VEGF promotes malaria-associated acute lung injury in mice

Abstract

The spectrum of the clinical presentation and severity of malaria infections is broad, ranging from uncomplicated febrile illness to severe forms of disease such as cerebral malaria (CM), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), pregnancy-associated malaria (PAM) or severe anemia (SA). Rodent models that mimic human CM, PAM and SA syndromes have been established. Here, we show that DBA/2 mice infected with P. berghei ANKA constitute a new model for malaria-associated ALI. Up to 60% of the mice showed dyspnea, airway obstruction and hypoxemia and died between days 7 and 12 post-infection. The most common pathological findings were pleural effusion, pulmonary hemorrhage and edema, consistent with increased lung vessel permeability, while the blood-brain barrier was intact. Malaria-associated ALI correlated with high levels of circulating VEGF, produced de novo in the spleen, and its blockage led to protection of mice from this syndrome. In addition, either splenectomization or administration of the anti-inflammatory molecule carbon monoxide led to a significant reduction in the levels of sera VEGF and to protection from ALI. The similarities between the physiopathological lesions described here and the ones occurring in humans, as well as the demonstration that VEGF is a critical host factor in the onset of malaria-associated ALI in mice, not only offers important mechanistic insights into the processes underlying the pathology related with malaria but may also pave the way for interventional studies.

Figure 1. Infection of C57BL/6, BALB/c and DBA/2 mice with P. berghei ANKA.

(A) Survival and (B) parasitemia curves are shown for C57BL/6 (B6) (n = 7), BALB/c (n = 9) and DBA mice (n = 9) and mice. Parasitemias are shown as mean ± standard deviation. (C, D) Penh (enhanced pause) values for non-infected (NI) versus P. berghei ANKA infected DBA mice (n = 21). (E) Respiratory frequency values for non-infected (NI) versus P. berghei ANKA-infected DBA mice (n = 21). ALI and HP groups were defined at the end of each experiment according to cause of death. (F) PaO2/FI(O2) values for non-infected (NI) versus P. berghei ANKA infected DBA mice (n = 6). Values for ALI mice were obtained after the onset of ALI symptoms. Values for HP mice were obtained after day 12 of infection and on mice not displaying ALI symptoms. Results are shown as mean concentration ± standard deviation. (*P<0.05).

Figure 2. Infection of DBA/2 with P. berghei ANKA does not cause brain damage.

(A) Cranium and Hematoxylin-Eosin staining of brain sections of P. berghei ANKA-infected C57BL/6, B6 (CM), BALB/c, BALB (HP), and DBA mice, DBA (ALI) or DBA (HP) (5 µm). Images are representative of 9 mice in 3 independent experiments. (B) Quantification of cerebral hemorrhagic foci area in brain-sections of the same group of mice. Results are shown as mean concentration ± standard deviation (n = 9 animals per group).

Figure 3. Infection of DBA/2 with P. berghei ANKA leads to lung vascular permeabilization without compromising BBB.

(A, B) Assessment of BBB and lung vascular permeabilization by Evans Blue (EB) quantification, during the onset of ECM, i.e. C57BL/6 mice, B6 (CM), hyperparasitemia, i.e. BALB/c mice, BALB (HP), and ALI, i.e. DBA mice, DBA (ALI). Evans Blue quantification is shown as mean µg of Evans Blue (EB) per g of brain or lung tissue ± standard deviation (n = 5–11 animals per group).

Figure 4. Infection of DBA/2 with P. berghei ANKA constitutes a rodent model for malaria-associated acute lung injury (ALI).

(A–C) Hematoxylin-Eosin staining of lung sections (5 µm) and quantification of edema and haemorrhages, using a blinded scoring system (1-mild, 4-severe), during the onset of ECM, i.e. C57BL/6 mice, B6 (CM), hyperparasitemia, i.e. BALB/c mice, BALB (HP), and ALI, i.e. DBA mice, DBA (ALI). Images are representative of 9 mice in 3 independent experiments. The bar corresponds to 100 µm.

Figure 5. VEGF correlates with ALI onset during malaria infection.

(A) Levels of VEGF protein in the sera of P. berghei ANKA-infected DBA mice with ALI and hyperparasitemia (HP), compared to non-infected mice (NI). Results are shown as mean concentration ± standard deviation (n = 6, 23 and 46 mouse sera per group, for NI, ALI and HP, respectively). (B) Expression of VEGF mRNA levels in the lung, spleen, liver and kidney of P. berghei ANKA-infected DBA mice with ALI and HP (n = 15 animals per group), when compared to non-infected mice. (C) Correlation between VEGF protein levels in the serum and mRNA expression of VEGF in the spleen of P. berghei ANKA-infected DBA mice with ALI. (D) Levels of VEGF protein in the sera of different strains of mice (C57BL/6, BALB/c and DBA) infected with different Plasmodia (P. berghei ANKA, P. berghei NK65 - PbNK, P. yoelii yoelii 17X – Py, and P. chabaudi chabaudi AS - Pc) (n = 10 animals per group). Results are shown as mean concentration ± standard deviation. (*P<0.001).

Figure 6. Spleen is required for the onset of malaria-associated ALI.

(A) Survival and (B) parasitemia of splenectomised and control P. berghei ANKA-infected DBA mice. (C) Levels of VEGF in the serum of non-infected (NI) DBA mice, P. berghei ANKA-infected DBA mice with ALI and splenectomised P. berghei ANKA-infected DBA mice were taken on the same day as control DBA infected mice developed ALI. Results are shown as mean concentration ± standard deviation. (*P<0.01).

Figure 7. VEGF promotes ALI onset in mice with malaria.

(A) Levels of the soluble form of VEGF receptor (sFLT1) in the serum of P. berghei ANKA-infected DBA mice with ALI or mice in HP group. Since only small volumes of blood from the mouse tail vein can be used for this determination, the group classification was only performed by the end of each experiment by determining the cause of death. (B) Levels of the soluble form of VEGF receptor (sFLT1) in the serum of P. berghei ANKA-infected DBA mice after the administration, by intraperitoneal injection on day 3 and day 5 after infection, of LacZ and sFLT1-expressing adenoviruses (n = 11 animals per group). Results are shown as mean concentration ± standard deviation. (C) Survival of LacZ and sFLT1-expressing adenoviruses treated P. berghei ANKA-infected DBA mice. (D) Levels of VEGF in the serum of the control LacZ-expressing adenoviruses-treated P. berghei ANKA-infected DBA (ALI and non-ALI/HP) mice versus the sFLT1-expressing adenoviruses-treated P. berghei ANKA-infected DBA (non-ALI/HP) mice. Results are shown as mean concentration ± standard deviation. (*P<0.05).

Figure 8. Exposure to CO suppresses onset of malaria-associated ALI.

(A–B) Effect of CO inhalation on P. berghei ANKA-infected DBA mice survival (A) and parasitemia (B). CO inhalation (starting at day 2 after infection and during 72 h) was compared to normal atmosphere. Parasitemias are shown as mean ± standard deviation (n = 10 animals per group). (C) Levels of VEGF in the sera of P. berghei ANKA-infected DBA mice exposed to air or CO. Results are shown as mean concentration ± standard deviation (n = 3 mice per group). (*P<0.01) (D–F) Quantification of edema and haemorrhages of hematoxylin-eosin-stained lung sections of non-infected DBA mice (NI DBA) versus P. berghei ANKA-infected DBA mice exposed to air, I DBA (ALI), or CO, I DBA+CO, using a blinded score system (1-mild, 4-severe). Images are representative of 6 mice in 2 independent experiments.