Minjun Ji

Schistosoma japonicum egg antigens stimulate CD4 CD25 T cells and modulate airway inflammation in a murine model of asthma.

A number of epidemiological and clinical studies have suggested an inverse association between allergy and helminth infection, such as Schistosomiasis. Therefore, we hypothesize that Schistosoma japonicum egg antigens, a type of native antigen, can induce production of CD4+ CD25+ T cells with regulatory activity, modulating airway inflammation and inhibiting asthma development. The frequency of CD4+ CD25+ T cells was determined by flow cytometry for mice treated with ovalbumin (OVA), CD25+ depletion/OVA, schistosome egg antigens, schistosome egg antigens/OVA and for control mice. The ability of CD25+ T cells from these mice to suppress T‐cell proliferation and cytokine production was investigated both in vivo and in vitro. Results showed that the CD4+ CD25+ T cells of OVA‐treated mice exhibited impaired control of dysregulated mucosal T helper 2 responses compared to the controls (P < 0·05). Depletion of CD25+ cells accelerated OVA‐induced airway inflammation and increased the expression of interleukin (IL)‐5 and IL‐4. Treatment with schistosome egg antigens increased the number and suppressive activity of CD4+ CD25+ T cells, which made IL‐10, but little IL‐4. In a murine model of asthma, S. japonicum egg antigens decreased the expression of Th2 cytokines, relieved antigen‐induced airway inflammation, and inhibited asthma development. Thus, we provided evidence that S. japonicum egg antigens induced the production of CD4+ CD25+ T cells, resulting in constitutive immunosuppressive activity and inhibition of asthma development. These results reveal a novel form of protection against asthma and suggest a mechanistic explanation for the protective effect of helminth infection on the development of allergy.

Application of DNA-based diagnostics in detection of schistosomal DNA in early infection and after drug treatment

BackgroundResearch is now focused on identification of sensitive and specific diagnostic tests for early identification of schistosomal infection and evaluation of chemotherapy in field situations in China.ResultsThis study compared loop-mediated isothermal amplification (LAMP) with conventional PCR as DNA-based diagnostic techniques for the early detection of schistosomal DNA and the evaluation of chemotherapy. The results showed that both PCR and LAMP assays targeting a 301 base pair (bp) sequence of the highly repetitive retrotransposon, SjR2, amplified DNA from schistosomes but were unable to distinguish between schistosome species. LAMP and conventional PCR were shown to amplify the target sequence of the SjR2-pCR2.1 recombinant plasmid template with limits of detection of 10-4 ng and 10-2 ng, respectively, thus demonstrating the superior sensitivity of the LAMP method. Schistosoma japonicum DNA was detected in all serum samples obtained from the three experimental groups at 1 week post-infection by LAMP assay, while the rate of detection by conventional PCR ranged from 50% to 66%. The potential application of PCR and LAMP assays for the evaluation of artesunate and praziquantel chemotherapy was investigated. PCR was shown to be less sensitive for detection of schistosomal DNA in drug-treated rabbit sera than the LAMP method.ConclusionsThe data presented here indicate that LAMP is suitable for the detection of early infection in the groups primarily infected with Schistosoma japonicum, such as migrants, travellers, military personnel and the younger age groups. However, it is less suitable for evaluation of the efficacy of chemotherapy in the early stages because of its high sensitivity.

Immune Events Associated with High Level Protection against Schistosoma japonicum Infection in Pigs Immunized with UV-Attenuated Cercariae

Background The vaccination of radiation-attenuated Schistosoma japonicum cercariae can induce effective protection in artiodactyl, but the immune events related to protective immunity are not fully understood. To provide a paradigm for a human recombinant antigen vaccine, we have undertaken a vaccination and challenge experiment in pigs, which was recognized as an appropriate animal model in this type of study because of their similarity to human in immunology, and investigated the relative immune events induced by the radiation-attenuated S. japonicum cercariae. Methods and Findings We found that pigs immunized once with 400 µw UV-irradiated cercariae exhibited 63.84% and 71.82% reductions in worm burden and hepatic eggs respectively. Protective immunity in vaccinated pigs was associated with high level productions of IgM, total IgG, IgG1 and IgG2; IgG2 was significantly increased in the acute infection. IFN-γ levels could be elicited by immunization. At week 6 post-infection, IFN-γ, IL-4 and IL-10 levels also showed a dramatic rise synchronously in vaccinated pigs. Moreover, the granzyme b, nk-lysin, ifnγ, il4 and il10 mRNA levels in early skin-draining lymph nodes of immunized pigs were higher than those in pigs with non-irradiated cercariae infection. In addition, cytotoxicity-related genes in the mesenteric lymph nodes were significantly upregulated in vaccinated pigs in the acute infection. Conclusion/Significance Our results demonstrated that IFN-γ and IgG2 antibody production, as well as genes related to cytotoxicity are associated with the high level protection induced by UV-irradiated Schistosoma japonicum vaccine. These findings indicated that optimal vaccination against S. japonicum required the induction of IFN-γ, IgG2 antibody related to Th1 responses and cytotoxicity effect.

