小鼠骨髓間葉幹細胞介白素-1beta的分泌機制與alpha型防禦素間相關性之探討

Abstract

近來研究指出間葉幹細胞(mesenchymal stem cells; MSCs)在發炎環境中扮演著調節者的腳色，針對不同種類的發炎刺激，會引發細胞內完全相反的反應。本研究針對小鼠骨髓間葉幹細胞中介白素-1beta(IL-1beta)的基因表現及細胞內製造途徑進行調查。 在BALB/c小鼠模式中，取其腹腔巨噬細胞(peritoneal macrophage; pM)、骨髓衍生巨噬細胞(bone marrow derived macrophage; BMDM)作為對照組，以LPS (lipopolysaccharide)與第二刺激物 (Alum或ATP)的組合模擬NLRP3發炎複合體活化及IL-1beta製造途徑。實驗結果發現，間葉幹細胞在不受刺激的情況下即製造少量IL-1beta，製造途徑仍然透過NLRP3發炎複合體活化。但是，製造出來的IL-1beta並不如預期在經後轉譯修飾後隨即被釋放出細胞外，而是堆積在間葉幹細胞的細胞質內，表達未知的生理意義。經文獻回顧，我們假設一種抗微生物胜肽(antimicrobial peptide) alpha型防禦素 (alpha-defensin)能在不影響製造途徑的前提下，調控IL-1bera分泌的能力。比諸作為對照組的巨噬細胞，alpha型防禦素的製造在間葉幹細胞內旺盛許多。並且，在使用免疫沉澱(immunoprecipitation)的成果中，我們也看到alpha型防禦素與IL-1beta間具直接相互結合的能力。然而，關於兩者之間關係的直接證據，尚待繼續研究。 總結而言，本篇研究在間葉幹細胞中發現了自發性活化的NLRP3發炎複合體及在該細胞中IL-1beta具有獨特的分布現象。我們進一步假設了alpha型防禦素在發炎環境中的腳色與IL-1beta的分泌相關。本研究的主要貢獻在於報導一個全新且獨特存在於間葉幹細胞中的反應途徑，使我們對於該細胞在發炎環境中的反應有更深一層的認識。

In recent studies, the role of mesenchymal stem/stromal cells (MSCs) has become a modulator of inflammation in response to different inflammatory signals. Among all, we focus on the profile of NLRP3 inflammasome and its main product cytokine IL-1beta. Secretion and processing pathway of IL-1beta in BALB/c mice thioglycollate-elicited peritoneal macrophages (pMs), bone marrow derived macrophages (BMDMs) and BM-MSCs were investigated at both protein and RNA level after lipopolysaccharide (LPS) and a secondary stimulator (Alum or ATP) treatment. Interestingly, we found the NLRP3-caspase 1-IL-1beta pathway autonomously launched without LPS stimulation in MSCs. Moreover, MSCs maintain this small amount of IL-1beta in their cytosol for unknown physiological purpose. Since this discovery is apart from our knowledge of secretion right after production, we further postulated that mouse alpha-defensins, cryptdin 1 and cryptdin 4, might play a modulatory role in blocking the release of IL-1beta based on the previous reports and our observation of their larger amount in MSCs compared to pMs. Furthermore, the immunopreicipitation data also supported alpha-defensins would bind with IL-1beta in the cell. In conclusion, we demonstrated autonomous NLRP3 inflammasome activation in MSCs and a possible new role of alpha-defensins involved in inflammation. We believed that this investigation here might shed light on further understanding the mechanism of IL-1beta release and also therapeutic strategies of high-efficacy MSC-based therapy in autoimmune diseases.