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Spotlight
Targeting the olfactory bulb during experimental cerebral malaria Laurent Re´nia and Shanshan Wu Howland Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), Biopolis, 138648 Singapore
Malaria is responsible for over 500 million clinical cases and over 500 000 deaths annually. Fatalities arise from a range of overlapping syndromes, such as cerebral malaria, whose pathogenesis is still incompletely understood. In a new study, Coban and colleagues provide new clues on the involvement of the olfactory bulb during experimental cerebral malaria in mice that open the way to testable hypotheses and potentially earlier intervention in humans. Human cerebral malaria (HCM) is the most severe pathology induced by Plasmodium infection (primarily Plasmodium falciparum). It encompasses a wide range of neurological complications with seizure and impaired consciousness being the more prominent [1]. Owing to ethical constraints, studies to understand HCM pathogenesis have largely relied on post-mortem autopsy brain samples, in which the accumulation of P. falciparum-infected red blood cells has frequently been observed. This has led to the dogma that parasite sequestration is the main culprit behind HCM [2] and that the best treatments are antimalarial drugs that rapidly reduce parasite load. However, even when treated with fast-acting artemisinin combination therapies, a significant fraction of P. falciparum-infected patients succumb to HCM [3]. These studies have suggested that the mechanisms leading to HCM are more complex and probably involve immunological components [4], and that adjunct therapies need to be developed. Because invasive experimental studies are impossible in humans, parasite dynamics and distribution in vivo in the brain and their interactions with brain-residing and immune host cells cannot be investigated. This has led scientists to turn to animal models that can help to test hypotheses. Susceptible mice such as C57BL/6 infected with Plasmodium berghei develop brain pathology and die with overt neurological signs between 6 and 14 days post-infection [5]. Called experimental cerebral malaria (ECM), this model has been instrumental in uncovering the role of many immune mediators in the development of this pathology. However, it has been argued that there are irreconcilable differences between HCM and ECM, mainly based on the observation that in histopathological brain samples from mice dying of ECM, leukocytes are sequestered as opposed to parasitized erythrocytes as seen in humans. However, Corresponding author: Re´nia, L. ([email protected]). Keywords: cerebral malaria; Plasmodium; brain; pathology; olfactory bulb. 1471-4922/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2014.05.002
this view has been challenged recently. Bioluminescence imaging of luciferase-expressing transgenic P. berghei has clearly demonstrated that some infected red blood cells do sequester in the brains of infected animals [6], in part by their capacity to cytoadhere to endothelial cells [7]. In an effort to provide a more detailed and dynamic picture of the modifications occurring in the brain during ECM, Coban and colleagues, in a study published this month in Cell Host and Microbe [8] have used two-photon imaging (TPI) and magnetic resonance imaging (MRI) on live mice. TPI allows tissues to be analyzed with increased depth and with fewer drawbacks such as photobleaching and phototoxicity. By contrast, MRI allows precise visualization of brain anatomy. Their most important finding is that the olfactory bulb is the first brain structure to be affected. Five days post-infection, mice developed bleeding in the olfactory bulb (OLB) as seen by MRI. Using TPI, they further showed that P. berghei-infected red blood cells are sequestered in and even occlude the OLB capillaries. This accumulation was accompanied by the recruitment of activated CD8+ T cells that crawl along OLB capillaries and even reach the OLB parenchyma. Although the exact nature and consequences of OLB capillary–CD8+ T cell interaction remain unclear, it is highly suggestive that brain capillaries to cross-present Plasmodium antigen during ECM while cognate cytolytic CD8+ T cells accumulate intracerebrally. Did morphological dysfunction lead to physiological dysfunction of the OLB? Using a simple behavioral test measuring the time taken for mice to find buried food, the authors demonstrated that olfaction deteriorates as