利用HL-60細胞模型探討多白胺酸重複激酶2在 嗜中性球功能中所參與的機制

Abstract

發炎性腸道疾病（inflammatory bowel disease, IBD）為腸道慢性發炎疾病的統稱，全球盛行率正逐年上升。雖然其病因尚未完全釐清，但研究指出免疫失調、基因、腸道微生物組成失衡與環境因子皆為重要影響因素。IBD患者腸道組織中常見中性球大量浸潤及中性球胞外網狀結構（neutrophil extracellular traps, NETs）的形成。此外，IBD 患者腸道菌叢中也常見真菌失衡，尤其是白色念珠菌（Candida albicans）大量增生，顯示真菌數量與嗜中性球相關的發炎反應可能存在關聯。基因方面的研究亦發現多個與 IBD 有關的基因，其中包括 lrrk2，此基因編碼多白胺酸重複激酶 LRRK2（Leucine-Rich Repeat Kinase 2）。LRRK2 為具有多重結構域的蛋白質，含有激酶功能，並在免疫調控與細胞恆定中扮演關鍵角色。LRRK2 在多種免疫細胞中表現豐富，尤其在嗜中性球中表現最為明顯，且與 IBD 密切相關。本研究旨在探討 LRRK2 在嗜中性球中的表現如何影響其功能，進而可能參與 IBD 的病程。實驗發現，使用基因改造的HL-60細胞，分別建立LRRK2基因剔除（knockout, KO）與激酶失活突變（K1906M）株，經Dimethyl sulfoxide (DMSO)分化為類中性球（neutrophil-like cells, NLCs）後分別探討其嗜中性球的功能，包括去顆粒化、吞噬能力、釋放NETs的能力。在吞噬能力上，結果顯示，相較於野生型（WT）細胞，LRRK2 敲除（KO）HL-60 細胞的吞噬能力顯著下降，而激酶失活突變株 K1906M 的表現則與 WT 相近。另外，我評估了 NETs 的形成，這是嗜中性球的重要效應功能之一，並越來越多地被認為與 IBD 發炎機制有關。透過 ionomycin 或 C. albicans 刺激，可成功誘導 NETs 生成。LRRK2 KO 細胞產生的 NETs 數量顯著高於 WT，而 K1906M 突變細胞則無顯著差異。鑑於 NETs 與腸道發炎之相關性，我進一步針對 C. albicans 誘導 NET 的機制進行探討。結果顯使用 cytochalasin D 抑制吞噬作用可顯著降低 NET 的產生，說明白色念珠菌被吞噬後的胞內訊號傳導對於 NET 誘導至關重要。有研究指出，在巨噬細胞中，當白色念珠菌破壞吞噬體膜時，LRRK2 會磷酸化下游的 Rab8A，而磷酸化後的 Rab8A 可召集運輸所需的內體分選複合體(endosomal sorting complexes required for transport, ESCRT)成員 多帶電泡體蛋白(Charged multivesicular body protein 4b, CHMP4B)，進行膜修復。在本研究中觀察在白色念珠菌於刺激一小時，Rab8A 的磷酸化程度(作為 LRRK2 激酶活性的指標)在 KO 與 K1906M 細胞中皆顯著下降顯示其修復吞噬體的相關訊號可能受損。雖然 CHMP4B 的體積像素量化結果未顯示顯著差異，整體結果仍支持我們的假說模型：在嗜中性球中，LRRK2 可能透過其激酶活性磷酸化 Rab8A，參與早期膜修復機制；而在 LRRK2 缺失或激酶功能喪失的情況下，修復機制失效可能導致吞噬體膜破裂，進而使原本局限於吞噬體內的顆粒蛋白（如 MPO、NE）洩漏至細胞質中，最終促發 NET 的形成。

Inflammatory bowel disease (IBD) is a chronic condition marked by recurrent inflammation of the digestive tract, with a rising global prevalence. While its causes remain unclear, key factors include immune system dysregulation, genetic predisposition, gut microbiome, and environmental influences. Studies have highlighted significant neutrophil infiltration and NET formation in IBD patients. In addition, fungal dysbiosis—particularly the overgrowth of C. albicans—has been frequently reported in the gut microbiota of IBD patients, suggesting a possible link between fungal burden and neutrophil-driven inflammation. Genetic studies have identified multiple IBD-associated genes, including lrrk2, which encodes the leucine-rich repeat kinase 2 (LRRK2). LRRK2 is a multi-domain protein, including a kinase domain, that plays a critical role in immune regulation and cellular homeostasis. LRRK2 is highly expressed in immune cells, particularly neutrophils, and is strongly associated with IBD. My research aims to investigate how LRRK2 expression in neutrophils affects their function, potentially influencing the progression of IBD. By understanding this mechanism, we may uncover novel insights into IBD pathogenesis and identify new therapeutic targets. In my study, I used gene-modified HL-60 cell lines. After differentiating HL-60 cells into neutrophil-like cells (NLCs) using DMSO, I first examined phagocytic function across different LRRK2 genotypes. Compared to wild-type (WT) cells, LRRK2 knockout (KO) NLCs exhibited significantly reduced phagocytic activity, while kinase-dead K1906M mutants showed phagocytosis levels similar to WT. I then assessed NET formation, an important neutrophil effector function increasingly recognized for its involvement in IBD pathogenesis. NETs were induced using ionomycin or C. albicans. LRRK2 KO cells produced significantly more NETs than WT, whereas K1906M mutants showed no significant difference from WT. Because NETs are more closely linked to intestinal inflammation in IBD, I further investigated how LRRK2 regulates NET formation in response to C. albicans, a fungal species commonly enriched in the gut microbiota of IBD patients. Blocking phagocytosis with cytochalasin D significantly reduced NET release, suggesting that intracellular signaling following fungal uptake is essential for NET induction. Previous research has shown that in macrophages, when C. albicans damages the phagosomal membrane, LRRK2 is activated and phosphorylates its downstream target Rab8A. Phosphorylated Rab8A subsequently recruits components of the endosomal sorting complexes required for transport (ESCRT), particularly charged multivesicular body protein 4B (CHMP4B), to initiate membrane repair. In this study, I observed that upon C. albicans stimulation for one hour, Rab8A phosphorylation—used as an indicator of LRRK2 kinase activity—was significantly reduced in both LRRK2 knockout (KO) and kinase-dead (K1906M) cells, suggesting impaired activation of phagosomal repair pathways. Although CHMP4B voxel intensity quantification did not reveal significant differences among genotypes, the overall results support our proposed model: LRRK2 may contribute to early membrane repair by phosphorylating Rab8A through its kinase activity. In the absence of functional LRRK2, this repair mechanism may fail, leading to phagosomal membrane rupture and subsequent leakage of granule proteins, such as into the cytosol, ultimately triggering NET formation.