Immunology & Innovative Cell therapy Unit, Dasman Diabetes Institute, Al-Soor Street, P.O. Box 1180, Dasman, 15462 Kuwait.
J Inflamm (Lond). 2015 Apr 18;12:32. doi: 10.1186/s12950-015-0077-0. eCollection 2015.
MMP-9 is crucial for a normal immune response, but excessive release of this enzyme leads to severe tissue damage. Listeria monocytogenes (LM) is an opportunistic food-borne pathogen causing listerosis, meningitis and sepsis. Heat killed Listeria monocytogenes (HKLM) activates immune system and leads production of cytokines and chemokines. However, nothing is known about the involvement of HKLM in MMP-9 regulation. Therefore we investigated the role of HKLM in the regulation of MMP-9 gene expression in THP-1 cells.
Commercially available heat killed Listeria monocytogenes was used in this study. HKLM-induced MMP-9 expression was assessed with quantitative real-time qPCR and ELISA. Action of HKLM in different signaling pathways were studied by using THP-1-XBlue™ cells (THP-1-cells with NF-κB/AP-1 reporter construct), THP-1-XBlue™-defMyD cells (MyD88(-/-) THP-1 cells), anti-TLR2 mAb and pharmacological inhibitors. Phospho and total proteins were determined by Western blotting.
Increased MMP-9 production (mRNA: 395-Fold; Protein: 8141 pg/ml; P < 0.05) was observed in HKLM stimulated THP-1 cells as compared to the un-stimulated THP-1 cells. This production of MMP-9 was completely abrogated by anti-TLR2 blocking mAb (P = 0.0024). Furthermore, THP-1-XBlue™-defMyD cells were unable to produce MMP-9 in response to HKLM. HKLM- induced activation of NF-kappaB/AP-1 was also observed in THP-1-XBlue™ Cells. In addition, inhibitors of JNK (SP600125), MEK/ERK (U0126; PD98056), p38 MAPK (SB203580) and NF-kappaB (BAY 11-7085, Triptolide and Resveratrol) significantly suppressed (P < 0.05) HKLM-stimulated MMP-9 expression.
Our results indicate that HKLM activates TLR2 and NF-κB/AP-1 signaling pathways, leading to up-regulation of MMP-9 production in THP-1 cells. Thus, MMP-9 could be an appropriate therapeutic target to stop severe tissue damage caused by infection or chronic inflammation.
MMP-9 对于正常的免疫反应至关重要，但这种酶的过度释放会导致严重的组织损伤。李斯特菌（Listeria monocytogenes）是一种机会性食源性病原体，可引起李斯特菌病、脑膜炎和败血症。热灭活李斯特菌（Heat killed Listeria monocytogenes，HKLM）可激活免疫系统并导致细胞因子和趋化因子的产生。然而，目前尚不清楚 HKLM 是否参与 MMP-9 的调节。因此，我们研究了 HKLM 在 THP-1 细胞中 MMP-9 基因表达调节中的作用。
本研究使用市售的热灭活李斯特菌。通过实时 qPCR 和 ELISA 评估 HKLM 诱导的 MMP-9 表达。通过 THP-1-XBlue™ 细胞（带有 NF-κB/AP-1 报告构建体的 THP-1 细胞）、THP-1-XBlue™-defMyD 细胞（MyD88(-/-) THP-1 细胞）、抗 TLR2 mAb 和药理学抑制剂研究 HKLM 在不同信号通路中的作用。通过 Western 印迹测定磷酸化和总蛋白。
与未刺激的 THP-1 细胞相比，HKLM 刺激的 THP-1 细胞中 MMP-9 的产生（mRNA：395 倍；蛋白：8141 pg/ml；P <0.05）增加。抗 TLR2 阻断 mAb 完全阻断了 MMP-9 的产生（P = 0.0024）。此外，THP-1-XBlue™-defMyD 细胞无法对 HKLM 产生反应而产生 MMP-9。还观察到 HKLM 诱导的 NF-kappaB/AP-1 激活。此外，JNK（SP600125）、MEK/ERK（U0126；PD98056）、p38 MAPK（SB203580）和 NF-kappaB（BAY 11-7085、Triptolide 和 Resveratrol）抑制剂显著抑制（P <0.05）HKLM 刺激的 MMP-9 表达。
我们的结果表明，HKLM 激活 TLR2 和 NF-κB/AP-1 信号通路，导致 THP-1 细胞中 MMP-9 产生的上调。因此，MMP-9 可能是阻止感染或慢性炎症引起的严重组织损伤的合适治疗靶点。
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