Deficiency in tumor necrosis factor alpha activity does not impair early protective Th1 responses against blood-stage malaria

Blood-stage Plasmodium chabaudi AS infection was controlled by 4 weeks in mice with deletion of tumor necrosis factor p55 and p75 receptors (TNFR-knockout [KO]) and control wild-type (WT) mice, although female TNFR-KO mice showed slightly but significantly higher parasitemia immediately following the peak. Serum interleukin 12 (IL-12) p70 and gamma interferon (IFN-gamma) levels were similar but tumor necrosis factor alpha levels were significantly higher in TNFR-KO mice than in WT controls. Splenic IL-12 receptor beta1 and beta2 and IFN-gamma mRNA expression, as well as spleen cell production of IFN-gamma and IL-4, were comparable in both mouse types, but IL-10 production was significantly higher in cells from TNFR-KO mice than in cells from WT mice. Lipopolysaccharide-induced NO secretion by splenic macrophages in vitro was significantly reduced but systemic NO3- levels were similar in infected TNFR-KO and WT mice.     

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.     

Interferon-gamma and tumor necrosis factor-alpha determine resistance to Paracoccidioides brasiliensis infection in mice

To investigate the role of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in the resistance to Paracoccidioides brasiliensis (Pb) infection, mice with homologous disruption of the IFN-gamma (GKO) or TNF-alpha receptor p55 (p55KO) were infected with the parasite. GKO and p55KO, but not wild-type (WT) mice, were unable to control the growth of yeast cells and the mice succumbed to infection by days 16 and 90 after infection, respectively. Typical inflammatory granulomas were found only in WT mice. In contrast, knockout mice presented an inflammatory infiltrate composed of a few neutrophils, mononuclear, epithelioid, and multinuclear giant cells forming incipient granulomas in GKO mice and without granuloma formation in p55KO mice. Besides, both groups of knockout mice exhibited elevated numbers of yeast forms in agreement with colony-forming unit counts in organs. Compared with WT, splenocytes from infected GKO mice cultured with the Pb F1 fraction produced lower TNF-alpha levels, whereas leukocytes from infected p55KO mice produced similar amounts of TNF-alpha but higher levels of IFN-gamma. Moreover, splenocytes from infected WT mice produced higher levels of nitric oxide (NO) resulting in a lower T-cell proliferative response to Con A than uninfected WT, or infected p55KO and GKO mice. On the contrary, the addition of IFN-gamma to splenocytes from infected GKO mice resulted in higher NO production and lower T cell proliferation. Taken together, these findings suggests that endogenous TNF-alpha, acting through the p55 receptor, and IFN-gamma mediate resistance to Pb infection and induce NO production that determines marked T cell unresponsiveness.     

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.

We investigated whether gamma interferon (IFN-gamma; a Th1 cytokine), tumor necrosis factor alpha (TNF-alpha), and interleukin-4 (IL-4; a Th2 cytokine) modulate nitric oxide (NO) production in vivo during blood stage infection with Plasmodium chabaudi AS. Treatment of resistant C57BL/6 mice, which resolve infection with P. chabaudi AS and produce increased levels of IFN-gamma, TNF-alpha, and NO early during infection, with anti-IFN- gamma plus anti-TNF-alpha monoclonal antibodies (MAbs) resulted in a reduction of both splenic inducible NO synthase mRNA and serum NO3- levels by 50 and 100%, respectively. Treatment with the anti-TNF-alpha MAb alone reduced only serum NO3- levels by 35%, and treatment with the anti-IFN-gamma MAb alone had no effect on NO production by these mice during infection. Susceptible A/J mice, which succumb to infection with P. chabaudi AS and produce increased levels of IL-4 but low levels of IFN-gamma, TNF-alpha, and NO early during infection, were treated with an anti-IL-4 MAb. The latter treatment had no effect on NO production by this mouse strain during infection. In addition, our results also demonstrate that treatment of resistant C57BL/6 mice with anti-IFN-gamma plus anti-TNF-alpha MAbs affects, in addition to NO production, other traits of resistance to P. chabaudi AS malaria such as the peak level of parasitemia and the development of splenomegaly. Furthermore, the change in spleen weight was shown to be an IFN-gamma-independent effect of TNF-alpha. Treatment of susceptible A/J mice during infection with an anti IL-4 MAb had no effect on these markers of resistance. Thus, these results demonstrate that TNF-alpha and IFN-gamma are critical in the regulation of NO production and other traits of resistance during P. chabaudi AS malaria in C57BL/6 mice. These data also indicate that treatment with an anti-IL-4 antibody alone is not able to induce NO production or confer resistance to A/J mice against P. chabaudi AS malaria.     

