Mixed parasite infections are common in many parts of the world, but little is known of the effects of concomitant parasite infections on the immune response or severity of clinical disease. We have used the nonlethal malaria infection model of Plasmodium chabaudi AS in combination with the gastrointestinal nematode Heligmosomoides bakeri polygyrus to investigate the impact of nematode infections on malarial morbidity and antimalarial immunity. The data demonstrate that wild-type C57BL/6 mice coinfected with both parasites simultaneously exhibit a striking increase in mortality, while mice deficient in IFN-γ or IL-23 survive coinfection. The increase in mortality in wild-type mice was associated with severe liver pathology characterized by extensive coagulative necrosis and an increase in hepatic IFN-γ, IL-17, and IL-22 mRNA expression. This is the first demonstration of increased malaria-associated pathology associated with a switch toward a proinflammatory environment, involving not only IFN-γ but also the IL-17/IL-23 axis, as a result of coinfection with a gastrointestinal helminth.

Helminth infections are among the most common infections of humans. Most helminth infections, such as gastrointestinal nematode infections, are chronic infections, where the worms are long-lived and may survive within their host for many years. To survive such extended periods of time without provoking a strong inflammatory immune response, these organisms have developed sophisticated survival strategies involving secretion of immune modulatory substances that induce anti-inflammatory and/or regulatory immune responses (1). This ability of helminth parasites to modulate immune responses and immune responsiveness have recently generated a great deal of interest, and beneficial effects of helminth infections and/or products have been demonstrated using experimental models of conditions such as inflammatory bowel disease, diabetes, and allergy (2, 3, 4). The majority of such studies have used the adult stages of helminth infections and little is known about how the impact of infections with juvenile stages of helminths may differ from that of adult worms. Importantly, the immune response to larval stages of helminth infections can differ significantly from adult infections (5, 6, 7, 8, 9). This may be of importance in the human field situation during conditions such as migration where previously unexposed individuals may be exposed to primary infections with several pathogens simultaneously.

Experimental models of helminth-malaria coinfections have suggested a role for helminths in altering immune responsiveness and disease progression of malaria (10, 11, 12). In human helminth-malaria coinfections, the picture is less clear (reviewed in Refs. 13 and 14). To investigate the effects of larval infections rather than established adult helminth infections, we have focused our studies on how a concurrent larval intestinal nematode infection may alter the course of a blood-stage malaria infection in vivo. We have used the nonlethal rodent malaria Plasmodium chabaudi chabaudi AS along with larval infection of the gastrointestinal nematode Heligmosomoides bakeri polygyrus (=Nematospiroides dubius). The underlying immunological responses required to clear a blood-stage P. chabaudi AS infection have been well described and involve the sequential activation of NK cells and Th1 cells, followed by Th2 cells and Ab-producing B cells (15, 16, 17, 18). H. bakeri polygyrus is a well-known murine model of gastrointestinal nematode infection. Established adult infections with this worm is characterized by production of Th2 cytokines, IL-10, TGF-β, and expansion of T regulatory cells (1, 19, 20, 21), and this infection model has been commonly used to investigate the effects of chronic helminth infection on immune reactivity (12, 22, 23, 24, 25, 26, 27, 28). However, no previous studies have focused on the effects of early larval-stage infections.

Our data show that when mice were coinfected with both pathogens at the same time a striking increase in mortality was detected. This was associated with severe liver pathology characterized by extensive necrosis with increased levels of proinflammatory cytokines and liver-associated transaminase in serum. Splenic cytokine responses were unaltered, demonstrating that the dysregulated responses were organ specific. Mice deficient in TNF-α or lymphotoxin (LT)3 α were as susceptible to coinfection as wild-type (WT) animals. However, IFN-γ-, IL-12/23p40-, and IL-23p19-deficient animals were resistant, demonstrating that the mortality was associated with organ-specific overproduction of Th1- and Th17-type cytokines. This is the first demonstration of increased malaria-associated pathology as a result from coinfection with larval stages of a gastrointestinal helminth and appears to be mediated by proinflammatory cytokine pathways involving IL-23/IL-17 and IFN-γ.

Six- to 8-wk-old female C57BL/6 mice were bred at the animal unit of the London School of Hygiene and Tropical Medicine (London, U.K.). Female mice deficient in IL-12/23p40 (29), TNF-α (30), LT-α (31), and IFN-γ (32) on a C57BL/6 background were all originally obtained from the Jackson Laboratory and bred at the animal unit of the London School of Hygiene and Tropical Medicine under specific pathogen-free conditions. IL-23p19-deficient (33) mice were originally obtained from Schering-Plough Biopharma and were provided by Dr. B. Stockinger (National Institute of Medical Research, London, U.K.). All experiments were performed under the regulations of the Home Office Scientific Procedures Act (1986).

Experimental animals (8–20 per group) were infected with 106 asexual blood-stage P. chabaudi chabaudi AS malaria parasites i.p and/or 200 H. polygyrus L3 larvae by oral gavage. Maintenance and infection of P. chabaudi AS were as described previously (10). H. bakeri polygyrus were originally obtained from Prof. J. M. Behnke (University of Nottingham, Nottingham, U.K.) and maintained as stock infection in CD-1 mice. New stock was prepared every 2 mo. Infective L3 larvae were cultured as described previously (34). Control groups receiving single infections were infected in parallel for each experiment. Malaria parasitemia was determined on Giemsa-stained thin blood smears and the levels of blood hemoglobin were analyzed using a Hemocue hemoglobin analyzer according to the manufacturer’s instructions.

