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Plasmodium yoelii: Distinct CD4+CD25+ regulatory T cell responses during the early stages of infection in susceptible and resistant mice
Experimental Parasitology 115 (2007) 301–304 www.elsevier.com/locate/yexpr
Plasmodium yoelii: Distinct CD4+CD25+ regulatory T cell responses during the early stages of infection in susceptible and resistant mice Yi Wu a,b, Qing-hui Wang a, Li Zheng a, Hui Feng a, Jun Liu a, Shi-hong Ma a, Ya-ming Cao a,¤ a
Department of Immunology, College of Basic Medical Sciences, China Medical University, Shenyang 110001, China b Department of Internal Medicine, Liaoning University of Traditional Chinese Medicine, Shenyang 110032, China Received 3 March 2006; received in revised form 12 September 2006; accepted 21 September 2006 Available online 7 November 2006
Abstract The outcome of experimental murine infection with diVerent strains of malaria parasites, ranging from spontaneous cure to death, depends largely on the establishment of eVective Th1 responses during the early stages of infection. Here we describe the disparity in CD4+CD25+ regulatory T cell (Treg) responses during the early stages of infection with the highly virulent Plasmodium yoelii 17XL strain in susceptible (BALB/c) and resistant (DBA/2) mice. An increased proportion of Tregs 3–4 days post inoculation, co-occurring with elevated IL-10 levels, is observed in BALB/c but not in DBA/2 mice. These Wndings suggest that Treg proliferation might be causally associated with the suppression of Th1 responses during early malaria infection, leading to increase parasitemia and mortality in BALB/c mice, possibly in an IL-10-dependent manner. © 2006 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Plasmodium yoelii; Immunoregulation; ANOVA, analysis of variance; IFN-, interferon gamma; IL-10, interleukin10; i.p., intraperitoneal; NO, nitric oxide; p.i., post inoculation; Py17XL, lethal strain of Plasmodium yoelii 17X; TGF-, transforming growth factor-beta; Tregs, CD4+CD25+ regulatory T cells.
Malaria is one of the most prevalent infectious diseases worldwide, with over three billion people at risk of infection and 500 million clinical cases reported annually (Snow et al., 2005). However, the interaction between the parasites and the host is extremely complicated. Mice infected with rodent malaria parasites may provide a valuable animal model for dissecting protective immunity against human malaria. DiVerent secretion proWles of Th1-associated cytokines have been observed in Plasmodium chabaudi chabaudi (AS) infected mice from diVerent genetic backgrounds, which lead to distinct consequences—spontaneous cure and death (Jacobs et al., 1995), indicating that eVective Th1 response in the early stages of infection may critically inXuence the later development and Wnal outcome of malaria infection (Jacobs et al., 1996; Malhotra et al., 2005). Potential regulatory mechanisms including regulatory T cells
*
Corresponding author. Fax: +86 24 23264417. E-mail address: [email protected] (Y.-m. Cao).
0014-4894/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2006.09.015
have been shown to signiWcantly modify cellular immune responses to various protozoan infections, including malaria (Riley et al., 2006). A better understanding of modulating mechanisms of defense against the Plasmodium parasites may provide new targets for therapeutic intervention. CD4+CD25+ regulatory T cells (Tregs), intensively studied during the last decade, are widely accepted as potent suppressors to play important roles in immunotolerance, autoimmunity, and transplantation (Lee et al., 2005; Martin et al., 2004; Rouse and Suvas, 2004; Sakaguchi et al., 1995; Shevach, 2002). Interleukin (IL)-10 and transforming growth factor beta (TGF-) were thought to be implicated in Tregs-mediated suppression (Kingsley et al., 2002; Ho et al., 1998; Omer et al., 2003; Yamagiwa et al., 2001). Recently, Tregs have also been reported to maintain the persistence of parasites including Leishmania major and Plasmodium yoelii (Belkaid et al., 2002; Hisaeda et al., 2004), while the potential role of Tregs and their related mechanisms in such chronic infections remain largely unclear. Therefore, in the present study we compared the
302
Y. Wu et al. / Experimental Parasitology 115 (2007) 301–304
Fig. 1. Course of infection and IFN- production in BALB/c and DBA/2 mice infected with Plasmodium yoelii 17XL strain. Both BALB/c and DBA/2 mice (3 mice for each group) were inoculated with 106 parasitized erythrocytes intraperitoneally. (A) Parasitemia was monitored by microscopic evaluation of thin blood Wlms with Giemsa staining. (B) Survival rates were calculated within seven days post inoculation. (C) Parasiteinfected mice were dissected on days 3 to 6 and splenocytes were cultured. IFN- production was monitored by measuring the amount of IFN- in the splenocyte culture supernatants in duplicated wells using ELISA kits (R& D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Values represent the mean and the standard error (n D 3). Results are representative of three independent experiments. Statistical analysis of the diVerences was examined by analysis of variance (ANOVA) and asterisks indicate signiWcant diVerence (P < 0.05).
