The TLR8 agonist Selgantolimod regulates Kupffer cell differentiation status and indirectly impairs HBV entry into hepatocytes via an IL-6-dependent mechanism (PHH) [Kupffer cells]

Background & Aims: In spite of universal vaccination programs, HBV remains a global burden due to the limited therapeutic options available. Therefore, achieving the goal of HBV cure will require a continuous effort to develop novel molecules and combination therapies. In this context, the Toll-like receptor 8 (TLR8) agonist selgantolimod (SLGN) has shown promising results during its clinical evaluation. Although the effect of SLGN has been explored in the peripheral immune compartment, little is known regarding its intrahepatic response. Therefore, we characterized the transcriptomic changes and intercellular communication events associated with SLGN in the liver microenvironment. Approach & Results: The analysis of single-cell RNA sequencing (scRNA-seq) data allowed us to identify that hepatic TLR8 expression takes place in the myeloid compartment. Therefore, we stablished a method for the isolation of human Kupffer cells (KCs). We found that in vitro treatment of KCs with SLGN induces the upregulation of monocyte markers (e.g., EREG, S100A12) and the downregulation of genes associated with the KC identity (e.g., SPIC, FOLR2). Moreover, treatment of hepatocytes with SLGN-conditioned media (CM) led to the downregulation of NTCP and impaired HBV entry. Finally, co-treatment with SLGN-CM and an IL-6-inhibitory antibody identified this cytokine as mediating the reduced HBV entry. Conclusions: Our results suggest that in addition to its previously described therapeutic activity against HBV, SLGN also presents a prophylactic effect. Furthermore, our characterization of SLGN sheds light into the transcriptional programs regulating KC activation and underscores the importance of considering cell states when annotating cell populations based on scRNA-seq data. RNA-seq profiles from human Kupffer cells (KCs) exposed (24 h) to selgantolimod (SLGN, 150 nM, n=3) or DMSO (n=3).

研究背景与目的：尽管已实施全民疫苗接种计划，但乙型肝炎病毒（hepatitis B virus, HBV）仍因可用治疗手段有限而成为全球公共卫生负担。因此，实现HBV治愈目标，需要持续投入研发新型分子药物与联合治疗方案。在此背景下，Toll样受体8（Toll-like receptor 8, TLR8）激动剂司格妥莫德（selgantolimod, SLGN）在临床评估中展现出良好应用前景。尽管已有研究探索了SLGN在外周免疫区室中的作用，但人们对其肝内应答机制仍知之甚少。为此，本研究对肝脏微环境中与SLGN相关的转录组变化及细胞间通讯事件进行了表征分析。
研究方法与结果：通过对单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）数据的分析，我们发现肝脏中的TLR8表达主要定位于髓系区室。为此我们建立了人类库普弗细胞（Kupffer cells, KCs）的分离方法。实验结果显示，用SLGN体外处理库普弗细胞，可诱导单核细胞标志物（如EREG、S100A12）的上调，并下调与库普弗细胞特征相关的基因（如SPIC、FOLR2）。此外，用SLGN处理后的条件培养基（conditioned media, CM）培养肝细胞，可导致钠离子-牛磺胆酸共转运多肽（sodium taurocholate cotransporting polypeptide, NTCP）的下调并损害HBV的入侵过程。最后，通过联合使用SLGN-CM与白细胞介素6（interleukin-6, IL-6）抑制性抗体，我们证实该细胞因子介导了HBV入侵能力的降低。
研究结论：我们的研究结果表明，除此前已报道的抗HBV治疗活性外，SLGN还具备预防作用。此外，我们对SLGN的表征分析阐明了调控库普弗细胞活化的转录程序，并强调了在基于scRNA-seq数据注释细胞群时考虑细胞状态的重要性。本数据集包含经SLGN（150 nM，n=3）或二甲基亚砜（dimethyl sulfoxide, DMSO，n=3）处理24小时的人类库普弗细胞的RNA测序谱数据。