Impaired macrophage responses may contribute to exacerbation of blood-stage Plasmodium chabaudi chabaudi malaria in interleukin-12-deficient mice.

Aiming to clarify the role of endogenous interleukin-12 (IL-12) in protective immunity against blood stages of Plasmodium chabaudi chabaudi (AS), we evaluated the course of infection in IL-12p40 gene knockout (IL-12p40KO) and wild-type (WT) C57BL/6 mice, focusing (1) on the ability of T cells to develop adequate type 1 responses and (2) on the potentiality of macrophages to respond to parasites, interferon-gamma (IFN-gamma), or both. We observed that IL-12p40KO mice develop significantly higher parasitemias during the acute infection, although mice from both groups clear the parasites within a month and similarly eliminate a secondary challenge. Thus, fully protective immunity to P. c. chabaudi can be generated in the absence of IL-12. However, this cytokine may promote parasite control during the early phase of infection. The increased acute parasitemia of IL-12p40KO mice was associated with both impaired IFN-gamma and nitric oxide (NO) response by spleen cells. Because stimulation with recombinant IFN-gamma (rIFN-gamma) failed to improve the NO response in IL-12p40KO macrophages, we investigated whether these cells have an intrinsic defect. Analysis of peritoneal macrophages revealed that IL-12p40KO cells produce higher levels of transforming growth factor-beta1 (TGF-beta1) compared with WT cells and respond to infected erythrocytes or rIFN-gamma by releasing little NO. Moreover, IL-12p40KO macrophages had a severely impaired ability to internalize opsonized infected erythrocytes, suggesting that the low effector profile assumed by these cells may compromise antibody-mediated immunity. Taken together, our results support the idea that the absence of IL-12p40 not only affects IFN-gamma production but also has deep consequences in macrophage effector functions that may contribute to exacerbation of the early phase of P. c. chabaudi malaria.     

Role of endogenous gamma interferon in host response to infection with blood-stage Plasmodium chabaudi AS.

The role of gamma interferon (IFN-gamma), a pluripotent lymphokine capable of activating macrophages, in acquired immunity to blood-stage malaria was investigated. C57BL-derived, lipopolysaccharide-resistant C57BL/10ScN mice, which were found to be resistant to intraperitoneal (i.p.) infection with 10(6) Plasmodium chabaudi AS parasitized erythrocytes, were treated with monoclonal anti-IFN-gamma antibody (MAb). Two MAbs were used: R4-6A2, a rat anti-mouse, neutralizing immunoglobulin G1, which was prepared against natural murine IFN-gamma, and DB-1, a murine anti-rat immunoglobulin G1 prepared against recombinant rat IFN-gamma, which can neutralize the murine molecule as well as the rat molecule. C57BL/10ScNH mice were injected i.p. with 200 micrograms of R4-6A2 1 day before infection and every 3 days through day 21. Control mice were treated with normal rat serum. In separate experiments, DB-1 (1.0 mg per week for 4 weeks) was administered i.p. to C57BL/10ScNH mice beginning on the day of infection; control mice were untreated. Control and MAb-treated mice were infected i.p. with 10(6) P. chabaudi AS parasitized erythrocytes, and the course and outcome of infection were determined. Control mice exhibited a course of infection that was characterized by a peak parasitemia between 30 and 40% parasitized erythrocytes and elimination of the parasite by 4 weeks. MAb-treated mice exhibited a significantly greater parasitemia 1 to 2 days before the peak parasitemia as well as a significantly greater peak parasitemia but also completely cleared the infection by 4 weeks. Thus, these results suggest that treatment with anti-IFN-gamma MAb impairs but does not completely abrogate host resistance to P. chabaudi AS. We also examined the kinetics of IFN-gamma production by spleen cells cultured in vitro with malaria antigen or concanavalin A. Spleen cells were recovered from individual C57BL/6 mice at various times after i.p. infection with 10(6) P. chabaudi AS parasitized erythrocytes. The amount of IFN-gamma produced was quantitated by enzyme-linked immunosorbent assay. In each case, the peak of IFN-gamma production occurred just before the peak parasitemia, followed by a decrease to little or no IFN-gamma production through 42 days postinfection. There was thus a parallel between the kinetics of production of IFN-gamma in vitro by spleen cells from infected animals and the requirement in vivo for the endogenous molecule just before and at the time of peak parasitemia. In conclusion, these results suggest that IFN-gamma-dependent and -independent mechanisms contribute to host resistance to P. chabaudi AS.     

