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

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1 INFECTION AND IMMUNITY, Sept. 2003, p Vol. 71, No /03/$ DOI: /IAI Copyright 2003, American Society for Microbiology. All Rights Reserved. Pathology of Plasmodium chabaudi chabaudi Infection and Mortality in Interleukin-10-Deficient Mice Are Ameliorated by Anti-Tumor Necrosis Factor Alpha and Exacerbated by Anti-Transforming Growth Factor Antibodies Ching Li, 1 Latifu A. Sanni, 1 Fakhreldin Omer, 2 Eleanor Riley, 2 and Jean Langhorne 1 * Division of Parasitology, National Institute for Medical Research, 1 and Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, 2 London, United Kingdom Received 4 February 2003/Returned for modification 7 March 2003/Accepted 27 May 2003 Interleukin-10 (IL-10)-deficient (IL-10 / ) mice infected with Plasmodium chabaudi (AS) suffer a more severe disease and exhibit a higher rate of mortality than control C57BL/6 mice. Here, we show that a drop in body temperature to below 28 C and pronounced hypoglycemia of below 3 mm are reliable indicators of a lethal infection. Elevated inflammatory responses have been shown to accompany pathology in infected IL-10 / mice. We show that neutralization of tumor necrosis factor alpha (TNF- ) in IL-10 / mice abolishes mortality and ameliorates the hypothermia, weight loss, and anemia but does not affect the degree of hypoglycemia. These data suggest that TNF- is involved in some of the pathology associated with a P. chabaudi infection in IL-10 / mice but other factors play a role. IL-10 / mice that survive a primary infection have been shown to control gamma interferon (IFN- ) and TNF- production, indicating that other cytokines or mechanisms may be involved in their down-regulation. Significantly higher levels of transforming growth factor (TGF- ), a cytokine with such properties, are present in the plasma of infected IL-10 / mice at a time that coincides with the disappearance of IFN- and TNF- from the blood. Neutralization of TGF- in IL-10 / mice resulted in higher circulating amounts of TNF- and IFN-, and all treated IL-10 / mice died within 12 days with increased pathology but with no obvious increase in parasitemia. Our data suggest that a tight regulation of the balance between regulatory cytokines such as IL-10 and TGF- and inflammatory cytokines such as IFN- and TNF- is critical for survival in a mouse malaria infection. Interleukin-10 (IL-10), a cytokine known to suppress or down-regulate inflammatory responses, has been shown to control immunopathology in several infectious diseases. In malaria, IL-10 protects mice from developing cerebral malaria (CM) in a Plasmodium berghei (ANKA) infection (12) by down-regulating gamma interferon (IFN- ) and tumor necrosis factor alpha (TNF- ). In Plasmodium chabaudi infections, female mice in which the IL-10 gene has been inactivated by gene-targeting (IL-10 / mice) have enhanced levels of TNF- and IFN- (18, 20). Although there is no significant difference in the course of parasitemia, up to 50% of female IL-10 / mice die within 17 days and the infection is associated with more severe weight loss, hypoglycemia, and hypothermia than usually observed in C57BL/6 or other resistant strains of mice (18, 20). These data suggest that the severe pathological changes and increased mortality in P. chabaudi-infected IL- 10 / mice might be the result of the enhanced inflammatory responses, as is the case for Trypanosoma cruzi and Toxoplasma gondii infections in these mice (5, 9, 11). A clear relationship between IFN- and malaria-related pathology could not be demonstrated in P. chabaudi infections of IL-10 / mice (18), although removal of IFN- by antibodies and gene targeting of * Corresponding author. Mailing address: Division of Parasitology, National Institute for Medical Research, The Ridgeway, London NW7 1AA, United Kingdom. Phone: Fax: jlangho@nimr.mrc.ac.uk. the IFN- receptor did rescue IL-10 / mice from an otherwise lethal infection (18). This suggested that there might be IFN- -independent mechanisms contributing to pathology in P. chabaudi infections. One intriguing observation in the surviving IL-10 / mice infected with P. chabaudi was that the inflammatory cytokines