SARM1 is required for macrophage immunophenotype switching that is essential for nerve repair

This study reveals that SARM1 is essential in both macrophages and neurons for efficient peripheral nerve repair by enabling macrophage immunophenotype switching, myelin debris clearance, and the regulation of inflammatory pathways necessary for axon regeneration.

The Gist

Imagine your body's nervous system as a vast, intricate network of electrical wires (axons) connecting your brain to your muscles. When one of these wires gets cut or crushed (like in a car accident), the part of the wire far from the brain dies off. This is called Wallerian degeneration.

In the past, scientists thought this cleanup process was just a mechanical event: the wire breaks, it rots, and the body cleans it up. But this new research reveals that the cleanup crew is actually a highly organized team of macrophages (a type of immune cell), and they have a specific "manager" they need to do their job right. That manager is a protein called SARM1.

Here is the story of what happens when that manager is missing, explained through simple analogies.

1. The Cleanup Crew and the "Switch"

Think of macrophages as the janitors of your body. When a nerve is injured, these janitors rush to the scene. To do their job effectively, they need to be able to switch uniforms.

• The "Attack" Uniform (M1): They need to be aggressive to clear away toxic debris and signal that there is an emergency.
• The "Repair" Uniform (M2): Once the mess is cleared, they need to switch to a gentle, healing mode to help the nerve grow back.

The Problem: In mice without the SARM1 protein, the janitors get stuck in a state of confusion. They can't flip the switch properly.

• They seem to be stuck in "healing mode" (M2) even when they need to be "attacking" (M1).
• It's like a firefighter who shows up to a burning building but immediately starts trying to water the plants in the garden instead of putting out the fire. They are well-meaning, but they are in the wrong mode for the job.

2. The "Foamy" Janitors

One of the most critical jobs of these immune cells is to eat up the broken pieces of the nerve's insulation (myelin).

• Normal Mice: The janitors eat the debris, digest it, and throw it away. The path is clear for the new wire to grow.
• Mice without SARM1: The janitors can eat the debris, but they get stuffed. They become "foamy" cells—full of undigested garbage that they can't process.
• The Analogy: Imagine a garbage truck that picks up trash but has a broken compactor. It fills up with trash, but it can't crush it down or take it to the dump. The truck sits there, full and useless, blocking the road. Because the road (the nerve) is still blocked by this undigested trash, the new nerve wire cannot grow through.

3. The Confused Signal

The researchers found that SARM1 acts like a traffic light controller for these cells.

• When a nerve is cut, it sends out chemical signals (like a siren).
• In a normal body, SARM1 helps the macrophages understand the siren and change their behavior accordingly.
• Without SARM1, the macrophages hear the siren but don't know how to react. They stay in a "chill" state, failing to ramp up the inflammation needed to start the repair process.

4. The Big Surprise: Who is the Real Boss?

For a long time, scientists thought SARM1 was only important inside the nerve cell itself (the wire). They thought if the wire broke, the wire's own SARM1 would trigger the cleanup.

The Twist: The researchers created two special groups of mice:

1. Mice missing SARM1 only in their nerves: The nerves broke, but the janitors (macrophages) were normal. Result: The cleanup happened, and the nerve healed almost normally.
2. Mice missing SARM1 only in their janitors: The nerves were fine, but the janitors were confused. Result: The cleanup failed, the trash piled up, and the nerve couldn't heal.

The Lesson: The nerve doesn't need its own SARM1 to heal; it needs the janitors to have SARM1. The nerve is just the victim; the immune system is the one that needs the right manager to fix the problem.

Why Does This Matter?

This discovery changes how we might treat nerve injuries in the future.

• Old Idea: We need to fix the nerve cell itself to make it grow.
• New Idea: We need to help the immune system's "janitors" switch their uniforms correctly. If we can teach these cells how to stop being "foamy" and start clearing the trash efficiently, we might be able to help people with nerve damage (like from car accidents or diabetes) recover much faster.