Involvement of IFN-gamma receptor-medicated signaling in pathology and anti-malarial immunity induced by Plasmodium berghei infection

IFN-gamma has been implicated in the pathogenesis of experimental cerebral malaria (ECM). We have used mice lacking the alpha chain of the IFN-gamma receptor (KO mice) to define its role in the pathogenesis of ECM. Infected KO mice did not develop ECM and showed no leukocyte or parasite sequestration in the brain, and no hemorrhages. The resistance of KO mice to ECM was associated with the absence of any increases of TNF-alpha and ICAM-1 proteins in the brain, which are both essential for ECM. Wild-type (WT) mice which do not develop ECM, despite increased local production of TNF-alpha protein, showed no leukocyte accumulation in the brain and this was correlated with the absence of ICAM-1 protein from brain microvessels. KO mice infected with 106 parasitized erythrocytes (PE) of Plasmodium berghei ANKA (PbA) did not develop ECM, but they had high parasitemia and died earlier than WT mice which did not develop ECM. However, KO mice did not develop higher parasitemia than WT mice when both groups were infected with a lower dose (5x10(5) PE) of PbA-infected red blood cells. This indicates that different doses of PE may trigger different IFN-gamma responses and that there may be a threshold concentration for protection against parasitemia.     

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.     

Intrapulmonary response to Histoplasma capsulatum in gamma interferon knockout mice.

We examined the immunobiological responses to Histoplasma capsulatum in lungs of gamma interferon (IFN-gamma) knockout mice (GKO mice). Naive GKO mice succumbed by day 9 to intranasal challenge with 2.5 x 10(6) yeasts, whereas all wild-type (WT) mice survived for 45 days. Compared to lungs of WT mice, the lungs of acutely infected GKO mice exhibited dramatically elevated numbers of CFU in lungs and significantly higher levels of tumor necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) but not interleukin-12 (IL-12) or IL-4. To determine if IFN-gamma is necessary in reexposure histoplasmosis, GKO and WT mice were inoculated with 10(4) yeasts intranasally and given amphotericin B for 3 weeks. Six weeks later, mice were rechallenged with 2.5 x 10(6) yeasts. All GKO mice died by day 6, whereas all WT mice survived for 45 days. Lungs of GKO mice contained substantially elevated numbers of CFU and higher TNF-alpha and GM-CSF levels but not IL-12 or IL-4. Thus, IFN-gamma is requisite for control of pulmonary histoplasmosis in naive and reexposed mice.     

Role of interferon-gamma and tumor necrosis factor in host resistance to Plasmodium chabaudi AS

The contribution of the T cell- and macrophage-derived cytokines, interferon-gamma (IFN-gamma) and tumor necrosis factor (TNF), respectively, in the cell-mediated mechanisms leading to acquired immunity to blood-stage Plasmodium chabaudi AS was investigated. To examine the contribution of IFN-gamma, resistant C57BL-derived mice were treated during infection with two different neutralizing, anti-murine IFN-gamma mAbs. Such treatment impaired the ability of the host to limit parasite multiplication just before and at the time of the peak parasitemia but did not abrogate the development of acquired immunity resulting in control and elimination of acute infection. The requirement of endogenous IFN-gamma around the time of the peak parasitemia was confirmed by quantification of IFN-gamma production in vitro by spleen cells from infected animals in response to malaria antigen. To investigate the role of TNF, resistant C57BL/6 and susceptible A/J mice were treated with rTNF during P. chabaudi AS infection. Treatment with 10(3) or 10(5) U rTNF resulted in increased resistance in A/J hosts (that is, increased survival and a less severe course of infection); there was no difference between control and treated C57BL/6 mice in the course of infection but there was increased mortality among the animals treated with rTNF. Splenic macrophages harvested from C57BL/6 mice during infection were found to produce high levels of TNF from day 3 to day 28 post-infection. In conclusion, both IFN-gamma and TNF appear to contribute to host resistance to blood-stage infection with P. chabaudi AS.     

