探討嗜中性球胞外網狀結構中會影響腸道上皮細胞屏障完整性的成份

Abstract

發炎性腸道疾病 (Inflammatory bowel disease, IBD) 是一種腸道的自體免疫疾病，因為腸道免疫系統對腸道共生微生物產生不正常的免疫反應，使得腸道發炎伴隨腹瀉、直腸出血等症狀。近期有研究指出在潰瘍性大腸炎(Ulcerative colitis)患者的腸子內，可以看到嗜中性球（neutrophil）的浸潤的上升，並且根據蛋白質體分析結果，發現許多跟嗜中性白血球細胞外網狀結構(Neutrophil extracellular traps , NETs)相關蛋白，在潰瘍性大腸炎患者的大腸中比正常受試者的數量多很多，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。 嗜中性白血球細胞外網狀結構(Neutrophil extracellular traps , NETs) 會將自身含有抗微生物蛋白的DNA吐出釋放到胞外形成網狀結構，以限制並進一步清除外來微生物，此網狀結構主要包含嗜中性球本身DNA骨架和纏繞在上的抗微生物蛋白例如：組蛋白（histone）、骨髓過氧化酶（myeloperoxidase, MPO）和嗜中性球彈性蛋白酶（neutrophil elastase, NE）。我們實驗室先前的研究發現當使用葡聚糖硫酸（dextran sulfate sodium, DSS）或三硝基苯磺酸（2,4,6-trinitrobenzene sulfonic acid, TNBS）誘發C57BL/6小鼠產生似人類潰瘍性結腸炎的症狀時，如果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，腸道上皮細胞的破壞程度明顯比沒有注射去氧核醣核酸酶 I的老鼠來的輕微，腸道通透度也比較低，指出在小鼠C57BL/6大腸內嗜中性球胞外網狀結構雖然是一種抵抗微生物的免疫防禦機制，但卻對腸道上皮會具有破壞的現象。 由於以上研究皆為活體內實驗，為了探討在細胞層面嗜中性白血球胞外網狀結構對腸道上皮的破壞，和找出其造成腸道上皮破壞的成分，本篇研究利用人類大腸上皮細胞株Caco-2進行實驗。在Caco-2中加入嗜中性白血球細胞外網狀結構後，代表細胞凋亡訊號的乳酸去氫酶(lactate dehydrogenase, LDH)釋放，和代表上皮細胞通透度的跨上皮電阻值(transepithelial electrical resistance, TEER)變化、穿透過細胞的螢光異硫氰酸鹽(Fluorescein isothiocyanate, FITC)都有顯著增加。嗜中性白血球胞外網狀結構主要為去氧核醣核酸、組蛋白、骨髓過氧化酶和嗜中性球彈性蛋白酶，在Caco-2中加入預先用高溫去除蛋白活性的嗜中性白血球胞外網狀結構，可以明顯看到細胞凋亡信號和通透度的回復，但加入預先用去氧核醣核酸酶 I切割DNA的的嗜中性白血球胞外網狀結構還是維持很高的細胞凋亡信號跟通透度變化，顯示嗜中性白血球胞外網狀結構中的蛋白質而非DNA造成上皮細胞的傷害造成上皮細胞的傷害。而在嗜中性球細胞外網中抑制組蛋白後，上皮細胞受損的現象明顯減緩，但抑制骨髓過氧化酶和嗜中性球彈性蛋白酶後，就沒有這樣的現象，顯示嗜中性白血球胞外網狀結構中的組蛋白是造成上皮細胞的傷害的主要成分。同樣的，我們在Caco-2中直接加入純化的組蛋白後，可以看到明顯上升的細胞毒性和通透度，但在加入骨髓過氧化酶和嗜中性球彈性蛋白酶就沒有這樣的現象。 並且從螢光染色圖跟西方墨點法測量蛋白表現量後可以看到上皮細胞的細胞間連接蛋白（junction protein）在加入嗜中性白血球細胞外網狀結構和組蛋白後被破壞。而在嗜中性球細胞外網狀結構中預先抑制組蛋白，上皮細胞就可以恢復正常連接蛋白的型態和分佈，但預先抑制骨髓過氧化酶和嗜中性球彈性蛋白酶，上皮細胞仍維持被破壞的型態，說明嗜中性球細胞外網狀結構透過它的成份組蛋白去影響上皮細胞間的連接蛋白。最後，在直腸注射組蛋白到小鼠體內後發現，組蛋白也會影響小鼠腸道免疫細胞浸潤、通透度、連接蛋白表現和上皮細胞的凋亡。 總結我們的發現，嗜中性白血球細胞外網狀結構狀結構透過它的成份組蛋白造成細胞的傷害、腸道上皮細胞通透度上升和連接蛋白的破壞。

Inflammatory bowel disease (IBD) is an autoimmune disease characterized by chronic inflammation that causes diarrhea, abdominal pain, and fecal bleeding to patients. Previous studies have observed that the numbers of neutrophils and neutrophil extracellular traps (NETs)-related proteins are highly elevated in the inflamed intestine during colitis, suggesting a prominent role of NET in colitis development. Recently we have shown that DNase I treatments that dissociate NET structure prevent colonic epithelial cell death in mice with Dextran Sulfate Sodium (DSS)-induced colitis. However, the specific NET components that are responsible for cell damage in the intestinal epithelium remains unknown. Given that NETs are mainly composed of DNA, histone, myeloperoxidase (MPO), and neutrophil elastase (NE), here we utilized human intestinal epithelial cell line, Caco-2 to interrogate the impact of each above component on cell cytotoxicity and permeability. Upon incubation with Caco-2 monolayers, NETs induced lactate dehydrogenase (LDH) level, reduced transepithelial electrical resistance (TEER) and Fluorescein isothiocyanate (FITC) dextran leakage. However, protein denaturation in NETs protected the Caco-2 monolayers from the increased cell cytotoxicity and permeability. In contrast, there was no difference in Caco-2 cytotoxicity and permeability when the DNA backbone of NETs was digested. These results indicate that protein is responsible for the cell damage of NETs. Moreover, inhibition of histone by polysialic acid (PSA) in NETs protected the Caco-2 monolayers from the increased cell cytotoxicity and permeability while there was no difference in Caco-2 cytotoxicity and permeability when the activity of MPO or NE was inhibited. Consistently, there was a marked increase in cell cytotoxicity and permeability of Caco-2 monolayers incubated with purified histone but not MPO or NE. Furthermore, immunofluorescence staining and western blot also found that incubation with NETs significantly destroyed tight junction and adherens junction proteins in Caco-2 cells that can be rescued by the inhibition of histones in NETs. In vivo studies further demonstrated that intra-rectal administration of histones in mice altered intestinal permeability, tight junction and adherens junction integrity, epithelial cell death, and immune cell infiltration. In summary, our results suggest that NETs cause cell damage and increase the permeability of colon epithelial cells, particularly through histones.