In short: SARM1 is the essential manager that tells the body's cleanup crew when to fight, when to heal, and how to actually throw away the trash. Without it, the cleanup crew gets stuck, the road stays blocked, and the nerve can't grow back.

Technical Summary

1. Problem Statement

Peripheral nerve injury (PNI) triggers Wallerian degeneration, a process where distal axons degenerate and are cleared by macrophages (M$\phi$) to facilitate regeneration. The protein SARM1 is a known executor of Wallerian degeneration in axons via its NADase activity; global knockout of Sarm1 in mice delays axon degeneration by weeks. However, Sarm1 knockout mice also exhibit delayed nerve regeneration and functional recovery. While SARM1's role in axons is established, its role in non-neuronal cells, specifically macrophages, during the injury response remains unclear. It is unknown whether the impaired regeneration in Sarm1 global knockouts is solely due to delayed axonal degeneration (indirect effect) or if SARM1 plays a cell-autonomous role in macrophage function, such as immunophenotype switching (M1/M2 polarization), phagocytosis, and debris clearance.

2. Methodology

The study employed a multi-faceted approach combining in vitro cell culture, in vivo injury models, and genetic manipulation:

• Animal Models:
  • Global Knockout: Sarm1^-/- mice vs. Wild Type (WT).
  • Conditional Knockouts (cKO):
    • Macrophage-specific (Mac-cKO): Sarm1^fx/fx crossed with LysM-Cre.
    • Neuron-specific (Neu-cKO): Sarm1^fx/fx crossed with Syn1-Cre.
  • Reporter Lines: ROSA26-tdTomato mice for cell tracking.
• Cell Culture & Assays:
  • Primary Macrophages: Isolated from spleens of WT and Sarm1^-/- mice.
  • Polarization: M$\phi$s were treated with m-CSF (homeostatic), IL-4 (M2/anti-inflammatory), or LPS/IFN$\gamma$ (M1/pro-inflammatory).
  • Functional Assays:
    • Morphology Analysis: Quantification of cell shape (round, elongated, stellate).
    • Wound Healing/Invasion: 2D scratch assays and 3D Matrigel invasion assays.
    • Phagocytosis/Clearance: Myelin uptake assays using OilRedO staining to distinguish between uptake and clearance.
    • Co-culture: DRG neurons co-cultured with polarized M$\phi$s to measure neurite outgrowth.
    • Molecular Analysis: qRT-PCR, Western Blotting (iNOS, Arginase-1, pSTAT6, p65), and NAD/NADH Glo assays.
• In Vivo Models:
  • Sciatic Nerve Crush (SNC): Performed on WT, Sarm1^-/-, Mac-cKO, and Neu-cKO mice.
  • Cell Injection: Polarized WT or Sarm1^-/- M$\phi$s were injected into the distal stump of injured sciatic nerves in Sarm1^-/- or WT mice to test rescue effects.
  • Behavioral Analysis: BlackBox Bio system used to assess walking distance, rearing, weight bearing, and toe spread ratio over 8 weeks.
  • Histology: Immunostaining for SCG10 (regenerating axons), MBP (myelin), ATP8A2 (eat-me signal), F4/80 (macrophages), and Degenotag (degenerating axons).

3. Key Contributions

• Discovery of Macrophage-Specific SARM1 Function: The study identifies SARM1 as a critical regulator of macrophage immunophenotype switching, distinct from its known role in axonal NAD depletion.
• Mechanism of Regeneration Failure: It demonstrates that the failure of nerve regeneration in Sarm1 global knockouts is not solely due to delayed axonal degeneration but is significantly driven by macrophage dysfunction.
• Cell-Autonomous vs. Non-Cell-Autonomous Roles: Through conditional knockouts, the authors dissect the specific contributions of neuronal vs. macrophage SARM1, revealing that macrophage SARM1 is essential for myelin clearance and functional recovery, whereas neuronal SARM1 is not strictly required for the initial inflammatory response if macrophages are functional.