Mesenteric lymph node and spleen cells were removed from uninfected and infected animals and resuspended in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM l-glutamine, 0.05 mM 2-ME (all from Invitrogen), and 25 μg/ml gentamicin (Sigma-Aldrich). Liver mononuclear cells were prepared by collagenase digestion and centrifugation over Ficoll. Cells were cultured at 37°C and 5% CO2 in flat-bottom 96-well plates (Nunc) at a final concentration of 5 × 106/ml in a final volume of 0.2 ml/well. Cells were stimulated with H. polygyrus crude worm Ag, P. chabaudi lysate (both at 25 μg/ml), or plate-bound anti-CD3 Ab (mAb145-2C11, 10 μg/ml; American Type Culture Collection). Cell-free supernatants were harvested after 48 h and stored at −80°C.

Cytokine analyses were conducted using routine sandwich ELISAs for IL-4, IL-10, and IFN-γ (Mabtech). IL-13 and TNF-α were analyzed using Ab pairs from R&D Systems. LBP in sera was analyzed by a commercial ELISA kit (Hycult Biotechnology) according to the manufacturer’s instructions.

To measure the liver-associated enzyme AST in serum, a modified protocol of the standard colorimetric end-point method was used. Briefly, 20 μl of serum was added to 100 μl of 0.2 M dl-aspartate and 1.8 M α-ketoglutaric acid in PBS, mixed, and incubated (37°C, 1 h), followed by the addition of 100 μl of 2,4-dinitrophenylhydrazine. The mixture was incubated for an additional 20 min at room temperature. The reaction was stopped by addition of 1 ml of 0.4 M NaOH and absorbance was read at 490 nm after 5 min.

Livers were fixed in neutral-buffered formalin, histologically processed using standard methods, and sections were stained with H&E.

Tissues were harvested and stored in RNAlater (Qiagen) at −80°C until processing. RNA was purified with an RNeasy mini kit from Qiagen according to the manufacturer’s instructions using the additional DNase treatment step (Qiagen). Reverse transcription was performed using the Omniscript RT kit (Qiagen). Real-time PCR was performed in an Applied Biosystems 7000 sequence detection system using the SYBR Green PCR Master Mix (Qiagen). Results were normalized to the housekeeping gene HPRT and expressed as fold increase compared with tissue from naive, uninfected controls (given an arbitrary value of 1).

Significant differences (p < 0.05) between experimental groups were determined using the Student t test.

To investigate whether an intestinal nematode infection can alter the course of a malaria infection in vivo, we infected female C57BL/6 mice (10–20 per group) with 200 H. polygyrus L3 larvae by oral gavage and/or 106 asexual blood-stage P. chabaudi chabaudi AS malaria parasites i.p. Control groups receiving single infections were infected in parallel for each experiment. Malaria parasitemia was determined on Giemsa-stained thin blood smears and blood hemoglobin levels were analyzed using a Hemocue hemoglobin analyzer. The results in Fig. 1 show that simultaneous coinfection of H. polygyrus and P. chabaudi malaria dramatically increased malaria mortality. P. chabaudi AS is normally a nonlethal malaria infection in most strains of mice, including the C57BL/6 strain, which was used in these experiments. Results in Fig. 1,D show that the survival rate of mice infected with either H. polygyrus or P. chabaudi alone was 100%. However, mice coinfected with H. polygyrus and P. chabaudi developed severe symptoms of disease and, by day 7 postinfection (p.i.), 12 of 20 animals (60%) succumbed to the infection. By day 9 p.i., the mortality rate had risen to 70%. The experiment was repeated five times with a similar outcome (data not shown). When the experiment was performed in BALB/c mice, the mortality reached 100% in the coinfected mice by day 9 p.i. (Fig. 1 E).

FIGURE 1.

Malaria and helminth coinfection results in high mortality without increasing malaria parasitemia. Malaria parasitemia (A), hemoglobin (B), body weight (C), and survival (D) during P. chabaudi-H. polygyrus coinfection in C57BL/6 mice. Mice were infected orally with 200 H. polygyrus L3 larvae (▴), 106 asexual blood-stage P. chabaudi AS parasites i.p. (▪), or both (□) at day 0. Mean of 10–20 mice per group and SEM are shown. Asterisks indicate statistically significant difference between groups of mice with P.c. or P.c. + H. p. infection (p < 0.05). Data from one representative experiment of five are shown. Mortality (E) during H. polygyrus-P. chabaudi coinfection in BALB/c mice infected with P.c. AS (▪) or H.p. + P.c. (▴) at day 0 (eight mice per group). Data from one representative experiment of two are shown. Mortality (F) during H. polygyrus-P. chabaudi coinfection in C57BL/6 mice infected with P. chabaudi AS only at day 0 (▪) or P. chabaudi at day 0 plus H. polygyrus at the indicated time points (day 0 or 7 or 14 days before P. chabaudi) (five to seven mice per group). Data from one representative experiment of three are shown.

