Cytokine production in lethal and non-lethal murine malaria

Levels of IFN-gamma, IL-2 and IL-4 were measured in vitro during the course of non-lethal Plasmodium chabaudi adami and lethal P. chabaudi strain 1309 infections in BALB/cByJ mice. Spleen cells from mice infected with the non-lethal Plasmodium had a higher initial response to P. chabaudi antigens than mice infected with P. chabaudi strain 1309, as determined by measuring all three lymphokines. We conclude that both Th1 and Th2 subsets of T helper lymphocytes are activated during P. chabaudi adami infection but that these responses are suppressed in mice infected with the more virulent P. chabaudi strain 1309.     

Effects of splenectomy on antibody-independent immunity to Plasmodium chabaudi adami malaria.

Splenectomy of B-cell-deficient mice and immunologically intact mice before infection with Plasmodium chabaudi adami led to the development of significant parasitemias which eventually resolved in the latter mice. Whereas both eusplenic B-cell-deficient mice and immunologically intact mice resolved their acute P. chabaudi adami infection, only B-cell-deficient mice subsequently developed chronic low-grade malaria. Splenectomy of B-cell-deficient mice with chronic malaria led to recrudescing infections, suggesting that the expression of antibody-independent immunity to reinfection was spleen dependent. When dispersed spleen cells were injected into splenectomized mice before challenge with P. chabaudi adami, the kinetics of the resulting infection resembled that seen in splenectomized mice which had not been grafted with normal spleen cells. This finding indicates that immunity to P. chabaudi adami requires the presence of an architecturally intact spleen.     

T-cell-dependent immunity and thrombocytopenia in rats infected with Plasmodium chabaudi.

Normal, splenectomized, and athymic Fischer rats were infected with Plasmodium chabaudi. In normal rat infections, acute-phase infection resolved rapidly and completely. In splenectomized rats, infection resulted in high parasitemia and ultimately death. In nude rats, parasite growth was reduced compared with normal rats, and a persistent parasitemia (between 20 and 45%) was observed for several months. Complete resolution of the infection was achieved after adoptive transfer of T lymphocytes, even when transfer occurred during the course of infection. These results indicated that an acquired, T-lymphocyte-dependent immunity was necessary for the complete recovery observed in normal rats. In normal rats, thrombocytopenia and splenomegaly occurred during infection. By contrast, in nude rats, both of these pathological manifestations were only observed after thymus grafting. Thrombocytopenia was also absent in the splenectomized animals. Despite an increase in platelet-associated immunoglobulin levels during the infection, thrombocytopenia was not transferred by injection of infected rat serum to normal recipients. It has been concluded that the nude rat infection can be regarded as a novel and useful model for studying the T-cell-dependent effector and pathological mechanisms and to investigate the anti-P. chabaudi immune response.     

Humoral and cell-mediated immune mechanisms in the production of pathology in avirulent Semliki Forest virus encephalitis

Seven days after peripheral inoculation with an avirulent strain of Semliki Forest virus, the brains of CBA and nude mice exhibited a mononuclear inflammation and spongiform degeneration. Mice that had received cyclophosphamide (150 mg/kg) 24 h after infection showed no pathology until day 11. However, immunofluorescence studies of the brains of immunosuppressed, infected mice demonstrated viral antigen within the soma and processes of neurons at earlier periods. The brain lesions could be reconstituted on day 7 in immunosuppressed, infected recipients with 6-day immune spleen cells. Immune spleen cells depleted of T lymphocytes, the non-immunoglobulin-bearing population deficient in B lymphocytes, or immune sera plus nonimmune bone marrow cells could also reconstitute the lesions. However, inflammation and spongiform changes were reduced when donor immune cells were depleted of either T or B lymphocytes. When both T and B lymphocytes were removed from the donor immune population, recipient brains did not show pathology. The results demonstrate that either antibody or immune T cells can trigger pathology, but there is also participation of nonimmune bone marrow-derived mononuclear cells, probably of the monocyte-macrophage lineage.     

Differential requirements for an intact spleen in induction and expression of B-cell-dependent immunity to Plasmodium chabaudi AS.

