Inflammasome activation drives gasdermin-independent plasma membrane rupture by clustering ninjurin-1 in macrophages

This study reveals that inflammasome activation drives gasdermin-independent plasma membrane rupture and inflammatory cell death in macrophages by triggering the oligomerization and clustering of ninjurin-1 (NINJ1), a process that is further facilitated by phosphatidylserine exposure via Xkr8.

The Gist

The Big Picture: A "Plan B" for Cell Suicide

Imagine your body's immune system as a highly trained security force. When they detect a threat (like bacteria or dangerous crystals), they need to sound the alarm and call for backup. To do this, they sometimes have to sacrifice one of their own cells.

Usually, we thought there was only one way for these cells to self-destruct and release the alarm signals. This method involved a specific "key" protein called Gasdermin. Think of Gasdermin as a master key that unlocks a door in the cell's wall, letting the alarm signals (like IL-1β) escape and causing the cell to burst.

The Big Discovery:
This paper reveals that the security force has a secret "Plan B." Even if you take away the master key (Gasdermin), the cells can still burst open and release the alarm. They use a different tool entirely: a protein called NINJ1.

---

The Story in Three Acts

Act 1: The Failed Lockpick (Gasdermin is Missing)

Scientists created mice whose immune cells were missing the "master key" proteins (Gasdermin D and Gasdermin E). They expected that when these cells were triggered to die, they would stay sealed tight and not release any alarm signals.

What happened?
Surprisingly, the cells still burst open! They swelled up and popped, releasing the inflammatory signals just like normal cells.

• The Analogy: Imagine a bank vault with a broken lock (missing Gasdermin). You'd expect the vault to stay closed. Instead, the robbers (the immune system) just smashed the wall down with a sledgehammer. The money (inflammatory signals) still got out.

Act 2: The Real Culprit (NINJ1)

The researchers asked: "If it's not the master key, what is breaking the wall?"

They found a protein called NINJ1 (Nine-J-1).

• How it works: In a healthy cell, NINJ1 is like a group of construction workers standing quietly on the cell's surface. When the cell gets the "suicide signal," these workers suddenly link arms, form a tight cluster, and pull the cell wall apart, causing it to rupture.
• The Proof: When the scientists stopped NINJ1 from working (using a substance called glycine, which acts like a "glue" to keep the workers apart), the cells didn't burst. The alarm signals stayed trapped inside.
• The Analogy: If the master key is missing, the security team sends in a demolition crew (NINJ1). If you handcuff the demolition crew, the building stays standing.

Act 3: The Trigger (Why do they link up?)

The scientists wanted to know why NINJ1 decided to link up and break the wall. They looked at the cell's surface, which is like a lipid (fatty) bilayer.

They found that before the wall breaks, the cell flips a specific type of fat (Phosphatidylserine) from the inside to the outside. Think of this like flipping a "Surrender" flag or a red warning light on the outside of the cell.

• The Mechanism: A protein called Xkr8 acts as the switch that flips this flag. Once the flag is up, the NINJ1 workers see it, link arms, and tear the wall down.
• The Analogy: The cell flips a red "Danger" flag (Xkr8). The demolition crew (NINJ1) sees the flag, rushes over, and pulls the building down. If you remove the person who flips the flag (Xkr8), the demolition crew never shows up, and the cell stays safe.

---

Why Does This Matter?

1. It changes the rules of the game.
For years, scientists thought that if you blocked the "Gasdermin" pathway, you could stop dangerous inflammation (like in gout, heart disease, or Alzheimer's). This paper says: "Not so fast!" Even if you block Gasdermin, the body has a backup plan (NINJ1) that will still cause inflammation.

2. New targets for medicine.
If we want to stop these inflammatory diseases, we can't just block the "master key" (Gasdermin). We might need to block the "demolition crew" (NINJ1) or the "flag flipper" (Xkr8).

