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    Influence of Nod proteins on susceptibility to cerebral malaria. The 8–12-week-old C57BL/6 mice (+/+) and nod1nod2 −/− (−/−, backcrossed 9 times to the C57BL/6 background) mice were infected with 1 × 106 Plasmodium berghei ANKA parasitized erythrocytes by intraperitaneal injection. Parasitemia was measured every 2 days by thin blood smear. (A) Survival data are combined data from three experiments (N = 21 mice per group; Log rank test: P = 0.62). (B) Parasitemia (N = 10 mice per group; mean ± SEM; Student’s t test on sum values of parasitemia over time for each animal: P = 0.94). Data shown are representative of three independent experiments with a minimum of 5 animals per group.

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    Plasma cytokine levels during Plasmodium berghei ANKA infection. The 8–12-week old C57BL/6 mice (+/+) and nod1nod2−/− (−/−) mice were infected with 1 × 106 parasitized erythrocytes. Plasma was collected on days 1, 5, and 6, and cytokines measured using the CBA mouse inflammation kit (BD Pharmingen) and ELISA IL1-β and KC kits (R&D Systems). Parametric data were analyzed using Student’s t test, whereas the Mann–Whitney test was used for non-parametric data. Data are representative of two independent experiments (with N ≥ 5 mice per group). (A) Plasma levels of interferon-γ (IFN-γ) (left, data are represented as mean ± SEM, P = 0.03, Mann–Whitney test on sum values of cytokine level over time for each animal, N = 10 per group), and IL1-β (right, data are represented as mean ± SEM, P = 0.001, Students t test on sum values of cytokine level over time for each animal, N = 10 per group) over the course of infection. (B) Plasma levels of tumor necrosis factor (TNF) (left, data are represented as mean ± SEM, P = 0.08, Students t test on sum values of cytokine level over time for each animal, N = 10 per group), and IL-6 (right, data are represented as mean ± SEM, P = 0.79, Students t test on sum values of cytokine level over time for each animal, N = 10 per group) over the course of infection. (C) Plasma levels of MCP-1 (left, data are represented as mean ± SEM, P = 0.01, Mann–Whitney test on sum values of cytokine level over time for each animal, N = 10 per group), and KC (right, data are represented as mean ± SEM, P = 0.002, Mann–Whitney test on sum values of cytokine level over time for each animal, N = 10 per group) over the course of infection.

  • 1

    World Health Organization, Communicable Diseases Cluster, 2000. Severe falciparum malaria. Trans R Soc Trop Med Hyg 94 (Suppl 1):S1–S90.

    • Search Google Scholar
    • Export Citation
  • 2

    Lou J, Lucas R, Grau GE, 2001. Pathogenesis of cerebral malaria: recent experimental data and possible applications for humans. Clin Microbiol Rev 14 :810–820.

    • Search Google Scholar
    • Export Citation
  • 3

    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.

    • Search Google Scholar
    • Export Citation
  • 4

    Rest JR, 1982. Cerebral malaria in inbred mice. I. A new model and its pathology. Trans R Soc Trop Med Hyg 76 :410–415.

  • 5

    de Kossodo S, Grau GE, 1993. Role of cytokines and adhesion molecules in malaria immunopathology. Stem Cells 11 :41–48.

  • 6

    Grau GE, Fajardo LF, Piguet PF, Allet B, Lambert PH, Vassalli P, 1987. Tumor necrosis factor (cachectin) as an essential mediator in murine cerebral malaria. Science 237 :1210–1212.

    • Search Google Scholar
    • Export Citation
  • 7

    Grau GE, Piguet PF, Vassalli P, Lambert PH, 1989. Tumor-necrosis factor and other cytokines in cerebral malaria: experimental and clinical data. Immunol Rev 112 :49–70.

    • Search Google Scholar
    • Export Citation
  • 8

    Lucas R, Lou JN, Juillard P, Moore M, Bluethmann H, Grau GE, 1997. Respective role of TNF receptors in the development of experimental cerebral malaria. J Neuroimmunol 72 :143–148.

    • Search Google Scholar
    • Export Citation
  • 9

    Piguet PF, Kan CD, Vesin C, 2002. Role of the tumor necrosis factor receptor 2 (TNFR2) in cerebral malaria in mice. Lab Invest 82 :1155–1166.

    • Search Google Scholar
    • Export Citation
  • 10

    Gowda DC, 2007. TLR-mediated cell signaling by malaria GPIs. Trends Parasitol 23 :596–604.

  • 11

    Lyke KE, Burges R, Cissoko Y, Sangare L, Dao M, Diarra I, Kone A, Harley R, Plowe CV, Doumbo OK, Sztein MB, 2004. Serum levels of the proinflammatory cytokines interleukin-1 beta (IL-1beta), IL-6, IL-8, IL-10, tumor necrosis factor alpha, and IL-12(p70) in Malian children with severe Plasmodium falciparum malaria and matched uncomplicated malaria or healthy controls. Infect Immun 72 :5630–5637.

    • Search Google Scholar
    • Export Citation
  • 12

    Malaguarnera L, Musumeci S, 2002. The immune response to Plasmodium falciparum malaria. Lancet Infect Dis 2 :472–478.

  • 13

    Riley EM, Wahl S, Perkins DJ, Schofield L, 2006. Regulating immunity to malaria. Parasite Immunol 28 :35–49.

  • 14

    Stevenson MM, Riley EM, 2004. Innate immunity to malaria. Nat Rev Immunol 4 :169–180.

  • 15

    Aderem A, 2003. Phagocytosis and the inflammatory response. J Infect Dis 187 (Suppl 2):S340–S345.

  • 16

    Akira S, Uematsu S, Takeuchi O, 2006. Pathogen recognition and innate immunity. Cell 124 :783–801.

