Reg3β removes aged neutrophils after myocardial infarction

This study reveals that Reg3β promotes cardiac repair after myocardial infarction by selectively binding to aged neutrophils via paucimannosylated proteins, triggering their lysosome-mediated death and subsequent clearance by macrophages to resolve inflammation.

The Gist

Imagine your heart is a bustling city. When a major disaster strikes—a heart attack, or **Myocardial Infarction **(MI)—the city is damaged, and debris is everywhere. To clean up, the city calls in its emergency cleanup crew: neutrophils.

These neutrophils are like a highly aggressive, hyper-active SWAT team. Their job is to rush in, destroy the damaged tissue, and fight off any potential infections. They are essential for the initial cleanup. But here's the problem: if this SWAT team stays too long or gets too rowdy, they start smashing up the healthy parts of the city, causing even more damage and potentially leading to the city's collapse (heart rupture).

Usually, the city has a system to tell these cleanup crews when to stand down and leave. But in this new study, scientists discovered a specific "off-switch" protein called REG3β that acts as a bouncer for these cells.

Here is the story of how REG3β works, broken down into simple steps:

1. The "Bad" Neutrophils Get a Badge

When neutrophils have been working hard for a few days, they get "aged" and "hyper-activated." Think of them as the cleanup crew members who have been working overtime, are covered in grime, and are starting to lose control.

The scientists found that these specific "aged" neutrophils develop a unique sticky badge on their surface. This badge is made of a specific sugar molecule called paucimannose. It's like a neon sign that says, "I am old, I am tired, and I am dangerous. Please remove me."

2. The Bouncer (REG3β) Arrives

While the heart is healing, the heart muscle cells themselves produce a protein called REG3β. You can think of REG3β as a specialized bouncer standing at the door of the heart's "clean zone."

This bouncer has a very specific job: it only looks for the neutrophils wearing that specific "aged" sugar badge. It ignores the fresh, young neutrophils that are still needed for the initial cleanup.

3. The Trap: The "Eat-Me" Signal

When REG3β spots a neutrophil with the badge, it grabs onto it. But it doesn't just push them away; it actually pulls them inside the cell.

• The Analogy: Imagine the bouncer grabbing the rowdy crew member and pulling them into a small, locked room inside the building (the cell's lysosome, which is like a trash compactor).
• Once inside this "trash compactor," the REG3β protein causes the walls of the room to crack (a process called lysosomal membrane permeabilization).
• This releases a flood of "digestive enzymes" (cathepsins) that essentially dissolve the neutrophil from the inside out. It's a rapid, self-destruct sequence.

4. The Clean-Up Crew (Macrophages) Takes Over

Once the neutrophil is dissolved, it leaves behind a signal (like a flag) that says, "I am dead, come get me."
This signals the next phase of the cleanup crew: macrophages. These are the "garbage collectors" of the immune system. They come in, eat the remains of the dissolved neutrophils, and turn the whole situation into a peaceful, healing environment.

Why Does This Matter?

The researchers tested this by removing the REG3β protein from mice.

• Without the bouncer: The "aged" neutrophils stayed in the heart way too long. They kept attacking healthy tissue, causing the heart to rupture (burst) and leading to heart failure.
• With the bouncer: The aged neutrophils were removed quickly. The heart healed properly, and the mice survived.

The Big Picture

This discovery is like finding a missing piece of the puzzle for how our bodies heal. We knew the immune system sends in the troops, but we didn't fully understand how it stops them from going too far.

REG3β is the "Stop" signal. It ensures that the aggressive cleanup crew is removed exactly when they are no longer needed, preventing them from destroying the very organ they were trying to save. This could lead to new treatments for heart attacks, where doctors might be able to boost this "bouncer" protein to ensure the heart heals cleanly without scarring or rupture.

In short: REG3β is the heart's way of saying, "Good job, cleanup crew. You're fired. Go home before you break the house."

Technical Summary

1. Problem Statement

Following a myocardial infarction (MI), the heart undergoes a complex wound-healing process involving the recruitment of immune cells, primarily neutrophils. While neutrophils are essential for clearing debris and initiating repair, their persistence and accumulation in the infarcted tissue are deleterious.

• The Gap: While mechanisms for neutrophil recruitment are well-characterized, the signals controlling their removal and resolution are poorly understood.
• The Consequence: Failure to clear neutrophils leads to maladaptive remodeling, cardiac rupture, and increased mortality. Previous studies suggested that Regenerating-islet derived protein 3 beta (REG3β) deficiency leads to cardiac rupture and prolonged neutrophil persistence, but the molecular mechanism by which REG3β regulates neutrophil clearance was unknown.

2. Methodology

The authors employed a multi-disciplinary approach combining in vivo mouse models, ex vivo cell culture, advanced imaging, and multi-omics technologies:

• Animal Models: Used Wild-Type (WT) and Reg3b-deficient (Reg3b-/-) mice subjected to permanent left anterior descending (LAD) coronary artery ligation to induce MI.
• Flow Cytometry & Imaging: Multicolor flow cytometry for neutrophil quantification and viability (7-AAD/Annexin V). Light-sheet fluorescence microscopy and immunofluorescence were used for 3D spatial mapping of neutrophils and REG3β.
• Omics Analysis:
  • Bulk RNA-seq: Compared gene expression in REG3β-negative (REG3βneg) vs. REG3β-positive (REG3βpos) neutrophils.
  • Single-cell RNA-seq (scRNA-seq): Analyzed neutrophil heterogeneity, aging trajectories, and cluster-specific gene expression.
  • Glycoproteomics & Glycomics: Mass spectrometry (LC-MS/MS) and N-glycan analysis to identify surface carbohydrate motifs.
• Functional Assays:
  • In vitro cytotoxicity assays using recombinant REG3β on peritoneal, bone marrow, and blood neutrophils.
  • Enzymatic treatments (PNGase F, EndoD, EndoH, EndoF2) to test glycan dependence.
  • Inhibitor studies (chlorpromazine for endocytosis; cathepsin inhibitors E-64d, Pepstatin A, CA-074) to dissect cell death pathways.
  • Electron microscopy (TEM/SEM) and expansion microscopy for ultrastructural analysis.
• Genetic Tools: Used Catchup mice (Ly6G-TdTomato reporter) to track neutrophil-macrophage interactions and efferocytosis.