TNF- and IFN- were down-regulated during the resolution phase of infection, despite the lack of IL-10, suggesting that some other negative feedback mechanism was operating. One cytokine that could play this role may be transforming growth factor (TGF- ). TGF- is produced by many cell types, including macrophages (35), and CD4 T regulatory cells, whose major function is to regulate both Th1 and Th2 responses elicited by an antigen or a pathogen (16, 17). TGF- is detectable in the plasma during mouse malaria infections. Low levels of TGF- have been correlated with the severity of malarial disease (26), and recombinant TGF- given to mice infected with P. berghei prolonged survival time (26). In addition, BALB/c mice given neutralizing antibody against TGF- during a nonlethal P. chabaudi infection succumbed to a lethal infection (26). In human malaria, low levels of IL-12 and TGF- 1 and high levels of TNF- have been shown to be associated with high parasitemia, severe anemia, and CM in children (28, 36). High ratios of IL-12, IFN-, or TNF- to TGF- have been correlated with increased risk of fever and clinical malaria (3). However, high levels of TGF- are found at the site of pathology in patients who died of CM (2). These 4850

2 VOL. 71, 2003 TNF- IN P. CHABAUDI-INFECTED IL-10-DEFICIENT MICE 4851 studies suggest that regulatory cells and cytokines may be important in controlling pathology and that their relative amounts and locations could be relevant. In this study, we have investigated whether neutralization of TNF- with antibodies in vivo during the acute phase of a P. chabaudi infection could rescue IL-10 / mice from death and ameliorate the severity of malaria-associated pathology. In addition, we have determined whether TGF- has any role in the pathology of a P. chabaudi infection in IL-10 / mice. Anti- TNF- antibody treatment allowed all IL-10 / mice to recover from infection and significantly ameliorated pathology. By contrast, administration of anti-tgf- antibodies in vivo results in higher levels of IFN- and TNF- in plasma, exacerbated pathology, and increased mortality. MATERIALS AND METHODS Mice and parasites. IL-10 / mice (seven times backcrossed onto C57BL/6) were bred in the animal facilities at the National Institute for Medical Research (NIMR), Mill Hill, London, United Kingdom. The line is maintained by crossing homozygous (IL-10 / ) males with heterozygous (IL-10 / ) female mice. The genotype of female IL-10 / mice used in experiments was verified by PCR as described previously (18). C57BL/6 mice used as wild-type (WT) controls were bred in the specific-pathogen-free unit at the NIMR. All experimental animals were kept on sterile bedding, food, and water. A cloned line of the malaria parasite P. chabaudi chabaudi (AS) and stocks thereof were maintained as described previously (32). Female IL-10 / and C57BL/6 mice aged 6 to 12 weeks were infected by injecting 10 5 parasitized erythrocytes intraperitoneally (i.p.). The course of infection was monitored by thin blood smears every 2 days. Neutralization of TNF- and TGF- in vivo. Female IL-10 / and WT mice were injected daily with 100 g of rabbit anti-mouse TNF- immunoglobulin G (IgG) (AB-410-NA; R&D Systems, Abington, United Kingdom) i.p. between days 5 and 8 of the infection. Another group of IL-10 /, and WT mice also received 100 g of the control rabbit IgG (AB-105-C; R&D Systems) at the same time. Plasma samples were taken 30 min after schizont rupture from these mice at days 6, 7, 8, and 9 to measure levels of IFN- and TNF-. The administration of anti-tgf- antibody has been described elsewhere (26). Briefly, female IL-10 / and WT mice were injected i.p. with 50 g of monoclonal anti-tgf- antibody (MAB1835; R&D Systems) 1 day before and every 2 days thereafter until day 11 of the infection. Plasma samples were collected 30 min after schizont rupture between days 6 and 9 to measure levels of IFN-, TNF-, and TGF-. Groups of IL-10 /, and WT mice were treated with a control monoclonal antibody of the same isotype (MAB002; R&D Systems). Groups of IL-10 / and WT mice were also infected with 10 5 parasites i.p. and left untreated. Blood samples were taken from all groups of IL-10 / and WT mice throughout 21 days of infection for the