| OBJECTIVE:To investigate the anti-hepatitis B virus immune responses in BALB/c mice immunized with uric acid and dendritic cells (DCs) pulsed with HBV surface antigen or HBS28-39 epitope peptide. And the immune effect of uric acid was studied on the maturation and the biological function of murine bone-marrow derived dendritic cells (BMDCs) in vitro and its possible mechanism was explored involving TLRs mRNA and MAPKs or NF-κB signal pathway;METHODS:1.DC were cultured combinated rmGM-CSF, rmIL-4 with uric acid or LPS or RMPI-1640. Cell phenotypes were analyzed by flow cytometry. MTT was used to detect the allogeneic T cell proliferation. IL-12p70 released by DC was detected using ELISA method.2.After treatment with uric acid for 48 hours, The total RNA of DC were were extracted using TRIzol Reagent according to the manufacturer's protocol. The mRNA expression of TLR2, TLR3, TLR4 and IL-12p70 in DCs was performed by RT-PCR method.3.After treatment with NF-κB inhibitor PDTC or MAPKs inhibitors SB203580, PD98059 or SP600125 for 30 min, immature DCs were stimulated with uric acid (200μg) for 15, 30 or 45 min, cytoplasmic or nuclear extracts were collected and were subjected to immunoblot analysis with Abs specific for NF-κB p65 or phosphorylated forms of p38,ERK1/2,JNK. Cell lysates from DCs treatment with LPS or DMSO were serves as control. After treatment with uric acid and PDTC or SB203580, PD98059 or SP600125 for 48 hours, DC were collected. Cell surface marks were analyzed by flow cytometry. IL-12p70 production in cultured cells supernatants was detected using ELISA.4. DCs were pulsed or unpulsed with HBsAg protein or HBS28-39 (HBsAg -DCs, HBS28-39-DCs or unpulsed-DCs) in vitro. Then BABL/c mice were immunized with HBsAg-DCs or HBS28-39-DCs (1×106 per mouse) and uric acid, injected through the tail vein of each mouse. The doses of uric acid were 100, 200 or 400μg. The mice in control groups were immunized with HBsAg-DCs or HBS28-39-DCs alone, unpulsed-DCs alone or 200μg uric acid alone or PBS alone. The immunization was repeated after 7 days. HBsAg-specific cytotoxic T lymphocytes (CTLs) in vivo were determined by the CFSE labed spleen lysis assay. Spleen cells or spleen T cells were isolated, and re-stimulated in vitro with HBsAg or HBS28-39 for 72h. Production of IFN-γand IL-4 secreted by Spleen cells were determined by ELISA method; proliferations of spleen T cells were detected by flow cytometry.RESULTS:1.DC were cultured successfully and were identified. Uric acid (100, 200, 400μg/ml) could increase the CD83, CD86, IA/IE molecules on DC and the IL-12p70 level in the culture supernatants(P<0.05). Uric acid (100, 200, 400μg/ml) can promote the allogeneic T cell proliferation at the T: DC rate 5:1,10:1,20:1( P<0.05). But uric acid (70μg/ml) has not those effect (P>0.05).2. The expression of TLR2 mRNA,TLR3 mRNA,TLR4 mRNA reduced significantly in DC treated with uric acid (200μg/ml)( P<0.05, vs negative control) . Uric acid could increase the IL-12p70 mRNA expression of DC (P<0.05 , vs negative control). The effect of uric acid was similar with LPS stimulation on DC (P<0.05).3.(1)Increased expression of NF-κB p65 and the phosphorylation of the p38,ERK1/2 and JNK in DCs nuclear or cytoplasmic extracts, within 15 min of uric acid conditioning of immature DC. In 30 min, the peak of these proteins was appear, then in 45 min, the expression was inclined. Pretreatment of DC with SB203580,SP600125, PD98059 or PDTC, blocked the expression of NF-κBp65 and phosphorylation of p38, ERK1/2 and JNK, respectively, in response to uric acid stimulation.(2)Pretreatment of DC with SB203580,SP600125 or PDTC, reduced the uric acid induced-CD83,CD86,IA/IE up-regulation and inhibit the effect of uric acid on IL-12p70 secretion(P<0. 05 or 0.01). SB203580 and PDTC possessed a significant inhibition effect on uric acid. Otherwise, PD98059 increase the up- regulation of CD83,CD86,IA/IE and IL-12p70 production induced by uric acid(P<0.05).4. The cytotoxicities of HBsAg-specific CTLs, generated after immunization of HBsAg-DCs and uric acid, were significantly stronger than that in the control groups(p<0.01=. The percent of lysis in the mice treatment with uric acid(400μg) and HBsAg-DCs was 32.45%±11.63%, 74.56%±12.38%, respectively, in the first or second week. Compared with control groups, immunization with uric acid and HBsAg-DCs, enhanced proliferations activity of spleen T cells (p<0.01), to HBsAg re-stimulation, the level of IFN-γsecreted by spleen cells was increased(p<0.01) and IL-4 level was decreased(p<0.01). Uric acid could enhance the immune effect to HBsAg-DCs in a dose depentment manner. Also, uric acid could promote the anti-HBV immune effect to HBS28-39-DCs, which could elicit a similar degree immune response with HBsAg-DCs.CONCLUSIONS1.Uric acid could promote the phaenotype maturation,and the immune function maturation, including allostimulatory capacity and the secretion capacity of IL-12p70; while the activities of uric acid was in relation to its dose range.2.Uric acid could down-regulate the mRNA expression of TLR2, TLR3, TLR4, and up-regulate the IL-12 p70 mRNA expression. DC maturation to uric acid maybe involve TLRs.3.The p38 MAPK,ERK1/2,JNK and NF-κB signal pathways were activated after uric acid signaling of immature DC . Blocking of p38,JNK,NF-κB pathway inhibited uric acid-induced surface marker changes and IL-12p70 cytokine production. Blocking of ERK1/2 enhaced the stimulation effect of uric acid on DC . Uric acid control the balance of phosphorylation of the three MAPK and NF-κB, A possible mechanism of the DC maturation to uric acid may involve modulation of the threshold and duration of MAPK and NF-κB signaling.4. Uric acid can strongly enhance anti-HBV immune response to DCs vaccine, including HBsAg-specific CTLs and Th1 immune response. Uric acid may serve as an effective adjuvant of DCs vaccine against HBV infection.
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