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Cerebral malaria (CM) is a serious and fatal malaria-associated syndrome caused by the development of an overwhelming proinflammatory response. Vitamin D (Vit.D; cholecalciferol) has regulatory functions associated with both innate and adaptive immune responses. Prevention is better than cure, in this experiment, we evaluated prophylactic oral Vit.D as a means of preventing CM presentation before infection of C57BL/6 mice with Plasmodium berghei ANKA (PbA) by modulating the host proinflammatory response. Mice that were supplemented with oral Vit.D has reduce death rate and ameliorated the integrity of the blood brain barrier. Prophylactic oral vitamin D relieved the symptoms of brain malaria and avoided death, gained valuable time for the diagnosis and treatment post infection. The robust Th1 response was attenuated in the Vit.D + PbA group. Furthermore, T-cell trafficking to the brain was diminished before PbA infection using Vit.D. The results suggest that Vit.D supplementation mediates the development of an anti-inflammatory environment that improves CM severity. In summary, the use of Vit.D as a nutritional supplement in malaria-endemic regions may help reduce the severity and mortality of CM.

Interleukin-10 (IL-10)-producing regulatory B (Breg) cells were found to be induced in a variety of infectious diseases. However, its importance in the regulation of immune response to malaria is still unclear. Here, we investigated the dynamics, phenotype, and function of Breg cells using Plasmodium chabaudi chabaudi AS-infected C57BL/6 and BALB/c mice. BALB/c mice were more susceptible to infection and had a stronger IL-10 response in spleen than C57BL/6 mice. Analysis of the surface markers of IL-10-producing cells with flow cytometry showed that CD19 + B cells were one of the primary IL-10-producing populations in P. c. chabaudi AS-infected C57BL/6 and BALB/c mice, especially in the latter one. The Breg cells had a heterogeneous phenotype which shifted during infection. The well-established Breg subset, CD19 + CD5 + CD1d hi cells, accounted for less than 20% of IL-10-producing B cells in both strains during the course of infection. Most Breg cells were IgG + and CD138 − from day 0 to day 8 postinfection. Adoptive transfer of Breg cells to C57BL/6 mice infected with P. c. chabaudi AS led to a transient increase of parasitemia without an impact on survival rate. Our finding reveals that B cells play an active and important regulatory role in addition to mediating humoral immunity in immune response against malaria, which should be paid more attention in developing therapeutic or vaccine strategies against malaria involving stimulation of B cells.

Chloroquine (CQ), a well-known anti-malarial drug, has long been used for the treatment of autoimmune diseases because of its profound immunomodulatory effects. However, whether this drug modifies anti-malaria immune response is still not clear. Here we studied the immunomodulatory role of CQ in a mouse model of malaria. DBA/2 mice were infected with Plasmodium yoelii (Py) parasite (intraperitoneal injection of parasitized erythrocytes) and divided into three groups. Two groups received single dose of CQ (gavage administration) at 6 hours after Py infection (post-6h) and 3 days after Py infection (post-3d), respectively. The third group received saline as control. The course of disease was monitored and the changes of immune response were investigated. It is shown that mice from the post-6h group took longer time to clear the parasites compared with those of the post-3d group. The activation of T helper cells, macrophages, and B cells was significantly suppressed in mice with post-6h CQ treatment as compared with control mice on day 3 and day 5 after infection. In contrast, no such changes were found in mice from the post-3d group. Dendritic cells (DCs) from the post-6h CQ treated mice were less mature as compared with those from control mice as well as those from the post-3d group. Taken together, our data suggest that treatment with CQ early in infection inhibits protective immune response against Py infection possibly via mechanisms involving the modulation of DC's function. Our finding provided important information for reasonable use of CQ in malaria chemotherapy.