3. Key Contributions & Mechanisms

The study establishes REG3β as a critical local immune checkpoint that specifically targets and eliminates aged, hyperactive neutrophils via a unique cell death pathway.

A. Specificity for Aged/Hyperactive Neutrophils

• REG3β does not bind to all neutrophils. It selectively binds to a subset characterized as aged and hyperactivated.
• These cells exhibit high expression of activation markers (CD54, TLR2, CD63, TREM2), ROS production (NOX2), and phagocytic activity.
• scRNA-seq revealed that REG3βpos neutrophils cluster with "aged" subsets (high Cxcr4, Siglecf, low Sell) and show upregulation of cell death pathways.

B. The Ligand: Paucimannosylated Proteins

• Discovery: REG3β binds to paucimannosidic N-glycans (specifically M2F: Mannose2-Fucose1-N-Acetylglucosamine2) on the neutrophil surface.
• Mechanism of Exposure: These glycans are attached to proteins normally stored in azurophilic granules (e.g., Myeloperoxidase/MPO, Neutrophil Elastase/ELANE, Neutrophilic Granule Protein/NGP).
• Translocation: Upon neutrophil activation and aging, these granule proteins translocate to the plasma membrane via degranulation, exposing the paucimannose motifs that serve as the "eat-me" signal for REG3β.

C. Cell Death Pathway: Lysosomal Membrane Permeabilization

• Uptake: Upon binding, REG3β is internalized via clathrin-mediated endocytosis and accumulates in lysosomes.
• Mechanism: REG3β induces Lysosomal Membrane Permeabilization (LMP). This is distinct from classical apoptosis (no Caspase-3/7 activation) and necroptosis.
• Execution: LMP causes the release of lysosomal cathepsins (Cathepsin B and D) into the cytosol, triggering cell death.
• Clearance: The dying neutrophils externalize phosphatidylserine (Annexin V+), facilitating their rapid clearance by macrophages via efferocytosis.

4. Key Results

1. Phenotype of Reg3b-/- Mice:

   • Neutrophil recruitment (Days 1–2) is normal, but clearance is impaired (Days 3–4).
   • Neutrophils persist and expand from the infarct zone (IZ) into the remote zone (RZ).
   • Increased levels of neutrophil-derived proteases (MPO, ELANE, MMP-9) in the remote zone.
   • High mortality due to cardiac rupture and impaired cardiac function (reduced ejection fraction).
   • Rescue: Depleting neutrophils in Reg3b-/- mice using anti-Ly6G antibodies restored survival and heart function, confirming neutrophil accumulation as the cause of pathology.

2. Direct Cytotoxicity:

   • Recombinant REG3β induces rapid death in activated/peritoneal neutrophils but not in quiescent bone marrow neutrophils.
   • This cytotoxicity is abolished by removing N-glycans (PNGase F) or specifically cleaving paucimannose (EndoD), or by competing with Mannose.

3. Molecular Pathway Validation:

   • REG3βpos neutrophils show lysosomal depletion (Lysotracker negative) and Galectin-1 puncta formation (indicating LMP).
   • Cathepsin release is confirmed via immunofluorescence (diffuse cytosolic Cathepsin B/D).
   • Inhibiting cathepsins or blocking clathrin-mediated endocytosis (chlorpromazine) prevents REG3β-induced death.

4. Efferocytosis:

   • In Reg3b-/- mice, macrophage-neutrophil hybrids (indicative of efferocytosis) are significantly reduced.
   • Macrophages in Reg3b-/- hearts remain in a pro-inflammatory state (MHC-IIhi/Ly6Clo) rather than transitioning to a reparative phenotype.

5. Significance

• Novel Mechanism of Resolution: The paper identifies a previously unknown, tissue-instructed mechanism for neutrophil resolution that is distinct from apoptosis, necroptosis, or NETosis. It defines a "lysoptosis"-like pathway driven by lysosomal cathepsin release.
• Glycan-Based Targeting: It highlights paucimannosylation as a critical, age-dependent "tag" on neutrophils that allows the immune system to distinguish between functional and senescent/dangerous cells.
• Therapeutic Potential:
  • Understanding this pathway offers new targets for treating MI and preventing cardiac rupture.
  • It suggests that manipulating neutrophil glycosylation or REG3β signaling could help resolve inflammation without broadly suppressing the immune system.
  • The mechanism parallels the function of RegIIIγ in the gut (separating microbiota from epithelium), suggesting an evolutionary conserved role for REG proteins in creating spatial barriers against inflammatory cells.

In summary, this work redefines the role of cardiomyocyte-derived REG3β as a critical regulator of the innate immune response, acting as a "molecular switch" that selectively eliminates aged neutrophils to ensure effective cardiac repair and prevent maladaptive remodeling.