抗甲型腫瘤壞死因子製劑對類風溼性關節炎相關細胞激素的影響及表觀基因調控

The Immunomodulatory Effects of Tumor Necrosis Factor-Alpha Inhibitors on Cytokine/Chemokine Expression and Epigenetic Regulation in Rheumatoid Arthritis

林宜靜(Yi-Ching Lin)

指導教授 ： 洪志興

高雄醫學大學/醫學院/臨床醫學研究所/博士(2017年)

摘要

背景:類風濕性關節炎是一種自體免疫疾病，其確切病因仍未完全知曉，目前認為可能與遺傳及環境因子有關。表觀基因的調控被認為是環境因子與遺傳信息DNA之間的橋樑。許多免疫細胞，包括T淋巴細胞、單核細胞以及巨噬細胞，都與類風溼性關節炎的疾病發展有關。抗甲型腫瘤壞死因子製劑是目前在類風濕性關節炎治療中最常使用且效果顯著的生物製劑，但目前對於其在人類免疫細胞的調控仍不完全清楚，且因為在治療效果的異質性以及伺機性感染等嚴重的副作用，類風溼性關節炎的治療上也在尋找其他可能的新標的。

假說:抗甲型腫瘤壞死因子製劑會對人類免疫細胞產生調節作用，進而影響相關細胞激素的表現，此調節作用包含對表觀基因的調控。
目標一:抗甲型腫瘤壞死因子對Th17細胞及相關細胞激素的影響及調控途徑。
目標二:抗甲型腫瘤壞死因子對單核球及趨化激素CCL2的影響及調控途徑。

研究方法:利用人類單核球細胞株THP-1、並招募健康受試者與類風濕性關節炎病人，分離其周邊血液T細胞及單核球，測定這些細胞在給予免疫刺激後加入抗甲型腫瘤壞死因子製劑，對細胞功能及細胞激素的影響及細胞內作用機轉。細胞激素與趨化激素以酵素免疫分析法測定。細胞內調控機制則以受體及路徑抑制劑、西方點墨法、聚合酵素鏈鎖反應、染色質免疫沉澱法進行研究。

研究結果:兩種臨床上常用的抗甲型腫瘤壞死因子製劑，恩博(etanercept)以及復邁(adalimumab)，會抑制類風濕性關節炎病人血中Th17細胞分泌細胞激素IL-17A, IL-17F以及IL-22的產生，其機制主要來自NFκB、MAPK細胞內路徑以及組織蛋白乙醯化對RORγt基因的調控。我們也發現抗甲型腫瘤壞死因子會透過控制組織蛋白特定位置乙醯化(Histone H3, H4 acetylation)及三甲基化（Histone H3K4，H3K9，H3K36，H3K79 trimethylation）來調控人類單核球趨化激素CCL2的產生。

結論:抗甲型腫瘤壞死因子製劑會對人類Th17細胞以及單核球細胞產生調節作用，進而影響相關細胞激素及趨化激素的表現，此調節作用包含對組織蛋白乙醯化及三甲基化的表觀基因調控。此研究成果可提供未來對於類風溼性關節炎等自體免疫疾病或其他慢性發炎疾病的致病機轉及新藥開發的重要基礎。

關鍵字

抗甲型腫瘤壞死因子 ； 表觀基因調控 ； 細胞激素 ； 趨化激素 ； 類風濕性關節炎

並列摘要

Background: Rheumatoid arthritis (RA) is an autoimmune, inflammatory joint disease whose exact cause is still not completely known. Genetic involvement and environmental components are responsible for the development of RA. Epigenetic factors react to external stimuli and form bridges between the environment and the genetic information. Anti-tumor necrosis factor-alpha (anti-TNF-α, also called TNF-α inhibitor) is now in the standard treatment of patients with RA. Despite the extensive use of TNF-α inhibitors in RA treatment, our understanding of the immunomodulatory effects on immune cells and how treatment failures and adverse effects occur remains limited.

Hypothesis: We supposed the TNF-α inhibitors have immunomodulatory effects on the cytokines/chemokines production in human immune cells.

Aim: We aimed to investigate the effects of two common TNF-α inhibitors, etanercept and adalimumab, on IL-17 related cytokines in human Th17-polarized cells and CCL2 production in monocytes, and explore the possible intracellular mechanisms of these effects including epigenetic regulation.

Materials and Methods: THP-1 cell line, the human immune cells (including CD4+ T cells and CD14+ monocytes) were collected from peripheral blood of enrolled healthy subjects and patients with RA. These cells were pretreated with etanercept or adalimumab, followed by immune stimulation or cytokine polarization, to investigate the immunomodulatory effects and possible mechanism. The levels of cytokines and chemokines were determined by enzyme-linked immunosorbent assay. Intracellular signaling was investigated using receptor antagonists, pathway inhibitors, western blot, RT-PCR and chromatin immunoprecipitation.

Results: Th17-polarized cells from patients with RA produced more IL-17A, IL-17F and IL-22 than those from healthy subjects. Etanercept and adalimumab suppressed IL-17A, IL-17F and IL-22 levels in Th17-polarized cells from healthy subjects and patients with RA by decreasing mitogen-activated protein kinase (MAPK)-phospho-p38, nuclear factor-κB (NF-κB)-phospho-p65, phospho- activator of transcription 3 (STAT3) and retinoid-related orphan receptor γ-T (RORγt) expression. Etanercept and adalimumab decreased histone (H)3 and H4 acetylation in the RORγt gene promotor region by decreasing the recruitment of the acetyltransferases p300, CREB-binding protein (CBP) and p300/CBP-associated factor (PCAF). In THP-1 cells and human primary monocytes, etanercept and adalimumab decreased CCL2 production, and these changes in the CCL2 levels were independent of the TNF-α levels. Etanercept and adalimumab suppressed MAPK-phospho-p38, phospho-JNK, phospho-ERK and NF-κB-phospho-p65, and down-regulated acetylation of histone H3 and H4 in the CCL2 promoter region by decreasing the recruitment of the NF-κB associated acetyltransferases p300, CBP and PCAF. Etanercept and adalimumab also down-regulated trimethylation of H3K4, H3K27, H3K36and H3K79 in the CCL2 promoter region by decreasing the expression of the related methyltransferases WDR5 and Smyd2.

Conclusion: TNF-α inhibitors exert suppressive effects on IL-17A, IL-17F, IL-22 and CCL2 expression via MAPKs, NF-κB and epigenetic modifications. These findings broaden the knowledge of the mechanisms underlying the immunomodulatory effects of TNF-α inhibitors and provide novel therapeutic targets for RA.

並列關鍵字

TNF-α inhibitor ； epigenetic regulation ； cytokine ； chemokine ； rheumatoid arthritis

參考文獻

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