Listeria monocytogenes (Lm) has been used as the adjuvant or vector for tumor and viral vaccine for its capability of eliciting all aspects of cell-mediated immunity including T cell activation and interferon-gamma (IFN-γ) production. These effector components play critical roles in the protection against Plasmodium infection in both human malaria and mouse models. Therefore, immune response induced by Lm infection may benefit the defense against malaria. To test this hypothesis, we employed blood-stage Plasmodium yoelii (PyL) infected mice and challenged them with Lm. C57BL/6 and BALB/c mice that are sensitive to PyL infection were used in experiments. These two strains are resistant and sensitive, respectively, to Lm infection. The outcomes of double infection with PyL and Lm and the changes of immune response were investigated. We found that live Lm inoculation inhibited PyL multiplication in both C57BL/6 and BALB/c mice. Lm inoculation increased production of IFN-γ, infiltration of CD11b-positive macrophages and generation of nitric oxide in the spleen of C57BL/6 mice at day 5 after parasite infection. Both CD4- and CD8- positive T cells contributed to IFN-γ production induced by Lm inoculation in PyL-infected mice. The protective effect of Lm against PyL infection depended on the viability of the bacteria. Live Lm, rather than heat-killed Lm, stimulated early IFN-γ production which provided essential cytokine environment for the development of Th1 response in PyL-infected mice. Our data show for the first time that Lm inoculation has protective effect against blood-stage murine malaria, which provides a new clue for enhancing anti-Plasmodium immunity.

Background Transmission-blocking vaccine (TBV) is a promising strategy for interrupting the malaria transmission cycle. Current TBV candidates include both pre- and post-fertilization antigens expressed during sexual development of the malaria parasites. Methods We tested whether a TBV design combining two sexual-stage antigens has better transmission-blocking activity. Using the rodent malaria model Plasmodium yoelii, we pursued a DNA vaccination strategy with genes encoding the gametocyte antigen Pys48/45 and the major ookinete surface protein Pys25. Results Immunization of mice with DNA constructs expression either Pys48/45 or Pys25 elicited strong antibody responses, which specifically recognized a ~45 and ~25 kDa protein from gametocyte and ookinete lysates, respectively. Immune sera from mice immunized with DNA constructs expressing Pys48/45 and Pys25 individually and in combination displayed evident transmission-blocking activity in in vitro ookinete culture and direct mosquito feeding experiments. With both assays, the Pys25 sera had higher transmission-blocking activity than the Pys48/45 sera. Intriguingly, compared with the immunization with the individual DNA vaccines, immunization with both DNA constructs produced lower antibody responses against individual antigens. The resultant immune sera from the composite vaccination had significantly lower transmission-blocking activity than those from Pys25 DNA immunization group, albeit the activity was substantially higher than that from the Pys48 DNA vaccination group. Conclusions This result suggested that vaccination with the two DNA constructs did not achieve a synergistic effect, but rather caused interference in inducing antigen-specific antibody responses. This result has important implications for future design of composite vaccines targeting different sexual antigens.

CD4+ CD25+ regulatory T (Treg) cells expressing Foxp3+ play a critical role in maintaining immune homoeostasis and controlling excessive immune responses. However, controversy about the immunoregulatory role of Treg cells exists in malaria studies. Given the role of maintenance of Foxp3 expression in Treg cells’ activities, we investigated whether anti-CD25 mAb (7D4 clone) treatment affects Foxp3 expression in CD4+ T cells in DBA/2 mice infected with Plasmodium chabaudi chabaudi AS (P. c. chabaudi AS). We found that DBA/2 mice succumbed to P. c. chabaudi AS infection, which was accompanied by increased expression of Foxp3 in CD4+ T cells at the peak parasitemia. In contrast, Foxp3 expression was impaired in CD25-depleted mice with 7D4 mAb treatment, leading to delayed parasitemia and extended survival of infected mice. Production of IFN-γ, TNF-α and IL-6, as well as NO was significantly enhanced in CD25-depleted mice. The majority of CD4+ CTLA-4+ cells expressed high levels of Foxp3 (Foxp3hi cells) in control mice with P. c. chabaudi AS infection. However, the number of CD4+ Foxp3hiCTLA-4+ cells was reduced in CD25-depleted mice. Together, these data suggest that CD4+ Foxp3hiCTLA-4+ cells may be involved in regulating the intensity of pro-inflammatory responses via CTLA-4.

