促炎性激素引發腸道上皮細胞內吞細菌之機制

Abstract

背景﹕促炎性激素如﹕IFNγ、TNFα和IL-1β可造成腸道屏障功能損壞，像是高濃度IFNγ可造成腸道上皮細胞緊密連結破壞，而低濃度IFNγ導致腸道上皮細胞內吞細菌。由先前文獻發現，MLCK活化皆參與間細胞(透過細胞間空隙)或穿細胞(直接穿越細胞)途徑其中，然而MLCK是如何分別調控兩條途徑仍尚未清楚。此外，近期發現TNF superfamily之一TL1A和其受器DR3或DcR3結合後，會影響IFNγ產量，然而TL1A是否有參與腸道屏障功能調控未知。目的﹕探討促炎性激素刺激下，引發腸道上皮細胞內吞細菌之機制。方法材料﹕利用C57BL/6小鼠將其遠端小腸利用機械性打結引發腸阻塞，手術後6小時或24小時收取其腸道上皮細胞，測量細菌內吞之情形。此外，分離上皮細胞或收取黏膜層組織利用西方墨點法和反轉錄酶聚合酶鏈反應觀察TL1A和DR3表現。利用人類大腸癌細胞株Caco-2 cells給予IFNγ (100 IU/ml) 或TL1A (10 ng/ml)刺激，及在其上層加入非致病菌E. coli測量細菌內吞情形及蛋白表現改變，以及測量跨細胞電阻和緊密連結蛋白occludin變化。另外，將細胞轉染siMLCK或siMLCK1之後，或是先加入MLCK抑制劑和ROCK抑制劑，再給予激素之刺激。 結果﹕由動物實驗結果顯示，在腸阻塞動物中，黏膜層TL1A和DR3表現量增加與細菌內吞至腸道上皮細胞內有密切相關，然而MLC異構體無變化。 此外，我們發現在人類大腸癌細胞株Caco-2 和HT-29常態性表現TL1A、DR3和DcR3，並且受到E. coli暴露後，細胞TL1A蛋白表現量上升，由此我們推測腸道上皮細胞是TL1A製造來源。我們證明IFNγ和TL1A增加MLCK蛋白表現促使終末端網絡MLC磷酸化，並且無破壞緊密連結，且利用抑制劑證明TL1A引發細胞內吞細菌過程是藉由MLCK而非ROCK調控。而Caco-2細胞常態性表現MLCK異構體MLCK1和MLCK2及MLC異構體Myl12a、Myl12b，若細胞受到IFNγ或TL1A刺激，會增加MLCK2表現而其餘不改變。此外，我們利用siMLCK看到細菌內吞現象顯著降低，然而siMLCK1則不影響TL1A引發細菌內吞之情形，推測由MLCK2參與細菌內吞之機制。相反地，siMLCK1可抑制高濃度IFNγ而導致Caco-2細胞之跨細胞電阻降低。結論﹕低濃度IFNγ和TL1A引發MLCK活化造成腸道上皮細胞內吞細菌，且其過程中無緊密連結破壞和MLCK1之參與；低濃度IFNγ和TL1A增加long MLCK2表現量，但不影響MLCK1和Myl異構體的表現。高濃度IFNγ導致緊密連結破壞且其現象和MLCK1相關。

Background: Pro-inflammatory cytokines, such as IFNγ, TNFα, and IL-1β, causes intestinal barrier defects. High dose IFNγ induced tight junctional disruption, whereas low dose IFNγ triggered bacterial endocytosis. Both paracellular and transcellular permeability are dependent on activation of myosin light chain kinase (MLCK), through which the differential regulatory mechanisms remain elusive. A novel member of TNF superfamily TNF-like 1A (TL1A) is recently identified from monocytic cell lineages, and binds to death receptor 3 (DR3) and death decoy receptor (DcR3) for modulation of IFNγ production. It remains unclear whether TL1A also causes epithelial barrier damage. Aim: To investigate the regulatory mechanisms of bacterial endocytosis by epithelial cells under proinflammatory stress. Methods: Distal small intestines of C57BL/6 mice were obstructed by loop ligation to induce barrier damage. After obstruction for 6 or 24 hours, intestinal epithelial cells were isolated for measuring bacterial endocytosis. Isolated epithelial cells and mucosal samples were examined for TL1A and DR3 expression by Western blotting and RT-PCR. Human Caco-2 cells (clone C2BBe) were treated with TL1A (10 ng/ml) or IFNγ (100 IU/ml) for assessment of transepithelial electrical resistance (TER) and dextran flux as tight junctional changes, and apically exposed to nonpathogenic and noninvasive E.coli for measuring bacterial endocytosis. In separate experiments, cells were transfected with siMLCK or siMLCK1, or pretreated with pharmacological inhibitors to MLCK or Rho-associated kinase (ROCK), prior to the cytokine treatment. Results: Our murine study showed that following IO, increased mucosal expression of TL1A and DR3 correlated with augmented bacteria endocytosis by epithelial cells. No change in epithelial Myl isoforms was seen. Constitutive expressions of TL1A, DcR3 and DR3 mRNA were found in Caco-2 and HT-29 cells, and bacterial exposure increased TL1A protein expression, suggesting epithelial sources and autocrine actions of TL1A. We demonstrated that IFNγ and TL1A increased MLCK expression and induced MLC phosphorylation in terminal web regions without tight junctional damage in Caco-2 cells. TL1A-induced bacterial endocytosis was inhibited by pretreatment with ML-7 (a specific inhibitor to MLCK) but not Y27632 (a specific inhibitor to ROCK). Splicing variants of long MLCK-1 and -2, as well as MLC isoforms encoded by Myl12a and Myl12b, but not Myl9, were constitutively expressed in Caco-2 cells. Stimulation with IFNγ or TL1A induced an increase in the transcript levels of MLCK-2, and no change in MLCK-1, Myl12a, or Myl12b. Moreover, TL1A-induced bacterial endocytosis was inhibited by siMLCK but not siMLCK1, suggesting the involvement of MLCK-2 in transcellular pathways. In contrast, siMLCK-1 inhibited TER decrease caused by high dose IFNγ (3000 IU/ml). Conclusion: Low dose IFNγ and TL1A induced MLCK-dependent bacterial endocytosis by intestinal epithelial cells, uncoupled with tight junctional damage or MLCK1 activation. Low dose IFNγ and TL1A upregulated the long MLCK2 transcripts, but had no effect on MLCK1 levels or MLC isoforms. High dose IFNγ caused tight junction disruption in a MLCK1-dependent fashion.