Kunisuke Himeno

Escape of malaria parasites from host immunity requires CD4+ CD25+ regulatory T cells.

Infection with malaria parasites frequently induces total immune suppression, which makes it difficult for the host to maintain long-lasting immunity. Here we show that depletion of CD4+CD25+ regulatory T cells (Treg) protects mice from death when infected with a lethal strain of Plasmodium yoelii, and that this protection is associated with an increased T-cell responsiveness against parasite-derived antigens. These results suggest that activation of Treg cells contributes to immune suppression during malaria infection, and helps malaria parasites to escape from host immune responses.

Nippocystatin, a cysteine protease inhibitor from Nippostrongylus brasiliensis, inhibits antigen processing and modulates antigen-specific immune response.

ABSTRACT During infection, parasites evade the host immune system by modulating or exploiting the immune system; e.g., they suppress expression of major histocompatibility complex class II molecules or secrete cytokine-like molecules. However, it is not clear whether helminths disturb the immune responses of their hosts by controlling the antigen-processing pathways of the hosts. In this study, we identified a new cysteine protease inhibitor, nippocystatin, derived from excretory-secretory (ES) products of an intestinal nematode,Nippostrongylus brasiliensis. Nippocystatin, which belongs to cystatin family 2, consists of 144 amino acids and is secreted as a 14-kDa mature form. In vivo treatment of ovalbumin (OVA)-immunized mice with recombinant nippocystatin (rNbCys) profoundly suppressed OVA-specific proliferation of splenocytes but not non-antigen-specific proliferation of splenocytes. OVA-specific cytokine production was also greatly suppressed in rNbCys-treated mice. Although the serum levels of both OVA-specific immunoglobulin G1 (IgG1) and IgG2a were not affected by rNbCys treatment, OVA-specific IgE was preferentially downregulated in rNbCys-treated mice. In vitro rNbCys inhibited processing of OVA by lysosomal cysteine proteases from the spleens of mice. Mice with anti-nippocystatin antibodies became partially resistant to infection with N. brasiliensis. Based on these findings, N. brasiliensis appears to skillfully evade host immune systems by secreting nippocystatin, which modulates antigen processing in antigen-presenting cells of hosts.

Hyperproduction of Proinflammatory Cytokines by WSX-1-Deficient NKT Cells in Concanavalin A-Induced Hepatitis

Administration of Con A induces liver injury that is considered to be an experimental model for human autoimmune or viral hepatitis, where immunopathology plays roles mediated by activated lymphocytes, especially NK1.1+ CD3+ NKT cells, and inflammatory cytokines, including IFN-γ and IL-4. In the present study we investigated the role of WSX-1, a component of IL-27R, in Con A-induced hepatitis by taking advantage of WSX-1 knockout mice. WSX-1-deficient mice were more susceptible to Con A treatment than wild-type mice, showing serum alanine aminotransferase elevation and massive necrosis in the liver. Although the development of NKT cells appeared normal in WSX-1 knockout mice, purified NKT cells from the knockout mice produced more IFN-γ and IL-4 than those from wild-type mice in response to stimulation with Con A both in vitro and in vivo. In addition, hyperproduction of proinflammatory cytokines, including IL-1, IL-6, and TNF-α, was observed in the knockout mice after Con A administration. These data revealed a novel role for WSX-1 as an inhibitory regulator of cytokine production and inflammation in Con A-induced hepatitis.

Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice

We previously reported that CA074, a specific inhibitor of cathepsin B, modulates specific immune responses from the T helper 2 (Th2) type to Th1 type in BALB/c mice infected with Leishmania major. In the present study, we found that a similar type of immune deviation was also induced in mice immunized with ovalbumin (OVA). However, treatment of mice with pepstatin A, a specific cathepsin D inhibitor, suppressed the OVA‐specific proliferation of lymphocytes and blocked the development of both Th1 and Th2 cellular responses. These inhibitors did not appear to have any direct influence in vitro on functions of naive lymphocytes. OVA antigen (47u2003000 MW) was digested mainly into 40u2003000 MW protein in vitro by lysosomal proteases from naive BALB/c mice, and its digestion was markedly inhibited by the addition of CA074, but not by addition of pepstatin A, during incubation. However, pepstatin A strongly suppressed the degradation of the major histocompatibility complex class II‐associated invariant chain (Ii) molecule in vivo and in vitro. Thus, cathepsin B appears to process antigens directed to preferential activation of Th2 cells, while cathepsin D may be responsible for the degradation of Ii, the processing of which is essential in initiating the antigen‐specific activation of Th1 and Th2 CD4+ T cells. These lysosomal proteases may have different functions in regulating immune responses.

