The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2008, 180, 6877 -6884
Copyright © 2008 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Niikura, M.
Right arrow Articles by Kobayashi, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Niikura, M.
Right arrow Articles by Kobayashi, F.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Substance via MeSH
Medline Plus Health Information
*Malaria

Coinfection with Nonlethal Murine Malaria Parasites Suppresses Pathogenesis Caused by Plasmodium berghei NK65

Mamoru Niikura1,*,{ddagger}, Shigeru Kamiya{dagger}, Kiyoshi Kita{ddagger} and Fumie Kobayashi1,{dagger}

* Institute of Laboratory Animals, Graduate School of Medicine and {dagger} Department of Infectious Diseases, Faculty of Medicine, Kyorin University, Tokyo; and {ddagger} Department of Biomedical Chemistry, Graduate School of Medicine, University of Tokyo, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mixed infection with different Plasmodium species is often observed in endemic areas, and the infection with benign malaria parasites such as Plasmodium vivax or P. malariae has been considered to reduce the risk of developing severe pathogenesis caused by P. falciparum. However, it is still unknown how disease severity is reduced in hosts during coinfection. In the present study, we investigated the influence of coinfection with nonlethal parasites, P. berghei XAT (Pb XAT) or P. yoelii 17X (Py 17X), on the outcome of P. berghei NK65 (Pb NK65) lethal infection, which caused high levels of parasitemia and severe pathogenesis in mice. We found that the simultaneous infection with nonlethal Pb XAT or Py 17X suppressed high levels of parasitemia, liver injury, and body weight loss caused by Pb NK65 infection, induced high levels of reticulocytemia, and subsequently prolonged survival of mice. In coinfected mice, the immune response, including the expansion of B220intCD11c+ cells and CD4+ T cells and expression of IL-10 mRNA, was comparable to that in nonlethal infection. Moreover, the suppression of liver injury and body weight loss by coinfection was reduced in IL-10–/– mice, suggesting that IL-10 plays a role for a reduction of severity by coinfection with nonlethal malaria parasites.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Malaria is the infectious disease that causes incidence estimates of 2–3 million deaths and 300–500 million clinical cases in the world (1). There are four species of Plasmodium that infect humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale. P. falciparum is the major human parasite responsible for high morbidity and mortality, and infection with P. falciparum is associated with developing fever, a high number of parasites in the blood, and pathogenesis, including severe anemia, body weight loss, and cerebral malaria in humans (2). The sensitive PCR-based techniques have revealed that coinfection with different Plasmodium species is common in developing countries (3, 4). In particular, the simultaneous presence of P. vivax or P. malariae during P. falciparum infection is often observed when the prevalence of Plasmodium infections in humans is analyzed in endemic areas (5, 6, 7) and it is known to reduce the risk of developing a high number of parasites in the blood as well as pathogenesis (8, 9, 10, 11).

Murine malaria models have been used for understanding the induction of immune interaction in hosts and investigating factors associated with malarial defense mechanism. Coinfection with two different species and/or strains of murine malaria parasites has been shown to influence the parasitemia or mortality of each other (12). The development of experimental cerebral malaria caused by Plasmodium berghei (Pb)2 ANKA was inhibited by the simultaneous presence of Plasmodium yoelii yoelii or P. berghei K173 (13, 14). However, it is still unknown how the disease severity is suppressed in simultaneous infection.

In the present study, we investigated the influence of simultaneous infection with nonlethal parasites, P. berghei XAT (Pb XAT) or P. yoelii 17X (Py 17X), on the outcome of P. berghei NK65 (Pb NK65) lethal infection, which causes high levels of parasitemia and pathogenesis such as body weight loss and liver injury in mice. First, we found that Pb XAT-immunized mice acquired resistance to Pb NK65 infection, although Py 17X-immunized mice were susceptible to Pb NK65 infection. By using these three species and strains, we examined how Pb XAT or Py 17X nonlethal infection modulated the immune responses such as cytokine production and cellular expansion during Pb NK65 lethal infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Female C57BL/6 (B6) mice were purchased from CLEA Japan and used at 5–6 wk of age. IL-10–/– mice on B6 background were purchased from The Jackson Laboratory. We used 20- to 24-wk-old female IL-10–/– mice (experiment 1), 5- to 6-wk-old male or female IL-10–/– mice (experiment 2), and age-matched female B6 mice in these studies. The genotype of female IL-10–/– mice used in experiments was verified by PCR. The experiments were approved by the Experimental Animal Ethics Committee at Kyorin University, and all experimental animals were kept on the specific pathogen-free unit at the animal facility with sterile bedding, food, and water.

Parasites and infections

Malaria parasites were stored as frozen stocks in liquid nitrogen. Pb NK65 is a high-virulence strain and was originally obtained from Dr. M. Yoeli (New York University Medical Center, New York, NY). Pb XAT is a low-virulence derivative from Pb NK65 (15). A nonlethal isolate of Py 17X was originally obtained from Dr. J. Finnerty (National Institutes of Health, Bethesda, MD) and cloned by limiting dilution. Parasitized RBCs (pRBCs) of Pb NK65, Pb XAT, and Py 17X were generated in donor mice inoculated i.p. with each frozen stock of parasites. The donor mice were monitored for parasitemia daily and bled for experimental infection in ascending periods of parasitemia. Experimental mice were infected i.v. with 1 x 104 pRBCs of a given parasite species or strain. Therefore, when mice were coinfected with two species/strains of parasites, a total of 2 x 104 pRBCs (1 x 104 of each parasite species/strain) were inoculated.

