Tolle Leslie, Yu Fu-shin, Kovach Melissa A, Ballinger Megan N, Newstead Michael W, Zeng Xianying, Nunez Gabriel, Standiford Theodore J
Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Michigan Medical Center, Ann Arbor, Mich., USA.
J Innate Immun. 2015;7(2):177-86. doi: 10.1159/000367790. Epub 2014 Nov 12.
Flagellin is the major structural component of flagella expressed by Pseudomonas aeruginosa (PA) and other bacteria. This protein has been shown to activate the Toll-like receptor TLR5 and the Nod-like receptor Nlrc4/Ipaf, culminating in the expression of innate cytokines and antimicrobial molecules. In this study, we tested the hypothesis that TLR5 and Nlrc4 in combination are required for maximal protective lung innate mucosal immunity against PA. To test this hypothesis, we compared innate immune responses in wild-type (WT) C57B6 mice challenged with PA intratracheally to those observed in mice genetically deficient in TLR5 (TLR5(-/-)) or Nlrc4 (Nlrc4(-/-)) alone or in combination (TLR5/Nlrc4(-/-)). As compared to WT, TLR5(-/-) and Nlrc4(-/-) mice, we observed a significant increase in mortality in TLR5/Nlrc4(-/-) mice, which was associated with a >5,000-fold increase in lung PA colony-forming units and systemic bacterial dissemination. The increased mortality observed in double-deficient mice was not attributable to differences in lung leukocyte influx or lung injury responses. Levels of biologically active IL-1β and IL-18 were reduced in the bronchoalveolar lavage fluid from PA-infected Nlrc4(-/-) and TLR5/Nlrc4(-/-) but not TLR5(-/-) mice, indicating the requirement for Nlrc4-dependent caspase-1 activation. Similarly, decreased production of biologically active IL-1β and activation of caspase-1 was observed in PA-stimulated pulmonary macrophages isolated from Nlrc4(-/-) and TLR5/Nlrc4(-/-) but not TLR5(-/-) mice, whereas the expression of iNOS and the production of NO were significantly reduced in cells from double-mutant but not single-mutant mice. Collectively, our findings indicate that TLR5 and Nlrc4 have both unique and redundant roles in lung antibacterial mucosal immunity, and the absence of both pathogen recognition receptors results in an increase in susceptibility to invasive lung infection.
鞭毛蛋白是铜绿假单胞菌（PA）和其他细菌表达的鞭毛的主要结构成分。该蛋白已被证明可激活Toll样受体TLR5和Nod样受体Nlrc4/Ipaf，最终导致先天性细胞因子和抗菌分子的表达。在本研究中，我们测试了以下假设：TLR5和Nlrc4共同作用对于针对PA的最大程度的肺部先天性黏膜免疫保护是必需的。为了验证这一假设，我们比较了经气管内注射PA攻击的野生型（WT）C57B6小鼠与单独或联合缺乏TLR5（TLR5(-/-)）或Nlrc4（Nlrc4(-/-)）的基因缺陷小鼠的先天性免疫反应。与WT、TLR5(-/-)和Nlrc4(-/-)小鼠相比，我们观察到TLR5/Nlrc4(-/-)小鼠的死亡率显著增加，这与肺部PA菌落形成单位增加>5000倍以及全身细菌播散有关。在双缺陷小鼠中观察到的死亡率增加并非归因于肺白细胞流入或肺损伤反应的差异。来自PA感染的Nlrc4(-/-)和TLR5/Nlrc4(-/-)但不是TLR5(-/-)小鼠的支气管肺泡灌洗液中生物活性IL-1β和IL-18水平降低，表明需要Nlrc4依赖性半胱天冬酶-1激活。同样，在从Nlrc4(-/-)和TLR5/Nlrc4(-/-)但不是TLR5(-/-)小鼠分离的PA刺激的肺巨噬细胞中观察到生物活性IL-1β的产生减少和半胱天冬酶-1的激活，而在双突变小鼠而非单突变小鼠的细胞中iNOS的表达和NO的产生显著降低。总体而言，我们的研究结果表明，TLR5和Nlrc4在肺部抗菌黏膜免疫中具有独特和冗余的作用，并且两种病原体识别受体的缺失导致对侵袭性肺部感染的易感性增加。
