Pulmonary Clinical Science, Department of Anaesthesia and Critical Care Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland.
Department of Biology, Maynooth University, Maynooth, Kildare, Ireland.
Front Immunol. 2024 Oct 18;15:1452393. doi: 10.3389/fimmu.2024.1452393. eCollection 2024.
The role of neutrophils in host defense involves several cell processes including phagocytosis, degranulation of antimicrobial proteins, and the release of neutrophil extracellular traps (NETs). In turn, dysregulated cell activity is associated with the pathogenesis of airway and rheumatic diseases, in which neutrophil-derived enzymes including peptidyl-arginine deiminases (PADs) play a role. Known physiological functions of PADs in neutrophils are limited to the activity of PAD isotype 4 in histone citrullination in NET formation. The aim of this study was to extend our knowledge on the role of PADs in neutrophils and, specifically, bacterial killing within the confines of the phagocytic vacuole. Human neutrophils were fractionated by sucrose gradient ultracentrifuge and PADs localized in subcellular compartments by Western blot analysis. Direct interaction of PADs with () was assessed by flow cytometry and Western blot overlay. The participation of neutrophil PAD2 and PAD4 in killing of was assessed by inclusion of PAD-specific inhibitors. , bactericidal activity of recombinant human PAD2 or PAD4 enzymes against was determined by enumeration of colony-forming units (CFU). Together with neutrophil elastase (NE), PAD2 and PAD4 were localized to primary granules and, following activation with particulate stimuli, were degranulated in to the phagocytic vacuole. , PAD2 and PAD4 bound (p = 0.04) and significantly reduced bacterial survival to 49.1 ± 17.0 (p < 0.0001) and 48.5 ± 13.9% (p < 0.0001), respectively. Higher antibacterial activity was observed at neutral pH levels with the maximum toxicity at pH 6.5 and pH 7.5, comparable to the effects of neutrophil bactericidal permeability increasing protein. In phagosomal killing assays, inclusion of the PAD2 inhibitor, AFM-30a, or PAD4 inhibitor, GSK484, significantly increased survival of (AFM-30a, p = 0.05; and GSK484, p = 0.0079). Results indicate that PAD2 and PAD4 possess antimicrobial activity and are directly involved in the neutrophil antimicrobial processes. This study supports further research into the development of PAD-based antimicrobials.
中性粒细胞在宿主防御中的作用涉及几个细胞过程，包括吞噬作用、抗菌蛋白的脱颗粒作用和中性粒细胞胞外陷阱（NETs）的释放。反过来，细胞活动的失调与气道和风湿性疾病的发病机制有关，其中中性粒细胞衍生的酶，包括肽基精氨酸脱亚氨酶（PADs），发挥作用。已知 PAD 在中性粒细胞中的生理功能仅限于 PAD 同工型 4 在 NET 形成中的组蛋白瓜氨酸化活性。本研究旨在扩展我们对 PAD 在中性粒细胞中的作用的认识，特别是在吞噬小泡内的细菌杀伤作用。通过蔗糖梯度超速离心分离人中性粒细胞，通过 Western blot 分析将 PAD 定位在亚细胞隔室中。通过流式细胞术和 Western blot 覆盖评估 PAD 与 的直接相互作用。通过包含 PAD 特异性抑制剂来评估中性粒细胞 PAD2 和 PAD4 对 的杀伤作用。通过对集落形成单位（CFU）的计数来确定重组人 PAD2 或 PAD4 酶对 的杀菌活性。与中性粒细胞弹性蛋白酶（NE）一起，PAD2 和 PAD4 被定位到初级颗粒中，并且在用颗粒性刺激物激活后，在吞噬小泡中脱颗粒。与 NE 不同，PAD2 和 PAD4 与 （p = 0.04）结合，并分别将细菌存活率显著降低至 49.1 ± 17.0%（p < 0.0001）和 48.5 ± 13.9%（p < 0.0001）。在中性 pH 水平下观察到更高的抗菌活性，最大毒性在 pH 6.5 和 pH 7.5，与中性粒细胞杀菌通透性增加蛋白的作用相当。在吞噬体杀伤测定中，包含 PAD2 抑制剂 AFM-30a 或 PAD4 抑制剂 GSK484，显著增加 的存活率（AFM-30a，p = 0.05；和 GSK484，p = 0.0079）。结果表明，PAD2 和 PAD4 具有抗菌活性，并直接参与中性粒细胞抗菌过程。本研究支持进一步研究基于 PAD 的抗菌剂的开发。
