Introduction

Acetaminophen (APAP) overdose causes severe acute liver failure. Impaired liver repair and regeneration after APAP hepatotoxicity leads to failed recovery and mortality. Accumulating evidence indicates that macrophages play an important role in liver repair after APAP-induced liver injury; however, underlying mechanisms of involvement of macrophages remain unknown.

Aims & Methods

We examined the role of endogenous thromboxane A₂ (TXA₂) in macrophages in liver repair after APAP-induced liver injury.
APAP (300 mg/kg, ip) was administered to macrophage-specific thromboxane prostanoid receptor (TP) deficient mice (TP^△mac) and control mice (Cont).

Results

Compared with Cont, TP^△mac exhibited severe liver injury as indicated by increased levels of ALT, necrotic area, and pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and decreased expression of hepatocyte growth factor (HGF) and PCNA, a marker of hepatocyte proliferation at 48 h after APAP treatment. CD68-positive macrophages less accumulated in the livers from TP^△mac, accompanied by reduced expressions of chemokines such as CCL2, CCL7 and CCL9. Flow cytometry analysis revealed that the numbers of pro-inflammatory macrophages (M1) in TP^△mac were higher than Cont, while the numbers of reparative macrophages (M2) in TP^△mac were lower than Cont. In cultured bone marrow-derived macrophages from TP^△mac, M1-related gene expressions were increased and M2-related gene expressions were decreased. HGF treatment in TP^△mac attenuated liver injury and promoted liver repair after APAP-induced liver injury.

Conclusion

TP receptor signaling in macrophages promoted liver repair after APAP-induced liver injury by accumulation of reparative macrophages and production of HGF in the livers.

Introduction

Acetaminophen-induced liver failure (AILF) has a high fatality rate and limited therapeutic approaches at present. Type 1 conventional dendritic cells (cDC1) are one of the major subgroups of cDCs in the liver, which is not only an important type in initiating adaptive immune response, but also can promote the generation, stability and function of Regulatory T (T_reg) cells through various mechanisms. cDC1 maintains liver immune homeostasis by regulating the balance between immune activation and tolerance. Nevertheless, the precise involvement and mechanistic contributions of hepatic cDCs in AILF remain to be elucidated.

Aims & Methods

Multiplex Immunohistochemistry (mIHC), flow cytometry, spatial transcriptomics (ST), single-cell RNA sequencing (scRNA-seq) and intravital 3D imaging were used to characterize the distribution and frequency of the cDC1. Bulk RNA-seq, metabolomic analyses, Cleavage Under Targets and Tagmentation (CUT&Tag) and Chromatin immunoprecipitation (ChIP)-qPCR assay were employed to explore the molecular mechanisms. CD11c^creXCR1^f/f, CD11c^creIDO1^f/f, CD11c^creP300^f/f and Foxp3^DTR mice, various mouse primary cells, human liver and serum samples were applied to verify the discoveries of bioinformatics analysis. Intravenous delivery of tolerogenic dendritic cells (tolDCs) was used to elucidate their regulatory effects on T_reg cells and roles in the AILF process.

Results

Our analyses revealed that the amount of cDC1 was significantly reduced across both clinical AILF cohorts and mouse AILF models, with the magnitude of depletion exhibiting dose- and duration-dependent associations with APAP exposure. mIHC, ST, scRNA-seq and intravital 3D imaging delineated that cDC1 depletion impaired T_reg cells priming capacity, ultimately disrupting hepatic immune homeostasis and exacerbating AILF. Intravenous tolDCs injection demonstrated therapeutic efficacy by orchestrating hepatic T_reg cells infiltration, attenuating inflammatory responses, potentiating liver regeneration, and ultimately reversing AILF-associated pathological manifestations. The therapeutic effect of intravenous tolDCs administration was abolished after diphtheria toxin-mediated T_reg cells depletion in Foxp3^DTR mice. CD11c^creXCR1^f/f mice andwild-type mice clearing cDC1 with XCL1-antibody had exacerbated liver pathology. Mechanistically, EP300-mediated histone 3 lysine 27 acetylation (H3K27ac) in IDO1 promoter region enforced stable tolerogenic states of cDC1, these epigenetically programmed cDC1 subsequently initiated tryptophan catabolism, generating kynurenine metabolites that triggered both differentiation and functional maturation of T_reg cells. Both the DC-specific knockout and inhibition of EP300 could reduce the H3k27ac levels of IDO1. And the CD11c^creIDO1^f/f mice with APAP exposure had less T_reg cells recruitment and worse phenotype.

Conclusion

This study elucidated that EP300 orchestrated tolDCs differentiation via H3K27ac/IDO1 signaling axis, this epigenetic-metabolic cascade licensed tolDCs to recruit and activate T_reg cells, thereby preserving hepatic immune homeostasis while promoting hepatocyte proliferation and tissue repair. These findings highlight novel therapeutic targets for reversing AILF pathogenesis.

