- 學位論文
表皮角質細胞在皮膚疾病的免疫調節角色:著重於化學致癌及異位性皮膚炎
Immunoregulation of epidermal keratinocytes in cutaneous diseases: focusing on chemical carcinogenesis and atopic dermatitis
摘要
皮膚為個體重要的阻絕外界病原體或過敏原的物理屏障，其中組成皮膚最多的表皮角質細胞多方位參與固有的免疫調控，可以分泌很多細胞激素及趨化激素，表現Toll-like receptor (TLR)，分泌Antimicrobial peptides，而角質細胞的分化不良合併發炎則是皮膚癌化的前期反應。角質細胞除了調節腫瘤免疫反應，也是許多免疫反應攻擊的對象。因此，本研究應用兩種模式，包括化學致癌及異位性皮膚炎，來深究角質細胞在癌症及發炎的角色。 化學性致癌: 砷為重要的環境危害物質能導致人類癌症。砷可引起原位皮膚癌，又稱波文氏病(As-BD)，也是最為常見之皮膚砷癌，其微觀特徵是表皮分化不良、凋亡、表皮增厚和發炎細胞浸潤。儘管砷致癌機轉已有很多文獻，惟宿主免疫如何調節砷致癌進程仍多所未知。As-BD患者的接觸過敏反應是降低的，在砷癌中，T細胞的活化受抑制的機轉已被廣泛報告。但是樹突狀細胞（DC）為調節先天免疫的關鍵細胞，其如何調節砷癌免疫反應仍不清楚。本研究使用人類As-BD患者及正常對照的周邊血骨髓樹突狀細胞（MDDC），以及有或無砷餵養小鼠的骨髓樹突狀細胞（BMDC），比較有無砷存在時樹突狀細胞的遷移情形。結果顯示As-BD患者MDDC的CCL21引導的體外移動能力降低；BMDCs注入小鼠腳底後，餵養砷小鼠膕窩淋巴結的樹突狀細胞移動明顯較低。比較As-BD與正常對照皮膚，STAT3的活化增加。砷也可促進角質細胞STAT3的活化及VEGF製造，但以STAT3阻斷劑JSI-124或是RNA干擾阻斷可降低砷引起VEGF的增加。雖然VEGF可些微誘導MDDC的CD86和MHC-II的表達，但砷處理可以阻斷此VEGF對MDDC的作用。結果發現在As-BD砷癌的微環境中，角質細胞中的STAT3-VEGF的路徑可抑制樹突狀細胞的移動，顯示細胞間的相互作用在調節砷癌症過程中扮演重要角色。 異位性皮膚炎: IL-9和IL-9R在氣喘的發病機轉中扮演重要角色，而IL-9/IL-9R在異位性皮膚炎（AD）的作用機轉文獻並不多，包括核苷酸多樣性，在患病者的皮膚IL-9和IL-9R的轉錄訊號增加，以及血中IL-9的濃度與AD臨床嚴重程度的關聯性。但很少探討有關IL-9 / IL-9R在AD皮膚的病生理調控機轉。我們研究IL-9R是否表現在表皮角質細胞，及IL-9R的活化在角質細胞有什麼功能性，其細胞因子的產生和胞內信號通路是如何調控的。本研究經由免疫螢光的檢查，測量和比較AD患者皮膚和對照組皮膚的IL-9R的表現。對IL-9處理的角質細胞，並以流式細胞儀測量IL-9R的表現， 及以西方墨點法測量mTOR，S6K，ERK，p38和STAT3的活化，以ELISA測量細胞因子的產生，再以免疫螢光染色測量STIM1的表現。結果發現，IL-9R確實表現在角質細胞上，但纖維母細胞沒有。IL-4能增加IL-9R在角質細胞的表現，但TGF-β1則無影響。IL-9誘導角質細胞產生IL-8，但CXCL16、CCL22、TSLP及IL-33不會被誘發。IL-9能誘導形成STIM1點狀聚集，及能誘導ERK磷酸化有劑量和時間依賴性，不會活化mTOR、S6K、P38或STAT3。以ERK抑制劑U0126，而非mTOR抑制劑rapamycin可以去除IL-9誘導的IL-8產生。STIM1的抑制劑BTP2或SKF96265處理可以去除IL-9導致的ERK磷酸化和IL-8的產生。本研究首次證明角質細胞中IL-9-STIM1-ERK-IL-8路徑調節機轉，及此路徑在AD的病生理學中扮演的重要角色，具學術及臨床的參考價值。 綜上所述，角質細胞不僅是人體的物理性屏障，而且是重要多元的環境傳感器(environmental sensor) ，可以在皮膚癌發生和炎症過程中擔任活性的免疫調節作用。治療干擾角質細胞的功能和作用途徑及免疫反應可能是未來治療各種皮膚疾病的好方法。
並列摘要
Skin is an important physical barrier for the host to defend foreign pathogens or barricade allergens. Epidermal keratinocytes are the predominant cells in skin that regulate innate immunity in a multifaceted way. Kerationcytes could produce a robust of cytokines and chemokines, express toll-like receptors, and secrete antimicrobial peptides. The abnormal differentiation of keratinocytes along with the inflammation, on the other hand, is an early event in skin carcinogenesis. Besides in the regulation of tumor immunity, keratinocytes are also the targets of many immune diseases and reactions. Therefore, this study would like to investigate the role of keratinocytes in skin carcinogenesis and inflammation, using the models of chemical carcinogenesis and atopic dermatitis, respectively. For arsenic carcinogenesis, arsenic remains an important environmental hazard that causes several human cancers. Arsenic-induced Bowen's disease (As-BD), a skin carcinoma in situ, is the most common arsenical cancer. While great strides have been made in our understanding of arsenic carcinogenesis, how host immunity contributes to this process remains unknown. Patients with As-BD have an impaired contact hypersensitivity response. Although impaired T cell activation has been well-documented in arsenical cancers, how dendritic cell (DC), the key cell regulating innate immunity, regulates the immune response in