Impairment of protective immunity to blood-stage malaria by concurrent nematode infection

Helminthiases, which are highly prevalent in areas where malaria is endemic, have been shown to modulate or suppress the immune response to unrelated antigens or pathogens. In this study, we established a murine model of coinfection with a gastrointestinal nematode parasite, Heligmosomoides polygyrus, and the blood-stage malaria parasite Plasmodium chabaudi AS in order to investigate the modulation of antimalarial immunity by concurrent nematode infection. Chronic infection with the nematode for 2, 3, or 5 weeks before P. chabaudi AS infection severely impaired the ability of C57BL/6 mice to control malaria, as demonstrated by severe mortality and significantly increased malaria peak parasitemia levels. Coinfected mice produced significantly lower levels of gamma interferon (IFN-gamma) during P. chabaudi AS infection than mice infected with malaria alone. Concurrent nematode infection also suppressed production of type 1-associated, malaria-specific immunoglobulin G2a. Mice either infected with the nematode alone or coinfected with the nematode and malaria had high transforming growth factor beta1 (TGF-beta1) levels, and concurrent nematode and malaria infections resulted in high levels of interleukin-10 in vivo. Splenic CD11c(+) dendritic cells (DC) from mice infected with malaria alone and coinfected mice showed similarly increased expression of CD40, CD80, and CD86, but DC from coinfected mice were unable to induce CD4(+) T-cell proliferation and optimal IFN-gamma production in response to the malaria antigen in vitro. Importantly, treatment of nematode-infected mice with an anthelmintic drug prior to malaria infection fully restored protective antimalarial immunity and reduced TGF-beta1 levels. These results demonstrate that concurrent nematode infection strongly modulates multiple aspects of immunity to blood-stage malaria and consequently impairs the development of protective antimalarial immunity.     

A Defect in Interleukin-10 Leads to Enhanced Malarial Disease in Plasmodium chabaudi chabaudi Infection in Mice

Infection of interleukin-10 (IL-10)-nonexpressing (IL-10(-/-)) mice with Plasmodium chabaudi chabaudi (AS) leads to exacerbated pathology in female mice and death in a proportion of them. Hypoglycemia, hypothermia, and loss in body weight were significantly greater in female IL-10(-/-) mice than in male knockout mice and all wild-type (WT) mice during the acute phase of infection. At this time, both female and male IL-10(-/-) mice produced more gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and IL-12p40 mRNA than their respective WT counterparts. Inactivation of IFN-gamma in IL-10(-/-) mice by the injection of anti-IFN-gamma antibodies or by the generation of IL-10(-/-) IFN-gamma receptor(-/-) double-knockout mice resulted in reduced mortality but did not affect body weight, temperature, or blood glucose levels. The data suggest that IFN-gamma-independent pathways may be responsible for these pathological features of P. chabaudi malaria and may be due to direct stimulation of TNF-alpha by the parasite. Since male and female knockout mice both produce more inflammatory cytokines than their WT counterparts, it is likely that the mortality seen in females is due to the nature or magnitude of the response to these cytokines rather than the amount of IFN-gamma or TNF-alpha produced.     