FIGURE 1.

Malaria and helminth coinfection results in high mortality without increasing malaria parasitemia. Malaria parasitemia (A), hemoglobin (B), body weight (C), and survival (D) during P. chabaudi-H. polygyrus coinfection in C57BL/6 mice. Mice were infected orally with 200 H. polygyrus L3 larvae (▴), 106 asexual blood-stage P. chabaudi AS parasites i.p. (▪), or both (□) at day 0. Mean of 10–20 mice per group and SEM are shown. Asterisks indicate statistically significant difference between groups of mice with P.c. or P.c. + H. p. infection (p < 0.05). Data from one representative experiment of five are shown. Mortality (E) during H. polygyrus-P. chabaudi coinfection in BALB/c mice infected with P.c. AS (▪) or H.p. + P.c. (▴) at day 0 (eight mice per group). Data from one representative experiment of two are shown. Mortality (F) during H. polygyrus-P. chabaudi coinfection in C57BL/6 mice infected with P. chabaudi AS only at day 0 (▪) or P. chabaudi at day 0 plus H. polygyrus at the indicated time points (day 0 or 7 or 14 days before P. chabaudi) (five to seven mice per group). Data from one representative experiment of three are shown.

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The development of malaria parasitemia was similar in the single and coinfected groups before (days 3–5 p.i.) and at the peak of parasitemia (days 6 and 7 p.i.; Fig. 1,A). However, the malaria parasitemia was higher in the small proportion of coinfected mice that survived infection, after the peak of parasitemia (at days 9 and 10 p.i.) than in mice infected with malaria alone (Fig. 1 A).

As a measure of the severity of disease, we also measured the levels of hemoglobin in blood and changes in body weight during the course of infection. The coinfected group developed anemia at the same rate as the control group, but the small group of surviving coinfected animals had significantly lower hemoglobin levels than the singly infected animals at days 9 and 12 p.i. (Fig. 1,B), correlating with the higher parasitemia detected in this group at these time points (Fig. 1,A). Weight loss was similar in single infected and surviving coinfected animals and was maximal at times of peak parasitemia (Fig. 1,C). Surviving coinfected animals regained weight at a similar rate to the malaria alone-infected group (Fig. 1 C). As expected, mice infected with H. polygyrus alone developed neither anemia nor weight loss and survived indefinitely.

We further investigated the importance of the timing of H. polygyrus infection. When mice were inoculated with H. polygyrus 7 or 14 days before P. chabaudi infection, there was only a minor, nonsignificant, effect on P. chabaudi infection (Fig. 1 F), demonstrating that it was the larval stages rather than adult stages of the helminth infection that were responsible for the increased malaria mortality.

To investigate whether the increased mortality was due to an imbalance in cytokine production in the spleen, the main organ for antimalarial immune responses, we analyzed cytokine mRNA expression by quantitative real- time PCR. The data presented in Fig. 2 show that P. chabaudi infection alone strongly increased the expression of IFN-γ by day 4 p.i. and to a lesser degree IL-12p40, IL-12p35, TNF-α, IL-10, IL-13, and IL-4. The expression of IL-10 continued to increase by day 7 p.i. and a concomitant decrease in the mRNA expression of all other cytokines at this time point was observed in animals infected with malaria only, in agreement with other studies showing the switch away from a Th1 response around the time of peak parasitemia (15, 16, 17, 18). P. chabaudi infection alone did not increase expression of IL-23p19 at any time point. Interestingly, H. polygyrus infection alone strongly increased IFN-γ and IL-23p19 already at day 4 p.i. and this was followed by an increased expression of IL-4 and IL-13 by day 7 p.i. H. polygyrus alone did not increase expression of IL-12p35 or TNF-α and only induced a very moderate increase in IL-10. Spleens from coinfected animals showed a similar cytokine profile to P. chabaudi-infected animals, demonstrating that the concurrent helminth infection did not inhibit or enhance the cytokine response to the malaria infection. We also measured protein levels of IL-4, IFN-γ, and IL-10 from in vitro- restimulated spleen cell cultures by ELISA and found no differences in the malaria-specific response between malaria- only infected and helminth-coinfected animals (Fig. 3). The ELISA data also confirm that the malaria-specific response had little effect on worm-specific responses on day 4, but by day 7 the malaria-specific responses had come to dominate, resulting in down-regulation of worm-specific IFN-γ and IL-4 responses (but not worm-specific IL-10) in the spleens of coinfected animals (Fig. 3).

FIGURE 2.

Normal splenic cytokine mRNA expression during P. chabaudi-H. polygyrus coinfection. C57BL/6 mice were infected orally with 200 H. polygyrus L3 larvae (▴), 106 asexual blood-stage P. chabaudi AS parasites i.p (▪), or both (□) at day 0. RNA was prepared from freshly isolated spleen tissue at various time points p.i., reverse transcribed, and cytokine mRNA expression was analyzed by real-time PCR. Mean of four to six mice per group and SEM are shown. Data from one representative experiment of three are shown.

FIGURE 2.

