Integrating single-cell RNA sequencing with Visium HD spatial atlas reveals immune–stromal cell interactions in Echinococcus multilocularis metacestode infection

Abstract Background Alveolar echinococcosis (AE), caused by the larval stage of the tapeworm Echinococcus multilocularis , is a fatal zoonotic disease characterized by progressive hepatic granulomatous inflammation and fibrosis. However, the mechanisms underlying the pathological transition from early inflammation to chronic immunosuppression and fibrosis remain unclear, particularly regarding the spatial regulation of immune–stromal interactions. Methods Mice were subjected to an experimental secondary hepatic infection model of E. multilocularis . Liver tissues from uninfected, 2‑week and 3‑month infected mice were analyzed by flow cytometry, immunofluorescence, single‑cell RNA sequencing (scRNA‑seq), and Visium HD spatial transcriptomics (ST). Results Flow cytometry indicated a significant increase in eosinophils, macrophages, and CD4⁺ T cells in the livers of mice two weeks after infection; by three months, these populations declined, but the levels of eosinophils and macrophages remained higher than those of the control group. scRNA-seq combined with Visium HD ST analysis suggested that at two weeks after infection, stromal cell subsets had already infiltrated into the granulomatous areas. These included three transcriptionally distinct endothelial cell populations (ECs1, ECs2, and ECs3) and a marked accumulation of myofibroblasts. By the chronic phase (three months), myofibroblasts significantly upregulated expression of the pro-fibrotic markers Timp1 and Spp1. Mononuclear phagocytes exhibited significant heterogeneity: monocyte-derived macrophages differentiate into Arg1⁺Spp1⁺ and Ccr7⁺Itgax⁺ macrophage subpopulations, while Kupffer cell subpopulations significantly reduced inflammatory cytokine expression levels during chronic infection. In this model, at two weeks after infection, eosinophils were specifically enriched in the cores of granulomas, displayed an immunoregulatory transcriptional program, and were spatially associated with IL-33⁺ cells within the same area. CD8⁺ T cells diversified into two functionally complementary subsets—an Ifng⁺ effector population and a Gzmk⁺ subset with migratory and homeostatic characteristics—both predominantly localized to nongranulomatous areas. Spatial deconvolution coupled with cell–cell communication analysis further identified the collagen‑rich granuloma as a critical niche where myofibroblast‑derived signals (e.g., COL1A1–CD44) are predicted to interface with lymphocytes. Conclusions This study provides a spatially resolved atlas of immune–stromal interactions in a murine protoscolex‑based hepatic infection model of E. multilocularis , suggesting that myofibroblasts play an important role in fibrotic and immunosuppressive processes. Our findings provide a cellular and spatial framework for understanding the immune–stromal regulatory network during E. multilocularis infection, and may serve as a foundation for future studies of disease pathogenesis.