Aim: To evaluate the capacity of P. falciparum infected erythrocytes to prompt neutrophil extracellular traps (NETs) formation by human neutrophils; and to assess the presence of NETs in the intervillous space of malaria-infected placentas.
Methods: Neutrophils isolated from healthy donors were co-cultured with P. falciparum infected erythrocytes and then stained with NET markers (myeloperoxidase and neutrophil elastase) and nuclear stain DAPI, and examined by confocal microscopy. To assess the presence of NETs in the intervillous space of malaria-infected placentas, paraffin-embedded placental tissues were stained with Giemsa and immunohistochemistry then examined by microscopy for the presence of leukocytes, neutrophils and NETs in the intervillous space. Immunofluorescence staining was performed to confirm the contents of NETs by observing the overlay of myeloperoxidase, neutrophil elastase and DAPI.
Result: Neutrophil incubation with infected erythrocytes revealed structures that resembled NETs trapping infected erythrocytes which were more pronounced on IgG-opsonized infected erythrocytes. Examination of Giemsa stained slides showed the total number of leukocytes and NETs-like structures in the placental intervillous space is higher in malaria infected placentas as compared to non-infected groups (P< 0.05 & P= 0.01 respectively). The number of neutrophils was similar between the two groups. Immunohistochemistry and immunofluorescence staining showed many extracellular granular proteins (myeloperoxidase, neutrophil elastase overlaps) in infected placentas.
In Conclusion, In vitro results suggest there is release of NETs when infected erythrocytes interact with neutrophils. Ex vivo results suggest malaria infected erythrocytes cause infiltration of neutrophils and formation of NETs in the intervillous space. This is the first examination of the role of NETs in placental malaria.