PAD4+ neutrophils promote hepatic stellate cell activation and accelerate MASH fibrosis progression viaNET-DNA/TAOK1/MAPK pathways
Jiajia Shen, Shanshan Huang, Yaohui Wang, Qingyuan Wang, Shibo Lin, Wei Guan, Yingyun Gong, Yiming Si, Ming Zhao, Hongwen Zhou, Hui Liang
Jiajia Shen, Shanshan Huang, Yaohui Wang, Qingyuan Wang, Shibo Lin, Wei Guan, Yingyun Gong, Yiming Si, Ming Zhao, Hongwen Zhou, Hui Liang
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Research Article Cell biology Hepatology Metabolism

PAD4+ neutrophils promote hepatic stellate cell activation and accelerate MASH fibrosis progression viaNET-DNA/TAOK1/MAPK pathways

Abstract

Neutrophils play a pivotal role in the progression of metabolic dysfunction–associated steatohepatitis (MASH) by mediating inflammatory responses. However, the heterogeneity of neutrophil subsets in MASH and their specific contributions to disease progression remain unclear. In this study, analysis of liver biopsies from 265 patients revealed a strong association between elevated neutrophil counts and MASH severity, particularly fibrosis. Five distinct neutrophil subsets were identified in human liver tissue, with PAD4+ neutrophils serving as key drivers in MASH progression. Mechanistically, PAD4+ neutrophils generate neutrophil extracellular traps (NETs) and activate hepatic stellate cells via the TAOK1-dependent MAPK signaling pathway. Inhibition of PAD4+ neutrophils in vivo attenuated the progression of liver fibrosis without exacerbating liver injury. Collectively, these findings elucidate the pivotal involvement of PAD4+ neutrophils in MASH progression and identify them as promising therapeutic targets for mitigating fibrosis and inflammation.

Authors

Jiajia Shen, Shanshan Huang, Yaohui Wang, Qingyuan Wang, Shibo Lin, Wei Guan, Yingyun Gong, Yiming Si, Ming Zhao, Hongwen Zhou, Hui Liang

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Figure 8

The critical role of NET-DNA and TAOK1 interaction in MAPK pathway activation.

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The critical role of NET-DNA and TAOK1 interaction in MAPK pathway activ...
(A) Neutrophils isolated from MASH patient peripheral blood were induced to form NETs upon stimulation with IL-8, G-CSF, and TNF-β. Proteomic analysis using liquid chromatography/mass spectrometry (LC-MS) identified the amounts of proteins in these NETs, which are depicted in a Venn diagram. (B) A heatmap illustrates the top 20 protein components identified in the NETs by LC-MS, highlighting key proteins associated with NET formation. (C) Western blot analysis was performed to assess the impact of MPO inhibitor (PF1355), NE inhibitor (ONO5046), and DNase I treatment on MAPK pathway activation in LX-2 cells exposed to NETs. (D and E) Quantitative grayscale analysis of Western blot bands for α-SMA and TIMP-1 expression in response to NETs and inhibitors. (F) LC-MS analysis of enriched proteins associated with the MAPK pathway following NET-DNA pull-down, with key MAPK pathway components highlighted. (G) Coculture of neutrophils or NETs with LX-2 cells for varying durations (0, 30 minutes, 1, 2, 4, and 6 hours), followed by Western blot detection of TAOK1 and MAPKAPK5 expression levels. (H) Immunofluorescence microscopy showed changes in TAOK1 expression during co-cultivation of NETs and LX-2 cells (TAOK1 shown in red, DAPI for cell nuclei in blue). Scale bar: 100 μm. (I) Quantification of TAOK1+ staining intensity using ImageJ software, showing the increase in TAOK1 expression in LX-2 cells following NET exposure. (J) Western blot analysis after TAOK1 inhibitor treatment to examine the impact of NETs on MAPK pathway activation in LX-2 cells. (K) Schematic diagram illustrating the mechanism by which NET-DNA activates the MAPK pathway through TAOK1 interaction, promoting HepSC activation and fibrosis (created with BioRender; biorender.com). Statistical analyses were performed using 1-way ANOVA. Data are shown as the mean ± SEM.