Study Of Der P2Recombinant BCG Modulating Dendritic Cells’phenotype And Function In Mouse Asthma Model

At present, Th1/Th2imbalance and Th2bias are considered as one of themost important immunological pathogenesis of asthma. In recent years, immuneintolerance is a hot research topic of the pathogenesis of asthma. Dendritic cells(DCs) are effective antigen presenting cells (APCs). They could induce T cellsto differentiate and proliferate, and regulate immune tolerance effectively. Afterantigen recognition and uptake, the levels of MHC-II, costimulatory moleculesand multiple chemokines were elevated in DCs. The mature DCs interacted withT cells through MHC-peptide and T-cell receptor (TCR) complex, and inducednaive Th0cells to differentiate into Th subtype cells. With further research,more and more DC subsets were found in vivo. Because different subtypes ofDCs have different capacities of inducing T cells differentiation andproliferation, modulation of DC’s functions and phenotypes has been consideredas promising biological targets for asthma treatment. There were several researches demonstrating that modulation of functions and phenotypes of DCsby gene transfection or medical drugs could inhibit pathologies of asthma. Butthose methods have some serious problems in safety and operability in vivo.Therefore, it is important to explore new strategy with safety and effectivenessto modulate DCs’ phenotypes and functions for restoring the Th cell balance inasthma patients.BCG vaccine, which is the attenuated M. bovis strain vaccine, has been usedworld widely. The safety and immune stabilization of BCG have been confirmed.Evidences demonstrated that vaccination of BCG early could inhibit thedevelopment of allergic diseases in genetic susceptible population. T cells playcentral role in asthma pathological process, while the DCs are important inmodulation of T cells. Several studies confirmed that BCG’s immunomodulatoryeffects are closely related to its function of regulating DCs. DCs collected fromthe spleen of mice immunized by BCG could inhibit airway inflammation ofasthma model. Meanwhile, BCG killed by extended freeze-drying may increaseplasmacytoid dendritic cells in spleen, which regulate lung in ammation. Fromthis, it is feasible to explore asthma vaccines based on BCG. Since Der p2antigen of house dust mites are important allergen of asthma, we constructed arecombinant BCG which expressed the Der p2of house dust mites (Der p2rBCG). It was demonstrated in our previous study that Der p2rBCG couldsuppress asthmatic airway in ammation more effectively than BCG. Accordingto the results, we speculated that the Der p2rBCG was more effective than BCGin directing T cell differentiation and suppressing asthma symptoms throughmodulating DCs’ functions and phenotypes.The Notch signaling pathway is an important pathway that regulatesdevelopment with high conservation. In mammals, there are four Notch receptors (Notch1,2,3,4) and five ligands (Jagged-1, Jagged-2, and Delta-likeligands1,3,4). When the Notch receptors bind with the ligands, the Notchintracellular domain (NICD) translocates into the nucleus, where it interactswith transcription factor RBP-J. The proteins become a co-activatingtranscription complex when they recruit MAML1. Then, the complex regulatesthe transcriptions of the target genes. Notch receptors and ligands were involvedin the interaction between DCs and T cells. The effects produced by the differentNotch receptors and ligands are different. It has been reported that Delta-like1,4could promote naive T cells differentiate into Th1; while in the pro-Th2environment, the level of Jagged-2on the DC’s surface was elevated. Notch3induces Th1differentiation and Notch1,2were associated with Th2differentiation. According to the literatures, Mycobacteria infection could affectthe levels of Notch receptors. And researchers have found that BCG couldrecognize TLR9on DCs through their PRMPs. Then BCG affects theexpressions of Notch ligands in DCs through MyD88-dependent pathway, andthe regulation of Th cell differentiation and development.Whether Der p2rBCG’s immunological regulation was related to altering the Notch receptorsand ligands in DCs or not? It is worthy of further study.C-kit is a type III tyrosine kinase receptorts ligand stem cell factor (SCF)expressed on a variety of cell surface with a wide range of biological functions.They have an important impact on the differentiation of immune cells.The levelof c-kit and SCF were all elevated when the antigens stimulated the DCs. Recentresearches highlight an important role of c-kit in the regulation of expressions oftwo molecules in dendritic cells (DCs), interleukin-6(IL-6) and Jagged-2, whichcould promote Th2cell differentiation. There were few researches about therelationship between BCG, c-kit and SCF。