Multi-omics and spatial proteomic profiling reveal novel AGXT2− PYCR3− macrophages and their phenotypes in metabolic dysfunction-associated steatotic liver disease
6 affiliations
- 1Department of Endocrinology, Jiangsu Province Hospital of Chinese Medicine (Affiliated Hospital of Nanjing University of Chinese Medicine), Nanjing, China
- 2The First School of Clinical Medicine, Nanjing University of Chinese Medicine, Nanjing, China
- 3Department of General Surgery, Jiangsu Province Hospital of Chinese Medicine (Affiliated Hospital of Nanjing University of Chinese Medicine), Nanjing, China
- 4Hepatobiliary Center, Jiangsu Province Hospital (First Affiliated Hospital of Nanjing Medical University), Nanjing, China
- 5Department of Cardiology, Jiangsu Province Hospital of Chinese Medicine (Affiliated Hospital of Nanjing University of Chinese Medicine), Nanjing, China
- 6Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China
Abstract
Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global liver disorder with a rising incidence. Early-to-middle-stage MASLD remains amenable to clinical intervention; without timely management, it may progress to fibrosis and cirrhosis. Therefore, identifying novel targets through integrated multi-technical approaches is critical for preventing and treating MASLD.
Methods: We developed an integrative multi-omics and spatial proteomic strategy combined with in vitro validation. CyTOF was applied to high-dimensional immunophenotyping of the MASLD liver microenvironment. Key cell populations were then sorted accordingly for mass spectrometry to uncover their core signaling. Focusing on identified key amino acid metabolic enzymes, we performed IMC for spatial single-cell proteomic profiling to localize metabolic aberrant subsets. Finally, functional phenotypes and mechanisms were validated in cell models.
Results: CyTOF revealed significant expansion of myeloid-derived cells. Thus, by targeting myeloid-derived cells, proteomic and phosphoproteomic analyses identified two severely impaired amino acid metabolic pathways: the glycine metabolic pathway regulated by AGXT2 and the proline metabolic pathway regulated by PYCR3. We then focused on AGXT2 and PYCR3 for spatial exploration via IMC and ultimately identified a macrophage subset negative for both AGXT2 and PYCR3. These macrophages were significantly elevated within MASLD and exhibited high spatial colocalization with inflammatory cells and fibrotic cells. Compared with dysregulated M1 subsets (M1-C3), dysregulated M2 subsets (M2-C1) indicated stronger pro-inflammatory and pro-fibrotic potential. In vitro models using human and murine cells confirmed that AGXT2⁻PYCR3⁻ macrophages exhibited enhanced proliferation and migration, secreted higher levels of inflammatory cytokines, chemokines, and classic fibrotic proteins, and exerted strong inductive effects on hepatic fibrotic cells, with downregulation of intercellular GSH. Mechanistically, the NF-κB and MAPK/AP-1 pathways were involved in inflammatory responses, while the p-SMAD3(T8)/TGFβ pathway contributed to the fibrotic phenotype. However, the abnormal biological functions could be effectively rescued by supplementation with corresponding amino acids, accompanied by the reversal of aberrant signaling pathway activation.
Conclusions: In summary, our study explores an initial link between amino acid metabolism and early-to-middle-stage MASLD. AGXT2⁻PYCR3⁻ macrophages are significantly enriched in MASLD with pro-inflammatory and pro-fibrotic potential, and amino acid supplementation can rescue their aberrant phenotypes in vitro, warranting further validation of their treatment potential.
Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1186/s13073-026-01716-9.
