Cerebral Edema and Cerebral Hemorrhages in Interleukin-10-Deficient Mice Infected with Plasmodium chabaudi

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1 INFECTION AND IMMUNITY, May 2004, p Vol. 72, No /04/$ DOI: /IAI Copyright 2004, American Society for Microbiology. All Rights Reserved. Cerebral Edema and Cerebral Hemorrhages in Interleukin-10-Deficient Mice Infected with Plasmodium chabaudi Latifu A. Sanni, William Jarra, Ching Li, and Jean Langhorne* Division of Parasitology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom Received 19 December 2003/Returned for modification 13 January 2004/Accepted 27 January 2004 During a Plasmodium chabaudi infection in interleukin-10 (IL-10) knockout mice, there is greater parasite sequestration, more severe cerebral edema, and a high frequency of cerebral hemorrhage compared with infection of C57BL/6 Anti-tumor necrosis factor alpha treatment ameliorated both cerebral edema and hemorrhages, suggesting that proinflammatory responses contributed to cerebral complications in infected IL-10 / It is well established that pro-inflammatory cytokines, such as gamma interferon (IFN- ), tumor necrosis factor alpha (TNF- ), and lymphotoxin, are involved in some of the complications of malaria infection in humans and mice (3, 4, 6, 10). Lower interleukin-10 (IL-10) plasma concentration (9) and IL-10/TNF- ratios (17) in children with malarial anemia suggest that an imbalance in pro- and anti-inflammatory cytokines, such as IL-10, may be related to the development of disease. In support of this, IL-10 knockout mice (IL-10 / ) are more susceptible to a lethal infection with Plasmodium chabaudi, develop a more severe anemia and hypoglycemia, and have higher concentrations of IFN- and TNF- in plasma than infected C57BL/6 mice (11 13). Treatment of infected IL-10 / mice with antibodies to IFN- and TNF- eliminates mortality and some of the pathological sequelae (11, 12), suggesting that proinflammatory cytokines play a role in the pathogenesis of malaria in P. chabaudi infections of One of the severe complications of malaria infection that has not been described for P. chabaudi infection is cerebral involvement. It is possible that under conditions where proinflammatory cytokines are upregulated, P. chabaudi infection may induce responses that result in cerebral complications. Therefore, we have examined P. chabaudi infections in IL- 10 / mice for evidence of cerebral involvement. Female IL-10 / mice (8), back-crossed at least eight times onto the C57BL/6 background, and control C57BL/6 mice (6 to 12 weeks), bred and maintained as described previously (11), were infected with P. chabaudi chabaudi (AS) (P. chabaudi) and monitored as described previously (21). The courses of infection, associated anemia, hypoglycemia, loss in body * Corresponding author. Mailing address: Division of Parasitology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom. Phone: 44 (0) Fax: 44 (0) jlangho@nimr.mrc.ac.uk. Present address: Department of Histopathology, The General Infirmary at Leeds, Leeds LS2 9JT, United Kingdom. Present address: Kennedy Institute of Rheumatology Division, Imperial College London, Faculty of Medicine, London W6 8LH, United Kingdom. weight, and mortality were all as described previously for IL- 10 / and C57BL/6 mice (11 13). Mice were checked for signs of neurological impairment, such as gait disturbance, convulsions, hemiplegia, and coma; 3 out of 27 of the IL-10 / mice showed signs of a wobbling gait suggestive of limb paralysis. Convulsions, tonic or clonic, were not observed in any of the Sequestration of P. chabaudi (AS) was determined by measuring total and differential peripheral blood parasitemia over a 24-h period on days 7 to 8 postinfection (p.i.) for IL-10 / and C57BL/6 Parasitemias of IL-10 / and C57BL/6 over this time period are shown in Fig. 1a and b. We observed a transient reduction in the total parasitemia and in the percentage of trophozoite-infected red blood cells (RBC) in the blood of both groups of Mean reduction of peripheral parasitemia was significantly greater for IL-10 / mice than for IL-10 / mice (87% compared with 61% for trophozoiteinfected RBC; 24% compared with 9% in the total parasitemia for IL-10 / and IL-10 / mice, respectively [P 0.05 for each; n 5; Mann-Whitney test]). To determine if blood-brain barrier breakdown and cerebral edema occur during P. chabaudi infection, the extent of leakage of Evans Blue dye into the brain was determined as described previously (22). Leakage of Evans Blue dye into the brains of IL-10 / and IL-10 / mice was observed between 6 and 10 days p.i. (Fig. 2a). In two independent experiments, the level of dye leakage was significantly higher for the IL-10 / mice than for the IL-10 / mice (Fig. 2b): in experiment 1 on days 6, 8, and 10 p.i. (P 0.05, Mann-Whitney test) and experiment 2 on day 8 p.i. (P 0.05). To investigate the role of TNF- in the development of cerebral edema in infected IL-10 / mice, groups of mice were injected intraperitoneally with 1 mg of either anti-tnf- (Mp6-XT22) monoclonal antibody (Alexis Corporation, Nottingham, United Kingdom) (16) or immunoglobulin G1 isotype control antibody (GL113) on day 0 and day 5 or 6 during infection. At intervals in the infection, mice were injected with Evans Blue as described above and dye leakage into the brain was quantified. The degree of cerebral edema in the brain was 3054