Interleukin-3-Deficient Mice Have Increased Resistance to Blood-Stage Malaria

The contribution of interleukin-3 (IL-3), a hematopoietic growth factor and immunoregulatory cytokine, to resistance to blood-stage malaria was investigated by infecting IL-3-deficient (knockout [KO]) mice with Plasmodium berghei NK65. Male IL-3 KO mice, but not female mice, were more resistant to infection than wild-type (WT) mice, as evidenced by lower peak parasitemia and prolonged survival. Both male and female IL-3 KO mice had increased splenomegaly and were more anemic than corresponding WT mice. Anemia was compensated for by an increase in bone marrow and splenic erythropoiesis in IL-3 KO mice, as evidenced by higher levels of erythroid progenitors. Plasma levels of gamma interferon (IFN-γ) and CXCL9 (monokine induced by IFN-γ [MIG]) were found to be significantly reduced in IL-3 KO mice during early stages of infection. In contrast, granulocyte colony-stimulating factor (G-CSF) levels were significantly higher, and the percentage of peripheral blood neutrophils lower, in infected IL-3 KO mice than in WT counterparts. Overall, our results indicate that IL-3 plays a critical role in suppressing protective immunity to P. berghei NK65 infection and that it is involved in inhibiting the development of splenomegaly, anemia, and erythropoiesis. IL-3 also influences IFN-γ, CXCL9, and G-CSF production in response to infection. The abnormal responses seen in infected IL-3 KO mice may be due to the lack of IL-3 during development, to the lack of IL-3 in the infected mature mice, or to both.     

Pathology of Plasmodium chabaudi chabaudi infection and mortality in interleukin-10-deficient mice are ameliorated by anti-tumor necrosis factor alpha and exacerbated by anti-transforming growth factor beta antibodies

Interleukin-10 (IL-10)-deficient (IL-10(-/-)) mice infected with Plasmodium chabaudi (AS) suffer a more severe disease and exhibit a higher rate of mortality than control C57BL/6 mice. Here, we show that a drop in body temperature to below 28 degrees C and pronounced hypoglycemia of below 3 mM are reliable indicators of a lethal infection. Elevated inflammatory responses have been shown to accompany pathology in infected IL-10(-/-) mice. We show that neutralization of tumor necrosis factor alpha (TNF-alpha) in IL-10(-/-) mice abolishes mortality and ameliorates the hypothermia, weight loss, and anemia but does not affect the degree of hypoglycemia. These data suggest that TNF-alpha is involved in some of the pathology associated with a P. chabaudi infection in IL-10(-/-) mice but other factors play a role. IL-10(-/-) mice that survive a primary infection have been shown to control gamma interferon (IFN-gamma) and TNF-alpha production, indicating that other cytokines or mechanisms may be involved in their down-regulation. Significantly higher levels of transforming growth factor beta (TGF-beta), a cytokine with such properties, are present in the plasma of infected IL-10(-/-) mice at a time that coincides with the disappearance of IFN-gamma and TNF-alpha from the blood. Neutralization of TGF-beta in IL-10(-/-) mice resulted in higher circulating amounts of TNF-alpha and IFN-gamma, and all treated IL-10(-/-) mice died within 12 days with increased pathology but with no obvious increase in parasitemia. Our data suggest that a tight regulation of the balance between regulatory cytokines such as IL-10 and TGF-beta and inflammatory cytokines such as IFN-gamma and TNF-alpha is critical for survival in a mouse malaria infection.     

Granulocyte-macrophage colony-stimulating factor: involvement in control of Trypanosoma cruzi infection in mice.

Several cytokines play crucial roles in Trypanosoma cruzi infection in mice, but the involvement of endogenous granulocyte-macrophage colony-stimulating factor (GM-CSF) is poorly documented. This report shows that T. cruzi infection of mice triggered an early and sharp increase in plasma GM-CSF during the ascending phase of parasitemia. The plasma GM-CSF concentration remained stable at the peak of parasitemia and subsequently increased in those mice that survived to the acute phase. GM-CSF level increased again sharply, while parasitemia was rapidly decreasing. Finally, GM-CSF was undetectable, soon after the disappearance of circulating parasites. Injection of T. cruzi-infected mice with neutralizing anti-GM-CSF monoclonal antibodies induced the early appearance of parasitemia and aggravated cumulative mortality. In contrast, recombinant mouse GM-CSF (rmGM-CSF) caused sharp decreases in both parasitemia and cumulative mortality in T. cruzi-infected mice. Peritoneal macrophages from rmGM-CSF-treated and infected or uninfected mice were less infected ex vivo than those from control mice. Taken together these data demonstrate the protective action of endogenous GM-CSF in T. cruzi infection. Neutralization of endogenous GM-CSF aggravates infection, while exogenous rmGM-CSF decreases both parasitemia and host mortality.     

