Role of Interleukin-10 in Malaria: Focusing on Coinfection with Lethal and Nonlethal Murine Malaria Parasites

Interleukin- (IL-) 10, anti-inflammatory cytokine, is known to inhibit the protective immune responses against malaria parasites and to be involved in exacerbating parasitemia during Plasmodium infection. In contrast, IL-10 is regarded as necessary for suppressing severe pathology during Plasmodium infection. Here, we summarize the role of IL-10 during murine malaria infection, focusing especially on coinfection with lethal and nonlethal strains of malaria parasites. Recent studies have demonstrated that the major sources of IL-10 are subpopulations of CD4+ T cells in humans and mice infected with Plasmodium. We also discuss the influence of innate immunity on the induction of CD4+ T cells during murine malaria coinfection.


Introduction
Malaria, caused by protozoan parasites of the genus Plasmodium, is the major parasitic disease in tropical and subtropical regions, including parts of the Americas, Asia, and Africa. With more than 200-300 million clinical cases globally and approximately 1 million deaths per year, malaria represents the most important infectious disease worldwide. Four species of Plasmodium infect humans: P. falciparum, P. vivax, P. malariae, and P. ovale.
Human malarial parasites develop through two stages in humans: a liver stage and a blood stage. The asexual blood stage of the parasite is the cause of malarial pathologies. Therefore, it is important to prevent the replication of this stage of parasite. Particularly, P. falciparum causes severe pathologies such as cerebral malaria, severe anemia, and respiratory injury in the blood stage. It is necessary to understand the mechanism of protective immunity against the blood stage of the parasite during malaria infection. Nevertheless, it is difficult to investigate the human in vivo immune response against malaria parasite for many reasons. Consequently, murine malaria models with P. berghei, P. yoelii, and P. chabaudi have been used to elucidate the immune interaction in hosts and to demonstrate many factors associated with malarial defense mechanisms [1].

Parasite Killing: The Role of Proinflammatory Cytokines during Human and Murine Malaria Infection
Interferon-(IFN-) γ and Interleukin-(IL-) 12 play a crucial role in the clearance of intracellular pathogens [2][3][4][5]. Low levels of IFN-γ and IL-12 production have been observed in young African children with severe anemia during infection with P. falciparum [6]. The IFN-γ-mediated responses have been shown to be involved in protection against infection with P. falciparum [7]. In murine malaria, IFN-γ produced by CD4 + T cells has been shown to play a pivotal role in protective immunity against P. chabaudi (Pc) AS [8], nonlethal P. berghei (Pb) XAT [9], and P. yoelii (Py) 17XNL [10] infection. Actually, IFN-γ-depleted mice infected with murine malaria parasites show high levels of parasitemia and eventually die. IL-12 is a necessary factor for clearance of nonlethal Pc AS [11], Pb XAT [12], and Py 17XNL [13], suggesting that IL-12 plays an important role in protective immunity via IFN-γ production in murine malaria. Production of IFN-γ and IL-12 is suppressed by anti-inflammatory cytokines such as IL-10. It is possible that enhancement of IL-10 production contributes to suppression of parasite killing, considering that IL-10 plays a detrimental role during P. falciparum infection.

Detrimental Effect of IL-10 on the Outcome of Human and Murine Malaria Infection
High levels of IL-10 and TNF in plasma have been characteristic of young African children with malarial anemia and high levels of parasitemia [32][33][34][35][36][37][38][39]. In common IL-10 promoter variants, the -1082A/-819T/-592A (ATA) haplotype has been associated with increased susceptibility to severe anemia [39]. Their IL-10 : IL-12 ratio was higher than that in the non-ATA haplotype. On the other hand, the -1082G/-819C/-592C (GCC) haplotype has been associated with protection against severe anemia [39]. The IL-10 : IL-12 ratio in the GCC haplotype was lower than that in the ATA haplotype. These findings suggest that a high IL-10 : IL-12 ratio is associated with the downregulation of IFN-γ production and that it causes development of severe anemia during P. falciparum infection.
Lethal Py 17XL-infected mice show higher levels of IL-10 and TGF-β production than nonlethal Py 17XNL-infected mice early in infection [40,41]. High levels of IL-10 and TGF-β are associated with inhibition of proinflammatory response, resulting in high levels of parasitemia, severe anemia by which RBCs ruptured, causing parasite replication and the death of infected mice. Depletion or deficiency of IL-10 [40,42], or the blockade of IL-10 receptor [41] regulates parasitemia during lethal Py 17XL infection and prolongs survival of infected mice. Couper et al. [42] reported that the major source of IL-10 in lethal Py 17XL-infected mice is CD4 + Tr1 cells, just as it is in toxoplasmosis [43] and cutaneous leishmaniasis [44] (Figure 1).
In nonlethal Py 17XNL-infected mice, the production of IL-10 and TGF-β is induced in the late phase of infection [41]. The population of CD4 + Tr1 cells has been shown to be the major source of IL-10 in nonlethal Py 17XNL as well as lethal Py 17XL infection. Moreover, IL-10-deficient mice show marked suppression of the replication of parasites compared with that in wild-type mice [42] (Figure 1). These findings suggest that enhanced-IL-10 production suppresses inflammatory response against malaria parasites, resulting in high levels of parasitemia and anemia by replication of parasites in infected mice. Results show that IL-10 plays a detrimental role during human and murine malaria infection.