2 VOL. 72, 2004 NOTES 3055 FIG. 1. Sequestration of parasitized RBC in IL-10 / and IL-10 / mice infected with P. chabaudi. (a and b) Parasitemias, total ( ), trophozoites (F), schizonts (Œ), and rings ( ), in a representative C57BL/6 (a) and IL-10 / (b) mouse over a 24-h period on day 8 of infection. Percentage parasitemias were determined on Giemsa-stained blood films taken hourly for 24 h. Schizont rupture generally took place at approximately midnight. A parasite was considered to be at the schizont stage when at least two nuclei could be identified clearly. (c and d) Drop in parasitemia of maturing trophozoites prior to schizont rupture, expressed as the mean percentage reduction of the total parasitemia (c) (filled bars) and trophozoites (d) (hatched bars). The reduction was calculated as percentage reduction of parasitemia: [average parasitemia before drop (lowest parasitemia/average parasitemia before drop)] 100. An asterisk indicates that the differences in pathology or reduction in parasitemia are significant between IL-10 / and IL-10 / mice (P 0.05 [Mann-Whitney test; n 5 mice]). determined on day 8 p.i. Treatment with anti-tnf- antibody significantly decreased the amount of cerebral edema in the P. chabaudi-infected IL-10 / mice (Fig. 2c [P 0.05, Mann- Whitney test]). Cerebral hemorrhages were observed in 60% of IL-10 / mice between days 8 and day 12 of infection, whereas no hemorrhages were observed in IL-10 / mice by either gross or microscopic examination at any time of infection (Fig. 3a and 4). Light microscopy confirmed the gross anatomical findings of brain hemorrhage. Hemorrhages ranged from microscopic to petechial and larger-size hemorrhages (Fig. 4c to f) and were mainly in the cerebrum, but any region of the brain could be affected (data not shown). Uninfected and salineinjected IL-10 / or infected IL-10 / mice sacrificed before day 8 had no evidence of cerebral hemorrhages, suggesting that this manifestation is related to infection with P. chabaudi and is not the result of spontaneous hemorrhaging in IL-10 / Treatment of infected IL-10 / mice with anti-tnf- antibody reduced the frequency of hemorrhages by approximately 50%; 5 of 21 anti-tnf- -treated mice had cerebral hemorrhages compared with 11 of 22 control immunoglobulin treated mice at days 8 and 9 p.i. (Fig. 3b). This study shows that in addition to the higher mortality, greater body weight loss, anemia, and hypoglycemia observed previously (11 13), acute P. chabaudi infection in IL-10 / mice is also accompanied by increased sequestration of parasites, cerebral hemorrhages, and cerebral edema. Similar to the mortality and other features of malarial disease (11, 12) in IL-10 / mice, both cerebral edema and cerebral hemorrhages are related to the increased production of TNF-. The observation of cerebral involvement during P. chabaudi infection is consistent with a previous report that IL-10 is protective against the development of cerebral malaria (CM) in Plasmodium berghei ANKA infection (7) and supports the view that regulation of inflammatory cytokines may be an important factor in the pathogenesis of cerebral complications (2, 3, 18, 20). The role of cerebral edema in the pathogenesis of CM is controversial. Several publications support such a role in human (1, 14) and mouse (19, 20) CM. However, others (23) argue against the permeability hypothesis. We show here that cerebral edema occurs in IL-10 / mice as early as day 6 p.i., earlier than any clinical signs of illness or cerebral hemorrhages in these The exact cause of increased cerebral microvascular permeability during P. chabaudi infection in IL-