Parasitemia

Parasitized RBCs were observed by microscopic examination of methanol-fixed tail blood smears stained for 45 min with 1% Giemsa diluted in phosphate buffer (pH 7.2). The number of pRBCs in 250 RBCs was enumerated when parasitemia exceeded 10%, whereas 1 x 104 RBCs were examined when mice showed lower parasitemia. The percentage of parasitemia was calculated as follows: [(No. of pRBCs)/(Total no. of RBCs counted)] x 100.

Measurement of body weights, hematocrits, and circulating reticulocytes

Body weights were measured by balance for animals (KN-661; Natume), and body weight loss was expressed as a percentage of the day 0 value. For hematocrit measurement, tail blood (50 µl) was collected into a heparinized capillary tube and centrifuged at 13,000 x rpm for 5 min with a micro hematocrit centrifuge (HC-12A; Tomy). The hematocrit value was expressed as a percentage of the total blood volume. Reticulocytes in 250 RBCs were counted when reticulocytemia exceeded 20%, whereas 1 x 104 RBCs were examined when mice showed lower reticulocytemia. The percentage of reticulocytemia was calculated as follows: [(No. of reticulocytes)/(Total no. of RBCs counted)] x 100.

Histological examination and measurement of parameters of liver injury

Livers were obtained from infected mice on day 9 postinfection and fixed in 10% buffered formalin and embedded in paraffin. Six-micrometer-thick sections were stained with H&E. The blood was obtained from infected mice on day 9 and centrifuged at 500 x g for 10 min. The resulting supernatants were stored at –20°C and used as plasma. The levels of aspartic aminotransferase (AST) and alanine aminotransferase (ALT) in plasma were determined at Nagahama Life Science Laboratory (Shiga, Japan).

Flow cytometry

Flow cytometric analysis was performed on single-cell suspensions of spleen and peripheral blood cells as described previously (16). Total spleen cell numbers in uninfected and infected mice are shown in Table I. The following mAbs were used for analysis: FITC-conjugated anti-CD3{epsilon} mAb (clone 145-2C11; eBioscience) and anti-CD4 mAb (clone RM4–5; eBioscience); PE-conjugated anti-CD11c mAb (clone N418; Miltenyi Biotec); allophycocyanin-conjugated anti-CD3{epsilon} mAb (clone 145–2C11; eBioscience); and biotin-conjugated anti-CD45R mAb (B220; clone RA3–6B2; BD Pharmingen). MAbs were added to cells in FACS buffer (1% BSA, 0.1% sodium azide in PBS) and incubated at 4°C for 30 min and the cells were washed with cold FACS buffer by centrifugation at 250 x g for 2 min. Biotinylated mAbs were followed by streptavidin-conjugated allophycocyanin (4°C, 30 min). After washing with FACS buffer, cells were fixed with 1% paraformaldehyde. Two-color flow cytometry was performed and analyzed with a FACSCalibur (BD Biosciences) using a FlowJo software (version 7.1.3, for Windows).


View this table:
[in this window]
[in a new window]

  		Table I. Total spleen cell number in uninfected and infected mice (x108)a

 
Detection of cytokine mRNA in spleens

Spleens were removed from infected mice on day 9 postinfection and total RNA was isolated by Isogen (Nippon Gene) according to the manufacturer’s protocol. The splenic RNA was reverse-transcribed by murine leukemia virus reverse transcriptase (Applied Biosystems) using random hexamer primers, and reverse transcriptase reaction was preformed at 70°C for 10 min, at 25°C for 10 min, and at 42°C for 30 min. The reaction was terminated by heating at 99°C for 5 min, and the cDNA products were stored at –20°C until use. The 50 µl PCR mixture contained 1 x TaKaRa Ex Taq buffer, 2.5 mM dNTP, 1 µl cDNA products, 5 U/µl TaKaRa Ex Taq DNA polymerase, and 0.25 µM of PCR primers. The primers used for PCR amplification were as follows: IL-10, 5'-GTG AAG ACT TTC TTT CAA ACA AAG, 3'-CTG CTC CAC TGC CTT GCT CTT ATT; IFN-{gamma}, 5'-TAC TGC CAC GGC ACA GTC ATT GAA, 3'-GCA GCG ACT CCT TTT CCG CTT CCT T; β-actin, 5'-CCA GCC TTC CTT CCT GGG TA, 3'-CTA GAA GCA TTT GCG GTG CA. Thirty cycles of PCR were performed on a thermal cycler (iCycler; Bio-Rad). Each cycle consisted of 30 s of denaturation at 94°C, 30 s of annealing at 60°C, and 1 min of extension at 72°C. The PCR products were analyzed on a 2% agarose gel stained with ethidium bromide.

Cytokine assay

An ELISA for the detection of IFN-{gamma} or IL-10 in plasma was conducted as described previously (16). A rat anti-mouse IFN-{gamma} (clone R4–6A2; eBioscience) and a rat anti-mouse IL-10 (clone JES5–16E3; eBioscience) were used as the capture Abs, and a biotin-coupled rat anti-mouse IFN-{gamma} (clone XMG1.2; eBioscience) and IL-10 (clone JES5-2A5; eBioscience) were used as the detecting Abs. The concentration of cytokines in plasma was calculated from standard curves prepared with known quantities of murine recombinant IFN-{gamma} (Genzyme) and murine recombinant IL-10 (Pierce).

