NP30 stimulates Th17 differentiation through DC in Schistosomiasis Japonicum

Summary The murine monoclonal anti‐idiotypic antibody, NP30, is a potential vaccine candidate against Schistosoma japonicum. Previous studies have revealed that NP30 has an immunoregulatory effect, but the underlying mechanism for this effect remains unknown. This study shows that NP30 induces dendritic cell (DC) maturation and increases the production of pro‐inflammatory cytokines. The expression of CD86 and MHC II was upregulated in DCs following stimulation with NP30 in vitro. Moreover, NP30 induced Th17 polarization by increasing the production of IL‐6 and TGF‐β. In vivo, Th17 differentiation was induced by the production of key pro‐inflammatory cytokines, including IL‐6and TGF‐β, from DCs of NP30‐immunized mice. These results indicate that NP30 promotes Th17 polarization through DC activation, preventing serious schistosomiasis.


| INTRODUC TION
In China, Schistosomiasis japonicum has been one of the most dangerous parasitic diseases for more than 2000 years. 1 For decades, praziquantel has been the only drug widely used for the clinical treatment and chemotherapy of Schistosomiasis japonicum in China. Although the application of praziquantel has greatly reduced the number of infected people, potential resistance against praziquantel limits its use. According to the survey data of 2013, there were still more than 185 000 cases of schistosomiasis and approximately 68 million individuals at risk of infection in China. 2 Considering the difficulty in blocking the transmission of S. japonicum with the sole use of praziquantel, looking for more effective candidates against S. japonicum infection is urgent. Recently, many researchers have been interested in the study of the vaccine of schistosomiasis.
Since Niels Kaj Jerne proposed the immune network theory, 3 it has been widely demonstrated that anti-idiotypic antibody strategy can be used to mimic cytokines by mimicking its receptor binding epitopes and the kind of anti-idiotypic antibody the "internal image" of antigen. Taking advantage of their exquisite specificity and high affinity, anti-idiotypic antibodies have been used as a strong tool in the treatment of infectious diseases and antipathologic vaccine research. 4 NP30 is the anti-idiotypic antibody of Schistosoma japonicum gut-associated antigen (GAA), which is a kind of IgM secreted by the hybridoma cells, according to Guan. 5 Based on the theory of the immune network, NP30 belongs to the family of β-class antiidiotype antibodies, which not only bind to the paratope but also represent a three-dimensional inversion of the nominal antigen and can therefore be used as surrogate antigens, for example for further immunizations or in ligand-binding assay applications. 6 In addition to being an "antigen reagent" in the diagnostic assays of Schistosomiasis japonicum for years in China, NP30 has also induced a protection rate of 50.46% against the challenge of Schistosoma japonicum cercariae. 7,8 The transfer of NP30 results in smaller granulomas around parasite eggs and lower portal pressure in vivo, which suggested that the anti-idiotypic antibody had the potential for the treatment of schistosome infection through an immune regulation mechanism. Nevertheless, to date, there are few reports on monoclonal anti-idiotypic antibodies for the vaccination of schistosomiasis due to the shortage of related research on mechanisms. 5 Depending on the production of many different associated antigens, Schistosoma japonicum stimulates the secretion of some proinflammatory cytokines to induce Th1 and Th2 cells, which play key roles in the infection immune responses. 9 During the acute stage of S. japonicum infection, schistosome antigens induce Th1-dominant cell-mediated immune response in the host. During the chronic infection stage, Th1-type cellular immunity shifts to Th2-type cellular immunity. 10 Particularly, some recent studies have revealed that Th17 cells play crucial roles in the pathology in schistosomiasis. 11 Moreover, in the context of severe egg-induced immunopathology, this differentiation of Th17 cells stimulates antigen-presenting cells (APCs) to secret some pro-inflammatory cytokines. 12,13 APCs, especially DCs, are useful for studying the mechanisms underlying the immune regulation against schistosomiasis. It has been reported that some anti-idiotypic antibodies upregulate the coreceptors of DCs and sustain CD4 + lymphocyte activation through binding to DCs. 14 In previous studies, we found that the immunization of NP30 can enhance not only Th2 but also Th1 differentiation, and at the same time, the binding of DC with NP30 was detected. However, the outcome of DC exposure to NP30 and the differentiation of Th17 have not yet been documented. Our hypothesis is that NP30 may stimulate Th17 differentiation through increasing the expression of some particular surface molecules of DCs.
In this study, we detect the expressions of costimulatory molecules on DCs' cytokine productions and the differentiation of CD4 + T cell cultured with dendritic cells taken from normal or NP30immunized mice. The results indicate the restricted activation state of DCs stimulated with NP30 and production of nonpathogenic Th17.

