Emergency Department, Minhang Hospital, Fudan University, Shanghai, China.
Cell Commun Signal. 2024 Aug 14;22(1):399. doi: 10.1186/s12964-024-01768-7.
Lipopolysaccharide (LPS)-activated pro-inflammatory responses play a critical role in sepsis, a life-threatening condition. This study investigates the role of origin recognition complex subunit 6 (ORC6) in LPS responses in macrophages and monocytes. Silencing ORC6 using targeted shRNA significantly reduced LPS-induced expression and production of IL-1β (interleukin-1 beta), TNF-α (tumor necrosis factor alpha), and IL-6 (interleukin-6) in THP-1 human macrophages, peripheral blood mononuclear cells (PBMCs), and bone marrow-derived macrophages (BMDMs). Additionally, ORC6 knockout (KO) via the CRISPR/Cas9 method in THP-1 macrophages inhibited LPS-induced pro-inflammatory responses, while ectopic overexpression of ORC6 enhanced LPS-induced expression and production of pro-inflammatory cytokines. ORC6 is crucial for the activation of the nuclear factor kappa B (NFκB) signaling cascade in macrophages and monocytes. LPS-induced NFκB activation was largely inhibited by ORC6 silencing or KO, but potentiated following ORC6 overexpression. Mechanistically, ORC6 associated with nuclear p65 after LPS stimulation, an interaction necessary for NFκB activation. Overexpression of ORC6 did not recover the reduced pro-inflammatory response to LPS in THP-1 macrophages with silenced p65. Furthermore, the NFκB inhibitor BMS-345,541 nearly eliminated the pro-inflammatory response enhanced by ORC6 overexpression in response to LPS. Further studies revealed that ORC6 depletion inhibited NFκB activation induced by double-stranded RNA (dsRNA) and high mobility group box 1 (HMGB1) in THP-1 macrophages. In vivo experiments demonstrated that macrophage-specific knockdown of ORC6 protected mice from LPS-induced septic shock and inhibited LPS-stimulated production of IL-1β, TNF-α, and IL-6 in mouse serum. ORC6 silencing also inhibited LPS-induced NFκB activation in ex vivo cultured PBMCs following macrophage-specific knockdown of ORC6. These findings highlight ORC6 as a pivotal mediator in LPS-induced NFκB activation and the pro-inflammatory response in sepsis, suggesting that targeting ORC6 could be a novel therapeutic strategy for managing sepsis and related inflammatory conditions.
脂多糖 (LPS) 激活的促炎反应在败血症（一种危及生命的病症）中起着关键作用。本研究探讨了起始识别复合物亚基 6 (ORC6) 在巨噬细胞和单核细胞中 LPS 反应中的作用。使用靶向 shRNA 沉默 ORC6 可显著降低 THP-1 人巨噬细胞、外周血单核细胞 (PBMC) 和骨髓来源巨噬细胞 (BMDM) 中 LPS 诱导的 IL-1β（白细胞介素-1β）、TNF-α（肿瘤坏死因子-α）和 IL-6（白细胞介素-6）的表达和产生。此外，通过 CRISPR/Cas9 方法在 THP-1 巨噬细胞中敲除 ORC6 抑制 LPS 诱导的促炎反应，而 ORC6 的异位过表达增强 LPS 诱导的促炎细胞因子的表达和产生。ORC6 对于巨噬细胞和单核细胞中核因子 kappa B (NFκB) 信号级联的激活至关重要。LPS 诱导的 NFκB 激活被 ORC6 沉默或敲除大大抑制，但在 ORC6 过表达后增强。在机制上，ORC6 在 LPS 刺激后与核 p65 相关，这种相互作用对于 NFκB 激活是必需的。ORC6 的过表达不能恢复沉默 p65 的 THP-1 巨噬细胞对 LPS 的促炎反应降低。此外，NFκB 抑制剂 BMS-345,541 几乎消除了 ORC6 过表达对 LPS 反应增强的促炎反应。进一步的研究表明，ORC6 耗竭抑制了 THP-1 巨噬细胞中双链 RNA (dsRNA) 和高迁移率族蛋白 1 (HMGB1) 诱导的 NFκB 激活。体内实验表明，巨噬细胞特异性敲低 ORC6 可保护小鼠免受 LPS 诱导的败血症休克，并抑制 LPS 刺激的小鼠血清中 IL-1β、TNF-α 和 IL-6 的产生。ORC6 沉默还抑制了巨噬细胞特异性敲低 ORC6 后 ex vivo 培养的 PBMC 中 LPS 诱导的 NFκB 激活。这些发现强调了 ORC6 作为 LPS 诱导的 NFκB 激活和败血症中促炎反应的关键介质，表明靶向 ORC6 可能是管理败血症和相关炎症疾病的一种新的治疗策略。
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