Normal splenic cytokine mRNA expression during P. chabaudi-H. polygyrus coinfection. C57BL/6 mice were infected orally with 200 H. polygyrus L3 larvae (▴), 106 asexual blood-stage P. chabaudi AS parasites i.p (▪), or both (□) at day 0. RNA was prepared from freshly isolated spleen tissue at various time points p.i., reverse transcribed, and cytokine mRNA expression was analyzed by real-time PCR. Mean of four to six mice per group and SEM are shown. Data from one representative experiment of three are shown.

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FIGURE 3.

Ag-specific splenic cytokine secretion during P. chabaudi-H. polygyrus coinfection. C57BL/6 mice were infected orally with 200 H. polygyrus L3 larvae (▴), 106 asexual blood-stage P. chabaudi AS parasites i.p. (▪), or both (□) at day 0. Spleen cell suspensions were prepared at the indicated time points and restimulated with H. polygyrus Ag (H.poly Ag) or lysate of P. chabaudi-infected RBC (pRBC) for 48 h. IFN-γ, IL-4, and IL-10 levels in supernatants were analyzed by sandwich ELISA. Mean of four to six mice per group and SEM are shown. Data from one representative experiment of three are shown.

FIGURE 3.

Ag-specific splenic cytokine secretion during P. chabaudi-H. polygyrus coinfection. C57BL/6 mice were infected orally with 200 H. polygyrus L3 larvae (▴), 106 asexual blood-stage P. chabaudi AS parasites i.p. (▪), or both (□) at day 0. Spleen cell suspensions were prepared at the indicated time points and restimulated with H. polygyrus Ag (H.poly Ag) or lysate of P. chabaudi-infected RBC (pRBC) for 48 h. IFN-γ, IL-4, and IL-10 levels in supernatants were analyzed by sandwich ELISA. Mean of four to six mice per group and SEM are shown. Data from one representative experiment of three are shown.

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We next investigated whether the appearance of inflammatory mediators in serum, which normally accompanies acute P. chabaudi infection, was altered by the presence of larval nematode infection. Animals infected with P. chabaudi showed a moderate, but significant, increase in serum IFN-γ (day 4 p.i.) and TNF-α (days 4 and 7 p.i.) compared with sera from uninfected animals (Fig. 4, A and B). No such increase could be detected in animals with only H. polygyrus infection. Coinfected animals, however, had significantly higher levels of serum IFN-γ (days 4 and 7 p.i.) and TNF-α (day 7 p.i.) (Fig. 4, A and B) than animals infected with malaria alone. Mice with only P. chabaudi infection had significantly elevated levels of IL-10 in the circulation already from day 2 p.i. The levels of IL-10 then continued to increase as the infection progressed (Fig. 4,C). The coinfected group, however, had significantly lower levels of circulating IL-10 throughout the infection (Fig. 4,C), which were similar to the modest up-regulation of IL-10 seen in animals infected with H. polygyrus alone. When expressing the data as the ratio of IFN-γ vs IL-10 in serum, it is clear that the coinfected mice had a very strong bias toward circulating IFN-γ, both at days 4 and 7, compared with the groups with single malaria or nematode infections (Fig. 4 D).

FIGURE 4.

Increased levels of serum cytokines, LBP, and liver enzyme during malaria-helminth coinfection. C57BL/6 mice were infected as above. Serum levels of IFN-γ (A), TNF-α (B), IL-10 (C), and LBP (F) were measured by sandwich ELISA and AST (E) using a colorimetric assay. Mean of four to eight mice per group and SEM are shown. Data from one representative experiment of three are shown. Asterisks indicate statistically significant difference between groups of mice with P.c. or P.c + H.p. infection (p < 0.05).

FIGURE 4.

Increased levels of serum cytokines, LBP, and liver enzyme during malaria-helminth coinfection. C57BL/6 mice were infected as above. Serum levels of IFN-γ (A), TNF-α (B), IL-10 (C), and LBP (F) were measured by sandwich ELISA and AST (E) using a colorimetric assay. Mean of four to eight mice per group and SEM are shown. Data from one representative experiment of three are shown. Asterisks indicate statistically significant difference between groups of mice with P.c. or P.c + H.p. infection (p < 0.05).

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The liver is well known as the site of pre-erythrocytic development of Plasmodium parasites. However, the liver also participates in the killing and removal of infected RBC from the circulation (35, 36) and signs of T cell-mediated liver injury were present during blood-stage infection (35, 37, 38, 39). We therefore measured serum levels of the liver-associated enzyme AST in mice with single infection and coinfection (Fig. 4,E). AST is normally contained within hepatocytes, but when liver damage occurs, AST is released, resulting in elevated levels of the enzyme in the circulation. The data in Fig. 4,E show that coinfected mice displayed greatly increased levels of AST in serum already at day 2 after malaria infection. The levels increased further on days 4 and 7 p.i. (Fig. 4 E), indicating severe liver damage during H. polygyrus/P. chabaudi coinfection but not during single infection with either pathogen.

LBP acts to amplify the cellular response to LPS and can be used as a marker for acute phase responses and sepsis-like syndromes (40). We analyzed the levels of LBP in serum and found that the coinfected mice had greatly enhanced LBP levels at day 7 p.i. compared with the singly infected mice, again suggesting a severe, acute endotoxin-like response during the malaria-nematode infection (Fig. 4 F).

