贝氏蛔虫迁移期幼虫中的一种新型半胱氨酸蛋白酶抑制剂通过 TLR4-ROS-NLRP3 途径调节炎症小体激活。

A novel cysteine protease inhibitor in Baylisascaris schroederi migratory larvae regulates inflammasome activation through the TLR4-ROS-NLRP3 pathway.

机构信息

Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, 611130, China.

Department of Chemistry, College of Life and Basic Science, Sichuan Agricultural University, Wenjiang, 611130, China.

出版信息

Parasit Vectors. 2022 Sep 23;15(1):334. doi: 10.1186/s13071-022-05466-6.

DOI:10.1186/s13071-022-05466-6

PMID:36151570

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9508711/

Abstract

BACKGROUND

Giant pandas (Ailuropoda melanoleuca) are the obligate host of the parasitic roundworm Baylisascaris schroederi. The infection of giant pandas with B. schroederi is very common. At present, little is known about the mechanism of immune interaction between B. schroederi and the host. As an important component of innate immunity, the NOD-like receptor 3 (NLRP3) inflammasome plays an important role in host immune response and the occurrence and development of infectious diseases.

METHODS

We analyzed the regulation of NLRP3 inflammasome activation in monocyte-derived macrophages (MDMs) by the recombinant B. schroederi migratory larvae cysteine protease inhibitor rBsCPI-1, knowing from a previous study that the CPI-1 is highly expressed in B. schroederi migratory larvae. We first determined the effects of rBsCPI-1 and excretory-secretory products of B. schroederi migratory larvae on cell proliferation using the CCK-8 and LDH release assays. We then analyzed NLRP3 inflammasome activation, pyroptosis and pro-inflammatory cytokine release by quantitative-PCR, western blotting and enzyme-linked immunosorbent assay. The signaling pathway of rBsCPI-1 to activate NLRP3 inflammasomes was analyzed in activation and inhibition experiments. Finally, the effects of rBsCPI-1 on inflammasome activation in mice immunized with rBsCPI-1 were analyzed.

RESULTS

The activation and inhibition experiments revealed that rBsCPI-1 induced inflammasome activation through the TLR4-ROS-NLRP3 signaling pathway, with reactive oxygen species (ROS) not only functioning as an activator of the NLRP3 inflammasome, but also an activation product of the NLRP3 inflammasome. rBsCPI-1 promoted the activation and assembly of the NLRP3 inflammasome, which further converted the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 into mature active forms. At the same time, caspase-1 cleaved gasdermin D to trigger cell pyroptosis. The results of animal immunization experiments further confirmed that rBsCPI-1 could induce the activation of the NLRP3 inflammasome.

CONCLUSIONS

rBsCPI-1 activates the inflammasome through the TLR4-ROS-NLRP3 signaling pathway and further induces the pyroptosis of MDMs and release of pro-inflammatory factors IL-1β and IL-18, thus promoting the occurrence and development of the inflammatory response in the host.

摘要

背景

大熊猫（Ailuropoda melanoleuca）是寄生蛔虫巴氏副蛔虫的专性宿主。大熊猫感染巴氏副蛔虫非常普遍。目前，人们对巴氏副蛔虫与宿主之间免疫相互作用的机制知之甚少。作为先天免疫的重要组成部分，NOD 样受体 3（NLRP3）炎性小体在宿主免疫反应和传染病的发生和发展中发挥着重要作用。

方法

我们分析了先前研究中高度表达于巴氏副蛔虫迁移幼虫的重组巴氏副蛔虫迁移幼虫半胱氨酸蛋白酶抑制剂 rBsCPI-1 对单核细胞衍生的巨噬细胞（MDMs）中 NLRP3 炎性小体激活的调节作用。我们首先使用 CCK-8 和 LDH 释放测定法确定 rBsCPI-1 和巴氏副蛔虫迁移幼虫的排泄-分泌产物对细胞增殖的影响。然后，通过定量 PCR、western blot 和酶联免疫吸附测定分析 NLRP3 炎性小体的激活、细胞焦亡和促炎细胞因子的释放。在激活和抑制实验中分析 rBsCPI-1 激活 NLRP3 炎性小体的信号通路。最后，分析 rBsCPI-1 对用 rBsCPI-1 免疫的小鼠中炎性小体激活的影响。

结果

激活和抑制实验表明，rBsCPI-1 通过 TLR4-ROS-NLRP3 信号通路诱导炎性小体激活，其中活性氧（ROS）不仅作为 NLRP3 炎性小体的激活剂，而且作为 NLRP3 炎性小体的激活产物。rBsCPI-1 促进 NLRP3 炎性小体的激活和组装，进而将促炎细胞因子白细胞介素（IL）-1β和 IL-18 转化为成熟的活性形式。同时，半胱天冬酶-1 切割 gasdermin D 引发细胞焦亡。动物免疫实验的结果进一步证实，rBsCPI-1 可诱导 NLRP3 炎性小体的激活。