Regulatory Effect Of MicroRNA-449B On HMGB1-mediated Immunodysfunction Of Dendritic Cell In Septic Mouse

Objective: To investigate the roles of miR-449 b in immunoregulation of dendritic cell(DC)mediated by high mobility group box-1 protein(HMGB1),and further explore the significance of miR-449 b as the key regulatory molecuole in immunodysfunction in mouse models of sepsis. Methods: 1. DCs isolated from the spleen of normal mice were treated with HMGB1 of different dose(10,100 and 1000ng/ml) for different duration(24, 48, and 72 h). Expressions of co-stimulatory molecules including CD80 and CD86, and MHC-II on the surface of DCs were analyzed with flow cytometry(FCM). Cytokines, including IL-12 and IL-10, in the culture supernatant of DCs were analyzed with ELISA. Meanwhile expression level of miR-449 b in DCs was measured with QPCR. 2. DCs were transfected with miR-449 b mimics(miR-449b), negative control mimics(miR-NC), miR-449 b inhibition(In-miR-449b) and negative control inhibition(In-miR-NC). After stimulated with HMGB1(100ng/ml for 48 h), the expression level of costimulatory molecules and MHC-II on DC surface were analyzed with FCM. The apoptosis rate of DC was also analysised with FCM. Levels of IL-12 and IL-10 in culture supernatant were analyzed with ELISA. Moreover, the capacity of DC to modulate the proliferation and differentiation of T cells was assessed with mixed lymphocyte reaction. Further,expression levels of Notch1 and Sirt1 were assessed with western blotting as the target proteins of miR-449 b. 3. Experimental sepsis was induced in mice using the cecal ligation and puncture(CLP) method. miR-449 b antagomir and antagomir NC were injected 48 h and 120 h respectly after procedure via the tail vein at dose of 8mg/kg body weight in 300 μl volumes. LPS was injected intraperitoneally 7 days later.The proportion of matural DC in spleen mononuclear cells, the level of cytokines including IL-10, IL-12 in serum were analyzed 6 h after the second attack with LPS.Moreover, the expression of co-stimulate moleculars including MHC-II, CD80, CD86 on surface, the apoptosis rate, as well as the capacity to stimulate the proliferation and differentiation of T cells were analysed in splenic DC from model mice. And the expression of Notch1 and Sirt1 were analyzed with western blotting. Results: 1. the co-stimulatory molecules expressions and the level of IL-12 were markedly upregulated after DC were treated with HMGB1(100 ng/m L at 48h),thus they were decreased after DC were treated with HMGB1(1000 ng/mL at 72h).The apoptosis rateand the level of IL-10 were elevated with the increase of dosage and duration(P<0.05). The expression of miR-449 b in DC was decreased at 12 h and 24 h, but increased at 48h(P<0.05). 2. miR-449 b over-expressing could up-regulate significantly the expression level of CD86(P<0.05) and increase the apoptosis of DC(P<0.01). the capacity to modulate T-cell proliferation and differentiation was also significantly inhibited(P<0.05). Further, the expression of Notch1 and Sirt1, the target proteins of miR-449 b, was inhibited. 3. In vivo inhibiting of miR-449 b by antagomiRs improves late sepsis survival. Furthermore, inhibiting miR- 449 b via antagomir injection can obviously increase the proportion of DC derived from spleens of mouse late sepsis model. Meanwhile, DC from mmu-miR-449 b antagomir group have higher expression of MHC-II and, CD80(all P<0.05) and more production of IL-12(P<0.05). The rate of apoptosis and level of IL-10 decreased in mmu-miR-449 b antagomir group(P<0.05). The ability of DC from mmu-miR-449 b antagomir group to promote T cell proliferation and polarize to Th1 was enhanced. Furthermore, the target proteins, Notch1 and Sirt1, were elevated in miR-449 b antagomir group.Conclusions: miR-449 b could negatively regulate DC maturation and activation induced by HMGB1 and promote the apoptosis. Down-regulation of miR-449 b could effectively improve the immunodysfunction of DC from the late sepsis mice model,and thus improve late sepsis survival. The experimental results indicate that miR-449 b might be a potential therapeutic target for modulating immune dysfunction in sepsis.