TLR8 agonist selgantolimod regulates Kupffer cell differentiation status and impairs HBV entry into hepatocytes via an IL-6-dependent mechanism

Abstract

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.

Keywords: HEPATITIS B; IMMUNOTHERAPY; KUPFFER CELL.

Figure 1. Hepatic TLR8 expression takes place primarily in the myeloid compartment. (A) Expression of TLR8 mRNA in each cell population of the human liver microenvironment (GSE192742). (B) Expression of CD163 mRNA in the myeloid compartment. Violin plots represent mean expression values. (C) Expression of CD163 protein in each cell population of the human liver microenvironment. (D) KC isolation method based on a two-phase iodixanol gradient and CD163-positive selection. (E) Immunofluorescence image of KCs isolated with the protocol described in (D), showing positive staining for nuclear DNA (DAPI, blue), CD68 (red) and CD163 (green). (F) Western blot showing expression of TLR8 protein in human KCs as compared with PHH from the same donor. cDCs, classical dendritic cells; HSCs, hepatic stellate cells; KCs, Kupffer cells; Mig.cDCs, migratory cDCs; moKCs, monocyte-derived KCs; NK cells, natural killer cells; LAMs, lipid-associated macrophages; LSECs, liver sinusoidal endothelial cells; Pat.Mono, patrolling monocytes; pDCs, plasmacytoid dendritic cells; PHH, primary human hepatocytes; TLR8, toll-like receptor 8.

Figure 2. SLGN treatment leads to morphological and transcriptomic changes associated with KC differentiation status. (A) Microscopic image of human KCs treated with SLGN (150 nM) or DMSO for 24 hours. (B) Volcano plot depicting significantly downregulated (SLGN-DOWN, n=869) and upregulated (SLGN-UP, n=966) KC genes in response to SLGN (150 nM) for 24 hours (n=3, FDR<0.05, log₂FC>1). (C) SLGN treatment of KCs (B) induces the upregulation of monocyte markers and the downregulation of KC genes and TFs. (D) Expression of the SLGN-DOWN KC signature in each population of the hepatic myeloid compartment (GSE192742). Violin plots represent mean expression values. (E) GSEA showing significantly upregulated pathways in KCs treated with SLGN (n=3, FDR<0.05). (F) Concentration of cytokines from culture supernatants of KCs stimulated with SLGN (150 nM) or DMSO for 24 hours (n=4, Mann-Whitney test). Bars represent mean±SEM. (G) Mean expression levels of the SLGN-UP signature in liver transcriptomic data from HBV-infected patients at different disease stages (Kruskal-Wallis test, GSE230397). AQP9, aquaporin 9; CCL, C-C motif chemokine ligand; CD5L, CD5 molecule like; DNMT3A, DNA methyltransferase 3 alpha; ENCHB, HBeAg-positive chronic hepatitis B; ENCI, HBeAg-negative chronic infection; EPCHB, HBeAg-positive chronic hepatitis B; EPCI, HBeAg-positive chronic infection; EREG, epiregulin; FOLR2, folate receptor beta; GSEA, gene set enrichment analysis; HC, healthy control; ID3, inhibitor of DNA binding 3; IFN, interferon; IL, interleukin; KCs, Kupffer cells; moKCs, monocyte-derived KCs; NES, normalised enrichment score; LAMs, lipid-associated macrophages; S100A, S100 calcium binding protein A; SLC, solute carrier family; SLGN, selgantolimod; SPIC, Spi-C transcription factor; TFs, transcription factors; TFEC, transcription factor EC; TIMD4, T cell immunoglobulin and mucin domain containing 4; TNF-α, tumour necrosis factor alpha; TLR, toll-like receptor; VSIG4, V-set and immunoglobulin domain containing 4.

Figure 3. In vivo activation of TLR8 signalling modulates the expression of liver transcriptomic programmes associated with KC differentiation status. (A) Cynomolgus macaques (n=6 per group) received weekly doses of TLR8 agonist (0.1, 0.5 and 2.5 mg/kg) during a 4-week period. Liver samples were obtained 4 hours after the last dose in order to perform transcriptomic analyses. (B) TLR8 agonist treatment induces the upregulation of monocyte markers and the downregulation of KC genes and TFs in liver tissues from cynomolgus macaques. (C–D) Mean expression levels of the (C) SLGN-DOWN and (D) SLGN-UP KC signatures in liver tissues of macaques treated with TLR8 agonist (Kruskal-Wallis test). (E) GSEA showing significantly upregulated pathways in liver tissues of cynomolgus macaques treated with TLR8 agonist (2.5 mg/kg vs vehicle control, FDR<0.05). AQP9, aquaporin 9; CD5L, CD5 molecule like; DNMT3A, DNA methyltransferase 3 alpha; EREG, epiregulin; FOLR2, folate receptor beta; GSEA, gene set enrichment analysis; ID3, inhibitor of DNA binding 3; KCs, Kupffer cells; NES, normalised enrichment score; S100A, S100 calcium binding protein A; SLC, solute carrier family; SLGN, selgantolimod; SPIC, Spi-C transcription factor; TFs, transcription factors; TFEC, transcription factor EC; TIMD4, T cell immunoglobulin and mucin domain containing 4; TLR8, toll-like receptor 8; VSIG4, V-set and immunoglobulin domain containing 4.

