Objective Achieving HBV cure will require novel combination therapies of direct-acting antivirals and immunomodulatory agents. In this context, the toll-like receptor 8 (TLR8) agonist selgantolimod (SLGN) has been investigated in preclinical models and clinical trials for chronic hepatitis B (CHB). However, little is known regarding its action on immune effectors within the liver. Our aim was to characterise the transcriptomic changes and intercellular communication events induced by SLGN in the hepatic microenvironment. Design We identified TLR8 -expressing cell types in the human liver using publicly available single-cell RNA-seq data and established a method to isolate Kupffer cells (KCs). We characterised transcriptomic and cytokine KC profiles in response to SLGN. SLGN’s indirect effect was evaluated by RNA-seq in hepatocytes treated with SLGN-conditioned media (CM) and quantification of HBV parameters following infection. Pathways mediating SLGN’s effect were validated using transcriptomic data from HBV-infected patients. Results Hepatic TLR8 expression takes place in the myeloid compartment. SLGN treatment of KCs upregulated monocyte markers (eg, S100A12 ) and downregulated genes associated with the KC identity (eg, SPIC ). Treatment of hepatocytes with SLGN-CM downregulated NTCP and impaired HBV entry. Cotreatment with an interleukin 6-neutralising antibody reverted the HBV entry inhibition. Conclusion Our transcriptomic characterisation of SLGN sheds light into the programmes regulating KC activation. Furthermore, in addition to its previously described effect on established HBV infection and adaptive immunity, we show that SLGN impairs HBV entry. Altogether, SLGN may contribute through KCs to remodelling the intrahepatic immune microenvironment and may thus represent an important component of future combinations to cure HBV infection. Data are available in a public, open access repository. RNA-seq data from KCs treated with SLGN or DMSO (n=3), PHH treated with SLGN-CM or Mock-CM (n=3) and cynomolgus macaques treated with TLR8 agonist (n=6 per group) can be found in the GEO accessions GSE240054, GSE240529 and GSE261125, respectively.

中文翻译：

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TLR8 激动剂 Selgantolimod 通过 IL-6 依赖性机制调节 Kupffer 细胞分化状态并损害 HBV 进入肝细胞

目的 实现乙型肝炎治愈需要直接作用抗病毒药物和免疫调节剂的新型联合疗法。在此背景下，Toll 样受体 8 (TLR8) 激动剂 Selgantolimod (SLGN) 已在慢性乙型肝炎 (CHB) 的临床前模型和临床试验中进行了研究。然而，人们对其对肝脏内免疫效应器的作用知之甚少。我们的目的是表征 SLGN 在肝脏微环境中诱导的转录组变化和细胞间通讯事件。设计 我们使用公开的单细胞 RNA-seq 数据鉴定了人肝脏中表达 TLR8 的细胞类型，并建立了一种分离 Kupffer 细胞 (KC) 的方法。我们对 SLGN 反应的转录组和细胞因子 KC 谱进行了表征。通过在用 SLGN 条件培养基 (CM) 处理的肝细胞中进行 RNA-seq 评估 SLGN 的间接作用，并对感染后的 HBV 参数进行定量。使用 HBV 感染患者的转录组数据验证了介导 SLGN 效应的途径。结果 肝脏 TLR8 表达发生在髓样区室中。 KC 的 SLGN 处理上调单核细胞标记（例如 S100A12 ）并下调与 KC 身份相关的基因（例如 SPIC ）。用 SLGN-CM 治疗肝细胞可下调 NTCP 并削弱 HBV 进入。与白细胞介素 6 中和抗体共同治疗可恢复 HBV 进入抑制。结论 我们对 SLGN 的转录组特征揭示了调节 KC 激活的程序。此外，除了之前描述的对已建立的 HBV 感染和适应性免疫的影响外，我们还发现 SLGN 还会损害 HBV 进入。总而言之，SLGN 可能通过 KC 重塑肝内免疫微环境，因此可能代表未来治疗 HBV 感染的组合的重要组成部分。数据可在公共、开放访问存储库中获取。来自用 SLGN 或 DMSO 处理的 KC（n=3）、用 SLGN-CM 或 Mock-CM 处理的 PHH（n=3）以及用 TLR8 激动剂处理的食蟹猴（每组 n=6）的 RNA-seq 数据可以在GEO 登记号分别为 GSE240054、GSE240529 和 GSE261125。