Statistical analysis

For time-series comparisons, Student’s t test and one- and two-way ANOVAs with Fisher’s PLSD post hoc test were performed using Statcel program (OMS). Survival curves were compared using a log-rank test. p < 0.05 was set as statistical significance of differences.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Infection with Pb XAT but not Pb 17X induces protective immunity to Pb NK65

It has been shown that mice infected with Pb NK65 develop severe parasitemia and die within 2 wk, although mice infected with Pb XAT or Py 17X cure spontaneously around 3 wk of infection (15, 17). To examine whether primary infection with each of the two nonlethal parasites can induce protective immunity against Pb NK65 lethal infection, groups of C57BL/6 (B6) mice were infected with Pb XAT or Py 17X then challenged with Pb NK65 on day 30 after primary infection. As expected, mice cured from Pb XAT infection (Pb XAT-immunized mice) showed extremely low levels of parasitemia after secondary infection with Pb NK65 (Fig. 1A). On the contrary, mice cured from Py 17X infection (Py 17X-immunized mice) showed high levels of parasitemia, with some delay in onset of parasitemia, and eventually died after Pb NK65 infection (Fig. 1B). These results suggest that protective immunity to Pb NK65 is induced by immunizing mice with Pb XAT but not with heterologous Py 17X.


Figure 1
View larger version (25K):
[in this window]
[in a new window]

  		FIGURE 1. Immunization with Pb XAT but not Py 17X induces protective immunity to Pbi NK65. C57BL/6 mice were infected with 1 x 104 pRBCs of Pb XAT (A) or Py 17X (B) (day 0, open arrows). On day 30 after primary infection (filled arrows), both groups of mice were challenged with 1 x 104 pRBCs of Pb NK65. A, Course of parasitemia in immunized mice with Pb XAT ({diamond}). B, Course of parasitemia in immunized mice with Py 17X ({diamondsuit}). Course of parasitemia of unimmunized mice infected with Pb NK65 is inserted to figures (shaded triangles). Results are expressed as mean percentage parasitemia ± SD of three mice. Experiments were performed three times with similar results.

 
The pathogenesis during Pb NK65 infection is reduced by simultaneous infection with nonlethal malaria parasites

To investigate whether the existence of nonlethal malaria parasite affects the outcome of Pb NK65 infection, B6 mice were infected with Pb NK65 and nonlethal parasites simultaneously. When mice were coinfected with Pb NK65 and Pb XAT (Pb NK65/Pb XAT), they showed lower levels of parasitemia than did Pb NK65 singly infected mice during early infection (Fig. 2A) and survived significantly longer than did Pb NK65 singly infected mice (Fig. 2B) (p = 0.0013). Moreover, the body weight loss of the coinfected mice was prevented early in infection (Fig. 2C) (p < 0.0005 compared with Pb NK65-infected mice on days 9–10). Next, we examined the influence of coinfection with nonlethal Py 17X on the outcome of Pb NK65 infection. Although Py 17X immunization did not affect the outcome of Pb NK65 infection greatly (Fig. 1B), simultaneous infection with Py 17X (Pb NK65/Py 17X) suppressed severe parasitemia, mortality (p = 0.0005), and the body weight loss (p < 0.0005 on days 6–10) observed in Pb NK65 singly infected mice (Fig. 2, D–F).


Figure 2
View larger version (33K):
[in this window]
[in a new window]

  		FIGURE 2. Coinfection of nonlethal Pb XAT or Py 17X suppresses the acute severe parasitemia and body weight loss caused by Pb NK65 infection in mice and prolonged their survival. C57BL/6 mice were inoculated with 1 x 104 pRBCs of Pb NK65, Pb XAT, or Py 17X. When mice were coinfected with two species/strains of parasites, a total of 2 x 104 pRBCs were inoculated (Pb NK65/Pb XAT or Pb NK65/Py 17X). Results of coinfection are shown for Pb NK65/Pb XAT (A–C) or Pb NK65/Py 17X (D and E). A and D, Course of parasitemia. Asterisks indicate statistically significant differences (*, p < 0.001 as compared with Pb NK65-infected mice). B and E, Survival rates. Differences between Pb NK65 singly infected mice and coinfected mice are statistically significant (p < 0.001). C and F, Body weights. Asterisks indicate statistically significant differences (*, p < 0.001 as compared with Pb NK65-infected mice). Results are expressed as means ± SD of five mice. Experiments were performed three times with similar results.

 
Coinfection with nonlethal malaria parasites induces reticulocytemia

To examine whether the existence of nonlethal malaria parasites affects the severe anemia caused by Pb NK65 infection, we determined the hematocrit in mice during Pb NK65 single infection and coinfection with Pb XAT or Py 17X. Coinfection with Pb XAT caused acute anemia as severe as did Pb NK65 single infection on day 9 postinfection, and the levels of hematocrit were also low on day 15 (Fig. 3A). Mice infected with Pb XAT did not cause acute severe anemia. In contrast, mice coinfected with Pb NK65/Py 17X did not cause as severe anemia as for Pb NK65-infected mice on day 9, and their reducing pattern of hematocrit was similar to that in Py 17X singly infected mice (Fig. 3C).


Figure 3
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 3. Coinfection with nonlethal malaria parasites induces reticulocytemia. Mice were infected with malarial parasites as described in the legend to Fig. 2. A and C, Blood (50 µl) was collected from infected mice on days 3, 6, 9, and 15 after infection and hematocrit values were determined. B and D, Reticulocytemia was determined on days 3, 5, 7, 9, 11, 13, and 15 after infection. The percentage of reticulocytemia was calculated as follows: [(number of reticulocytes)/(total number of RBCs counted)] x 100. Asterisks indicate a statistically significant difference (*, p < 0.001 as compared with Pb NK65-infected mice). Results are expressed as means ± SD of three mice. Experiments were performed three times with similar results.

