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Am. J. Trop. Med. Hyg., 79(6), 2008, pp. 823-825
Copyright © 2008 by The American Society of Tropical Medicine and Hygiene

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SHORT REPORT


Failure of Two Distinct Anti-apoptotic Approaches to Reduce Mortality in Experimental Cerebral Malaria

Andrew J. Helmers, Fiona E. Lovegrove, John M. Harlan, Kevin C. Kain{dagger}, AND W. Conrad Liles*,{dagger}
Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada; McLaughlin-Rotman Centre for Global Health, McLaughlin Centre for Molecular Medicine, University of Toronto, Toronto, Ontario, Canada; Department of Medicine, University of Washington, Seattle, Washington; Division of Infectious Diseases, Department of Medicine, University of Toronto, Toronto, Ontario, Canada; Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada

 

ABSTRACT

Cerebral malaria is responsible for a high proportion of mortality in human Plasmodium falciparum infection. Previous studies have reported the presence of apoptosis in endothelial cells, astrocytes, neurons, and glial cells in experimental murine cerebral malaria caused by infection with Plasmodium berghei ANKA. Using this model, we tested two strategies, which have been shown to improve survival in murine models of sepsis: 1) treatment with z-VAD, a pancaspase inhibitor; and 2) overexpression of Bcl-2 using transgenic mice expressing human Bcl-2 (which prevents the release of apoptotic mediators from the mitochondria) from a myeloid cell promoter. Neither of these anti-apoptotic strategies, previously shown to provide therapeutic benefit in sepsis, improved survival in experimental cerebral malaria.

Plasmodium falciparum infection causes the most severe form of human malaria, and cerebral malaria (CM) accounts for a significant proportion of associated morbidity and mortality. The CM is more common in children, who lack the protective benefit of partial immunity. The pathophysiology of CM resembles that of sepsis-related multiple organ dysfunction syndrome, in which exaggerated, dysregulated host immune responses (including the upregulation of pro-inflammatory cytokines, such as interferon [IFN-{gamma}], tumor necrosis factor [TNF], and lymphotoxin-{alpha}) have been implicated as important pathogenic mechanisms.1,2 Indeed, malaria is a leading cause of non-bacterial sepsis in the developing world.3 A subset of children who survive CM develop long-term neurologic deficits, mostly paresis and ataxia.4 Infection of mice with Plasmodium berghei ANKA (PbA) mimics many of the clinical features of human CM and serves as an established and well-characterized animal model of this disease.5

Although apoptosis has been observed in experimental murine CM, largely in endothelial cells, astrocytes, neurons, and glial cells, its precise role in the pathogenesis of CM is largely unknown.6,7 Two principal mechanisms of apoptosis have been characterized: 1) the extrinsic pathway; and 2) the intrinsic pathway. The former involves activation of Fas (CD95) or other death receptors by their cognate ligands. Previous studies have demonstrated that lpr/lpr and gld/gld mice (deficient in Fas and Fas ligand [FasL; CD178], respectively) are resistant to experimental CM, suggesting a role for the Fas/FasL system of apoptosis in the pathogenesis of CM.6,8 Interestingly, z-VAD-FMK (z-Val-Ala-Asp-fluoromethylketone), a pancaspase inhibitor, has also been shown to improve survival in the cecal ligation and puncture (CLP) murine model of sepsis.9 The intrinsic pathway is initiated when cytochrome c is released from the mitochondria and interacts with Apaf-1 and caspase-9 to form the apoptosome. Mitochondrial permeability is regulated by a family of proteins containing the Bcl-2-homology (BH) domain, including the pro-apoptotic Bax and BH3-only proteins and the anti-apoptotic proteins Bcl-2 and Bcl-xL.10 Mitochondrial dysfunction and the induction of the pro-apoptotic protein Bax have been reported in the cerebellum of mice with CM caused by PbA infection.11 Furthermore, in the murine model of CLP-related sepsis, survival was improved by a trans effect in transgenic hMRP8-Bcl-2 mice, engineered to express the human Bcl-2 peptide in myeloid cells, compared with wild-type mice.12