Multiple vaccinations with UV- attenuated cercariae in pig enhance protective immunity against Schistosoma japonicum infection as compared to single vaccination

BackgroundSchistosomiasis japonica is a major public health problem in the endemic areas of China, the Philippines, and Indonesia. To date, a vaccine has not been developed against this disease but immunization with UV-attenuated cercariae can induce a high level of protective immunity in Landrace/Yorkshire/Duroc crossbred pigs. To compare the efficacy of a single vaccination and multiple vaccinations with UV-attenuated Schistosoma japonicum cercariae, two groups of pigs received either one or three exposures to 10,000 cercariae attenuated with 400 μw UV.ResultsPigs with a single immunization had a 59.33% reduction in adult worm burden, a 89.87% reduction in hepatic eggs and a 86.27% reduction in fecal eggs at eight weeks post-challenge (P < 0.01). After three immunizations, protection increased to 77.62%, 88.8% and 99.78% reduction in adult worms, hepatic eggs and fecal eggs, respectively (P < 0.01). Humoral and cellular immunological parameters measured indicated that schistosome-specific IgG1 and IgG2 levels in the vaccinated groups were higher than in the infection-control group. Triple vaccinations resulted in higher levels of antibodies, especially IgG2, compared with a single vaccination and IFN-γ levels increased with repeated immunization with UV-irradiated cercariae.ConclusionThe high levels of protection against S. japonicum infection can be achieved with a UV-attenuated vaccine in pigs, and that three vaccinations were possibly more effective than a single vaccination. Moreover, triple vaccinations evoked a more vigorous IFN-γ response and a stronger antibody-mediated response, especially an increase in the levels of IgG2 antibodies.

Toll‐like receptor (TLR) 2 and TLR4 deficiencies exert differential in vivo effects against Schistosoma japonicum

Little is known about the functions of Toll‐like receptor 2 (TLR2) and TLR4 in innate/acquired responses to Schistosoma japonicum. Through in vivo study, the contributions of TLR2 and TLR4 to host immune responses during S. japonicum infection were investigated. Early infection experiments showed higher protein and mRNA levels of IL‐12, IFN‐γ and IL‐4 in dermal tissues and retroauricular draining lymph nodes respectively in TLR2−/− mice on day four post‐infection and opposite changes in TLR4−/− mice. In the acute infection with S. japonicum for 6 weeks, TLR2−/− mice manifested lower egg burden as well as the enhancement of T cell activation and upregulated expression of some cytotoxic genes, as assayed by Th1/Th2 cytokine secretion and DNA microarray analysis. Also, the opposite parasitological and immunological effects were observed in TLR4−/− mice. These results demonstrate that during S. japonicum infection, TLR2 and TLR4 might direct distinct adaptive immune responses since the early stage, which may lead to different infection outcomes.

Deficiency in TLR2 but not in TLR4 impairs dendritic cells derived IL-10 responses to schistosome antigens.

The purpose of this study was to observe the diverse functions of Toll-like receptors (TLRs) in responses to specific schistosome antigens. Bone marrow-derived dendritic cells (BMDCs) from TLR2-deficient (TLR2(-/-)) or TLR4-deficient (TLR4(-/-)) mice were activated with soluble schistosomule antigen (SSA) or soluble egg antigen (SEA). TLR2 mRNA expression was significantly increased in B6 BMDCs following SEA stimulation. TLR2-deficient BMDCs showed enhanced MHCII expression following SSA and SEA stimulation. TLR2-deficient but not TLR4-deficient BMDC failed to produce IL-12p70 and IL-10 in response to schistosome antigens. TLR2-deficient BMDCs induced a stronger CD4(+) T cell proliferative response. IL-4 and IL-10 expression was inhibited in CD4(+) T cells primed with TLR2-deficient BMDCs, while enhanced in TLR4-deficient BMDCs-primed CD4(+) T cells. These results suggest that TLR2 is essential for the establishment of the DC production of IL-12p70 and IL-10.

TLR2 directing PD-L2 expression inhibit T cells response in Schistosoma japonicum infection.

Toll-like receptor 2 (TLR2) was shown to be an important immune receptor involved in the recognition of schistosome antigens, especially soluble egg antigen (SEA). In mice models with Schistosoma japonicum acute infection, we observed enhanced T cell-mediated immune responses in TLR2 knock out (TLR2−/−) mice compared with B6 mice. In Schistosoma japonicum chronic infection models, programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2) expression as well as TLR2 expression gradually increased in B6 mice, while only PD-L2 expression significantly decreased in TLR2−/− mice. Meanwhile, Programmed Death 1(PD-1) expression on CD4+T cells was down-regulated in TLR2−/− mice after a large number of egg appeared. We also found that stimulation with schistosome antigens, especially SEA, could up-regulate PD-L2 expression on BMDCs in a TLR2-dependent manner in vitro. Schistosome antigens primed-BMDCs with impaired expression of TLR2 or PD-L2 could induce CD4+T cells to produce low level of IL-10 or high level of IFN-γ. Our results indicated that TLR2 signaling can direct PD-L2 expression on DCs, which binds to PD-1 mainly on CD4+T cells, to help inhibit T cells response in Schistosoma japonicum infection.

