Splenic architecture disruption and parasite-induced splenocyte activation and anergy in Plasmodium falciparum-infected Saimiri sciureus monkeys

Abstract

Background: The understanding of the mechanisms of immunity in malaria is crucial for the rational development of interventions such as vaccines. During blood stage infection, the spleen is considered to play critical roles in both immunity and immunopathology of Plasmodium falciparum infections.

Methods: Saimiri sciureus monkeys were inoculated with blood stages of P. falciparum (FUP strain) and spleens removed during acute disease (days 7 and 13 of infection) and during convalescence (15 days after start of chloroquine treatment). Cytokine (IFNγ, TNFα, IL2, IL6, IL10, and IL12) responses of splenocytes stimulated with P. falciparum-parasitized red blood cells were assessed by real-time PCR using specific Saimiri primers, and histological changes were evaluated using haematoxylin-eosin and Giemsa-stained slides.

Results: Early during infection (day 7, 1-2% parasitaemia), spleens showed disruption of germinal centre architecture with heavy B-cell activation (centroblasts), and splenocytes showed increased expression of IFNγ, IL6 and IL12 upon in vitro stimuli by P. falciparum-parasitized red blood cells (pRBC). Conversely, 15 days after treatment of blood stage infection with chloroquine, splenocytes showed spontaneous in vitro expression of TNFα, IL2, IL6, IL10, and IL12, but not IFNγ, and stimulation with P. falciparum pRBC blocked the expression of all these cytokines. During the acute phase of infection, splenic disarray with disorganized germinal centres was observed. During convalescence, spleens of the chloroquine-treated animals showed white pulp hyperplasia with extensive lymphocyte activation and persistency of heavily haemozoin-laden macrophages throughout the red pulp.

Conclusions: Inability to eliminate haemozoin is likely involved in the persistent lymphocyte activation and in the anergic responses of Saimiri splenocytes to P. falciparum pRBC, with important negative impact in immune responses and implications for the design of malaria vaccine.

Figure 1

Efficiency and kinetics of Saimiri sciureus cytokine cDNA amplification. (A) Efficiency of cytokine (IFNγ, IL2, IL6, IL10, IL12, TNFα) cDNA amplification by real-time PCR using pair of primers specifically designed for Saimiri sciureus sequences. The Figure shows the average Ct values after cytokine amplification using serial five-fold dilutions of cDNA from Saimiri peripheral blood mononuclear leukocytes stimulated with ionomycin-PMA. (B) Kinetics of cytokine (IFNγ, IL2, IL6, IL10, IL12, TNFα) cDNA amplification by real-time PCR following in vitro mitogen stimulation. Peripheral blood mononuclear leukocytes from non-infected Saimiri were stimulated in vitro with ionomycin-PMA and mRNA amplification was determined at various timepoints. Results are shown as fold increases in cytokine expression in relation to unstimulated leukocytes.

Figure 2

Course of parasitaemia. Six Saimiri sciureus monkeys were inoculated with 5×10⁷ P. falciparum parasitized red blood cells. Two monkeys were splenectomized and treated with chloroquine at d7 (solid lines). The four remaining monkeys were treated with chloroquine at d13, and two of them were splenectomized on day 13 (dashed lines) and two on day 28 (dotted lines).

Figure 3

Cytokine expression by real-time PCR in Saimiri sciureus splenocytes. Animals were splenectomized at d7 (two monkeys, rising parasitaemia) and d28 (two monkeys, 15 days after start of chloroquine therapy) post-inoculation with 5×10⁷ P. falciparum pRBC. Splenocytes were stimulated in vitro with P. falciparum pRBC or culture medium. Fold increases in cytokine expression were calculated in relation to unstimulated splenocytes from two uninfected control Saimiri monkeys. *: p <0.05; **: p <0.01; ***: p <0.001 (two-way ANOVA with Bonferroni post test).

Figure 4

Changes in spleen histology during Plasmodium falciparum infection. 4A-C: Spleens of non-infected control animals, showing well-defined limits between the red and white pulps (A), quiescent T-cell zones without immunoblasts (B); a central arteriole is denoted by an asterisk) and mostly resting B-cell follicles (C). Clusters of weakly stained cells, suggestive of macrophages, were observed (A, C, arrows). D-F: At d7 of infection, haemozoin was observed throughout an engorged red pulp (D, asterisk). Follicles showed numerous phagocytosis centers (D, arrows). Limits between the red and white pulps were blurred and follicles presented light-stained nuclei cells surrounded by small lymphocytes (E), and the B-cell follicles showed penetration by RBCs (E, asterisks). Large cells with weakly stained nuclei suggestive of centroblasts were widespread (central darker area of the image), and mitosis (black arrows) and apoptosis foci (red arrows) were abundant (F). All sections were stained with Lennert’s Giemsa.

Figure 5

Changes in spleen histology during Plasmodium falciparum infection. 5A-D: At d13 of infection (just before chloroquine treatment), the red pulp was further enlarged in relation to controls and d7 of infection and laden with haemozoin (A, asterisk). The follicles were also enlarged and limits between the red and white pulp were blurred (A, arrow). Phagocytes were heavily laden with pigment and strings of cells suggestive of plasmacytogenesis cords were observed near the T-cell zone (B). Follicles showed penetrating RBCs and limits between red and white pulp were blurred (C). Follicles were interspersed with cells of different morphology, showing phagocytes with malaria pigment, centroblasts and mainly small lymphocytes in the periphery (D). E, F: At d28 (15 days after start of chloroquine treatment), the red pulp phagocytes were heavily laden with malaria pigment (E, F). The distribution of haemozoin-containing phagocytes was better defined in relation to d13, with individual cells containing more compacted, less granulous pigment (E, F). The follicles were further enlarged in relation to d13, with large follicles touching each other with little red pulp in between (E) and showed mostly activated cells (F). All sections were stained with Lennert’s Giemsa.