  • 17

    Herskovits AA, Auerbuch V, Portnoy DA, 2007. Bacterial ligands generated in a phagosome are targets of the cytosolic innate immune system. PLoS Pathog 3 :e51.

    • Search Google Scholar
    • Export Citation
  • 18

    Medzhitov R, Janeway C Jr, 2000. Innate immune recognition: mechanisms and pathways. Immunol Rev 173 :89–97.

  • 19

    Stuart LM, Ezekowitz RA, 2005. Phagocytosis: elegant complexity. Immunity 22 :539–550.

  • 20

    Chamaillard M, Girardin SE, Viala J, Philpott DJ, 2003. Nods, Nalps and Naip: intracellular regulators of bacterial-induced inflammation. Cell Microbiol 5 :581–592.

    • Search Google Scholar
    • Export Citation
  • 21

    Inohara, Chamaillard, McDonald C, Nuñez G, 2005. NOD-LRR proteins: role in host-microbial interactions and inflammatory disease. Annu Rev Biochem 74 :355–383.

    • Search Google Scholar
    • Export Citation
  • 22

    Medzhitov R, 2001. Toll-like receptors and innate immunity. Nat Rev Immunol 1 :135–145.

  • 23

    Le Bourhis L, Benko S, Girardin SE, 2007. Nod1 and Nod2 in innate immunity and human inflammatory disorders. Biochem Soc Trans 35 :1479–1484.

    • Search Google Scholar
    • Export Citation
  • 24

    Fritz JH, Ferrero RL, Philpott DJ, Girardin SE, 2006. Nod-like proteins in immunity, inflammation and disease. Nat Immunol 7 :1250–1257.

    • Search Google Scholar
    • Export Citation
  • 25

    Coban C, Ishii KJ, Kawai T, Hemmi H, Sato S, Uematsu S, Yamamoto M, Takeuchi O, Itagaki S, Kumar N, Horii T, Akira S, 2005. Toll-like receptor 9 mediates innate immune activation by the malaria pigment hemozoin. J Exp Med 201 :19–25.

    • Search Google Scholar
    • Export Citation
  • 26

    Krishnegowda G, Hajjar AM, Zhu J, Douglass EJ, Uematsu S, Akira S, Woods AS, Gowda DC, 2005. Induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols of Plasmodium falciparum: cell signaling receptors, glycosylphosphatidylinositol (GPI) structural requirement, and regulation of GPI activity. J Biol Chem 280 :8606–8616.

    • Search Google Scholar
    • Export Citation
  • 27

    Parroche P, Lauw FN, Goutagny N, Latz E, Monks BG, Visintin A, Halmen KA, Lamphier M, Olivier M, Bartholomeu DC, Gazzinelli RT, Golenbock DT, 2007. Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to Toll-like receptor 9. Proc Natl Acad Sci USA 104 :1919–1924.

    • Search Google Scholar
    • Export Citation
  • 28

    Coban C, Ishii KJ, Uematsu S, Arisue N, Sato S, Yamamoto M, Kawai T, Takeuchi O, Hisaeda H, Horii T, Akira S, 2007. Pathological role of Toll-like receptor signaling in cerebral malaria. Int Immunol 19 :67–79.

    • Search Google Scholar
    • Export Citation
  • 29

    Griffith JW, O’Connor C, Bernard K, Town T, Goldstein DR, Bucala R, 2007. Toll-like receptor modulation of murine cerebral malaria is dependent on the genetic background of the host. J Infect Dis 196 :1553–1564.

    • Search Google Scholar
    • Export Citation
  • 30

    Lepenies B, Cramer JP, Burchard GD, Wagner H, Kirschning CJ, Jacobs T, 2007. Induction of experimental cerebral malaria is independent of TLR2/4/9. Med Microbiol Immunol 179 :39–44.

    • Search Google Scholar
    • Export Citation
  • 31

    Togbe D, Schofield L, Grau GE, Schnyder B, Boissay V, Charron S, Rose S, Beutler B, Quesniaux VF, Ryffel B, 2007. Murine cerebral malaria development is independent of Toll-like receptor signaling. Am J Pathol 170 :1640–1648.

    • Search Google Scholar
    • Export Citation
  • 32

    Khor CC, Chapman SJ, Vannberg FO, Dunne A, Murphy C, Ling EY, Frodsham AJ, Walley AJ, Kyrieleis O, Khan A, Aucan C, Segal S, Moore CE, Knox K, Campbell SJ, Lienhardt C, Scott A, Aaby P, Sow OY, Grignani RT, Sillah J, Sirugo G, Peshu N, Williams TN, Maitland K, Davies RJ, Kwiatkowski DP, Day NP, Yala D, Crook DW, Marsh K, Berkley JA, O’Neill LA, Hill AV, 2007. A Mal functional variant is associated with protection against invasive pneumococcal disease, bacteremia, malaria and tuberculosis. Nat Genet 39 :523–528.

    • Search Google Scholar
    • Export Citation
  • 33

    Mockenhaupt FP, Cramer JP, Hamann L, Stegemann MS, Eckert J, Oh NR, Otchwemah RN, Dietz E, Ehrhardt S, Schroder NW, Bienzle U, Schumann RR, 2006. Toll-like receptor (TLR) polymorphisms in African children: common TLR-4 variants predispose to severe malaria. Proc Natl Acad Sci USA 103 :177–182.

    • Search Google Scholar
    • Export Citation
  • 34

    Carneiro LA, Magalhaes JG, Tattoli I, Philpott DJ, Travassos LH, 2008. Nod-like proteins in inflammation and disease. J Pathol 214 :136–148.