early as 4 days after infection, 1–3 days before overt neurological signs manifest. One application of this behavioral test is to facilitate early diagnosis of ECM under experimental conditions where not all mice develop neurological signs. If the same dysfunction also happens in HCM, olfaction tests could revolutionize triage of malaria patients, particularly in resource-poor settings. Another important application of these behavioral tests is for the development of adjunct therapy. Up to now, the utility of studying adjunct therapy with the ECM model has been a serious point of contention in the malaria community. Because of the absence of predictive markers, many treatments were initiated before the onset of ECM and thus confounded effects on parasite growth and ECM pathogenesis. In addition, to be relevant to HCM where patients only seek medical care when symptoms develop, adjunct treatment should be initiated only when mice exhibit neurological signs. This poses a difficulty as mice Trends in Parasitology xx (2014) 1–2
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Spotlight frequently die within 24 h of the onset of these symptoms. With this study, treatments can now be tested as soon as the mice display olfaction deficits in behavioral tests. New adjunct therapies initially tested in mice could have clinical benefit even if olfaction alterations turn out to be irrelevant in humans, as long as another early HCM diagnosis criterion is identified. Another noteworthy finding of this study is that mice infected with P. berghei develop fever. Using an elegant set-up with a sensitive thermal camera and continuous recording, they show that P. berghei-infected mice, but not mice infected with a non-ECM-inducing parasite strain, have a peak of fever at the onset of ECM before developing hypothermia. In previous studies with manual recording of temperature, only hypothermia was reported because fever was obscured by the circadian rhythm. In humans, recurrent fever is characteristic of Plasmodium infection, and the absence of fever in mice was previously put forward to dismiss mouse models in general. This new finding brings ECM closer to its human counterpart. Another striking finding of this study is that fever seems to occur by a mechanism different from those commonly proposed [9]. Fever caused by the bacterial molecule lipopolysaccharide (LPS) has been thoroughly described in mice and humans and is mediated by cytokines such as interleukin 1b (IL-1b), tumor necrosis factor (TNF)-a, and IL-6. Of these, only IL-6 levels were elevated at the time of ECMassociated fever. Lastly, it remains unclear in this study how damage in OLB and fever are related. These results will open new lines of research to understand the mechanisms of fever during malaria and HCM.
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In conclusion, this study using cutting-edge technologies has uncovered specific mechanisms leading to ECM that can be tested in HCM and substantiates the informed use of the ECM model [10].
Acknowledgments This work was supported by an intramural grant from Singapore’s Agency for Science, Technology and Research (A*STAR).
References 1 World Health Organization (2000) Severe falciparum malaria. World Health Organization, Communicable Diseases Cluster. Trans. R. Soc. Trop. Med. Hyg. 94, S1–S90 2 White, N.J. et al. (2013) Lethal malaria: marchiafava and bignami were right. J. Infect. Dis. 208, 192–198 3 Dondorp, A.M. et al. (2005) Artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial. Lancet 366, 717–725 4 Schofield, L. and Grau, G.E. (2005) Immunological processes in malaria pathogenesis. Nat. Rev. Immunol. 5, 722–735 5 Engwerda, C.R. et al. (2005) Experimental models of cerebral malaria. Curr. Top. Immunol. 297, 103–143 6 Claser, C. et al. (2011) CD8+ T cells and IFN-gamma mediate the timedependent accumulation of infected red blood cells in deep organs during experimental cerebral malaria. PLoS ONE 6, e18720 7 El-Assaad, F. et al. (2013) Cytoadherence of Plasmodium bergheiinfected red blood cells to murine brain and lung microvascular endothelial cells in vitro. Infect. Immun. 81, 3984–3991 8 Zhao, H. et al. (2014) Olfactory plays a key role in spatiotemporal pathogenesis of cerebral malaria. Cell Host Microbe http://dx.doi.org/ 10.1016/j.chom.2014.04.008 9 Netea, M.G. et al. (2000) Circulating cytokines as mediators of fever. Clin. Infect. Dis. 31, S178–S184 10 Renia, L. et al. (2010) Cerebral malaria: in praise of epistemes. Trends Parasitol. 26, 275–276