Role of Anti-Inflammatory Cytokines during Murine Malaria Infection
Reportedly, a low IL-10/TNF ratio is associated with severe malarial anemia [36][37][38]. These results suggest that low levels of IL-10 production are associated with enhancement of TNF production, followed by increased IFN-γ production. The enhancement of TNF production might be associated with the aggravation of disease severity, such as severe anemia, by which phagocytosis of uninfected RBC occurs [45], or dyserythropoiesis [46]. Moreover, results obtained using mouse models have suggested that IL-10 plays a protective role in the host during murine malaria infection. Although IL-10-deficient mice show lower levels of parasitemia than wild-type mice do during murine malaria infection, they indicate severe diseases such as hepatic pathology [42,47,48] and cerebral pathology [49,50]. Actually, inflammation, which is involved in parasite killing, is upregulated in IL-10-deficient mice, but excessive inflammation, such as the increase of IFN-γ production, also presents the risk of developing hepatic pathology and/or cerebral pathology. Therefore, it seems that IL-10 might be necessary for suppression of hepatic pathology and cerebral pathology in the host during infection. B6 mice infected with Pb NK65 display hepatic pathology and die within 2 weeks. The development of severe hepatic pathology is involved in IL-12 [11], IFN-γ, and CD8 + T cells [9]. The IL-12 production is induced through a MyD88dependent pathway in DCs or macrophage and engenders hepatic pathology in a perforin/granzyme-dependent manner during Pb NK65 infection [51]. Coinfection with nonlethal Pb XAT or Py 17XNL prevents the development of hepatic pathology caused by Pb NK65 infection and prolongs survival of mice [47]. In fact, IL-10 KO mice coinfected with nonlethal Pb XAT or Py 17XNL showed severe hepatic pathology, suggesting that IL-10 is involved in suppression of disease severity during coinfection [47] (Figure 2(b)). During lethal Py 17XL or nonlethal Py 17XNL infection, IL-10, which is derived from CD4 + Tr1 cells, is also necessary for the prevention of hepatic pathology [42]. Nevertheless, it remains unclear whether IFN-γ and CD8 + T cells are   [52,53]. Proinflammatory cytokines, such as IFNγ and lymphotoxin-α, are known to accelerate ECM development [54] (Figure 2(c)). In contrast, anti-inflammatory cytokines, such as IL-10, prevent ECM development [55,56]. However, it remains unclear how IL-10 suppresses ECM development, because high levels of IL-10 production were observed in spleen [57] and plasma [58] of mice singly infected with Pb ANKA.
The ECM development is suppressed by the simultaneous presence of murine AIDS during Pb ANKA infection [55]. Results demonstrated that murine AIDS-mediated protection of ECM is dependent on IL-10, which is produced by splenic CD4 + T cells, with the use of anti-IL-10 mAb. It is particularly interesting that coinfection with parasites such as nonlethal Pb XAT [50] or Filaria [59] has also been shown to prevent ECM development. The suppressive effect of coinfection with nonlethal Pb XAT or Filaria on ECM during Pb ANKA infection was abrogated in IL-10 KO mice [50,59], suggesting that IL-10 plays a crucial role in the suppression of ECM during coinfection with other parasites (Figure 2(b)).
In contrast to coinfection with nonlethal Pb XAT, the suppressive effect of coinfection with nonlethal Py 17XNL on ECM during Pb ANKA infection is independent of IL-10 [50,60]. A recent study demonstrated that Treg cells, which are expanded by IL-2/anti-IL-2 complexes, suppress the recruitment of pathogenic CD4 + and CD8 + T cells to brains and protect mice from developing ECM during Pb ANKA [61]. The IL-2/anti-IL-2 complexes enhanced the levels of Foxp3 and CTLA-4 expression and increased the levels of IL-10 production from Treg cells during Pb ANKA infection. However, the suppression of ECM by Treg cells was dependent on CTLA-4 but not on IL-10 [61] (Figure 2(c)). The suppressive effect of coinfection with nonlethal Py 17XNL on ECM during Pb ANKA infection was not reversible by depleting antibodies against CD25 + -bearing CD4 + T cells or CTLA4 + -bearing CD4 + T cells [60]. A key factor that has a suppressive effect on ECM by coinfection with nonlethal Py 17XNL has not yet been discovered. Development of severe disease such as hepatic pathology and cerebral pathology generally involves excessive inflammation in murine malaria parasites. Little is known about the differences between the developmental mechanisms of hepatic pathology and cerebral pathology during Pb NK65 and Pb ANKA infection, respectively. However, IL-10 can downregulate excessive inflammation during Pb NK65 or Pb ANKA infection. It is associated with the suppression of hepatic pathology and cerebral pathology. Results show that IL-10 plays a protective role in the host during P. falciparum infection.