    • Search Google Scholar
    • Export Citation
  • 35

    Girardin SE, Boneca IG, Carneiro LA, Antignac A, Jehanno M, Viala J, Tedin K, Taha MK, Labigne A, Zahringer U, Coyle AJ, DiStefano PS, Bertin J, Sansonetti PJ, Philpott DJ, 2003. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 300 :1584–1587.

    • Search Google Scholar
    • Export Citation
  • 36

    Girardin SE, Boneca IG, Viala J, Chamaillard M, Labigne A, Thomas G, Philpott DJ, Sansonetti PJ, 2003. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem 278 :8869–8872.

    • Search Google Scholar
    • Export Citation
  • 37

    Kobayashi KS, Chamaillard M, Ogura Y, Henegariu O, Inohara N, Nunez G, Flavell RA, 2005. Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract. Science 307 :731–734.

    • Search Google Scholar
    • Export Citation
  • 38

    Viala J, Chaput C, Boneca IG, Cardona A, Girardin SE, Moran AP, Athman R, Memet S, Huerre MR, Coyle AJ, DiStefano PS, Sansonetti PJ, Labigne A, Bertin J, Philpott DJ, Ferrero RL, 2004. Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag pathogenicity island. Nat Immunol 5 :1166–1174.

    • Search Google Scholar
    • Export Citation
  • 39

    Ockenhouse CF, Hu WC, Kester KE, Cummings JF, Stewart A, Heppner DG, Jedlicka AE, Scott AL, Wolfe ND, Vahey M, Burke DS, 2006. Common and divergent immune response signaling pathways discovered in peripheral blood mononuclear cell gene expression patterns in presymptomatic and clinically apparent malaria. Infect Immun 74 :5561–5573.

    • Search Google Scholar
    • Export Citation
  • 40

    John CC, Panoskaltsis-Mortari A, Opoka RO, Park GS, Orchard PJ, Jurek AM, Idro R, Byarugaba J, Boivin MJ, 2008. Cerebrospinal fluid cytokine levels and cognitive impairment in cerebral malaria. Am J Trop Med Hyg 78 :198–205.

    • Search Google Scholar
    • Export Citation
  • 41

    John CC, Park GS, Sam-Agudu N, Opoka RO, Boivin MJ, 2008. Elevated serum levels of IL-1ra in children with Plasmodium falciparum malaria are associated with increased severity of disease. Cytokine 41 :204–208.

    • Search Google Scholar
    • Export Citation
  • 42

    Ouma C, Davenport GC, Awandare GA, Keller CC, Were T, Otieno MF, Vulule JM, Martinson J, Ong’echa JM, Ferrell RE, Perkins DJ, 2008. Polymorphic variability in the interleukin (IL)-1beta promoter conditions susceptibility to severe malarial anemia and functional changes in IL-1beta production. J Infect Dis 198 :1219–1226.

    • Search Google Scholar
    • Export Citation
  • 43

    Raices RM, Kannan Y, Sarkar A, Bellamkonda-Athmaram V, Wewers MD, 2008. A synergistic role for IL-1beta and TNFalpha in monocyte-derived IFNgamma inducing activity. Cytokine 44 :234–241.

    • Search Google Scholar
    • Export Citation
  • 44

    Yanez DM, Manning DD, Cooley AJ, Weidanz WP, van der Heyde HC, 1996. Participation of lymphocyte subpopulations in the pathogenesis of experimental murine cerebral malaria. J Immunol 157 :1620–1624.

    • Search Google Scholar
    • Export Citation
  • 45

    Amani V, Vigario AM, Belnoue E, Marussig M, Fonseca L, Mazier D, Renia L, 2000. Involvement of IFN-gamma receptor-medicated signaling in pathology and anti-malarial immunity induced by Plasmodium berghei infection. Eur J Immunol 30 :1646–1655.

    • Search Google Scholar
    • Export Citation
  • 46

    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.

    • Search Google Scholar
    • Export Citation
  • 47

    Pedra JH, Sutterwala FS, Sukumaran B, Ogura Y, Qian F, Montgomery RR, Flavell RA, Fikrig E, 2007. ASC/PYCARD and caspase-1 regulate the IL-18/IFN-gamma axis during Anaplasma phagocytophilum infection. J Immunol 179 :4783–4791.

    • Search Google Scholar
    • Export Citation
  • 48

    Mitchell AJ, Hansen AM, Hee L, Ball HJ, Potter SM, Walker JC, Hunt NH, 2005. Early cytokine production is associated with protection from murine cerebral malaria. Infect Immun 73 :5645–5653.

    • Search Google Scholar
    • Export Citation
  • 49

    Ferwerda G, Girardin SE, Kullberg BJ, Le Bourhis L, de Jong DJ, Langenberg DM, van Crevel R, Adema GJ, Ottenhoff TH, Van der Meer JW, Netea MG, 2005. NOD2 and Toll-like receptors are nonredundant recognition systems of Mycobacterium tuberculosis. PLoS Pathog 1 :279–285.

    • Search Google Scholar
    • Export Citation
  • 50

    Rosenstiel P, Fantini M, Brautigam K, Kuhbacher T, Waetzig GH, Seegert D, Schreiber S, 2003. TNF-alpha and IFN-gamma regulate the expression of the NOD2 (CARD15) gene in human intestinal epithelial cells. Gastroenterology 124 :1001–1009.

    • Search Google Scholar
    • Export Citation
  • 51

    Takahashi Y, Isuzugawa K, Murase Y, Imai M, Yamamoto S, Iizuka M, Akira S, Bahr GM, Momotani E, Hori M, Ozaki H, Imakawa K, 2006. Up-regulation of NOD1 and NOD2 through TLR4 and TNF-alpha in LPS-treated murine macrophages. J Vet Med Sci 68 :471–478.