On the basis of the aforementioned similarities between the pathophysiology of CM and sepsis, and the potential involvement of apoptosis in CM pathogenesis, we hypothesized that anti-apoptotic therapeutic strategies with demonstrated efficacy in murine models of sepsis would improve outcome in experimental CM. To test this hypothesis, we examined the effect of z-VAD on experimental CM induced by PbA infection in C57BL/6 mice, and evaluated the outcome of PbA infection in hMRP8-Bcl-2 mice. Treatment with z-VAD failed to affect survival in wild-type mice. Similarly, mortality related to CM was not different in hMRP8-Bcl-2 mice, with enforced myeloid expression of anti-apoptotic Bcl-2, compared with wild-type mice.

All animal experiments were carried out with the approval of the University of Toronto Animal Care Committee, in accordance with institutional guidelines. The C57BL/6 wild-type mice were obtained from Charles River Laboratories (Saint-Constant, QC) and housed under pathogen-free conditions with a 12-hour light cycle. The z-VAD (MP Biomedicals, Solon, OH) was dissolved in 100% endotoxin-free dimethyl sulfoxide (DMSO) (Sigma, Oakville, ON) and diluted in sterile phosphate buffered saline (PBS) (2.2% vol/vol) to a final concentration of 0.44 mM—the preparation and treatment protocol for z-VAD was based on previous studies involving this compound.9,13 The PbA parasites (MR4, Manassas, VA) were cultivated by passage through C57BL/6 mice and experimental infections were introduced (Day 0) by the intraperitoneal injection of 5 x 105 parasitized erythrocytes in male mice (7–9 weeks old). The z-VAD treatment was initiated 72 hours after parasite inoculation, with each mouse (N = 10) receiving 4 mg/kg IP every 12 hours. The vehicle-treated group (N = 10) received PBS with 2.2% DMSO. Injections were continued until seven days after the malaria challenge.

Treatment with z-VAD had no effect on survival, yielding a Kaplan-Meier survival curve (Figure 1Go) with a P value of 0.34 (log-rank test). No mice survived in either the control or z-VAD-treated group by Day 9 post-inoculation. These results contrast with those reported from a study by Piguet and others, in which there was a survival benefit (40% survival in z-VAD-treated mice versus no survival in the control group), after treating PbA-infected C57BL/6 mice with z-VAD on Days 7 and 8 post-inoculation.14 It should also be noted that z-VAD has been shown to reduce blood-brain barrier (BBB) permeability, brain edema and brain endothelial apoptosis (in a rat model of endovascular perforation), but it does not cross the BBB; although disruption of the BBB is a recognized feature of experimental CM,15 IP delivery of z-VAD might have a limited ability to affect neuronal apoptosis, especially at earlier stages of the infection.16,17 This pharmacologic drawback is overcome in lpr/lpr and gld/gld mice, where Fas or Fas ligand are genetically absent from neural cells.6 A further consideration is the potential for z-VAD to enhance TNF-mediated toxicity, which might offset any beneficial effects in the severe phase of the infection.18


Figure 1
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  		FIGURE 1. Seven- to nine-week-old male C57BL/6 mice were treated every 12 hours (i.p. injection) with 4 mg/kg of z-VAD or vehicle (N = 10 for each group), beginning 72 hours after Plasmodium berghei ANKA (PbA) inoculation. The 5.0 x 105 PbA parasitized erythrocytes were injected i.p., and mice were monitored at least twice daily during the infection. No significant difference in survival was seen (log-rank test: {chi}2 = 0.90, P = 0.34).

