Predictive Criteria to Study the Pathogenesis of Malaria-Associated ALI/ARDS in Mice

Malaria-associated acute lung injury/acute respiratory distress syndrome (ALI/ARDS) often results in morbidity and mortality. Murine models to study malaria-associated ALI/ARDS have been described; we still lack a method of distinguishing which mice will develop ALI/ARDS before death. This work aimed to characterize malaria-associated ALI/ARDS in a murine model and to demonstrate the first method to predict whether mice are suffering from ALI/ARDS before death. DBA/2 mice infected with Plasmodium berghei ANKA developing ALI/ARDS or hyperparasitemia (HP) were compared using histopathology, PaO2 measurement, pulmonary X-ray, breathing capacity, lung permeability, and serum vascular endothelial growth factor (VEGF) levels according to either the day of death or the suggested predictive criteria. We proposed a model to predict malaria-associated ALI/ARDS using breathing patterns (enhanced pause and frequency respiration) and parasitemia as predictive criteria from mice whose cause of death was known to retrospectively diagnose the sacrificed mice as likely to die of ALI/ARDS as early as 7 days after infection. Using this method, we showed increased VEGF levels and increased lung permeability in mice predicted to die of ALI/ARDS. This proposed method for accurately identifying mice suffering from ALI/ARDS before death will enable the use of this model to study the pathogenesis of this disease.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ São Paulo, Inst Ciencias Biomed, Dept Imunol, BR-05508900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, BR-09972270 Diadema, SP, BrazilUniv São Paulo, Inst Med Trop São Paulo, BR-05403000 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Exatas & Terra, BR-09972270 Diadema, SP, BrazilUniv São Paulo, Inst Ciencias Biomed, Dept Parasitol, BR-05508000 São Paulo, BrazilUniv São Paulo, Fac Med Vet & Zootecn, Dept Cirurgia, BR-05508270 São Paulo, BrazilUniv São Paulo, Fac Med Vet & Zootecn, Dept Med Vet Prevent & Saude Anim, BR-05508270 São Paulo, BrazilUniv São Paulo, Fac Ciencias Farmaceut, Dept Anal Clin & Toxicol, BR-05508000 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, BR-09972270 Diadema, SP, BrazilUniversidade Federal de São Paulo, Dept Ciencias Exatas & Terra, BR-09972270 Diadema, SP, BrazilFAPESP: 2009/53256-7FAPESP: 2009/53889-0CNPq: 306668/2012-2CNPq: 470590/2009-2Web of Scienc

Correction: Targeting Neutrophils to Prevent Malaria-Associated Acute Lung Injury/Acute Respiratory Distress Syndrome in Mice.

[This corrects the article DOI: 10.1371/journal.ppat.1006054.]

Targeting Neutrophils to Prevent Malaria-Associated Acute Lung Injury/Acute Respiratory Distress Syndrome in Mice

<div><p>Malaria remains one of the greatest burdens to global health, causing nearly 500,000 deaths in 2014. When manifesting in the lungs, severe malaria causes acute lung injury/acute respiratory distress syndrome (ALI/ARDS). We have previously shown that a proportion of DBA/2 mice infected with <i>Plasmodium berghei</i> ANKA (PbA) develop ALI/ARDS and that these mice recapitulate various aspects of the human syndrome, such as pulmonary edema, hemorrhaging, pleural effusion and hypoxemia. Herein, we investigated the role of neutrophils in the pathogenesis of malaria-associated ALI/ARDS. Mice developing ALI/ARDS showed greater neutrophil accumulation in the lungs compared with mice that did not develop pulmonary complications. In addition, mice with ALI/ARDS produced more neutrophil-attracting chemokines, myeloperoxidase and reactive oxygen species. We also observed that the parasites <i>Plasmodium falciparum</i> and PbA induced the formation of neutrophil extracellular traps (NETs) <i>ex vivo</i>, which were associated with inflammation and tissue injury. The depletion of neutrophils, treatment with AMD3100 (a CXCR4 antagonist), Pulmozyme (human recombinant DNase) or Sivelestat (inhibitor of neutrophil elastase) decreased the development of malaria-associated ALI/ARDS and significantly increased mouse survival. This study implicates neutrophils and NETs in the genesis of experimentally induced malaria-associated ALI/ARDS and proposes a new therapeutic approach to improve the prognosis of severe malaria.</p></div

Neutrophil interactions in malaria-associated ALI/ARDS.

<p>Following <i>P</i>. <i>berghei</i> ANKA infection in DBA/2 mice, neutrophils promote the pathogenesis of ALI/ARDS. In particular, the release of myeloperoxidase and reactive species of oxygen and the formation of neutrophil extracellular traps determines the cause of death of these mice.</p

Recombinant human DNase 1 (Pulmozyme) and an elastase inhibitor (Sivelestat) improve outcomes of malaria-associated ALI/ARDS.

<p>(A) Lung sections of DBA/2 mice infected with 10⁶ <i>P</i>. <i>berghei</i> ANKA-iRBCs on the day of death (9^th dpi) were stained for histone (red), myeloperoxidase (MPO) (green), and DNA (blue). Overlapping colors indicate cloud-like structures. White arrowheads show selected cells forming NETs (400x, scale bar 25 μm). (B) Survival, (C) parasitemia, (D) enhanced respiratory pause (Penh) and (E) respiratory frequency (RF) on the 7^th days post-infection (dpi) in <i>P</i>. <i>berghei</i> ANKA-infected mice treated with Pulmozyme (50 μg/mouse) or saline solution (control group) on the 3^rd and 6^th dpi. Data are representative of three independent experiments. (F) Survival, (G) parasitemia, (H) enhanced respiratory pause (Penh) and (I) respiratory frequency (RF) on the 7^th dpi in <i>P</i>. <i>berghei</i> ANKA-infected mice treated with elastase inhibitor (30 mg/kg) or saline solution (control group) on the 3^rd and 6^th dpi. Data are representative of one experiment. (J) Lung tissue on the day of death from representative control mice (died on the 10^th dpi with ALI/ARDS), Pulmozyme-treated mice (died on the 20^th dpi), and Sivelestat-treated mice (died on the 20^th dpi) stained with HE (400x, scale bar 25 μm). Data are expressed as the mean ± SEM (C, log-rank test and Wilcoxon-Gehan-Breslow, p<0.05; Mann Whitey-test, *** p <0.001; n = 10–15 mice/experiment). The dashed line represents the mean value of non-infected mice (NI).</p

<i>Plasmodium berghei</i>-iRBCs promote more NETs than RBCs in mouse neutrophils.

<p>(A) Immunofluorescence of non-stimulated (NS) DBA/2 mouse neutrophils and of neutrophils stimulated with PMA [positive control (PC)], red blood cells (RBCs), and <i>P</i>. <i>berghei</i> ANKA (iRBCs) stained with Sytox Green (630x, scale bar 20 μm). (B) Quantification of extracellular DNA (NETs) using Sytox Green based on units of arbitrary fluorescence (UAF). Measurements were performed after 60, 120 and 180 minutes of stimulation. The data are representative of three independent experiments with mean ± SEM (Kruskal-Wallis test where *** p <0.001, between iRBC and RBC).</p