    • Search Google Scholar
    • Export Citation
  • 52

    Hanum PS, Hayano M, Kojima S, 2003. Cytokine and chemokine responses in a cerebral malaria-susceptible or -resistant strain of mice to Plasmodium berghei ANKA infection: early chemokine expression in the brain. Int Immunol 15 :633–640.

    • Search Google Scholar
    • Export Citation

 

 

 

Disruption of Nod-like Receptors Alters Inflammatory Response to Infection but Does Not Confer Protection in Experimental Cerebral Malaria

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  • 1 Tropical Disease Unit, Division of Infectious Diseases, Department of Medicine, Toronto General Hospital, University Health Network, Toronto, Ontario, Canada; McLaughlin-Rotman Centre for Global Health, McLaughlin Centre for Molecular Medicine, University of Toronto, Toronto, Ontario, Canada; Department of Immunology, University of Toronto, Toronto, Ontario, Canada

Research relating to host inflammatory processes during malaria infection has focused on Toll-like receptors, membrane-bound receptors implicated in innate sensing, and phagocytosis of parasitized erythrocytes by host cells. This is the first study to examine the role of Nod proteins, members of the Nod-like receptor (NLR) family of cytoplasmic proteins involved in pathogen recognition, in a murine model of cerebral malaria (Plasmodium berghei ANKA, PbA). Here, we find that nod1nod2−/− mice infected with PbA show no difference in survival or parasitemia compared with wild-type infected animals. However, cytokine levels, notably those associated with NLR activation including interleukin (IL)1-β, KC, and MCP-1, and proteins linked to malaria pathogenesis, such as interferon-γ (IFN-γ), were decreased in the nod-1nod2−/− animals. We therefore demonstrate for the first time that Nod proteins are activated in response to parasites, and they play a role in regulating host inflammatory responses during malaria infection.

Severe malaria can present as a variety of clinical syndromes, including both severe malarial anemia and cerebral malaria (CM), to which the majority of deaths are attributable.1 Plasmodium berghei ANKA infection represents an experimental model of CM, with associated neurologic symptoms. 2,3 Mice susceptible to this pathogen (e.g., C57BL/6 strain) succumb to infection within 6–10 days4 as a result of excess, dysregulated inflammation mediated by pro-inflammatory cytokines, such as tumor necrosis factor (TNF)/lymphotoxin-α59 and interferon-γ (IFN-γ). 5,7 Although pro-inflammatory cytokines are essential for parasite clearance, the overproduction of cytokines through the stimulation of immune cells by parasite products can contribute to CM and fatal outcomes. 1014 Current research is focused on discovering the host receptors responsible for mediating this immunopathology, with particular attention to pattern recognition receptors.

Pattern recognition receptors play a critical role in pathogen sensing and the activation of the host innate immune response. 1519 Membrane-associated Toll-like receptors (TLRs) and cytosolic Nod-like receptors (NLRs) represent two important families of microbial sensor proteins, and as such have been identified as key host molecules in innate immune recognition and the inflammatory response to microbial products. 2022 Indeed, the stimulation of these receptors on host cells results in the activation of signaling pathways leading to the activation of transcription factors such as NF-γB, 22,23 and ultimately the production of pro-inflammatory cytokines and chemokines. 22,24

The roles of TLRs and NLRs in generating/amplifying this host inflammatory response during malaria infection, however, have yet to be fully defined. TLR2, 4, and 9 have all been reported to recognize various Plasmodium-associated molecules, including the malaria toxin glycophosphotidylionositol (GPI) and malarial DNA complexed to the parasite waste product hemozoin, leading to the secretion of pro-inflammatory cytokines by host cells. 2527 However, at present their contribution to the inflammatory response remains controversial. Coban and colleagues demonstrated that the survival of susceptible mice infected with P. berghei ANKA was increased in animals lacking tlr2 or tlr9, but not tlr428; these results have since been contested. 2931 Furthermore, in human studies polymorphisms in the tlr4 and tlr9, but not tlr2 genes, have so far been associated with disease severity, 32,33 whereas a functional variant of MAL, a signaling molecule in the TLR2/4 pathway, has been linked to protection from severe malaria. 33 Thus, although it is clear that TLRs do play a role during malaria infection, the nature and extent of their function remain to be elucidated.

As with TLRs, activation of NLRs has been shown to contribute to pathogenesis in several infectious diseases. 34 More specifically, both Nod1 and Nod2 recognize bacterial molecules produced during the synthesis and/or degradation of peptidoglycan, 20,21,35,36 and as such, mice lacking these proteins show increased susceptibility to and impaired clearance of bacteria. 37,38 However, little is known about the involvement of NLRs during malaria infection. Recent studies have shown that Nod proteins are upregulated when peripheral blood mononuclear cells (PBMCs) are exposed to malaria sporozoites, 39 but the role of NLRs during blood-stage infection, and their contribution to severe malaria pathogenesis, have not yet been examined. This was the focus of our study, and using the P. berghei ANKA murine model of CM, we found that Nod1 and Nod2 have no direct effect on survival (Figure 1A) or parasitemia (Figure 1B) of infected C57BL/6 mice. However, levels of cytokines/chemokines associated with NLR activation (IL-1β, KC, and MCP-1) and, interestingly, malaria pathogenesis (IFN-γ) were influenced by the absence of Nod proteins (Figure 2). As such, this is the first study to demonstrate NLR signaling during a parasitic infection, specifically implicating Nod proteins in malaria disease processes but not outcome.

The production of IL1-β, KC, and MCP-1, known to be induced by Nod1 and Nod2, 34 was observed during malaria infection in wild-type mice. The nod1nod2−/− animals, however, had reduced levels of IL1-β (baseline differences observed at D0 are not significantly different, Student’s t test, P = 0.12), KC, and MCP-1 over the course of infection (Figures 2A and 2C), indicating that Nod proteins are activated by malaria infection, through currently unknown pathways or in response to as yet unidentified parasite-derived agonists. The reduced IL1-β levels observed in nod1nod2−/− mice (Figure 2A) during infection are of particular interest in light of recent publications implicating IL1-β and IL1-β signaling in the pathogenesis of human cerebral malaria 40,41 and severe malarial anemia. 42 Therefore, Nod proteins may be involved in malaria pathogenesis through their strong induction of IL1-β.

