Plasmodium berghei XAT: contribution of gammadelta T cells to host defense against infection with blood-stage nonlethal malaria parasite

We examined a potential role of gammadelta T cells in protective immunity to blood-stage Plasmodium berghei XAT infection. Plasmodium berghei XAT is an attenuated variant of the lethal strain P. berghei NK65 and its infection is self-resolving in immune competent mice. To determine whether gammadelta T cells are essential for the resolution of P. berghei XAT malaria, mice were depleted of gammadelta T cells with anti-TCRgammadelta antibody treatment. Although mice that had received control antibody resolved infections, mice received anti-TCRgammadelta antibody could not control their infections and eventually died. Spleen cells from infected mice produced IFN-gamma and nitric oxide (NO) within the first week of infection, however, levels of IFN-gamma and NO in gammadelta T cell-depleted mice were significantly lower than in control mice. To examine whether gammadelta T cells are involved in the antibody production, malarial-specific antibodies of the various isotypes were measured in the sera of gammadelta T cell-depleted mice and control mice. Serum levels of IgG2a, which was known to be a protective antibody in P. berghei XAT malaria, were significantly lower in gammadelta T cell-depleted mice than in control mice, whereas levels of IgG1 were comparable to those in control mice. Our results indicated that the presence of the gammadelta T cell subset was essential for resolution of blood-stage P. berghei XAT malaria and played a modulatory role in the development of Th1 response and host defense against this malarial parasites.     

The role of IL-18 in blood-stage immunity against murine malaria Plasmodium yoelii 265 and Plasmodium berghei ANKA

A possible protective role of IL-18 in host defense against blood-stage murine malarial infection was studied in BALB/c mice using a nonlethal strain, Plasmodium yoelii 265, and a lethal strain, Plasmodium berghei ANKA. Infection induced an increase in mRNA expression of IL-18, IL-12p40, IFN-gamma, and TNF-alpha in the case of P. yoelii 265 and an increase of IL-18, IL-12p40, and IFN-gamma in the case of P. berghei ANKA. The timing of mRNA expression of IL-18 in both cases was consistent with a role in the induction of IFN-gamma protein expression. Histological examination of spleen and liver tissues from infected controls treated with PBS showed poor cellular inflammatory reaction, massive necrosis, a large number of infected parasitized RBCs, and severe deposition of hemozoin pigment. In contrast, IL-18-treated infected mice showed massive infiltration of inflammatory cells consisting of mononuclear cells and Kupffer cells, decreased necrosis, and decreased deposition of the pigment hemozoin. Treatment with rIL-18 increased serum IFN-gamma levels in mice infected with both parasites, delayed onset of parasitemia, conferred a protective effect, and thus increased survival rate of infected mice. Administration of neutralizing anti-IL-18 Ab exacerbated infection, impaired host resistance and shortened the mean survival of mice infected with P. berghei ANKA. Furthermore, IL-18 knockout mice were more susceptible to P. berghei ANKA than were wild-type C57BL/6 mice. These data suggest that IL-18 plays a protective role in host defense by enhancing IFN-gamma production during blood-stage infection by murine malaria.     

Transgenic parasites: improving our understanding of innate immunity to malaria

Clinical immunity to Plasmodium falciparum malaria takes years to develop and is never complete. One explanation for these observations is that antigenic variation enables malaria parasites to evade humoral immunity; another is that P. falciparum induces immune dysregulation, which inhibits the development of protective cellular immunity. Research described by D'Ombrain et al. in this Cell Host & Microbe issue probes how the parasite's main virulence factor PfEMP-1 might significantly alter human innate immune responses.     

The contribution of Plasmodium chabaudi to our understanding of malaria

Malaria kills close to a million people every year, mostly children under the age of five. In the drive towards the development of an effective vaccine and new chemotherapeutic targets for malaria, field-based studies on human malaria infection and laboratory-based studies using animal models of malaria offer complementary opportunities to further our understanding of the mechanisms behind malaria infection and pathology. We outline here the parallels between the Plasmodium chabaudi mouse model of malaria and human malaria. We will highlight the contribution of P. chabaudi to our understanding of malaria in particular, how the immune response in malaria infection is initiated and regulated, its role in pathology, and how immunological memory is maintained. We will also discuss areas where new tools have opened up potential areas of exploration using this invaluable model system.     