3056 NOTES INFECT. IMMUN. FIG. 2. Cerebral edema in P. chabaudi-infected IL-10 / (a) Photograph showing a bluish discoloration of the brain of a P. chabaudi-infected IL-10 / mouse on day 8 p.i. caused by leakage of Evans Blue into the extravascular brain tissue due to a breakdown of the blood-brain barrier.

(b) The histograms represent two independent experiments in which Evans Blue dye leakage into the brains of IL-10 / (filled bars) and IL-10 / (open bars) mice is measured by spectrophotometry on days

3 3056 NOTES INFECT. IMMUN. FIG. 2. Cerebral edema in P. chabaudi-infected IL-10 / (a) Photograph showing a bluish discoloration of the brain of a P. chabaudi-infected IL-10 / mouse on day 8 p.i. caused by leakage of Evans Blue into the extravascular brain tissue due to a breakdown of the blood-brain barrier. The brain of an IL-10 / wild-type mouse on day 8 p.i. is shown for comparison. (b) The histograms represent two independent experiments in which Evans Blue dye leakage into the brains of IL-10 / (filled bars) and IL-10 / (open bars) mice is measured by spectrophotometry on days 6, 8, and 10 p.i. The values shown are the means standard errors of the means for four to five mice per group at each time point. (c) Effect of anti-tnf- treatment on Evans Blue dye leakage into the brain of IL-10 / mice infected with P. chabaudi. The values are means standard errors of the means (n 5) of Evans Blue dye (micrograms of dye per gram of wet brain tissue) for anti-tnf- -treated and control antibody-treated IL-10 / mice on day 8 p.i. Brains of uninfected C57BL/6 mice not injected with Evans Blue were used as a reference, and the amount of dye present in the supernatants is calculated from a standard curve of Evans Blue dye. An asterisk indicates that the difference in dye leakage into the brains of IL-10 / mice treated with anti-tnf- is significant compared with results for IL-10 / mice treated with control antibody (P 0.05 [Mann-Whitney test]). FIG. 3. Frequency of brain hemorrhage in P. chabaudi-infected IL-10 / (a) The frequency of brain hemorrhages determined by gross anatomical examination or light-microscopic examination in IL- 10 / Brain hemorrhages were not seen in IL-10 / The data are representative of 15 mice per group. (b) Reduction in the frequency of brain hemorrhages as determined by gross anatomical observation in IL-10 / mice treated with anti-tnf- or control antibody on day 0 and day 5 or 6 as described in Materials and Methods. The values shown represent the frequency of brain hemorrhages in 21 anti-tnf- -treated mice and 22 control immunoglobulin-treated 10 / mice is unknown, although its amelioration by treatment of mice with anti-tnf- antibody supports the idea that inflammatory mediators might contribute. Development of brain hemorrhages is a hallmark of human CM. However, despite the occurrence of brain hemorrhages in infected IL-10 / mice, they did not show overt clinical signs of cerebral involvement, apart from occasional signs of hind limb paralysis. Therefore, although, IL-10 / mice show some of the pathological features of CM in humans, they do not provide the complete model of the disease spectrum seen in humans. This host-parasite combination, however, might prove useful in dissecting the contributory roles of the inflammatory cytokines, IFN- and TNF-, in the development of CM. Although we observed increased sequestration of P. chabaudi in IL-10 / mice, gross histological examination of brain sections did not reveal large numbers of parasitized RBC on brain endothelium (data not shown). However, studies by Mota et al. (15) using electron microscopy have clearly demonstrated P. chabaudi-infected RBC in the brains of infected CBA/ca It would therefore be of great importance to determine the relative extent of parasite sequestration in IL- 10 / and C57BL/6 mice by this method, and by more sensitive and quantitative PCR approaches for the measurement of total parasite load (5) in the brain and other organs. Such studies are under way.