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BibTeX
@article{lv2026multiomics,
title = {{Multi-omics and spatial proteomic profiling reveal novel AGXT2− PYCR3− macrophages and their phenotypes in metabolic dysfunction-associated steatotic liver disease}},
author = {Tiansu Lv and Hongshan Dai and Shihu Zhang and Ee Chang and Ming Ni and Jing Ge and Yutian Cao and Zhe Cheng and Yixian He and Jiaxuan Huai and Wenlin Ma and Yiran Zhu and Xinzi Xu and Qianhua Yan and Zhuyuan Fang and Jiangyi Yu and Feng Zhang and Xiqiao Zhou},
journal = {Genome Medicine},
year = {2026},
month = jul,
doi = {10.1186/s13073-026-01716-9},
url = {https://doi.org/10.1186/s13073-026-01716-9},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - JOUR
TI - Multi-omics and spatial proteomic profiling reveal novel AGXT2− PYCR3− macrophages and their phenotypes in metabolic dysfunction-associated steatotic liver disease
AU - Tiansu Lv
AU - Hongshan Dai
AU - Shihu Zhang
AU - Ee Chang
AU - Ming Ni
AU - Jing Ge
AU - Yutian Cao
AU - Zhe Cheng
AU - Yixian He
AU - Jiaxuan Huai
AU - Wenlin Ma
AU - Yiran Zhu
AU - Xinzi Xu
AU - Qianhua Yan
AU - Zhuyuan Fang
AU - Jiangyi Yu
AU - Feng Zhang
AU - Xiqiao Zhou
PY - 2026
DA - 2026/07/13
JO - Genome Medicine
DO - 10.1186/s13073-026-01716-9
UR - https://doi.org/10.1186/s13073-026-01716-9
AB - Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global liver disorder with a rising incidence. Early-to-middle-stage MASLD remains amenable to clinical intervention; without timely management, it may progress to fibrosis and cirrhosis. Therefore, identifying novel targets through integrated multi-technical approaches is critical for preventing and treating MASLD. Methods: We developed an integrative multi-omics and spatial proteomic strategy combined with in vitro validation. CyTOF was applied to high-dimensional immunophenotyping of the MASLD liver microenvironment. Key cell populations were then sorted accordingly for mass spectrometry to uncover their core signaling. Focusing on identified key amino acid metabolic enzymes, we performed IMC for spatial single-cell proteomic profiling to localize metabolic aberrant subsets. Finally, functional phenotypes and mechanisms were validated in cell models. Results: CyTOF revealed significant expansion of myeloid-derived cells. Thus, by targeting myeloid-derived cells, proteomic and phosphoproteomic analyses identified two severely impaired amino acid metabolic pathways: the glycine metabolic pathway regulated by AGXT2 and the proline metabolic pathway regulated by PYCR3. We then focused on AGXT2 and PYCR3 for spatial exploration via IMC and ultimately identified a macrophage subset negative for both AGXT2 and PYCR3. These macrophages were significantly elevated within MASLD and exhibited high spatial colocalization with inflammatory cells and fibrotic cells. Compared with dysregulated M1 subsets (M1-C3), dysregulated M2 subsets (M2-C1) indicated stronger pro-inflammatory and pro-fibrotic potential. In vitro models using human and murine cells confirmed that AGXT2⁻PYCR3⁻ macrophages exhibited enhanced proliferation and migration, secreted higher levels of inflammatory cytokines, chemokines, and classic fibrotic proteins, and exerted strong inductive effects on hepatic fibrotic cells, with downregulation of intercellular GSH. Mechanistically, the NF-κB and MAPK/AP-1 pathways were involved in inflammatory responses, while the p-SMAD3(T8)/TGFβ pathway contributed to the fibrotic phenotype. However, the abnormal biological functions could be effectively rescued by supplementation with corresponding amino acids, accompanied by the reversal of aberrant signaling pathway activation. Conclusions: In summary, our study explores an initial link between amino acid metabolism and early-to-middle-stage MASLD. AGXT2⁻PYCR3⁻ macrophages are significantly enriched in MASLD with pro-inflammatory and pro-fibrotic potential, and amino acid supplementation can rescue their aberrant phenotypes in vitro, warranting further validation of their treatment potential. Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1186/s13073-026-01716-9.
SN - 1756-994X
ER -