 
In addition to z-VAD, we also evaluated the outcome of PbA infection in hMRP-Bcl-2 mice, generated from a C57BL/6 background as described previously.12 These mice were bred with wild-type C57BL/6 females, and pups were genotyped to ensure heterozygosity for the Bcl-2 gene prior to experimentation. The mice (sex-matched) were 8–12 weeks old, with 16 in each group (wild-type, hMRP8-Bcl-2). The PbA parasites (MR4, Manassas, VA) were cultivated by passage through C57BL/6 mice. Experimental infections were introduced (Day 0) by intraperitoneal injection of 5 x 105 (for males) or 1 x 106 (for females) parasitized erythrocytes.

Brains from hMRP8-Bcl-2 (male, N = 3) and wild-type (male, N = 3) C57BL/6 mice were removed on Day 7 post-infection (PbA) and fixed in 4% paraformaldehyde/PBS. Fixed brains were sliced into four coronal sections (~0.5 cm) and embedded in paraffin, after which the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay was performed on the brain sections as described previously.19 For each mouse, TUNEL-positive cells were counted for two brain sections, each containing four cross-sectional slices of brain, and the average number of cells was recorded. Samples were analyzed and scored in a blinded fashion and a t test was carried out on the TUNEL assay results (Graphpad Prism 4.0b, San Diego, CA).

Previous studies suggest that apoptosis occurs in the brains of CM-susceptible mice, indicating that anti-apoptotic treatment may be a viable adjunctive therapy in CM.20 The use of hMRP8-Bcl-2 mice expressing human Bcl-2 peptide in myeloid cells resulted in no difference in survival compared with wild-type C57BL/6 mice after PbA infection (Figure 2AGo). Both hMRP8-Bcl-2 and wild-type mice developed CM and died by Day 10 post-inoculation. The Kaplan-Meier survival curve had a P value of 0.86—comparing mice of the same sex, the P values were 0.73 (female, hMRP8-Bcl-2 versus wild-type) and 0.32 (male, hMRP8-Bcl-2 versus wild-type). The TUNEL staining was performed on brains from hMRP8-Bcl-2 and wild-type C57BL/6 mice on Day 7 post-inoculation. The average number of TUNEL-positive cells counted for each section is shown (Figure 2BGo). Thus, there was no improvement in survival or reduction in neuronal apoptosis in hMRP8-Bcl-2 mice infected with PbA.


Figure 2
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  		FIGURE 2. A, Wild-type (N = 16, 8 male and 8 female) and hMRP8-Bcl-2 (N = 16, 8 male and 8 female) C57BL/6 mice 8–12 weeks of age were injected i.p. with 5.0 x 105 (male) or 1.0 x 106 (female) Plasmodium berghei ANKA parasitized erythrocytes. The mice were monitored at least twice daily during the infection. No significant difference in survival was seen (log-rank test: {chi}2 = 0.030, P = 0.86). B, hMRP8-Bcl-2 mice had no difference in neural apoptosis as analyzed by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay. A TUNEL assay was performed on brains from wild-type (N = 3) and hMRP8-Bcl-2 (N = 3) C57BL/6 mice, on Day 7 post-infection with P. berghei. Brains were cut into four sections, and two slices were counted for each mouse—the number of apoptotic cells is an average of the total number of TUNEL-stained cells counted for all four sections of each slice. Error bars represent the standard deviation; P = 0.50 (unpaired t test).

 
The hMRP8-Bcl-2 strategy was shown in an earlier study to rescue mice from CLP sepsis, leading us to hypothesize a protective role for this transgene in experimental CM, which involves a similar systemic inflammatory response.12 However, survival was not affected, and TUNEL staining revealed that this strategy had no effect upon neuronal apoptosis—we limited ourselves to the determination of brain cell apoptosis because of its aforementioned implication in CM pathogenesis. Different delivery strategies for Bcl-2 could be explored, such as administering a recombinant peptide or placing the transgene under a neural promoter (such as in the model created by Farlie and others), to modulate the dose, time-course and/or location of the intervention.21

In summary, two anti-apoptotic therapeutic strategies were tested and neither resulted in improved survival in experimental CM. As noted earlier, both strategies have been shown to be effective in improving outcome in a CLP model of sepsis. The lack of protection in experimental CM may reflect the prolonged time course (7–10 days before mortality) in CM pathogenesis compared with sepsis (mortality within 72 hours).9,12 One possible factor in the negative results seen for these two anti-apoptotic strategies is the balance between necrosis and apoptosis. Necrosis, a pro-inflammatory event, can be triggered in some cases by strategies designed to inhibit programmed cell death.22 Thus, any beneficial effects of anti-apoptotic treatment might be offset by the concomitant occurrence of necrosis.