Dendritic cells subsets mediated immune response during Plasmodium berghei ANKA and Plasmodium yoelii infection

The roles of dendritic cells (DCs) in mediating immunity against Plasmodium infection have been extensively investigated, but immune response during pathogenesis of malaria is still poorly understood. In the present study, we compared the splenic DCs phenotype and function during P. berghei ANKA (PbA) or P. yoelii (P. yoelii) infection in Swiss mice. We observed that PbA-infected mice developed more myeloid and mature DCs capable of secreting IL-12, while P. yoelii-infected mice had more plasmacytoid and immature DCs secreting higher levels of IL-10. Expression of FoxP3, IL-17, TGF-β and IL-6 were also different between these two infections. Thus, these results suggest that the phenotypic and functional subsets of splenic DCs are key factors for regulating immune responses to PbA and P. yoelii infections.     

Plasmodium berghei: antibody response and protective immunity induced by vaccination

The authors have studied the behaviour of Swiss mice and of 5 inbred strains of mice in order to investigate: the protective effect, in the homologous infection test, of six vaccine inoculations of irradiated parasites belonging to two strains of Plasmodium berghei: ISTISAN and K173; the capacity to produce humoral antibodies after vaccine treatments and during infection; the probable correlation between the high antibody titre and the protection against infection. The results of the present study show that the antibody response plays a precise role in the immunity induced by vaccination. There is a certain degree of correlation, which is more evident for K173 vaccine, between the level of antibody response during infection and the protective efficacy of vaccination.     

Plasmodium berghei: characterization of antigens and their role in inducing immunity to infection

In vitro, Plasmodium berghei infected erythrocytes incorporated 35S-methionine into 31 polypeptides with molecular weights from 21 kd to 300 kd. Hemoglobin and additional smaller molecular weight polypeptides were labelled with 35S-methionine by a population of uninfected, reticulocyte-rich rat erythrocytes. 3H-glucosamine was incorporated into at least 3 components by Plasmodium berghei infected erythrocytes. Uninfected, reticulocyte-rich rat erythrocytes did not incorporate 3H-glucosamine. Rabbit antisera against small, free plasmodia formed complexes which contained between 12 and 22 of the 31 labelled polypeptides in the 35S-methionine labelled antigen preparation. Rabbit antisera against soluble antigens washed from small, free plasmodia formed complexes containing many of the same labelled plasmodial polypeptides, however the reactions were particularly strong with those components which yielded polypeptides with molecular weights of 25 kd and 31 kd. Rabbit origin antisera against the 2 preparations did not form detectable complexes with the 3H-glucosamine labelled plasmodial components. Sera from rats undergoing progressive P. berghei infection formed complexes containing an increasing number of 35S-methionine labelled plasmodial polypeptides. Hyperimmune rat serum, the only serum protective upon passive transfer into mice, formed complexes containing 7 polypeptides with molecular weights of 35 kd, 75 kd, 80 kd, 92 kd, 100 kd, 150 kd and 190 kd. Antigens containing 1 or more of these polypeptides may be important in the induction of a protective antibody response against the parasite.     

IL-12 is required for antibody-mediated protective immunity against blood-stage Plasmodium chabaudi AS malaria infection in mice.

