Liver-specific Inflammatory Signatures Predict Clinically Significant Liver Damage

Background and Aims Inflammation drives progression of chronic liver disease. However, the triggers of inflammation remain undefined during chronic hepatitis B (CHB) because hepatic flares are spontaneous and difficult to capture. We used nucleoside analogue (NA) withdrawal to investigate early inflammatory events because liver damage after stopping therapy occurs in a predictable time frame. 11 CHB patients underwent 192 weeks of NA therapy before a protocol defined stop. Liver fine-needle aspirates (FNAs) were collected at baseline and 4-weeks post-withdrawal and analyzed using flow cytometry and single-cell RNA sequencing (scRNA-seq). Intrahepatic mononuclear cells (IHMCs) from uninfected livers were used to validate transcriptomic findings. At 4 weeks post NA-withdrawal, HBV DNA rebounded but alanine aminotransferase (ALT) levels remained normal, 7/11 patients developed ALT elevations (>2xULN) at later timepoints. There were no changes in cell frequencies between baseline and viral rebound. ScRNA-seq revealed upregulation of IFN stimulated genes (ISGs) and pro-inflammatory cytokine MIF upon viral rebound. In vitro experiments confirmed the type I IFN-dependent ISG profile whereas MIF was induced primarily by IL-12. MIF exposure further amplified inflammatory cytokine production by myeloid cells. Our data show that innate immune activation is detectable in the liver before clinically-significant liver damage is detectable in the serum. ### Competing Interest Statement Conflict of interest statement: R.T.C. received funding or consulting fees from AbbVie, Gilead, Janssen, Merck, BMS, GSK and Boehringer. J.J.F. received funding or consulting fees from AbbVie, Arbutus, Gilead, Janssen, Eiger, Enanta, and GSK. H.L.A.J. received funding or consulting fees from AbbVie, Arbutus, Gilead, Janssen, Merck, Roche, GSK, BMS, VIR, Aligos, Arena, Eiger, Enyo, Regulus, VBI Vaccines and Viroclinics. A.J.G received funding or consulting fees from Gilead, Janssen, GSK, VIR, Finch Therapeutics, and BlueJay Therapeutics. The other authors declare no competing interests.