Treg responses in BALB/c and DBA/2 mice infected with lethal strain of P. yoelii 17X (Py17XL), to gain an insight into the importance of Tregs on the induction of establishing protective Th1 immunity during early malaria infection. First, we compared the course of infection and Th1-type immune responses, as evaluated by the production of interferon gamma (IFN-) and nitric oxide (NO), in the two mouse strains. Py17XL infected BALB/c mice showed a rapid increase of parasitemia from day 4 to day 6 post inoculation (p.i.) with a peak around 60–70% (Fig. 1A) and all mice died within seven days (Fig. 1B). While parasiteinfected DBA/2 mice developed a moderate parasitemia
which maintained approximately 10–30% by day 4–6 p.i. (Fig. 1A) and all mice survived on day seven p.i. (Fig. 1B). These observations indicated that BALB/c mice were susceptible, whereas DBA/2 mice were resistant to Py17XL infection. Furthermore, in cultured splenocyte supernatants from DBA/2 mice, IFN- increased rapidly to reach the peak of 7.4 § 0.8 ng/ml on day 3 p.i. and then decreased slowly during the course of infection, but still kept signiWcantly elevated level until day 6 p.i. (Fig. 1C). BALB/c mice also showed a peak for IFN- production on day 3 p.i. However, productions of IFN- in DBA/2 mice were signiWcantly higher than those in BALB/c mice on days 3 to 5 (Fig. 1C). Consistently, the diVerence of increased magnitude of NO (data not shown) was also observed in both mouse strains. These data, thus, demonstrated that successful resistance to blood stage malaria infections in mice depended on their ability to induce an early eVective Th1 cellular immunity characterized by a predominant IFN- secretion, which could promote NO production to control parasite growth as described previously (Jacobs et al., 1995, 1996). Next, we were interested in elucidating whether Tregs had roles in aVecting the host Th1 immunity during the early stages of infection. Flow cytometric analysis was introduced to enumerate Tregs obtained from splenocytes of BALB/c and DBA/2 mice infected with Py17XL by double staining with anti-CD4 and anti-CD25 monoclonal antibodies. The population of Tregs signiWcantly increased (approximately 2-fold of pre-infection level) on days 3 and 4 p.i. in susceptible BALB/c mice; whereas the percentage of Tregs still stood at pre-infection level on day 3 p.i. and didn’t increase until day 5 p.i. in resistant DBA/2 mice (Fig. 2A). To demonstrate the potential mechanisms of immunosuppression by Tregs, we evaluated the Th1 inhibitory cytokines IL-10 and TGF-1 in supernatants of splenocytes from Py17XL infected mice. The levels of IL-10 markedly increased and were kept at elevated levels from day 3 to day 6 in BALB/c mice, whereas there was no change of IL-10 levels in DBA/2 mice (Fig. 2B); By contrast, TGF-1 secretion did not show any signiWcant change in both mouse strains (data not shown). Thus, these data showed a marked diVerence in kinetics of Tregs’ proliferation and the production of immunoregulatory cytokine IL-10 between susceptible and resistant mouse strains during the early stages of Py17XL infection. The suppression of Tregs in the malaria infection has been addressed in rodent model by presenting an increased survival associated with higher T-cell responsiveness against parasite-derived antigens after depletion of Tregs (Hisaeda et al., 2004). Tregs could exert their function in an IL-10-dependent manner (Belkaid et al., 2002). Moreover, a recent human malaria research reported the close relationship of increased percentages of Tregs and elevated IL-10 responses to antigen of P. falciparum in cord blood mononuclear cells (Brustoski et al., 2006). In this study, we developed a rodent model to dissect the role of Tregs in modulating Th1 cellular immunity. Data presented here
Y. Wu et al. / Experimental Parasitology 115 (2007) 301–304
303
Fig. 2. CD4+CD25+ regulatory T cell (Treg) proportion and IL-10 production in Py17XL-infected BALB/c and DBA/2 mice. (A) Percentage of Tregs in total spleen CD4+T cell population was measured by Xow cytometry in BALB/c and DBA/2 mice after inoculation. Cells were stained with FITC-conjugated anti-CD4 monoclonal antibody (GK1.5; BD PharMingen, San Diego, CA, USA) and phycoerythrin-conjugated anti-CD25 monoclonal antibody (3C7; BD PharMingen). (B) IL-10 production. Parasite-infected mice were dissected on days 3 to 6 and splenocytes were cultured. IL-10 production was monitored by measuring the amount of IL-10 in the splenocyte culture supernatants in duplicated wells using ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Values represent the mean and the standard error (n D 3). Results are representative of three independent experiments. Statistical analysis of the diVerences was examined by analysis of variance (ANOVA) and asterisks indicate signiWcant diVerence (P < 0.05).
