Shirey Kari Ann, Romerio Alessio, Shaik Mohammed Monsoor, Leake David S, Palmer Charys, Skupinska Natalia, Paton Jules, Pirianov Grisha, Blanco Jorge Cg, Vogel Stefanie N, Peri Francesco
Department of Microbiology and Immunology, University of Maryland, School of Medicine, Baltimore, Maryland, USA.
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy.
Innate Immun. 2025 Jan-Dec;31:17534259241313201. doi: 10.1177/17534259241313201.
Acute Lung Injuries (ALI) are a severe consequence of influenza-induced cytokine storm that can cause respiratory failure and death. It has been demonstrated that Toll-like Receptor 4 (TLR4) is involved in cytokine storm and that TLR4 mice are protected against ALI. Therefore, TLR4 is a prime target for protection against ALI. FP12 is a known TLR4 antagonist that reduces TLR4-dependent immune activation and it is a promising lead compound for the treatment of innate immunity related pathologies.
We present here the preclinical development of FP12 as an anti-inflammatory lead compound acting on influenza-induced ALI.
We pre-treated THP-1 cells with FP12 (10 μM) for 0.5 h, then exposed to LPS (100 ng/ml) for 0 to 16 h. In some experiments, cells were simultaneously incubated with FP12 and LPS, or FP12 was added 30 min after LPS. Cytokine levels were measured by Western blot and ELISA assays. WT C57BL/6J mice were infected with mouse-adapted influenza virus (PR8). Two days after infection, mice received either vehicle, FP7 (200 µg/mouse), or FP12 (200 µg/mouse) once daily (Day 2 to Day 6). Mice were monitored daily for survival for 14 days. Data were collected through histological staining, qRT-PCR, and ELISA assay.
FP12 treatment inhibited both LPS- and HMGB1-induced TLR4 intracellular pathways (MyD88 and TRIF) leading to significantly reduced levels of a variety of proinflammatory cytokines including Type I interferon (IFN-β), highlighting its effectiveness in controlling proinflammatory protein production and reducing inflammation. FP12 protected mice therapeutically from influenza virus-induced lethality and reduced both cytokine gene expression and High Mobility Group Box 1 (HMGB1) levels in the lungs as well as ALI.
FP12 can antagonize TLR4 activation in vitro and protects mice from severe influenza infection, most likely by reducing the TLR4-dependent cytokine storm mediated by danger-associated molecular patterns (DAMPs).
急性肺损伤（ALI）是流感诱导的细胞因子风暴的严重后果，可导致呼吸衰竭和死亡。已有研究表明，Toll样受体4（TLR4）参与细胞因子风暴，且TLR4基因敲除小鼠可免受ALI影响。因此，TLR4是预防ALI的主要靶点。FP12是一种已知的TLR4拮抗剂，可降低TLR4依赖性免疫激活，是治疗先天性免疫相关疾病的一种有前景的先导化合物。
本文介绍FP12作为一种作用于流感诱导的ALI的抗炎先导化合物的临床前开发情况。
我们用FP12（10 μM）预处理THP-1细胞0.5小时，然后用脂多糖（LPS，100 ng/ml）刺激0至16小时。在一些实验中，细胞同时与FP12和LPS孵育，或在LPS刺激30分钟后加入FP12。通过蛋白质免疫印迹法和酶联免疫吸附测定法检测细胞因子水平。野生型C57BL/6J小鼠感染适应小鼠的流感病毒（PR8）。感染后两天，小鼠每天接受一次溶剂对照、FP7（200 μg/小鼠）或FP12（200 μg/小鼠）（第2天至第6天）。每天监测小鼠存活情况，持续14天。通过组织学染色、定量逆转录聚合酶链反应和酶联免疫吸附测定法收集数据。
FP12处理可抑制LPS和高迁移率族蛋白B1（HMGB1）诱导的TLR4细胞内信号通路（髓样分化因子88（MyD88）和TIR结构域衔接蛋白诱导干扰素β（TRIF）），导致多种促炎细胞因子水平显著降低，包括I型干扰素（IFN-β），突出了其在控制促炎蛋白产生和减轻炎症方面的有效性。FP12在治疗上可保护小鼠免受流感病毒诱导的致死性影响，并降低肺部细胞因子基因表达和高迁移率族蛋白B1（HMGB1）水平以及ALI。
FP12可在体外拮抗TLR4激活，并保护小鼠免受严重流感感染，最可能是通过减少由危险相关分子模式（DAMPs）介导的TLR4依赖性细胞因子风暴实现的。