measurement of TGF-. IFN-, TNF-, and TGF- ELISA. IFN- enzyme-linked immunosorbent assay (ELISA) was carried out as described previously (18, 32). Briefly, the monoclonal antibody R4-6A2 was used as the capture antibody and biotinylated AN18 was used as the detection antibody. Recombinant IFN- (MG-IFN; Genzyme, Kent, United Kingdom) was used as a standard. The sensitivity of this ELISA was 6 pg/ml. The TNF- ELISA was carried out according to the manufacturer s instructions, with the monoclonal antibody TN3 (BD Pharmingen, Oxford, United Kingdom) as the capture antibody and biotinylated anti-mouse TNF- (BD Pharmingen) as the detection antibody. Recombinant TNF- (BD Pharmingen) was used as a standard. The sensitivity of the TNF- ELISA was 9 to 15 pg/ml. The TGF- ELISA was carried out as described previously (26) and as described in the manufacturer s instructions (DuoSet, DY240; R&D Systems) with slight modifications. Briefly, the plasma samples were diluted 100-fold and acidified in 0.1 M HCl for 10 min at room temperature. One molar NaOH was used to neutralize the samples to ph 7 to 8. Samples were then added to the capture antibody, either chicken anti-tgf- 1 (AB-101; R&D Systems) or a pan-specific mouse anti-tgf- 1,2,3 (MAB1835; R&D Systems) within 10 to 15 min after neutralization. The other antibody in each case was used as the detecting antibody as described. Recombinant human TGF- (240-B-002; R&D Systems) was used as a standard. The sensitivity of the assay was 30 pg/ml. Measurement of pathological indicators. Body weight was measured with a top-pan electric balance. The results were presented as the percentage of weight lost or gained with respect to the day 0 weight. Anal temperature was measured with an electronic thermometer (VWR, Poole, Dorset, United Kingdom). Blood glucose levels were measured in millimolar concentrations with a commercially available glucose monitor and BM Accu-test strips (Roche, Lewes, East Sussex, United Kingdom). Hematocrit was used as a measure of anemia. Tail blood (10 l) was collected into a heparinized capillary tube. The blood was centrifuged at 13,000 g for 3 min in a microhematocrit centrifuge. The hematocrit value was expressed as a percentage of the total blood volume. Statistics. Student s t tests and Mann-Whitney nonparametric tests were used to analyze the significance of differences in weight loss, temperature, blood glucose levels, cytokine levels, and parasitemia. The log rank test was used to test the equality of survival function across the different groups. RESULTS Indicators of pathology. Female IL-10 / mice infected with P. chabaudi suffer more severe malaria disease than infected C57BL/6 mice (18). Furthermore, although a P. chabaudi infection is not normally lethal in C57BL/6 mice, 30 to 50% of female IL-10 / mice on this genetic background died within 17 days, without increased parasitemia (18). To determine whether any of the pathological parameters used to monitor malarial disease in infection, i.e., weight loss, anemia, hypothermia, and hypoglycemia, could be used as indicators of a lethal infection in IL-10 / mice, we have compared their magnitude in IL-10 / mice which have survived acute infection and those which have succumbed to a lethal infection. Body weight loss, anemia (hematocrit), hypoglycemia, and body temperature were compared on days 8 to 14 of infection (Fig. 1). Only blood glucose levels and body temperatures were significantly different between those mice that survived and those that did not. Of the IL-10 / mice that survived infection, body temperature did not drop below 28 C (Fig. 1B) and blood glucose did not drop below 3 mm (Fig. 1C), whereas 13 of 20 of those mice that subsequently died had a body temperature lower than 28 C (P , Mann-Whitney test) and 5 of 20 had a blood glucose level lower that 3 mm (P , Mann-Whitney test). In all subsequent experiments, a blood glucose level below 3 mm and a body temperature of below 28 C was taken to indicate that mice were unlikely to survive infection and those mice were culled for humane reasons. Neutralization of TNF- prevents mortality and ameliorates malarial associated pathology. Higher levels of IFN- and TNF- have been detected in IL-10 / mice infected with P. chabaudi