Macrophages orchestrate tail regeneration in lizards Scincella tsinlingensis through spatiotemporal control of immunity, oxidative stress, and myogenesis.

BACKGROUND

Macrophages facilitate blastema and wound epithelium formation by clearing debris and remodeling the immune and extracellular matrix. The M1-to-M2 transition is well documented in amphibians, but its occurrence and timing in diverse lizards remain unclear. This study examined macrophage function during tail regeneration in Scincella tsinlingensis.

METHODS

A tail amputation model was established in S. tsinlingensis, and macrophages were depleted using clodronate liposomes (L-Clodronate). Transcriptomic analysis was performed during the wound epithelialization phase, followed by qRT-PCR and immunohistochemistry to assess immune responses, chemokine signaling, oxidative balance, myogenic regulation, and matrix metalloproteinase (MMP) activity. MMP function was further examined using GM6001 inhibition.

RESULTS

Macrophage depletion severely impaired tail regeneration. By 21 days post-amputation (dpa), control regenerates reached 6.1 ± 2.2 mm, whereas depleted tails grew only 0.5 ± 0.1 mm (P < 0.0001). Depletion delayed wound epithelialization and prevented blastema formation. Macrophage recruitment peaked later in depleted animals (86.8 ± 13.8 at 7 dpa) than in controls (148.4 ± 7.1 at 3 dpa). CTSK positive osteoclasts were reduced by ∼47% (53 ± 5.6 vs. 100.3 ± 18.1, P < 0.05). Transcriptomic analysis identified 341 DEGs, with downregulated genes enriched in type I interferon, desmosome, and TLR/NOD pathways. TLR4 was a hub gene. qPCR and immunohistochemistry confirmed disrupted M1-to-M2 transition, suppressed cytokines (TNFα, IL-6, IL-10, TGFβ1), dysregulated CXCL12/CXCR4, oxidative imbalance (gp91phox), failed myogenesis (decreased MYOD1, persistent PAX7), and reduced Ctsk, MMP2, MMP9. GM6001 matrix metalloproteinase inhibition delayed regeneration (10.2 ± 1.2 vs. 1.5 ± 0.1 mm at 28 dpa, P < 0.0001) with compensatory upregulation of MMP2/3/9.

CONCLUSION

Macrophages serve as central timing regulators of tail regeneration in S. tsinlingensis. Their depletion disrupts the early inflammatory switch, impairs blastema formation, and severely limits outgrowth within the 21-day observation period, whereas MMP inhibition only slows regeneration. Macrophages coordinate the immune, myogenic, and matrix remodeling programs essential for successful tissue restoration, highlighting their non-redundant role in reptile regeneration.