In this study, we investigated the role of endogenous IL-12 in protective immunity against blood-stage P. chabaudi AS malaria using IL-12 p40 gene knockout (KO) and wild-type (WT) C57BL/6 mice. Following infection, KO mice developed significantly higher levels of primary parasitemia than WT mice and were unable to rapidly resolve primary infection and control challenge infection. Infected KO mice had severely impaired IFN-gamma production in vivo and in vitro by NK cells and splenocytes compared with WT mice. Production of TNF-alpha and IL-4 was not compromised in infected KO mice. KO mice produced significantly lower levels of Th1-dependent IgG2a and IgG3 but a higher level of Th2-dependent IgG1 than WT mice during primary and challenge infections. Treatment of KO mice with murine rIL-12 during the early stage of primary infection corrected the altered IgG2a, IgG3, and IgG1 responses and restored the ability to rapidly resolve primary and control challenge infections. Transfer of immune serum from WT mice to P. chabaudi AS-infected susceptible A/J mice completely protected the recipients, whereas immune serum from KO mice did not, as evidenced by high levels of parasitemia and 100% mortality in recipient mice. Furthermore, depletion of IgG2a from WT immune serum significantly reduced the protective effect of the serum while IgG1 depletion had no significant effect. Taken together, these results demonstrate the protective role of a Th1-immune response during both acute and chronic phases of blood-stage malaria and extend the immunoregulatory role of IL-12 to Ab-mediated immunity against Plasmodium parasites.     

Protection from experimental cerebral malaria with a single dose of radiation-attenuated, blood-stage Plasmodium berghei parasites

Background

Whole malaria parasites are highly effective in inducing immunity against malaria. Due to the limited success of subunit based vaccines in clinical studies, there has been a renewed interest in whole parasite-based malaria vaccines. Apart from attenuated sporozoites, there have also been efforts to use live asexual stage parasites as vaccine immunogens.

Methodology and Results

We used radiation exposure to attenuate the highly virulent asexual blood stages of the murine malaria parasite P. berghei to a non-replicable, avirulent form. We tested the ability of the attenuated blood stage parasites to induce immunity to parasitemia and the symptoms of severe malaria disease. Depending on the mouse genetic background, a single high dose immunization without adjuvant protected mice from parasitemia and severe disease (CD1 mice) or from experimental cerebral malaria (ECM) (C57BL/6 mice). A low dose immunization did not protect against parasitemia or severe disease in either model after one or two immunizations. The protection from ECM was associated with a parasite specific antibody response and also with a lower level of splenic parasite-specific IFN-γ production, which is a mediator of ECM pathology in C57BL/6 mice. Surprisingly, there was no difference in the sequestration of CD8+ T cells and CD45+ CD11b+ macrophages in the brains of immunized, ECM-protected mice.

Conclusions

This report further demonstrates the effectiveness of a whole parasite blood-stage vaccine in inducing immunity to malaria and explicitly demonstrates its effectiveness against ECM, the most pathogenic consequence of malaria infection. This experimental model will be important to explore the formulation of whole parasite blood-stage vaccines against malaria and to investigate the immune mechanisms that mediate protection against parasitemia and cerebral malaria.     

Transfer of immunity to Plasmodium berghei by spleen and lymph node immune RNA.

The RNA from spleens and lymph nodes of Lewis rats immune to Plasmodium berghei protected A/J mice against a lethal challenge of the blood stages of P. berghei, NK65. The RNA was extracted by the hot phenol procedure from freshly removed spleens and lymph nodes. Protection was measured by survival and level of parasitemia as compared to controls. The levels of RNA administered were 10, 50, and 100 μg of RNA. There was observed 100% survival with 50 and 100 μg of immune spleen RNA. The maximum percentage of parasitemia was not reduced below that of the controls in the groups given immune RNA from lymph nodes, but was significantly reduced below that of the controls in the groups given immune RNA from spleens.     