Proteomic analysis of schistosomiasis japonica vaccine candidate antigens recognized by UV-attenuated cercariae-immunized porcine serum IgG2

Many studies have showed that the radiation-attenuated cercariae (RAC) vaccine could induce the high protection of laboratory animals to resist the schistosoma infection by cellular and humoral mechanism. Here, we aimed to identify possible vaccine antigens by using specific IgG2 antibody from RAC-vaccinated pigs or vaccination and challenge pigs. The antigens from the schistosomal soluble worm antigen preparation (SWAP) recognized by the porcine IgG2 antibody were obtained using immunoprecipitation technique. These antigens were separated by 2-D electrophoresis, and 116 spots were successfully identified by MALDI-TOF MS from about 400 putative spots in gels. Among these spots, 113 spots could match to the Schistosoma japonicum. These identified proteins in four groups were classified by Gene Ontology (Go) database, and the mainly functions of these proteins were involved in binding, catalytic activity (thioredoxin peroxidase-2, et al.), signal transduction class (MAP Kinase, et al.), cell process (the heat shock 70-kDa protein 9B, et al.), and the intracellular component (tektin, et al.). Our methods suggested that it was possible to pull-down the interesting proteins recognized by specific antibodies. Our results may provide new clues for exploring the mechanism of high protection induced by RAC and shed some light on the research for anti-schistosomiasis japonica vaccine.

Immune responses result in misdiagnosis of Schistosoma japonicum by immunodiagnosis kits in egg-positive patients living in a low schistosomiasis transmission area of China.

BackgroundIn recent field surveys, we failed to detect the presence of specific antibody against Schistosoma japonicum in some egg-positive patients by commonly used immunodiagnostic kits. To find out whether low levels of specific antibody truly exist among egg-positive individuals and elucidate the underlying immune mechanisms, we carried out a cross-sectional epidemiologic study in a S. japonicum low transmission endemic area of Poyang Lake region, China and compared the humoral and cellular immune characteristics between S. japonicum high and low antibody responders.MethodsKato–Katz thick smear assay was used to determine the schistosomiasis status of 3,384 participants residing in two Poyang Lake region villages, Jiangxi, China. Among the 142 stool egg-positive participants, we identified low and high S. japonicum antibody responders with soluble egg antigen (SEA) and adult worm antigen (AWA) specific IgG levels by adopting ROC curve analysis. To compare the humoral and cellular immune responses between high and low S. japonicum antibody responders, serum specific antibody levels as well as the percentage of T lymphocyte subpopulation in PMBC, and cell stimulated cytokines (IFN- gamma and interlukin-10) were detected.ResultsEight S. japonicum egg-positive participants were defined as low antibody responders. Although the percentage of CD3+T cells in low responders was slightly higher and the percentage of CD4+ T cells, CD8+ T cells, the ratio of CD4+/CD8+ and CD4+ CD25+ Treg cells were lower than those in high responders, the differences between the two groups were not significant (P > 0.05). AWA -stimulated interlukin-10 level was significantly higher in high responders, while other cytokines did not show differences between two groups. For antibody profiles, except AWA specific IgA, significant differences of each antibody isotype between low and high responders were detected (P < 0.05).ConclusionsOur study confirmed that there are S. japonicum antibody low responders among schistosome egg-positive residents in S. japonicum low-transmission areas in China. Thus, mis-diagnosis using immune-diagnosis kits do exist. Significant differences of responding antibody levels between low and high responders were detected, while no major cellular response changes were observed.

Schistosoma japonicum infection induces macrophage polarization

Abstract The role of macrophages (Mφ) as the first line of host defense is well accepted. These cells play a central role in orchestrating crucial functions during schistosomal infection. Thus, understanding the functional diversity of these cells in the process of infection as well as the mechanisms underlying these events is crucial for developing disease control strategies. In this study, we adopted a Mφ polarization recognition system. M1 macrophage was characterized by expressing CD16/32, IL-12 and iNOS. M2 macrophage was characterized by expressing CD206, IL-10 and arg-1. In vivo (mouse peritoneal macrophages of different infection stages were obtained) and in vitro (different S. japonicum antigens were used to stimulate RAW264.7) were characterized by using the above mentioned system. NCA and ACA stimulated RAW264.7 express significantly higher levels of IL-12 while significantly higher levels of IL-10 were detected after soluble egg antigen (SEA) stimulation. The results showed that dramatic changes of antigen in the microenvironment before and after egg production led to macrophage polarization. Furthermore, through TLR blocking experiments, the TLR4 signaling pathway was found to play a role in the process of macrophage polarization toward M1. Our data suggest that macrophage polarization during S. japonicum infection had significant effects on host immune responses to S. japonicum.