further indicate that just at the critical phase (days 3 and 4 p.i.) when the eVective Th1 immune response is supposed to be established, the activation of Tregs accompanied with high level of IL-10 counteracts the production of IFN- and NO, contributing to the exacerbation of infection and Wnal death in susceptible BALB/c mice. On the contrary, at the same time point the failure of Tregs activation would favor in setting up an eVective Th1 immune response, which controls parasitemia around 10–30%, and allows the resistant DBA/2 mice to survive. More importantly, the timing of Treg activation seems to be critical for the outcome of malaria infection. Considering the expansion of Tregs in resistant DBA/2 mice on the later time point (day 5–6 p.i.), we speculate that the proliferation might limit the excessive Th1 reaction or modify subsequent antibody responses to abrogate the parasites. The possible mechanisms remain to be clariWed. In conclusion, the signiWcant Wnding in this report is the observation of distinct Treg responses during the early stages of infection with Py17XL in susceptible BALB/c and resistant DBA/2 mice, which raised the possibility that IL-10-dependent Tregs might play an immunosuppressive role in establishment of early eVective Th1 cellular immunity, leading to the persistent rise of parasitemia and mortality as a Wnal consequence. The clariWcation of the mechanisms of immune eVect of Tregs will favor in developing both eVective malarial vaccine and anti-malarial drugs. Acknowledgments We thank Dr. Motomi Torii, Ehime University Graduate School of Medicine, Japan, for his guidance of this research and providing malaria parasite strain of Py17XL; Critical readings of the manuscript by Drs. Takafumi Tsuboi and Osamu Kaneko are gratefully acknowledged. This work was supported by Grant 05L547 from the Educational Department of Liaoning province, China.
References Belkaid, Y., Piccirillo, C.A., Mendez, S., Shevach, E.M., Sacks, D.L., 2002. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420, 502–507. Brustoski, K., Moller, U., Kramer, M., Hartgers, F.C., Kremsner, P.G., Krzych, U., Luty, A.J., 2006. Reduced cord blood immune eVector-cell responsiveness mediated by CD4+ cells induced in utero as a consequence of placental Plasmodium falciparum infection. The Journal of Infectious Diseases 193, 146–154. Hisaeda, H., Maekawa, Y., Iwakawa, D., Okada, H., Himeno, K., Kishihara, K., Tsukumo, S., Yasutomo, K., 2004. Escape of malaria parasites from host immunity requires CD4+CD25+ regulatory T cells. Nature Medicine 10, 29–30. Ho, M., Schollaardt, T., Snape, S., Looareesuwan, S., Suntharasamai, P., White, N.J., 1998. Endogenous interleukin-10 modulates proinXammatory response in Plasmodium falciparum malaria. The Journal of Infectious Diseases 178, 520–525. Jacobs, P., Radzioch, D., Stevenson, M.M., 1995. Nitric oxide expression in the spleen, but not in the liver, correlates with resistance to blood-stage malaria in mice. Journal of Immunology 155, 5306–5313. Jacobs, P., Radzioch, D., Stevenson, M.M., 1996. In vivo regulation of nitric oxide production by tumor necrosis factor alpha and gamma interferon, but not by interleukin-4, during blood stage malaria in mice. Infection and Immunity 64, 44–49. Kingsley, C.I., Karim, M., Bushell, A.R., Wood, K.J., 2002. CD25+CD4+ regulatory T cells prevent graft rejection: CTLA-4- and IL-10-dependent immunoregulation of alloresponses. Journal of Immunology 168, 1080–1086. Lee, K.S., Sen, G., Snapper, C.M., 2005. Endogenous CD4+CD25+ regulatory