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 调节 Kupffer 细胞分化状态，并通过 IL-6 依赖性机制损害 HBV 进入肝细胞


目的 实现 HBV 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，关于它对肝脏内免疫效应物的作用知之甚少。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 我们使用公开可用的单细胞 RNA-seq 数据在人肝脏中鉴定了表达 TLR8 的细胞类型，并建立了一种分离 Kupffer 细胞 （KCs） 的方法。我们表征了响应 SLGN 的转录组和细胞因子 KC 谱。通过 RNA-seq 在用 SLGN 条件培养基 （CM） 处理的肝细胞中评估 SLGN 的间接影响，并在感染后量化 HBV 参数。使用 HBV 感染患者的转录组数据验证介导 SLGN 效应的通路。结果 肝脏 TLR8 表达发生在骨髓区室。SLGN 治疗 KCs 上调单核细胞标志物 （如S100A12 ） 并下调与 KC 身份相关的基因 （如，SPIC）。用 SLGN-CM 治疗肝细胞下调 NTCP 并损害 HBV 进入。与白细胞介素 6 中和抗体的联合治疗逆转了 HBV 进入抑制。结论我们对 SLGN 的转录组学特征揭示了调节 KC 激活的程序。此外，除了其先前描述的对已建立的 HBV 感染和适应性免疫的影响外，我们还表明 SLGN 会损害 HBV 进入。 总而言之，SLGN 可能通过 KC 有助于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。数据在公共、开放访问存储库中可用。来自 SLGN 或 DMSO 处理的 KCs （n=3）、用 SLGN-CM 或 Mock-CM 处理的 PHH （n=3） 和用 TLR8 激动剂处理的食蟹猴 （每组 n=6） 的 RNA-seq 数据分别在 GEO 种质 GSE240054、GSE240529 和 GSE261125 中找到。