Outer membrane protein P2 (OmpP2) of the virulent Haemophilus (Glaesserella) parasuis has been shown to induce the release of proinflammatory cytokines. The OmpP2 protein is composed of eight or nine surface-exposed loops, but it is unclear which of them participates in the OmpP2-induced inflammatory response. In this study, we synthesized linear peptides corresponding to surface-exposed loops L1–L8 of OmpP2 from the virulent H. parasuis SC096 strain to stimulate porcine alveolar macrophages (PAMs) in vitro. We found that both L7 and L8 significantly upregulated the mRNA expression of interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-17, and IL-23 and the chemokines CCL-4 and CCL-5 in a time- and dose-dependent manner. Additionally, we constructed ompP2ΔLoop7 and ompP2ΔLoop8 mutant SC096 strains and extracted their native OmpP2 proteins to stimulate PAMs. These mutant proteins induced significantly less mRNA expression of inflammatory cytokines than SC096 OmpP2. Next, the amino acid sequences of L7 and L8 from 15 serovars of H. parasuis OmpP2 were aligned. These sequences were relatively conserved among the most virulent reference strains, suggesting that L7 and L8 are the most active peptides of the OmpP2 protein. Furthermore, L7 and L8 significantly upregulated the NF-κB and AP-1 activity levels based on luciferase reporter assays in a dose-dependent manner. Therefore, our results demonstrated that both surface-exposed loops L7 and L8 of H. parasuis OmpP2 induced the expression of proinflammatory cytokines possibly by activating the NF-κB and MAPK signalling pathways in cells infected by H. parasuis.

中文翻译：

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表面暴露的环L7和L8的嗜血杆菌（Glaesserella）副猪嗜血杆菌OmpP2有助于促炎细胞因子的表达在猪肺泡巨噬细胞

副猪嗜血嗜血杆菌（Glaesserella）的外膜蛋白P2（OmpP2）已显示可诱导促炎性细胞因子释放。OmpP2蛋白由八个或九个表面暴露的环组成，但尚不清楚它们中的哪一个参与OmpP2诱导的炎症反应。在这项研究中，我们从副猪嗜血杆菌SC096菌株合成了与OmpP2的表面暴露环L1-L8相对应的线性肽，以体外刺激猪肺泡巨噬细胞（PAM）。我们发现L7和L8均显着上调白介素（IL）-1α，IL-1β，IL-6，IL-8，IL-17和IL-23的mRNA表达以及趋化因子CCL-4和CCL-5以时间和剂量依赖的方式。此外，我们构建了ompP2ΔLoop7和ompP2ΔLoop8突变SC096菌株，并提取了它们的天然OmpP2蛋白来刺激PAM。与SC096 OmpP2相比，这些突变蛋白诱导的炎症细胞因子的mRNA表达明显更少。接下来，比对来自副猪嗜血杆菌OmpP2的15个血清型的L7和L8的氨基酸序列。这些序列在最强毒的参考菌株中相对保守，表明L7和L8是OmpP2蛋白最活跃的肽。此外，基于萤光素酶报告基因分析，L7和L8以剂量依赖性方式显着上调NF-κB和AP-1活性水平。因此，我们的结果表明，副猪嗜血杆菌OmpP2的两个表面暴露环L7和L8都可能通过激活副猪嗜血杆菌感染的细胞中的NF-κB和MAPK信号通路来诱导促炎性细胞因子的表达。