GEF-H1調控嗜中性白血球胞外網狀結構的形成

Abstract

白色念珠菌(Candida albicans)是人類常見的共生真菌，然而當免疫能力失調時，它也會轉變成伺機型病原菌導致疾病。嗜中性白血球(Neutrophils)是在哺乳類體內中數量最多的白血球，且在對抗真菌的免疫反應扮演非常重要的角色。近期研究發現嗜中性白血球有個特殊的殺菌機制，會將細胞核的染色體結構解旋後釋放出去，稱為嗜中性胞外網狀結構（Neutrophil Extracellular Traps, NETs），這種網狀結構會纏繞病原菌，使其無法擴散，然後促進其清除和分解。由於微小管(Microtubules)和肌動蛋白(Actins)會調控NETs的形成，且Guanine nucleotide exchange factor H1 (GEF-H1)可以藉由調節RhoA GTPases的活性，進而影響下游微小管和肌動蛋白的動態平衡，因此本論文欲探討GEF-H1在NETs形成時所扮演的角色。藉由使用缺乏GEF-H1的基因改造小鼠，發現GEF-H1並不會影響嗜中性白血球在骨髓中的生成，也不會改變嗜中性白血球內的微小管與肌動蛋白的結構。利用Phorbol 12-myristate 13-acetate (PMA)刺激嗜中性白血球讓其產生NETs，發現在GEF-H1缺陷的小鼠嗜中性白血球的活性氧化物質(Reactive oxygen species, ROS)形成量不受影響，但Neutrophil elastase (NE)、Myeloperoxidase (MPO)和瓜氨酸化組蛋白H3 (Citrullinated histone H3, citH3)在細胞核內的表現量卻減少，且進而影響到NETs形成明顯下降的現象。當將菌絲狀(hyphae)的白色念珠菌與嗜中性白血球一同培養時，和正常的嗜中性白血球相比，缺少GEF-H1的嗜中性白血球形成較少的NETs。綜合以上實驗結果，可推論GEF-H1會調控NETs形成。

Candida albicans is common commensal fungi in human. However, they can also cause fetal disease in immunocompromised patients. Neutrophils are the most abundant type of white blood cells in most mammals, and play a critical role in defense of fungal infection. Previous studies have shown that neutrophils trap and kill a variety of pathogens by neutrophil extracellular traps (NETs), whose formation depends on histone citrullination and dynamic microtubule networks. The guanine nucleotide exchange factor H1 (GEF-H1) is crucial in coupling microtubule dynamics to RhoA GTPase activation in a variety of normal biological situations. It is also a newly defined component of cellular defenses for the detection of microbial effectors during cell invasion by pathogens. However, it remains unknown whether GEF-H1 regulates NET formation in response to pathogen infection. Here I show that GEF-H1 did not affect granulopoiesis of neutrophils in the bone marrow. The actin and microtubule networks were comparable between mouse naïve wild-type and GEF-H1-deficient neutrophils. In naive state, the level of citrullinated histone 3 was reduced in GEF-H1–deficient neutrophils. After activation by phorbol 12-myristate 13-acetate (PMA), the rate of NET release was reduced in GEF-H1–deficient neutrophils compared to wild-type neutrophils. The reduced NET formation in GEF-H1-deificient neutrophils was not to due to the impaired reactive oxygen species (ROS) production. The decreased translocation of neutrophil elastase (NE) and myeloperoxidase (MPO) into nucleus and histone H3 citrulination was observed in GEF-H1-deficient neutrophils. I further found that GEF-H1 deficiency lead to impaired NET formation, ROS production and antifungal immunity in response to Candida albicans infection. Overall, our results suggested a potential role for GEF-H1 in the regulation of NET formation.