探討感染性心內膜炎中轉糖鏈球菌誘導嗜中性白血球胞外網狀結構形成機制

Abstract

嗜中性白血球作為先天免疫上第一線的防禦來抵禦感染，目前提出有三種嗜中性白血球的機制可以抑制病原菌的擴散，包括吞噬作用、去顆粒作用以及形成嗜中性白血球胞外陷阱(Neutrophil extracellular traps，NETs)。NETs被認為可以防止細菌與真菌的擴散，然而，在一些調控異常的情況下，它們也會影響疾病的惡化，例如自體免疫疾病以及癌症。我們先前的研究證明了NETs在感染性心內膜炎(Infective Endocarditis，IE)的病理機轉中扮演重要角色，同時也顯示IE的致病菌-轉糖鏈球菌(Streptococcus mutans)不僅可以直接誘導嗜中性白血球組蛋白瓜氨酸化，也會提升血小板上P-selectin的表現量，進而提供另一個訊號來促進NETosis發生。在本篇研究中，我們發現轉糖鏈球菌誘導的NETosis需要依靠嗜中性白血球彈性蛋白酶的活性，而不需要PAD4介導的組蛋白瓜氨酸化。此外，在沒有血小板的條件下，轉糖鏈球菌的感染會誘導嗜中性白血球LC3B-II表現，而經由bafilomycin A1這個液胞H+-ATPase抑制劑的處理之下，則會阻止轉糖鏈球菌誘導的LC3B-II表現，並促進組蛋白瓜氨酸化、彈性蛋白酶易位以及NET形成。在共軛焦顯微鏡的圖像中顯示Rubicon與LC3B具有共定位，意味著在轉糖鏈球菌的感染下，會誘導LC3相關吞噬作用(LC3-associated phagocytosis，LAP)的形成。在bafilomycin A1的處理之下，會降低嗜中性白血球吞噬殺死轉糖鏈球菌的能力，證實LAP在嗜中性白血球對抗轉糖鏈球菌感染中的重要性。綜合來說，這些研究結果顯示當利用bafilomycin A1抑制轉糖鏈球菌誘導的LAP形成，將會促進組蛋白瓜氨酸化、彈性蛋白酶易位以及NET形成。

Neutrophils are the first line of innate immune defense against infection. Three mechanisms of neutrophils to restrain pathogen spreading are proposed: phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs). NETs are thought to prevent bacterial and fungal dissemination; however, in some unregulated condition, they also play roles in disease progression, such as autoimmune diseases and tumor. Our previous study demonstrated the important role of NETs in the pathogenesis of infective endocarditis (IE) and showed that Streptococcus mutans (S. mutans), an IE-pathogen, not only directly induced neutrophil histone citrullination, but also up-regulated the expression of P-selectin on platelets to provide another signal to promote the NETosis. In this study, we found S. mutans-induced NETosis relied on neutrophil elastase activity and was independent of PAD4-mediated histone citrullination. In addition, S. mutans infection induced LC3B-II expression in neutrophils, and treatment with bafilomycin A1, a vacuolar H+-ATPase inhibitor, blocked S. mutans-induced LC3B-II expression, and promoted histone citrullination, elastase translocation and NET formation in the absence of platelets. Confocal microscopy image showed the colocalization of Rubicon and LC3B, suggesting S. mutans infection induced the formation of the LC3-associated phagocytosis (LAP). Treatment with bafilomycin A1 reduced neutrophil phagocytic killing activity against S. mutans, confirmed the role of LAP in neutrophil against S. mutans infection. Taken together, these data suggested inhibition of S. mutans-induced LAP formation by bafilomycin A1 will promote neutrophil histone citrullination, elastase translocation and NET formation.