嗜中性球胞外網狀結構對DSS誘發小鼠結腸炎模式之影響

Abstract

嗜中性球（Neutrophil）為哺乳類血液中最主要的白血球種類，以吞噬作用（Phagocytosis）、去顆粒化（Degranulation）以及嗜中性球胞外網狀結構（Neutrophil extracellular traps, NETs）等機制，於第一時間參與宿主對入侵病原的防禦並招募其他免疫細胞至發炎處執行後續的免疫功能。嗜中性球胞外網狀結構由網狀組蛋白（Chromatin）與其他顆粒蛋白或細胞質蛋白在細胞內組成複合體後，伴隨著細胞膜的破裂被釋放到細胞外捕捉並破壞病原。 發炎性腸道疾病（Inflammatory Bowel Disease, IBD）主要可分為兩種型態，潰瘍性結腸炎（Ulcerative colitis, UC）與克隆氏症（Crohn’s disease, CD），近期有一蛋白質體分析之研究發現，相較健康的受試者，潰瘍性結腸炎病患的腸道組織切片中，嗜中性球細胞外網相關蛋白伴隨炎症有上升的現象。另於臨床研究中更發現嗜中性白血球浸潤與發炎區域重疊，並且在潰瘍性結腸炎病患的檢體中偵測到較多嗜中性球細胞外網構造。以先前研究的結果初步指出嗜中性球細胞外網對於發炎性腸道疾病而言可能是個不利的因素。 然而，嗜中性球細胞外網本為嗜中性白血球抵抗入侵病原的防禦機制，且在潰瘍性結腸炎的病理情況下，腸道粘膜破損會造成腸道共生微生物有直接入侵宿主的機會，因此嗜中性球細胞外網在發炎的不同時期可能扮演著不同的角色。本篇研究以dextran sulfate sodium（DSS）誘發C57BL/6小鼠產生似人類潰瘍性結腸炎的症狀，再以尾靜脈注射去氧核醣核酸酶 I（Deoxyribonuclease I, DNase I）作為抑制嗜中性球細胞外網結構之藥物，施打後觀察到潰瘍性結腸炎的臨床症狀有減緩的趨勢，並且大腸長度的恢復、發炎細胞浸潤以及腸道結構破損程度上有明顯改善。此外，免疫螢光染色觀察下，給予DNase I組別的小鼠有較少髓過氧化物酶（Myeloperoxidase, MPO）與瓜氨酸化組蛋白 H3（Citrullinated histone H3, Cit H3）之表現，大腸組織的凋亡細胞比例也明顯降低。於腸粘膜細胞凋亡比率上升與腸道通透度增加的結果可推測嗜中性球細胞外網可能直接造成腸道屏障功能受損、促進發炎反應，並導致較嚴重的臨床與病理指標。綜合本篇研究的成果，可以推測嗜中性球細胞外網在急性潰瘍性結腸炎中應扮演著傷害宿主以及過度促炎的角色。

Both the most abundant leukocytes and the first line defense in mammals, neutrophils possess several antimicrobial functions, such as phagocytosis, degranulation, and neutrophil extracellular traps (NETs). They can stop invasive pathogens from spreading by recruiting other immune cells to the site of inflammation and promoting further immune responses for eliminating threats. NETs consisting of web-like chromatin decorated with cytosolic and granule proteins are released by neutrophils to capture and kill the pathogens. A recent proteome analysis of intestinal biopsies from ulcerative colitis (UC) patients indicates an increased neutrophil abundance and aberrant NET formation in the inflamed colon tissues. Another clinical study shows overlapping of infiltrated neutrophils at the damaged mucosa in UC patients and detects higher NET formation. These results indicate that NETs serve as a detrimental factor in IBD. However, NETs are a defensive mechanism utilized by neutrophils to exclude invasive pathogens during inflammation. In this case, ulcerative colitis causes damage on the epithelial layer and breaches the commensal microorganisms. Excessive inflammation under IBD seems to be related to NET formation to shift to the development of overly active inflammation. Here we demonstrated that reducing NET formation by DNase I administration suppressed the severity of colitis symptoms in mice during DSS-induced colitis. The DNase I treatment also alleviated colon shrinkage, levels of inflammatory marker and pro-inflammatory cytokines, and apoptotic cell coverage in colon tissue. Overall, our results suggested that excessive NETs in colitis are capable of worsening inflammation, promoting apoptosis, and interfering with gut barrier function. Furthermore, the administration of DNase I as a NET inhibitor by intravenous injection can effectively reduce colitis symptoms implying that DNase I can act as a potential treatment or reliever of IBD.