Figure 4. SLGN indirectly downregulates NTCP expression and impairs HBV entry into hepatocytes. (A) Liver transcriptomic data from HBV-infected patients showing a correlation of TLR8 expression with genes implicated in the HBV cycle (n=83, Spearman correlation, GSE65359). (B) Volcano plot depicting significantly downregulated (SLGN-CM-DOWN, n=48) and upregulated (SLGN-CM-UP, n=141) PHH genes in response to SLGN-CM (n=3, FDR<0.05, FC>1). (C) Treatment of PHH with SLGN-CM (1/50, 72 hours) produced in KCs induces the downregulation of NTCP and HSPA1A (RNA-seq). (D) Treatment of PHH with SLGN-CM leads to a dose-dependent downregulation of NTCP, as assessed by qPCR (Mann-Whitney test, n=3). Bars represent mean±SEM. (E–F) Treatment of PHH (E) or differentiated HepaRG cells (F) with SLGN-CM (1/50) 72 hours prior HBV infection leads to a significant decrease in HBsAg, HBeAg, 3.5 kb RNA and cccDNA levels at day 6 postinoculation (one-way analysis of variance, n=5). Bulevirtide (100 nM, 24 hours) was used as positive control. Bars represent mean±SEM. APOBEC3G, apolipoprotein B mRNA editing enzyme catalytic subunit 3G; cccDNA, covalently closed circular DNA; CEBPA, CCAAT enhancer binding protein alpha; CM, conditioned media; EGFR, epidermal growth factor receptor; EZH2, enhancer of zeste 2 polycomb repressive complex 2 subunit; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HDAC1, histone deacetylase 1; HSPA1A, heat shock protein family A (Hsp70) member 1A; NEDD8, NEDD8 ubiquitin-like modifier; NTCP, sodium/taurocholate cotransporting polypeptide; LIG3, DNA ligase 3; PHH, primary human hepatocytes; PRPF31, pre-mRNA processing factor 31; RXRA, retinoid X receptor alpha; SDC2, syndecan 2; SLGN, selgantolimod; SPIN1, spindlin 1; STAT1, signal transducer and activator of transcription 1; TLR8, toll-like receptor 8.

Figure 5. SLGN indirectly activates STAT3 signalling in hepatocytes. (A) GSEA of liver transcriptomic data from HBV-infected patients according to high TLR8 mRNA expression (FDR<0.05, n=83, GSE65359). (B) NicheNet ligand activity scores for soluble KC factors potentially mediating the transcriptomic profile observed in PHH treated with SLGN-CM. (C) Treatment of PHH (15 min) with decreasing dilutions of SLGN-CM leads to an increased STAT3 Y705 phosphorylation. Quantification of band intensities for p-STAT3 and total STAT3 are presented as ratio means±SEM (Mann-Whitney test, n=3). (D) Treatment of PHH (72 hours) with decreasing dilutions of SLGN-CM leads to an increased SOCS3 expression (Mann-Whitney test, n=3). Bars represent mean±SEM. (E) GSEA of PHH treated with SLGN-CM (1/50, 72 hours) showing a positive enrichment of the IL-6/STAT3 signalling pathway (FDR<0.0001). ADM, adrenomedullin; CM, conditioned media; GSEA, gene set enrichment analysis; HPX, haemopexin; IFNG, interferon gamma; IL, interleukin; JAK, Janus kinase; KCs, Kupffer cells; NES, normalised enrichment score; OSM, oncostatin M; SLGN, selgantolimod; SOCS3, suppressor of cytokine signalling 3; STAT3, signal transducer and activator of transcription 3; TGFB1, transforming growth factor beta 1; TNF, tumour necrosis factor; TLR8, toll-like receptor 8.

Figure 6. SLGN indirectly impairs HBV entry via an IL-6-dependent mechanism. (A) Prioritisation of KC ligands predicted by NicheNet, based on expression of KC ligands (left), fold change in response to SLGN treatment (centre) and receptor expression for these ligands in PHH (right). Expression levels represent log-transformed base means. (B) Experimental protocol for the treatment of PHH with SLGN-CM (1/50, 72 hours) in combination with an IL-6-neutralising antibody prior HBV inoculation (6 days). (C-G) Treatment of PHH with an IL-6-neutralising antibody prior HBV infection is able to prevent the decrease in HBsAg, HBeAg, 3.5 kb RNA and cccDNA levels observed with SLGN-CM alone (one-way analysis of variance, n=4). IL-6 (5 ng/mL) was used as positive control. Bars represent mean±SEM. (H) The TLR8 agonist SLGN regulates KC differentiation status and indirectly impairs HBV entry into hepatocytes via an IL-6-dependent mechanism. ADM, adrenomedullin; cccDNA, covalently closed circular DNA; CM, conditioned media; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HPX, haemopexin; IFNG, interferon gamma; IL-6, interleukin 6; KCs, Kupffer cells; NTCP, sodium/taurocholate cotransporting polypeptide; OSM, oncostatin M; PHH, primary human hepatocytes; SLGN, selgantolimod; TGFB1, transforming growth factor beta 1; TNF, tumour necrosis factor.