4. Key Results

A. Macrophage Phenotype and Morphology

• Baseline State: Sarm1^-/- macrophages exhibit a skewed "M2-like" (anti-inflammatory/wound-healing) phenotype even at baseline, characterized by increased stellate and elongated morphologies and higher expression of CD163.
• Impaired Switching: Upon stimulation with pro-inflammatory cues (LPS/IFN$\gamma$), Sarm1^-/- macrophages fail to fully transition to an "M1" (pro-inflammatory) state. They display mixed phenotypes, failing to upregulate M1 markers (iNOS) and downregulate M2 markers (Arginase-1, Dectin-1) effectively.
• Gene Expression: Sarm1^-/- macrophages show dysregulated gene expression, with simultaneous upregulation of opposing pathways (e.g., both Nos2 and Arg1), indicating a loss of transcriptional precision.

B. Functional Deficits

• Migration and Invasion: While Sarm1^-/- macrophages migrate well in 2D, they fail to invade 3D matrices when polarized with LPS or IFN$\gamma$, suggesting an inability to navigate the tissue environment during a pro-inflammatory response.
• Myelin Clearance: Sarm1^-/- macrophages can phagocytose myelin debris (uptake is intact or enhanced in IL-4 conditions) but fail to clear it. They accumulate lipid droplets (OilRedO positive), resembling "foamy" macrophages, indicating a defect in intracellular processing and degradation.
• Neurite Outgrowth: In co-culture, Sarm1^-/- macrophages enhance neurite outgrowth regardless of polarization (unlike WT, where LPS suppresses growth). However, in vivo injection of Sarm1^-/- macrophages into injured nerves failed to rescue regeneration in Sarm1^-/- mice and actually inhibited regeneration in WT mice. This suggests that the inability to switch phenotypes creates a "hostile" microenvironment that hinders axon growth.

C. Conditional Knockout Analysis

• Macrophage-specific KO (Mac-cKO): Phenocopied the global knockout regarding myelin clearance. At 7 days post-injury, Mac-cKO nerves showed intact myelin (MBP) and a lack of myelin breakdown signals (ATP8A2) within macrophages, confirming that macrophage SARM1 is required for debris clearance.
• Neuron-specific KO (Neu-cKO): Surprisingly, Neu-cKO mice showed robust myelin breakdown and macrophage activation similar to WT, despite delayed axonal degeneration (Degenotag signal). This indicates that neuronal SARM1 loss alone does not block the inflammatory cascade if macrophages are functional.
• Functional Recovery: Both Mac-cKO and Neu-cKO mice showed functional recovery (walking distance, weight bearing) similar to WT, unlike global Sarm1^-/- mice which showed severe deficits. This suggests that having SARM1 in either cell type is sufficient for functional recovery, but the combination of defects in global knockouts is catastrophic.

5. Significance

• Redefining SARM1's Role: This study expands the understanding of SARM1 beyond a neuronal NADase. It establishes SARM1 as a crucial regulator of macrophage plasticity, specifically the ability to switch between pro-inflammatory and anti-inflammatory states.
• Therapeutic Implications: The findings suggest that therapeutic strategies for nerve repair must consider macrophage metabolism and phenotype switching. Simply delaying axonal degeneration (by inhibiting SARM1 in neurons) may not be sufficient if macrophages cannot clear debris or switch phenotypes effectively.
• Mechanism of "Foamy" Macrophages: The study provides a mechanistic link between SARM1 loss and the "foamy" macrophage phenotype observed in chronic injury, where cells ingest but cannot process lipid debris, leading to a hostile environment for regeneration.
• Cell-Autonomous vs. Non-Cell-Autonomous: The work clarifies that while SARM1 in neurons triggers degeneration, SARM1 in macrophages is essential for the subsequent inflammatory and clearance phases. The failure of regeneration in global knockouts is a compound effect of delayed degeneration and macrophage dysfunction.

In conclusion, SARM1 is essential for macrophages to adopt the correct immunophenotype in response to injury cues. Without SARM1, macrophages remain stuck in a dysregulated, mixed state that impairs myelin clearance and creates a non-permissive environment for axon regeneration.