Department of Respiratory, The Second Hospital of Shanxi Medical University, Taiyuan 030001, PR China.
Mol Med Rep. 2013 Mar;7(3):921-6. doi: 10.3892/mmr.2013.1268. Epub 2013 Jan 10.
Triggering receptor expressed on myeloid cells-2 (TREM-2) has been shown to attenuate inflammatory responses in various cell lines including bone marrow-derived macrophages, hepatic macrophages, osteoclasts and dendritic cells. However, its effects on alveolar macrophages remain unknown. Lentivirus-mediated RNA interference (RNAi) is a post-transcriptional gene silencing method, which is capable of degrading target genes specifically and efficiently. In this study, we silenced TREM-2 in murine alveolar macrophages by using lentivirus-mediated short hairpin RNA (shRNA) and evaluated the effects of TREM-2 silencing on expression of toll-like receptor-4 (TLR-4), tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in response to lipopolysaccharide (LPS). Alveolar macrophages were transfected with shRNA targeting TREM-2 by use of lentivirus vector, non-sense shRNA as a negative control or empty lentivirus vector as a blank control. Silencing of TREM-2 was assessed by real‑time fluorescence quantitative PCR and flow cytometry. Following LPS stimulation, the levels of TLR-4, TNF-α and IL-10 expressed in alveolar macrophages were measured by real-time PCR, flow cytometry or ELISA. TREM-2 expression on alveolar macrophages was downregulated significantly by lentivirus-mediated shRNA treatment at the transcriptional and translational levels. However, alveolar macrophages that received non-sense shRNA or empty lentivirus vectors showed no effects on TREM-2 expression. Silencing of TREM-2 enhanced expression of TLR-4, as well as TNF-α and IL-10, by alveolar macrophages following LPS stimulation. These results indicate a significant effect of TREM-2 on attenuating the LPS-induced inflammatory response of murine alveolar macrophages, which may be dependent on TLR-4.
触发受体表达在髓样细胞-2（TREM-2）已被证明减弱在各种细胞系的炎症反应，包括骨髓来源的巨噬细胞，肝巨噬细胞，破骨细胞和树突状细胞。然而，其对肺泡巨噬细胞的影响尚不清楚。慢病毒介导的 RNA 干扰（RNAi）是一种转录后基因沉默的方法，其能够特异性和有效地降解靶基因。在本研究中，我们通过慢病毒介导的短发夹 RNA（shRNA）沉默了鼠肺泡巨噬细胞中的 TREM-2，并评估了 TREM-2 沉默对脂多糖（LPS）刺激后 TLR-4、肿瘤坏死因子-α（TNF-α）和白细胞介素-10（IL-10）表达的影响。肺泡巨噬细胞用靶向 TREM-2 的 shRNA 通过慢病毒载体转染，用无意义 shRNA 作为阴性对照或空慢病毒载体作为空白对照。通过实时荧光定量 PCR 和流式细胞术评估 TREM-2 的沉默。在 LPS 刺激后，通过实时 PCR、流式细胞术或 ELISA 测量肺泡巨噬细胞中 TLR-4、TNF-α和 IL-10 的表达。TREM-2 在转录和翻译水平上通过慢病毒介导的 shRNA 处理显著下调。然而，接受无意义 shRNA 或空慢病毒载体的肺泡巨噬细胞对 TREM-2 表达没有影响。TREM-2 沉默增强了 LPS 刺激后肺泡巨噬细胞中 TLR-4 以及 TNF-α和 IL-10 的表达。这些结果表明 TREM-2 对减轻 LPS 诱导的鼠肺泡巨噬细胞炎症反应有显著影响，这可能依赖于 TLR-4。