Changes in the cytokine profile of lupus-prone mice (NZB/NZW)F1 induced by Plasmodium chabaudi and their implications in the reversal of clinical symptoms

We have previously observed that aged lupus-prone (NZB/NZW)Fl (BWF1) mice when infected with Plasmodium chabaudi show an improvement in their clinical lupus-like symptoms. In order to study the mechanisms involved in the long-lasting protective effect of the P. chabaudi infection in lupus-prone mice we analysed specific aspects of the cellular response, namely the profiles of cytokine mRNA expression and cytokine secretion levels in old BWF1 mice, in comparison with uninfected age-matched BWF1 mice and infected or uninfected BALB/c mice. Two months after infection, cells from BWF1 mice were stimulated with concanavalin A (Con A) and demonstrated a recovery of T cell responsiveness that reached the levels obtained with BALB/c cells. Old BWF1 mice showed high levels of interferon-gamma (IFN-gamma) and IL-5 production and correspondingly low levels of IL-2 and IL-4 secretion before infection with P. chabaudi. Infection did not modify the IFN-gamma levels of BWF1 T cells, whereas it considerably increased the secretion of the Th2-related cytokines IL-4, IL-5 and IL-10. In addition, only BWF1 T cells showed increased mRNA expression of tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta). This counter-regulatory cytokine network of infected BWF1 mice may be involved in the improvement of their lupus symptoms. The results of our investigations using the complex model of P. chabaudi infection can be extended and, by using more restricted approaches, it may be possible to explain the multiple regulatory defects of lupus-prone mice.     

Tumor necrosis factor alpha p55 receptor is important for development of memory responses to blood-stage malaria infection

Tumor necrosis factor alpha (TNF-alpha) is associated with malarial pathology in both humans and mice. In Plasmodium chabaudi chabaudi (AS) infections, the production of TNF-alpha and reactive metabolites from macrophages are also thought to play a role in controlling acute parasitemia. Since many of the biological functions of TNF-alpha are effected through the p55 receptor (p55R), mice made defective in this receptor via a targeted gene disruption (p55R(-/-)) have been used to study its involvement in the immune response against P. chabaudi chabaudi and in the pathology associated with this infection. In the absence of the p55R, mice could overcome their primary infection, although higher acute-blood-stage parasitemias and more significant recrudescences were observed. Hypoglycemia, hypothermia, loss of erythrocytes, and loss of body weight, which occur transiently in this infection, were exacerbated by the lack of the p55R, but the differences were small, suggesting that other factors affect these symptoms. In contrast to wild-type (WT) mice, a second challenge infection in p55R(-/-) mice resulted in a course of infection similar to a primary infection. The malaria-specific immunoglobulin G antibody response of p55R(-/-) mice was lower than that of WT mice and was not increased by the second challenge infection. These data suggest that p55R(-/-) mice do not develop an efficient memory B-cell response against malarial infection and that this antibody response is important in immunity to reinfection.     

A dichotomous role for nitric oxide in protection against blood stage malaria infection

Nitric oxide (NO) is cytotoxic and cytostatic to blood stage malaria parasites in vitro, but the precise mechanism(s) by which it mediates an effect in vivo is not known. In particular, whether or not control of acute parasitemia depends on the presence of NO is unclear. We have shown previously that blocking NO synthesis at the time of its induction may cause an increase in peak primary parasitemia during infection of mice with Plasmodium chabaudi, suggesting that NO may be parasiticidal in vivo. However, as recent data indicate that NO suppresses Th1 cell proliferation in vitro by downregulating IL-2 production, we have investigated whether this immunoregulatory function of NO affects its capacity for anti-malarial activity. Treatment of P. chabaudi-infected mice with the iNOS inhibitor aminoguanidine hemisulfate (AG) starting just prior to the peak of primary parasitemia caused a significant elevation and extension of the acute infection and led to a partial but significant abrogation of the suppression of spleen cell proliferation to both mitogen and specific antigen observed when NO synthesis was not blocked. In the absence of NO, levels of IL-2, but not of IFN-gamma, TNF-alpha, or of any Th2-regulated cytokines examined, increased significantly. However, when AG treatment was brought forward to the early ascending phase of primary parasitemia, significantly increased levels of IFN-gamma and TNF-alpha, as well as of IL-2, were observed over those for infected control mice similarly treated with phosphate-buffered saline. Moreover, despite the absence of NO, parasitemias of AG-treated mice were not significantly elevated. The effect of AG therefore appeared to be dependent upon the timing of its administration in vivo. We propose that during malaria infections, there is a dynamic balance between the regulatory and anti-parasitic roles of NO. While the immunosuppressive function of NO leads to a downregulation in vivo of production of IL-2, and indirectly of IFN-gamma and TNF-alpha, this perceived weakening of the host cell-mediated immune response is in part masked by the protective anti-malarial effects of NO itself.     