Spotlight
Targeting the olfactory bulb during experimental cerebral malaria Laurent Re´nia and Shanshan Wu Howland Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), Biopolis, 138648 Singapore
Malaria is responsible for over 500 million clinical cases and over 500 000 deaths annually. Fatalities arise from a range of overlapping syndromes, such as cerebral malaria, whose pathogenesis is still incompletely understood. In a new study, Coban and colleagues provide new clues on the involvement of the olfactory bulb during experimental cerebral malaria in mice that open the way to testable hypotheses and potentially earlier intervention in humans. Human cerebral malaria (HCM) is the most severe pathology induced by Plasmodium infection (primarily Plasmodium falciparum). It encompasses a wide range of neurological complications with seizure and impaired consciousness being the more prominent [1]. Owing to ethical constraints, studies to understand HCM pathogenesis have largely relied on post-mortem autopsy brain samples, in which the accumulation of P. falciparum-infected red blood cells has frequently been observed. This has led to the dogma that parasite sequestration is the main culprit behind HCM [2] and that the best treatments are antimalarial drugs that rapidly reduce parasite load. However, even when treated with fast-acting artemisinin combination therapies, a significant fraction of P. falciparum-infected patients succumb to HCM [3]. These studies have suggested that the mechanisms leading to HCM are more complex and probably involve immunological components [4], and that adjunct therapies need to be developed. Because invasive experimental studies are impossible in humans, parasite dynamics and distribution in vivo in the brain and their interactions with brain-residing and immune host cells cannot be investigated. This has led scientists to turn to animal models that can help to test hypotheses. Susceptible mice such as C57BL/6 infected with Plasmodium berghei develop brain pathology and die with overt neurological signs between 6 and 14 days post-infection [5]. Called experimental cerebral malaria (ECM), this model has been instrumental in uncovering the role of many immune mediators in the development of this pathology. However, it has been argued that there are irreconcilable differences between HCM and ECM, mainly based on the observation that in histopathological brain samples from mice dying of ECM, leukocytes are sequestered as opposed to parasitized erythrocytes as seen in humans. However, Corresponding author: Re´nia, L. ([email protected]). Keywords: cerebral malaria; Plasmodium; brain; pathology; olfactory bulb. 1471-4922/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2014.05.002
this view has been challenged recently. Bioluminescence imaging of luciferase-expressing transgenic P. berghei has clearly demonstrated that some infected red blood cells do sequester in the brains of infected animals [6], in part by their capacity to cytoadhere to endothelial cells [7]. In an effort to provide a more detailed and dynamic picture of the modifications occurring in the brain during ECM, Coban and colleagues, in a study published this month in Cell Host and Microbe [8] have used two-photon imaging (TPI) and magnetic resonance imaging (MRI) on live mice. TPI allows tissues to be analyzed with increased depth and with fewer drawbacks such as photobleaching and phototoxicity. By contrast, MRI allows precise visualization of brain anatomy. Their most important finding is that the olfactory bulb is the first brain structure to be affected. Five days post-infection, mice developed bleeding in the olfactory bulb (OLB) as seen by MRI. Using TPI, they further showed that P. berghei-infected red blood cells are sequestered in and even occlude the OLB capillaries. This accumulation was accompanied by the recruitment of activated CD8+ T cells that crawl along OLB capillaries and even reach the OLB parenchyma. Although the exact nature and consequences of OLB capillary–CD8+ T cell interaction remain unclear, it is highly suggestive that brain capillaries to cross-present Plasmodium antigen during ECM while cognate cytolytic CD8+ T cells accumulate intracerebrally. Did morphological dysfunction lead to physiological dysfunction of the OLB? Using a simple behavioral test measuring the time taken for mice to find buried food, the authors demonstrated that olfaction deteriorates as early as 4 