嗜中性球胞外陷阱形成的調控及其在急性發炎的消解作用：以HL-60細胞模型分析

Abstract

在人體免疫系統中，多形核嗜中性白血球（polymorphonuclear neutrophils）是循環中數量最為豐富的白血球，也是先天免疫的重要防線。除了已被廣泛認可的促發炎功能外，多形核嗜中性白血球亦逐漸被視為發炎終止及組織修復的主動調節者。在適當刺激下，多形核嗜中性白血球能經由嗜中性球胞外陷阱形成（neutrophil extracellular trap formation）釋放嗜中性球胞外陷阱（neutrophil extracellular traps, NETs），其結構包含了以抗菌蛋白裝飾之網狀染色質，可協助清除病原並調節發炎反應。為了解析 NETosis 的分子機制，我們以全反式視黃酸（all‑trans retinoic acid）聯合顆粒性白血球生長因子（granulocyte colony‑stimulating factor）將 HL‑60 細胞分化為具嗜中性球特性的細胞（neutrophil‑like cells），並作為 多形核嗜中性白血球的替代模型。隨後，我們建立以HL‑60 細胞模擬臨床中單鈉尿酸晶體（monosodium urate crystal）誘發之急性痛風性關節炎（acute gouty arthritis）之模型，探討 嗜中性球胞外陷阱可能的調控功能；該疾病通常於 7至10 日內自限性緩解。藉由此細胞模式，我們得以剖析 嗜中性球胞外陷阱誘導與終止途徑，並進一步瞭解嗜中性球於痛風病理生理中兼具促發炎與緩解發炎的雙重角色。 先前研究顯示，全反式視黃酸誘導分化之 HL‑60 細胞在以佛波醇-12-十四烷醯-13-乙酸酯（phorbol 12-myristate 13-acetate）刺激時，相較於原代嗜中性球，呈現較弱的 NETosis 及較低的活性氧（reactive oxygen species）產生量。可能被忽略的一個變因為實驗室中標準培養基與人體血漿成分之差異甚大，例如培養基缺乏人類免疫球蛋白 G（immunoglobulin G），可能減弱依賴 Fc γ 受體（Fc gamma receptor, FcγR）的訊號傳遞及下游效應功能。免疫球蛋白 G為人體血清中含量最高之免疫球蛋白，對適應性免疫反應具有關鍵角色。在本研究中，人類單體免疫球蛋白 G，特別是免疫球蛋白 G1 與免疫球蛋白 G2 亞類，本身對分化之HL-60細胞誘發之 NETosis 影響甚微；然而，當 免疫球蛋白 G 與 FcγRIII 結合並再以 佛波醇-12-十四烷醯-13-乙酸酯刺激時，會以劑量相依方式顯著增強 嗜中性球胞外陷阱形成。此增強現象與脾臟酪胺酸激酶（spleen tyrosine kinase）及細胞外訊號調節激酶（extracellular signal‑regulated kinase）磷酸化增加、細胞內活性氧產生上升，以及促發炎細胞激素介白素‑8（interleukin‑8）與腫瘤壞死因子‑α（tumor necrosis factor‑alpha）分泌增加有關。值得注意的是，使用專一性中和抗體阻斷 FcγRIII 後，此增強之嗜中性球胞外陷阱形成顯著減弱，更加證實該受體在免疫球蛋白 G驅動嗜中性球活化中的關鍵調控角色。以上結果突顯免疫球蛋白 G透過 FcγRIII 之免疫調節潛力，並強調此途徑在調控嗜中性球媒介之發炎反應中的重要性；同時，也再次驗證分化之HL-60細胞作為探討嗜中性球胞外陷阱形成之分子機制是便利且具生理相關性的模型。 為探討嗜中性球之抗發炎功能，我們以 單鈉尿酸晶體活化分化之HL-60細胞並確認其釋放嗜中性球胞外陷阱；這些 嗜中性球胞外陷阱可捕捉 單鈉尿酸晶體形成大型、有組織的嗜中性球胞外陷阱-單鈉尿酸晶體聚集體。促發炎細胞激素介白素‑8與腫瘤壞死因子‑α以及抗發炎的介白素‑1受體拮抗劑（interleukin‑1 receptor antagonist）皆為急性痛風性關節炎之關鍵調節因子。我們定義相應的估計發炎嚴重度評分指數（estimated inflammation score；詳見材料與方法、第 13 節），計算方式為有／無單鈉尿酸晶體刺激時細胞激素濃度之比值。在此急性痛風細胞模型中，對聚集體的量化分析顯示，培養 22 小時後估計發炎嚴重度評分指數可達 19.40，反映顯著的發炎反應。有趣的是，延長培養時間或加入新的分化之HL-60細胞及 MSU 晶體以模擬臨床痛風發作中嗜中性球持續募集的現象，雖能導致嗜中性球胞外陷阱面積增加，估計發炎嚴重度評分指數卻顯著下降至 1.53，指出急性痛風發炎反應已趨於緩解。 而此轉向抗發炎狀態之變化，被發現伴隨著細胞外訊號調節激酶（extracellular signal‑regulated kinase）磷酸化降低，以及 Src 同源區域 2 結構域的蛋白質酪氨酸磷酸酶‑1（Src homology region 2 domain-containing phosphatase-1）磷酸化增加，同時細胞激素訊號負向調節子 CISH、SOCS2、SOCS3 之信使核糖核酸（mRNA）表現上調。此外，自該培養液收集之上清液體亦可增強細胞對單鈉尿酸晶體刺激之抗發炎反應。更進一步，大型嗜中性球胞外陷阱-單鈉尿酸晶體聚集體的形成促進嗜中性球由促發炎 N1 表型（CD54highCD182low）轉變為抗發炎 N2 表型（CD54lowCD182high）。可認為此表型極化之轉變在促成急性痛風發炎之自發性緩解中扮演關鍵角色，並有助於揭示體內限制發炎時組織損傷之內生調控機制。 綜合而言，我們的結果顯示，由 FcγRIII 結合所驅動之脾臟酪胺酸激酶–細胞外訊號調節激酶訊號與佛波醇-12-十四烷醯-13-乙酸酯活化之蛋白質激酶 C（protein kinase C）間的交互作用，對 嗜中性球胞外陷阱形成具關鍵調控作用。此外，免疫球蛋白 G在佛波醇-12-十四烷醯-13-乙酸酯刺激過程中可幫助維持分化之HL-60細胞更具模擬嗜中性白血球表現之能力，從而有效率地促成嗜中性球胞外陷阱形成。另一方面，單鈉尿酸晶體誘導之嗜中性球胞外陷阱-單鈉尿酸晶體聚集體所引發的動態變化，於發炎的快速展及隨後而來之發炎主動緩解中，皆扮演核心角色。嗜中性球胞外陷阱兼具促進急性發炎及促進其終止的雙重功能，凸顯其複雜的免疫調節特性。這些發現不僅深度揭示嗜中性球行為及細胞激素訊號調控機制，亦顯示針對嗜中性球胞外陷阱相關途徑的治療策略，可能對調控過度或失衡的發炎反應之炎症與自體免疫疾病具臨床潛力。

