Fig 2: Neutrophil ICAM-1 is an important receptor for their killing ability of iRBCs A, BqRT-PCR analysis of the relative expression of ICAM-1 and EPCR mRNA in non-treated PLB985 cells (control), ICAM-1 knockdown (ICAM-1kd) and EPCR knockdown (EPCRkd) PLB985 cells.CFlow cytometric analysis (FACS) of surface ICAM-1 (a human mAb, see methods) expression in control non-treated PLB985, ICAM-1kd, and EPCRkd PLB985 cells.DFlow cytometry quantification of the percentage of neutrophils that became GFP+ following incubation with GFP+ iRBC of control (untreated PLB985 cells), ICAM-1kd and EPCRkd PLB985 cells.EShort-term (6 h) killing assay of NF54-luciferase iRBCs incubated with PLB985 neutrophils in the presence of anti-CD11b and anti-ICAM-1 blocking (mAb 15.2) and non-blocking (a human mAb, see methods) antibodies.FInhibition of PLB985 neutrophil (%) phagocytosis of GFP+ iRBCs in the presence of anti-CD11b and anti-ICAM-1 (mAb 15.2) blocking antibodies. The inhibition was quantified by flow cytometry (FACS).GShort-term (6 h) PLB985 neutrophil killing of NF54-luciferase iRBCs in the presence or absence of soluble ICAM-1-Fc fusion protein (sICAM-1-Fc). Soluble RAGE (sRAGE) protein was used as a negative control. Data information: Results represent the average of three biological replicates (n = 3) ± standard error of the mean. Statistical significance was determined using student t-test **P < 0.01.

Fig 3: The impact of phagocytosis on apoptosis and necrosis of PLB985 neutrophils AAnnexin V and propidium iodide (PI) flow cytometry (FACS) were used to analyze for apoptosis and necrosis of GFP- neutrophils that did not phagocytose (left panel) or GFP+ neutrophils that did phagocytose GFP-expressing P. falciparum iRBCs following a 6-h co-incubation. Neutrophils reduce the parasitemia in cultures.BPercentage of luciferase signal reduction during the short killing assay (6 h) of luciferase-expressing P. falciparum iRBCs with or without neutrophils.CiRBCs used for the short-term killing assay in (B) were put back into culture and the reduction in parasitemia was measured following 1 and 3 days of co-culture.DPercentage of parasitemia reduction on days 1 and 3 shown in B.EReduction in parasitemia at day 1 and 3 following neutrophils incubation with iRBC in the presence or absence of catalase. The impact of serum proteins on the neutrophil killing of iRBCs.FShort-term killing assay of AB+-serum opsonized (AB+ serum) and non-opsonized iRBCs (no serum) in the presence of anti-ICAM1 blocking antibody (mAb 15.2) and anti-CD11b control antibody. Data information: Results represent the average of three biological replicates (n = 3) ± standard error of the mean (SEM). Statistical significance was determined using student t-test *P ? 0.05; **P < 0.01.

Fig 4: The interaction of iRBC with endothelium and neutrophilsInfected RBCs can interact with both endothelial cells and neutrophils. Left panel, iRBCs adhere to the endothelium as a strategy for escaping removal by the spleen. The binding of iRBC to the endothelial cell is mediated by PfEMP1 and different endothelial receptors, for example, ICAM-1, CSA, CD36, EPCR. Right panel, neutrophil surface expresses ICAM-1 and binds to PfEMP1 on iRBC. This results to extracellular killing or the phagocytosis and killing of the iRBC (not shown)—see also Fig 2B.