Identification of hydroxyeicosatetraenoic acid components of schistosomal hemozoin

Abstract

During the late stages of Schistosoma mansoni infection, adult schistosomes catabolize host erythrocytic hemoglobin. In order to evade the toxic effects of free heme, the blood fluke biomineralizes dimeric heme into an inert crystalline pigment called hemozoin. In the present study, the chemical reactivity of schistosomal hemozoin (SmHz) toward lipid oxidation was examined, and the biological consequences of reactivity were investigated. Mass spectrometric analysis of polar lipid content associated with SmHz identified a variety of primary and secondary polyunsaturated fatty acid oxidation products, including hydroxyeicosatetraenoic acids. Furthermore, RAW 264.7 macrophage-like cells challenged with lipopolysaccharide prior to phagocytosis of SmHz experienced a decrease in nitric oxide production as compared to control experiments. The presence of these biologically active oxidation products suggests native SmHz is capable of modulating the innate immune response and may play a potential role in the pathogenesis of schistosomiasis.

Figure 1

Hemozoin from S. mansoni. (A) Adult female (left) and male (right) S. mansoni laden with hemozoin. Female worms accumulate a heavier hemozoin burden, as seen by their darker pigmentation, due to the nutritional demands of oogenesis. (B) SEM image of schistosomal hemozoin isolated from homogenized S. mansoni.

Figure 2

HETE-containing lipid coat of native SmHz. (A) Lipids were extracted from native SmHz using ethyl acetate, blown dry under N₂ (g) and redissolved in RP-LC-MS/MS mobile phase. All six hydroxylated fatty acid derivatives of arachidonic acid were identified (15-, 11-, 12-, 8-, 9- and 5-HETE). Insert shows the representative chiral resolution for 15-HETE (11.5 min. R and 14.5 min. S). (B) Representative MS/MS fragmentation for 12-HETE. All isomers were confirmed using authentic standards and extracted ion profiles from MS/MS (SI 2).

Figure 3

Hemozoin-mediated lipid peroxidation. Oxidation studies by incubation of 0.3 mg/mL BH or SmHz with 5.32 mM AA were conducted. The hydroxylated fatty acid isomers 15-, 11-, 12-, 8-, 9- and 5-HETE were identified in the reactions’ lipid extracts using RP-LC-MS/MS. The isomers were confirmed using authentic standards and extracted ion profiles from MS/MS.

Figure 4

Impact of native SmHz and components on iNOS activity. (A) RNS production in macrophage cells monitored following treatment with increasing concentrations of (●) 15-, (■) 12- and (▲) 5-S-HETE by the Griess assay. (B) Cells were treated with 0.1 mg/mL BH or SmHz, as well as 40 μM 15-S-HETE or HNE. The activity of cells stimulated with LPS was used as a control.