A critical role of Fc receptor-mediated antibody-dependent phagocytosis in the host resistance to blood-stage Plasmodium berghei XAT infection

Plasmodium berghei XAT is an irradiation-induced attenuated variant derived from the lethal strain P. berghei NK65, and its blood-stage parasites are spontaneously cleared in immune competent mice. In the present study, we studied the mechanism of host resistance to blood-stage malaria infection using P. berghei XAT. Infection enhanced Ab-dependent phagocytosis of PRBC by splenic macrophages in wild-type C57BL/6 mice. In contrast, FcR gamma-chain knockout (FcRgamma(-/-)) mice, which lack the ability to mediate Ab-dependent phagocytosis and Ab-dependent cell-mediated cytotoxicity through FcgammaRI, FcgammaRII, and FcgammaRIII, could not induce Ab-dependent phagocytic activity. These FcRgamma(-/-) mice showed increased susceptibility to the P. berghei XAT infection, with eventually fatal results, although they produced comparable amounts of IFN-gamma by spleen cells and anti-XAT Abs in serum. In addition, passive transfer of anti-XAT IgG obtained from wild-type mice that had recovered from infection into FcRgamma(-/-) mice could not suppress the increase in parasitemia, and almost all of these mice died after marked parasitemia. In contrast, passive transfer of anti-XAT IgG into control wild-type mice inhibited the increase in parasitemia. IFN-gamma(-/-) mice, which were highly susceptible to the P. berghei XAT infection, failed to induce Ab-dependent phagocytic activity and also showed reduced production of serum anti-XAT IgG2a isotype compared with control wild-type mice. These results suggest that FcR-mediated Ab-dependent phagocytosis, which is located downstream of IFN-gamma production, is important as an effector mechanism to eliminate PRBC in blood-stage P. berghei XAT infection.     

Innate immune responses to human malaria: heterogeneous cytokine responses to blood-stage Plasmodium falciparum correlate with parasitological and clinical outcomes

Taking advantage of a sporozoite challenge model established to evaluate the efficacy of new malaria vaccine candidates, we have explored the kinetics of systemic cytokine responses during the prepatent period of Plasmodium falciparum infection in 18 unvaccinated, previously malaria-naive subjects, using a highly sensitive, bead-based multiplex assay, and relate these data to peripheral parasite densities as measured by quantitative real-time PCR. These data are complemented with the analysis of cytokine production measured in vitro from whole blood or PBMC, stimulated with P. falciparum-infected RBC. We found considerable qualitative and quantitative interindividual variability in the innate responses, with subjects falling into three groups according to the strength of their inflammatory response. One group secreted moderate levels of IFN-gamma and IL-10, but no detectable IL-12p70. A second group produced detectable levels of circulating IL-12p70 and developed very high levels of IFN-gamma and IL-10. The third group failed to up-regulate any significant proinflammatory responses, but showed the highest levels of TGF-beta. Proinflammatory responses were associated with more rapid control of parasite growth but only at the cost of developing clinical symptoms, suggesting that the initial innate response may have far-reaching consequences on disease outcome. Furthermore, the in vitro observations on cytokine kinetics presented here, suggest that intact schizont-stage infected RBC can trigger innate responses before rupture of the infected RBC.     

The spleen CD4+ T cell response to blood-stage Plasmodium chabaudi malaria develops in two phases characterized by different properties

The pivotal role of spleen CD4(+) T cells in the development of both malaria pathogenesis and protective immunity makes necessary a profound comprehension of the mechanisms involved in their activation and regulation during Plasmodium infection. Herein, we examined in detail the behaviour of non-conventional and conventional splenic CD4(+) T cells during P. chabaudi malaria. We took advantage of the fact that a great proportion of CD4(+) T cells generated in CD1d(-/-) mice are I-A(b)-restricted (conventional cells), while their counterparts in I-A(b-/-) mice are restricted by CD1d and other class IB major histocompatibility complex (MHC) molecules (non-conventional cells). We found that conventional CD4(+) T cells are the main protagonists of the immune response to infection, which develops in two consecutive phases concomitant with acute and chronic parasitaemias. The early phase of the conventional CD4(+) T cell response is intense and short lasting, rapidly providing large amounts of proinflammatory cytokines and helping follicular and marginal zone B cells to secrete polyclonal immunoglobulin. Both TNF-α and IFN-γ production depend mostly on conventional CD4(+) T cells. IFN-γ is produced simultaneously by non-conventional and conventional CD4(+) T cells. The early phase of the response finishes after a week of infection, with the elimination of a large proportion of CD4(+) T cells, which then gives opportunity to the development of acquired immunity. Unexpectedly, the major contribution of CD1d-restricted CD4(+) T cells occurs at the beginning of the second phase of the response, but not earlier, helping both IFN-γ and parasite-specific antibody production. We concluded that conventional CD4(+) T cells have a central role from the onset of P. chabaudi malaria, acting in parallel with non-conventional CD4(+) T cells as a link between innate and acquired immunity. This study contributes to the understanding of malaria immunology and opens a perspective for future studies designed to decipher the molecular mechanisms behind immune responses to Plasmodium infection.     