when compared with C57BL/6 or WT controls (18). This suggested that enhanced inflammatory responses may contribute to the increased severity of disease and mortality seen in IL-10 / mice. Injection of anti-tnf- antibodies into IL-10 / mice eliminated mortality while 66% of control antibody-treated mice died by day 10 (Fig. 2B) (log rank test for equality of survival function, P 0.001). By contrast, the anti-tnf- treatment had only minimal effects on parasitemia during infection (Fig. 2A). The courses of parasitemia were similar to those reported previously (18, 20), except that IL-10 / mice treated with anti-tnf- showed a slightly more pronounced recrudescence of parasitemia at days 18 and 25 (data not shown) of infection compared with surviving untreated mice. Neutralization of TNF- in vivo ameliorated weight loss, hypothermia, and to a lesser extent, anemia (Fig. 2C, E, and

3 4852 LI ET AL. INFECT. IMMUN. FIG. 1. Comparison of pathological changes between IL-10 / mice that succumbed to infection (F, n 25) and those that survived (E, n 29). The distribution of the percentage of body weight loss (A), body temperature (B), blood glucose concentration (C), and percentage of packed red cell volume (D) are shown for each individual mouse. F). The greatest weight loss, hypoglycemia, hypothermia, and anemia (as measured by hematocrit) occurred between days 8 and 10 of the infection in IL-10 / mice with or without anti- TNF- treatment. The severity of these disease indicators in untreated IL-10 / mice was comparable with those described previously (18). Treatment with anti-tnf- resulted in less weight loss and a more rapid recovery of weight (Fig. 2C). Significant differences in weight loss between anti-tnf- and control antibody-treated mice was observed between days 2 and 5 and at day 10 of infection ( 7.815% in those treated with anti-tnf- antibody compared with 14.28% in those treated with control IgG, P , Student s t test). Similarly, neutralization of TNF- in IL-10 / mice significantly prevented the sharp drop of body temperature between days 8 and 12 (from 35 to 33 C in those treated compared with 35 to 31 C in those untreated, P 0.006, Student s t test) (Fig. 2E). The hematocrit on day 10 of infection was significantly greater in anti-tnf- -treated IL-10 / mice than in control IgG-treated mice (20.557% compared with %, P , Student s t test) (Fig. 2F). There was no significant difference in glucose levels (Fig. 2D) at any time between the two groups of mice. Anti-TNF- treatment reduces levels of TNF- but not of IFN- in plasma. Consistent with previous reports (18), IFN- and TNF- could only be detected in plasma between days 6 and 9 of a primary P. chabaudi infection (Fig. 3). TNF- concentrations were significantly lower in IL-10 / mice treated with anti-tnf- antibodies on day 8 (P 0.05, Mann- Whitney test; P 0.016, Student s t test) (Fig. 3B) and in 4 of 5 mice on day 9 of the infection. Neutralization of TNF- had no significant impact on the level of IFN- (Fig. 3A). Neutralization of TGF- exaggerated the severity of disease in IL-10 / mice. TGF- was measured at 2-day intervals throughout a primary P. chabaudi infection in IL-10 / and control C57BL/6 mice (Fig. 4). The levels of TGF- were essentially similar in both IL-10 / and control BL/6 mice during infection. However, significantly higher levels were transiently observed in IL-10 / mice on day 7 (experiment 2) and days 8 and 10 (experiment 1) (day 7, P , Mann- Whitney test, P 0.036, Student s t test; day 8, P , Mann-Whitney test, P , Student s t test; day 10, P , Mann-Whitney test, P , Student s t test). Treatment of IL-10 / mice with anti-tgf- antibodies significantly increased mortality from 60% in control antibodytreated mice to 100% within 12 days of infection (Fig. 5B) (log rank test for equality of survival function, P 0.001) without any significant difference in parasitemia during that time (Fig. 5A). Treatment of IL-10 / mice with anti-tgf- also resulted in a more severe anemia at day 10 (P , Student s t test) (Fig. 5F) and hypoglycemia from day 8 to 12 (P 0.006, Student s t test) (Fig. 5D). The anti-tgf- treatment did not significantly affect the weight loss (Fig. 5C) or hypothermia in IL-10 / mice (Fig. 5E). Anti-TGF- treatment of control C57BL/6 mice did not affect the survival (100%) or parasitemia (data not shown). Levels of IFN- and TNF- in plasma were transiently in-

