitized erythrocytes in the brain capillaries and associated with elevated plasma levels of TNF and IL-1beta, blood brain barrier (BBB) damage, mononuclear cell margination, ring hemorrhages and Dürck’s granulomas infiltrated with macrophages. Human monocytes avidly phagocytose native hemozoin (nHZ, malarial pigment), a lipid-enriched ferriprotoporphyrin IX produced by Plasmodium parasite after hemoglobin catabolism. Interestingly, phagocytosed nHZ was shown to increase expression and activity of MMP-9 and to enhance production of MMP-9-related cytokines TNF and IL-1beta. Present work investigated what hemozoin moiety (heme or lipid) was responsible for these effects, and explored the signaling mechanisms involved. METHODS. Adherent human monocytes were treated for 24-48 h with either nHZ or lipid-free HZ (synthetic, sHZ, and delipidized, dHZ). Alternatively, cells were treated with different doses (0.1-10 microM) of 15(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid (15-HETE), a potent lipoperoxidation derivative generated by HZ from arachidonic acid via heme-catalysis. In selected experiments, quercetin, artemisinin and parthenolide, three inhibitors of IkappaBalpha phosphorylation and subsequent degradation, NF-kappaB nuclear translocation, and NF-kappaB-p65 binding to DNA, respectively, were added. Therefore, MMP-9 activity by direct gelatine zymography; TNFalpha and IL-1beta by specific ELISA; cytosolic IkBalpha protein expression and phosphorylation by western blotting; and nuclear translocation of NFkappaB-p65 and –p50 subunits by electrophoresis mobility shift assay and additional western blotting were evaluated. RESULTS. nHZ enhancing effects on MMP-9 activity and TNFalpha/IL-1beta production were not reproduced by lipid-free HZ (sHZ/dHZ), while they were recapitulated dose-dependently by 15-HETE. Both nHZ/15-HETE, but not dHZ/sHZ, promoted cytosolic IkappaBalpha protein phosphorylation and degradation, and NF-kappaB complex migration to the nuclear fraction. All HZ/15-HETE effects on MMP-9 activity and TNF/IL-1beta production were abrogated by quercetin, artemisinin and parthenolide. CONCLUSIONS. Enhanced activation of MMP-9 and release of pro-inflammatory cytokines TNF and IL-1beta, a triad of effects dramatically involved in CM pathogenesis and elicited in human monocytes by HZ phagocytosis, is dependent on lipid moiety of malarial pigment, and 15-HETE seems to play a major role. Additionally, all these events appear to be causally connected to persisting activation of the NF-kappaB system.