VOL. 72, 2004 NOTES 3057 Downloaded from http://iai.asm.org/ FIG. 4. Hemorrhages in the brain of P.

4 VOL. 72, 2004 NOTES 3057 Downloaded from FIG. 4. Hemorrhages in the brain of P. chabaudi-infected IL-10 / (a and b) Hematoxylin and eosin-stained sections of the brain of an IL-10 / mouse on day 8 p.i. without hemorrhage (a) and the brain of an infected IL-10 / mouse on day 8 p.i. (b). There is hemorrhage in the right cerebral hemisphere (arrow) in the region of the motor cortex above the corpus callosum. (c) A normal meningeal vessel (magnification, 400) from an IL-10 / mouse on day 8 p.i. (d) Hemorrhage next to a collapsed meningeal vessel (magnification, 400) from an IL-10 / mouse on day 8 p.i. (e) Hemorrhage in the region of the white matter (magnification, 200) in an IL-10 / mouse on day 8 p.i. (f) Hemorrhage into the third ventricle (magnification, 200) in another IL-10 / mouse on day 8 p.i. on November 20, 2018 by guest We thank Alec Gallagher and Tracey Lamb for their help with the examination of cerebral hemorrhages, Frank Albano and Robin Stephens for their critical comments and careful reading of the manuscript, and Anne O Garra for helpful advice. This work was supported by the Medical Research Council, United Kingdom. REFERENCES 1. Brown, H., S. Rogerson, T. Taylor, M. Tembo, J. Mwenechanya, M. E. Molyneux, and G. Turner Blood-brain barrier function in cerebral malaria in Malawian children. Am. J. Trop. Med. Hyg. 64: Grau, G. E., L. F. Fajardo, P. F. Piguet, B. Allet, P. H. Lambert, and P. Vassalli Tumor necrosis factor (cachectin) as an essential mediator in murine cerebral malaria. Science 237: Grau, G. E., H. Heremans, P. F. Piguet, P. Pointaire, P. H. Lambert, A. Billiau, and P. Vassalli Monoclonal antibody against interferon gamma can prevent experimental cerebral malaria and its associated overproduction of tumor necrosis factor. Proc. Natl. Acad. Sci. USA 86: Grau, G. E., P. F. Piguet, P. Vassalli, and P. H. Lambert Tumornecrosis factor and other cytokines in cerebral malaria: experimental and clinical data. Immunol. Rev. 112: Hulier, E., P. Petour, G. Snounou, M.-P. Nivez, D. Mazier, and L. Renia A method for the quantitative assessment of malaria parasite development in organs of the mammalian host. Mol. Biochem. Parasitol. 77: Knight, J. C., I. Udalova, A. V. Hill, B. M. Greenwood, N. Peshu, K. Marsh, and D. Kwiatkowski A polymorphism that affects OCT-1 binding to

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Received 4 February 2003/Returned for modification 7 March 2003/Accepted 27 May 2003

Received 4 February 2003/Returned for modification 7 March 2003/Accepted 27 May 2003 INFECTION AND IMMUNITY, Sept. 2003, p. 4850 4856 Vol. 71, No. 9 0019-9567/03/$08.00 0 DOI: 10.1128/IAI.71.9.4850 4856.2003 Copyright 2003, American Society for Microbiology. All Rights Reserved. Pathology