L-Arginine (L-Arg), the substrate for nitric oxide (NO) synthase, has been used to treat malaria to reverse endothelial dysfunction in adults. However, the safety and efficacy of L-Arg remains unknown in malaria patients under the age of five, who are at the greatest risk of developing cerebral malaria (CM), a severe malaria complication. Here, we tested effects of L-Arg treatment on the outcomes of CM using a mouse model. Experimental cerebral malaria (ECM) was induced in female C57BL/6 mice infected with Plasmodium berghei ANKA, and L-Arg was administrated either prophylactically or after parasite infection. Surprisingly, both types of L-Arg administration caused a decline in survival time and raised CM clinical scores. L-Arg treatment increased the population of CD4(+)T-bet(+)IFN-γ(+) Th1 cells and the activated macrophages (F4/80(+)CD36(+)) in the spleen. The levels of pro-inflammatory cytokines, IFN-γ and TNF-α, in splenocyte cultures were also increased by L-Arg treatment. The above changes were accompanied with a rise in the number of dendritic cells (DCs) and an increase in their maturation. However, L-Arg did not affect the population of regulatory T cells or the level of IL-10 in the spleen. Taken together, these data suggest that L-Arg may enhance the Th1 immune response, which is essential for a protective response in uncomplicated malaria but could be lethal in CM patients. Therefore, the prophylactic use of L-Arg to treat CM, based on the assumption that restoring the bioavailability of endothelial NO improves the outcome of CM, may need to be reconsidered especially for children.

Chloroquine (CQ), a well-known anti-malarial drug, has long been used for the treatment of autoimmune diseases because of its profound immunomodulatory effects. However, whether this drug modifies anti-malaria immune response is still not clear. Here we studied the immunomodulatory role of CQ in a mouse model of malaria. DBA/2 mice were infected with Plasmodium yoelii (Py) parasite (intraperitoneal injection of parasitized erythrocytes) and divided into three groups. Two groups received single dose of CQ (gavage administration) at 6 hours after Py infection (post-6h) and 3 days after Py infection (post-3d), respectively. The third group received saline as control. The course of disease was monitored and the changes of immune response were investigated. It is shown that mice from the post-6h group took longer time to clear the parasites compared with those of the post-3d group. The activation of T helper cells, macrophages, and B cells was significantly suppressed in mice with post-6h CQ treatment as compared with control mice on day 3 and day 5 after infection. In contrast, no such changes were found in mice from the post-3d group. Dendritic cells (DCs) from the post-6h CQ treated mice were less mature as compared with those from control mice as well as those from the post-3d group. Taken together, our data suggest that treatment with CQ early in infection inhibits protective immune response against Py infection possibly via mechanisms involving the modulation of DC's function. Our finding provided important information for reasonable use of CQ in malaria chemotherapy.

In animal models of experimental cerebral malaria (ECM), neuropathology is associated with an overwhelming inflammatory response and sequestration of leukocytes and parasite-infected RBCs in the brain. In this study, we explored the effect of vitamin D (VD; cholecalciferol) treatment on host immunity and outcome of ECM in C57BL/6 mice during Plasmodium berghei ANKA (PbA) infection. We observed that oral administration of VD both before and after PbA infection completely protected mice from ECM. VD administration significantly dampened the inducible systemic inflammatory responses with reduced circulating cytokines IFN-γ and TNF and decreased expression of these cytokines by the spleen cells. Meanwhile, VD also resulted in decreased expression of the chemokines CXCL9 and CXCL10 and cytoadhesion molecules (ICAM-1, VCAM-1, and CD36) in the brain, leading to reduced accumulation of pathogenic T cells in the brain and ultimately substantial improvement of the blood–brain barriers of PbA-infected mice. In addition, VD inhibited the differentiation, activation, and maturation of splenic dendritic cells. Meanwhile, regulatory T cells and IL-10 expression levels were upregulated upon VD treatment. These data collectively demonstrated the suppressive function of VD on host inflammatory responses, which provides significant survival benefits in the murine ECM model.

