Department of Infectious Diseases, Institute for Viral Hepatitis, Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Second Affiliated Hospital of Chongqing Medical University, Chongqing, China.
Department of Clinical Laboratory, The People's Hospital of Leshan, Chongqing, China.
Virol J. 2022 Feb 10;19(1):28. doi: 10.1186/s12985-022-01753-x.
Some cytokine signaling pathways can interact with interferon (IFN)-α pathway and thus regulate cell responses to IFN-α. Levels of the pro-inflammatory cytokine interleukin-17A (IL-17A) were found to be elevated in both the peripheral blood and liver in chronic hepatitis B (CHB) patients. However, how IL-17A affects the anti-HBV activity of IFN-α remains unclear.
The effects of IL-17A on anti-HBV activity of IFN-α were evaluated in HBV-expressing HepG2 cells (HepG2-HBV1.3) with IL-17A pretreatment and IFN-α stimulation. Culture supernatant levels of HBsAg, HBeAg, and HBV DNA, or intracellular expression of HBsAg and HBcAg were detected by ELISA, real-time quantitative PCR (RT-qPCR), or western blotting (WB). The expression of canonical IFN-α signaling pathway components, including the interferon-α/β receptor (IFNAR), Janus Kinase 1 (JAK1), Tyrosine Kinase 2 (TYK2), the Interferon Stimulated Gene Factor 3 complex (ISGF3) and IFN-stimulated genes (ISGs), was also examined by RT-qPCR, Immunofluorescence or WB. The effects of IL-17A were further investigated by the suppression of the IL-17A pathway with a TRAF6 inhibitor.
Compared to IFN-α stimulation alone, IL-17A pretreatment followed by IFN-α stimulation increased the levels of HBsAg, HBeAg, and HBV DNA, and decreased the levels of ISGF3 complex (phosphorylated (p)-signal transducer and activator of transcription (STAT1)/p-STAT2/IRF9) and antiviral-related ISGs (ISG15, ISG20 and Mx1). Interestingly, IL-17A pretreatment increased the expression of suppressor of cytokine signaling (SOCS) 1, SOCS3 and USP18, which were also the ISGs negatively regulating activity of ISGF3. Moreover, IFNAR1 protein expression declined more sharply in the group with IL-17A pretreatment than in the group with IFN-α stimulation alone. Blocking the IL-17A pathway reversed the effects of IL-17A on the IFN-α-induced activation of ISGF3 and anti-HBV efficacy.
Our results demonstrate that IL-17A pretreatment could attenuate IFN-α-induced anti-HBV activity by upregulating negative regulators of the critical transcriptional ISGF3 complex. Thus, this might be a potential target for improving response to IFN-α therapy.
某些细胞因子信号通路可以与干扰素 (IFN)-α 途径相互作用，从而调节细胞对 IFN-α 的反应。研究发现，慢性乙型肝炎 (CHB) 患者的外周血和肝脏中促炎细胞因子白细胞介素-17A (IL-17A) 的水平升高。然而，IL-17A 如何影响 IFN-α 的抗 HBV 活性尚不清楚。
通过 IL-17A 预处理和 IFN-α 刺激，评估 IL-17A 对表达 HBV 的 HepG2 细胞 (HepG2-HBV1.3) 中抗 HBV 活性的影响。通过 ELISA、实时定量 PCR (RT-qPCR) 或 Western blot (WB) 检测 HBsAg、HBeAg 和 HBV DNA 的培养上清水平，或通过 ELISA、HBsAg 和 HBcAg 的实时定量 PCR (RT-qPCR)、免疫荧光或 WB 检测细胞内表达。通过 RT-qPCR、免疫荧光或 WB 检测经典 IFN-α 信号通路成分，包括干扰素-α/β受体 (IFNAR)、Janus 激酶 1 (JAK1)、酪氨酸激酶 2 (TYK2)、干扰素刺激基因因子 3 复合物 (ISGF3) 和 IFN 刺激基因 (ISGs) 的表达。用 TRAF6 抑制剂抑制 IL-17A 通路进一步研究 IL-17A 的作用。
与单独 IFN-α 刺激相比，IL-17A 预处理后再用 IFN-α 刺激可增加 HBsAg、HBeAg 和 HBV DNA 的水平，并降低 ISGF3 复合物 (磷酸化 (p)-信号转导和转录激活因子 (STAT1)/p-STAT2/IRF9) 和抗病毒相关 ISGs (ISG15、ISG20 和 Mx1) 的水平。有趣的是，IL-17A 预处理可增加负调节 ISGF3 活性的细胞因子信号转导抑制剂 (SOCS) 1、SOCS3 和 USP18 的表达，这些也是 ISGs。此外，与单独 IFN-α 刺激相比，IL-17A 预处理组的 IFNAR1 蛋白表达下降更为明显。阻断 IL-17A 通路可逆转 IL-17A 对 IFN-α 诱导的 ISGF3 激活和抗 HBV 疗效的影响。
我们的结果表明，IL-17A 预处理可通过上调关键转录 ISGF3 复合物的负调节剂来减弱 IFN-α 诱导的抗 HBV 活性。因此，这可能是改善 IFN-α 治疗反应的潜在靶点。