Summary in One Sentence

Even when the main "suicide key" (Gasdermin) is broken, immune cells can still burst open and cause inflammation by using a backup demolition crew (NINJ1) that is triggered by a red warning flag (Xkr8) on the cell surface.

Technical Summary

1. Problem Statement

The inflammasome is a critical molecular complex that activates caspase-1, leading to the processing of pro-inflammatory cytokines (IL-1β, IL-18) and the induction of pyroptosis, a form of inflammatory cell death.

• Current Paradigm: Pyroptosis is widely defined as a gasdermin-dependent process. Specifically, caspase-1 cleaves Gasdermin D (GSDMD) to form pores in the plasma membrane, causing cell lysis and the release of cytosolic contents. In the absence of GSDMD, Gasdermin E (GSDME) is often cited as a backup executor, processed by caspase-3/8 to induce secondary pyroptosis.
• The Knowledge Gap: It remains unclear whether the blockade of both GSDMD and GSDME is sufficient to completely prevent inflammasome-driven necrotic cell death and cytokine release. Previous studies have yielded conflicting results regarding the role of GSDME in this context. Furthermore, the molecular mechanism driving membrane rupture in the absence of gasdermins has not been fully elucidated.

2. Methodology

The authors employed a combination of genetic mouse models, cell engineering, live-cell imaging, and biochemical assays to dissect the mechanisms of cell death.

• Genetic Models:

  • Generated Gsdmd–/–Gsdme–/– (DKO) mice by crossing single-knockout strains to create a double-knockout background.
  • Used Casp1/11–/– mice as a negative control for caspase-1 activity.
  • Utilized Immortalized Bone Marrow-Derived Macrophages (iBMDMs) engineered with a dimerizer-inducible ASC oligomerization system (DmrBASC) to trigger inflammasome assembly independent of upstream PRRs.
  • Generated DKO iBMDMs lacking Gsdmd and Gsdme using CRISPR/Cas9 (enCas12a).
  • Created NINJ1-deficient and Phospholipid Scramblase-deficient (Xkr8, Ano6, Tmem63b) iBMDM lines using CRISPR/Cas9.

• Stimulation and Inhibition:

  • Stimuli: Nigericin (NLRP3 activator), Pam3CSK4 (priming), Cholesterol crystals (in vivo), and B/B dimerizer (to induce ASC oligomerization in DmrBASC cells).
  • Inhibitors: Glycine (NINJ1 inhibitor), VX-765 (caspase-1 inhibitor), Ferrostatin-1/Liproxstatin-1 (ferroptosis inhibitors), and GSK'872 (necroptosis inhibitor).

• Assays:

  • Cell Death: LDH release assays, SYTOX Green/Deep Red uptake (membrane permeability), and live-cell confocal microscopy.
  • Cytokine Release: ELISA for IL-1β and IL-1α.
  • Molecular Analysis: Western blotting for caspase activation, gasdermin processing, and NINJ1 oligomerization.
  • Live Imaging: Time-lapse microscopy using Annexin V (Phosphatidylserine/PtdSer exposure), SYTOX dyes, and Flipper-TR (membrane tension).
  • In Vivo: Intraperitoneal injection of cholesterol crystals to assess neutrophil recruitment.

3. Key Contributions

1. Discovery of Gasdermin-Independent Pyroptosis: The study definitively demonstrates that inflammasome activation drives necrotic cell death and IL-1 release even in the complete absence of both GSDMD and GSDME.
2. Identification of NINJ1 as the Primary Executor: The authors identify Ninjurin-1 (NINJ1) as the critical mediator of plasma membrane rupture in the absence of gasdermins. They show that inflammasome activation triggers NINJ1 oligomerization and clustering independently of GSDMD/GSDME.
3. Mechanistic Link to PtdSer Exposure: The study establishes that Xkr8-dependent Phosphatidylserine (PtdSer) exposure precedes or coincides with membrane permeabilization and is essential for NINJ1-mediated rupture in this context.
4. Refinement of Pyroptosis Definition: The findings suggest that "pyroptosis" (caspase-1-dependent inflammatory death) can occur via a gasdermin-independent pathway, challenging the strict definition that requires gasdermin pore formation.