4 VOL. 71, 2003 TNF- IN P. CHABAUDI-INFECTED IL-10-DEFICIENT MICE 4853 FIG. 2. Course of parasitemia (A), survival rate (B) and pathological changes (C to F) of IL-10 / mice treated with anti-tnf- ( ) or control IgG antibodies ( ) during a primary erythrocytic-stage P. chabaudi infection. Parasitemias are presented as the geometric means standard errors of the means of pooled data from 32 mice from four independent experiments. Body weight (C), blood glucose concentration (D), temperature (E), and hematocrit (F) changes were measured during the course of infection as described in the text. These graphs show means standard errors of the means of the values for 32 mice pooled from four independent experiments. Standard errors of the means of less than 10% of the means are not shown. *, significant differences are observed between two groups of mice. creased in those IL-10 / mice treated with anti-tgf- antibodies (Fig. 5G and H). DISCUSSION Proinflammatory cytokines such as TNF- have long been associated with the immunopathology of malaria infection in both animal models and in humans. It was previously shown that IL-10 / mice infected with P. chabaudi, in which cytokines such as TNF-, IFN-, and IL-12 are elevated, suffer a more severe malaria infection than their WT control littermates or C57BL/6 mice (18). Approximately 50% of female mice die without increased parasitemia, and all mice display increased weight loss, hypothermia, hypoglycemia, and anemia during the acute phase of infection compared with control mice. Here, we show that a glucose concentration of less than 3 mm and a temperature lower than 28 C generally indicated a poor prognosis in P. chabaudi-infected IL-10 / mice. Treatment of IL-10 / mice with antibodies to neutralize TNF- in vivo allowed all mice to survive a primary infection and also ameliorated hypothermia and weight loss to levels normally found in the infected control C57BL/6 mice (18). This contrasts somewhat to the effects of IFN- neutralization in these mice, where it has been shown that although IFN- contributed to the death of P. chabaudi-infected IL-10 / mice, its removal did not affect any of the other symptoms (18). In addition, plasma levels of TNF- were not diminished by anti-ifn- antibody treatment. These data together suggest that TNF- can be produced in P. chabaudi infections by IFN- -independent pathways and that this TNF- may be important in inducing weight loss and hypothermia. The inability of anti-tnf- treatment to eliminate weight loss and hypo-

5 4854 LI ET AL. INFECT. IMMUN. FIG. 3. Concentrations of IFN- (A) and TNF- (B) in the plasma of individual IL-10 / mice treated with either anti-tnf- (, n 5) or control IgG (, n 5) during the course of a primary P. chabaudi infection. Each point represents data from one animal. thermia completely suggests either that TNF- -mediated mechanisms are not the only ones involved in the pathological sequelae of P. chabaudi infection or that TNF- is not completely neutralized. This latter explanation is supported by the finding that low levels of TNF- in the plasma of treated mice on day 8 of infection. The minimal effects of anti-tnf- treatment on hypoglycemia and anemia indicate that TNF- may not be directly involved in these complications. Experiments with double knockout mice defective in both IL-10 and TNF- or TNF- receptors would address these issues. In another mouse model of malaria pathology, experimental CM caused by P. berghei (ANKA), neutralization by anti- TNF- antibodies, or inactivation of TNF- receptors by gene targeting could prevent the development of cerebral complications (7, 21, 22). However, anti-tnf- antibodies given to children with severe malaria decreased the level of fever (14) but failed to ameliorate established CM (14, 34). Polymorphism of the TNF- gene promoter have been shown to associate with different aspects of malarial disease in humans; the TNF -308 A allele is associated with a high level of TNF- production and CM (23), whereas the TNF -238 A allele is associated with severe anemia (24). Other field studies have shown that