Introduction
There is an urgent and unmet need for novel therapeutic approaches of acetaminophen-induced liver injury (AILI) in current clinical practice. We recently has revealed the beneficial role of hepatocyte-specific Mas activation dependent fatty acid oxidation in mouse AILI[1], which is the first study of Mas signaling in AILI and also highlighted by the editorial of Journal of Hepatology[2]. However, considering the broad expression pattern of Mas, it is important to investigate whether targeting other cell types could also have protective effects against AILI, which has important implications for the efficacy and safety of future drug development.
Aims & Methods
Multiple cell-specific Mas1-knockout mice (LysMcreMas1f/f, AlbcreMas1f/f, Cdh5creMas1f/f, LratcreMas1f/f and Cd4creMas1f/f mice) were used for in vivo analyses. Single-cell RNA sequencing (scRNA-seq), spatial transcriptomics (ST) and intravital imaging were used to reveal cellular interactions in complex pathophysiological microenvironments. Cdh5crePkmf/+mice, metabolic flux analysis, pharmacological compounds, genetic tools, cell co-culture and human liver samples were used to validate the discoveries of bioinformatics analysis.
Results
By generating multiple cell-specific Mas1-knockout mice and using multi-omic analysis, we have further identified the myeloid-Mas signaling as the critical driver of immunometabolic landscape in the progression of AILI. Notably, the AILI-imprinted immunometabolic landscape, consisting of CD31+MYC+CD63+ endothelial cells (ECs), F4/80+MMP12+ macrophages (Mψ) and monocytes, which is firstly described and visualized by the hour-long intravital imaging in living mice. By incorporating the paradigm with a computational imaging framework, a novel intravital microscopy that fulfills the unmet requirement for hour-long, high-speed three-dimension (3D) subcellular observations in vivo[3], we demonstrated that evaluating the behaviors of immune cells in situ is an effective way to reveal complicated immune processes and understand disease pathogenesis. The deficiency of myeloid-Mas could cause a significant up-regulation of TNF and NF-κB signaling in monocytes, and the subsequent release of TNF-α to induce the pro-inflammatory phenotype of ECs possibly through metabolic reprogramming. Human blood monocytes (CD14+ cells) were isolated to verify the clinical relevance. Moreover, MYC+ ECs could produce abundant glycolytic metabolites to enhance glycolysis in adjacent Mψ and regulate their function. Glycolysis was demonstrated to be the key metabolic pathway for intercellular interactions. Importantly, the myeloid-Mas driven landscape established in mouse AILI, was perfectly mapped and enriched in human AILI, whose landscape also consists of three cell counterparts including MYC+ ECs, MMP12+ Mψ and CD14+ monocytes. Then, the k-means algorithm was applied to conduct an unsupervised clustering, suggesting the probability of 83% to identify advanced AILI by the microenvironmental characteristic.
Conclusion
Our findings offer a new insight into Mas-targeted therapeutic strategies against AILI, which should be designed based on the specific cellular and metabolic pathway. Our data show a great potential for the clinical transformation of “disease-imprinted microenvironment” into diagnostic and severity stratified models through artificial intelligence in the future. By combining scRNA-seq, ST and intravital imaging, our work provides a comprehensive paradigm for studying mammalian cellular interactions in complex pathophysiological microenvironments.
References
[1] Chen S, Lu Z, Jia H, et al. Hepatocyte-specific Mas activation enhances lipophagy and fatty acid oxidation to protect against acetaminophen-induced hepatotoxicity in mice. Journal of Hepatology, 2023, 78(3): 543-557.
[2] Nogueiras R, Aspichueta P. Expanding the roles of the renin–angiotensin system: drug-induced liver injury. Journal of Hepatology, 2023, 78(3): 457-459.
[3] Wu J, Lu Z, Jiang D, et al. Iterative tomography with digital adaptive optics permits hour-long intravital observation of 3D subcellular dynamics at millisecond scale. Cell, 2021, 184(12): 3318-3332. e17.