days after infection, 1–3 days before overt neurological signs manifest. One application of this behavioral test is to facilitate early diagnosis of ECM under experimental conditions where not all mice develop neurological signs. If the same dysfunction also happens in HCM, olfaction tests could revolutionize triage of malaria patients, particularly in resource-poor settings. Another important application of these behavioral tests is for the development of adjunct therapy. Up to now, the utility of studying adjunct therapy with the ECM model has been a serious point of contention in the malaria community. Because of the absence of predictive markers, many treatments were initiated before the onset of ECM and thus confounded effects on parasite growth and ECM pathogenesis. In addition, to be relevant to HCM where patients only seek medical care when symptoms develop, adjunct treatment should be initiated only when mice exhibit neurological signs. This poses a difficulty as mice Trends in Parasitology xx (2014) 1–2
1
TREPAR-1291; No. of Pages 2
Spotlight frequently die within 24 h of the onset of these symptoms. With this study, treatments can now be tested as soon as the mice display olfaction deficits in behavioral tests. New adjunct therapies initially tested in mice could have clinical benefit even if olfaction alterations turn out to be irrelevant in humans, as long as another early HCM diagnosis criterion is identified. Another noteworthy finding of this study is that mice infected with P. berghei develop fever. Using an elegant set-up with a sensitive thermal camera and continuous recording, they show that P. berghei-infected mice, but not mice infected with a non-ECM-inducing parasite strain, have a peak of fever at the onset of ECM before developing hypothermia. In previous studies with manual recording of temperature, only hypothermia was reported because fever was obscured by the circadian rhythm. In humans, recurrent fever is characteristic of Plasmodium infection, and the absence of fever in mice was previously put forward to dismiss mouse models in general. This new finding brings ECM closer to its human counterpart. Another striking finding of this study is that fever seems to occur by a mechanism different from those commonly proposed [9]. Fever caused by the bacterial molecule lipopolysaccharide (LPS) has been thoroughly described in mice and humans and is mediated by cytokines such as interleukin 1b (IL-1b), tumor necrosis factor (TNF)-a, and IL-6. Of these, only IL-6 levels were elevated at the time of ECMassociated fever. Lastly, it remains unclear in this study how damage in OLB and fever are related. These results will open new lines of research to understand the mechanisms of fever during malaria and HCM.
2
Trends in Parasitology xxx xxxx, Vol. xxx, No. x
In conclusion, this study using cutting-edge technologies has uncovered specific mechanisms leading to ECM that can be tested in HCM and substantiates the informed use of the ECM model [10].
Acknowledgments This work was supported by an intramural grant from Singapore’s Agency for Science, Technology and Research (A*STAR).
References 1 World Health Organization (2000) Severe falciparum malaria. World Health Organization, Communicable Diseases Cluster. Trans. R. Soc. Trop. Med. Hyg. 94, S1–S90 2 White, N.J. et al. (2013) Lethal malaria: marchiafava and bignami were right. J. Infect. Dis. 208, 192–198 3 Dondorp, A.M. et al. (2005) Artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial. Lancet 366, 717–725 4 Schofield, L. and Grau, G.E. (2005) Immunological processes in malaria pathogenesis. Nat. Rev. Immunol. 5, 722–735 5 Engwerda, C.R. et al. (2005) Experimental models of cerebral malaria. Curr. Top. Immunol. 297, 103–143 6 Claser, C. et al. (2011) CD8+ T cells and IFN-gamma mediate the timedependent accumulation of infected red blood cells in deep organs during experimental cerebral malaria. PLoS ONE 6, e18720 7 El-Assaad, F. et al. (2013) Cytoadherence of Plasmodium bergheiinfected red blood cells to murine brain and lung microvascular endothelial cells in vitro. Infect. Immun. 81, 3984–3991 8 Zhao, H. et al. (2014) Olfactory plays a key role in spatiotemporal pathogenesis of cerebral malaria. Cell Host Microbe http://dx.doi.org/ 10.1016/j.chom.2014.04.008 9 Netea, M.G. et al. (2000) Circulating cytokines as mediators of fever. Clin. Infect. Dis. 31, S178–S184 10 Renia, L. et al. (2010) Cerebral malaria: in praise of epistemes. Trends Parasitol. 26, 275–276