| Ethics statement
All experiments were performed in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals, and all efforts were made to minimize animal suffering. All animal procedures were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University for the use of laboratory animals.

| Bone marrow-derived DC (BMDC)
Bone marrow cells were flushed from the femurs and tibias of BALB/c mice, RBC were lysed with Tris-ammonium chloride buffer, and the remaining cells were cultured at a concentration of 1 × 106 cells/ml in 10 mL of complete-RPMI 1640 medium containing 10% heat-inactivated FBS, 100 U/mL penicillin, 100 mg/mL streptomycin with 10 ng/mL GM-CSF and 5 ng/mL IL-4 (PeproTech, Rocky Hill, NJ, USA). Two days after seeding, an additional GM-CSF and IL-4 were added to cultures. At day 9, nonadherent/semi-adherent cells were harvested, CD11c + cells were sorted with anti-CD11c-Cy5.5 (eBioscience, San Diego, CA, USA) by FACS, and the purity of the sorting was approximately 95%.

| Statistical analysis
One-way ANOVA was used to determine the statistical analysis of the differences between groups. P values of <.05 were considered significant and were calculated with GraphPad Prism 5.0.

| NP30 binding to DC in vitro
In a first series of experiments, we evaluated the possible interaction of NP30 with immune cells. To this purpose, DCs and CD4 + T cells were mixed with NP30 for 24 hours, and after addition of IgM-FITC, NP30 surface binding and intracellular uptake were determined by cytofluorimetric analysis and expressed as MFI. The results showed that DCs showed the higher level of NP30 binding and uptake. On the contrary, CD4 + T cells did not bind NP30 significantly ( Figure 1).
Binding of NP30 resulted marginal (MFI ranging from 10 to 15) in each cell population evaluated.

| CD86 and MHCII expressions on DCs are upregulated following stimulation with NP30 in vitro
To detect whether NP30 exerts effects on DCs, the expressions of

| Pro-inflammatory cytokines are produced in response to NP30 in vitro
Only maturation is not sufficient to cause DCs capable of inducing T-cell differentiation. Another functional property of DCs is the production of inflammatory mediators. Considering the strong relationship of S. japonicum-induced immunopathology with high levels of IL-17, we detected the optimal Th17 cytokines, such as IL-6, IL-23 and TGFβ, in different groups of DCs. The results showed that DCs in the presence of NP30 or SEA produced much more IL-6 and TGFβ than control DCs, and the production of IL-23 was also higher than control DCs (Figure 3). Because optimal Th17 differentiation and IL-17 expression were promoted with the combination of IL-6, TGFβ and IL-23, these results suggest that DCs stimulated with NP30 and SEA have the ability to direct DCs towards a Th17 response.

| NP30 induces Th17/Th2 cell differentiation in DC-CD4 + T cell cocultures
To examine the effects of NP30 on the ability of DCs to stimulate CD4 + T cells, BMDCs and syngeneic naïve CD4 + T cells were incubated together with NP30 or SEA. The cytokine productions of different culture supernatants were analysed after 72 hours. Compared with control DCs, the stimulation of CD4 + T with DC NP30 resulted in more IL-4, IL-6 and IL-17 production, and IL-23 production was a little high ( Figure 4). The observation that DC NP30 is a potent stimulator of CD4 + T cells to produce more IL-4, IL-6, IL-17 and IL-23 reveals that the differentiation of Th17 and Th2 is induced by DC NP30 .