Because high levels of AST in serum are indicative of liver injury, we performed histological analysis on the livers from the different groups of mice. As can be seen in Fig. 5, severe liver damage characterized by centrilobular necrosis, inflammatory infiltration, and hemorrhaging was evident in the coinfected animals at day 7 p.i. (Fig. 5, D and F). H. polygyrus infection alone did not alter liver histology and there was no evidence of inflammation or eosinophil infiltration (Fig. 5,B). P. chabaudi infection alone resulted in inflammatory cell infiltration but no overt hepatic damage or necrosis was observed (Fig. 5, C and E). These results clearly demonstrate that malaria-H. polygyrus coinfection results in severe hepatic liver injury with increased levels of serum AST, hepatic inflammatory infiltrates, and centrilobular necrosis.

FIGURE 5.

Severe liver damage during malaria-helminth coinfection. Liver histology at day 7 p.i. C57BL/6 mice were infected as above. Livers were fixed in neutral-buffered formalin, processed, and stained with H&E using standard methodology. Liver from uninfected control (A). H. polygyrus alone day 7 p.i. (B), P. chabaudi alone day 7 p.i. (C and E), H. polygyrus plus P. chabaudi day 7 p.i. (D and F). A–D, ×100 original magnification; E and F, ×200 original magnification. Data from one representative experiment of three are shown.

FIGURE 5.

Severe liver damage during malaria-helminth coinfection. Liver histology at day 7 p.i. C57BL/6 mice were infected as above. Livers were fixed in neutral-buffered formalin, processed, and stained with H&E using standard methodology. Liver from uninfected control (A). H. polygyrus alone day 7 p.i. (B), P. chabaudi alone day 7 p.i. (C and E), H. polygyrus plus P. chabaudi day 7 p.i. (D and F). A–D, ×100 original magnification; E and F, ×200 original magnification. Data from one representative experiment of three are shown.

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Because coinfected animals displayed inflammatory infiltrates and severe pathology of the liver, we analyzed hepatic cytokine mRNA expression by quantitative real-time PCR. The data presented in Fig. 6 show that liver cytokine expression during P. chabaudi infection alone closely mirrored that of the mRNA expression in the spleen. There was a strong increase in the expression of IFN-γ and TNF-α already at day 4 p.i. However, by day 7, both IFN-γ and TNF-α expression were down-regulated. This down-regulation coincided with a strong up-regulation of IL-10. P. chabaudi infection alone did not increase liver expression of IL-23p19, IL-12p35, IL-4, IL-13, or the Th17 cytokines IL-17 and IL-22. H. polygyrus infection alone increased IL-4 and IL-13 expression and to a lesser degree IL-12p40, IL-23p19, and IFN-γ. H. polygyrus infection alone did not increase expression of IL-10, TNF-α, IL-12p35, IL-17, or IL-22. However, livers from coinfected animals expressed high levels of IFN-γ, TNF-α, IL-12/23p40, IL-23p19, IL-17, IL-22, and IL-4 mRNA with little or no increase in IL-12p35, IL-13, and IL-10 expression. Thus, P. chabaudi infection alone results in strong inflammatory cytokine responses in the liver, similar to the cytokine profile observed in the spleen, and this proinflammatory cytokine response appears to be controlled by IL-10. In contrast, the low levels of IL-10 and high levels of inflammatory cytokines in the livers of H. polygyrus-P. chabaudi coinfected animals suggest that coinfection may result in an uncontrolled proinflammatory response leading to enhanced liver pathology. The lack of IL-12p35 expression during both P. chabaudi infection alone and during coinfection, in conjunction with increased IL-12p40 and IL-23p19 mRNA expression, strongly suggests that the proinflammatory response is driven by IL-23 rather than IL-12. This is further supported by the increase in IL-17 and IL-22, cytokines produced by IL-23-driven Th17 cells (41).

FIGURE 6.

Altered liver cytokine mRNA expression during P. chabaudi-H. polygyrus coinfection. C57BL/6 mice were infected as above. RNA was prepared from freshly isolated liver tissue, reverse transcribed, and cytokine mRNA expression was analyzed by real-time PCR. Mean of four to six mice per group and SEM are shown. Data from one representative experiment of three are shown.

FIGURE 6.

Altered liver cytokine mRNA expression during P. chabaudi-H. polygyrus coinfection. C57BL/6 mice were infected as above. RNA was prepared from freshly isolated liver tissue, reverse transcribed, and cytokine mRNA expression was analyzed by real-time PCR. Mean of four to six mice per group and SEM are shown. Data from one representative experiment of three are shown.

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Cytokine secretion from purified liver mononuclear cells analyzed by ELISA confirmed the high IFN-γ and low IL-10 responses in livers from coinfected animals (IFN-γ: P. chabaudi (P.c.) infected, 3.42 ± 0.78 ng/ml vs P.c. plus H. polygyrus (H.p.) infected, 6.46 ± 0.46 ng/ml; IL-10: P.c. infected, 2.15 ± 0.09 ng/ml vs P.c. plus H.p. infected, 0.61 ± 0.10 ng/ml).

Thus, our data suggest that a larval intestinal nematode infection can disrupt the delicate and critical balance of pro- and anti-inflammatory responses needed to prevent excessive liver pathology during acute blood-stage malaria infection.