Plasmodium berghei: malaria infection causes increased cardiac output in rats, Rattus rattus

Thirty-two 4-week-old male Wistar rats were infected with Plasmodium berghei malaria. On Days 12 through 14, blood volume, arterial blood pressure, right ventricular pressure, heart rate, cardiac output, stroke volume, hematocrit, and vascular resistances were determined. All of the cardiovascular parameters measured, with the exception of calculated pulmonary vascular resistance, changed progressively as the peripheral blood parasitemia increased. With a rising parasitemia, cardiac output increased, despite a reduced heart rate. The highest parasitemia of 63% was accompanied by a doubling of the normal cardiac output. The relationship between parasitemia and cardiac output can be described by the equation, cardiac output = (6.14) x % parasitemia + 452 ml/min/kg. The mean arterial blood pressure was lower than controls when parasitemia exceeded 20%, whereas systolic right ventricular pressure was elevated only at the highest parasitemias. When noninfected control rats were compared with those animals having parasitemias greater than 40%, in the infected animals, mean arterial pressure was 28% lower (P less than 0.01) and systolic right ventricular pressure rose by 21% (P less than 0.02). A 50% decline was observed in the total peripheral vascular resistance (P less than 0.01), although the pulmonary resistance was apparently unchanged. With P. berghei infection, there is also a marked anemia, an increase in plasma volume, and a 16% increase in blood volume (% body weight). It is concluded from these results that although the hemodynamic changes previously reported in the literature indicate that infection with malaria may result in focal blockages in microvessels and poor tissue perfusion, the total systemic effect, in the rat, is an increase in cardiac output secondary to a reduced peripheral resistance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell- rather than antibody-mediated immunity leads to the development of profound thrombocytopenia during experimental Plasmodium berghei malaria

Experimental malarial thrombocytopenia can reach life-threatening levels and is believed to be due to Abs targeting platelets for destruction by the reticuloendothelial system. However, we report that Abs account for at most 15% of platelet destruction as Plasmodium berghei-infected B cell-deficient mice exhibited profound thrombocytopenia (83%) as did C57BL/6 controls (98%). Further, no significant increase in Abs bound to intact platelets was observed during infection. P. berghei infection can enhance the activity of anti-platelet Abs as indicated by a significantly (p < 0.005) increased thrombocytopenia on day 4 of infection in mice that were administered a low dose anti-CD41 mAb compared with rat IgG1-injected controls. RAG1-/- and CD4- plus CD8-deficient mice were markedly protected from thrombocytopenia (p < 0.005) and malarial pathogenesis. CD8- or TCRgammadelta-deficient mice were not protected from thrombocytopenia and CD4-deficient mice were modestly protected. RAG1-/- mice exhibited significantly (p < 0.05) lower levels of plasma TNF, IFN-gamma, and IL-12 during infection. IFNgamma-/- and IL-12-/- mice exhibited increased survival but similar thrombocytopenia to C57BL/6 controls. Collectively, these data indicate that thrombocytopenia is necessary but not sufficient for malarial pathogenesis and Abs are not the major contributors to malarial thrombocytopenia. Rather, we propose that both CD4+ and CD8+ T cell populations play key roles in malarial thrombocytopenia; a complex bidirectional interaction between cell-mediated immunity and platelets exists during experimental severe malaria that regulates both responses.