Cysteine Protease Inhibitor of Schistosoma japonicum – A Parasite-Derived Negative Immunoregulatory Factor
Abstract
Studies have shown that cysteine protease inhibitors from some parasites exert immunosuppressive effects on the host. We have previously cloned a novel cysteine protease inhibitor from Schistosoma japonicum and purified its recombinant form (protein named rSj-C). Its potential inhibitory effect on the host immune response had not been described prior to this study. Here, we demonstrate that rSj-C inhibits lysosomal cysteine protease of murine dendritic cells (DCs). When DCs were incubated with rSj-C and then with soluble adult worm antigen (AWA) of S. japonicum, the mean fluorescence intensity of MHC class II antigens on their surface decreased significantly, indicating suppression of exogenous antigen presentation by DCs. Further, eight weeks after infected mice were injected with rSj-C, the proportion of CD4+CD25+Foxp3+ regulatory T cells among CD4+CD25+ T cells increased significantly compared to controls. Additionally, production of IL-4 and TGF-β by T cells increased significantly. These findings clearly show that cysteine protease inhibitor from S. japonicum is a parasite-derived immunosuppressive factor.
Keywords: Schistosoma japonicum, cysteine protease inhibitor, recombinant cystatin, regulatory T cells, immunosuppression
Introduction
Cysteine protease inhibitors are widespread in nature and belong to the superfamily comprising the stefin, cystatin, and kininogen families. Those derived from parasites can inhibit antigen presentation by antigen-presenting cells such as dendritic cells, suppress T cell proliferation, and promote synthesis of immunosuppressive cytokines by binding to immune receptors. These observations suggest that parasite-derived cysteine protease inhibitors can suppress host immune responses. Whether the newly discovered cysteine protease inhibitor from S. japonicum has a similar effect and the mechanism of its action had been unknown.
We previously obtained genomic DNA and cDNA sequences of a novel cysteine protease inhibitor from S. japonicum and produced its recombinant protein, rSj-C. Expression analysis showed the gene is expressed in eggs, schistosomula, and adult worms. Immunohistochemical and immunofluorescent staining revealed localization mainly in miracidia within eggs and in the intestinal epithelium and body wall of adults, with release into the host circulatory system. During infection, adult worms continuously release soluble egg antigens containing cysteine protease inhibitor into the host. The host immune response gradually shifts from a Th1 to Th2 response and, by eight weeks, to an immunosuppressed state. Regulatory T cells are implicated in this suppression; however, whether it relates to continual release of parasite-derived cysteine protease inhibitor had yet to be determined.
This study aimed to test whether cysteine protease inhibitor from S. japonicum is a parasite-derived immunosuppressive protein involved in inhibiting cell-mediated immune responses to infection.
Materials and Methods
The rSj-C protein was prepared as previously described and used in experiments. Twenty-four female BALB/c mice were divided into four groups: normal control (N), rSj-C injection only (Ij), S. japonicum infection only (If), and infection plus rSj-C injection (If+Ij), six mice each. Mice in infected groups were exposed to cercariae via abdominal skin. Five weeks post-infection, mice in If+Ij received intraperitoneal rSj-C injections (50 μg in PBS) every other day for three weeks. Group Ij received rSj-C injections without infection; group If received PBS injections.
Spleen-derived DCs were prepared from mice by generating splenocyte suspensions, lysing red blood cells, and culturing with GM-CSF and IL-4 for seven days.
Recognition of rSj-C by DCs was determined by incubating DCs on coverslips with rSj-C, staining with specific antibodies, and visualizing by confocal microscopy.
The inhibitory effect of rSj-C on lysosomal cysteine protease activity in DCs was assessed by incubating DCs with rSj-C for various durations and using an MTS assay to measure cell viability and metabolic activity, serving as an indirect measure of protease activity.
The effect of rSj-C on antigen presentation was evaluated by incubating DCs with rSj-C followed by AWA, staining for MHC class II, and measuring fluorescence intensity by flow cytometry.
For induction of regulatory T cells, splenocytes were activated with anti-CD3, anti-CD28, IL-2, and anti-TGF-β antibodies. Flow cytometry was used to determine the proportion of CD4+CD25+Foxp3+ T cells.
Cytokine production (IL-4, IFN-γ, TGF-β) was stimulated with concanavalin A and quantified by ELISA from cell culture supernatants.
Statistical analyses were performed, with p < 0.05 indicating significant differences. Results rSj-C was taken up by DCs and localized to the cytoplasm, as shown by immunofluorescence microscopy. In comparison with controls, DCs incubated with rSj-C showed decreased viability, metabolic activity, and protease function, with maximal inhibition at three hours. Flow cytometry revealed that rSj-C reduced MHC class II surface expression on DCs exposed to AWA, indicating inhibited antigen presentation. In vivo, infection reduced the proportion of CD4+CD25+Foxp3+ T cells. rSj-C injection in infected mice significantly increased this proportion, while rSj-C alone in normal mice had no significant effect. Cytokine analysis showed that in infected mice, TGF-β was elevated. In infected mice injected with rSj-C, IL-4 and TGF-β were significantly increased compared to normal controls, indicating skewing toward Th2 and regulatory responses. Discussion DCs are key antigen-presenting cells, and cysteine proteases are critical in processing exogenous antigens for MHC II presentation. Cystatins, as natural protease inhibitors, can impair this process. The S. japonicum cysteine protease inhibitor studied here is recognized and internalized by DCs, inhibits their cysteine protease activity, and reduces MHC II expression, leading to suppressed antigen presentation. Regulatory T cells suppress immune responses through direct contact and cytokine secretion. Many parasites exploit this by inducing Tregs. In this study, rSj-C did not affect Tregs in normal mice but markedly increased them in infected hosts, suggesting a requirement for infection-related co-stimulatory signals. The parasite's modulation of the Th1/Th2 balance is another immune evasion strategy. Increases in Th2 cytokine IL-4 and immunosuppressive TGF-β in infected mice given rSj-C support a shift toward regulatory and Th2-mediated immunity, favoring parasite survival and reducing host pathology. Conclusion Cysteine protease inhibitor from Schistosoma japonicum is a parasite-derived immunosuppressive protein that can inhibit antigen presentation by DCs, promote regulatory T cell proliferation, and increase immunosuppressive cytokine production in infected hosts. This factor likely contributes to parasite immune evasion. Further work is needed to clarify the requirement for host co-stimulatory molecules in its MG-101 immunosuppressive function.