Babesia microti alleviates disease manifestations caused by Plasmodium berghei ANKA in murine co-infection model of complicated malaria.

Malaria remains one of the most significant health issues worldwide, accounting for 2.6% of the total global disease burden, and efforts to eliminate this threat continue. The key focus is to develop an efficient and long-term immunity to this disease via vaccination or therapeutic approach, and innovative strategies would enable us to achieve this target. Previously, using a mouse co-infection disease model, cross-protection was illustrated between Babesia microti and Plasmodium chabaudi. Hence, this study was planned to elucidate the impact of acute B. microti Peabody mjr and Plasmodium berghei ANKA co-infection on the consequence of complicated malaria in the C57BL/6J mouse model of malaria. Furthermore, immune response and pathological features were analyzed, and the course of the disease was compared among experimental groups. Our study established that acute B. microti infection activated immunity which was otherwise suppressed by P. berghei. The immunosuppressive tissue microenvironment was counteracted as evidenced by the enhanced immune cell population in co-infected mice, in contrast to P. berghei-infected control mice. Parasite sequestration in the brain, liver, lung, and spleen of co-infected mice was significantly decreased and tissue injury was ameliorated. Meanwhile, the serum levels of IFN-γ, TNF-α, and IL-12p70 were reduced while the secretion of IL-10 was promoted in co-infected mice. Eventually, co-infected mice showed an extended rate of survival. Hereby, the principal cytokines associated with the severity of malaria by P. berghei infection were TNF-α, IFN-γ, and IL-12p70. Moreover, it was evident from our flow cytometry results that innate immunity is crucial and macrophages are at the frontline of immunity against P. berghei infection. Our study recommended further investigations to shed light on the effects of babesiosis in suppressing malaria with the goal of developing Babesia-based therapy against malaria.

Fluctuations of Spleen Cytokine and Blood Lactate, Importance of Cellular Immunity in Host Defense Against Blood Stage Malaria Plasmodium yoelii.

Our previous studies of protective immunity and pathology against blood stage malaria parasites have shown that not only CD4+ T cells, but also CD8+ T cells and macrophages, are important for host defense against blood stage malaria infection. Furthermore, we found that Plasmodium yoelii 17XNL (PyNL) parasitizes erythroblasts, the red blood cell (RBC) precursor cells, which then express MHC class I molecules. In the present study, we analyzed spleen cytokine production. In CD8+ T cell-depleted mice, IL-10 production in early stage infection was increased over two-fold relative to infected control animals and IL-10+ CD3- cells were increased, whereas IFN-γ production in the late stage of infection was decreased. At day 16 after PyNL infection, CD8+ T cells produced more IFN-γ than CD4+ T cells. We evaluated the involvement of the immunoproteasome in induction of immune CD8+ T cells, and the role of Fas in protection against PyNL both of which are downstream of IFN-γ. In cell transfer experiments, at least the single molecules LMP7, LMP2, and PA28 are not essential for CD8+ T cell induction. The Fas mutant LPR mouse was weaker in resistance to PyNL infection than WT mice, and 20% of the animals died. LPR-derived parasitized erythroid cells exhibited less externalization of phosphatidylserine (PS), and phagocytosis by macrophages was impaired. Furthermore, we tried to identify the cause of death in malaria infection. Blood lactate concentration was increased in the CD8+ T cell-depleted PyNL-infected group at day 19 (around peak parasitemia) to similar levels as day 7 after infection with a lethal strain of Py. When we injected mice with lactate at day 4 and 6 of PyNL infection, all mice died at day 8 despite demonstrating low parasitemia, suggesting that hyperlactatemia is one of the causes of death in CD8+ T cell-depleted PyNL-infected mice. We conclude that CD8+ T cells might control cytokine production to some extent and regulate hyperparasitemia and hyperlactatemia in protection against blood stage malaria parasites.

Cytotoxic activities of CD8⁺ T cells collaborate with macrophages to protect against blood-stage murine malaria.

The protective immunity afforded by CD8(+) T cells against blood-stage malaria remains controversial because no MHC class I molecules are displayed on parasite-infected human erythrocytes. We recently reported that rodent malaria parasites infect erythroblasts that express major histocompatibility complex (MHC) class I antigens, which are recognized by CD8(+) T cells. In this study, we demonstrate that the cytotoxic activity of CD8(+) T cells contributes to the protection of mice against blood-stage malaria in a Fas ligand (FasL)-dependent manner. Erythroblasts infected with malarial parasites express the death receptor Fas. CD8(+) T cells induce the externalization of phosphatidylserine (PS) on the infected erythroblasts in a cell-to-cell contact-dependent manner. PS enhances the engulfment of the infected erythroid cells by phagocytes. As a PS receptor, T-cell immunoglobulin-domain and mucin-domain-containing molecule 4 (Tim-4) contributes to the phagocytosis of malaria-parasite-infected cells. Our findings provide insight into the molecular mechanisms underlying the protective immunity exerted by CD8(+) T cells in collaboration with phagocytes.

IL-23 protection against Plasmodium berghei infection in mice is partially dependent on IL-17 from macrophages.

