Age-dependent differences in SARS-CoV-2 entry-factor expression and innate immune responses between pediatric and adult liver organoids

Abstract

Since early 2022, a global surge in cases of severe acute hepatitis of unknown origin (AHUO) in children has been reported, with no definitive aetiology established. Epidemiological evidence suggests a potential link to SARS-CoV-2 infection, particularly during Omicron-predominant periods, yet the mechanisms underlying the apparently higher susceptibility of children to SARS-CoV-2-related liver injury remain poorly understood. This study used non-tumorous (tumor-adjacent) liver organoids derived from pediatric and adult donors to compare viral RNA accumulation and host immune responses to SARS-CoV-2 wild-type (WT) and Omicron (BA.1/BA.2) variants, aiming to explore the possible role of SARS-CoV-2 in pediatric acute hepatitis. Liver organoids were established from non-tumorous (tumor-adjacent) liver tissue of three adult patients with hepatocellular carcinoma and three pediatric patients with hepatoblastoma, collected between February and December 2022. Organoids were infected with SARS-CoV-2 WT, Omicron BA.1 or BA.2 at a multiplicity of infection (MOI) of 0.1. Viral loads in supernatants and intracellular viral RNA were quantified by RT-qPCR at 24 and 48 h post-infection (hpi). At 48 hpi, RNA-seq was performed to profile transcriptomic changes and pathway enrichment. Additionally, single-cell RNA-seq data were used to compare baseline expression of SARS-CoV-2 entry-associated genes between pediatric and adult hepatocytes. Pediatric hepatocytes showed significantly higher basal expression of entry-related genes, including ASGR1, KREMEN1, CTSB, CTSL, and VIM, than adult cells. Both pediatric and adult liver organoids were permissive to WT and Omicron, but Omicron BA.1/BA.2 yielded higher intracellular viral RNA levels than WT, with notably higher intracellular viral RNA burden in pediatric organoids. RNA-seq revealed that Omicron infection induced a more pronounced innate-immune-associated transcriptional response in pediatric organoids—encompassing interferon signaling, RIG-I-like receptor and Toll-like receptor pathways—alongside relative downregulation of adaptive immune-pathway transcripts. In adult organoids, transcriptomic divergence between Omicron and WT was less pronounced. Compared with adults, Omicron-infected pediatric organoids upregulated interferon-γ signaling and other innate immune-associated transcriptional programs, while downregulating adaptive immune-pathway transcripts; key receptors such as ACE2 and TMPRSS4 remained consistently higher in pediatric organoids across all viral strains. Pediatric liver organoids accumulate higher levels of Omicron viral RNA than WT-infected organoids and mount a stronger innate-immune transcriptional response alongside relative downregulation of adaptive immune-pathway transcripts, correlating with elevated expression of viral entry factors. These age-dependent in vitro differences raise the hypothesis that SARS-CoV-2, particularly Omicron, could contribute to hepatocyte injury in children; however, this organoid-based model does not include patients with acute hepatitis of unknown origin and cannot establish a causal or epidemiological link, which will require dedicated prospective clinical and mechanistic studies.