 
Next, we determined the reticulocytemia in infected mice. Pb NK65 singly infected mice did not show any reticulocytemia during infection (Fig. 3B). Mice coinfected with Pb NK65/Pb XAT showed the same levels of reticulocytemia as did Pb XAT singly infected mice on day 9 postinfection. However, coinfected mice showed much higher reticulocytemia than that in Pb XAT singly infected mice from day 11 postinfection (Fig. 3B). As shown in Fig. 3D, reticulocytemia in mice coinfected with Pb NK65/Py 17X increased moderately, and their kinetics were similar to those in Py 17X singly infection. These results indicate that the severe anemia caused by Pb NK65 infection is suppressed by coinfection with Py 17X but not with Pb XAT. It is suggested that high levels of reticulocytemia observed during Pb NK65/Pb XAT infection may be induced by severe anemia. The different outcome of the suppression of anemia and reticulocytemia between Pb NK65/Pb XAT- and Pb NK65/Py 17X-infected mice might be attributed to the difference in parasitemia during early infection.

Low levels of liver injury in mice coinfected with nonlethal malaria parasites

To investigate whether the existence of nonlethal malaria parasites affects the liver injury caused by Pb NK65 infection, we performed histological examination of livers from mice during Pb NK65 single infection and coinfection with Pb XAT or Py 17X. As shown in Fig. 4, focal necrosis of the liver cells (Fig. 4, B and F, arrowheads) and dense infiltration of inflammatory cells such as mononuclear cells around the portal tracts (Fig. 4F, arrows) were observed in Pb NK65-infected mice. Mice coinfected with Pb NK65/Pb XAT or Pb NK65/Py 17X also showed dense infiltration of inflammatory cells (Fig. 4, G and H, arrows), but focal necroses were not observed in the liver (Fig. 4, C and D).


Figure 4
View larger version (72K):
[in this window]
[in a new window]

  		FIGURE 4. The existence of nonlethal malaria parasites prevents the liver injury caused by Pb NK65 infection. Mice were infected with malarial parasites as described in the legend to Fig. 2. Livers and plasma were obtained from infected mice on day 9 after infection and from uninfected mice. A–H, Histological analysis was performed after staining with H & E. Typical results of uninfected mice (A and E), mice singly infected with Pb NK65 (B and F), and mice coinfected with Pb NK65/Pb XAT (C and G) or Pb NK65/Py 17X (D and H) are shown. A–D, The scale bar indicates 100 µm. Arrowheads indicate focal necrosis of the liver cells. E–H, The scale bar indicates 40 µm. Arrows indicate dense infiltration of inflammatory cells. I and J, Levels of AST and ALT. Asterisks indicate a statistically significant difference (*, p < 0.001 as compared with uninfected control mice). Results are expressed as means ± SD of three mice. Experiments were performed three times with similar results and the representative data are shown.

 
We determined the levels of AST and ALT, which are parameters of liver injury, in the plasma. Pb NK65-infected mice, in which the focal necroses of liver cells were observed, showed the significantly high concentration of AST and ALT compared with uninfected control mice (Fig. 4, I and J). The levels of AST and ALT in coinfected mice were quite low and almost the same as those in Pb XAT or Py 17X singly infected mice.

Coinfection with nonlethal parasites accelerates B220intCD11c+ cell expansion in spleen and peripheral blood

To examine the expansion of the CD11c+ cell populations during malaria, additional experiments were performed using peripheral blood and spleen obtained from infected mice by flow cytometry in each time point after infection. It was notable that the B220intCD11c+ cell population significantly increased in peripheral blood from Pb NK65/Pb XAT- or Pb NK65/Py 17X-coinfected mice on day 6 postinfection (Fig. 5A, upper panels). Their expansion was comparable to that observed in Pb XAT or Py 17X single infection, respectively (Fig. 5C). The B220intCD11c+ cell population in those four groups of mice decreased on day 9 postinfection (Fig. 5A, lower panels). Although B220intCD11c+ cells in Pb NK65-infected mice also expanded on day 6 postinfection, they were much less than those in coinfected or nonlethal parasite-infected mice. The cell population in Pb NK65-infected mice further expanded on day 9 postinfection, when no other groups of mice showed the expansion (Fig. 5A). The B220intCD11c+ cell population of spleen showed a similar pattern to that of peripheral blood (Fig. 5B), but the proportion of the cells in Pb NK65/Pb XAT-infected mice was lower than that in Pb XAT-infected mice on day 6 postinfection (Fig. 5D). These results suggested that coinfection with nonlethal parasites accelerated much more B220intCD11c+ cell expansion than did Pb NK65 single infection during the early phase of infection.


Figure 5
View larger version (47K):
[in this window]
[in a new window]

  		FIGURE 5. Coinfection with nonlethal parasites accelerates B220intCD11c+ cell expansion in spleen and peripheral blood. Peripheral blood and spleen were obtained from infected mice as described in the legend to Fig. 2 on days 6 and 9 after infection and from uninfected mice. Analyses of CD11c+ cell population in peripheral blood (A and C) and spleen (B and D) from infected mice were performed by flow cytometery. Expression of B220 and CD11c was analyzed in the gate of CD3–. A and B, Contour plots of B220intCD11c+ cell population (day 6, upper panels; day 9, lower panels). p.i., Post infection. Experiments were performed three times with similar results and the representative results are shown. C and D, The proportion of B220intCD11c+ cells in CD3– cells is shown (on day 6 postinfection). Asterisks indicate a statistically significant difference (*, p < 0.005; **, p < 0.001 as compared with Pb NK65-infected mice). Results are expressed as means ± SD of three mice.

