The Effect Of Suppressive Oligodeoxynucleotides Induced Tolerogenic Plasmacytoid Dendritic Cells In The Experimental Autoimmune Neuritis

Objective Guillain-Barrés syndrome(GBS) is a frequently-occurring and severity disease of the nervous department. The pathogenesis of GBS is very complex and still unkonwn. The relationship between inflammation and immune tolerance plays an key role in the mechanism of GBS. Elucidating the mechanism of unbalance immune tolerance will be helpful for our understanding of GBS and exploring new strategies for related autoimmune diseases. Experimental autoimmune neuritis(EAN)is a CD4+ T cell-mediated autoimmune disease and can be established in using P0180-199 emulsified with complete Freund's adjuvant. EAN is a classic model to study GBS. Dendritic cells(DCs) are professional APCs and can be divided into plasmacytoid DCs(p DCs) and other DC. PDCs selectively expressing Toll like receptor(TLR) 7 and TLR9, mainly modulate the extent of the immune response or the maintenance of immunological homeostasis. Researchs have demonstared that the ligant of TLR9 is ODN. Typically, agonists of TLR9, Cp G oligodeoxynucleotides(ODN), can induce the activation of p DCs and promote Th1-type cytokine production.Previous study demonstrated a increased expression of TLR9 in EAN. The study also demonstrated that s ODN could ameliorate the clinical signs of an anminal model of RA. In this study, Quantitative PCR was performed to detect the m RNA expression of CD40, CD80 and CD86 and western blot was used to analyze expression of TLR9 and MyD 88. ELISA was used to detect the secretion of TFN-?, IL12,IL-4 and IL-6 in the supernatants. We are aimed to investigate weather suppressive ODN can induce the tolerogenic plasmacytoid cells and explore the possible mechanism. We also aim to study the effect of tolerogenic p DCs in the EAN.Method In this study, we isolated the p DCs from spleen of C57BL/6 mice using magnetic beads. Flow cytometry was used to test the purity of p DCs. Cp G ODN,suppressive ODN and control ODN were respectively added into the culture of P0180-199-treated p DCs. After 48 h, Quantitative PCR was performed to detect the m RNA expression of CD40, CD80 and CD86 in the p DCs with different treatment.p DCs were harvested to extract both cytoplasmic protein and nuclear protein.Western blot was used to analyze expression of TLR9,MyD 88,MAPK(p38,ERK1/2,JNK),PI3K/Akt, NF-?B and their phosphorylations.ELISA was used to detect the secretion of IFN-?,IL12 and IL-6 in the supernatants of p DCs cultures. The EANmodel was established in C57BL/6 mice using P0180-199 emulsified with complete Freund's adjuvant. On the 10 th day after the immunization, p DCs stimulated with different ODN were injected into the EAN model. The EAN model was evaluated daily. The survival curves was used to compere the surviving percentage.Result 1. PDCs were detected by Flow cytometry to determine the purity. The purity of p DCs were between 90%-93%. 2. Western blot was used to investigate the mechanism by which TLR9 signaling modulates p DCs. Results showed that s ODN modulated the immune response in P0180–199 peptides-stimulated p DCs via TLR9/NF-?B signaling pathway. Neither MAPK molecules(ERK, JNK and p38) nor PI3K/Akt were involved in the TLR9/NF- ?B signaling pathway. MyD 88 is necessary for s ODN but not for Cp G ODN to modulating the immune response in P0180–199peptides-stimulated p DCs. 3. The results of q PCR showed that P0180–199 peptides significantly upregulated the expression of CD40, CD80 and CD86 m RNAs in p DCs.Cp G ODN-treatment significantly enhanced the effects of P0180–199 peptides on p DCs while s ODN exerted the opposite effect. The IDO m RNA in P0180–199-stimulated p DCs was significantly upregulated by the treatment of s ODN. 4. The isolated p DCs were stimulated with P0180–199 peptides. Cp G ODN, s ODN or the control ODN were,respectively, added into the culture. After 48 h, the supernatants were harvested to investigate the effects of different ODN on secretion of Th1-type cytokines including IL-12p40, IL-6 and TNF-?using ELISA. The results showed that P0180–199 peptides significantly induced secretion of Th1-type cytokines in p DCs and Cp G ODN enhanced the effect of P0180–199 peptides on secre-tion of Th1-type cytokines. s ODN significantly inhibited the secretion of Th1-type cytokines. 5. On the 10 th day after the immunization, p DCs stimulated with different ODN were injected into the EAN model. s ODN-treated p DCs significantly promoted the recovery of EAN mice. The EAN mice were monitored daily until the 28 th day after the immunization. The survival curves showed the surviving percentage of animals was significantly lower in Cp G ODN group than EAN groups(p= 0.02). The clinical score of animals in s ODN group was significantly lower than EAN groups(p<0.05). The result showed that time to reach complete recovery(a score of <0.5 or less) was significantly less in s ODN group than EAN group, log-rank test, p < 0.001.Conclusion 1. p DCs participate in EAN via TLR9. 2. s ODN induce tolerogenic p DCs via TLR9/NF-?B signaling and the MyD 88 played a key role in this signaling.3.s ODN can induce tolerogenic p DCs via TLR9/NF- ?B signaling and ameliorated the experimental autoimmune neuritis. Because therapeutic applications of TLR9 agonists are available, our study provides new sights for the therapy of GBS and p DC-related autoimmune diseases.