The requirement for an architecturally intact spleen in the afferent and efferent arms of immunity to the murine malaria parasite Plasmodium chabaudi AS was analyzed. C57BL/6 mice with intact spleens develop a single, patent parasitemia and resolve the infection. In contrast, surgically splenectomized mice experience persistent waves of patent parasitemia interrupted briefly by periods of parasitologic crises. Transfer of spleen cells from immune donors, but not transfer of spleen cells from normal mice, into splenectomized mice enabled the recipients to resolve the infection similar to mice with intact spleens. B-cell depletion, but not T-cell depletion, of spleen cells prior to transfer abrogated the ability of splenectomized recipients to resolve the infection. Compared with mice with intact spleens, splenectomized mice exhibited a delayed antibody response whereas all groups of immune cell recipients had an accelerated antibody response. Nevertheless, splenectomized mice and recipients of B-cell-depleted cells failed to resolve infections, despite the development of high-titer antibodies late during the course of infection. Analysis of immunoglobulin G isotype responses showed a lower representation of immunoglobulin G2a in mice which failed to resolve infections. The latter mice had characteristic histopathologic changes in the liver. These observations indicate a unique role of the splenic microenvironment for the induction and development of an effective B-cell-dependent response against malarial parasites.     

Resolution of acute malarial infections by T cell-dependent non-antibody-mediated mechanisms of immunity.

While it is generally accepted that acute blood stage malarial infections are resolved through the actions of protective antibodies, we observed that resistance to acute infection with Plasmodium chabaudi adami was mediated by T cell-dependent cellular immune mechanisms independent of antibody. We now report that acute blood stage infections caused by three additional murine hemoprotozoan parasites, Plasmodium vinckei petteri, Plasmodium chabaudi chabaudi, and Babesia microti, appear to be controlled by similar T cell-dependent mechanisms of immunity. Mice rendered B cell deficient by lifelong treatment with goat anti-mouse immunoglobulin M (IgM) had IgM levels in serum of less than 0.6 micrograms/ml and contained precipitating amounts of goat anti-mouse IgM. When these B cell-deficient mice were infected with blood stage P. vinckei petteri, P. chabaudi chabaudi, or B. microti, they resolved their infections with kinetics similar to those seen in immunologically intact mice. Infected B cell-deficient mice did not produce antiparasite antibodies. As assayed by immunofluorescence, significant titers of parasite-specific antibody were present only in the sera of infected immunocompetent mice. In addition, only sera from infected immunocompetent mice immunoprecipitated metabolically labeled parasite antigens. In contrast to B cell-deficient mice, athymic nude mice failed to resolve acute P. vinckei petteri or B. microti infections. These data suggest that antibody-independent, T cell-mediated immune mechanisms play a more significant role in resisting acute blood stage infections caused by hemoprotozoa than was recognized previously.     

CD4+ T cells and B cells are necessary for the transfer of protective immunity to Plasmodium chabaudi chabaudi.

It is shown here that B cells, in addition to CD4+ T cells, are necessary for the development of protective immunity to Plasmodium chabaudi chabaudi (P. chabaudi) in mice. Reconstitution of severe combined immunodeficient (SCID) mice with immune or normal CD4+ T cells protected the majority of mice against an otherwise lethal challenge but the mice were unable to clear their parasitemias. By contrast, transfer of the same T cell populations into athymic nu/nu mice enabled the recipients to control and clear their infections, immune CD4+ T cells being most effective. Furthermore, SCID mice given CD4+ T cells from immune and normal donors simultaneously with immune B cells also could eliminate their infection. Clearance of parasitemia correlated with the presence of malaria-specific antibodies in the serum. The role of B cells and CD4+ T cells in the protective immune response to P. chabaudi is discussed.     

Cellular mechanisms in the immune response to malaria in Plasmodium vinckei-infected mice.