4. Key Results

• GSDMD/GSDME are Dispensable for Late-Phase Necrosis:

  • In Gsdmd–/–Gsdme–/– macrophages, nigericin-induced cell death was delayed in the early phase but occurred robustly in the later phase (3–6 hours), accompanied by cell swelling and LDH release.
  • This necrotic death was caspase-1 dependent (blocked by VX-765) but independent of ferroptosis or necroptosis pathways.
  • IL-1 Release: Despite the lack of gasdermins, Gsdmd–/–Gsdme–/– macrophages released significant amounts of IL-1β and IL-1α upon inflammasome activation.

• NINJ1 Mediates Membrane Rupture:

  • Oligomerization: NINJ1 oligomerization was detected in Gsdmd–/–Gsdme–/– macrophages upon stimulation.
  • Glycine Sensitivity: Glycine, known to inhibit NINJ1, completely blocked LDH release and SYTOX entry in Gsdmd–/–Gsdme–/– cells. In contrast, glycine blocked LDH release but failed to prevent SYTOX entry in WT cells (where GSDMD pores form first), indicating distinct mechanisms.
  • Knockout Validation: In DmrBASC-Gsdmd/e DKO iBMDMs, NINJ1 deficiency prevented ASC oligomerization-induced membrane rupture and permeabilization. NINJ1-mNeonGreen imaging showed NINJ1 clustering on the plasma membrane during cell death.

• Role of PtdSer and Xkr8:

  • Live imaging revealed that PtdSer exposure (Annexin V+) precedes or coincides with membrane permeabilization (SYTOX+).
  • Xkr8 Dependency: Among phospholipid scramblases (Xkr8, Ano6, Tmem63b), only Xkr8 deficiency attenuated PtdSer exposure and subsequent membrane rupture in Gsdmd/e DKO cells. Ano6 and Tmem63b knockouts had no effect.
  • Cell Volume/Tension: The study ruled out cell swelling or increased membrane tension as the primary triggers for NINJ1 activation in this specific context, as cell volume and Flipper-TR tension measurements remained unchanged prior to rupture.

• In Vivo Relevance:

  • In a mouse model of cholesterol crystal-induced inflammation, Gsdmd–/–Gsdme–/– mice exhibited neutrophil recruitment comparable to WT mice, confirming that gasdermin-independent inflammation occurs in vivo.

5. Significance

• Therapeutic Implications: The findings suggest that targeting GSDMD alone may be insufficient to treat inflammasome-driven diseases (e.g., atherosclerosis, gout, neurodegeneration). Therapeutic strategies must also consider NINJ1 and Xkr8 as potential targets to fully block inflammatory cell death and cytokine release.
• Redefining Cell Death Mechanisms: This study challenges the dogma that gasdermins are the sole executors of inflammasome-mediated pyroptosis. It highlights a backup, gasdermin-independent pathway where caspase-1 activation directly drives NINJ1 clustering via Xkr8-mediated lipid scrambling.
• Mechanistic Insight: The work provides a detailed temporal sequence: Inflammasome activation $\rightarrow$ Caspase-1 activation $\rightarrow$ Xkr8 activation $\rightarrow$ PtdSer exposure $\rightarrow$ NINJ1 clustering $\rightarrow$ Plasma membrane rupture. This clarifies the molecular hierarchy of regulated necrosis in macrophages.

In conclusion, the paper establishes that NINJ1 is a critical, gasdermin-independent executor of inflammasome-driven necrotic cell death, regulated by Xkr8-dependent PtdSer exposure, offering new avenues for anti-inflammatory drug development.