| NP30 regulates the secretion of proinflammatory cytokines from DCs in vivo
Thus far, the data reveal that NP30 induces potent Th17 and Th2 responses in vitro, and Th17 and Th2 may be major contributors to the schistosomiasis-associated pathology of NP30-immunized mice. To

| NP30 alters antigen-specific T-cell polarization primed by DCs in vivo
Naïve CD4 + T cells were stimulated with syngeneic DC NP30 collected from NP30-immunized mice sacrificed at 0 (normal group were not infected with cercariae), 4 or 7 weeks after infection. As shown in in IL-10 production in nonpathogenic Th17 cells in vivo, we isolated CD4 + T cells from mice. As shown in Figure 6E, NP30 immunization induced an increase in IL-10 + IL-17 + cells (within total CD4 + IL-17 + T cells) along with an increase in total IL-17 + cells ( Figure 6B and E).   was not only a potential vaccine candidate against S. japonicum but also had potential for the treatment of schistosomiasis. 5 Although we have already noticed that NP30 has the ability to improve Th1 and Th2 differentiation, these results are far from a satisfactory answer to the question of how NP30 produces significant protection to S. japonicum.

| D ISCUSS I ON
During the past few years, many studies have proven that through the expression of chemokines and the secretion of cytokines, the responses of Th17 cells dominate and play key roles in the host defence in response to S. japonicum. 15,16 Because Th17 has become an important supplement to Th1/Th2 paradigm, 10  In the present study, we demonstrated that NP30 binds to DC, possibly produces phenotypic and functional changes on DC, modulating DC capacity to induce an important response of CD4 + T cells.
To further clarify the mechanism that leads to Th17 cell differentiation after exposure to NP30, we used BMDCs, which were cultured with NP30, SEA or LPS in vitro. BMDC exposure to LPS induced an increase in the expressions of costimulatory (CD40, CD80 and CD86) molecules. NP30 enhanced the expressions of CD86 and MHCII even higher than LPS; however, significant differences in CD40 and CD80 expressions between the NP30 treatment group and control group were not observed. Previous articles reported that SEA stimulated a minor increase in MHCII on DC, but not a major enhancement  In the 7-week post-infection groups, cells from NP30-immunized mice stimulated more IL-5 and IFNγ but less IL-6 than control mice. The results are shown as the mean of four experiments+standard error of the mean. *P < .05 and **P < .01 compared to uninfected control group mice immunized with PBS As we know, naïve CD4 + T cells can be activated by some costimulatory signals expressed on the surface of DCs. 29 Prior studies showed that CD80 and CD86 play key roles in the initiation of immune responses, especially some immune-mediated diseases. The exposure of costimulatory factors (CD80 or CD86) to TCR (T-cell   receptor) can produce T-cell differentiation, such as Th1 and Th2. Recently, some studies have revealed that mice deficient in both CD86 and CD80 failed to stimulate the differentiation of Th17, although the mechanisms are still not clear. 30 However, another report indicated that CD86 but not CD80 enhanced the secretion of IL-17, and the blockade of CD86 significantly suppressed splenocyte IL-17 production. 31 Our data suggest that NP30 stimulates the expression of CD86, but not CD80, in DCs and induces Th17 and Th2 differentiation. Therefore, it is possible that NP30 promotes the differentiation of Th17 by upregulating the expression of CD86.  32 Our hypothesis is that the differentiation of Th17 induced by NP30 without the expression of IL-23 produces only mild pathogenic appearances. Therefore, we examined the expression of IL-23 produced by DCs and CD4 + T cells from NP30-immunized mice. In agreement with our hypothesis, our findings indicate that NP30 immunization increased the expression of IL-17 cells with high increased IL-23 level. Although the relative contributions of IL-6, TGFβand IL-23 to Th17 differentiation are confirmed, our data indicate that the Th17 polarization induced by NP30 accompanied only the increased expression of IL-6 and TGFβ without much IL-23. These results confirm that the differentiation of Th17 stimulated by NP30 without the presence of much IL-23 induces a mild pathogenic form. Additionally, nonpathogenic Th17 cells differentially also express anti-inflammatory IL-10. 34 Although the fraction of IL-17A-producing cells within the IL-10-producing cell population was relatively small, this fraction represented up to 30% of all IL-17A producing T cells. 35  However, the mechanism should be clarified in further studies.
In summary, the present study indicates that NP30 stimulates naïve CD4 + T cell differentiation by modulating DC costimulatory molecule expression and cytokine production, leading to an upregulation of the nonpathologic Th17-mediated immune response. These findings may increase our understanding of the role of NP30 as a potential therapeutic target.

ACK N OWLED G EM ENTS
This work was supported by National Natural Science Foundation of China grant 30972573. The authors declare that they have no competing interests.