Chlamydia abortus Pmp18.1 Induces IL-1β Secretion by TLR4 Activation through the MyD88, NF-κB, and Caspase-1 Signaling Pathways.
Chlamydia abortus Pmp18.1 Induces IL-1β Secretion by TLR4 Activation through the MyD88, NF-κB, and Caspase-1 Signaling Pathways.
复制标题
DOI：
10.3389/fcimb.2017.00514
复制
发表时间：
2017
影响因子：
5.7
通讯作者：
Eko FO
中科院分区：
文献类型：
--
作者：
Pan Q;Zhang Q;Chu J;Pais R;Liu S;He C;Eko FO
多态性膜蛋白D（Pmp 18 D）是一种保守的160 kDa外膜蛋白，在流产衣原体的发病机制中起着重要作用。我们已经确定了一个N-末端片段Pmp 18 D（指定Pmp18.1）作为一个可能的亚单位疫苗抗原。在这项研究中，我们通过研究Pmp18.1在树突状细胞中诱导先天性免疫应答的能力以及参与rPmp18.1诱导的IL-1β分泌的信号通路来评估Pmp18.1的疫苗潜力。接下来，我们研究了VCG与更成熟的Th 1促进佐剂CpG和FL相比对rPmp 18.1介导的先天免疫激活的免疫调节作用。最后，观察靶向TLR 4、MyD 88、NF-κB p50和Caspase-1 mRNA的siRNA对DC分泌IL-1β细胞因子的影响。在存在或不存在VCG或CpG或FL的情况下，用rPmp 18.1刺激骨髓来源的树突状细胞（BMDC），并使用多重细胞因子ELISA测定来评估产生的细胞因子的大小。通过流式细胞术分析共刺激分子和Toll样受体（TLR）的表达。通过Western免疫印迹分析评估髓样分化因子88（MyD 88）、核因子κ β（NF-κB p50/p65）和Caspase-1的细胞内水平定量，同时通过共聚焦显微镜评估NF-κB p65核转位。结果显示，用rPmp 18.1刺激DC引起促炎细胞因子的分泌，并上调与DC成熟相关的TLR和共刺激分子的表达。此外，rPmp 18.1可激活经处理的DC中MyD 88、NF-κB p50和Caspase-1的表达以及NF-κB p65的核表达。此外，靶向TLR 4、MyD 88、NF-κB p50和Caspase-1 mRNA的siRNA显著降低了它们在BMDCs中的表达水平，导致IL-1β细胞因子分泌减少，强烈表明它们参与了rPmp 18.1诱导的IL-1β细胞因子分泌。这些结果表明，C. Pmp 18.1通过MyD 88、NF-κB和Caspase-1信号通路激活TLR 4，诱导IL-1β分泌，可能是一种潜在的C.流产疫苗候选人。随后将在适当的动物模型中，使用VCG作为免疫调节剂，在免疫和攻击后评价Pmp 18.1的疫苗潜力。
The polymorphic membrane protein D (Pmp18D) is a 160-kDa outer membrane protein that is conserved and plays an important role in Chlamydia abortus pathogenesis. We have identified an N-terminal fragment of Pmp18D (designated Pmp18.1) as a possible subunit vaccine antigen. In this study, we evaluated the vaccine potential of Pmp18.1 by investigating its ability to induce innate immune responses in dendritic cells and the signaling pathway(s) involved in rPmp18.1-induced IL-1β secretion. We next investigated the immunomodulatory impact of VCG, in comparison with the more established Th1-promoting adjuvants, CpG and FL, on rPmp18.1-mediated innate immune activation. Finally, the effect of siRNA targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in DCs on IL-1β cytokine secretion was also investigated. Bone marrow-derived dendritic cells (BMDCs) were stimulated with rPmp18.1 in the presence or absence of VCG or CpG or FL and the magnitude of cytokines produced was assessed using a multiplex cytokine ELISA assay. Expression of costimulatory molecules and Toll-like receptors (TLRs) was analyzed by flow cytometry. Quantitation of intracellular levels of myeloid differentiation factor 88 (MyD88), nuclear factor kappa beta (NF-κB p50/p65), and Caspase-1 was evaluated by Western immunoblotting analysis while NF-κB p65 nuclear translocation was assessed by confocal microscopy. The results showed DC stimulation with rPmp18.1 provoked the secretion of proinflammatory cytokines and upregulated expression of TLRs and co-stimulatory molecules associated with DC maturation. These responses were significantly (p ≤ 0.001) enhanced by VCG but not CpG or FL. In addition, rPmp18.1 activated the expression of MyD88, NF-κB p50, and Caspase-1 as well as the nuclear expression of NF-κB p65 in treated DCs. Furthermore, targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in BMDCs with siRNA significantly reduced their expression levels, resulting in decreased IL-1β cytokine secretion, strongly suggesting their involvement in the rPmp18.1-induced IL-1β cytokine secretion. Taken together, these results indicate that C. abortus Pmp18.1 induces IL-1β secretion by TLR4 activation through the MyD88, NF-κB as well as the Caspase-1 signaling pathways and may be a potential C. abortus vaccine candidate. The vaccine potential of Pmp18.1 will subsequently be evaluated in an appropriate animal model, using VCG as an immunomodulator, following immunization and challenge.
登录
查看更多内容
影响因子：
3.6
作者：
Hodgson A;Wan F
通讯作者：
Wan F
影响因子：
7.3
作者：
Corridoni D;Arseneau KO;Cifone MG;Cominelli F
通讯作者：
Cominelli F
影响因子：
4.4
作者：
Eko, FO;He, Q;Igietseme, JU
通讯作者：
Igietseme, JU
影响因子：
2.7
作者：
Jorgensen, Astrid Sissel;Rosenkilde, Mette M.;Hjorto, Gertrud M.
通讯作者：
Hjorto, Gertrud M.
影响因子：
5.5
作者：
Eko, F. O.;Ekong, E.;Igietseme, J. U.
通讯作者：
Igietseme, J. U.