Infection of mice with the malaria parasite Plasmodium vinckei vinckei is 100% lethal. However, after two infections followed by drug cure, BALB/c mice develop a solid immunity which is antibody independent but mediated by CD4+ T cells. To elucidate the mechanisms of this immunity, spleen cells from immune mice were challenged in vitro with lysates of P. vinckei-infected or uninfected erythrocytes. The parasite antigen induced proliferation of T cells from immune mice but not from nonimmune mice. When gamma interferon production by cells from immune mice was assayed at the single-cell level, 1 to 3 cells per 1,000 cells were found to release this cytokine when exposed to antigen. In contrast, the numbers of interleukin 4 (IL-4)-producing cells from both immune and control mice were < or = 4 per 10(6) cells, regardless of antigen exposure. Investigation in a bioassay showed that P. vinckei antigen induced the release of IL-4 from spleen cells of immune mice but not from those of control mice. Nevertheless, that IL-4 is of minor significance in this system is also suggested by the absence of elevation of immunoglobulin E levels in blood samples from these mice, in contrast to what is seen with P. chabaudi infection, in which IL-4-producing Th2 cells are of major importance for immunity during later phases of infection. Taken together, the present results indicate that immunity to P. vinckei is a Th1 response, with gamma interferon being an important protective factor. Whether or not the Th1 response, through overproduction of tumor necrosis factor alpha, is also responsible for pathology and death in this infection remains to be clarified.     

CD28 costimulation is required for the expression of T-cell-dependent cell-mediated immunity against blood-stage Plasmodium chabaudi malaria parasites

Mice suppress the parasitemia of acute blood-stage Plasmodium chabaudi malaria by an antibody- or T-cell-dependent cell-mediated mechanism of immunity (AMI and CMI, respectively) or by both mechanisms. To determine whether CD28 costimulation is required for expression of these polar immune responses, we first compared the time courses of P. chabaudi malaria in CD28-deficient (CD28(-/-)) and CD28-intact (CD28(+/+)) mice. Acute infections in both knockout (KO) and control mice followed similar time courses, with the period of descending parasitemia being prolonged approximately 2 weeks in KO mice followed by intermittent low-grade chronic parasitemia. Infected CD28(-/-) mice produced primarily the immunoglobulin M antibody, which upon passive transfer provided partial protection against P. chabaudi challenge, suggesting that the elimination of blood-stage parasites by CD28(-/-) mice was achieved by AMI. To determine whether CD28(-/-) costimulation is required for the expression of CMI against the parasite, we compared the time courses of parasitemia in B-cell-deficient double-KO (J(H)(-/-) x CD28(-/-)) mice and control (J(H)(-/-) x CD28(+/+)) mice. Whereas control mice suppressed parasitemia to subpatent levels within approximately 2 weeks postinoculation, double-KO mice developed high levels of parasitemia of long-lasting duration. Although not required for the suppression of acute P. chabaudi parasitemia by AMI, CD28 costimulation is essential for the elimination of blood-stage parasites by CMI.     

Proteins with molecular masses of 25 to 40 kilodaltons elicit optimal protective responses against Plasmodium chabaudi adami infection.

The presence of the CD4+ T cell has been shown to be crucial for resolution of acute infection in the Plasmodium chabaudi adami murine malaria model. This model is, therefore, suitable for the isolation of malaria antigens that are capable of activating protective T cells. In light of this, we set out to identify P. chabaudi adami molecules that activate protective responses in this model. Denatured P. chabaudi adami proteins were isolated by continuous-flow electrophoresis on the basis of their apparent molecular masses and then sequentially assessed for the ability to protect mice in immunization experiments. We report here that low-molecular-mass P. chabaudi adami polypeptides in the range from 25 to 40 kDa are most effective at immunizing mice against a challenge infection with viable P. chabaudi adami. The method used to obtain these proteins could also be applied to identify molecules that activate protective cell-mediated responses in other infectious disease models.     

Exacerbation of Plasmodium chabaudi malaria in mice by depletion of TCR alpha beta+ T cells, but not TCR gamma delta+ T cells.

