Early Activation of MAP Kinases by Influenza A Virus X-31 in Murine Macrophage Cell Lines

Early molecular responses to Influenza A (FLUA) virus strain A/X-31 H3N2 in macrophages were explored using J774.A1 and RAW 264.7 murine cell lines. NF-kappa B (NFκB) was reported to be central to FLUA host-response in other cell types. Our data showed that FLUA activation of the classical NFκB dependent pathway in these macrophages was minimal. Regulator proteins, IkappaB-alpha and –beta (IκBα, IκBβ), showed limited degradation peaking at 2 h post FLUA exposure and p65 was not observed to translocate from the cytoplasm to the nucleus. Additionally, the non-canonical NFκB pathway was not activated in response to FLUA. The cells did display early increases in TNFα and other inflammatory cytokine and chemokine production. Mitogen activated phosphokinase (MAPK) signaling pathways are also reported to control production of inflammatory cytokines in response to FLUA. The activation of the MAPKs, cJun kinases 1 and 2 (JNK 1/2), extracellular regulated kinases 1 and 2 (ERK 1/2), and p38 were investigated in both cell lines between 0.25 and 3 h post-infection. Each of these kinases showed increased phosphorylation post FLUA exposure. JNK phosphorylation occurred early while p38 phosphorylation appeared later. Phosphorylation of ERK 1/2 occurred earlier in J774.A1 cells compared to RAW 264.7 cells. Inhibition of MAPK activation resulted in decreased production of most FLUA responsive cytokines and chemokines in these cells. The results suggest that in these monocytic cells the MAPK pathways are important in the early response to FLUA.

本研究以J774.A1与RAW 264.7两种鼠源细胞系为模型，探究了甲型流感病毒（Influenza A, FLUA）A/X-31 H3N2毒株感染巨噬细胞后的早期分子应答反应。既往研究表明，核因子κB（NFκB）在其他细胞类型的甲型流感病毒宿主应答过程中扮演核心调控角色。本研究数据显示，上述巨噬细胞中，甲型流感病毒对经典NFκB依赖通路的激活程度极低：调节蛋白IκBα与IκBβ的降解幅度有限，且仅在病毒暴露后2小时达到峰值；同时未观察到p65蛋白从细胞质向细胞核发生转位。此外，非经典NFκB通路也未被甲型流感病毒激活。但实验细胞确实可检测到肿瘤坏死因子α（TNFα）及其他炎性细胞因子、趋化因子的早期表达上调。另有研究证实，丝裂原活化蛋白激酶（MAPK）信号通路可调控甲型流感病毒感染诱导的炎性细胞因子产生。本研究在感染后0.25至3小时的时间范围内，对两种细胞系中的MAPK家族激酶——c-Jun氨基末端激酶1/2（JNK1/2）、细胞外调节蛋白激酶1/2（ERK1/2）及p38的活化状态进行了检测。结果显示，上述所有激酶在病毒暴露后均出现磷酸化水平升高：其中JNK的磷酸化发生较早，而p38的磷酸化则出现较晚；与RAW 264.7细胞相比，J774.A1细胞中ERK1/2的磷酸化启动更早。抑制MAPK通路的激活，可显著降低该类细胞中多数甲型流感病毒应答性细胞因子与趋化因子的产生量。上述结果表明，在这类单核细胞系中，MAPK信号通路在甲型流感病毒感染的早期应答过程中发挥关键调控作用。