In view of the fact that the coinfected mice expressed high levels of IFN-γ, TNF-α, IL-12p40, and IL-23p19 in the liver, we investigated the importance of these cytokines in the increased mortality seen during coinfection. Groups of IFN-γ, TNF-α, LT-α, IL-12p40, IL-23p19 knockout (KO), and C57BL/6 WT mice were infected with P. chabaudi alone, H. polygyrus alone, or coinfected. The data in Fig. 7,A show that the C57BL/6 WT mice developed severe pathology and >80% of the animals had died by day 9 p.i. Surprisingly, the TNF-α and LT-α KO mice succumbed to the coinfection at a similar rate as the WT mice, demonstrating that the mortality associated with coinfection is not mediated by TNF-α or LT-α (Fig. 7,A). Importantly, the majority of the IL-12p40, IL-23p19, and IFN-γ KO mice survived coinfection, demonstrating that the mortality was dependent on IL-12/23 and IFN-γ (Fig. 7,A). None of the mice died of malaria infection or H. polygyrus infection alone during the time course studied (21 days; data not shown). There were no major differences in malaria parasitemia between malaria-only and coinfected animals for each strain of KO mice used (Fig. 7,B). IFN-γ and IL-12p40 KO mice had increased peak parasitemia and delayed clearance from the bloodstream compared with WT mice, in agreement with other studies using female mice (42, 43), and coinfection did not alter this (Fig. 7 B). TNF-α and LT-α KO mice did not exhibit altered parasitemia compared with WT mice, again in agreement with previous studies (44).

FIGURE 7.

Increased mortality during H. polygyrus-P. chabaudi coinfection is dependent on IL-12p40, IFN-γ, and IL-23p19, but not on TNF-α or LT-α. Survival rates (A) and malaria parasitemia (B) in various KO strains coinfected with P. chabaudi and H. polygyrus. C57BL/6 WT mice, IFN-γ, IL-12p40, TNF-α, LT-α, and IL-23p19KO mice were infected as above (five to eight mice per group). Data from one representative experiment of three (two for p19KO experiment) are shown.

FIGURE 7.

Increased mortality during H. polygyrus-P. chabaudi coinfection is dependent on IL-12p40, IFN-γ, and IL-23p19, but not on TNF-α or LT-α. Survival rates (A) and malaria parasitemia (B) in various KO strains coinfected with P. chabaudi and H. polygyrus. C57BL/6 WT mice, IFN-γ, IL-12p40, TNF-α, LT-α, and IL-23p19KO mice were infected as above (five to eight mice per group). Data from one representative experiment of three (two for p19KO experiment) are shown.

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Serum cytokine analysis at day 7 p.i. demonstrated that the susceptible WT and TNF-α KO mice had high ratios of IFN-γ vs IL-10 in serum, while the resistant IL-12p40 KO mice managed to retain balanced levels of these two cytokines (Fig. 8,A). Measurement of AST levels in serum confirmed that the liver damage was dependent on IL-12p40, IL-23p19, and IFN-γ but not on TNF-α (Fig. 8,B). Furthermore, liver histology at day 7 p.i. again confirmed that WT and TNF-α KO mice developed extensive centrilobular necrosis and inflammatory infiltrates while no such histopathology could be detected in IFN-γ and IL-12p40 KO mice (Fig. 9).

FIGURE 8.

Liver damage during H. polygyrus-P. chabaudi coinfection is dependent on IL-12p40, IFN-γ, and IL-23p19, but not on TNF-α or LT-α. Serum cytokines (A) and AST levels in serum (B) in various KO strains coinfected with P. chabaudi and H. polygyrus. C57BL/6 WT mice, IFN-γ, IL-12p40, TNF-α, LT-α, and IL-23p19KO mice were infected as above (five to eight mice per group). Data from one representative experiment of three (two for p19KO experiment) are shown. nd, Not detectable. Asterisks indicate statistically significant difference between groups of mice with P.c. or P.c. + H.p. infection (p < 0.05).

FIGURE 8.

Liver damage during H. polygyrus-P. chabaudi coinfection is dependent on IL-12p40, IFN-γ, and IL-23p19, but not on TNF-α or LT-α. Serum cytokines (A) and AST levels in serum (B) in various KO strains coinfected with P. chabaudi and H. polygyrus. C57BL/6 WT mice, IFN-γ, IL-12p40, TNF-α, LT-α, and IL-23p19KO mice were infected as above (five to eight mice per group). Data from one representative experiment of three (two for p19KO experiment) are shown. nd, Not detectable. Asterisks indicate statistically significant difference between groups of mice with P.c. or P.c. + H.p. infection (p < 0.05).

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FIGURE 9.

Liver histology at day 7 p.i. C57BL/6 WT mice, IFN-γ, IL-12p40, and TNF-α KO mice were coinfected as above. Livers were fixed in neutral-buffered formalin, processed, and stained with H&E using standard methodology. C57BL/6 WT (A), TNF-α KO (B), IFN-γ KO (C), and IL-12p40 KO (D). A–D, ×200 original magnification. Data from one representative experiment of three are shown.

FIGURE 9.