 
Coinfection with nonlethal parasites induces CD4+ T cell expansion in spleen

We analyzed the kinetics of CD4+ T cell expansion in spleen during single and mixed infection (Fig. 6). Significant expansion of splenic CD4+ T cells in Pb XAT- or Py 17X-infected mice was observed from day 9 postinfection. In contrast, Pb NK65-infected mice did not show the increased levels of CD4+ T cells even on day 9 postinfection. Mice coinfected with Pb NK65/Pb XAT or Pb NK65/Py 17X had almost the same number of splenic CD4+ T cells as did Pb XAT- or Py 17X-infected mice, respectively.


Figure 6
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 6. Coinfection with nonlethal parasites induces CD4+ T cell expansion in spleen. Spleens were obtained from infected mice as described in the legend to Fig. 2 on days 6, 9, and 15 after infection and from uninfected mice. Splenic CD3+CD4+ cells were analyzed by flow cytometry and total numbers of CD4+ T cells in spleen were calculated. Asterisks indicate a statistically significant difference (*, p < 0.05; **, p < 0.005 as compared with uninfected control mice). Results are expressed as means ± SD of three mice. Experiments were performed three times with similar results.

 
Enhanced levels of IL-10 mRNA during coinfection and nonlethal infection

IFN-{gamma} and IL-10 have been shown to be associated with protection and exacerbation during P. berghei and P. yoelii malaria (17, 18). To examine whether these cytokines are associated with the suppression of Pb NK65-caused pathogenesis by coinfection with the nonlethal malaria parasites, we determined the levels of cytokines in plasma and cytokine mRNA in spleens from singly infected or coinfected mice (Fig. 7). Pb NK65 singly infected mice showed a high level of IFN-{gamma} in plasma on day 9 compared with that in uninfected mice (Fig. 7A). Although the plasma IFN-{gamma} levels in coinfected mice or nonlethal singly infected mice on days 6 and 9 were not different from those in uninfected mice, these mice showed significantly lower levels of IFN-{gamma} than did uninfected mice on day 15. In contrast, strong IFN-{gamma} mRNA expression was detected in the spleen from mice singly infected with Py 17X and coinfected with Pb NK65/Pb XAT and Pb NK65/Py 17X, compared with that observed in uninfected mice on day 9 (Fig. 7C). However, Pb NK65 singly infected mice did not show high levels of IFN-{gamma} mRNA expression. These results suggested that spleen might not be a main organ for production of IFN-{gamma}, which was involved in severe pathogenesis during Pb NK65 single infection (18), but the association of IFN-{gamma} with suppressive pathogenesis by coinfection was still unclear.


Figure 7
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 7. Enhanced levels of IL-10 mRNA during coinfection and nonlethal infection. A and B, Levels of IFN-{gamma} or IL-10 were determined by ELISA. Plasma was collected from uninfected mice and infected mice on days 6, 9, and 15 post infection. A, Levels of IFN-{gamma} in plasma. B, Levels of IL-10 in plasma. Asterisks indicate a statistically significant difference as compared with uninfected mice (p < 0.001). C, Total RNA was isolated from spleen of uninfected and infected mice as described in the legend to Fig. 2 on day 9 and subjected to RT-PCR using cytokine-specific primers. The samples without RNA template were used as negative control. Note that coinfected mice (Pb NK65/Pb XAT, Pb NK65/Py 17X) show IL-10 mRNA expression that is comparable to nonlethal parasite-infected mice (Pb XAT, Py 17X). Experiments were performed three times with similar results.

 
The levels of IL-10 in plasma from single or coinfected mice on days 6 and 9 were not different from uninfected mice, but coinfected mice as well as nonlethal Py 17X singly infected mice showed elevated levels of IL-10 on day 15 (Fig. 7B). Although Pb NK65 singly infected mice showed only faint expression of IL-10 mRNA on day 9, mice coinfected with Pb NK65/Pb XAT or Pb NK65/Py 17X showed strong IL-10 mRNA expression, which was comparable to that observed in mice during Pb XAT or Py 17X single infection (Fig. 7C). IL-10 mRNA was not detected in spleen from uninfected mice. These results led us to hypothesize that the enhanced levels of IL-10 may be involved in suppression of pathogenesis during coinfection.

IL-10-deficient mice fail to receive benefits by coinfection with nonlethal malaria parasites

To examine whether IL-10 is associated with the suppression of the pathogenesis caused by coinfection, we determined the parasitemia, mortality, and the body weight of Pb NK65-infected IL-10–/– mice coinfected with Pb XAT or Py 17X. Pb NK65/Pb XAT-coinfected wild-type mice survived by day 21 (Fig. 8D), confirming the data obtained in Fig. 2B. In contrast, IL-10–/– mice coinfected with Pb NK65/Pb XAT began to die from day 10, and all mice died by day 21 postinfection (Fig. 8D) (p = 0.034). Moreover, their body weights were significantly lower than coinfected wild-type mice (Fig. 8E) (p < 0.001 on days 9, 11, and 13), although their parasitemia did not increase from day 11 (Fig. 8F). Similarly, Pb NK65/Py 17X-coinfected IL-10–/– mice began to die earlier than did wild-type mice (Fig. 8G), and their body weights were also lower than those of wild-type mice (Fig. 8H) (p < 0.001 on days 9, 13, and 18). During the period when coinfected IL-10–/– mice began to die, they developed liver injury (Fig. 8, M and O), which was not observed in coinfected wild-type mice (Fig. 8, L and N). In contrast, the parasitemia, mortality, the body weight, and development of liver injury of Pb NK65 singly infected IL-10–/– mice were not different from those of wild-type mice (Fig. 8, A-C, J, and K). Altogether, these results suggest that IL-10 may be involved in the suppressive effect of coinfection with nonlethal malaria parasites on the outcome of lethal Pb NK65 infection.


