Integrated murine and human transcriptomics prioritizes a multicellular mitochondrial signature associated with immune and parenchymal features in NAFLD/MASLD

Abstract Metabolic dysfunction-associated steatotic liver disease (MASLD), historically termed nonalcoholic fatty liver disease (NAFLD), involves metabolic dysregulation, mitochondrial stress, and immune remodeling. However, disease-associated multicellular transcriptional programs and their spatial context remain incompletely defined. We integrated exploratory murine single-cell RNA sequencing (scRNA-seq), human bulk transcriptomics, murine spatial transcriptomics, and RT-qPCR in an FFA-treated human hepatocyte model. The murine scRNA-seq discovery dataset comprised one control and two NAFLD samples. Monocyte-focused single-cell analyses were used to prioritize candidate genes, which were evaluated in human bulk transcriptomic datasets, spatially contextualized in murine liver sections, and assessed for transcriptional responsiveness to hepatocyte lipotoxic stress. Exploratory analysis of 37,220 murine single cells suggested relatively higher monocyte proportions in the two available NAFLD samples than in the single control sample. Cell-level monocyte differential expression analysis and hdWGCNA prioritized a seven-gene mitochondrial set comprising Atp5k, Atp5e, Cox7a2, Cox7c, Cox6c, Cox5b, and Cox6b1, corresponding to ATP5ME, ATP5F1E, COX7A2, COX7C, COX6C, COX5B, and COX6B1 in humans. In human bulk transcriptomes, the orthologous gene set was associated with oxidative phosphorylation-related, DNA repair, p53, TGF-β, PI3K-AKT-mTOR, and immunoregulatory transcriptional features, without establishing direct regulatory relationships between individual genes and these pathways. Spatial analyses indicated inferred neighborhood relationships among multiple cell-type-enriched multicellular regions, including macrophage-enriched and hepatocyte-enriched regions, and broad expression of the hub genes across multicellular spots. RT-qPCR showed increased expression of all seven human orthologues in FFA-treated HepLi5 cells, indicating transcriptional responsiveness to hepatocyte lipotoxic stress. This exploratory cross-species study prioritizes a multicellular mitochondrial signature associated with immune and parenchymal features in NAFLD/MASLD. The gene set was initially prioritized through monocyte-focused murine analyses but was not restricted to monocytes and showed substantial parenchymal-associated expression. The signature provides a hypothesis-generating framework for future cell-resolved studies of mitochondrial stress in NAFLD/MASLD; however, its biomarker utility, prognostic value, and therapeutic relevance require validation in larger clinically annotated cohorts and functional studies.