Background Malaria and schistosomiasis are endemic and co-exist in the same geographic areas, even co-infecting the same host. Previous studies have reported that concomitant infection with Schistosoma japonicum could offer protection against experimental cerebral malaria (ECM) in mice. This study was performed to evaluate whether alterations in parasite density could alter this protective effect. Methods Mice were inoculated with 100 or 200 S. japonicum cercariae followed by infection with high or low density of Plasmodium berghei ANKA strain eight weeks after the first infection. Then, parasitaemia, survival rate and blood–brain-barrier (BBB) damage were assessed. Interferon-gamma (IFN-γ), interleukin (IL)-4, IL-5, IL-13, IL-10, and TGF-β levels were determined in splenocyte supernatants using enzyme-linked immunosorbent assay (ELISA). Cell surface/intracellular staining and flow cytometry were used to analyse the level of CD4+/CD8+ T cells, CD4+CD25+Foxp3+ Tregs, IL-10-secreting Tregs, and IL-10+Foxp3-CD4+ T cells in the spleen, and CD4+/CD8+ T cells infiltrating the brain. Results Co-infection with low density P. berghei and increased S. japonicum cercariae significantly increased the levels of IL-4, IL-5, IL-13, TGF-β and Tregs, but significantly decreased the levels of IFN-γ and the percentage of CD4+ T cells and CD8+ T cells in the spleen and CD8+ T cell infiltration in the brain. Increased worm loads also significantly decreased mortality and BBB impairment during ECM. When challenged with higher numbers of P. berghei and increased cercariae, the observed cytokine changes were not statistically significant. The corresponding ECM mortality and BBB impairment also remained unchanged. Conclusions This study demonstrates that protection for ECM depends on the numbers of the parasites, S. japonicum and P. berghei, during co-infection. Alterations in the regulatory response appear to play a key role in this adaptation.

The present study aimed to investigate the signal transduction mechanism of the NZW type interleukin (IL)‑10R1 and the effect on the function of B lymphocytes. Vectors for the expression of wild‑type, NZW and G1146A IL‑10R1 were constructed and transfected into Ba/F3 cells. The cell proliferation curve was determined using an MTT assay. CD32 and leukocyte-endothelial cell adhesion molecule 1 (LECAM‑1) expression levels were assessed using flow cytometry. The phosphorylation of Janus kinase 1 (Jak1), tyrosine kinase 2 (Tyk2) and signal transducer and activator of transcription 3 (Stat3) were measured by western blot analysis. As compared with wild‑type IL‑10R1 cells, the proliferation ability of NZW IL‑10R1 cells was increased following stimulation with IL‑10, which was even greater for G1146A cells. CD32 levels in NZW IL‑10R1 cells were lower, while the expression of LECAM‑1 was higher compared with wild‑type IL‑10R1 cells, which was also observed for G1146A cells. The phosphorylation levels of Jak1 and Stat3 in NZW IL‑10R1 and G1146A IL‑10R1 cells were significantly lower compared with the wild‑type IL‑10R1 cells. However, the phosphorylation levels in the three cell types were not significantly different. In conclusion, the deficiency in Jak1‑Stat3 signal transduction in NZW IL‑10R1 cells induces a loss in the inhibition ability of proliferation and activation as well as a migration tendency of B lymphocytes, which is hypothesized to be associated with the occurrence and development of the autoimmune disease systemic lupus erythematosus (SLE).

Listeria monocytogenes (Lm) has been used as the adjuvant or vector for tumor and viral vaccine for its capability of eliciting all aspects of cell-mediated immunity including T cell activation and interferon-gamma (IFN-γ) production. These effector components play critical roles in the protection against Plasmodium infection in both human malaria and mouse models. Therefore, immune response induced by Lm infection may benefit the defense against malaria. To test this hypothesis, we employed blood-stage Plasmodium yoelii (PyL) infected mice and challenged them with Lm. C57BL/6 and BALB/c mice that are sensitive to PyL infection were used in experiments. These two strains are resistant and sensitive, respectively, to Lm infection. The outcomes of double infection with PyL and Lm and the changes of immune response were investigated. We found that live Lm inoculation inhibited PyL multiplication in both C57BL/6 and BALB/c mice. Lm inoculation increased production of IFN-γ, infiltration of CD11b-positive macrophages and generation of nitric oxide in the spleen of C57BL/6 mice at day 5 after parasite infection. Both CD4- and CD8- positive T cells contributed to IFN-γ production induced by Lm inoculation in PyL-infected mice. The protective effect of Lm against PyL infection depended on the viability of the bacteria. Live Lm, rather than heat-killed Lm, stimulated early IFN-γ production which provided essential cytokine environment for the development of Th1 response in PyL-infected mice. Our data show for the first time that Lm inoculation has protective effect against blood-stage murine malaria, which provides a new clue for enhancing anti-Plasmodium immunity.