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Background <p>Endoplasmic reticulum stress (ERS) plays a pivotal role in regulating immune cell fate. While ERS is known to induce apoptosis, its contribution to the recently defined inflammatory cell death pathway, PANoptosis, remains unclear. This study aimed to elucidate how tunicamycin-induced ERS regulates Z-DNA binding protein 1 (ZBP1)-mediated PANoptosis in macrophages and mouse spleen.</p> Methods <p>RAW264.7 macrophages were treated with tunicamycin to induce ERS. Cell viability, proliferation, migration, and invasion were assessed by CCK-8, colony formation, wound-healing, and Transwell assays. ERS-related and PANoptosis-associated molecules were analyzed by RT-qPCR and Western blotting. Reactive oxygen species (ROS) generation, apoptosis, and cytokine secretion were quantified by flow cytometry and ELISA. The ER stress inhibitor 4-phenylbutyric acid (4-PBA) and RNA sequencing were used to explore regulatory mechanisms. An in vivo tunicamycin mouse model was established to validate ERS-induced PANoptosis in spleen tissue.</p> Results <p>Tunicamycin significantly suppressed macrophage proliferation, migration, and invasion by activating the GRP78/PERK/eIF2α/ATF4/CHOP pathway and increasing ROS levels. ERS upregulated ZBP1 and RIPK3 expression, accompanied by activation of apoptotic (Caspase-3/7/8), necroptotic MLKL (mixed lineage kinase domain-like protein)/p-MLKL, and pyroptotic GSDMD (gasdermin D)/GSDME (gasdermin E) markers, and increased secretion of IL-6, TNF-α, IL-1β, and IL-18, indicating PANoptosis induction. 4-PBA alleviated ERS, reduced ZBP1 and CHOP expression, and restored cell viability. RNA-seq analysis confirmed enrichment of ERS and inflammatory death pathways. In vivo, tunicamycin triggered ZBP1 upregulation and PANoptotic features in mouse spleen.</p> Conclusion <p>These findings demonstrate that tunicamycin-induced ERS activates the GRP78/PERK/eIF2α/ATF4/CHOP pathway and provides the first evidence that ER stress alone can trigger ZBP1-mediated PANoptosis in macrophages and mouse spleen, highlighting the ER stress–ZBP1–PANoptosis axis as a novel mechanism linking ERS to inflammation and immune dysregulation.</p>

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Tunicamycin-induced endoplasmic reticulum stress triggers ZBP1-Mediated PANoptosis in macrophages and mouse spleen

  • 期刊论文

  • 发表日期：

    2026年4月24日

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  • Hongkun Jiang,
  • Chen Qian,
  • Xiaoting Lin,
  • Ximei Huang,
  • Zila Wang,
  • Faquan Lin

摘要

Background

Endoplasmic reticulum stress (ERS) plays a pivotal role in regulating immune cell fate. While ERS is known to induce apoptosis, its contribution to the recently defined inflammatory cell death pathway, PANoptosis, remains unclear. This study aimed to elucidate how tunicamycin-induced ERS regulates Z-DNA binding protein 1 (ZBP1)-mediated PANoptosis in macrophages and mouse spleen.

Methods

RAW264.7 macrophages were treated with tunicamycin to induce ERS. Cell viability, proliferation, migration, and invasion were assessed by CCK-8, colony formation, wound-healing, and Transwell assays. ERS-related and PANoptosis-associated molecules were analyzed by RT-qPCR and Western blotting. Reactive oxygen species (ROS) generation, apoptosis, and cytokine secretion were quantified by flow cytometry and ELISA. The ER stress inhibitor 4-phenylbutyric acid (4-PBA) and RNA sequencing were used to explore regulatory mechanisms. An in vivo tunicamycin mouse model was established to validate ERS-induced PANoptosis in spleen tissue.

Results

Tunicamycin significantly suppressed macrophage proliferation, migration, and invasion by activating the GRP78/PERK/eIF2α/ATF4/CHOP pathway and increasing ROS levels. ERS upregulated ZBP1 and RIPK3 expression, accompanied by activation of apoptotic (Caspase-3/7/8), necroptotic MLKL (mixed lineage kinase domain-like protein)/p-MLKL, and pyroptotic GSDMD (gasdermin D)/GSDME (gasdermin E) markers, and increased secretion of IL-6, TNF-α, IL-1β, and IL-18, indicating PANoptosis induction. 4-PBA alleviated ERS, reduced ZBP1 and CHOP expression, and restored cell viability. RNA-seq analysis confirmed enrichment of ERS and inflammatory death pathways. In vivo, tunicamycin triggered ZBP1 upregulation and PANoptotic features in mouse spleen.

Conclusion

These findings demonstrate that tunicamycin-induced ERS activates the GRP78/PERK/eIF2α/ATF4/CHOP pathway and provides the first evidence that ER stress alone can trigger ZBP1-mediated PANoptosis in macrophages and mouse spleen, highlighting the ER stress–ZBP1–PANoptosis axis as a novel mechanism linking ERS to inflammation and immune dysregulation.