A Different Type of DC Induced by Lethal and Nonlethal Murine Malaria Infection
How are different subsets of CD4 + T cells, such as pathological CD4 + T cells [62,63], IL-10-producing CD4 + T cells [42], and Treg cells [64], induced between lethal and nonlethal murine malaria infection? The development of CM is inhibited completely by the simultaneous presence of nonlethal Py 17XNL [60], lethal Pb K173 [57], and nonlethal Pb XAT [50]. However, protection from CM was not induced in mice when they were infected with Py 17XNL on day 4 after Pb ANKA infection [60]. Similarly, coinfection with Pb XAT on day 1 or day 3 after infection with Pb ANKA failed to protect mice from cerebral malaria (Niikura et al. unpublished data). In simultaneous infection with Pb ANKA and Pb K173, suppression of ECM was associated with the induction of cytokines such as IFN-γ, IL-10, and IL-12 on day 1 after infection [57]. These findings suggest that the presence of other parasites might modulate some key factors/cells that are involved in innate immunity in early infection with Pb ANKA. Actually, DCs are important for initiating immune responses against malaria parasites. It is possible that immune responses induced by DCs produce protective and pathological effects, respectively, when mice are infected with nonlethal and lethal parasites. Therefore, DCs might contribute to the determination of the virulence of malaria parasites. In coinfection, a type of DC activated by nonlethal malaria parasites before activation by lethal parasites might fail to expand pathological CD4 + T cells and subsequently fail to suppress severe disease. Wykes et al. [13] showed that although DCs from mice infected with nonlethal Py 17XNL were fully functional, DCs from mice infected with lethal Py YM were unable to produce IL-12 or present antigens to T cells. Apparently, lethal malaria causes a failure of DC function, resulting in the suppression of Th1 immune responses (Figure 1). Similar to lethal Py 17XL infected mice, it is possible that mice infected with lethal Py YM induce IL-10-producing CD4 + T cells. Although IL-10 might inhibit the DC function, such as antigen presentation and release of proinflammatory cytokines, little is known about whether IL-10 associates with a different type of DC induced between lethal and nonlethal murine malaria infection. Toll-like receptors (TLRs) play an important role in the innate immune system against pathogens [65]. Therefore, TLRs might be associated with disease severity during malaria infection. During lethal Py 17XL infection, TLR9 signaling in DCs is known to be crucial for the activation of Treg cells that suppress Th1 immune responses, causing high levels of parasitemia [64]. In contrast, MyD88, but not TLR signaling, has been shown to be necessary for elimination of parasites in mice infected with nonlethal Py 17XNL [66]. Accordingly, a different type of DC induced between lethal and nonlethal murine malaria infection might induce different subsets of CD4 + T cells, such as IL-10-producing CD4 + T cells or Treg cells.

Is IL-10 Necessary for Host Protection against Murine Malaria Parasites?
Although IL-10-deficient mice suppressed an increase of parasitemia during coinfection with lethal and nonlethal parasites, mice were unable to eliminate parasites completely and eventually died [47,50]. These results suggest that the lethal strains of malaria parasites may modulate the induction of adaptive immunity independent of IL-10. Millington et al. [67] demonstrated that Plasmodium infection inhibits the induction of adaptive immunity to heterologous antigens by modulating DC function. According to their paper, hemozoin (HZ), rather than infected RBC membranes, was a key factor involved in the suppression of murine DC function. On the other hand, it has been shown that repeated stimulation through TLR9, which is the receptor for HZ, engenders tolerance to signaling through TLR4 [68]. In fact, HZ activates DCs through the TLR9-MyD88 pathway [69]. A recent study has demonstrated that parasite protein-DNA complex, but not HZ, plays a crucial role in TLR9-mediated activation of DCs during infection [70]. Stimulation through TLR9 might be associated with development of severe hepatic pathology, because MyD88 pathway, which is activated by TLR9 stimulation, is known to be involved in severe hepatic pathology caused by Pb NK65 [51]. Coban et al. [71] and Griffith et al. [72] reported that the TLRs-MyD88 signaling pathway might play a critical role in ECM during lethal Pb ANKA infection. It has been shown that ECM is prevented by oral treatment with E6446, which is a synthetic antagonist of nucleic acid-sensing TLRs [73].
In contrast, it is demonstrated that murine cerebral malaria development is independent of Toll-like receptor signaling [74,75]. It remains controversial whether TLRs-MyD88 signaling pathway is associated with ECM development.
In summary, IL-10 is necessary for suppression of hepatic pathology or ECM in the host although IL-10 entails a risk of downregulation of protective immunity against malaria parasites. CD4 + T cells of different kinds, such as pathological CD4 + T cells, IL-10-producing CD4 + T cells, or Treg cells, are induced during different kinds of Plasmodium spp infection. To induce a more effective immune response in host defense against Plasmodium spp, it is necessary to elucidate the interaction of innate and acquired immune cells such as DCs, αβ T cells, and γδ T cells.