T cells play no apparent role in the acute humoral response to intact Streptococcus pneumoniae. Infection and Immunity 73, 4427–4431. Malhotra, I., Mungai, P., Muchiri, E., Ouma, J., Sharma, S., Kazura, J.W., King, C.L., 2005. Distinct Th1- and Th2-Type prenatal cytokine responses to Plasmodium falciparum erythrocyte invasion ligands. Infection and Immunity 73, 3462–3470. Martin, B., Banz, A., Bienvenu, B., Cordier, C., Dautigny, N., Becourt, C., Lucas, B., 2004. Suppression of CD4+ T lymphocyte eVector functions by CD4+ CD25+ cells in vivo. Journal of Immunology 172, 3391–3398. Omer, F.M., de Souza, J.B., Riley, E.M., 2003. DiVerential induction of TGF-beta regulates proinXammatory cytokine production and determines the outcome of lethal and nonlethal Plasmodium yoelii infections. Journal of Immunology 171, 5430–5436. Riley, E.M., Wahl, S., Perkins, D.J., SchoWeld, L., 2006. Regulating immunity to malaria. Parasite Immunology 28, 35–49. Rouse, B.T., Suvas, S., 2004. Regulatory cells and infectious agents: detentes cordiale and contraire. Journal of Immunology 173, 2211–2215.
304
Y. Wu et al. / Experimental Parasitology 115 (2007) 301–304
Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M., Toda, M., 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. Journal of Immunology 155, 1151–1164. Shevach, E.M., 2002. CD4+CD25+ suppressor T cells: more questions than answers. Nature Reviews Immunology 2, 389–400.
Snow, R.W., Guerra, C.A., Noor, A.M., Myint, H.Y., Hay, S.I., 2005. The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature 434, 214–217. Yamagiwa, S., Gray, J.D., Hashimoto, S., Horwitz, D.A., 2001. A role for TGF-beta in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood. Journal of Immunology 166, 7282–7289.
Plasmodium yoelii: Distinct CD4+CD25+ regulatory T cell responses during the early stages of infection in susceptible and resistant mice Yi Wu a,b, Qing-hui Wang a, Li Zheng a, Hui Feng a, Jun Liu a, Shi-hong Ma a, Ya-ming Cao a,¤ a
Department of Immunology, College of Basic Medical Sciences, China Medical University, Shenyang 110001, China b Department of Internal Medicine, Liaoning University of Traditional Chinese Medicine, Shenyang 110032, China Received 3 March 2006; received in revised form 12 September 2006; accepted 21 September 2006 Available online 7 November 2006
Abstract The outcome of experimental murine infection with diVerent strains of malaria parasites, ranging from spontaneous cure to death, depends largely on the establishment of eVective Th1 responses during the early stages of infection. Here we describe the disparity in CD4+CD25+ regulatory T cell (Treg) responses during the early stages of infection with the highly virulent Plasmodium yoelii 17XL strain in susceptible (BALB/c) and resistant (DBA/2) mice. An increased proportion of Tregs 3–4 days post inoculation, co-occurring with elevated IL-10 levels, is observed in BALB/c but not in DBA/2 mice. These Wndings suggest that Treg proliferation might be causally associated with the suppression of Th1 responses during early malaria infection, leading to increase parasitemia and mortality in BALB/c mice, possibly in an IL-10-dependent manner. © 2006 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Plasmodium yoelii; Immunoregulation; ANOVA, analysis of variance; IFN-, interferon gamma; IL-10, interleukin10; i.p., intraperitoneal; NO, nitric oxide; p.i., post inoculation; Py17XL, lethal strain of Plasmodium yoelii 17X; TGF-, transforming growth factor-beta; Tregs, CD4+CD25+ regulatory T cells.