Although gamma delta T cells are found in increased numbers in the spleens of humans and mice infected with malaria, it is not known if these cells are necessary components of an effective immune response. The surface phenotype of spleen cells obtained from mice infected with avirulent Plasmodium chabaudi adami or virulent Plasmodium chabaudi chabaudi were examined using anti-delta or anti-alpha beta T-cell-specific reagents and flow cytometry. Levels of parasitaemia, red blood cell (RBC) counts, and survival times were followed in mice depleted of tumour necrosis factor (TCR)gamma delta+ or TCR alpha beta+ T cells. Numbers of gamma delta T cells increased in the spleens of control antibody-treated infected mice, but not in mice depleted of TCR gamma delta+ or TCR alpha beta+ T cells. Mice depleted of gamma delta T cells had levels of parasitaemia, RBCs, and survival rates similar to control antibody-treated mice. However, mice depleted of TCR alpha beta+ T cells had higher levels of parasitaemia, lower RBC counts, and decreased survival rates. These results indicate that TCR alpha beta+ but not TCR gamma delta+ T cells play an essential role in host defense against P. chabaudi infection in mice.     

CD4+ and CD8+ T lymphocytes both contribute to acquired immunity to blood-stage Plasmodium chabaudi AS.

In the present study, the contribution of CD4+ and CD8+ T lymphocytes to acquired immunity to blood-stage infection with the murine malaria species Plasmodium chabaudi AS was investigated. C57BL/6 mice, which are genetically resistant to infection with this hemoprotozoan parasite and exhibit a transient course of infection, were treated intraperitoneally with monoclonal antibodies to T-cell epitopes, either anti-Thy-1, anti-CD4, or anti-CD8. After intraperitoneal infection with 10(6) parasitized erythrocytes, control C57BL/6 mice exhibited a peak parasitemia on day 9 of approximately 35% parasitized erythrocytes and eliminated the infection within 4 weeks. Mice depleted of Thy-1+ or CD4+ T cells had significantly higher parasitemias on day 7 as well as significantly higher peak parasitemias. These mice were unable to control the infection and developed a persistent, high parasitemia that fluctuated between 40 and 60% until the experiment was terminated on day 56 postinfection. Depletion of CD8+ T lymphocytes was found to have no effect on the early course of parasitemia or on the level of peak parasitemia. However, mice depleted of CD8+ T cells experienced two recurrent bouts of parasitemia during the later stage of the infection and required more than 5 weeks to eliminate the parasites. After the peak parasitemia, which occurred in control and experimental animals on day 9, there was a sharp drop in parasitemia coinciding with a wave of reticulocytosis. Therefore, the contribution of the influx of reticulocytes, which are not the preferred host cell of this hemoprotozoan parasite, to limiting the parasitemia was also examined by determining the course of reticulocytosis during infection in control and T cell-depleted animals. Early in infection, there was a marked and comparable reticulocytosis in the peripheral blood of control and T cell-depleted mice; the reticulocytosis peaked on day 12 and coincided with the dramatic and sudden reduction in parasitemia occurring in all groups. In both control and CD8-depleted mice the percentage of reticulocytes decreased as the infection was resolved, whereas in CD4-depleted mice marked reticulocytosis correlated with high, persistent parasitemia. These results thus demonstrate that both CD4+ and CD8+ T cells are involved in acquired immunity to blood-stage P. chabaudi AS and that the influx of reticulocytes into the blood that occurs just after the peak parasitemia may contribute temporarily to limiting the parasitemia.     

Reconstitution of B-cell-depleted mice with B cells restores Th2-type immune responses during Plasmodium chabaudi chabaudi infection.

In mice depleted of B cells from birth by treatment with anti-immunoglobulin M(mu) antibodies, progression from a Th1- to a Th2-regulated immune response during primary infection with Plasmodium chabaudi chabaudi fails to occur. While Th1-type immunity limits parasitemia, in the absence of B cells, chronic low-grade infections persist. Here, we show that reconstituting immune, and to a lesser extent naive, B cells to mice rendered deficient in B-cell function through anti-immunoglobulin M(mu) pretreatment restores the CD4+ T-cell response to the Th2 type later in P. c. chabaudi infection and with it the capacity to eliminate infection. This finding provides clear evidence that B cells are required for switching the balance of immune regulation between CD4+ T cells from Th1 to Th2 during P.c. chabaudi infection and supports the concept that B cells, through antibody production, are needed for effective antimalarial immunity.     

