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.