DataSheet_1_Vibrio alginolyticus Triggers Inflammatory Response in Mouse Peritoneal Macrophages via Activation of NLRP3 Inflammasome.pdf

Vibrio alginolyticus is a food-borne marine Vibrio that causes gastroenteritis, otitis media, otitis externa, and septicemia in humans. The pathogenic mechanisms of V. alginolyticus have previously been studied in aquaculture animals; however, the underlying mechanisms in mammals remain unknown. In this study, an in vitro model of mouse peritoneal macrophages infected with V. alginolyticus was established. qPCR results revealed that V. alginolyticus induced the transcription levels of various cytokines, including IL-1β, IL-12, IL-18, TNF-α, IL-17, IL-6, IFN-γ, and IL-10, and the secretion level of IL-1β is the most significant. Inhibition assays with Ac-YVAD-CHO (a caspase-1 inhibitor) and Z-VAD-FMK (a pan-caspase inhibitor) were conducted to determine whether caspase-1 or caspase-11 is involved in V. alginolyticus-triggered IL-1β secretion. Results showed that IL-1β secretion was partly inhibited by Ac-YVAD-CHO and absolutely blocked by Z-VAD-FMK. To explore the sensed pattern recognition receptors, several NLR family members and the AIM2 receptor were detected and many receptors were upregulated especially NLRP3. Moreover, the NLRP3 protein displayed a puncta-like surrounding cell nucleus, which signified that the NLRP3 inflammasome was activated in response to V. alginolyticus infection. Inhibition assays with glyburide and CA-074 methyl ester (K+ outflow inhibitor and cathepsin B inhibitor) blocked IL-1β secretion, which demonstrated the essential role of the NLRP3 inflammasome in inflammatory response. To better understand how V. alginolyticus affects IL-1β release, the NLRP3 inflammasome was detected with doses ranging from 0.1 to 10 MOIs and time periods ranging from 3 to 12 h. Results showed that V. alginolyticus-mediated NLRP3 inflammasome activation was in a time- and dose-dependent manner and IL-1β release peaked at MOI of 1 for 12 h. Most importantly, blocking the NLRP3 inflammasome with inhibitors and the use of NLRP3-/- and caspase-1/11-/- mice could attenuate pro-inflammatory cytokine secretion, such as IL-1β, IL-6, IL-12, and TNF-α. Taken together, our study first found that the NLRP3 inflammasome plays vital roles in V. alginolyticus triggered inflammatory response in mouse peritoneal macrophages. This may provide reference information for the development of potential anti-inflammatory treatments against V. alginolyticus infection.

溶藻弧菌（Vibrio alginolyticus）是一种食源性海洋弧菌，可引发人类胃肠炎、中耳炎、外耳炎及败血症。既往研究已在水产养殖动物中解析了溶藻弧菌的致病机制，但哺乳动物体内的潜在致病通路仍未阐明。本研究构建了溶藻弧菌感染小鼠腹腔巨噬细胞的体外感染模型。实时荧光定量PCR（quantitative real-time PCR, qPCR）结果显示，溶藻弧菌可诱导多种细胞因子的转录水平显著上调，包括白细胞介素-1β（interleukin-1β, IL-1β）、白细胞介素-12（interleukin-12, IL-12）、白细胞介素-18（interleukin-18, IL-18）、肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）、白细胞介素-17（interleukin-17, IL-17）、白细胞介素-6（interleukin-6, IL-6）、干扰素-γ（interferon-γ, IFN-γ）及白细胞介素-10（interleukin-10, IL-10），其中IL-1β的分泌上调幅度最为显著。为明确半胱天冬酶-1（caspase-1）或半胱天冬酶-11（caspase-11）是否参与溶藻弧菌触发的IL-1β分泌过程，本研究分别采用Ac-YVAD-CHO（半胱天冬酶-1特异性抑制剂）与Z-VAD-FMK（泛半胱天冬酶抑制剂）开展抑制实验。结果显示，Ac-YVAD-CHO可部分抑制IL-1β的分泌，而Z-VAD-FMK则可完全阻断该分泌过程。为探究该过程中识别病原体的模式识别受体，本研究检测了多个NLR家族成员及AIM2受体的表达水平，发现多数受体均出现上调，其中NLR家族含pyrin结构域蛋白3（NLR family pyrin domain containing 3, NLRP3）的表达上调尤为显著。进一步观察发现，NLRP3蛋白呈现出围绕细胞核的点状聚集形态，提示溶藻弧菌感染可激活NLRP3炎症小体。为验证NLRP3炎症小体在炎症反应中的核心作用，本研究采用格列本脲（glyburide，钾离子外流抑制剂）与CA-074甲酯（CA-074 methyl ester，组织蛋白酶B抑制剂）进行抑制实验，结果显示二者均可阻断IL-1β的分泌，证实了NLRP3炎症小体在该炎症应答中的关键功能。为进一步明确溶藻弧菌调控IL-1β释放的具体特征，本研究设置了0.1至10的感染复数（multiplicity of infection, MOI）梯度以及3至12小时的感染时间梯度，检测NLRP3炎症小体的激活情况。结果表明，溶藻弧菌介导的NLRP3炎症小体激活呈时间与剂量依赖性，且当感染复数为1、感染时长为12小时时，IL-1β的释放达到峰值。尤为关键的是，通过抑制剂阻断NLRP3炎症小体，以及使用NLRP3基因敲除（NLRP3-/-）与半胱天冬酶-1/11双基因敲除（caspase-1/11-/-）小鼠，均可显著减弱IL-1β、IL-6、IL-12及TNF-α等促炎细胞因子的分泌。综上，本研究首次证实NLRP3炎症小体在溶藻弧菌感染小鼠腹腔巨噬细胞引发的炎症反应中发挥关键调控作用。该研究成果可为开发针对溶藻弧菌感染的潜在抗炎治疗策略提供重要参考依据。