Fig 1.
P. falciparum-infected erythrocytes induce NETs.
(A) Neutrophil elastase and circulating nucleosomes (B) in plasma from human patients diagnosed with uncomplicated malaria (U.M., n = 42) or severe malaria (S.M., n = 23). Mann-Whitney was performed for statistical significance between groups. P values are indicated in each graph. (C) Fluorimetric determination of NET production by human neutrophils in the presence of P. falciparum-infected red blood cells (iRBC) or uninfected RBC (cRBC) at varying red blood cell:neutrophil ratios. (D) Fluorimetric determination of NET production by human neutrophils in the presence of P. falciparum-infected red blood cells (iRBC) or uninfected RBC (cRBC) at different time-points. Data in C and D are presented as means ± S.E.M. of the fold induction of extracellular DNA signal relative to resting neutrophils. (E) Visualization by fluorescence microscopy of NETs produced by human neutrophils in the presence of P. falciparum-infected (iRBC) or uninfected (cRBC) red blood cells for 3 hours. DNA is stained in blue (Hoesch), neutrophil elastase is stained in green (elastase) and myeloperoxidase is stained in red (MPO). Unstimulated human neutrophils were used as controls. (F) Representative images of the effect of DNAse treatment on NET signal as visualized by fluorescence microscopy. Human neutrophils were incubated with iRBC or cRBC for 3 hours in the presence of DNAse. DNA was stained with Hoesch. (G) Quantification of data derived from (F). Data are presented as means ± S.E.M. of the fold induction of extracellular DNA signal relative to resting neutrophils. * P< 0.05 and *** P<0.001 relative to controls incubated with cRBC, # P< 0.01 relative to untreated control.
Fig 2.
Involvement of ROS, MPO, NE and PAD4 on NET production in response to infected erythrocytes.
Human neutrophils were treated with DPI (10 μg/mL) (A), MPO inhibitor (MPOi, 10 μg/mL) (B), neutrophil elastase inhibitor (NEi, 10 μg/mL) (C) or Cl-amidine (12 μM) (E) for 30 minutes and then incubated with P. falciparum-infected red blood cells (iRBC) for 3 hours. NET production was determined by fluorimetry. Uninfected red blood cells (cRBC) were used as control. Data are presented as means ± S.E.M. of the fold induction of extracellular DNA signal relative to resting neutrophils. (D) Representative westernblot image of citrullinated histone H3 in extracts of human neutrophils incubated for 3 hours in the presence of infected (iRBC) or uninfected (cRBC) red blood cells. β-actin was used as loading control. * P< 0.05 relative to controls incubated with cRBC, # P< 0.01 relative to untreated control.
Fig 3.
Involvement of CD18, ICAM-1, CD36 and phagocytosis on NET production in response to infected erythrocytes.
Human neutrophils were treated with neutralizing antibodies to CD18 (20μg/mL) (A), ICAM-1 (20μg/mL) (B) or CD36 (20μg/mL) (C) for 30 minutes and then incubated with P. falciparum-infected red blood cells (iRBC) at a 1:5 ratio for 3 hours. (D) Human neutrophils were treated with cytochalasin D (8 μM) for 30 minutes and then incubated with P. falciparum-infected red blood cells (iRBC) at a 1:5 ratio for 3 hours. NET production was determined by fluorimetry. Uninfected red blood cells (cRBC) were used as control. Data are presented as means ± S.E.M. of the fold induction of extracellular DNA signal relative to resting neutrophils. * P< 0.05 relative to controls incubated with cRBC.
Fig 4.
Involvement of CXCR4-MIF axis on NET production in response to infected RBC.
