Tianqian Zhang

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.

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 (47 000 MW) was digested mainly into 40 000 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.

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.

Different cytokines are required for induction and maintenance of the Th2 type response in DBA/2 mice resistant to infection with Leishmania major

Experimental cutaneous leishmaniasis is a useful model in studying the mechanism regulating immune responses between T helper type 1 (Th1) and Th2. Mice susceptible to Leishmania major infection such as BALB/c (H-2(d)) are associated with the induction of the disease-promoting Th2 response, while the resistant mice such as DBA/2 (H-2(d)) develop the protective Th1 response. To understand the induction mechanism of Th1 and Th2 responses, it is necessary to establish an immunization scheme by which the induction of each Th response can be easily and experimentally controlled. Adjuvants are known to enhance the immune responses through the combined effect of several factors: prolonged release of antigen, migration of cells, mitogenic effect and so forth. When the genetically resistant DBA/2 mice were immunized twice with soluble leishmanial antigen (SLA), emulsified in incomplete Freunds adjuvant (IFA) before L. major inoculation, these mice mounted a Th2 cell response and suffered from progressive infection. While IL-4 and IL-13 were upregulated early after the infection in both healer and non-healer groups of mice, IL-5 and IL-10 were upregulated only in non-healer mice. From these results, IL-5 and IL-10 appear to have an important role, at least in the early phases of the infection, rather than IL-4 and IL-13 in establishing the disease-promoting Th2 response in leishmaniasis. Further, IL-9 was found to be expressed in both BALB/c and DBA/2 mice immunized with IFA/SLA. This cytokine may support the establishment of a Th2 response in these mice. Therefore it is suggested that Th2 cytokines play different roles between priming and maintaining the Th2 immune response after the infection.

Granule-dependent killing of Toxoplasma gondii by CD8+ T cells.

Immunization of mice with live bradyzoites of a low‐virulent Beverley strain of Toxoplasma gondii has been shown to increase CD8+ T‐cell mediated immunity against a highly virulent RH strain. We found that preimmunization with an RH homogenate further enhanced this immunity. Using this model, we investigated the mechanism of CD8+ T‐cell mediated protection against T. gondii infection. Splenic cells from mice immunized with RH homogenate and live bradyzoites stimulated apoptosis of RH‐infected J774A.1 macrophages in vitro, and at the same time, the immunization significantly suppressed the proliferation of parasites within macrophages, as assessed by measuring 3H‐uracil uptake by the parasites. Splenic cells from the immunized mice produced larger amounts of interferon‐γ (IFN‐γ) than did naive splenic cells; however, the production of nitric oxide (NO) by RH‐infected macrophages was not enhanced. The elimination of CD8+ T cells from splenic cells significantly reduced their inhibitory action on parasite proliferation as well as their cytotoxic activity against RH‐infected macrophages, but it did not affect the production of IFN‐γ. Treatment of CD8+ T‐enriched splenic cells from the immunized mice with concanamycin A, but not an anti‐Fas ligand monoclonal antibody, significantly reduced their anti‐proliferative and killing capabilities, suggesting that the CD8+ T cells induced by immunization with RH antigen and live bradyzoites of the Beverley strain may exert protection against T. gondii infection at least in part through granule‐dependent cytotoxic activities.