Introduction

Acetaminophen (APAP) overdose is a primary cause of drug-induced liver injury (DILI). Although the inflammatory microenvironment plays a critical role in APAP-induced liver injury (AILI), its cellular interaction network and molecular regulatory mechanisms remain incompletely elucidated. Krüppel-like factor 4 (KLF4), a zinc finger transcription factor, is known to regulate monocyte differentiation, but its functional role in the AILI inflammatory microenvironment remains unclear. This study aims to investigate the regulatory effects of myeloid KLF4 deletion on the AILI microenvironment.

Aims & Methods

By integrating multidimensional omics analysis and a myeloid-specific Klf4 knockout mouse model, we dynamically dissected microenvironmental remodeling during AILI progression. Liver samples were collected for RNA sequencing, combined with single-cell transcriptomics, flow cytometry, and molecular interaction validation to clarify the KLF4-dependent cellular subpopulation interaction network. In vivo liver imaging, histology, biochemical assays, and molecular experiments were performed to validate KLF4-dependent intercellular interactions. Primary hepatocytes and hepatocyte cell lines were used for in vitro verification.

Results

Myeloid-specific KLF4 deletion led to an increase in CXCL2⁺CXCL3⁺CD24⁺ monocytes, which activated the NF-κB signaling pathway to secrete CXCL2/CXCL3 chemokines. These chemokines bound to the CXCR2 receptor on neutrophils, driving neutrophil infiltration and neutrophil extracellular traps (NETs) formation, while amplifying the TNF-α signaling pathway. Concurrently, myeloid KLF4 knockout induced enrichment of CXCL1⁺IL17RA⁺ endothelial cells. Neutrophil-derived NETs promoted endothelial phenotypic switching toward pro-inflammatory polarization, while endothelial-derived CXCL1 further recruited neutrophils, establishing a "monocyte-neutrophil-endothelial" cellular subpopulation interaction that perpetuated microenvironmental inflammation.

Conclusion

This study reveals the molecular mechanism by which KLF4 drives inflammatory cascades in the AILI microenvironment through regulation of the myeloid-endothelial interaction network. We propose therapeutic strategies targeting NETs formation or the CXCL2/CXCL3-CXCR2 axis, providing novel targets for precise intervention in AILI.

Introduction

Acetaminophen (APAP) is a major cause of drug-induced liver injury (DILI), once the latter evolves into liver failure, it significantly impacting clinical outcomes. Urokinase plasminogen activator receptor (uPAR) is a membrane protein that is distributed on various immune-active cell membranes, which is not only associated with cell adhesion, invasion and migration, but also can regulate the immune microenvironment. However, uPAR specific roles in the regulation of myeloid cell innate immunity and the maintenance of hepatic homeostasis during DILI remains unclear.

Aims & Methods

The expression pattern of uPAR in both mouse DILI models and DILI patients was determined. Multiplex Immunohistochemistry (mIHC), flow cytometry, spatial transcriptomics (ST), single-cell RNA sequencing (scRNA-seq), intravital 3D imaging, luciferase reporter gene assay and Cleavage Under Targets and Tagmentation (CUT&Tag) were used to explore molecular mechanisms. uPAR^-/-, Lyz2^creuPAR^f/f and AXL^-/- mice, various mouse primary cells, human liver and serum samples were used to verify the discoveries of bioinformatics analysis.

Results

We found that the expression level of uPAR in the liver of both mouse DILI models and DILI patients was significantly high, systemic and myeloid deficiency of uPAR could alleviate liver inflammation in mice after challenged with APAP. mIHC, flow cytometry, ST, scRNA-seq and intravital 3D imaging multidimensionally elucidated that macrophage efferocytosis in both uPAR^-/- and Lyz2^creuPAR^f/f mice was enhanced due to the upregulation of the AXL, leading to the accelerated clearance of senescent neutrophils and thereby promoting the resolution of liver inflammation. Luciferase reporter gene assay and CUT&Tag confirmed that transcription factor IRF7 directly bound to the promoter region of AXL in macrophages. Mechanistic studies showed that uPAR regulated AXL mediated efferocytosis through the STAT3/IRF7 pathway. Macrophage-specific overexpression of IRF7 in vivo enhanced macrophage efferocytosis and promoted the attenuation of liver inflammatory response in both mouse DILI and liver ischemia-reperfusion injury models. AXL^-/- mice had exacerbated phenotype, while AXL activator GAS-6 protein could alleviate liver injury.

Conclusion

uPAR/STAT3/IRF7/AXL signaling is critical to regulate macrophage efferocytosis and maintain hepatic tissue homeostasis in the progression of DILI. Its dual functionality as a liver homeostatic regulator and DILI therapeutic target warrants further investigation.

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 (LysM^creMas1^f/f, Alb^creMas1^f/f, Cdh5^creMas1^f/f, Lrat^creMas1^f/f and Cd4^creMas1^f/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. Cdh5^crePkm^f/+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.