Received May 22, 2008. Accepted for publication August 12, 2008.

Financial support: This work was supported by grants from the Canadian Institutes of Health Research (Team Grant in Malaria CTP 79842 [KCK, Principal Investigator] and MT-13721 [KCK]), and by Genome Canada through the Ontario Genomics Institute. AJH is supported by a CIHR research Studentship, FEL is supported by a CIHR MD/PhD Studentship, KCK by a CIHR Canada Research Chair in Molecular Parasitology, and WCL by a CIHR Canada Research Chair in Infectious Diseases and Inflammation.

* Address correspondence to W. Conrad Liles, Toronto General Hospital 13E 214, 200 Elizabeth Street, Toronto, Ontario M5G 2C4, Canada. E-mail: Conrad.liles{at}uhn.on.ca Back

{dagger} These authors contributed equally to this work. Back

Authors’ addresses: Andrew J. Helmers, Fiona E. Lovegrove, Kevin C. Kain, and W. Conrad Liles, Toronto General Hospital 13E 214, 200 Elizabeth Street, Toronto, Ontario M5G 2C4, Canada, Tel: 416-340-4800 ext. 3535, Fax: 416-340-3357, E-mail: kevin.kain{at}uhn.on.ca. John M. Harlan, Box 357710, University of Washington, Seattle, WA 98195-7710, Tel: 206-543-3360, Fax: 206-543-3560.

 