Figure 8
View larger version (80K):
[in this window]
[in a new window]

  		FIGURE 8. IL-10-deficient mice fail to receive benefits by coinfection with nonlethal malaria parasites. IL-10–/– mice and age-matched wild-type mice were singly infected with Pb NK65, coinfected with Pb NK65/Pb XAT, or coinfected with Pb NK65/Py 17X. Survival rates (A, D, and G), body weight (B, E and H), and course of parasitemia (C, F, and I) are shown. Asterisks indicate a statistically significant difference (*, p < 0.001 as compared with wild-type mice). Results are expressed as means ± SD of three to five mice. Histological analysis of liver was performed after staining with H & E (J–O). Livers were obtained from infected wild-type mice (J, L, and N) and IL-10–/– mice (K, M, and O) immediately after death from day 10 to 22 after infection. The scale bar indicates 100 µm. Arrowheads indicate focal necrosis of the liver cells. Experiments were performed twice with similar results and the representative data are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study, we investigated the influence of simultaneous infection with nonlethal murine malaria parasites, Pb XAT or Py 17X, on the outcome of the lethal Pb NK65 infection. Pb NK65 infection caused acute high parasitemia and pathogenesis, including body weight loss, severe anemia, and liver injury in mice. We found herein that the coinfection with nonlethal Pb XAT or Py 17X reduced such pathogenesis caused by Pb NK65 infection and prolonged survival of mice (Figs. 2–4). Because low levels of parasitemia and body weight loss in coinfected mice were observed from day 6 to 7 (Fig. 2), we postulated that T/B cell-mediated immunity would be involved in the suppressive effects of simultaneous infection with nonlethal parasites on lethal Pb NK65 infection and examined the response of dendritic cells and CD4+ T cells.

The large expansion of B220intCD11c+ cells was observed in spleen and peripheral blood from coinfected mice on day 6, which was comparable to that from nonlethal parasite-infected mice (Fig. 5). These results suggest that expansion of B220intCD11c+ cells in coinfected mice may be accelerated by nonlethal parasite relative to lethal parasite infection. It has been reported that CD11c+ dendritic cells are one of the professional APCs. As the murine plasmacytoid dendritic cell subset has been shown to coexpress CD11c and B220 (19, 20), one would speculate that B220intCD11c+ cells expanded during nonlethal infection or coinfection might be one of the murine plasmacytoid dendritic cell subpopulations. Further characterization of the B220intCD11c+ cells, however, is needed for identification of these cells. In Pb NK65-infected mice, the peak expansion of B220intCD11c+ cells was observed on day 9, when these cells began to decrease in coinfected mice as well as nonlethal Pb XAT- or Py 17X-infected mice. Because Pb NK65 parasites multiply quickly, especially in early phase of infection, earlier expansion of B220intCD11c+ cells may be the key for the suppression of pathogenesis during coinfection.

In contrast, mice coinfected with Pb NK65 and nonlethal Pb XAT or Py 17X showed increased levels of CD4+ T cells from day 9 that were comparable to nonlethal parasite-infected mice (Fig. 6). Dendritic cells have been shown to activate naive T cells and play a crucial role in the initiation of immune responses (21, 22, 23). It is possible that the expansion of splenic CD4+ T cells might be induced by B220intCD11c+ cells that had been expanded earlier (on day 6), and then the expanded CD4+ T cells might be involved in suppression of pathogenesis in coinfected mice. CD4+ T cells have been shown to play both protective and pathological roles during malaria infection (24, 25). However, it seems that CD4+ T cells would play protective roles during coinfection with lethal and nonlethal malaria parasites.

IL-10, which is produced by Th2 cells in CD4+ T cell categories, inhibits inflammatory cytokines such as IFN-{gamma}, TNF-{alpha} (26), and IL-12 (27). In malaria, IL-10 as well as TGF-β has been shown to be critical for host survival during P. berghei ANKA (28, 29) and P. chabaudi AS (30) infection. In the present study, Pb NK65/Pb XAT- or Pb NK65/Py 17X-coinfected mice showed high levels of IL-10 mRNA comparable to those in nonlethal Pb XAT- or Py 17X-infected mice (Fig. 7C), although Pb NK65-infected mice showed only a faint level of IL-10 mRNA. Moreover, high levels of IL-10 in plasma were followed by the IL-10 mRNA expression in coinfected mice on day 15 when IFN-{gamma} production was suppressed (Fig. 7). These results suggest that IL-10 may be involved in the suppression of pathogenesis in coinfected mice.

As expected, the suppressive effect of coinfection with nonlethal Pb XAT or Py 17X on severe body weight loss, liver injury, and mortality during Pb NK65 infection was reduced in IL-10–/– mice (Fig. 8), suggesting that IL-10 was involved in suppression of exacerbation of infection in simultaneous infection. The excessive inflammation has been shown to be able to account for body weight loss, liver injury, and mortality in mice infected with Pb NK65 (18, 31). Therefore, it is probable that enhancement of IL-10 would have suppressed the excessive inflammation caused by Pb NK65 and subsequently led to suppression of pathogenesis. In contrast, mortality as well as body weight loss in IL-10–/– mice during coinfection were not identical with those in Pb NK65 singly infected IL-10–/–mice, suggesting that other regulatory factors, such as TGF-β (30), may be involved in suppression of pathogenesis.

