A landmark studyfrom Prof. Bin Cao's team (China-Japan Friendship Hospital) in Signal Transduction and Targeted Therapy deciphers Ninjurin-1 (NINJ1) 's role in influenza A virus (IAV) - triggered immunopathology.

Severe IAV pneumonia is fueled by PANoptosis -- a hyperinflammatory programmed cell death integrating pyroptosis, apoptosis, andnecroptosis. While upstream targets (e.g., ZBP1) or pathway-specific executors (GSDMD, MLKL) have been explored, their protective effects in vivo remain inconsistent, often compromising antiviral immunity. NINJ1, a plasma membrane rupture (PMR) mediator in lytic cell death, had unknown functions in IAV-induced PANoptosis -- creating a critical knowledge gap this study aimed to fill.

Figure S6. Schematic ofNINJ1 oligomerization coupling to PANoptosis activation in IAV-infected BMDMs

Multidimensional Research Approach

The teamcombined in vitro mechanistic assays, in vivo murine models, and clinical single-cell profiling for comprehensive validation:

• Cellular Models: IAV-infected bone marrow-derived macrophages (BMDMs), THP-1 cells,and A549 cells; CRISPR-Cas9-generated knockout lines (Ninj1⁻/⁻, Gsdmd⁻/⁻,Zbp1⁻/⁻, etc.) to dissect NINJ1’s role.

• Functional Assays: BS³-crosslinking to detect NINJ1 oligomerization; immunoblottingfor PANoptosis markers (GSDMD cleavage, MLKL phosphorylation); LDH/cytokine (IL-1β, TNF-α) quantification to assess PMR and inflammation.

• In Vivo Validation: Lethal/non-lethal IAV challenge in Ninj1⁻/⁻, Zbp1⁻/⁻, andwild-type mice; survival curves, lung histopathology, and bronchoalveolarlavage fluid (BALF) analysis.

• Clinical Relevance: Single-cell RNA-seq of BALF from severe COVID-19 patients (survivors vs. non-survivors) to link NINJ1 expression with disease severity.

HySigen's Ninj1^⁻/⁻ iBMDMs: The Pivotal Tool

A cornerstone of the study’s in vitro success was the Ninj1⁻/⁻ induced BMDMs (iBMDMs) custom-developed by Haixing Bioscience (Suzhou, China). This cell model enabledthe team to make three defining observations:

• PMR Mediation: Ninj1⁻/⁻ iBMDMs showed abrogated LDH and HMGB1 release post-IAV infection, confirming NINJ1 as the final executor of PMR -- while cell death rates remained unchanged, ruling out a role in death initiation.

• Cytokine Regulation: Unlike Gsdmd⁻/⁻ cells, Ninj1⁻/⁻ iBMDMs exhibited reduced IL-1β secretion, revealing a unique, GSDMD-independent role of NINJ1 in inflammatory cytokine release during PANoptosis.

• Pathway Redundancy: Even in Gsdmd⁻/⁻Gsdme⁻/⁻Mlkl⁻/⁻ triple-knockout backgrounds, Ninj1⁻/⁻ iBMDMs blocked PMR -- proving NINJ1 acts downstream of all PANoptosis pathways, a finding unachievable with non-specific tools.

✨ Key Takeaway: Ninj1⁻/⁻ iBMDMs provided the genetic precision needed to validate NINJ1 as a “bottleneck” target—intervening here alleviates immunopathology without impairing viral clearance (unlike ZBP1 inhibition). In mice, Ninj1⁻/⁻ significantly improved survival and reduced lung injury; in COVID-19, monocyte/macrophage NINJ1 expression correlated with poor prognosis.

Publication: Xu Y, Zheng Y, Liu Y, et al. Ninjurin-1 mediates cell lysis and detrimental inflammation of PANoptosis during influenza A virus infection[J]. Signal Transduction and Targeted Therapy, 2025, 10(1): 1-13.https://doi.org/10.1038/s41392-025-02391-9

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