Although IL-12 is believed to contribute to protective immune responses, the role played by IL-23 (a member of the IL-12 family) in malaria is elusive. Here, we show that IL-23 is produced during infection with Plasmodium berghei NK65. Mice deficient in IL-23 (p19KO) had higher parasitemia and died earlier than wild-type (WT) controls. Interestingly, p19KO mice had lower numbers of IL-17-producing splenic cells than their WT counterparts. Furthermore, mice deficient in IL-17 (17KO) suffered higher parasitemia than the WT controls, indicating that IL-23-mediated protection is dependent on induction of IL-17 during infection. We found that macrophages were responsible for IL-17 production in response to IL-23. We observed a striking reduction in splenic macrophages in the p19KO and 17KO mice, both of which became highly susceptible to infection. Thus, IL-17 appears to be crucial for maintenance of splenic macrophages. Adoptive transfer of macrophages into macrophage-depleted mice confirmed that macrophage-derived IL-17 is required for macrophage accumulation and parasite eradication in the recipient mice. We also found that IL-17 induces CCL2/7, which recruit macrophages. Our findings reveal a novel protective mechanism whereby IL-23, IL-17, and macrophages reduce the severity of infection with blood-stage malaria parasites.

Resistance to malaria by enhanced phagocytosis of erythrocytes in LMP7-deficient mice.

General cellular functions of proteasomes occur through protein degradation, whereas the specific function of immunoproteasomes is the optimization of antigen processing associated with MHC class I. We and others previously reported that deficiency in subunits of immunoproteasomes impaired the activation of antigen-specific CD8(+) T cells, resulting in higher susceptibility to tumor and infections. We demonstrated that CD8(+) T cells contributed to protection against malaria parasites. In this study, we evaluated the role of immunoproteasomes in the course of infection with rodent malaria parasites. Unexpectedly, Plasmodium yoelii infection of mice deficient in LMP7, a catalytic subunit of immunoproteasomes, showed lower parasite growth in the early phase of infection and lower lethality compared with control mice. The protective characteristics of LMP7-deficient mice were not associated with enhanced immune responses, as the mutant mice showed comparable or diminished activation of innate and acquired immunity. The remarkable difference was observed in erythrocytes instead of immune responses. Parasitized red blood cells (pRBCs) purified from LMP7-deficient mice were more susceptible to phagocytosis by macrophages compared with those from wild-type mice. The susceptibility of pRBC to phagocytosis appeared to correlate with deformity of the membrane structures that were only observed after infection. Our results suggest that RBCs of LMP7-deficient mice were more likely to deform in response to infection with malaria parasites, presumably resulting in higher susceptibility to phagocytosis and in the partial resistance to malaria.

Induction of apoptosis of leukemic cells by TRUE gene silencing using small guide RNAs targeting the WT1 mRNA.

TRUE gene silencing is a technology to eliminate specific cellular RNAs by using tRNase Z(L) and small guide RNA (sgRNA). Here we investigated how WT1-mRNA-targeting sgRNAs affect leukemic cells. We showed that sgRNA can be easily taken up by cells without any transfection reagents, and that the naked sgRNAs targeting the WT1 mRNA can reduce its mRNA levels and WT1 protein amounts in the WT1-expressing leukemic cells. Concomitantly, these sgRNAs efficiently induced apoptosis in these cells but not in WT1-nonexpressing cells. We also demonstrated that the reduction in the WT1 mRNA level is caused by its cleavage by tRNase Z(L).

Enhancement of antigen presenting ability in the leukemic plasmacytoid dendritic cell line (PMDC05) by lentiviral vector-mediated transduction of CD80 gene.

PMDC05, a leukemic plasmacytoid dendritic cell (pDC) line which was established in our laboratory, showed a capacity of generating antigen-specific cytotoxic T lymphocytes (CTLs). In order to enhance an antigen presenting ability of PMDC05, PMDC05 was transduced with CD80 gene by lentiviral vector, which was named as PMDC11. PMDC11 displayed a strong antigen presenting ability even without any stimulation, and by culturing with stimulators such as calcium ionophore PMDC11 gained a more potent antigen presenting ability. Our data suggested PMDC11 could be applied as antigen presenting cells more efficiently in adoptive cellular immunotherapy for tumors and severe infections in comparison with PMDC05.

Generation of antigen-specific cytotoxic T lymphocytes using a leukemic plasmacytoid dendritic cell line as antigen presenting cells.

Establishment of a leukemia plasmacytoid dendritic cell line (PMDC05) and intra-lineage transformation from pDCs to mDCs in PMDC05 has been reported. In this paper, we show the applicability of PMDC05 for cellular immunotherapy. By stimulation with LPS, PMDC05 showed enhancement in expression of antigen presentation-associated surface molecules and production of cytokines (IL-12p70 and TNF-α). The antigen presenting ability was markedly increased in PMDC05 stimulated with LPS. By co-culturing of CD8(+) T cells with LPS-stimulated and WT1/CMVpp65 peptide-pulsed PMDC05, WT1/CMVpp65 tetramer(+) cytotoxic T lymphocytes were efficiently generated. These findings reveal the applicability of PMDC05 in cellular immunotherapy for tumor and severe viral infections.