Liver histology at day 7 p.i. C57BL/6 WT mice, IFN-γ, IL-12p40, and TNF-α KO mice were coinfected as above. Livers were fixed in neutral-buffered formalin, processed, and stained with H&E using standard methodology. C57BL/6 WT (A), TNF-α KO (B), IFN-γ KO (C), and IL-12p40 KO (D). A–D, ×200 original magnification. Data from one representative experiment of three are shown.

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Its is well established that the control and clearance of a nonlethal blood-stage P. chabaudi AS infection is dependent on a strong proinflammatory response involving NK cells and Th1 cells (15, 16, 17, 18) and that this proinflammatory response is under the critical control of the anti-inflammatory cytokines IL-10 and TGF-β (45, 46, 47). Although the spleen is of central importance in the immune response to blood-stage malaria, the liver is well known as the site of pre-erythrocytic development of Plasmodium parasites. However, the liver is also an important organ during blood-stage infection and participates in the killing and removal of infected RBC from the circulation (35, 36). Adoptive transfer experiments have shown that lymphomyeloid cells isolated from infected livers can confer protection to naive recipients (35), and there is an increase in the number of active Kupffer cells (48) during blood-stage infection. Furthermore, elevated levels of IFN-γ, IL-12p40, IL-18, and IL-10 in the liver have been demonstrated during blood-stage malaria (37, 49). As with the need for a critically balanced pro- and anti-inflammatory response in the spleen, the response in the liver needs to be similarly carefully balanced. Lethal strains of murine malaria (e.g., Plasmodium berghei NK65) induce signs of severe hepatotoxicity, mediated by CTLs and NK cells (50), while nonlethal strains (e.g., P. chabaudi AS) induce mild but clear signs of T cell-mediated liver injury (35, 37, 38, 39). Importantly, in vivo blockade of CTLA-4 results in a more severe liver injury and increased mortality, suggesting that regulatory activity is crucial in controlling hepatotoxicity during blood-stage infection (38). This is in agreement with our data showing that at the point of peak parasitemia in the bloodstream, the proinflammatory response in both liver and spleen is down-regulated and a concomitant increase in IL-10 is detected, representing an attempt to balance the proinflammatory response and to prevent immune-mediated pathology. However, despite the fact that H. polygyrus infection induced IL-10 production in the spleen in both singly and coinfected mice, the regulatory IL-10 response was absent from the liver in coinfected animals and a strong increase in proinflammatory cytokines was detected instead. This was in contrast to the cytokine profile in the spleen, where the pro- and anti-inflammatory cytokine response remained balanced during coinfection. Using gene-deficient mice, we have demonstrated that liver injury and mortality in this model is dependent on IL-12p40, IL-23p19, and IFN-γ, but not on TNF-α or LT-α. The IL-12p40 chain, along with IL-12p35, forms bioactive IL-12 and along with IL-23p19 form IL-23 (41). The observed up-regulation of IL-17 and IL-22 mRNA in the livers of coinfected mice further support a role for IL-23 in this system. IL-17 and IL-22 are both secreted by IL-23-driven Th17 cells and participate in inflammatory reactions (51, 52). Interestingly, a recent report shows that IL-22 plays a protective role in preventing hepatic tissue injury (53), and it is possible that the hepatic up-regulation of IL-22 detected in our model represents a host-protective response. This is to our knowledge is the first demonstration of the involvement of IL-23-driven responses in the pathogenesis of malaria infection. The inherent difficulties with coinfection models makes it complicated to pinpoint the exact functional mechanisms behind these observations, as experiments aimed at functional intervention often result in alterations of the course of the single infections, making data interpretation difficult. Be that as it may, future work will shed more light on the relative contribution of these particular cytokine responses in the development of immunity and immunopathology associated with protozoan and/or helminth infections.

Most studies aimed at investigating the immunomodulatory effects of concurrent H. polygyrus infection have focused on the adult phase of infection, i.e., >2 wk p.i. In most cases, the worm infection has been initiated several weeks, or even months, before coinfection or coadministration of Ag, thus allowing for the development of the strong Th2/regulatory response that is the hallmark of chronic H. polygyrus infection. By using this approach, studies have demonstrated the immunomodulatory effects of H. polygyrus on colitis, Citrobacter, Helicobacter and malaria infection, airway hyperreactivity, and diabetes (12, 22, 23, 24, 25, 26). As such, there is no doubt that an adult H. polygyrus infection is strongly immunomodulatory toward the Th2/regulatory spectrum of response. Previous experimental helminth-malaria coinfection studies have all focused on the possible counterregulatory antiparasite cytokine responses, where the Th1-dependent control of malaria parasitemia might be impaired by the Th2-dominated environment of an animal carrying a preexisting helminth infection. Indeed, several studies using different types of helminth infections have shown that an established dominant Th2 response can alter the disease progression of a murine malaria infection by diminishing the Th1 response (10, 11, 12, 54, 55, 56, 57, 58). However, the consequences of the early larval stages of helminth infections have received little attention. Our data show that a larval H. polygyrus infection on its own is sufficient to selectively induce the up-regulation of certain cytokines in the liver and spleen, including proinflammatory cytokines. Previous studies have reported Th1 responses in the intestine, mesenteric lymph nodes, and/or spleen during larval infections with several intestinal nematodes (5, 6, 7, 8, 9). Thus, our data are in agreement with previous studies showing that different life stages of helminth parasites may affect immune responsiveness differently depending on timing and anatomical site, a fact that is often overlooked in the literature.