In the late phase of infection, IL-10–/– mice coinfected with Pb NK65/Pb XAT or Pb NK65/Py 17X had lower levels of parasitemia than that in wild-type mice. These results suggest that although IL-10 plays an important role for suppression of liver injury, it may be also involved in suppression of clearance of malaria parasites and cause death by severe anemia in the late phase of coinfection. It has been shown that during Py 17XL lethal infection, IL-10 is involved in the exacerbation of infection because depletion of IL-10 prolonged survival of hosts and made some mice resolve the infection (17, 32, 33). IL-10 might have dual roles, protective and pathological, in mice coinfected with lethal and nonlethal malaria parasites.

Our findings showing the beneficial influence of coinfection with nonlethal Pb XAT or Py 17X to hosts during Pb NK65 infection indicate that suppression of disease severity induced by coinfection occurs in not only cerebral malaria but also pathogenesis such as body weight loss and liver injury. Our data suggest that the beneficial influence of coinfection with nonlethal malaria parasites may not be species-specific because a different species of malaria parasites, Py 17X, also induced protective immunity to Pb NK65 lethal infection by simultaneous infection (Fig. 2). In endemic areas, coinfections have made diagnosis and treatment difficult because host immune responses induced by each of the different Plasmodium spp. are mutually interfered with in a complicated manner. Results obtained from in vivo models of coinfection with murine malaria parasites would contribute to understand the host immune responses during mixed infection with different Plasmodium spp.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
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 Address correspondence and reprint requests to Dr. Fumie Kobayashi and Dr. Mamoru Niikura, Faculty of Medicine, Kyorin University, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan. E-mail addresses: fumfum{at}ks.kyorin-u.ac.jp and mniikura{at}ks.kyorin-u.ac.jp Back

2 Abbreviations used in this paper: Pb, Plasmodium berghei; Py, Plasmodium yoelii; pRBC, parasitized RBC; AST, aspartic aminotransferase; ALT, alanine aminotransferase. Back

