School of Biological Sciences, National Institute of Science Education and Research Bhubaneswar, Jatni, Odisha, India.
Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai, Maharashtra, India.
Front Immunol. 2023 Apr 20;14:1139808. doi: 10.3389/fimmu.2023.1139808. eCollection 2023.
Toll like receptor 4 (TLR4), a pathogen-associated molecular pattern (PAMP) receptor, is known to exert inflammation in various cases of microbial infection, cancer and autoimmune disorders. However, any such involvement of TLR4 in Chikungunya virus (CHIKV) infection is yet to be explored. Accordingly, the role of TLR4 was investigated towards CHIKV infection and modulation of host immune responses in the current study using mice macrophage cell line RAW264.7, primary macrophage cells of different origins and mice model. The findings suggest that TLR4 inhibition using TAK-242 (a specific pharmacological inhibitor) reduces viral copy number as well as reduces the CHIKV-E2 protein level significantly using p38 and JNK-MAPK pathways. Moreover, this led to reduced expression of macrophage activation markers like CD14, CD86, MHC-II and pro-inflammatory cytokines (TNF, IL-6, MCP-1) significantly in both the mouse primary macrophages and RAW264.7 cell line, . Additionally, TAK-242-directed TLR4 inhibition demonstrated a significant reduction of percent E2-positive cells, viral titre and TNF expression in hPBMC-derived macrophages, . These observations were further validated in TLR4-knockout (KO) RAW cells. Furthermore, the interaction between CHIKV-E2 and TLR4 was demonstrated by immuno-precipitation studies, and supported by molecular docking analysis, TLR4-dependent viral entry was further validated by an anti-TLR4 antibody-mediated blocking experiment. It was noticed that TLR4 is necessary for the early events of viral infection, especially during the attachment and entry stages. Interestingly, it was also observed that TLR4 is not involved in the post-entry stages of CHIKV infection in host macrophages. The administration of TAK-242 decreased CHIKV infection significantly by reducing disease manifestations, improving survivability (around 75%) and reducing inflammation in mice model. Collectively, for the first time, this study reports TLR4 as one of the novel receptors to facilitate the attachment and entry of CHIKV in host macrophages, the TLR4-CHIKV-E2 interactions are essential for efficient viral entry and modulation of infection-induced pro-inflammatory responses in host macrophages, which might have translational implication for designing future therapeutics to regulate the CHIKV infection.
Toll 样受体 4（TLR4）是一种病原体相关分子模式（PAMP）受体，已知在各种微生物感染、癌症和自身免疫性疾病中发挥炎症作用。然而，TLR4 是否参与基孔肯雅病毒（CHIKV）感染尚未得到探索。因此，本研究使用小鼠巨噬细胞系 RAW264.7、不同来源的原代巨噬细胞和小鼠模型，研究了 TLR4 在 CHIKV 感染和宿主免疫反应调节中的作用。研究结果表明，使用 TAK-242（一种特异性药理学抑制剂）抑制 TLR4 可减少病毒拷贝数，并通过 p38 和 JNK-MAPK 途径显著降低 CHIKV-E2 蛋白水平。此外，这导致在小鼠原代巨噬细胞和 RAW264.7 细胞系中，巨噬细胞活化标志物如 CD14、CD86、MHC-II 和促炎细胞因子（TNF、IL-6、MCP-1）的表达显著降低。此外，TAK-242 靶向 TLR4 抑制在人 PBMC 衍生的巨噬细胞中显著降低 E2 阳性细胞、病毒滴度和 TNF 表达。这些观察结果在 TLR4 敲除（KO）RAW 细胞中得到进一步验证。此外，通过免疫沉淀研究证实了 CHIKV-E2 与 TLR4 之间的相互作用，并通过分子对接分析得到支持。TLR4 依赖性病毒进入通过抗 TLR4 抗体介导的阻断实验进一步得到验证。研究结果表明，TLR4 是病毒感染早期事件所必需的，特别是在附着和进入阶段。有趣的是，研究结果还表明，TLR4 不参与宿主巨噬细胞中 CHIKV 感染的后期阶段。TAK-242 的给药通过减少疾病表现、提高存活率（约 75%）和减少小鼠模型中的炎症来显著降低 CHIKV 感染。总的来说，这项研究首次报道 TLR4 是促进 CHIKV 在宿主巨噬细胞中附着和进入的新型受体之一，TLR4-CHIKV-E2 相互作用对于有效的病毒进入和调节感染诱导的宿主巨噬细胞中的促炎反应至关重要，这可能对设计未来的治疗方法来调节 CHIKV 感染具有转化意义。