The decreased levels of IFN-γ recorded in the nod1nod2−/−animals could be attributed to IL1-β because this cytokine was implicated in the production of IFN-γ. 43 However, the lower levels of both plasma IL1-β and IFN-γ had no effect on plasma TNF or IL-6 levels, which remained similar in both wild-type and nod1nod2−/− animals over the course of infection.

Numerous studies have demonstrated that IFN-γ plays a critical mechanistic role in the pathogenesis of CM. High levels of this protein lead to excessive host inflammation and development of severe disease. 1214 Treatment with anti-IFN-γ antibody protects animals from CM,7 IFN−/−44 and IFNγR−/−45 mice show resistance to CM, and recent microarray analysis implicates IFN response mechanisms in susceptibility to CM. 46 The IFN-γ not only impacts the host inflammatory response, but is also required for the control of blood stage parasite replication during P. berghei ANKA infection.45 Previously, treatment with anti-IFN-γ antibody was shown to have no effect on parasitemia.7 However, later work conducted by Amani and colleagues using IFNγR−/− animals rather than anti-IFN-γ antibody treatment demonstrated that parasitemia is affected by IFN-γ levels, but only at high parasite burdens. 45

In concordance with our findings, other members of the NLR signaling pathway have been shown to regulate IFN-γ production during bacterial infection. 47 However, despite the decreased IFN-γ levels observed in our study, nod1nod2−/− animals did not possess a survival advantage over their wild-type counterparts, nor was their parasitemia affected. The low levels of IFN-γ detected in the nod1nod2−/− animals may therefore have been sufficient for initiating host inflammatory responses, ultimately leading to CM, without affecting parasitemia. Furthermore, the lack in early IFN-γ production in the nod1nod2−/− animals, which was shown to be protective against progression to CM in non-cerebral P. berghei models,48 may have contributed to CM pathology. Indeed, observing the brains of all animals collected upon euthanasia, no differences in pathology were observed between those of wild-type versus nod1nod2−/− mice (data not shown).

Our experiments indicate that Nod proteins are not responsible for the onset or course of CM, but that they are activated by infection. Therefore, our results are consistent with current thinking that inflammation during malaria occurs through mainly TLR signaling. 10,2628,33 Parasite–infected erythrocytes are recognized by the host immune system through the interaction between TLRs on the surface of host cells and parasite proteins. These interactions mediate inflammatory responses to infection and may augment phagocytosis, and contribute to clearance of parasites by host cells. Other models of pathogen:phagocyte interaction have demonstrated that NLRs can recognize their cognate ligands after pathogen internalization. This process could explain how host inflammation was altered during P. berghei ANKA infection in the nod1nod2−/− animals.

Activation of innate immune responses during bacterial infection requires both TLRs and NLRs, 49 and this may also be true during malaria infection. Several studies have linked Nod responses with TLR stimulation. Treatment of epithelial cells with TNF and IFN-γ upregulates the expression of nod2, amplifying their inflammatory response to TLR ligands, such as LPS. 50 Similarly, another study has shown that treating murine macrophages with LPS, IL-6, or TNF increases Nod2 protein and nod2 mRNA expression. 51 Finally, when using malaria sporozoites to stimulate PBMCs, examination of the transcriptional profile of these cells indicated elevated levels of nod2, tlr2, and myd88.39 Therefore, TLRs and NLRs may both contribute to CM, although to a different extent; teasing out their respective roles will prove vital in understanding how the dysregulated host inflammatory response to malaria parasites is generated. Future work should therefore consider disease pathology and cytokine levels in target organs, such as the brain, lung, spleen, and liver, which have proved informative in dissecting differences in susceptibility to CM, 52 and may yield more information as to the effect of Nod proteins at the inflammatory sites during malaria infection.

Figure 1.
Figure 1.

Influence of Nod proteins on susceptibility to cerebral malaria. The 8–12-week-old C57BL/6 mice (+/+) and nod1nod2 −/− (−/−, backcrossed 9 times to the C57BL/6 background) mice were infected with 1 × 106 Plasmodium berghei ANKA parasitized erythrocytes by intraperitaneal injection. Parasitemia was measured every 2 days by thin blood smear. (A) Survival data are combined data from three experiments (N = 21 mice per group; Log rank test: P = 0.62). (B) Parasitemia (N = 10 mice per group; mean ± SEM; Student’s t test on sum values of parasitemia over time for each animal: P = 0.94). Data shown are representative of three independent experiments with a minimum of 5 animals per group.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 5; 10.4269/ajtmh.2009.80.718

Figure 2.
Figure 2.