Human neutrophils were treated with AMD3100 (AMD, 100 ng/ml) (A), ISO-1 (ISO, 50μM) (B) or a neutralizing anti-MIF antibody (α-MIF, 20μg/mL) (C) for 30 minutes and then incubated with P. falciparum-infected red blood cells (iRBC) at a 1:5 ratio for 3 hours. NET production was determined by fluorimetry. Uninfected red blood cells (cRBC) were used as control. Data are presented as means ± S.E.M. of the fold induction of extracellular DNA signal relative to resting neutrophils. (D) Quantification of MIF levels on supernatants from P. falciparum-infected (iRBCsn) or uninfected (cRBCsn) red blood cells. (E) Human neutrophils were incubated with supernatants from P. falciparum-infected (iRBCsn) or uninfected (cRBCsn) red blood cells and NET production was determined by fluorimetry. (F) Human neutrophils were treated with neutralizing anti-MIF (α-MIF, 20μg/mL) or the appropriate isotype control (IgG) antibody and then incubated with supernatant from P. falciparum-infected (iRBCsn) or uninfected red blood cells (cRBCsn). NET production was determined by fluorimetry. Data are presented as means ± S.E.M. of the fold induction of extracellular DNA signal relative to resting neutrophils. * P< 0.05 relative to controls incubated with cRBC, # P< 0.01 relative to untreated control.
Fig 5.
Effect of NETs on P. falciparum dissemination in cultures of human erythrocytes.
NET-rich supernatants were added to cultures of P. falciparum-infected erythrocytes and the proportion of erythrocytes presenting intracellular ring structures (A) or the proportion of infected erythrocytes (B) were determined after 48 hours. Supernatants from unstimulated neutrophils were used as control. NET-rich supernatants were treated with DNAse (C), MPO inhibitor (MPOi, 10 μg/mL) (D) or NE inhibitor (NEi, 10 μg/mL) (E) 30 minutes before adding to erythrocyte cultures and the proportion of erythrocytes presenting ring structures was determined after 48 hours as in A. *** P< 0.001 relative to controls.
Fig 6.
P. berguei ANKA-infected erythrocytes induce NETs.
(A) Fluorimetric determination of NET production by murine neutrophils in the presence of P. berguei ANKA-infected mouse red blood cells (iRBC PbANKA) or uninfected RBC (cRBC) at varying red blood cell:neutrophil ratios. (B) Mouse neutrophils were pre-treated for 30 minutes with DPI and then incubated with PbANKA-infected RBC. NET production was determined by fluorimetry as before. Data in A and B are presented as means ± S.E.M. of the fold induction of extracellular DNA signal relative to resting neutrophils. (C) Visualization by fluorescence microscopy of NETs produced by murine neutrophils in the presence of PbANKA-infected (iRBC) or uninfected (cRBC) red blood cells. DNA is stained in blue (Hoesch), myeloperoxidase is stained in green (MPO) and citrullinated histone H3 (H3C) is stained in red. Data are presented as means ± S.E.M. of the percentage of infected red blood cells. * P< 0.05 and ** P<0.01 relative to untreated controls.
Fig 7.
Effect of DNAse treatment on the PbANKA model of cerebral malaria.
(A) Determination of circulating levels of DNA in plasma of P. berguei ANKA-infected C57BL6 mice 6 days after infection. (B) Mice were treated with DNAse (Pulmozyme, 5 mg/kg i.p., 1 hour before and every 8 hours for 6 days) and survival after PbANKA infection was monitored for 21 days. (C) Parasitemia of mice treated with DNAse (as in E) or vehicle and infected with PbANKA was monitored daily for 7 days. (D) IgG staining of brain tissue from PbANKA infected mice treated or not with DNAse (pulmozyme). Representative photomicrographs of coronal brain sections stained for IgG on the sixth day after P. berghei ANKA infection. Mice were treated with pulmozyme (PbANKA + Pulmozyme) or vehicle (PbANKA) as described. Non-infected mice were used as controls (uninfected). Scale bars: 500 μm. Data are presented as means ± S.E.M. of the percentage of infected red blood cells. * P< 0.05 and ** P<0.01 relative to untreated controls.