■目的是探索鞭毛蛋白-TLR5复合物信号是否可以通过TRIF-ERK1/2途径增强髓样DC的抗原呈递能力,以及该通路与肠黏膜炎症反应的相关性。
■将小鼠骨髓来源的DC系DC2.4分为四组:对照组(BC)为正常培养的DC2.4细胞;鞭毛蛋白单信号刺激组(DC2.4CBLB502)为培养过程中鞭毛蛋白衍生物CBLB502刺激的DC2.4细胞;TLR5-鞭毛蛋白复合物信号刺激组(TLR5过TRpeptlflag-TLLR5基因2.4-1ovWB检测各组细胞中TRIF和p-ERK1/2蛋白的表达;CCK8检测各组细胞增殖;流式细胞术检测表面分子MHCI、MHCII、各组细胞CD80、86;ELISA法检测各组细胞因子IL-12和IL-4的程度。
■与BC组相比,DC2.4+CBLB502组,和ov-TLR5-DC2.4+CBLB502+Pepinh-TRIFTFA组,在ov-TLR5-DC2.4+CBLB502组中,TRIF蛋白和p-ERK1/2蛋白的表达显著上调(TRIF:p=0.02,=0.007,=0.048)(ERK1:p<0.001,=0.0003,=0.0004;ERK2:p=0.0003,=0.0012,=0.0022).ov-TLR5-DC2.4+CBLB502组细胞增殖活性较其他组增强(p=0.0001,p<0.0001,p=0.0015);同时,表面分子MHCI,MHCII,DCs上CD80、86上调(p<0.05);IL-12和IL-4细胞因子的分泌增加,具有显著差异(IL-12:p<0.0001,p<0.0001,p=0.0005;IL-4:p=<0.0001,p=<0.0001,p=0.0001)。然而,ov-TLR5-DC2.4+CBLB502+Pepinh-TRIFTFA组,用TRIF信号干扰处理,显示细胞内TRIF蛋白和p-ERK1/2蛋白减少,以及细胞增殖能力和表面刺激分子的下降,IL-12和IL-4细胞因子的分泌减少(p<0.05)。
■鞭毛蛋白-TLR5复合信号刺激后,髓系树突状细胞中TRIF蛋白和p-ERK1/2蛋白表达显著上调,伴随着DCs增殖活性和成熟度的增加,增强抗原呈递功能,促炎细胞因子IL-12和IL-4的分泌增加。这一过程可以被TRIF信号的特异性抑制剂所抑制,提示TLR5-TRIF-ERK1/2通路可能在DCs中鞭毛蛋白介导的异常免疫应答和粘膜慢性炎症浸润中起重要作用,可以为我们后续的动物实验提供依据。
UNASSIGNED: The objective is to explore whether the flagellin-
TLR5 complex signal can enhance the antigen presentation ability of myeloid DCs through the TRIF-ERK1/2 pathway, and the correlation between this pathway and intestinal mucosal inflammation response.
UNASSIGNED: Mouse bone marrow-derived DC line DC2.4 was divided into four groups: control group (BC) was DC2.4 cells cultured normally; flagellin single signal stimulation group (DC2.4+CBLB502) was DC2.4 cells stimulated with flagellin derivative CBLB502 during culture;
TLR5-flagellin complex signal stimulation group (ov-
TLR5-DC2.4+CBLB502) was flagellin derivative CBLB502 stimulated ov-
TLR5-DC2.4 cells with
TLR5 gene overexpression; TRIF signal interference group (ov-
TLR5-DC2.4+CBLB502+Pepinh-TRIFTFA) was ov-
TLR5-DC2.4 cells with
TLR5 gene overexpression stimulated with flagellin derivative CBLB502 and intervened with TRIF-specific inhibitor Pepinh-TRIFTFA. WB was used to detect the expression of TRIF and p-ERK1/2 proteins in each group of cells; CCK8 was used to detect cell proliferation in each group; flow cytometry was used to detect the expression of surface molecules MHCI, MHCII, CD80, 86 in each group of cells; ELISA was used to detect the levels of IL-12 and IL-4 cytokines in each group.
UNASSIGNED: Compared with the BC group, DC2.4+CBLB502 group, and ov-TLR5-DC2.4+CBLB502+Pepinh-TRIFTFA group, the expression of TRIF protein and p-ERK1/2 protein in ov-TLR5-DC2.4+CBLB502 group was significantly upregulated (TRIF: p = 0.02, = 0.007, = 0.048) (ERK1: p < 0.001, =0.0003, = 0.0004; ERK2:p = 0.0003, = 0.0012, = 0.0022). The cell proliferation activity in ov-TLR5-DC2.4+CBLB502 group was enhanced compared with the other groups (p = 0.0001, p < 0.0001, p = 0.0015); at the same time, the expression of surface molecules MHCI, MHCII, CD80, 86 on DCs was upregulated (p < 0.05); and the secretion of IL-12 and IL-4 cytokines was increased, with significant differences (IL-12: p < 0.0001, p < 0.0001, p = 0.0005; IL-4: p = < 0.0001, p = < 0.0001, p = 0.0001). However, the ov-TLR5-DC2.4+CBLB502+Pepinh-TRIFTFA group, which was treated with TRIF signal interference, showed a decrease in intracellular TRIF protein and p-ERK1/2 protein, as well as a decrease in cell proliferation ability and surface stimulation molecules, and a decrease in the secretion of IL-12 and IL-4 cytokines (p < 0.05).
UNASSIGNED: After stimulation of flagellin protein-TLR5 complex signal, TRIF protein and p-ERK1/2 protein expression in myeloid dendritic cells were significantly up-regulated, accompanied by increased proliferation activity and maturity of DCs, enhanced antigen presentation function, increased secretion of pro-inflammatory cytokines IL-12 and IL-4. This process can be inhibited by the specific inhibitor of TRIF signal, suggesting that the TLR5-TRIF-ERK1/2 pathway may play an important role in abnormal immune response and mucosal chronic inflammation infiltration mediated by flagellin protein in DCs, which can provide a basis for our subsequent animal experiments.