Plasma cytokine levels during Plasmodium berghei ANKA infection. The 8–12-week old C57BL/6 mice (+/+) and nod1nod2−/− (−/−) mice were infected with 1 × 106 parasitized erythrocytes. Plasma was collected on days 1, 5, and 6, and cytokines measured using the CBA mouse inflammation kit (BD Pharmingen) and ELISA IL1-β and KC kits (R&D Systems). Parametric data were analyzed using Student’s t test, whereas the Mann–Whitney test was used for non-parametric data. Data are representative of two independent experiments (with N ≥ 5 mice per group). (A) Plasma levels of interferon-γ (IFN-γ) (left, data are represented as mean ± SEM, P = 0.03, Mann–Whitney test on sum values of cytokine level over time for each animal, N = 10 per group), and IL1-β (right, data are represented as mean ± SEM, P = 0.001, Students t test on sum values of cytokine level over time for each animal, N = 10 per group) over the course of infection. (B) Plasma levels of tumor necrosis factor (TNF) (left, data are represented as mean ± SEM, P = 0.08, Students t test on sum values of cytokine level over time for each animal, N = 10 per group), and IL-6 (right, data are represented as mean ± SEM, P = 0.79, Students t test on sum values of cytokine level over time for each animal, N = 10 per group) over the course of infection. (C) Plasma levels of MCP-1 (left, data are represented as mean ± SEM, P = 0.01, Mann–Whitney test on sum values of cytokine level over time for each animal, N = 10 per group), and KC (right, data are represented as mean ± SEM, P = 0.002, Mann–Whitney test on sum values of cytokine level over time for each animal, N = 10 per group) over the course of infection.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 5; 10.4269/ajtmh.2009.80.718

*

Address correspondence to Kevin C. Kain, Toronto General Hospital, University Health Network, 200 Elizabeth Street, EN 13-214, Toronto, Ontario, Canada M5G 2C4. E-mail: [email protected]

C. Finney and Z. Lu contributed equally to this work.

Authors’ addresses: Constance Finney and Ziyue Lu, McLaughlin-Rotman Centre, MaRS, TMDT, 101 College Street, Suite 10-401, Toronto, ON, Canada M5G 1L7. Lionel LeBourhis and Dana Philpott, Department of Immunology, Medical Sciences Building, University of Toronto, Toronto, ON, Canada M5S 1A8. Kevin C. Kain, Toronto General Hospital, University Health Network, 200 Elizabeth Street, EN 13-214, Toronto, Ontario, Canada M5G 2C4, Tel: 416-340-3535, Fax: 416-595-5826, E-mail: [email protected].

Financial support: This study was funded in part by a Canadian Institutes of Health Research (CIHR) Team Grant in Malaria (KCK), CIHR MT-13721 (KCK), Genome Canada through the Ontario Genomics Institute (KCK), CIHR Canada Research Chair (KCK).

Disclaimer: The funding agency had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

REFERENCES

  • 1

    World Health Organization, Communicable Diseases Cluster, 2000. Severe falciparum malaria. Trans R Soc Trop Med Hyg 94 (Suppl 1):S1–S90.

    • Search Google Scholar
    • Export Citation
  • 2

    Lou J, Lucas R, Grau GE, 2001. Pathogenesis of cerebral malaria: recent experimental data and possible applications for humans. Clin Microbiol Rev 14 :810–820.

    • Search Google Scholar
    • Export Citation
  • 3

    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.

    • Search Google Scholar
    • Export Citation
  • 4

    Rest JR, 1982. Cerebral malaria in inbred mice. I. A new model and its pathology. Trans R Soc Trop Med Hyg 76 :410–415.

  • 5

    de Kossodo S, Grau GE, 1993. Role of cytokines and adhesion molecules in malaria immunopathology. Stem Cells 11 :41–48.

  • 6

    Grau GE, Fajardo LF, Piguet PF, Allet B, Lambert PH, Vassalli P, 1987. Tumor necrosis factor (cachectin) as an essential mediator in murine cerebral malaria. Science 237 :1210–1212.

    • Search Google Scholar
    • Export Citation
  • 7

    Grau GE, Piguet PF, Vassalli P, Lambert PH, 1989. Tumor-necrosis factor and other cytokines in cerebral malaria: experimental and clinical data. Immunol Rev 112 :49–70.

    • Search Google Scholar
    • Export Citation
  • 8

    Lucas R, Lou JN, Juillard P, Moore M, Bluethmann H, Grau GE, 1997. Respective role of TNF receptors in the development of experimental cerebral malaria. J Neuroimmunol 72 :143–148.

    • Search Google Scholar
    • Export Citation
  • 9

    Piguet PF, Kan CD, Vesin C, 2002. Role of the tumor necrosis factor receptor 2 (TNFR2) in cerebral malaria in mice. Lab Invest 82 :1155–1166.

    • Search Google Scholar
    • Export Citation
  • 10

    Gowda DC, 2007. TLR-mediated cell signaling by malaria GPIs. Trends Parasitol 23 :596–604.

  • 11

    Lyke KE, Burges R, Cissoko Y, Sangare L, Dao M, Diarra I, Kone A, Harley R, Plowe CV, Doumbo OK, Sztein MB, 2004. Serum levels of the proinflammatory cytokines interleukin-1 beta (IL-1beta), IL-6, IL-8, IL-10, tumor necrosis factor alpha, and IL-12(p70) in Malian children with severe Plasmodium falciparum malaria and matched uncomplicated malaria or healthy controls. Infect Immun 72 :5630–5637.

    • Search Google Scholar
    • Export Citation
  • 12

    Malaguarnera L, Musumeci S, 2002. The immune response to Plasmodium falciparum malaria. Lancet Infect Dis 2 :472–478.

  • 13

    Riley EM, Wahl S, Perkins DJ, Schofield L, 2006. Regulating immunity to malaria. Parasite Immunol 28 :35–49.

  • 14

    Stevenson MM, Riley EM, 2004. Innate immunity to malaria. Nat Rev Immunol 4 :169–180.

  • 15

    Aderem A, 2003. Phagocytosis and the inflammatory response. J Infect Dis 187 (Suppl 2):S340–S345.

  • 16

    Akira S, Uematsu S, Takeuchi O, 2006. Pathogen recognition and innate immunity. Cell 124 :783–801.

  • 17

    Herskovits AA, Auerbuch V, Portnoy DA, 2007. Bacterial ligands generated in a phagosome are targets of the cytosolic innate immune system. PLoS Pathog 3 :e51.