A Th1-associated increase in tumor necrosis factor alpha expression in the spleen correlates with resistance to blood-stage malaria in mice.

We investigated the kinetics of tissue-specific mRNA expression and systemic production of tumor necrosis factor alpha (TNF-alpha) and the kinetics of splenic expression of mRNAs of gamma interferon (INF-gamma) and interleukin-4 (IL-4), cytokines that may regulate TNF-alpha production, during the early phase of blood-stage infection with Plasmodium chabaudi AS. Northern blot analysis revealed that resistant C57BL/6 mice, which clear the infection by 4 weeks, had higher levels of TNF-alpha mRNA in the spleen and liver early during infection that did susceptible A/J mice, which succumb to the disease 10 days after initiation of infection. Treatment of resistant mice with a polyclonal anti-TNF-alpha antibody confirmed the protective role of TNF-alpha early during the course of infection. Furthermore, resistant C57BL/6 mice also expressed high levels of mRNA of IFN-gamma (a Th1 marker) and low levels of mRNA of IL-4 (a Th2 marker) in the spleen, whereas susceptible A/J mice had low levels of IFN-gamma mRNA but high levels of TNF-alpha mRNA in the liver and had high levels of TNF-alpha protein in serum, as measured by enzyme-linked immunosorbent assay, later during infection just before death occurred. These results demonstrate that a Th1-associated increase in TNF-alpha mRNA expression in the spleen early during infection correlates with resistance to P. chabaudi AS, whereas increased TNF-alpha mRNA levels in the liver and excessive levels of the TNF-alpha protein in serum later during infection correlate with susceptibility. Thus, the role of the TNF-alpha during malaria appears to depend on the timing and site of its expression and the presence of cytokines regulating its production.     

Respiratory tract infection with Mycoplasma pneumoniae in interleukin-12 knockout mice results in improved bacterial clearance and reduced pulmonary inflammation

Mycoplasma pneumoniae is a leading cause of pneumonia and is associated with asthma. Evidence links M. pneumoniae respiratory disease severity with interleukin-12 (IL-12) concentration in respiratory secretions. We evaluated the microbiologic, inflammatory, and pulmonary function indices of M. pneumoniae pneumonia in IL-12 (p35) knockout (KO) mice and wild-type (WT) mice to determine the role of IL-12 in M. pneumoniae respiratory disease. Eight-week-old wild-type BALB/c mice and 8-week-old IL-12 (p35) KO BALB/c mice were inoculated once intranasally with 10(7) CFU of M. pneumoniae. Mice were evaluated at days 2, 4, and 7 after inoculation. Outcome variables included quantitative bronchoalveolar lavage (BAL) M. pneumoniae culture, lung histopathologic scores (HPS), BAL cytokine concentrations determined by enzyme-linked immunosorbent assay (tumor necrosis factor alpha [TNF-alpha], gamma interferon [IFN-gamma], IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor) and plethysmography, before and after methacholine, to assess airway obstruction (AO) and airway hyperreactivity (AHR). IL-12 (p35) KO mice infected with M. pneumoniae were found to have significantly lower BAL M. pneumoniae concentrations compared with M. pneumoniae-infected WT mice. Lung HPS and the parenchymal pneumonia subscores (neutrophilic alveolar infiltrate), as well as AO, were significantly lower in infected KO mice. No difference was found for AHR. Infected KO mice had significantly lower BAL concentrations of IFN-gamma than WT mice; a trend toward lower BAL concentrations was observed for IL-10 (P = 0.065) and TNF-alpha (P = 0.078). No differences were found for IL-1beta, IL-2, IL-4, IL-5, or IL-6. The lack of IL-12 in experimental M. pneumoniae pneumonia was associated with less severe pulmonary disease and more rapid microbiologic and histologic resolution.