Polymorphonuclear neutrophils (PMNs) are the most abundant leukocytes in human circulation and are critical effectors of innate immunity. Beyond their well‑established proinflammatory roles, they are increasingly recognized as active mediators of inflammatory resolution and tissue repair. Upon appropriate stimulation, PMNs can undergo neutrophil extracellular trap formation (NETosis), releasing neutrophil extracellular traps (NETs), which are web-like chromatin structures decorated with antimicrobial proteins that aid in pathogen clearance and modulate inflammation. To dissect the molecular mechanisms of NETosis, we differentiated HL‑60 cells into neutrophil‑like cells using all‑trans retinoic acid (ATRA) together with granulocyte colony‑stimulating factor and employed them as surrogate PMNs. We then interrogated the putative regulatory functions of NETs in the clinically relevant context of monosodium urate (MSU) crystal–induced acute gouty arthritis, a self‑limiting inflammatory condition that typically resolves spontaneously within 7–10 days. By using this cell-based model, we aimed to dissect the pathways involved in NET induction and resolution, offering insights into both the proinflammatory and inflammation-resolving roles. Prior studies have shown that ATRA-induced differentiated HL‑60 (dHL‑60) cells exhibit weaker NETosis and reduced ROS generation than primary neutrophils upon phorbol 12-myristate 13-acetate (PMA) stimulation. An often‑overlooked variable is that standard culture media differ substantially from human plasma, for example, they lack human Immunoglobulin G (IgG) which may dampen Fc gamma receptor (FcγR)‑dependent signaling and downstream effector functions. IgG is the most abundant immunoglobulin in human serum and plays a key role in adaptive immune responses. In our experiments, human monomeric IgG by itself induced negligible NETosis in dHL‑60 cells. However, their engagement with FcγRIII significantly enhanced NET formation when co-stimulated with PMA, in a dose-dependent manner. This enhancement was mechanistically associated with increased phosphorylation of spleen tyrosine kinase (Syk) and extracellular signal-regulated kinase (ERK), elevated intracellular reactive oxygen species (ROS) production, and upregulated secretion of pro-inflammatory cytokines such as interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α). Importantly, this augmented NET formation was attenuated by blocking FcγRIII engagement using a specific neutralizing antibody, further confirming the receptor's pivotal role in modulating IgG-driven neutrophil activation. These findings highlight the immunomodulatory potential of IgG subclasses through FcγRIII and underscore the relevance of this pathway in regulating neutrophil-mediated inflammation. They further validate dHL‑60 cells as a convenient and physiologically relevant model for mechanistic studies of NETosis. To investigate the anti-inflammatory functions of neutrophils, we activated dHL-60 