severe malarial anemia is not associated with the absolute levels of TNF- in plasma but rather with low ratios of IL-10 to TNF- (27). Taking all the evidence together, although a high concentration of TNF- may play a role in pathogenesis of malaria infections in mouse models and in FIG. 4. Concentration of TGF- in the plasma of IL-10 / (F) and WT (E) mice during a primary P. chabaudi infection. (A) Experiment 1, 5 mice per group; (B) experiment 2, 15 mice per group. Each data point represents one animal. *, significant differences between the two groups of mice. The horizontal line on each graph represents the amount of TGF- in uninfected control plasma. humans, this is not the only factor that contributes to the development of severe malaria. Recent findings of Engwerda et al. (4) in the P. berghei model of CM suggest that it is not the circulating TNF- itself that is responsible for disease but lymphotoxin- (LT), which can bind to the TNF receptor II (p75) (1) and is recognized by most anti-tnf- antibodies (30). It would therefore be of interest to determine whether LT is up-regulated and whether it plays a role in the pathology induced in IL-10 / mice infected with P. chabaudi. A role for LT in human malaria has not yet been demonstrated. Regulatory cytokines, such as IL-10 and TGF-, play important roles in maintaining the balance of inflammatory responses (16, 25). IL-10 is clearly important in the regulation of T-cell responses and in down-regulation of the inflammatory cytokines IFN- and TNF- in malaria infections (12, 18, 20) and in other infectious diseases (5, 11, 13). However, in IL- 10 / mice that survive a primary malaria infection, these cytokines are eventually down-regulated after the acute stage of infection. The levels of TNF- and IFN- decrease in the plasma, and CD4 Th2 responses to the parasite can be made (18). Therefore, it is likely that other cytokines can also control TNF- and IFN- in a P. chabaudi infection. TGF- has recently been shown to inhibit protective inflammatory responses in T. gondii, T. cruzi, and Leishmania infections (6, 10, 15, 29, 31) and to diminish the pathological inflammatory responses induced by Schistosoma mansoni (8) and P. berghei (26).

6 VOL. 71, 2003 TNF- IN P. CHABAUDI-INFECTED IL-10-DEFICIENT MICE 4855 FIG. 5. Survival rate and pathological changes in IL-10 / mice treated with anti-tgf- antibody (, n 10) or control IgG (, n 10) during a primary P. chabaudi infection. The data represent the means standard errors of the means of two independent experiments, with a total of 10 mice. Standard errors of the means of less than 10% of the mean are not shown. Representative levels of IFN- and TNF- in plasma on day 8 in IL-10 / mice treated with anti-tgf- antibody ( ) or control IgG ( ) are shown. Each bar represents an individual mouse. *, significant differences between the two groups of mice. IL-10 / mice infected with P. chabaudi showed transiently raised levels of TGF- in plasma during acute infection compared with infected WT controls (Fig. 4). This coincided with the disappearance of IFN- and TNF- from the circulation in IL-10 / mice. We therefore considered it possible that TGF- might be important in regulating proinflammatory cytokine production in the absence of IL-10, thus reducing the potential lethal effects of these cytokines. Removal of TGF- rendered IL-10 / mice completely susceptible to a P. chabaudi infection with 100% mortality within 12 days. This high mortality rate may have been due to the significantly increased weight loss and the rapid onset of anemia and increased hypoglycemia occurring in the treated mice. Unlike BALB/c mice (26), normal C57BL/6 mice infected with P. chabaudi and treated with anti-tgf- antibodies did not succumb to a lethal infection. This is consistent with earlier studies showing that TGF- had a less pronounced effect on mouse strains, such as C57BL/6, that were resistant to P. chabaudi infection (33). TGF- is secreted by CD4 T regulatory cells and macrophages and is able to regulate both Th1 and Th2 T-cell responses (35). Although the levels of TGF- are increased during a P. chabaudi infection, the source of this cytokine and its potential impor-

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