Received for publication July 12, 2007. Accepted for publication March 4, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Snow, R. W., M. H. Craig, U. Deichmann, D. le Sueur. 1999. A preliminary continental risk map for malaria mortality among African children. Parasitol. Today 15: 99-104. [Medline]
  2. Breman, J. G.. 2001. The ears of the hippopotamus: manifestations, determinants, and estimates of the malaria burden. Am. J. Trop. Med. Hyg. 64: 1-11. [Abstract]
  3. Siribal, S., S. Nakasiri, S. Looareesuwan, P. Chavalitshewinkoon-Petmitr. 2004. Identification of human malaria parasites and detection of mixed infection in Thai patients by nested PCR. Southeast Asian J. Trop. Med. Public Health 35: 5-9. [Medline]
  4. Imwong, M., S. Pukrittayakamee, A. C. Grüner, L. Rénia, F. Letourneur, S. Looareesuwan, N. J. White, G. Snounou. 2005. Practical PCR genotyping protocols for Plasmodium vivax using Pvcs and Pvmsp1. Malar. J. 4: 20[Medline]
  5. Mehlotra, R. K., K. Lorry, W. Kastens, S. M. Miller, M. P. Alpers, M. Bockarie, J. W. Kazura, P. A. Zimmerman. 2000. Random distribution of mixed species malaria infections in Papua New Guinea. Am. J. Trop. Med. Hyg. 62: 225-231. [Abstract]
  6. Pinto, J., C. A. Sousa, V. Gil, L. Gonçalves, D. Lopes, V. E. do Rosário, J. D. Charlwood. 2000. Mixed-species malaria infections in the human population of São Tomé island, west Africa. Trans. R. Soc. Trop. Med. Hyg. 94: 256-257. [Medline]
  7. Ebrahimzadeh, A., B. Fouladi, A. Fazaeli. 2007. High rate of detection of mixed infections of Plasmodium vivax and Plasmodium falciparum in south-east of Iran, using nested PCR. Parasitol. Int. 56: 61-64. [Medline]
  8. Mason, D. P., F. E. McKenzie, W. H. Bossert. 1999. The blood-stage dynamics of mixed Plasmodium malariae-Plasmodium falciparum infections. J. Theor. Biol. 198: 549-566. [Medline]
  9. Mason, D. P., F. E. McKenzie. 1999. Blood-stage dynamics and clinical implications of mixed Plasmodium vivax-Plasmodium falciparum infections. Am. J. Trop. Med. Hyg. 61: 367-374. [Abstract]
  10. McQueen, P. G., F. E. McKenzie. 2006. Competition for red blood cells can enhance Plasmodium vivax parasitemia in mixed-species malaria infections. Am. J. Trop. Med. Hyg. 75: 112-125. [Abstract/Free Full Text]
  11. Luxemburger, C., F. Ricci, F. Nosten, D. Raimond, S. Bathet, N. J. White. 1997. The epidemiology of severe malaria in an area of low transmission in Thailand. Trans. R. Soc. Trop. Med. Hyg. 91: 256-262. [Medline]
  12. Snounou, G., T. Bourne, W. Jarra, S. Viriyakosol, J. C. Wood, K. N. Brown. 1992. Assessment of parasite population dynamics in mixed infections of rodent plasmodia. Parasitology 105: 363-374. [Medline]
  13. Voza, T., A. M. Vigario, E. Belnoue, A. C. Gruner, J. C. Deschemin, M. Kayibanda, F. Delmas, C. J. Janse, B. Franke-Fayard, A. P. Waters, et al 2005. Species-specific inhibition of cerebral malaria in mice coinfected with Plasmodium spp. Infect. Immun. 73: 4777-4786. [Abstract/Free Full Text]
  14. Mitchell, A. J., A. M. Hansen, L. Hee, H. J. Ball, S. M. Potter, J. C. Walker, N. H. Hunt. 2005. Early cytokine production is associated with protection from murine cerebral malaria. Infect. Immun. 73: 5645-5653. [Abstract/Free Full Text]
  15. Waki, S., J. Tamura, M. Imanaka, S. Ishikawa, M. Suzuki. 1982. Plasmodium berghei: isolation and maintenance of an irradiation attenuated strain in the nude mouse. Exp. Parasitol. 53: 335-340. [Medline]
  16. Kobayashi, F., M. Niikura, S. Waki, T. Matsui, T. Fujino, T. Tsuruhara, S. Kamiya. 2007. Plasmodium berghei XAT: contribution of {gamma}{delta} T cells to host defense against infection with blood-stage nonlethal malaria parasite. Exp. Parasitol. 117: 368-375. [Medline]
  17. Kobayashi, F., T. Morii, T. Matsui, T. Fujino, Y. Watanabe, W. P. Weidanz, M. Tsuji. 1996. Production of IL-10 during malaria caused by lethal and nonlethal variants of Plasmodium yoelii yoelii. Parasitol. Res. 82: 385-391. [Medline]
  18. Waki, S., S. Uehara, K. Kanbe, K. Ono, M. Suzuki, H. Nariuchi. 1992. The role of T cells in pathogenesis and protective immunity to murine malaria. Immunology 75: 646-651. [Medline]
  19. Nakano, H., M. Yanagita, M. D. Gunn. 2001. CD11c+B220+Gr-1+cells in mouse lymph nodes and spleen display characteristics of plasmacytoid dendritic cells. J. Exp. Med. 194: 1171-1178. [Abstract/Free Full Text]
  20. Bjorck, P.. 2001. Isolation and characterization of plasmacytoid dendritic cells from Flt3 ligand and granulocyte-macrophage colony-stimulating factor-treated mice. Blood 98: 3520-3526. [Abstract/Free Full Text]
  21. Inaba, K., R. M. Steinman. 1984. Resting and sensitized T lymphocytes exhibit distinct stimulatory (antigen-presenting cell) requirements for growth and lymphokine release. J. Exp. Med. 160: 1717-1735. [Abstract/Free Full Text]
  22. Knight, S. C., A. J. Stagg. 1993. Antigen-presenting cell types. Curr. Opin. Immunol. 5: 374-382. [Medline]
  23. Metlay, J. P., M. D. Witmer-Pack, R. Agger, M. T. Crowley, D. Lawless, R. M. Steinman. 1990. The distinct leukocyte integrins of mouse spleen dendritic cells as identified with new hamster monoclonal antibodies. J. Exp. Med. 171: 1753-1771. [Abstract/Free Full Text]
  24. Yoneto, T., T. Yoshimoto, C. R. Wang, Y. Takahama, M. Tsuji, S. Waki, H. Nariuchi. 1999. Gamma interferon production is critical for protective immunity to infection with blood-stage Plasmodium berghei XAT but neither NO production nor NK cell activation is critical. Infect. Immun. 67: 2349-2356. [Abstract/Free Full Text]
  25. Langhorne, J., S. J. Meding, K. Eichmann, S. S. Cillard. 1989. The response of CD4+ T cells to Plasmodium chabaudi chabaudi. Immunol. Rev. 112: 71-94. [Medline]
  26. Mosmann, T. R., K. W. Moore. 1991. The role of IL-10 in crossregulation of TH1 and TH2 responses. Immunol. Today 12: A49-A53. [Medline]
  27. Xu, X., K. Sumita, C. Feng, X. Xiong, H. Shen, S. Maruyama, M. Kanoh, Y. Asano. 2001. Down-regulation of IL-12 p40 gene in Plasmodium berghei-infected mice. J. Immunol. 167: 235-241. [Abstract/Free Full Text]
  28. Tan, R. S., A. U. Kara, C. Feng, Y. Asano, R. Sinniah. 2000. Differential IL-10 expression in interferon regulatory factor-1 deficient mice during Plasmodium berghei blood-stage infection. Parasite Immunol. 22: 425-435. [Medline]
  29. Kossodo, S., C. Monso, P. Juillard, T. Velu, M. Goldman, G. E. Grau. 1997. Interleukin-10 modulates susceptibility in experimental cerebral malaria. Immunology 91: 536-540. [Medline]
  30. Li, C., L. A. Sanni, F. Omer, E. Riley, J. Langhorne. 2003. Pathology of Plasmodium chabaudi chabaudi infection and mortality in IL-10-deficient mice are ameliorated by anti-tumor necrosis factor {alpha} and exacerbated by anti-transforming growth factor β antibodies. Infect. Immun. 71: 4850-4856. [Abstract/Free Full Text]
  31. Yoshimoto, T., Y. Takahama, C. R. Wang, T. Yoneto, S. Waki, H. Nariuchi. 1998. A pathogenic role of IL-12 in blood-stage murine malaria lethal strain Plasmodium berghei NK65 infection. J. Immunol. 160: 5500-5505. [Abstract/Free Full Text]
  32. Omer, F. M., J. B. de Souza, E. M. Riley. 2003. Differential induction of TGF-β regulates proinflammatory cytokine production and determines the outcome of lethal and nonlethal Plasmodium yoelii infections. J. Immunol. 171: 5430-5436. [Abstract/Free Full Text]
  33. Kobayashi, F., H. Ishida, T. Matsui, M. Tsuji. 2000. Effects of in vivo administration of anti-IL-10 or anti-IFN-{gamma} monoclonal antibody on the host defense mechanism against Plasmodium yoelii yoelii infection. J. Vet. Med. Sci. 6: 583-587.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Niikura, M.
Right arrow Articles by Kobayashi, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Niikura, M.
Right arrow Articles by Kobayashi, F.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Substance via MeSH
Medline Plus Health Information
*Malaria

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS