View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Prolonged survival after treatment with antihistamine drugs. Wild-type C57BL/6 mice were left untreated (squares) or treated (triangles) with the histamine inhibitors levocetirizine, cimetidine, imetit, and JNJ7777123 (for H1R, H2R, H3R, and H4R, respectively) before and during infection with 106 infected erythrocytes per mouse. Survival (A) and parasitemia (B) were monitored over time. These data are from three independent experiments. No significant difference was observed for parasitemia between groups (7–15 mice per group). Data are shown as means ± SD.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 3. HDC–/– mice are resistant to lethal infection by two Pb strains of parasites. Mortality (A and C) and parasitemia (B and D) were recorded for wild-type C57BL/6 mice (squares) or HDC–/– mice (diamonds) infected via mosquito bites with Pb NK65 (A and B) or with Pb ANKA (C and D). Groups consisted of 10 mice. Differences in parasitemia are significant at each time point from day 12 until day 19 (P < 0.001) in B but not in D, and data are shown as means ± SD. Three independent experiments were performed with similar results.

The resistance of HDC^–/– mice to CM induced by the inoculation of parasites via mosquito bites prompted us to explore at what stage of parasite development histamine mediated this effect; thus, we assessed whether these mice were similarly resistant to infection with blood-stage parasites. After infection with 10⁶ infected erythrocytes, C57BL/6 mice were susceptible to CM, displaying characteristic neurological signs before dying within 8 d (median survival = day 7; Fig. 4). In contrast, HDC^–/– mice did not exhibit signs of CM, and death occurred on average at day 20, which was consistent with hyperparasitemia-induced anemia (Fig. 4). These data indicate that the resistance of HDC^–/– mice to CM does not depend on the stage of parasite development used for infection. These data also suggest that histamine signaling mediates its detrimental effect later in infection, but they do not exclude the possibility that histamine signaling also plays a role at the preerythrocytic stage.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 4. HDC–/– mice are resistant to lethal infection by blood-stage parasites. Survival rates (A) and parasitemia (B) were compared between wild-type C57BL/6 mice (squares) and HDC–/– mice (diamonds) that received 106Pb ANKA–infected erythrocytes i.p. per mouse. *, P < 0.02 for parasitemia. The cumulative results of four independent experiments (10 mice per group) are shown. Data are shown as means ± SD.

View larger version (60K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Preserved BBB and a lack of sequestration of infected erythrocytes in the brain of HDC–/– mice. (A) Wild-type C57BL/6 mice and HDC–/– mice were inoculated i.p. with 106 infected erythrocytes of Pb ANKA per mouse. At day 7 after infection, mice were injected with a solution of Evans blue dye and were perfused with PBS 1 h later, and macroscopic observation was made of the brains. Brains from uninfected mice were used as controls. (B) Sections from the brains of the same mice were stained with MGG. Blood-stage parasites associated with sequestered erythrocytes (panels 1 and 2) in the brain could be visualized by fluorescence because of their expression of GFP (white, arrows; panel 2). No fluorescence could be observed in brain sections from uninfected and infected HDC–/– mice (panels 5 and 6). Data shown are representative of six mice per group. (C) The density of erythrocyte aggregates in brain sections of infected and control HDC–/– and C57BL/6 mice were expressed as the mean of aggregates per field and counted in 50 consecutive microscopic fields. These data are from two different experiments. *, P < 0.05 between infected HDC–/– and C57BL/6 mice. Data are shown as means ± SD. Bar, 100 µm.

The resistance of HDC^–/– mice to the development of CM after infection with Pb ANKA correlates with a drastic reduction of brain-infiltrating T cells and decreased expression of adhesion molecules
During infection with Pb ANKA, the development of CM is strongly associated with the recruitment and activation of T cells in the brain (28, 29). Accordingly, we examined whether such T cell recruitment occurred in HDC^–/– mice. Brain-infiltrating leukocytes were isolated from C57BL/6 or HDC^–/– mice 7 d after infection with Pb ANKA, and the percentage and absolute numbers of CD4⁺ and CD8⁺ T cells were determined. Compared with uninfected control mice, infected C57BL/6 mice displayed a 3.6-fold increase in the number of CD8⁺ T cells sequestered in the brain, whereas HDC^–/– mice show only a 1.3-fold increase (Fig. 6). Regarding the CD4⁺ T cell sequestration, infected C57BL/6 mice displayed a 2.7-fold increase, whereas HDC^–/– mice had only a 1.18-fold increase.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Absence of sequestration of CD4+ and CD8+ T cells in the brains of infected HDC–/– mice. At the coma stage (day 7 after infection), brains from C57BL/6 and HDC–/– mice infected with 106 infected erythrocytes of Pb ANKA per mouse were taken, and leukocytes associated with cerebral tissue were analyzed for the presence of CD4+ and CD8+ T cells and expressed as a percentage of the total cell population and as absolute numbers per brain. Six mice per group were used. Values represent the means ± SD from three experiments.

View larger version (69K): [in this window] [in a new window] [Download PPT slide]   Figure 7. ICAM-1 expression in brain vessels of infected C57BL/6 and HDC–/– mice. (A) Histological section showing ICAM-1 expression indicated by immunohistochemical staining, using anti–ICAM-1 mAb, of brain sections from infected HDC–/– and C57BL/6 mice. Photographs from control noninfected mice (not depicted) show <0.5 positive vessels per field in both mice. (B) The number of ICAM-1–positive vessels per field in Pb ANKA–infected C57BL/6 and HDC–/– mice. No significant labeling could be detected in naive mice in both mouse strains (<0.5 positive vessels per field). The proportion of ICAM-1–positive vessels was counted from 10 biopsies for each sample, and three mice were used in each group. (C) Comparison of ICAM-1 mRNA transcripts between infected C57BL/6 and HDC–/– mice as measured by quantitative RT-PCR. The relative mRNA levels of ICAM-1 were measured using real-time RT-PCR in brains. The expression levels were normalized to the endogenous control gene GAPDH, and the relative expression levels were calculated using the uninfected animals as calibrators. Data are presented as the means ± SD from two independent experiments. Bar, 100 µm.

First, serum samples at 5 d after infection with Pb ANKA were analyzed by the LINCOplex protein array (Fig. 8 A) to evaluate differences in the expression of some relevant proteins between C57BL/6 and HDC^–/– mice. Of the proteins quantified, the most striking differences were observed in inflammation-associated proteins. Serum IL-5 levels in infected C57BL/6 mice were 10-fold higher than infected HDC^–/– mice (P < 0.05). Similarly, IFN- and keratinocyte-derived chemokine were, respectively, two- and threefold higher in infected C57BL/6 mice (P < 0.05). Although not statistically significant, IL-6 and monocyte chemotactic protein 1 were markedly reduced in infected HDC^–/– mice as compared with infected C57BL/6 mice.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Figure 8. Differential expression of immune signaling genes in C57BL/6 and HDC–/– mice infected with Pb ANKA. (A) Serum levels of specific immune and inflammatory proteins were quantified at day 5 after infection by LINCOplex protein assay, and proteins with the highest variation between groups are displayed. (B) Transcription of cytokines and chemokines in the brain (n > 6 per group) 7 d after infection as evaluated by real-time RT-PCR. Gene mRNA expression is normalized relative to uninfected controls for each mouse strain, and control mRNA levels did not differ significantly between groups. Data are shown as means ± SD. *, P < 0.05; #, P = 0.05.

The antigen-induced response of specific CD8⁺ and CD4⁺T cells is similarly affected by infection with Pb ANKAin C57BL/6 and HDC^–/– mice
Differences in the magnitude of the CD4 and CD8 responses to infection between C57BL/6 and HDC^–/– mice could result in dissimilar manifestations of CM between mice from these two strains. Therefore, in an attempt to assess whether infection with Plasmodium interferes differently with the development of T cell responses in C57BL/6 and HDC^–/– mice, the proliferative capacity of OVA-specific CD4 and CD8 cells in response to specific stimulation was examined in the syngeneic environment of either infected C57BL/6 or HDC^–/– mice. 3 d after infection with Pb ANKA, C57BL/6 and HDC^–/– mice were injected with either CD8 OT-1 or CD4 OT-2 cells labeled with CFSE and treated or not with OVA. The proliferation of these OVA-specific T cells, as reflected by a decrease in the mean fluorescence intensity (MFI) of CFSE-labeled cells, was determined by FACS analysis 3 d later and expressed as the ratio between the MFI of CFSE-labeled cells recovered from mice receiving OVA to the MFI of similarly labeled cells recovered from mice not injected with OVA. Fig. 9 A shows a FACS quadrant analysis of representative samples in which the numbers of CD4⁺ or CD8⁺ T cells were plotted against CFSE levels. This was quantitatively analyzed in Fig. 9 B, where no significant difference in the proliferative response of CD8 OT-1 cells could be observed between infected C57BL/6 and HDC^–/– mice (a 3 ± 0.09– and 2.9 ± 0.1–fold decrease in MFI, respectively). Similarly, there was no difference in the proliferative response of CD4 OT-2 cells between mice from both strains (a 3.1 ± 0.2– and 2.52 ± 0.01–fold decrease in MFI, respectively). In contrast, in noninfected mice from both strains, CD8 OT-1 cells and CD4 OT-2 cells exhibited a much higher proliferative response (a 27.7 ± 0.06– and 31.5 ± 0.9–fold decrease in MFI for C57BL/6 and HDC^–/– mice, respectively, for CD8⁺ T cells; and a 12.4 ± 0.09– and 16.4 ± 0.07–fold decrease in MFI for C57BL/6 and HDC^–/– mice, respectively, for CD4⁺ T cells). These data indicate that the specific responses of CD4⁺ and CD8⁺ T cells are equally affected by infection with Plasmodium in mice from both strains and, thus, render unlikely the possibility that differences in T cell expansion between mice from the two strains account for the observed dissimilarity in the susceptibility to develop CM.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Figure 9. Similar expansion of OT-1 and OT-2 cells in infected C57BL/6 and HDC–/– mice. Wild-type C57BL/6 and HDC–/– mice were infected i.p. with 106 infected erythrocytes of Pb ANKA per mouse, and 3 d later they received 5 x 106 CFSE-labeled OT-1 or OT-2 cells, followed by injection or not of 1 mg OVA 1 h later. At day 6 after infection, spleen cells were harvested and stained either with APC–anti-CD8 or APC–anti-CD4 mAb, and quadrant FACS analysis was performed (A). Numbers in the top right quadrants indicate the fraction of CFSE/CD8 or CFSE/CD4 double-positive cells and the MFI, respectively. The proliferative response of OT-1 and OT-2 cells was expressed as a ratio between the MFI of CFSE-labeled cells from mice that received OVA to the MFI of those that did not (B). These data were obtained using three mice per group, and the experiment was replicated twice. Values are expressed as the means ± SD.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Figure 10. In vivo depletion of CD4 or CD8 T cells did not alter resistance of HDC–/– mice to lethal infection by Pb ANKA. Wild-type C57BL/6 (A and C) and HDC–/– (B and D) mice were left untreated (squares) or treated with neutralizing anti-CD4 (triangles) or anti-CD8 mAb (diamonds) before and during infection with 106Pb ANKA–infected erythrocytes per mouse. Survival rates (A and B) and parasitemia (C and D) were recorded over time. Six mice per group were used, and data are shown as means ± SD. This experiment was replicated twice. Significant differences in A were observed between untreated and anti-CD8–treated (*, P < 0.001) and anti-CD4–treated (**, P < 0.0001) mice. A significant difference in B was observed only with anti-CD4–treated mice (*, P = 0.034).

	DISCUSSION

Top ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

Apart from the correlation with immunopathogenesis in other diseases, little is known of the role of histamine in malaria. In this study, we analyzed the influence of histamine on the course of disease in mice infected with Pb from two different strains that induce distinct pathologies. This work evaluated the hypothesis that the histamine signaling during infection with Plasmodium results in an inflammatory process that exacerbates disease by inducing immunopathology or facilitating the dissemination, adherence, and sequestration of parasites in tissues. Based on divergent approaches using H1R^–/– and H2R^–/– mice, antihistamine drugs, and histamine-deficient mice, we demonstrate that histamine signaling is associated with the severity of disease.

The mechanisms by which histamine contributes to disease severity and at which stage of the parasite life cycle they operate is unclear. Mice genetically deficient in H1R and H2R, as well as mice treated with H1 and H2 antihistamines, have delayed mortality as compared with similarly infected wild-type mice untreated with the antihistamines, suggesting that the production and binding of histamine to these two receptors are deleterious to the host. However, antagonizing the effects of histamine by interference only at the levels of H1R and H2R had a significant but limited effect with regard to the increase in the mean survival time, possibly because the other factors or histamine receptors, H3R and H4R, also play a role. This is unlikely, however, because imetit and JNJ7777120, H3 and H4 antihistamines known to target H3R and H4R, neither influenced parasitemia nor survival. Thioperamide, a nonselective H3/H4R inhibitor, was also tested and showed no protective effect (unpublished data). To unequivocally determine the effect of histamine on the course of infection with Plasmodium, histamine-free mice were used. HDC^–/– mice infected with Pb NK65 through mosquito bites were highly resistant to malaria disease (90–100% survival up to 35 d after infection), with only 30% of the mice developing blood-stage parasites, which were ultimately cleared. These data strongly suggest that the absence of histamine either resulted in the generation of efficient effector responses against the preerythrocytic and blood stages of the parasite or limited the immunopathology associated with the disease. To expand these results to CM, HDC^–/– mice were infected with parasites from Pb ANKA. Although HDC^–/– mice eventually succumbed from the infection with this strain of Pb, they died at much later times than similarly infected control C57BL/6 mice and did not exhibit any signs of CM. These data implicate histamine in the progression to CM, demonstrate that the enhanced resistance of histamine-free mice is not restricted to parasites from a particular strain, and suggest that histamine signaling could lead to enhanced immunopathological responses. Importantly, results showing comparable parasitemia between groups infected with parasites from Pb ANKA do not support the possibility that the inability of histamine-free mice to develop CM resulted from a reduced parasite burden.

In an attempt to better determine at which stage of parasite development histamine mediates its adverse effects, HDC^–/– mice were also infected with Pb ANKA–parasitized RBCs, thus bypassing the preerythrocytic phase. Identical to what was observed after infectious mosquito bites, histamine-free mice did not develop CM and survived significantly longer than similarly infected control C57BL/6 mice. These data indicate that although histamine is produced during all stages of infection with Plasmodium parasites (i.e., by histamine-producing MCs in the skin and in the liver during the preerythrocytic stage of parasites development, and by histamine-producing basophils in the blood where infected RBCs circulate), the pathogenic effects of histamine are likely prevailing in the latter stages of infection. However, the possibility that histamine also plays a role in the skin and/or the liver during the initial phases of the infection cannot be excluded. Indeed, during the initial phases of the infection, specifically the preerythrocytic stages of parasite development, histamine release may be triggered in the dermis when sporozoites are inoculated via mosquito bites. In this context, Anopheles saliva induces MC degranulation (1). During later phases of infection, particularly during the blood stage of parasite development, histamine production can be elicited from circulating basophils either via the cross-linking of parasite-specific IgE antibodies or by TCTP, a parasite-derived homologue of the histamine-releasing factor. TCTP has been found in the plasma of patients infected with P. falciparum and was shown to trigger histamine release from basophils and IL-8 secretion from eosinophils (37). The existence of a Plasmodium protein that stimulates histamine release lends support to the hypothesis that histamine signaling is advantageous to the pathogen and, thus, harmful to the host. These findings could provide a rational basis for higher levels of histamine in blood and in tissues during malaria as a result of the activity of vector- or parasite-derived constituents. Our results (Fig. 1) clearly revealed a progressive increase in histaminemia during the course of infection in C57BL/6 mice. Amplifying the host inflammatory response via histamine signaling may be a strategy developed by the parasites to create conditions advantageous for their own survival and persistence. Although the parasites from the two strains used in our study are lethal for mice, the disease courses that they elicit are quite different. Our results in HDC^–/– mice show that in the absence of histamine, the disease severity induced by both parasites was significantly attenuated. The adverse effect of histamine is highlighted by the survival induced after infection with Pb NK65 and a delayed non-CM death from Pb ANKA resulting from the interruption of histamine signaling. The reason that infection with Pb ANKA remains lethal in histamine-free HDC^–/– mice is unclear. It seems plausible that even though inhibiting histamine signaling may abrogate immunopathology, it cannot stop the adverse effects of hyperparasitemia. In addition to its distinctive efficiency in producing CM in mice, the possibility exists that Pb ANKA also elicits inflammatory mediators, other than histamine, that promote the progression of infection even in HDC^–/– mice. Indeed, analysis of several inflammatory proteins and other immune response–associated molecules, including IFN-, IL-6, IL-10, keratinocyte-derived chemokine (KC), monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1 (MIP-1), cytotoxic T lymphocyte–associated protein 4, and inducible co-stimulator (Fig. 8; and not depicted), indicates that an inflammatory response, although attenuated, was induced in HDC^–/– mice. Several studies indicate that the CM pathologies seen in mice after infection with Pb ANKA result from the induced proinflammatory immune response. In agreement, IFN-, which was shown to be implicated in the pathogenesis of experimental CM (38), reached significantly higher levels in the brain tissue and plasma of CM-sensitive C57BL/6 mice than in HDC^–/– mice (Fig. 8, A and B). Similarly, our results showing higher levels of IL-6 in the plasma and increased expression of IL-6 transcripts in the brain of CM-sensitive C57BL/6 than in HDC^–/– mice are also in agreement with studies showing higher production of IL-6 in response to TNF- by endothelial cells of brain capillaries of mice genetically sensitive to CM development (39). Our results, which diverge from murine CM experiments that suggest a protective effect for IL-10 in CM (40) but are in accordance with observations in humans that showed an increase in IL-10 with CM as compared with mild malaria patients (41), show a lower cerebral expression of IL-10 by HDC^–/– mice. IL-4 expression corresponds with CM in our model, in agreement with previous studies that demonstrate that deficiency in IL-4 or IL-4R leads to higher resistance to Pb ANKA (42).

Analogous to the present data that reveal the adverse effects of histamine for the host during infection with Plasmodium, a human study demonstrated that the concentration of histamine in plasma was increased by almost fivefold in children suffering from malaria (22). Additionally, in this study, higher levels of histamine were observed in the brains of children with severe malaria as compared to lesser disease presentations. Among the histamine-associated biological activities relevant to features predictive of severe CM in humans are increased intracerebral blood flow (43), increased vascular and BBB permeability (44), and edema (45). Strikingly, the resistance of histamine-deficient mice to CM was clearly identified by a preserved BBB integrity, in contrast to the greatly increased permeability of the brain vasculature with large clusters of infected erythrocytes observed in infected C57BL/6 mice.

In the absence of histamine signaling, profound alterations of the immune response may also occur. A characteristic feature of CM is the sequestration of CD8⁺ and CD4⁺ T cells in the brain capillaries (46–48). This was confirmed by results in this study showing a 3.7- and 2.7-fold increase of CD8⁺ and CD4⁺ T cells, respectively, in the brain of C57BL/6 mice infected with Pb ANKA. Such sequestration in the brain was not observed in similarly infected HDC^–/– mice (Fig. 6). These data suggest that the histamine produced after infection with Pb ANKA triggers inflammation that leads to the sequestration of T cells in brain capillaries, a prominent component of CM pathogenesis. In this context, it has been reported that histamine is essential for the recruitment of antigen-specific CD4⁺ and CD8⁺ T cells into the site of antigen delivery and the subsequent generation of inflammatory responses (12).

Several studies reported a crucial role of ICAM-1 in malaria pathogenesis, and notably, ICAM-1–deficient mice are protected from CM (49). In this model, ICAM-1^–/– mice survived >15 d despite a similar level of parasitemia in wild-type and knockout mice, a phenotype that is comparable to HDC^–/– mice. Results from our study confirmed the correlation between ICAM-1 expression, both at the protein and transcript levels, and susceptibility to CM. The low levels of ICAM-1 expression in brain microvascular endothelial cells in histamine-deficient HDC^–/– mice (Fig. 8) could represent a mechanism leading to the lack of T cell sequestration in the brain capillaries of these mice. It is interesting to note that ICAM-1^–/– and HDC^–/– mice, which express similar phenotypes, have in common a reduced synthesis of ICAM-1 molecules, suggesting a control mechanism exerted by histamine on ICAM-1 expression. Indeed, histamine has been shown to stimulate endothelial cells to express ICAM-1 and VCAM-1 (32), and cetirizine, a H1R antagonist, has recently been shown to have antiinflammatory properties through the inhibition of leukocyte recruitment and activation, and by the reduction of ICAM-1 expression on keratinocytes (50) and on conjunctival and nasal epithelial cells (33). Interestingly, a reduction in ICAM-1 and VCAM-1 expression was observed in the brain tissue of infected C57BL/6 mice treated with levocetirizine (unpublished data). In one study, VCAM-1 was identified by microarray analysis as a candidate gene that discriminates between CM-resistant and -sensitive mice in a Pb model of CM (51). Our data do not support the assertion that elevated VCAM-1 corresponds to CM, as we observed slightly higher VCAM-1 mRNA levels in HDC^–/– mice (P < 0.05) as compared with C57BL/6 mice, suggesting an uncertain or, perhaps, protective role for this adhesion molecule in our model. In accordance with our observation of a predominant role for ICAM-1 as compared with VCAM-1 in malaria pathogenesis, infusion of anti–ICAM-1 but not anti–VCAM-1 mAb prevents cytoadherence of infected erythrocytes in a P. yoelii model of CM (52).

A possible dissimilarity in the magnitude and quality of the CD4 and CD8 responses elicited by infection with Pb ANKA in C56BL/6 and HDC^–/– mice could account for the lack of CM in HDC^–/– mice. Such differences in the magnitude of the T cell responses could be the result of a divergent ability to suppress antigen-specific T cell responses in C57BL/6 and HDC^–/– mice.

Our results showing a similar effect of infection with Plasmodium on the specific proliferative response of OT-1 and OT-2 cells in C57BL/6 and HDC^–/– mice suggest that the magnitude of T cell responses is similarly affected in C57BL/6 and HDC^–/– mice. The effect of infection on the magnitude of the Pb-specific T cell response is currently being compared between C57BL/6 and HDC^–/– mice. Our results are consistent with previous findings showing that during an acute blood-stage malaria infection T cell responses to Plasmodium parasites and other bystander antigens are inhibited (53). Thus, the mechanism by which HDC^–/– mice resist CM does not appear to stem from a Pb ANKA–induced modification of the peripheral T cell responses, though one could argue that the absence of HDC expression may alter histamine-mediated inflammatory responses that are necessary for the manifestation of some effector functions exerted by CD4⁺ and CD8⁺ T cell responses.

To demonstrate directly and unequivocally the implication of histamine in the pathogenesis of CM, attempts were made to revert the CM-resistant phenotype (HDC^–/–) by frequent injections of histamine into Pb ANKA–infected mice. Such treatment failed to induce significant alterations in mortality (unpublished data). This method of repleting mice may, however, not be sufficient because of the low bioavailability and the lability of histamine (54). Nevertheless, our results with antihistamines and genetic knockouts strongly suggest that antihistamine could have a therapeutic value in the treatment of malaria infection, particularly by reducing the likelihood of adverse complications. This is supported by the fact that antihistaminic drugs such as chlorpheniramine potentiate the anti-Plasmodium effect of mefloquine, quinine, or pyronaridine (55). It should be pointed out that the antihistamine drugs do not very likely exert their effect directly on the parasite. We performed an experiment in which infected erythrocytes treated with the H1R blocker levocetirizine were compared with untreated infected erythrocytes for their capacity to infect mice. No significant difference regarding survival or parasitemia was observed between the two groups of mice, suggesting that H1 antihistamine drugs exert no direct effect on the parasite (unpublished data).

The present work represents the first comprehensive study documenting the role of histamine in the development of severe pathologies during infection with Plasmodium. Although malaria vaccine development remains a central and ultimate goal, alternative chemotherapy-based approaches such as histamine receptor antagonists have the potential to be highly valuable. Such treatment could be part of an integrated control strategy and could also be a useful candidate in intermittent preventive treatment strategies that target specific high-risk groups, such as children and pregnant women. Furthermore, given the availability of antihistamines, such treatment could be implemented rapidly to alleviate the burden of malaria in endemic areas.

	MATERIALS AND METHODS

Top ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Animals.
6–8-wk-old female C57BL/6 mice were purchased from Charles River Laboratories. HDC^–/– (56) and H1R and H2R knockout (H1R^–/– and H2R^–/–) (57, 58) mice were provided by H. Ohtsu and T. Watanabe, respectively. All knockout mice originated from the C57BL/6 background.

The mice, including OT-1 and OT-2 transgenic mice (59), were bred in our animal facility. OT-1 mice are transgenic for a TCR that recognizes the OVA peptide 257–264 in the context of H2K^b. OT-2 mice express a transgenic TCR, which recognizes the OVA peptide 323–339 in the context of I-A^b. All animal care and experimentation were conducted in accordance with the Pasteur Institute animal care and use committee guidelines.

An. stephensi (SDA 500 strain) mosquitoes were raised at the Center of Production and Infection of Anopheles (CEPIA) of the Institut Pasteur using standard procedures, as previously described (1).

Parasites and infection.
For all infections, Pb NK65 or Pb ANKA lines expressing GFP on circumsporozoite (60) or the hsp70 promoter (61), respectively, were used to allow the detection of sporozoites in living mosquitoes. Pb ANKA was provided by T. Ishino (Mie University School of Medicine, Edobashi, Tsu, Japan). This parasite induces experimental CM, characterized by paralysis, ataxia, convulsions, and coma between 7–9 d after infection. Pb NK65 induces lethal malaria without neurological symptoms and was provided by R. Ménard (Institut Pasteur, Paris, France). These parasites were maintained in a cycle between C57BL/6 mice and An. stephensi. The erythrocytic stages of the parasite were maintained in liquid nitrogen as parasitized RBCs (pRBCs) in Alsever's solution (Sigma-Aldrich) containing 10% glycerol. The infection was induced by i.p. injection of 10⁶ pRBCs or by exposure of mice to mosquito bites (seven to nine bites per mouse).

Treatment with antihistamine drugs.
The histamine receptor subclass-specific inhibitors cimetidine, imetit, and JNJ 7777120 (H2R, H3R, and H4R, respectively; Sigma-Aldrich) were injected i.p. daily at 10 mg/kg for 10 d, starting 24 h before infection. Levocetirizine (H1R), obtained from UCB, was used at 10 mg/kg. Histamineamia was determined using ELISA kits purchased from Neogen Corporation.

Preparation of brain cell suspensions.
The brains were obtained from wild-type–susceptible C57BL/6 mice at the coma stage of CM and at the same time from HDC^–/– mice (day 6). In brief, mice anesthetized with 600 mg/kg ketamine and 20 mg/kg xylazine were perfused with 150 ml PBS. Each brain was then removed and homogenized in RPMI 1640 medium (BioWhittaker) by passing through sterile meshes to obtain a single-cell suspension. Percoll (GE Healthcare) was added at a final concentration of 35% to the cell pellet and centrifuged at 400 g for 20 min at 20°C. The cell pellet was washed twice and analyzed via flow cytometry.

In vivo study of CFSE-labeled T cell proliferation.
Splenocytes were prepared from OT-1 and OT-2 mice. Cell suspensions were prepared by passing through sterile meshes. Erythrocytes were lysed using RBC lysis buffer (Sigma-Aldrich). Cells were washed twice in RPMI 1640 medium, and live cells were counted using Trypan blue exclusion staining and adjusted to 10⁷ cells per milliliter. CD8⁺ OT-1 or CD4⁺ OT-2 cells were purified with MACS beads according to the manufacturer's instructions (Miltenyi Biotec), and cells were stained with allophycocyanin (APC)-conjugated anti-CD8 or APC-conjugated anti-CD4 antibodies, and the purity was 95% as evaluated by FACS analysis. Staining of spleen cells with CFSE (Invitrogen) was performed according to the manufacturer's instructions. Recipient mice (C57BL/6, HDC^–/–) were inoculated 3 d after infection with 10⁶ pRBCs i.v. with either 5 x 10⁶ purified CFSE-CD8⁺ OT-1 or CFSE-CD4⁺ OT-2 cells. Mice were injected 1 h later with 1 mg OVA. Single-cell suspensions of spleens of the recipients were analyzed 3 d after adoptive cell transfer.

In vivo depletions.
Rat IgG2a anti-CD4 mAb (clone GK1.5; American Type Culture Collection) and rat IgG2b anti-CD8 mAb (clone 2.43; American Type Culture Collection) were provided by L. Rénia (Institut Cochin, Paris, France). Purified control rat antibodies were purchased from Sigma-Aldrich. Antibodies were purified from culture supernatants by ammonium sulfate precipitation. Mice were injected i.p. with 500 µg anti-CD8 mAb, anti-CD4 mAb, or control rat IgG on days 1 and 0 before challenge. More than 98% of blood CD8⁺ or CD4⁺ T cells were depleted by this procedure, as verified by cytofluorometry (FACScan; BD Biosciences) using anti-CD4 (clone H129-19; Sigma-Aldrich) and anti-CD8 (clone 53-6.7; BD Biosciences) mAbs that recognized epitopes different from those bound by the depleting mAbs. Mice were then injected with 10⁶ pRBCs infected with Pb ANKA, and 200 µg of antibodies was administered every other day until completion of the experiment.