Malaria is one of the most prevalent infectious diseases worldwide, with over three billion people at risk of infection and 500 million clinical cases reported annually (Snow et al., 2005). However, the interaction between the parasites and the host is extremely complicated. Mice infected with rodent malaria parasites may provide a valuable animal model for dissecting protective immunity against human malaria. DiVerent secretion proWles of Th1-associated cytokines have been observed in Plasmodium chabaudi chabaudi (AS) infected mice from diVerent genetic backgrounds, which lead to distinct consequences—spontaneous cure and death (Jacobs et al., 1995), indicating that eVective Th1 response in the early stages of infection may critically inXuence the later development and Wnal outcome of malaria infection (Jacobs et al., 1996; Malhotra et al., 2005). Potential regulatory mechanisms including regulatory T cells
*
Corresponding author. Fax: +86 24 23264417. E-mail address: [email protected] (Y.-m. Cao).
0014-4894/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2006.09.015
have been shown to signiWcantly modify cellular immune responses to various protozoan infections, including malaria (Riley et al., 2006). A better understanding of modulating mechanisms of defense against the Plasmodium parasites may provide new targets for therapeutic intervention. CD4+CD25+ regulatory T cells (Tregs), intensively studied during the last decade, are widely accepted as potent suppressors to play important roles in immunotolerance, autoimmunity, and transplantation (Lee et al., 2005; Martin et al., 2004; Rouse and Suvas, 2004; Sakaguchi et al., 1995; Shevach, 2002). Interleukin (IL)-10 and transforming growth factor beta (TGF-) were thought to be implicated in Tregs-mediated suppression (Kingsley et al., 2002; Ho et al., 1998; Omer et al., 2003; Yamagiwa et al., 2001). Recently, Tregs have also been reported to maintain the persistence of parasites including Leishmania major and Plasmodium yoelii (Belkaid et al., 2002; Hisaeda et al., 2004), while the potential role of Tregs and their related mechanisms in such chronic infections remain largely unclear. Therefore, in the present study we compared the
302
Y. Wu et al. / Experimental Parasitology 115 (2007) 301–304
Fig. 1. Course of infection and IFN- production in BALB/c and DBA/2 mice infected with Plasmodium yoelii 17XL strain. Both BALB/c and DBA/2 mice (3 mice for each group) were inoculated with 106 parasitized erythrocytes intraperitoneally. (A) Parasitemia was monitored by microscopic evaluation of thin blood Wlms with Giemsa staining. (B) Survival rates were calculated within seven days post inoculation. (C) Parasiteinfected mice were dissected on days 3 to 6 and splenocytes were cultured. IFN- production was monitored by measuring the amount of IFN- in the splenocyte culture supernatants in duplicated wells using ELISA kits (R& D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Values represent the mean and the standard error (n D 3). Results are representative of three independent experiments. Statistical analysis of the diVerences was examined by analysis of variance (ANOVA) and asterisks indicate signiWcant diVerence (P < 0.05).
Treg responses in BALB/c and DBA/2 mice infected with lethal strain of P. yoelii 17X (Py17XL), to gain an insight into the importance of Tregs on the induction of establishing protective Th1 immunity during early malaria infection. First, we compared the course of infection and Th1-type immune responses, as evaluated by the production of interferon gamma (IFN-) and nitric oxide (NO), in the two mouse strains. Py17XL infected BALB/c mice showed a rapid increase of parasitemia from day 4 to day 6 post inoculation (p.i.) with a peak around 60–70% (Fig. 1A) and all mice died within seven days (Fig. 1B). While parasiteinfected DBA/2 mice developed a moderate parasitemia
which maintained approximately 10–30% by day 4–6 p.i. (Fig. 1A) and all mice survived on day seven p.i. (Fig. 1B). These observations indicated that BALB/c mice were susceptible, whereas DBA/2 mice were resistant to Py17XL infection. Furthermore, in cultured splenocyte supernatants from DBA/2 mice, IFN- increased rapidly to reach the peak of 7.4 § 0.8 ng/ml on day 3 p.i. and then decreased slowly during the course of infection, but still kept signiWcantly elevated level until day 6 p.i. (Fig. 1C). BALB/c mice also showed a peak for IFN- production on day 3 p.i. However, productions of IFN- in DBA/2 mice were signiWcantly higher than those in BALB/c mice on days 3 to 5 (Fig. 1C). Consistently, the diVerence of increased magnitude of NO (data not shown) was also observed in both mouse strains. These data, thus, demonstrated that successful resistance to blood stage malaria infections in mice depended on their ability to induce an early eVective Th1 cellular immunity characterized by a predominant IFN- secretion, which could promote NO production to control parasite growth as described