NEUROIMMUNE CROSSTALK AND PHARMACOLOGICAL MODULATION OF SEGMENT REGENERATION

Scarless regeneration in annelids is critically regulated by the CNS and by free-floating coelomocytes, analogues of mammalian leukocytes that migrate and accumulate at the amputation site. However, the exact cellular and molecular mechanisms underlying this process remain largely unexplored. In vertebrates, immune cells participate in neuro-immune crosstalk by synthesizing, releasing, and responding to neurotransmitters. Here, we investigated whether annelid coelomocytes contain classical neurotransmitters and whether neurotransmitter signaling contributes to segment regeneration.
Using mass spectrometry and histochemistry, we detected GABA, Glut, DA, and 5-HT in coelomocytes of the earthworm model, Eisenia andrei. Transcriptomic analysis of coelomocytes revealed both evolutionarily conserved and lineage-specific features: transcripts encoding GAD, GABA-B receptor, 5-HT transporter, DA transporter, 5-HT receptors, and DA receptors were identified, whereas transcripts for TRH, TPH, AADC, GABA-A receptor, and GABA transporters were absent. The presence of GABAergic components suggests an ancient role for inhibitory signaling in immune regulation, while the lack of monoamine synthesis points to later evolutionary innovations in vertebrates. Quantitative mass spectrometry of coelomocytes collected at 0, 3, 7, and 14 days post-amputation revealed significant increases in Glut, 5-HT, and DA, accompanied by decreased GABA levels at days 3 and 7, implicating neurotransmitters in early regeneration. Pharmacological manipulation using different concentrations of haloperidol (D₁/D₂ antagonist), ondansetron (5-HT₃ antagonist), GABA, and their mixtures significantly impaired segment regeneration and tissue organization.
Together, these findings demonstrate that coelomocyte-mediated neurotransmitter homeostasis is essential for proper regeneration and support the idea that neuroimmune communication is an evolutionarily conserved mechanism with relevance to regenerative medicine.