Table_1_Agonistic Activation of Cytosolic DNA Sensing Receptors in Woodchuck Hepatocyte Cultures and Liver for Inducing Antiviral Effects.docx

Immune modulation for the treatment of chronic hepatitis B (CHB) has gained more traction in recent years, with an increasing number of compounds designed for targeting different host pattern recognition receptors (PRRs). These agonistic molecules activate the receptor signaling pathway and trigger an innate immune response that will eventually shape the adaptive immunity for control of chronic infection with hepatitis B virus (HBV). While definitive recognition of HBV nucleic acids by PRRs during viral infection still needs to be elucidated, several viral RNA sensing receptors, including toll-like receptors 7/8/9 and retinoic acid inducible gene-I-like receptors, are explored preclinically and clinically as possible anti-HBV targets. The antiviral potential of viral DNA sensing receptors is less investigated. In the present study, treatment of primary woodchuck hepatocytes generated from animals with CHB with HSV-60 or poly(dA:dT) agonists resulted in increased expression of interferon-gamma inducible protein 16 (IFI16) or Z-DNA-binding protein 1 (ZBP1/DAI) and absent in melanoma 2 (AIM2) receptors and their respective adaptor molecules and effector cytokines. Cytosolic DNA sensing receptor pathway activation correlated with a decline in woodchuck hepatitis virus (WHV) replication and secretion in these cells. Combination treatment with HSV-60 and poly(dA:dT) achieved a superior antiviral effect over monotreatment with either agonist that was associated with an increased expression of effector cytokines. The antiviral effect, however, could not be enhanced further by providing additional type-I interferons (IFNs) exogenously, indicating a saturated level of effector cytokines produced by these receptors following agonism. In WHV-uninfected woodchucks, a single poly(dA:dT) dose administered via liver-targeted delivery was well-tolerated and induced the intrahepatic expression of ZBP1/DAI and AIM2 receptors and their effector cytokines, IFN-β and interleukins 1β and 18. Receptor agonism also resulted in increased IFN-γ secretion of peripheral blood cells. Altogether, the effect on WHV replication and secretion following in vitro activation of IFI16, ZBP1/DAI, and AIM2 receptor pathways suggested an antiviral benefit of targeting more than one cytosolic DNA receptor. In addition, the in vivo activation of ZBP1/DAI and AIM2 receptor pathways in liver indicated the feasibility of the agonist delivery approach for future evaluation of therapeutic efficacy against HBV in woodchucks with CHB.

近年来，针对慢性乙型肝炎（CHB）的免疫调节治疗策略逐渐受到关注，越来越多的化合物被设计用以靶向不同的宿主模式识别受体（PRRs）。这些激动剂分子激活受体信号通路，引发先天免疫反应，最终塑造适应性免疫以控制乙型肝炎病毒（HBV）的慢性感染。尽管在病毒感染期间PRRs对HBV核酸的确定性识别尚需阐明，但包括7/8/9型Toll样受体和视黄酸诱导的基因-I样受体在内的几种病毒RNA传感受体已在临床前和临床研究中被探索作为可能的抗HBV靶点。病毒DNA传感受体的抗病毒潜力研究较少。在本研究中，使用HSV-60或聚（dA:dT）激动剂处理由慢性乙型肝炎动物产生的原代木地鼠肝细胞，导致干扰素-γ诱导蛋白16（IFI16）或Z-DNA结合蛋白1（ZBP1/DAI）及黑色素瘤2（AIM2）受体及其相应的适配分子和效应细胞因子的表达增加，而在这些细胞中这些受体表达缺失。细胞内DNA传感受体途径的激活与木地鼠肝炎病毒（WHV）在这些细胞中的复制和分泌减少相关。HSV-60与聚（dA:dT）的联合治疗相较于单一激动剂治疗表现出更优的抗病毒效果，这与效应细胞因子的表达增加有关。然而，通过提供额外的I型干扰素（IFNs）并不能进一步增强抗病毒效果，这表明这些受体在激动剂作用后效应细胞因子的产生已达到饱和水平。在未感染WHV的木地鼠中，通过肝脏靶向递送的单剂聚（dA:dT）被良好耐受，并诱导了ZBP1/DAI和AIM2受体及其效应细胞因子IFN-β和白细胞介素1β及18在肝脏内的表达。受体激动剂还导致外周血细胞的IFN-γ分泌增加。总体而言，在体外激活IFI16、ZBP1/DAI和AIM2受体途径对WHV复制和分泌的影响表明，靶向多个细胞内DNA受体具有抗病毒益处。此外，在肝脏中ZBP1/DAI和AIM2受体途径的体内激活表明，激动剂递送方法在未来评估CHB木地鼠对HBV的疗效方面具有可行性。