肌凝蛋白輕鏈激酶和Rho相關激酶參與細菌入侵腸道經由穿細胞與間細胞途徑所扮演的角色

Abstract

背景介紹:單層腸道上皮細胞透過緊密連接 (Tight junction, TJs) 作為物理性屏障來防衛細菌入侵。腸屏障功能失常導致異常細菌轉移和引發敗血倂發症的病理現象，可見於腸道疾病如腸阻塞（intestinal obstruction, IO）。目前腸阻塞導致異常細菌轉移經由穿細胞和間細胞機制尚不明瞭。過去研究指出腸道上皮細胞內吞細菌之現象可見於腸阻塞，且此病理現象與干擾素-γ（IFNγ）的產生有密切相關性。目前還不清楚共生細菌如何通過緻密的微絨毛根部的終末端網絡（terminal web, TW）而被內吞入腸道上皮細胞，而其分子機制是否依賴肌凝蛋白輕鏈激酶 (myosin light chain kinase, MLCK)活化刷狀緣扇形展開亦不明。另一方面，上皮細胞間通透性是藉由緊密連結蛋白重組和肌凝蛋白輕鏈 (myosin light chain, MLC)磷酸化的調節。誘導型一氧化氮合成酶 (inducible nitric oxide synthase, iNOS)大量表現是細菌轉移的病理變化之一，然而其分子機制並不清楚。研究目的: 利用腸阻塞模式來進一步探討共生細菌侵入腸道上皮細胞經由穿細胞(探討細菌內吞之分子和超微結構機制，著重於IFNγ依賴性MLCK的調控作用)與間細胞(本研究主要探討ROCK及PKCzeta在腸道上皮iNOS所造成的屏障損傷中所扮演的角色)途徑之分子機制。方法：在活體小鼠末端進行10公分結紮形成腸阻塞，由腸管腔注射入vehicle，ML-7 （MLCK抑制劑）或L-Nil （iNOS抑制劑）或Y27632 (ROCK抑制劑)。腸阻塞6或24小時後，將腸組織嵌在Ussing chamber上來測量大分子物質通通性。肝及脾組織用來評估細菌數量。Caco-2細胞給予100 IU/ml IFNγ之和1mM SNAP（NO供體）刺激後，進行跨上皮電阻和通通性評估。結果:我們的研究顯示將小鼠腸阻塞6小時後，腸道上皮細胞內吞細菌但無緊密連結蛋白的破壞。而終末端網絡處MLC磷酸化增加，拱形作用及刷狀緣扇形展開的現象，皆與細菌穿過微絨毛內空隙有相關性；此現象會被ML-7 和anti-IFNγ抑制, 但無法被Y27632抑制。此外，長型MLCK-210或IFNγ基因缺陷的小鼠腸阻塞6小時並未出現該現象。人類Caco-2BBe細胞受IFNγ刺激後，會先造成MLCK依賴性終末端網絡的拱形作用和刷狀緣扇形展開，之後才伴隨細菌經由caveolin-1所媒介富含胆固醇的脂筏被內吞至細胞。另一方面，小鼠腸阻塞24小時後，會造成腸道上皮緊密連結損害和上皮通透性增加的現象；若腸管腔注射給予L-Nil (iNOS抑制劑)或Y27632 (ROCK抑制劑) 則可減緩。L-Nil降低腸阻塞所造成MLC，MYPT1及PKCzeta磷酸化表現量，推測iNOS是ROCK及PKCzeta訊息的上游。在體外實驗顯示SNAP (一氧化碳供體) 造成跨上皮細胞電阻降低及探針通透性增加, 主要透過ROCK依賴性造成MLC磷酸化及PKCzeta依賴性活化造成緊密連結蛋白破壞。結論: 我們研究顯示腸阻塞6小時會造成細菌經由MLCK依賴性穿細胞途徑侵入腸道上皮細胞，而24小時則可見ROCK依賴性緊密連結損害和細菌經由間細胞進入黏膜。腸道上皮屏障功能失常會造成共生細菌經由穿細胞及間細胞途徑進入黏膜，而可能導致慢性腸道炎症和敗血症的誘發或復發。

Background: The single-layer epithelial cells linked by tight junctions (TJs) act as physical barriers to separate the microbiota from the lamina propria. Bacterial translocation (BT) and septic complications are documented in intestinal obstruction (IO). Whether bacterial crosses the epithelial layer through transcellular or intercellular routes in IO is poorly understood. Bacterial internalization to epithelial cells has been documented in IO, and is associated with interferon-gamma (IFNγ) production. It remains unclear how bacteria gain access to epithelial soma through densely packed microvilli rooted on terminal web (TW) and if myosin light chain kinase (MLCK)-dependent brush border (BB) fanning plays a role. On the other hand, intercellular epithelial permeability is regulated by TJ reorganization and myosin light chain (MLC) phosphorylation. Overexpression of inducible nitric oxide synthase (iNOS) is implicated in the pathogenesis of BT, of which the mechanism remains unclear. Aim: Our research objectives were to investigate the molecular mechanism of transcellular bacterial endocytosis (focusing on regulatory roles of IFNγ and MLCK) and intercellular tight junctional impairment (focusing on the roles of ROCK and PKCzeta in epithelial iNOS-mediated permeability increase) using a mouse model with IO. Methods: Mouse distal small intestines were obstructed by loop ligation in which vehicle, ML-7 (a MLCK inhibitor) or L-Nil (an iNOS inhibitor), or Y27632 (a ROCK inhibitor) was luminally administered. After obstruction for 6 or 24 hours, intestinal tissues were mounted on Ussing chambers for macromolecular flux. Liver and spleen tissues were assessed for bacterial counts. Caco-2 cells were exposed to 100 IU/ml IFNγ and 1 mM SNAP (a NO donor), and transepithelial resistance (TER) and permeability were evaluated. Results: Our results showed that following IO for 6 hrs, epithelial endocytosis was observed in the absence of TJ damage. Enhanced TW MLC phosphorylation, arc formation and BB fanning coincided with intermicrovillous bacterial penetration, which were inhibited by ML-7 (a MLCK inhibitor) and neutralizing anti-IFNγ, but not Y27632. The phenomena were not seen in mice deficient of long MLCK-210 or IFNγ. Stimulation of human Caco-2BBe cells with IFNγ caused MLCK-dependent BB fanning, which preceded caveolin-mediated bacterial internalization through cholesterol-rich lipid rafts. On the other hand, mice with IO for 24 hrs displayed epithelial permeability rise and BT, associated with TJ disruption and MLC phosphorylation. Enteric instillation of L-Nil (an iNOS inhibitor) and Y27632 (a ROCK inhibitor) attenuated the IO-induced barrier damage. L-Nil decreased IO-induced MLC, MYPT1 and PKCzeta phosphorylation, suggesting that iNOS is upstream of ROCK and PKCzeta signaling. In vitro studies showed that SNAP (a NO donor) induced transepithelial electrical resistance drop and permeability rise via ROCK-dependent MLC phosphorylation and PKCzeta-dependent TJ reorganization. In summary, we showed that epithelial iNOS activates two distinct signals, PKCzeta and ROCK, to disrupt TJs leading to bacterial influx. Conclusion: MLCK-dependent BB fanning and bacterial endocytosis were seen after 6 hrs of IO and ROCK-dependent TJ disruption in enterocytes and intercellular permeability increase were noted after 24 hrs of IO. Impaired epithelial barrier allowing intercellular and transcytotic penetration of bacteria may contribute to initiation or relapse of chronic gut inflammation and septic complications.