    • Search Google Scholar
    • Export Citation
  • 18

    Medzhitov R, Janeway C Jr, 2000. Innate immune recognition: mechanisms and pathways. Immunol Rev 173 :89–97.

  • 19

    Stuart LM, Ezekowitz RA, 2005. Phagocytosis: elegant complexity. Immunity 22 :539–550.

  • 20

    Chamaillard M, Girardin SE, Viala J, Philpott DJ, 2003. Nods, Nalps and Naip: intracellular regulators of bacterial-induced inflammation. Cell Microbiol 5 :581–592.

    • Search Google Scholar
    • Export Citation
  • 21

    Inohara, Chamaillard, McDonald C, Nuñez G, 2005. NOD-LRR proteins: role in host-microbial interactions and inflammatory disease. Annu Rev Biochem 74 :355–383.

    • Search Google Scholar
    • Export Citation
  • 22

    Medzhitov R, 2001. Toll-like receptors and innate immunity. Nat Rev Immunol 1 :135–145.

  • 23

    Le Bourhis L, Benko S, Girardin SE, 2007. Nod1 and Nod2 in innate immunity and human inflammatory disorders. Biochem Soc Trans 35 :1479–1484.

    • Search Google Scholar
    • Export Citation
  • 24

    Fritz JH, Ferrero RL, Philpott DJ, Girardin SE, 2006. Nod-like proteins in immunity, inflammation and disease. Nat Immunol 7 :1250–1257.

    • Search Google Scholar
    • Export Citation
  • 25

    Coban C, Ishii KJ, Kawai T, Hemmi H, Sato S, Uematsu S, Yamamoto M, Takeuchi O, Itagaki S, Kumar N, Horii T, Akira S, 2005. Toll-like receptor 9 mediates innate immune activation by the malaria pigment hemozoin. J Exp Med 201 :19–25.

    • Search Google Scholar
    • Export Citation
  • 26

    Krishnegowda G, Hajjar AM, Zhu J, Douglass EJ, Uematsu S, Akira S, Woods AS, Gowda DC, 2005. Induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols of Plasmodium falciparum: cell signaling receptors, glycosylphosphatidylinositol (GPI) structural requirement, and regulation of GPI activity. J Biol Chem 280 :8606–8616.

    • Search Google Scholar
    • Export Citation
  • 27

    Parroche P, Lauw FN, Goutagny N, Latz E, Monks BG, Visintin A, Halmen KA, Lamphier M, Olivier M, Bartholomeu DC, Gazzinelli RT, Golenbock DT, 2007. Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to Toll-like receptor 9. Proc Natl Acad Sci USA 104 :1919–1924.

    • Search Google Scholar
    • Export Citation
  • 28

    Coban C, Ishii KJ, Uematsu S, Arisue N, Sato S, Yamamoto M, Kawai T, Takeuchi O, Hisaeda H, Horii T, Akira S, 2007. Pathological role of Toll-like receptor signaling in cerebral malaria. Int Immunol 19 :67–79.

    • Search Google Scholar
    • Export Citation
  • 29

    Griffith JW, O’Connor C, Bernard K, Town T, Goldstein DR, Bucala R, 2007. Toll-like receptor modulation of murine cerebral malaria is dependent on the genetic background of the host. J Infect Dis 196 :1553–1564.

    • Search Google Scholar
    • Export Citation
  • 30

    Lepenies B, Cramer JP, Burchard GD, Wagner H, Kirschning CJ, Jacobs T, 2007. Induction of experimental cerebral malaria is independent of TLR2/4/9. Med Microbiol Immunol 179 :39–44.

    • Search Google Scholar
    • Export Citation
  • 31

    Togbe D, Schofield L, Grau GE, Schnyder B, Boissay V, Charron S, Rose S, Beutler B, Quesniaux VF, Ryffel B, 2007. Murine cerebral malaria development is independent of Toll-like receptor signaling. Am J Pathol 170 :1640–1648.

    • Search Google Scholar
    • Export Citation
  • 32

    Khor CC, Chapman SJ, Vannberg FO, Dunne A, Murphy C, Ling EY, Frodsham AJ, Walley AJ, Kyrieleis O, Khan A, Aucan C, Segal S, Moore CE, Knox K, Campbell SJ, Lienhardt C, Scott A, Aaby P, Sow OY, Grignani RT, Sillah J, Sirugo G, Peshu N, Williams TN, Maitland K, Davies RJ, Kwiatkowski DP, Day NP, Yala D, Crook DW, Marsh K, Berkley JA, O’Neill LA, Hill AV, 2007. A Mal functional variant is associated with protection against invasive pneumococcal disease, bacteremia, malaria and tuberculosis. Nat Genet 39 :523–528.

    • Search Google Scholar
    • Export Citation
  • 33

    Mockenhaupt FP, Cramer JP, Hamann L, Stegemann MS, Eckert J, Oh NR, Otchwemah RN, Dietz E, Ehrhardt S, Schroder NW, Bienzle U, Schumann RR, 2006. Toll-like receptor (TLR) polymorphisms in African children: common TLR-4 variants predispose to severe malaria. Proc Natl Acad Sci USA 103 :177–182.

    • Search Google Scholar
    • Export Citation
  • 34

    Carneiro LA, Magalhaes JG, Tattoli I, Philpott DJ, Travassos LH, 2008. Nod-like proteins in inflammation and disease. J Pathol 214 :136–148.

    • Search Google Scholar
    • Export Citation
  • 35

    Girardin SE, Boneca IG, Carneiro LA, Antignac A, Jehanno M, Viala J, Tedin K, Taha MK, Labigne A, Zahringer U, Coyle AJ, DiStefano PS, Bertin J, Sansonetti PJ, Philpott DJ, 2003. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 300 :1584–1587.