Murine malaria infection induces fetal loss associated with accumulation of Plasmodium chabaudi AS-infected erythrocytes in the placenta

Malarial infection in nonimmune women is a risk factor for pregnancy loss, but the role that maternal antimalarial immune responses play in fetal compromise is not clear. We conducted longitudinal and serial sacrifice studies to examine the pathogenesis of malaria during pregnancy using the Plasmodium chabaudi AS/C57BL/6 mouse model. Peak parasitemia following inoculation with 1,000 parasite-infected murine erythrocytes and survival were similar in infected pregnant and nonpregnant mice, although development of parasitemia and anemia was slightly accelerated in pregnant mice. Importantly, pregnant mice failed to maintain viable pregnancies, most aborting before day 12 of gestation. At abortion, maternal placental blood parasitemia was statistically significantly higher than peripheral parasitemia. Infected mice had similar increases in spleen size and cellularity which were statistically significantly higher than in uninfected mice. In contrast, splenocyte proliferation in response to mitogenic stimulation around peak parasitemia was statistically significantly reduced in both groups of infected mice compared to uninfected, nonpregnant mice, suggesting that lymphoproliferation is not a good indicator of the antimalarial immune responses in pregnant or nonpregnant animals. This study suggests that while pregnant and nonpregnant C57BL/6 mice are equally capable of mounting an effective immune response to and surviving P. chabaudi AS infection, pregnant mice cannot produce viable pups. Fetal loss appears to be associated with placental accumulation of infected erythrocytes. Further study is required to determine to what extent maternal antimalarial immune responses, anemia, and placental accumulation of parasites contribute to compromised pregnancy in this model.     

Adoptive transfer of immunity to Nocardia asteroides in nude mice.

Nude mice on a BALB/c background were adoptively transferred with unprimed spleen cells, Nocardia-primed spleen cells, or Nocardia-primed splenic T lymphocytes from syngeneic, heterozygous (nu/+) littermates. Two days later, these recipient mice and unmanipulated (control) nude mice were infected intravenously with a 50% lethal dose of Nocardia asteroides GUH-2 from an early stationary-phase culture. Antibody titers, spleen weights, percent mortality, and organ clearance of the microorganisms were measured at 3 h to 28 days after infection. Adoptively transferred nude mice had larger spleens and greater titers of anti-nocardial antibody 7 to 28 days after infection as compared with control nude mice. Adoptive transfer with either primed spleen cells or primed splenic T lymphocytes enhanced both the survival of recipient nude mice and their ability to eliminate N. asteroides from the liver and spleen. These data indicate that adoptive immunity to infection with N. asteroides can be transferred with either specifically primed spleen cells or splenic T lymphocytes. Thus, it appears that cell-mediated immunity and T lymphocytes are of uppermost importance in host resistance to nocardial infection.     

The A/T-specific DNA alkylating agent adozelesin inhibits Plasmodium falciparum growth in vitro and protects mice against Plasmodium chabaudi adami infection

There is an urgent need for new anti-malarial drugs to combat the resurgence of resistance to current therapies. To exploit the A/T richness of malaria DNA as a potential target for anti-malarial drugs we tested an A/T-specific DNA synthesis inhibitor, adozelesin, for activity against Plasmodium falciparum in vitro and Plasmodium chabaudi adami in mice. Adozelesin is a DNA alkylating agent that exhibits specificity for the motif A/T, A/T and A. In P. falciparum 3D7 cultures, adozelesin acts as a powerful inhibitor of parasite growth (IC(50) of 70 pM) and is equally potent at killing the drug-resistant strains FCR3 and 7G8. Using a real-time PCR assay, we show that treatment with adozelesin in vitro results in damage of P. falciparum genomic DNA. In synchronized cultures, adozelesin exhibits a concentration-dependent effect on parasitemia and on the development of parasites through the asexual cycle. In asynchronous cultures, parasites arrest at all stages of the asexual cycle suggesting that adozelesin exerts other anti-parasitic effects in addition to inhibiting DNA replication. These anti-parasite effects are irreversible since cultures exposed to adozelesin for more than 6h fail to recover upon removal of the drug. Furthermore, adozelesin is very effective at suppressing malaria infection in vivo; growth of P. c. adami DK in mice was highly impaired by a single injection of adozelesin (25 microg/kg) at 4 days post-infection. These results demonstrate that adozelesin irreversibly blocks parasite growth in vitro and suppresses parasite infection in vivo, suggesting that A/T-specific DNA damaging agents represent a new class of compounds with potential as anti-malarials.     

