NLRP12 通过负向调控 NF-κB 信号通路促进宿主抵抗铜绿假单胞菌角膜炎的炎症反应。

NLRP12 promotes host resistance against Pseudomonas aeruginosa keratitis inflammatory responses through the negative regulation of NF-κB signaling.

机构信息

Department of Laboratory, Longhua District Central Hospital, Longhua District, Shenzhen, China.

出版信息

Eur Rev Med Pharmacol Sci. 2018 Dec;22(23):8063-8075. doi: 10.26355/eurrev_201812_16496.

DOI:10.26355/eurrev_201812_16496

PMID:30556841

Abstract

OBJECTIVE

To investigate the role of NLRP12 in regulating Pseudomonas aeruginosa (P. aeruginosa) keratitis.

MATERIALS AND METHODS

Real-Time-PCR and Western blot were performed to measure the NLRP12 level in corneas and bone marrow-derived macrophages (BMDMs) of C57BL/6 (B6) mice. B6 mice received a subconjunctival injection of lentivirus expressing active NLRP12 (NLRP12-lentivirus) or Ctl-lentivirus (as control), followed by infection of P. aeruginosa. The clinical score, slit lamp and bacterial plate count of mice were evaluated. In addition, myeloperoxidase (MPO) was detected to assess the infiltration of polymorphonuclear neutrophil (PMN). Cytokine levels were measured by Real Time-PCR and ELISA. Meanwhile, the bacterial burden was also evaluated. The activation of NF-κB signaling was determined by pIκBα/IκBα levels based on Western blot and NF-κB-dependent Luciferase activity on the basis of Luciferase assays using 293T cells.

RESULTS

NLRP12 mRNA and protein levels were decreased in B6 corneas and BMDMs after P. aeruginosa infection. The over-expression of NLRP12 in B6 corneas significantly ameliorated the severity of corneal disease, bacterial burden, PMN infiltration and pro-inflammatory cytokine expression. In vitro analysis demonstrated that the up-regulation of NLRP12 suppressed pro-inflammatory cytokine production and enhanced bacterial clearance in RAW264.7 cells. The protein levels of pIκBα and IκBα were signiﬁcantly decreased after NLRP12-lentivirus treatment compared with that of Ctl-lentivirus. NF-κB-dependent Luciferase activity was potently inhibited by NLRP12 infected with P. aeruginosa or cotransfected with the downstream signaling molecules including IKKα and IKKβ in 293T cells.

CONCLUSIONS

NLRP12 decreases the severity of P. aeruginosa keratitis, reduces corneal inflammation and bacterial burden through the down-regulation of the NF-κB signaling pathway.

摘要

目的

研究 NLRP12 在调控铜绿假单胞菌（P. aeruginosa）角膜炎中的作用。

材料与方法

通过实时 PCR 和 Western blot 检测 C57BL/6（B6）小鼠角膜和骨髓来源巨噬细胞（BMDMs）中的 NLRP12 水平。B6 小鼠接受表达活性 NLRP12（NLRP12-慢病毒）或对照 Ctl-慢病毒（作为对照）的结膜下注射，然后感染铜绿假单胞菌。评估小鼠的临床评分、裂隙灯和细菌平板计数。此外，通过髓过氧化物酶（MPO）检测评估多形核中性粒细胞（PMN）的浸润。通过 Real Time-PCR 和 ELISA 测量细胞因子水平。同时，评估细菌负荷。通过 Western blot 基于 pIκBα/IκBα 水平确定 NF-κB 信号转导的激活，通过使用 293T 细胞的 Luciferase 测定基于 NF-κB 依赖性 Luciferase 活性确定 NF-κB 信号转导的激活。

结果

铜绿假单胞菌感染后，B6 角膜和 BMDMs 中的 NLRP12 mRNA 和蛋白水平降低。B6 角膜中 NLRP12 的过表达显著改善了角膜疾病的严重程度、细菌负荷、PMN 浸润和促炎细胞因子表达。体外分析表明，NLRP12 的上调抑制了 RAW264.7 细胞中促炎细胞因子的产生并增强了细菌清除。与 Ctl-慢病毒相比，NLRP12-慢病毒处理后 pIκBα 和 IκBα 的蛋白水平显著降低。NLRP12 感染或与下游信号分子（包括 IKKα 和 IKKβ）共转染后，NF-κB 依赖性 Luciferase 活性在 293T 细胞中被强烈抑制。