cells by MSU crystals and confirmed the release of NETs, which entrapped MSU crystals and formed large, structured NET-MSU aggregates. The pro‑inflammatory cytokines IL‑8 and TNF‑α, together with the anti‑inflammatory interleukin‑1 receptor antagonist (IL‑1RA), are key mediators in acute gouty arthritis. We defined a corresponding estimated inflammation score (EIS, detailed in Section 13 of the Materials and Methods) was calculated as the ratio of cytokine concentrations measured with versus without MSU stimulation. Using this cell model of acute gouty arthritis, the quantitative analysis of these aggregates revealed a pronounced inflammatory response, with a peak EIS of 19.40 after 22 hours of incubation. Interestingly, extending the incubation period or introducing fresh dHL-60 cells and MSU crystals into the culture—an approach designed to mimic the ongoing recruitment of neutrophils seen in clinical gout flares—resulted in increased NET area but markedly declined EIS to 1.53, suggesting resolution of acute gouty inflammation. This transition toward an anti-inflammatory state was associated with a decrease in phosphorylated ERK1/2 and a concomitant increase in phosphorylated Src homology region 2 domain-containing phosphatase-1, along with upregulation of negative regulators of cytokine signaling such as CISH, SOCS2, and SOCS3 mRNA expression. Furthermore, the supernatant derived from these cultures was found to amplify the anti‑inflammatory response to MSU stimulation. In addition, the formation of large NET-MSU aggregates also promoted neutrophil phenotypic switching from the pro-inflammatory N1 (CD54highCD182low) to the anti-inflammatory N2 (CD54lowCD182high) phenotype. This polarization of phenotype is believed to play a pivotal role in facilitating the spontaneous resolution of inflammation observed in acute gout, offering potential insights into endogenous regulatory mechanisms that limit tissue damage during inflammation. In conclusion, our data indicate that cross‑talk between FcγRIII‑engagement–driven Syk–ERK signaling and PMA‑activated protein kinase C critically regulates NETosis. Moreover, IgG sustains the neutrophil‑like phenotype of dHL‑60 cells during PMA stimulation. On the other hand, the dynamic changes triggered by MSU-induced NET-MSU aggregate formation play a pivotal role in both the rapid escalation and subsequent resolution of inflammation. This dual functionality of NETs—promoting acute inflammation while also facilitating its resolution—underscores their complex immunomodulatory nature. These findings not only provides valuable insights into the mechanisms governing neutrophil behavior and cytokine signaling but also highlight potential therapeutic strategies. Targeting NET-associated pathways may offer therapeutic potential in managing inflammatory diseases marked by dysregulated or excessive inflammation.