previously (Jacobs et al., 1995, 1996). Next, we were interested in elucidating whether Tregs had roles in aVecting the host Th1 immunity during the early stages of infection. Flow cytometric analysis was introduced to enumerate Tregs obtained from splenocytes of BALB/c and DBA/2 mice infected with Py17XL by double staining with anti-CD4 and anti-CD25 monoclonal antibodies. The population of Tregs signiWcantly increased (approximately 2-fold of pre-infection level) on days 3 and 4 p.i. in susceptible BALB/c mice; whereas the percentage of Tregs still stood at pre-infection level on day 3 p.i. and didn’t increase until day 5 p.i. in resistant DBA/2 mice (Fig. 2A). To demonstrate the potential mechanisms of immunosuppression by Tregs, we evaluated the Th1 inhibitory cytokines IL-10 and TGF-1 in supernatants of splenocytes from Py17XL infected mice. The levels of IL-10 markedly increased and were kept at elevated levels from day 3 to day 6 in BALB/c mice, whereas there was no change of IL-10 levels in DBA/2 mice (Fig. 2B); By contrast, TGF-1 secretion did not show any signiWcant change in both mouse strains (data not shown). Thus, these data showed a marked diVerence in kinetics of Tregs’ proliferation and the production of immunoregulatory cytokine IL-10 between susceptible and resistant mouse strains during the early stages of Py17XL infection. The suppression of Tregs in the malaria infection has been addressed in rodent model by presenting an increased survival associated with higher T-cell responsiveness against parasite-derived antigens after depletion of Tregs (Hisaeda et al., 2004). Tregs could exert their function in an IL-10-dependent manner (Belkaid et al., 2002). Moreover, a recent human malaria research reported the close relationship of increased percentages of Tregs and elevated IL-10 responses to antigen of P. falciparum in cord blood mononuclear cells (Brustoski et al., 2006). In this study, we developed a rodent model to dissect the role of Tregs in modulating Th1 cellular immunity. Data presented here
Y. Wu et al. / Experimental Parasitology 115 (2007) 301–304
303
Fig. 2. CD4+CD25+ regulatory T cell (Treg) proportion and IL-10 production in Py17XL-infected BALB/c and DBA/2 mice. (A) Percentage of Tregs in total spleen CD4+T cell population was measured by Xow cytometry in BALB/c and DBA/2 mice after inoculation. Cells were stained with FITC-conjugated anti-CD4 monoclonal antibody (GK1.5; BD PharMingen, San Diego, CA, USA) and phycoerythrin-conjugated anti-CD25 monoclonal antibody (3C7; BD PharMingen). (B) IL-10 production. Parasite-infected mice were dissected on days 3 to 6 and splenocytes were cultured. IL-10 production was monitored by measuring the amount of IL-10 in the splenocyte culture supernatants in duplicated wells using ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Values represent the mean and the standard error (n D 3). Results are representative of three independent experiments. Statistical analysis of the diVerences was examined by analysis of variance (ANOVA) and asterisks indicate signiWcant diVerence (P < 0.05).
further indicate that just at the critical phase (days 3 and 4 p.i.) when the eVective Th1 immune response is supposed to be established, the activation of Tregs accompanied with high level of IL-10 counteracts the production of IFN- and NO, contributing to the exacerbation of infection and Wnal death in susceptible BALB/c mice. On the contrary, at the same time point the failure of Tregs activation would favor in setting up an eVective Th1 immune response, which controls parasitemia around 10–30%, and allows the resistant DBA/2 mice to survive. More importantly, the timing of Treg activation seems to be critical for the outcome of malaria infection. Considering the expansion of Tregs in resistant DBA/2 mice on the later time point (day 5–6 p.i.), we speculate that the proliferation might limit the excessive Th1 reaction or modify subsequent antibody responses to abrogate the parasites. The possible mechanisms remain to be clariWed. In conclusion, the signiWcant Wnding in this report is the observation of distinct Treg responses during the early stages of infection with Py17XL in susceptible BALB/c and resistant DBA/2 mice, which raised the possibility that IL-10-dependent Tregs might play an immunosuppressive role in establishment of early eVective Th1 cellular immunity, leading to the persistent rise of parasitemia and mortality as a Wnal consequence. The clariWcation of the mechanisms of immune eVect of Tregs will favor in developing both eVective malarial vaccine and anti-malarial drugs. Acknowledgments We thank Dr. Motomi Torii, Ehime University Graduate School of Medicine, Japan, for his guidance of this research and providing malaria parasite strain of Py17XL; Critical readings of the manuscript by Drs. Takafumi Tsuboi and Osamu Kaneko are gratefully acknowledged. This work was supported by Grant 05L547 from the Educational Department of Liaoning province, China.
