Department of Molecular and Human Genetics, Institute of Science, Banaras Hindu University, Varanasi 221005, India.
Department of Obstetrics & Gynecology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India.
Int Immunopharmacol. 2024 Dec 5;142(Pt B):113165. doi: 10.1016/j.intimp.2024.113165. Epub 2024 Sep 19.
Neutrophil extracellular traps (NETs) being one of the predominant activities of neutrophils has become its key defense mechanism owing to its extensive role in inflammation and infection. However, the mechanisms regulating NET formation or NETosis still remains to be better understood. Our earlier whole genome transcriptomic data revealed two G-protein couple receptors (GPCRs) - complement component 5a receptor 1 (C5aR1) and leukotriene B4 receptor 1 (LTB4R1) were downregulated in term low birth weight (tLBW) newborns with deficient NET formation abilities. Neutrophils employ C5aR1 and LTB4R1 for mediating their immune responses, inflammation and antimicrobial activity. Hence, this study was aimed to explore the role of two GPCRs, C5aR1 and LTB4R1 including their downstream signaling molecules in NETs induction and regulation.
The validation of the transcriptomic data for C5aR1 and LTB4R1 was done using quantitative real time PCR. Pharmacological inhibition of C5aR1 and LTB4R1 using W-54011 and LY223982 on neutrophils of adults and newborns' was done to study their impact on NETosis. Extracellular DNA release, Reactive oxygen species (ROS) generation, expression of NET proteins, and signaling molecules downstream to C5aR1 and LTB4R1 were quantified using plate reader based assay, immunofluorescence, and western blotting. Myeloperoxidase (MPO)-DNA quantified by flow cytometry. Knockdown studies using siRNA against C5aR1 and LTB4R1 were done in HL-60 cells derived surrogate neutrophils and expression of downstream molecules of the two GPCRs, C5aR1 and LTB4R1 signaling axis along with NET proteins was quantified by western blotting.
The expression of C5aR1 and LTB4R1, extracellular DNA, ROS and NET associated proteins (NE, CitH3, PAD4 and MPO) was notably increased upon NET induction in healthy adults and normal birth weight (NBW) newborns' neutrophils. Pharmacological inhibition of these two GPCRs led to substantial reduction in NETosis, extracellular DNA, ROS generation, and expression of NET associated proteins like CitH3, NE, PAD4, MPO along with downstream signaling molecules Rap1a, B-Raf and pERK. Our observations suggest a precise role of C5aR1 and LTB4R1 on induction of NETs via Rap1a/B-Raf/ERK signaling axis.
The C5aR1 and LTB4R1 signaling via Rap1a/B-Raf/ERK axis acts as a signal-relay mechanism to regulate NET formation in neutrophils. Further, C5aR1 and LTB4R1 signaling cascade along with NET-associated proteins are remarkably downregulated in tLBW newborns' neutrophils leading to impaired NETosis in them. Therefore, C5aR1 and LTB4R1 and their signaling molecules could provide an effective therapeutic target for compromised NETosis like tLBW newborns.
中性粒细胞胞外诱捕网（NETs）是中性粒细胞的主要活性之一，由于其在炎症和感染中的广泛作用，已成为其关键防御机制。然而，调节 NET 形成或 NETosis 的机制仍有待更好地理解。我们之前的全基因组转录组数据显示，在具有 NET 形成能力缺陷的足月低出生体重（tLBW）新生儿中，两种 G 蛋白偶联受体（GPCR）-补体成分 5a 受体 1（C5aR1）和白三烯 B4 受体 1（LTB4R1）的表达下调。中性粒细胞利用 C5aR1 和 LTB4R1 来介导其免疫反应、炎症和抗菌活性。因此，本研究旨在探讨两种 GPCRs，即 C5aR1 和 LTB4R1 及其下游信号分子在 NETs 诱导和调节中的作用。
使用定量实时 PCR 验证 C5aR1 和 LTB4R1 的转录组数据。使用 W-54011 和 LY223982 对成人和新生儿的中性粒细胞进行 C5aR1 和 LTB4R1 的药理学抑制，以研究它们对 NETosis 的影响。使用平板读取器测定法、免疫荧光和 Western blot 定量测定细胞外 DNA 释放、活性氧（ROS）生成、NET 蛋白的表达以及 C5aR1 和 LTB4R1 下游的信号分子。用流式细胞术测定髓过氧化物酶（MPO）-DNA。在 HL-60 细胞来源的替代中性粒细胞中使用针对 C5aR1 和 LTB4R1 的 siRNA 进行敲低研究，并通过 Western blot 定量测定两种 GPCRs、C5aR1 和 LTB4R1 信号轴的下游分子以及 NET 蛋白的表达。
在健康成年人和正常出生体重（NBW）新生儿中性粒细胞的 NET 诱导中，C5aR1 和 LTB4R1 的表达、细胞外 DNA、ROS 和 NET 相关蛋白（NE、CitH3、PAD4 和 MPO）明显增加。这两种 GPCR 的药理学抑制导致 NETosis、细胞外 DNA、ROS 生成以及 CitH3、NE、PAD4、MPO 等 NET 相关蛋白的表达以及下游信号分子 Rap1a、B-Raf 和 pERK 显著减少。我们的观察结果表明，C5aR1 和 LTB4R1 通过 Rap1a/B-Raf/ERK 信号轴在诱导 NETs 中起着精确的作用。
C5aR1 和 LTB4R1 通过 Rap1a/B-Raf/ERK 信号通路充当调节中性粒细胞 NET 形成的信号中继机制。此外，tLBW 新生儿中性粒细胞中 C5aR1 和 LTB4R1 信号级联及其与 NET 相关的蛋白显著下调，导致 NETosis 受损。因此，C5aR1 和 LTB4R1 及其信号分子可为 NETosis 受损的患者（如 tLBW 新生儿）提供有效的治疗靶点。
