Integrated Multi-Omic Analysis Reveals Causal Relationships and Causal Mechanisms of Peripheral Lymphocyte-Driven Remodeling in the Intrahepatic Immune Microenvironment of Early-Stage MAFLD.

Background: Metabolic dysfunction-associated fatty liver disease (MAFLD), the most common chronic liver disease worldwide, is characterized as a chronic inflammatory disease within an altered immune microenvironment. Investigating the relationship between peripheral blood parameters and intrahepatic immune remodeling is significant for developing non-invasive diagnostic tools and targeted immunotherapies for early MAFLD. This study aimed to investigate the causal relationships and underlying mechanisms between peripheral blood cell indices and intrahepatic immune microenvironment remodeling in early-stage MAFLD. Methods: We performed bidirectional mendelian randomization analyses to assess the causal effects of peripheral blood cell traits on MAFLD risk. Mediation mendelian randomization was applied to identify key inflammatory mediators. Hepatic immune cell recruitment pathways were explored by integrating mendelian randomization findings with bulk RNA sequencing data. Single-cell RNA sequencing of mouse models that replicate metabolic syndrome-associated MAFLD pathology was employed to characterize lymphocyte-driven pathways. Key findings were validated using an independent public single-cell RNA sequencing dataset derived from human peripheral blood mononuclear cells and an independent public single-cell RNA sequencing dataset from a non-genetically modified murine model. Results: (1) Bidirectional Mendelian randomization revealed a unidirectional causal effect of elevated peripheral lymphocyte count on disease risk. (2) Mediation analysis implicated Cd5-mediated inflammatory pathways in this association. (3) Integration of mendelian randomization and bulk transcriptomic data linked lymphocytes to hepatic recruitment pathways. (4) Single-cell RNA sequencing of MAFLD models identified a novel Itgb1⁺Cd5⁺Cd4⁺T cell subset enriched in diseased livers. These cells interact with hepatic Vcam1highMmp12⁺Kupffer cells via Vcam1-Itgb1 signaling, initiating inflammation. (5) This pathogenic cell subset and interaction were conserved in an independent, non-genetically modified murine model. Furthermore, a corresponding Itgb1⁺Cd5⁺Cd4⁺T cell population was identified in the peripheral blood mononuclear cells of human MAFLD patients. Conclusion: We innovatively established that peripheral T lymphocytes exhibit a positive causal relationship with MAFLD development, mediated by Cd5 expression levels. Furthermore, liver-resident Vcam1highKupffer cells may facilitate immune microenvironment remodeling in early MAFLD by recruiting Itgb1⁺Cd5⁺Cd4⁺T cells through the Vcam1-Itgb1 adhesion pathway.