arsenical cancers remains unclear. Using myeloid derived DC (MDDC) from patients with As-BD and normal controls as well as bone marrow derived DC (BMDC) from mice fed with or without arsenic, we measured the migration of DC. As-BD patients showed an impaired CCL21-mediated MDDC migration in vitro. Arsenic-fed mice had defective DC migration towards popliteal lymph nodes when injected with allogenic BMDCs via foot pad. Using skin from As-BD and normal controls, we found an increased expression of STAT3, a transcriptional factor contributing to impaired DC activation. Arsenic induced STAT3 activation and the production of VEGF in keratinocytes. The increase in VEGF was blocked by inhibiting STAT3 with RNA interference or pharmaceutically with JSI-124. While VEGF by itself minimally induced the expression of CD86 and MHC-II in MDDC, arsenic induced-MDDC activation was abolished by VEGF pretreatment. We concluded that the STAT3-VEGF axis in keratinocytes inhibits DC migration in the microenvironment of As-BD, indicating that cellular interactions play an important role in regulating the disease course of arsenical cancers. For atopic dermatitis, IL-9 and its receptor play important roles in the pathogenesis of asthma. Its role in atopic dermatitis (AD) was examined in just a few studies, including nucleotide polymorphisms, increased transcriptional levels of IL-9 and IL-9R in diseased skin, and an association of blood IL-9 levels with clinical severity. Little was known about the pathophysiological regulation of IL-9/IL-9R in AD skin. We asked whether IL-9R was expressed in epidermal keratinocytes; if so, what the functional outcome, cytokine production, and signaling pathway of IL-9/IL-9R in keratinocytes are. We measured and compared the expression of IL-9R in skin from AD patients and controls by immunofluorescence. We also performed in vitro studies on the IL-9-treated primary keratinocytes, including flow cytometry for IL-9R expressions, Western blotting for mTOR, S6K, ERK, p38, and STAT3 activations, ELISA for cytokine levels, and immunofluorescence for STIM1. We found that IL-9R was indeed expressed in keratinocytes but not in fibroblasts. Its expression in keratinocytes was enhanced by IL-4 but not by TGF-beta1. IL-9 induced a moderate production of IL-8 but not CXCL16, CCL22, TSLP, nor IL-33. IL-9 induced formation of STIM1-puncta. IL-9 induced ERK phosphorylation both dose- and time-dependently, but not mTOR, S6K, p38, or STAT3. Pretreatment with U0126 (ERK inhibitor) but not rapamycin (mTOR inhibitor) abrogated the IL-9-mediated IL-8 production. Blockage of STIM1 with BTP2 or SKF96265 abrogated ERK phosphorylation and IL-8 production induced by IL-9. Conclusion: This study represents the first to show the regulation of the IL-9-STIM1-ERK-IL-8 axis in keratinocyte, and how the axis might play an important role in the pathophysiology of AD. In summary, keratinocytes not only contribute to the physical barrier but also plays an active immunoregulatory role in the process of skin carcinogenesis and inflammations. Therapies interfering keratinocyte functions, pathways, and immune reactions might be a good future approach in treating various skin diseases.