    • Search Google Scholar
    • Export Citation
  • 36

    Girardin SE, Boneca IG, Viala J, Chamaillard M, Labigne A, Thomas G, Philpott DJ, Sansonetti PJ, 2003. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem 278 :8869–8872.

    • Search Google Scholar
    • Export Citation
  • 37

    Kobayashi KS, Chamaillard M, Ogura Y, Henegariu O, Inohara N, Nunez G, Flavell RA, 2005. Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract. Science 307 :731–734.

    • Search Google Scholar
    • Export Citation
  • 38

    Viala J, Chaput C, Boneca IG, Cardona A, Girardin SE, Moran AP, Athman R, Memet S, Huerre MR, Coyle AJ, DiStefano PS, Sansonetti PJ, Labigne A, Bertin J, Philpott DJ, Ferrero RL, 2004. Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag pathogenicity island. Nat Immunol 5 :1166–1174.

    • Search Google Scholar
    • Export Citation
  • 39

    Ockenhouse CF, Hu WC, Kester KE, Cummings JF, Stewart A, Heppner DG, Jedlicka AE, Scott AL, Wolfe ND, Vahey M, Burke DS, 2006. Common and divergent immune response signaling pathways discovered in peripheral blood mononuclear cell gene expression patterns in presymptomatic and clinically apparent malaria. Infect Immun 74 :5561–5573.

    • Search Google Scholar
    • Export Citation
  • 40

    John CC, Panoskaltsis-Mortari A, Opoka RO, Park GS, Orchard PJ, Jurek AM, Idro R, Byarugaba J, Boivin MJ, 2008. Cerebrospinal fluid cytokine levels and cognitive impairment in cerebral malaria. Am J Trop Med Hyg 78 :198–205.

    • Search Google Scholar
    • Export Citation
  • 41

    John CC, Park GS, Sam-Agudu N, Opoka RO, Boivin MJ, 2008. Elevated serum levels of IL-1ra in children with Plasmodium falciparum malaria are associated with increased severity of disease. Cytokine 41 :204–208.

    • Search Google Scholar
    • Export Citation
  • 42

    Ouma C, Davenport GC, Awandare GA, Keller CC, Were T, Otieno MF, Vulule JM, Martinson J, Ong’echa JM, Ferrell RE, Perkins DJ, 2008. Polymorphic variability in the interleukin (IL)-1beta promoter conditions susceptibility to severe malarial anemia and functional changes in IL-1beta production. J Infect Dis 198 :1219–1226.

    • Search Google Scholar
    • Export Citation
  • 43

    Raices RM, Kannan Y, Sarkar A, Bellamkonda-Athmaram V, Wewers MD, 2008. A synergistic role for IL-1beta and TNFalpha in monocyte-derived IFNgamma inducing activity. Cytokine 44 :234–241.

    • Search Google Scholar
    • Export Citation
  • 44

    Yanez DM, Manning DD, Cooley AJ, Weidanz WP, van der Heyde HC, 1996. Participation of lymphocyte subpopulations in the pathogenesis of experimental murine cerebral malaria. J Immunol 157 :1620–1624.

    • Search Google Scholar
    • Export Citation
  • 45

    Amani V, Vigario AM, Belnoue E, Marussig M, Fonseca L, Mazier D, Renia L, 2000. Involvement of IFN-gamma receptor-medicated signaling in pathology and anti-malarial immunity induced by Plasmodium berghei infection. Eur J Immunol 30 :1646–1655.

    • Search Google Scholar
    • Export Citation
  • 46

    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.

    • Search Google Scholar
    • Export Citation
  • 47

    Pedra JH, Sutterwala FS, Sukumaran B, Ogura Y, Qian F, Montgomery RR, Flavell RA, Fikrig E, 2007. ASC/PYCARD and caspase-1 regulate the IL-18/IFN-gamma axis during Anaplasma phagocytophilum infection. J Immunol 179 :4783–4791.

    • Search Google Scholar
    • Export Citation
  • 48

    Mitchell AJ, Hansen AM, Hee L, Ball HJ, Potter SM, Walker JC, Hunt NH, 2005. Early cytokine production is associated with protection from murine cerebral malaria. Infect Immun 73 :5645–5653.

    • Search Google Scholar
    • Export Citation
  • 49

    Ferwerda G, Girardin SE, Kullberg BJ, Le Bourhis L, de Jong DJ, Langenberg DM, van Crevel R, Adema GJ, Ottenhoff TH, Van der Meer JW, Netea MG, 2005. NOD2 and Toll-like receptors are nonredundant recognition systems of Mycobacterium tuberculosis. PLoS Pathog 1 :279–285.

    • Search Google Scholar
    • Export Citation
  • 50

    Rosenstiel P, Fantini M, Brautigam K, Kuhbacher T, Waetzig GH, Seegert D, Schreiber S, 2003. TNF-alpha and IFN-gamma regulate the expression of the NOD2 (CARD15) gene in human intestinal epithelial cells. Gastroenterology 124 :1001–1009.

    • Search Google Scholar
    • Export Citation
  • 51

    Takahashi Y, Isuzugawa K, Murase Y, Imai M, Yamamoto S, Iizuka M, Akira S, Bahr GM, Momotani E, Hori M, Ozaki H, Imakawa K, 2006. Up-regulation of NOD1 and NOD2 through TLR4 and TNF-alpha in LPS-treated murine macrophages. J Vet Med Sci 68 :471–478.

    • Search Google Scholar
    • Export Citation
  • 52

    Hanum PS, Hayano M, Kojima S, 2003. Cytokine and chemokine responses in a cerebral malaria-susceptible or -resistant strain of mice to Plasmodium berghei ANKA infection: early chemokine expression in the brain. Int Immunol 15 :633–640.

    • Search Google Scholar
    • Export Citation
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