Department of Genetic Engineering, Beijing Institute of Biotechnology, Beijing, China.
Department of Medical Oncology, Beijing Tuberculosis and Thoracic Tumor Research Institute/Beijing Chest Hospital, Capital Medical University, Beijing, China.
Front Cell Infect Microbiol. 2023 Mar 2;13:1134511. doi: 10.3389/fcimb.2023.1134511. eCollection 2023.
Inflammation play important roles in the initiation and progression of acute lung injury (ALI), acute respiratory distress syndrome (ARDS), septic shock, clotting dysfunction, or even death associated with SARS-CoV-2 infection. However, the pathogenic mechanisms underlying SARS-CoV-2-induced hyperinflammation are still largely unknown.
The animal model of septic shock and ALI was established after LPS intraperitoneal injection or intratracheal instillation. Bone marrow-derived macrophages (BMDMs) from WT and BPOZ-2 KO mouse strains were harvested from the femurs and tibias of mice. Immunohistology staining, ELISA assay, coimmunoprecipitation, and immunoblot analysis were used to detect the histopathological changes of lung tissues and the expression of inflammatory factors and protein interaction.
We show a distinct mechanism by which the SARS-CoV-2 N (SARS-2-N) protein targets Bood POZ-containing gene type 2 (BPOZ-2), a scaffold protein for the E3 ubiquitin ligase Cullin 3 that we identified as a negative regulator of inflammatory responses, to promote NLRP3 inflammasome activation. We first demonstrated that BPOZ-2 knockout (BPOZ-2 KO) mice were more susceptible to lipopolysaccharide (LPS)-induced septic shock and ALI and showed increased serum IL-1β levels. In addition, BMDMs isolated from BPOZ-2 KO mice showed increased IL-1β production in response to NLRP3 stimuli. Mechanistically, BPOZ-2 interacted with NLRP3 and mediated its degradation by recruiting Cullin 3. In particular, the expression of was significantly reduced in lung tissues from mice infected with SARS-CoV-2 and in cells overexpressing SARS-2-N. Importantly, proinflammatory responses triggered by the SARS-2-N were significantly blocked by BPOZ-2 reintroduction. Thus, we concluded that BPOZ-2 is a negative regulator of the NLPR3 inflammasome that likely contributes to SARS-CoV-2-induced hyperinflammation.
炎症在急性肺损伤（ALI）、急性呼吸窘迫综合征（ARDS）、脓毒性休克、凝血功能障碍甚至与 SARS-CoV-2 感染相关的死亡的发生和进展中起着重要作用。然而，SARS-CoV-2 诱导的过度炎症的发病机制在很大程度上仍不清楚。
通过 LPS 腹腔注射或气管内滴注建立脓毒性休克和 ALI 动物模型。从 WT 和 BPOZ-2 KO 小鼠的股骨和胫骨中采集骨髓来源的巨噬细胞（BMDM）。免疫组织化学染色、ELISA 测定、共免疫沉淀和免疫印迹分析用于检测肺组织的组织病理学变化和炎症因子的表达及蛋白相互作用。
我们展示了一种独特的机制，即 SARS-CoV-2 N（SARS-2-N）蛋白靶向我们鉴定的 E3 泛素连接酶 Cullin 3 的支架蛋白 Bood POZ 包含基因类型 2（BPOZ-2），作为一种负调控炎症反应的负调节因子，促进 NLRP3 炎性体的激活。我们首次证明 BPOZ-2 敲除（BPOZ-2 KO）小鼠对脂多糖（LPS）诱导的脓毒性休克和 ALI 更敏感，并表现出血清 IL-1β 水平升高。此外，BPOZ-2 KO 小鼠分离的 BMDM 对 NLRP3 刺激表现出更高的 IL-1β 产生。在机制上，BPOZ-2 与 NLRP3 相互作用，并通过募集 Cullin 3 介导其降解。特别是，在感染 SARS-CoV-2 的小鼠的肺组织中和过表达 SARS-2-N 的细胞中，的表达明显降低。重要的是，BPOZ-2 的重新引入显著阻断了 SARS-2-N 触发的促炎反应。因此，我们得出结论，BPOZ-2 是 NLPR3 炎性体的负调节剂，可能有助于 SARS-CoV-2 诱导的过度炎症。