The life cycle of H. polygyrus, a trichostrongylid nematode commonly occurring in wild small rodents, is direct and involves both free-living and parasitic stages. The host is infected by the ingestion of infective third-stage larvae, which penetrate the anterior half of the small intestinal mucosa within 24 h of infection. The developing larvae form cysts in the intestinal wall, surrounded by mild inflammatory infiltrates (histotrophic phase) (59). Approximately 7–8 days p.i., the larvae finally molt to adult stages and move out to the gut lumen and egg production can be detected in feces ∼10 days p.i. (34). After the adult worms have left the cysts and migrated to the lumen, an inflammatory response involving neutrophils and macrophages develop around the empty cysts (59, 60). Since the early stages of H. polygyrus larval infection are invasive and induce localized inflammation, it is reasonable to believe that any secreted Ags will be transported directly from the intestine to the liver via the portal system, and once in the liver may affect T cell responses directly, or indirectly, by affecting Ag presentation. Studies have shown that during the development from L3 larvae to adult worms the parasite expresses a number of stage-specific Ags (61, 62), including several secreted larval-stage Ags (63). The identity and functions of these Ags are largely unknown but several have been suggested to act as immunosuppressors, facilitating the establishment and survival of the worm (64, 65). Considering that larval stages of nematodes are susceptible to Th2-induced immune-mediated resistance mechanisms (66), it would be beneficial for the larvae to interfere with the development of such immune responses at the earliest possible stage of infection, and this may result in the promotion of proinflammatory responses. Further detailed investigations into the identities and characteristics of stage-specific Ags and their effects on the immune system are needed to better understand how helminths can manipulate the immune system.

Another potential stimulus for the liver damage observed in the coinfected animals may be the leakage of bacteria or bacterial endotoxin (LPS) from the injured intestine to the portal system. The timing of mortality in the coinfected mice coincides with the emergence of adult worms from the intestinal cysts to the gut lumen. The inflammatory response that develops around the empty larval cysts in the intestinal wall (59, 60) suggest that bacteria may be involved, and it is possible that the bacteria itself, or LPS and/or LPS-like products, may reach the liver and cause some degree of inflammation. During H. polygyrus infection alone, this may not be sufficient to induce liver injury, but in combination with another liver-associated pathogen it may result in enhanced organ-specific pathology. Previous studies have shown that as a murine malaria infection progresses the mice become more resistant to systemic endotoxin challenge, probably due to the continuous stimulation by hemozoin through TLR 9 (67, 68). Nevertheless, it is possible that LPS leakage from the intestine, coinciding with an early inflammatory malaria response in the liver, could act synergistically to promote liver injury. Moreover, liver injury during blood-stage malaria infection is dependent on the MyD88 adapter molecule (69), a molecule intrinsic to the signaling pathway of TLRs, suggesting that increased stimulation through TLRs may trigger inflammation. Increased levels of pathology caused by intestinal “leakage” and reduced epithelial barrier function have previously been implicated in other models of helminth infection (70, 71).

The data from the current study demonstrate the striking and unanticipated results that concurrent exposure to the larval stages of an intestinal helminth and malaria infection may result in increased severity of malaria in a mouse model. Certainly, in the human field situation, individuals often carry chronic helminth infections from an early age and the likelihood of being exposed to concurrent primary larval stage helminth infections and malaria may not be very high. There are situations however when this may occur, such as during transmigration from nonendemic to endemic areas or from urban to rural areas. The high incidence of severe malaria reported in nonimmune adults during the first years of transmigration from Java to Irian Jaya (72) is particularly striking in this aspect. It is certainly possible that these adults were not only exposed to malaria for the first time but also to other parasitic infections, including intestinal helminths. Furthermore, with the current emphasis of anti-helminthic mass treatment in sub-Saharan Africa (73), reinfection situations where the host is exposed to repeated larval infections may coincide with malarial infection. As such, it will be of interest to investigate how drug-abbreviated infections, as well as trickle infections, function in this aspect of coinfection in the murine model.

Taken together, the data presented here demonstrate that an early stage intestinal nematode infection can enhance malaria-associated pathology without necessarily altering immune reactivity in the spleen. Larval infections may also alter disease progression in different ways to established adult worm infections. It is therefore of importance to consider a number of aspects of coinfections, such as the stage of the infection, the dose and the species involved, when interpreting data obtained from animal and human studies. Moreover, it is clear that further work is needed to clarify the exact role of cytokines, especially the IL-23/Th17 axis, and other mediators that play a role in the pathogenesis of nematode-malaria coinfections.

I thank Dr. Eleanor Riley and Dr. Quentin Bickle for data discussions and critical reading of this manuscript, Jenna Murdoch for technical support, Dr. Daniel Cua for the p19KO mice, and Dr. Brigitta Stockinger for helpful discussions, critical evaluation of this manuscript, and provision of mice.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This study was funded by the Wellcome Trust (Grant GR067320).

3

Abbreviations used in this paper: LT, lymphotoxin; WT, wild type; p.i., postinfection; AST, aspartate aminotransferase; LBP, LPS-binding protein; P.c., P. chabaudi; H.p., H. polygyrus; KO, knockout.

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