REFERENCES

  1. Hunt NH, Grau GE, 2003. Cytokines: accelerators and brakes in the pathogenesis of cerebral malaria. Trends Immunol 24: 491–499.[Web of Science][Medline]
  2. Abraham E, Singer M, 2007. Mechanisms of sepsis-induced organ dysfunction. Crit Care Med 35: 2408–2416.[Web of Science][Medline]
  3. Carcillo JA, 2005. Reducing the global burden of sepsis in infants and children: a clinical practice research agenda. Pediatr Crit Care Med 6: S157–S164.[Medline]
  4. van Hensbroek MB, Palmer A, Jaffar S, Schneider G, Kwiatkowski D, 1997. Residual neurologic sequelae after childhood cerebral malaria. J Pediatr 131: 125–129.[Web of Science][Medline]
  5. de Souza JB, Riley EM, 2002. Cerebral malaria: the contribution of studies in animal models to our understanding of immunopathogenesis. Microbes Infect 4: 291–300.[Web of Science][Medline]
  6. Potter SM, Chan-Ling T, Rosinova E, Ball HJ, Mitchell AJ, Hunt NH, 2006. A role for fas-fas ligand interactions during the late-stage neuropathological processes of experimental cerebral malaria. J Neuroimmunol 173: 96–107.[Medline]
  7. Lackner P, Burger C, Pfaller K, Heussler V, Helbok R, Morandell M, Broessner G, Tannich E, Schmutzhard E, Beer R, 2007. Apoptosis in experimental cerebral malaria: spatial profile of cleaved caspase-3 and ultrastructural alterations in different disease stages. Neuropathol Appl Neurobiol 33: 560–571.[Web of Science][Medline]
  8. Kim YS, Schwabe RF, Qian T, Lemasters JJ, Brenner DA, 2002. TRAIL-mediated apoptosis requires NF-{kappa}B inhibition and the mitochondrial permeability transition in human hepatoma cells. Hepatology 36: 1498–1508.[Web of Science][Medline]
  9. Hotchkiss RS, Tinsley KW, Swanson PE, Chang KC, Cobb JP, Buchman TG, Korsmeyer SJ, Karl IE, 1999. Prevention of lymphocyte cell death in sepsis improves survival in mice. Proc Natl Acad Sci USA 96: 14541–14546.[Abstract/Free Full Text]
  10. Chen M, Guerrero AD, Huang L, Shabier Z, Pan M, Tan TH, Wang J, 2007. Caspase-9-induced mitochondrial disruption through cleavage of anti-apoptotic BCL-2 family members. J Biol Chem. 282: 33888–33895.[Abstract/Free Full Text]
  11. Kumar KA, Babu PP, 2002. Mitochondrial anomalies are associated with the induction of intrinsic cell death proteins-bcl(2), bax, cytochrome-c and p53 in mice brain during experimental fatal murine cerebral malaria. Neurosci Lett 329: 319–323.[Medline]
  12. Iwata A, Stevenson VM, Minard A, Tasch M, Tupper J, Lagasse E, Weissman I, Harlan JM, Winn RK, 2003. Over-expression of bcl-2 provides protection in septic mice by a transeffect. J Immunol 171: 3136–3141.[Abstract/Free Full Text]
  13. Srivastava A, Henneke P, Visintin A, Morse SC, Martin V, Watkins C, Paton JC, Wessels MR, Golenbock DT, Malley R, 2005. The apoptotic response to pneumolysin is toll-like receptor 4 dependent and protects against pneumococcal disease. Infect Immun 73: 6479–6487.[Abstract/Free Full Text]
  14. Piguet PF, Kan CD, Vesin C, 2002. Thrombocytopenia in an animal model of malaria is associated with an increased caspase-mediated death of thrombocytes. Apoptosis 7: 91–98.[Medline]
  15. Pamplona A, Ferreira A, Balla J, Jeney V, Balla G, Epiphanio S, Choral A, Rodrigues CD, Gregoire IP, Cunha-Rodrigues M, Portugal S, Soares MP, Mota MM, 2007. Heme oxygenase-1 and carbon monoxide suppress the pathogenesis of experimental cerebral malaria. Nat Med 13: 703–710.[Web of Science][Medline]
  16. Wiessner C, Sauer D, Alaimo D, Allegrini PR, 2000. Protective effect of a caspase inhibitor in models for cerebral ischemia in vitro and in vivo. Cell Mol Biol (Noisy-le-grand) 46: 53–62.[Medline]
  17. Park S, Yamaguchi M, Zhou C, Calvert JW, Tang J, Zhang JH, 2004. Neurovascular protection reduces early brain injury after subarachnoid hemorrhage. Stroke 35: 2412–2417.[Abstract/Free Full Text]
  18. Cauwels A, Janssen B, Waeytens A, Cuvelier C, Brouckaert P, 2003. Caspase inhibition causes hyperacute tumor necrosis factor-induced shock via oxidative stress and phospholipase A2. Nat Immunol 4: 387–393.[Web of Science][Medline]
  19. Wijsman JH, Jonker RR, Keijzer R, van de Velde CJ, Cornelisse CJ, van Dierendonck JH, 1993. A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA. J Histochem Cytochem 41: 7–12.[Abstract]
  20. Lovegrove FE, Gharib SA, Patel SN, Hawkes CA, Kain KC, Liles WC, 2007. Expression microarray analysis implicates apoptosis and interferon-responsive mechanisms in susceptibility to experimental cerebral malaria. Am J Pathol 171: 1894–1903.[Abstract/Free Full Text]
  21. Farlie PG, Dringen R, Rees SM, Kannourakis G, Bernard O, 1995. Bcl-2 transgene expression can protect neurons against developmental and induced cell death. Proc Natl Acad Sci USA 92: 4397–4401.[Abstract/Free Full Text]
  22. Vandenabeele P, Vanden Berghe T, Festjens N, 2006. Caspase inhibitors promote alternative cell death pathways. Sci STKE 2006: pe44.[Abstract/Free Full Text]




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