Molecular characterization of a 2-Cys peroxiredoxin from the human malaria parasite Plasmodium falciparum

We have identified the 2-Cys peroxiredoxin (PfPrx-1) from the human malaria parasite Plasmodium falciparum. The PfPrx-1 showed the highest identity at amino acid level to the type II Prx among the currently known six subfamilies of mammalian Prx. The sequence identity between the PfPrx-1 and the previously reported 1-Cys Prx of P. falciparum (PfPrx-2), which corresponded to mammalian type VI Prx, was 25%. This suggests that the parasite possesses two Prx subfamilies. The PfPrx-1 showed significant sequence similarities with those of 2-Cys peroxiredoxins of plants in the BLASTX search. This may reflect the consequences of a genetic transfer from an algal endosymbiont to the parasite nucleus during evolution. The recombinant PfPrx-1 protein (rPfPrx-1) was expressed as a histidine fusion protein in Escherichia coli and purified with Ni chromatography. The rPfPrx-1 existed as dimers under non-reducing conditions and dissociated into monomers in the presence of dithiothreitol. The PfPrx-1 protein also exists as a dimer in the parasites themselves. The reduction of the oxidized enzyme by the donation of electrons from E. coli thioredoxin (Trx)/Trx reductase system was demonstrated in its reaction with H(2)O(2), using the rPfPrx-1 protein. These results suggested that the PfPrx-1 can act as a terminal peroxidase of the parasite Trx system. An elevated expression of the PfPrx-1 protein seen in the trophozoite, the stage with active metabolism, suggests an association of the parasite Trx system with its intracellular redox control.

Insights into the Roles of Cathepsins in Antigen Processing and Presentation Revealed by Specific Inhibitors

Abstract Eleven human cathepsins have been identified, however, the in vivo roles of individual cathepsins are still largely unknown. In this brief review we will summarize the functions of individual cathepsins in antigen processing and presentation, which are the initial steps of the immune response. Two general inhibitors of papainlike cysteine proteases, E-64 and pyridoxal phosphate, can completely suppress antigen presentation in vivo. To evaluate the contribution of individual cathepsins, specific inhibitors have been developed based on cathepsin tertiary structures: CA-074 for cathepsin B, CLIK-148 and -195 for cathepsin L, CLIK-60 for cathepsin S. Administration of CA-074, a cathepsin B inhibitor, suppresses the response to exogenous antigens, such as hepatitis B virus antigen, ovalbumin and Leishmania major antigen, and induces switching of the helper T cell responses from Th-2 to Th-1 of CD4+ T cells, thereby downregulating the production of IgE and IgG1. Administration of the cathepsin S inhibitor CLIK-60 impairs presentation of an autoantigen, α-fodrin, in Sjogrens syndrome and suppresses the Th-1 response and autoantibody production.

IL-18 gene therapy develops Th1-type immune responses in Leishmania major-infected BALB/c mice: is the effect mediated by the CpG signaling TLR9?

IL-18 regulates either Th1 or Th2 responses depending on the cytokine microenvironment. Administration of recombinant IL-18 (rIL-18) alone does not promote Th1 response, but rather induces Th2 response and exacerbates Leishmania major infection in susceptible BALB/c mice. Here, we treated BALB/c mice with an IL-18-expressing plasmid by using a gene gun weekly after L. major infection. This gene therapy resulted in improved pathogenic process and preferential induction of Th1 responses by inducing the expression of IL-12 p40, but treatment with rIL-18 did not. Notably, simultaneous administration of rIL-18 with an empty plasmid vector rendered BALB/c mice resistant to the infection, despite the fact that treatment with either rIL-18 alone or the plasmid vector alone did not influence the susceptibility. The synergistic role of the vector with rIL-18 was found to depend on CpG motifs, which enhanced expression of proinflammatory cytokines, especially IL-12, from APCs through Toll-like receptor (TLR) 9 ligation. Treatment with methylated plasmid vector in which CpG was disrupted could no longer prevent the disease development in coadministration with rIL-18. Taken together, IL-18 gene therapy was shown to develop Th1-type protective immunity in L. major-infected BALB/c mice without the requirement of exogenous IL-12, probably via CpG-TLR9 signaling pathway.