References Belkaid, Y., Piccirillo, C.A., Mendez, S., Shevach, E.M., Sacks, D.L., 2002. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420, 502–507. Brustoski, K., Moller, U., Kramer, M., Hartgers, F.C., Kremsner, P.G., Krzych, U., Luty, A.J., 2006. Reduced cord blood immune eVector-cell responsiveness mediated by CD4+ cells induced in utero as a consequence of placental Plasmodium falciparum infection. The Journal of Infectious Diseases 193, 146–154. Hisaeda, H., Maekawa, Y., Iwakawa, D., Okada, H., Himeno, K., Kishihara, K., Tsukumo, S., Yasutomo, K., 2004. Escape of malaria parasites from host immunity requires CD4+CD25+ regulatory T cells. Nature Medicine 10, 29–30. Ho, M., Schollaardt, T., Snape, S., Looareesuwan, S., Suntharasamai, P., White, N.J., 1998. Endogenous interleukin-10 modulates proinXammatory response in Plasmodium falciparum malaria. The Journal of Infectious Diseases 178, 520–525. Jacobs, P., Radzioch, D., Stevenson, M.M., 1995. Nitric oxide expression in the spleen, but not in the liver, correlates with resistance to blood-stage malaria in mice. Journal of Immunology 155, 5306–5313. Jacobs, P., Radzioch, D., Stevenson, M.M., 1996. In vivo regulation of nitric oxide production by tumor necrosis factor alpha and gamma interferon, but not by interleukin-4, during blood stage malaria in mice. Infection and Immunity 64, 44–49. Kingsley, C.I., Karim, M., Bushell, A.R., Wood, K.J., 2002. CD25+CD4+ regulatory T cells prevent graft rejection: CTLA-4- and IL-10-dependent immunoregulation of alloresponses. Journal of Immunology 168, 1080–1086. Lee, K.S., Sen, G., Snapper, C.M., 2005. Endogenous CD4+CD25+ regulatory T cells play no apparent role in the acute humoral response to intact Streptococcus pneumoniae. Infection and Immunity 73, 4427–4431. Malhotra, I., Mungai, P., Muchiri, E., Ouma, J., Sharma, S., Kazura, J.W., King, C.L., 2005. Distinct Th1- and Th2-Type prenatal cytokine responses to Plasmodium falciparum erythrocyte invasion ligands. Infection and Immunity 73, 3462–3470. Martin, B., Banz, A., Bienvenu, B., Cordier, C., Dautigny, N., Becourt, C., Lucas, B., 2004. Suppression of CD4+ T lymphocyte eVector functions by CD4+ CD25+ cells in vivo. Journal of Immunology 172, 3391–3398. Omer, F.M., de Souza, J.B., Riley, E.M., 2003. DiVerential induction of TGF-beta regulates proinXammatory cytokine production and determines the outcome of lethal and nonlethal Plasmodium yoelii infections. Journal of Immunology 171, 5430–5436. Riley, E.M., Wahl, S., Perkins, D.J., SchoWeld, L., 2006. Regulating immunity to malaria. Parasite Immunology 28, 35–49. Rouse, B.T., Suvas, S., 2004. Regulatory cells and infectious agents: detentes cordiale and contraire. Journal of Immunology 173, 2211–2215.
304
Y. Wu et al. / Experimental Parasitology 115 (2007) 301–304
Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M., Toda, M., 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. Journal of Immunology 155, 1151–1164. Shevach, E.M., 2002. CD4+CD25+ suppressor T cells: more questions than answers. Nature Reviews Immunology 2, 389–400.
Snow, R.W., Guerra, C.A., Noor, A.M., Myint, H.Y., Hay, S.I., 2005. The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature 434, 214–217. Yamagiwa, S., Gray, J.D., Hashimoto, S., Horwitz, D.A., 2001. A role for TGF-beta in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood. Journal of Immunology 166, 7282–7289.