Cloned T cells provide help for malaria-specific polyclonal antibody responses

Athymic mice grafted with antigen-reactive cloned T cells and challenged with Plasmodium chabaudi adami produced antibodies against multiple parasite antigens. Antibody reactivities were similar to those seen in infected euthymic mice and contrasted with their absence in infected athymic mice. These results suggest that it will not be possible to determine the antigenic specificity of clonal T cell populations in malarial infections by their capacity to provide help to restricted populations of B lymphocytes.     

Central role of endogenous gamma interferon in protective immunity against blood-stage Plasmodium chabaudi AS infection

The role of endogenous gamma interferon (IFN-gamma) in protective immunity against blood-stage Plasmodium chabaudi AS malaria was studied using IFN-gamma gene knockout (GKO) and wild-type (WT) C57BL/6 mice. Following infection with 10(6) parasitized erythrocytes, GKO mice developed significantly higher parasitemia during acute infection than WT mice and had severe mortality. In infected GKO mice, production of interleukin 12 (IL-12) p70 and tumor necrosis factor alpha in vivo and IL-12 p70 in vitro by splenic macrophages was significantly reduced compared to that in WT mice and the enhanced nitric oxide (NO) production observed in infected WT mice was completely absent. WT and GKO mice had comparable numbers of total nucleated spleen cells and B220(+) and Mac-1(+) spleen cells both before and after infection. Infected WT mice, however, had significantly more F4/80(+), NK1.1(+), and F4/80(+)Ia(+) spleen cells than infected GKO mice; male WT had more CD3(+) cells than male GKO mice. In comparison with those from WT mice, splenocytes from infected GKO mice had significantly higher proliferation in vitro in response to parasite antigen or concanavalin A stimulation and produced significantly higher levels of IL-10 in response to parasite antigen. Infected WT mice produced more parasite-specific immunoglobulin M (IgM), IgG2a, and IgG3 and less IgG1 than GKO mice. Significant gender differences in both GKO and WT mice in peak parasitemia levels, mortality, phenotypes of spleen cells, and proliferation of and cytokine production by splenocytes in vitro were apparent during infection. These results thus provide unequivocal evidence for the central role of endogenous IFN-gamma in the development of protective immunity against blood-stage P. chabaudi AS.     

Roles of CD4- and CD8-bearing T lymphocytes in the immune response to the erythrocytic stages of Plasmodium chabaudi.

The possible role of CD4- and CD8-bearing T lymphocytes in parasite clearance in vivo was investigated, using Plasmodium chabaudi in C57BL/6 mice as a model. Monoclonal antibodies specific for the CD4 and CD8 molecules were administered in vivo to deplete selectively the appropriate subset of T cells. The efficacy of depletion was ascertained by flow cytometry and functional studies. These mice were then infected with P. chabaudi, and the course of infection was followed. The control groups had maximum parasitemias of approximately 30% 10 days after infection, and the infection was cleared within 27 days. Mice without CD4+ cells had significantly higher parasitemias which they were unable to reduce below 20% for the duration of the experiment. Mice without CD8 cells had slightly higher parasitemias which were cleared after 34 days. Because of the possibility that CD8+ cells alone could not be activated in the absence of growth factors, exogenous interleukin-2 was administered to the mice depleted of CD4 cells. This did not significantly affect parasitemias, and the mice were still unable to clear their infections. The data suggest that CD4+ T cells play a crucial role in the protective immune response to the erythrocytic stages of P. chabaudi.