Gene gun-mediated delivery of an interleukin-12 expression plasmid protects against infections with the intracellular protozoan parasites Leishmania major and Trypanosoma cruzi in mice

An interleukin‐12 (IL‐12) expression plasmid was transferred, using a gene gun, to mice infected with Leishmania major or Trypanosoma cruzi. Transfer of the IL‐12 gene to susceptible BALB/c mice resulted in regression of lesion size and reduced the number of parasites in draining lymph nodes (LN) at the site of L. major infection. Coincident with these protective effects, the T‐helper type (Th) response shifted towards Th1, as evaluated by cytokine production in vitro and L. major‐specific antibody responses. Protective effects of the IL‐12 gene were also observed in T. cruzi infection. Treatment of BALB/c mice infected with T. cruzi enhanced the production of interferon‐γ (IFN‐γ) by spleen cells, while suppressed production of interleukin‐10 (IL‐10) compared with control mice. Administration of anti‐CD4 or anti‐CD8 monoclonal antibody (mAb) abolished the protective immunity against T. cruzi infection, and treatment with the IL‐12 gene could not restore the resistance in these mice. Mice depleted of natural killer (NK) cells with anti‐asialo GM1 also became susceptible to infection, while the resistance was restored when these mice were treated with the IL‐12 gene. Thus, target cells for the treatment appear to be CD4+ and CD8+ T cells, which are ordinarily activated by NK cells. These results suggest that the transfer of cytokine genes using a gene gun is an effective method for investigating the roles of cytokines and gene therapy in infectious diseases.

Gene gun-based co-immunization of merozoite surface protein-1 cDNA with IL-12 expression plasmid confers protection against lethal Plasmodium yoelii in A/J mice

The carboxyl-terminal region of the merozoite surface protein-1 (MSP1) is a leading candidate for a vaccine against malaria in the erythrocytic stage. In this study, we investigated the utility of interleukin-12 (IL-12) cDNA as an adjuvant for malaria DNA vaccine in a mouse challenge model. We found that co-immunization of expression plasmids encoding a C-terminal 15-kDa fragment of MSP1 (MSP1-15) with the IL-12 gene using a gene gun significantly increased the protective immunity against malaria as compared with MSP1-15 DNA immunization alone. Co-immunization of IL-12 DNA potentiated MSP1-15-specific T helper (Th)1-type immune responses as evaluated by in vivo antibody (Ab) responses and in vitro cytokine profiles. After the Plasmodium yoelii challenge, mice immunized with MSP1-15 plus IL-12 DNA showed a higher level of interferon gamma (IFN-gamma) production than did other groups of mice. In vivo neutralization of IFN-gamma or depletion of CD4(+) T cells completely abolished this protective immunity. Macrophages, but not nitric oxide (NO), were found to play an important role in this effector mechanism. The sera from mice in which the infection had been cleared by the vaccination showed strong protection against P. yoelii infection. Thus, in addition to cellular immune responses, Abs against parasites induced in the course of infection are essential for protection against P. yoelii. The results indicate that combined vaccination with DNA encoding antigenic peptides plus IL-12 DNA provides a strategy for improving the prophylactic efficacy of a vaccine for malaria infection.

Roles of NKT cells in resistance against infection with Toxoplasma gondii and in expression of heat shock protein 65 in the host macrophages.

We investigated the roles of gamma delta T, NK, and NK1.1(+) T-like (NKT) cells in protective immunity against infection with Toxoplasma gondii. gamma delta T cells, NKT and NK cells, and NK cells in BALB/c mice were depleted by treatment with anti-TCR-gamma delta monoclonal antibody (mAb), anti-interleukin-2 receptor beta chain (IL-2R beta) mAb, and anti-asialoGM1 Ab, respectively, and these mice were infected with T. gondii. Treatment of mice with anti-TCR-gamma delta mAb aggravated toxoplasmosis, while treatment with anti-asialoGM1 Ab had no effects. Treatment with anti-IL-2R beta mAb enhanced the expression of heat shock protein 65 (HSP65) and gamma interferon (IFN-gamma) mRNA, while it inhibited interleukin-4 (IL-4) mRNA expression, ameliorating toxoplasmosis. In addition to NK cells, anti-IL-2R beta mAb eliminated cells expressing IL-2R beta and intermediate levels of CD3 (IL-2R beta(+) CD3(int)). Mice treated with anti-IL-2R beta mAb decreased the number of DX5(+) CD3(int) cells, which are considered to be equivalent to NK1.1(+)T cells in NK1.1 allele-negative strains. IL-2R beta(+) CD3(int) cells isolated from splenic and hepatic lymphoid cells were confirmed to express the TCR-V alpha 14 transcript. The magnitude of HSP65 induction in macrophages correlated with the protective potential against T. gondii infection after treatment with the antibodies, supporting our previous finding that gamma delta T cells play an essential role in the induction of HSP65 in host macrophages. Interestingly, NKT cells suppressed the expression of gamma delta T cell-induced HSP65 and IFN-gamma. Furthermore, depletion of IL-2R beta(+) CD3(int) cells suppressed the IL-4 mRNA expression. These results suggest that NKT cells may be the cells responsible for suppression of protective immunity against T. gondii infection by interfering with the gamma delta T cell-induced HSP65 expression, possibly through the generation of IL-4.