It deserves further study whether Der p2rBCG affects Th cell differentiation through regulating DC’s cytokine levelsand adjusting the jagged-2expression or not. Our previous studies showed thatDer p2rBCG could suppress asthmatic airway in ammation. There are twopossible mechanisms. One is that Der p2rBCG elicits immune deviation fromTh2to Th1. The other is that Der p2rBCG induces antigen-specified regulatoryT cells. However, the functions of Der p2rBCG on the dendritic cells (DCs) inthis protective effect and in reprogramming T cell commitment still need furtherstudy. In this study, we explored the mechanisms of Der p2rBCG in modulatingDCs’ phenotypes and functions, in inducing Th cell differentiation and itssuppressing effect in asthma. These works provide the experimental theoreticalbases for the development of vaccine in clinical practice of asthma.The main results are as follows:1. Der p2rBCG modulates different DCs’ phenotypes and functions ofmouseFirstly, DCs originated from mice’s bone marrow, spleen and lung wereprimarily cultured. Then they were stimulated with Der p2rBCG for72hrsbefore being collected and analyzed by FACS. The results showed that Der p2rBCG could elevate the levels of MHC-II, CD80and CD86. It could activateDCs effectively. There are no significant differences between Der p2rBCG andBCG in the activation of DCs. Supernatants of DCs were collected and analyzedby ELISA. The results showed that both of Der p2rBCG and BCG could elevatethe expression level of IL-12in different DCs, but Der p2rBCG could inducemore TGF-β and IL-10than BCG. In addition, BCG could elevate more IL-6than Der p2rBCG. Mice were immunized with Der p2rBCG at day0. On day42, BMDC, Spleen DC, and lung DCs were collected for FACS analyzing ofCD80/CD86/MHCII. It was indicated that Der p2rBCG had the similar capacity of activating spleen DCs and lung DCs to BCG. But it has no significant effecton BMDC. After6days of culture, we collected the supernatants of spleen DCsand lung DCs and then analyzed the levels of several cytokines. The trends ofIL-12，TGF-β and IL-10were similar with studies in vitro. BCG could inducemore IL-6in Spleen DCs（p＜0.05）, but it did not influence IL-6in Lung DCs（p＞0.05）. There were no significant differences in IL-6of supernatants fromcultured spleen DCs and lung DCs between PS group and Der p2rBCG group.Furthermore, we found that Der p2rBCG immunization could recruit morepDCs in mice’s abdomen DLNs, while BCG could not.2. Der p2rBCG could regulate DCs’ function in inducing Th0todifferentiate into different subtypes of Th cells.Naive T cells were isolated from spleen in normal mice by CD4+CD62L+magnetic beads kits. The purity of the target cells was above85%. Th0cellswere co-cultured with DCs, which have been treated with PS/BCG/Der p2rBCG. Der p2antigen was added at the beginning of the DC-T cell co-culture.After72hours, T cells were collected and analyzed by FACS. Results showedthat the Der p2rBCG could effectively promote Th1cell differentiation andinhibit excessive Th2cell differentiation. Meanwhile, we found that BCG couldinduce Th17cell differentiation, while Der p2rBCG could not. Furthermore,Der p2rBCG induced more CD4+CD25+Foxp3+Tregs than BCG. In animalstudies, mice were immunized with PS/BCG/Der p2rBCG on day0.42dayslater, we set up asthma models with the mice. Lung draining lymph nodes werecollected and minced. FACS analyzing results showed that Der p2rBCG andBCG could decrease Th2excessive differentiation and increase Th1. Besides,we found Der p2rBCG could inhibit Th17differentiation, while BCG couldinduce it. It is worth noting that Der p2rBCG could induce more CD4~+CD25~+Foxp3~+Tregs than BCG.3. The mechanism of Der p2rBCG modulating DCs functionThe two parts mentioned above preliminarily demonstrated that Der p2rBCGcould modulate DC’s functions and phenotypes, and regulate Th0differentiatinginto different Th subtypes. Then, we investigated the exact molecularmechanism of this process. Real-Time PCR and Western blot results showed thatDll-4of DCs was elevated by Der p2rBCG and BCG, while the expressions ofDll-1,3were not influenced. Der p2rBCG was more efficient than BCG indown-regulating jagged-2. TLR-2and4antagonists could inhibit these changes.Der p2rBCG was weaker than BCG in inducing expression of c-kit in DCs.4. Der p2rBCG-DCs inhibits airway inflammation through immunologicalregulationTo ensure the immuno-regulating functions of DCs treated with Der p2rBCGin vivo, we did DCs transfer experiments. Firstly, mice were immunized withPS/BCG/Der p2rBCG. Then the DLNs’ DCs were collected and adoptivelytransferred into Der p2-sensitized mice, mice were all challenged by Der p2antigen. There are six groups in our experiment, including control group, asthmamodel group, Der p2rBCG-DC group, BCG-DC group, PS-DC group and Derp2rBCG-DC~+PC61group. The HE and AB-PAS results showed that Der p2rBCG-DCs inhibit airway inflammation, hypersecretion and AHR. Der p2rBCG-DCs could effectively elevate Th1cytokines and inhibit excessive Th2cytokines. The expression level of IL-10and TGF-β were increased by Der p2rBCG-DCs transferring. FACS analyzing results showed that the ratios andabsolute number of Th1cells and CD4~+CD25~+Foxp3~+Tregs were elevated inDer p2rBCG-DCs group, while the Th2cells decreased. It is worth noting thatthis effect was reversed by PC61. Conclusion1) Both of Der p2rBCG and BCG could activate DCs effectively byelevating the expression levels of MHC-II, CD80and CD86. Besides,Der p2rBCG immunization could increase the ratio and absolutenumber of pDC in mice’s abdomen DLNs.2) Der p2rBCG could modulate the DC’s capacities of inducing Th0differentiating into Th cells. DCs treated with Der p2rBCG or BCGcould produce more Th1cytokines than PS. But Der p2rBCG facilitateDCs to produce more regulatory cytokines such as TGF-β and IL-10andinduce CD4+CD25+Foxp3+T cells. Der p2rBCG has weaker capacity ofinducing IL-6than BCG.3) Der p2rBCG may regulate the level of Notch ligands and influence theTh1/2ratio through TLR signaling pathway.4) Der p2rBCG has weaker capacity of increasing IL-6through c-kit thanBCG. Finally, it induced less Th17differentiation.