白細胞介素17A和NLRP3發炎體在葛瑞芙氏眼病變致病機轉中所扮演之角色

研究目的: 葛瑞芙氏眼病變Graves’ ophthalmopathy (簡稱GO) 和自體免疫疾病相關聯。病變位於眼窩脂肪、結締組織及眼外肌，臨床表現為眼瞼攣縮、突眼、眼周水腫、眼外肌活動受限等，然而葛瑞芙氏眼病變的致病機轉至今尚未完全明瞭。最近的研究發現，白細胞介素17A (Interleukin-17A, IL-17A)、發炎體和相關的發炎細胞激素可能在葛瑞芙氏眼病變的自體免疫上參與其中。因此，我們設法瞭解IL-17A和NLRP3發炎體在葛瑞芙氏眼病變致病機轉中所扮演之角色，以提供將來治療的新方向。研究方法: 葛瑞芙氏眼病變患者和非葛瑞芙氏眼病變者各30名，前者為GO實驗組，後者為對照組，取其眼窩脂肪結締組織當作實驗檢體，並進行眼窩纖維母細胞培養。比較兩組之NLRP3發炎體免疫組織化學染色 (immunohistochemical staining)；以IL-17A刺激纖維母細胞後，利用即時反轉錄聚合酶鏈鎖反應 (real-time RT-PCR)、西方墨點法 (Western blot)、及酵素連結免疫吸附法 ( ELISA) 分析兩組pro- IL-1β、IL-1β、NLRP3及caspase-1濃度變化。接著在眼窩纖維母細胞培養中加入caspase-1抑制劑，並使用小分子干擾核糖核酸 (siRNA) knockdown NLRP3發炎體後，觀察兩組相關細胞激素 (cytokine) 變化情形。統計分析方法為One-way ANOVA及2-tailed Student’s t tests。研究結果: 免疫組織化學染色及細胞培養染色顯示在葛瑞芙氏眼病變患者的眼窩結締組織中相較於健康對照組，有較明顯的NLRP3發炎體表現。IL-17A能誘導GO眼窩纖維母細胞增加pro- IL-1β mRNA表現及白細胞介素1β (IL-1β) 蛋白質濃度。此外，IL-17A能活化caspase-1及NLRP3發炎體表現，表示NLRP3發炎體的激活和IL-17A息息相關。再者，我們使用caspase-1的專一抑制劑Z-YVAD-fmk，發現其能有效抑制IL-1β分泌，且和它的濃度呈正相關性。當siRNA轉染 (transfection) 纖維母細胞，能夠向下調節NLRP3 mRNA及蛋白質的表現。同時，NLRP3發炎體的knockdown也顯著地抑制IL-17A所調節之pro-IL-1β表現量。結論: 我們的研究發現IL-17A能夠透過眼窩纖維母細胞NLRP3發炎體的活化，刺激IL-1β的生成，進而促進相關發炎細胞激素分泌，導致後續自體免疫發展和葛瑞芙氏眼病變的形成。同時，也給予我們未來治療葛瑞芙氏眼病變藥物發展之新線索。

關鍵字

葛瑞芙氏眼病變；甲狀腺眼疾；白細胞介素17A ； NLRP3發炎體；白細胞介素1β

並列摘要

Objective: The development of Graves’ ophthalmopathy (GO) is associated with autoimmune dysfunction. Recent findings in GO indicate that IL-17A, inflammasome and related cytokines may also be involved in the autoimmunity of GO. We sought to investigate the pathogenic functions of IL-17A and NLRP3 inflammasome in GO. Methods: Orbital fat specimens were collected from 30 GO patients and 30 Non-GO controls. Immunohistochemical staining of orbital fat tissues and cell cultures of orbital fibroblasts were conducted in GO and Non-GO groups. Then, IL-17A was added to the fibroblast cultures, and cytokine expression, signaling pathways, and inflammasome mechanism were investigated using real-time RT-PCR, ELISA, Western blot, and small interfering RNA (siRNA). Results: Immunohistochemical staining of orbital fat specimen showed more expression of NLRP3 in orbital tissue of GO, compared with normal subjects. Interleukin-17A upregulated the mRNA levels of pro-IL-1β and the protein level of IL-1β in GO group. Furthermore, IL-17A was validated to enhance caspase-1 and NLRP3 protein expression in orbital fibroblasts, suggesting NLRP3 inflammasome activation. Inhibiting caspase-1 activity could also decrease IL-1β secretion. When siRNA transfected the orbital fibroblasts, NLRP3 expression was knockdown significantly and IL-17A-mediated pro-IL-1β mRNA release was also downregulated. Conclusions: Our observations illustrate that IL-17A could promote IL-1β production from orbital fibroblasts via the NLRP3 inflammasome in Graves’ ophthalmopathy, and subsequent cytokines may induce more inflammation and autoimmunity. This gives us the clues to target therapeutic potential for GO in the future.

並列關鍵字

Graves’ ophthalmopathy ； IL-17A ； NLRP3 inflammasome ； IL-1β

參考文獻

Antonelli A, Saracino A, Alberti B, et al. High-dose intravenous immunoglobulin treatment in Graves' ophthalmopathy. Acta Endocrinol (Copenh) 1992;126:13-23.

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