Inhibition of hypoxia-associated response and kynurenine production in response to hyperbaric oxygen as mechanisms involved in protection against experimental cerebral malaria

Abstract

Cerebral malaria (CM) is a multifactorial syndrome involving an exacerbated proinflammatory status, endothelial cell activation, coagulopathy, hypoxia, and accumulation of leukocytes and parasites in the brain microvasculature. Despite significant improvements in malaria control, 15% of mortality is still observed in CM cases, and 25% of survivors develop neurologic sequelae for life-even after appropriate antimalarial therapy. A treatment that ameliorates CM clinical signs, resulting in complete healing, is urgently needed. Previously, we showed a hyperbaric oxygen (HBO)-protective effect against experimental CM. Here, we provide molecular evidence that HBO targets brain endothelial cells by decreasing their activation and inhibits parasite and leukocyte accumulation, thus improving cerebral microcirculatory blood flow. HBO treatment increased the expression of aryl hydrocarbon receptor over hypoxia-inducible factor 1-α (HIF-1α), an oxygen-sensitive cytosolic receptor, along with decreased indoleamine 2,3-dioxygenase 1 expression and kynurenine levels. Moreover, ablation of HIF-1α expression in endothelial cells in mice conferred protection against CM and improved survival. We propose that HBO should be pursued as an adjunctive therapy in CM patients to prolong survival and diminish deleterious proinflammatory reaction. Furthermore, our data support the use of HBO in therapeutic strategies to improve outcomes of non-CM disorders affecting the brain.-Bastos, M. F., Kayano, A. C. A. V., Silva-Filho, J. L., Dos-Santos, J. C. K., Judice, C., Blanco, Y. C., Shryock, N., Sercundes, M. K., Ortolan, L. S., Francelin, C., Leite, J. A., Oliveira, R., Elias, R. M., Câmara, N. O. S., Lopes, S. C. P., Albrecht, L., Farias, A. S., Vicente, C. P., Werneck, C. C., Giorgio, S., Verinaud, L., Epiphanio, S., Marinho, C. R. F., Lalwani, P., Amino, R., Aliberti, J., Costa, F. T. M. Inhibition of hypoxia-associated response and kynurenine production in response to hyperbaric oxygen as mechanisms involved in protection against experimental cerebral malaria.

Keywords: aryl-hydrocarbon receptor; brain; endothelial cell activation; tryptophan metabolism.

Figure 1

HBO reduces parasite and leukocyte accumulation in the brain of infected mice after intracardiac perfusion. Mice infected with PbA (PbA group) were exposed daily, or not, to HBO (PbA-HBO) conditions (100% O₂, 3 ATA, 1 h). A, B) On d 6–7 p.i., when mice showed signs of ECM, brains were collected in nonflushed (A) or flushed (B) animals, and P. berghei 18S rRNA levels were quantified by qRT-PCR. Values represent the means of specific 18S gene expression normalized to HPRT ± sd. **P < 0.01 (Student’s t test). C, D) With the use of intravital microscopy, leukocyte adhesion to brain microvasculature and microcirculation velocity of groups of 4–6 mice, NI or infected with PbA and exposed to HBO (PbA-HBO) or not HBO-treated (PbA) were assessed on d 5 p.i. Results are expressed as the means of adhered leukocytes per 100 µm in at least 4 vessels ± sd. **P < 0.01, ***P < 0.001 (ANOVA test). Representative brain microcirculation photomicrographs of NI, PbA, and PbA-HBO mice show leukocyte adhesion (C) and microcirculation flow velocity (D) of rhodamine 6G-labeled cells. The triangles indicate cells with high speed, diamonds represent cells with low speed that drag on the endothelium, and the red dots represent the mean velocities of labeled cells. E, F) On d 6–7 p.i., when mice showed signs of ECM, brains were collected and Lfa1 and Perforin levels were quantified by qRT-PCR. Results were normalized to HPRT, and means ± sd of biologic triplicates are shown compared with NI mice. Ns, nonsignificant, **P < 0.01, ***P < 0.001 (ANOVA test).

Figure 2

Genetic expression of integrins in peripheral blood- or spleen-derived leukocytes. Groups of 5–7 animals infected with 10⁶ PbA iEs (PbA group) were daily exposed, or not, to HBO (PbA-HBO) conditions (100% O₂, 3 ATA, 1 h). On d 6–7 p.i., when PbA mice showed signs of ECM, leukocytes were isolated from the peripheral blood [peripheral blood mononuclear cells (PBMCs)] (A–C) or from the spleen and itga4: ITGA4 (D); itgb1: ITGB1 (E); and itgb2: ITGB2 (F) levels were assessed by qRT-PCR. Results were normalized to HPRT, and means and sd of biologic triplicates are shown compared with NI mice. *P < 0.05.

Figure 3

HBO reduces endothelial activation in the brain of infected animals. Total brain mRNA from PbA-infected mice or PbA mice exposed to HBO (PbA-HBO) was submitted to qRT-PCR for Icam1(A), Epcr (B), and tissue factor (C) genes. Results were normalized to HPRT, and means ± sd of biologic triplicates are shown compared with NI mice. *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA test).

Figure 4

HBO reduces hypoxia in the brain of infected animals. A) Representative images of HIF-1α⁺ cells in brains of NI, PbA, and PbA-HBO mice. Values represent means ± sd of 6 mice per group and are representative of 2 independent experiments with similar results. *P < 0.05, ***P < 0.001 (ANOVA test). B) On d 6–7 p.i., brains were collected and AhR expression was assessed by Western blot. Values represent means ± sd of 6 mice per group and are representative of 2 independent experiments with similar results. *P < 0.05 (ANOVA test). C) Hmox1 brain expression levels in PbA and PbA-HBO mice were determined by qRT-PCR. Results were normalized to HPRT, and means and sd of biologic triplicates are shown compared with NI mice. ***P < 0.001 (ANOVA test). D) Quantification of free heme was determined, at d 4–6 in the plasma of infected mice exposed, or not, to HBO. The results represent the average of 6–8 animals per group ± sd. *P < 0.05 (ANOVA test).

Figure 5

Lack of HIF-1α expression in endothelial cells is protective against ECM. A–D) Groups of 10–15 wild-type (WT) or Tie2cre HIF-1α^fl/fl mice lacking HIF-1α in endothelial cells were infected with 10⁶ PbA-iEs. On d 6–7 p.i., Ahr (A), Hmox (B), il12a (C), and il12b (D) levels were assessed by qRT-PCR. As controls, brains of NI mice were used. Values were expressed as the means of specific Ahr-, Hmox-, il12a-, or il12b-normalized expression of 10 mice ± sd. **P < 0.01, *P < 0.05 (ANOVA test). E) Tissue factor protein expression was determined by the mean fluorescence intensity (MFI) in CD31⁺CD11b⁻ (endothelial cells). The results represent the average of 10 animals per group ± sd. ***P < 0.001 (ANOVA test). F) Survival of the indicated PbA-infected mouse strains.

Figure 6

HBO treatment inhibits the KP of the Trp metabolism. A) Ido1 mRNA brain expression levels from PbA-iE or PbA-HBO mouse groups were determined by qRT-PCR. The NI mouse group was used as control. *P < 0.05, ***P < 0.001 (ANOVA test). B–D) Concentration of Trp (B), Kyn (µm) (C), and Kyn:Trp (D) ratio were determined in the serum of the different mouse groups. Values represent means ± sd of 7 mice per group and are representative of 2 independent experiments with similar results. **P < 0.01, ***P < 0.001 (ANOVA test).