Flow cytometric analysis of brain and spleen leukocytes.
Spleen and brain cells were stained for FACS analysis according to standard protocols in cold PBS containing 2% FCS and 0.01% sodium azide (FACS buffer) with the following antibodies: APC-labeled CD4, APC-labeled anti-CD8, and PE-labeled antiperforin. RBCs were eliminated using cell lysis buffer, and cells were washed in FACS buffer. After staining with anti-CD4 or anti-CD8 antibodies, cells were washed and resuspended in fixation/permeabilization solution for 20 min at 4°C. Fixed/permeabilized cells were resuspended in Perm/Wash buffer (BD Biosciences) for before flow cytometric analysis. A total of 4 x 10⁴ and 10⁵ living cells for brain and spleen, respectively, were analyzed using a four-color flow cytometer (FACSCalibur) with CellQuest Pro software (both from BD Biosciences).

Permeability of the BBB.
When mice infected with Pb ANKA began showing neurological symptoms, usually at day 6–7 after infection, a 200-µl volume of a 2% (wt/vol) solution of Evans blue in PBS was injected into the mice retroorbitally. 1 h later, mice were perfused with PBS after anesthesia with 600 mg/kg ketamine and 20 mg/kg xylazine. Each brain was removed and photographed.

Animal perfusion and histological analysis.
For the May-Grünwald Giemsa (MGG) staining procedure and immunohistochemical studies, the protocols used to obtain 5-µm-thick brain sections were described previously (62). Detection of blood-stage parasites associated with sequestered erythrocytes in the brain could be visualized either by MGG staining or by fluorescence because of their expression of GFP. Images of randomly selected endplates were collected using an upright microscope (BX61; Olympus) equipped with an oil immersion lens (60x) and a cooled video camera (Retiga 2000R; QImaging) with a color conversion filter. Digitizing was performed with a computer using the image analysis system Image-Pro Plus (Media Cybernetics). Detection of VCAM-1 and ICAM-1 was performed by using specific mAbs (clones 429 and 3E2, respectively), followed by biotin-conjugated goat anti–rat IgG-specific polyclonal antibody (for VCAM-1) and mouse anti–hamster antibody (for ICAM-1). Streptavidin peroxidase and 3-amino-9-ethylcarbazole, as substrates, were added thereafter. All antibodies were obtained from BD Biosciences.

Cytokine and chemokine quantification in the brain and serum.
To evaluate shifts of immune mediators in serum, LINCOplex protein arrays were performed on day 5 serum samples from HDC^–/– and C57BL/6 mice, according to the standard protocol. Cytokine and chemokine expression in the brain at day 7 was analyzed by real-time RT-PCR. RNA used for these assays was isolated by means of a two-step extraction process. First, brains were surgically removed from mice, in Preparation of brain cell suspensions, and placed immediately in RNAlater (Ambion) at 4°C overnight. After RNAlater infused the samples, it was removed and samples were maintained at –80°C until processing. Brains were thawed in 1 ml of TRIZOL and subjected to bead disruption in a polytron three times for 2 min at a setting of 30 cycles per second. Samples were spun at high speed (10,000 g) for 3 min to remove debris and lipids. Half of the sample was transferred to a new tube and mixed with 500 µl of TRIZOL reagent by vortexing. After this step, RNA extraction proceeded according to the manufacturer's protocol. Precipitated RNA was resuspended in 100 µl of RNase-free water. The second step of this extraction was in accordance with the manufacturer's protocol for RNA cleanup, including steps for the removal of protein and DNA (RNeasy kit; QIAGEN). Samples were eluted with 50 µl of RNase-free water, and quality and quantity were assured by photospectroscopy. Real-time RT-PCR used various primer-probe sets and standard Taqman protocols (Applied Biosystems), as previously described (63).

Statistical analysis.
Statistical significance was assessed by the Student's t test, except for survival. Significant differences in survival were evaluated by the generation of Kaplan-Meier plots and log-rank analysis. P < 0.05 was considered statistically significant.