Dissecting bi-directional neuroimmune crosstalk in an in vitro model relevant to apical periodontitis

Inflammatory response associated with Apical Periodontitis (AP) is shaped by neuroimmune signaling mediated by neuropeptides and cytokines that regulate interactions among sensory neurons, immune cells, and stromal cells in the periapical microenvironment. To investigate these mechanisms, this study employed an in vitro multicellular culture system modeling key neuroimmune features relevant to AP rather than anatomical lesion formation. Neuron-like cells (NLCs) were generated from periodontal ligament cells (PDLCs) using dual-SMAD inhibition, and bi-directional paracrine and juxtacrine signaling between NLCs, PDLCs, and THP-1-derived macrophages (MQs) was examined under lipopolysaccharide (LPS)-induced inflammatory conditions. Neuropeptides released by NLCs, Substance P (SP) and Calcitonin Gene-Related Peptide (CGRP), enhanced LPS-driven inflammatory responses in PDLC-MQ paracrine systems, characterized by increased macrophage expression of CD68 and CD80 and elevated secretion of IL-1β and TGF-β1. In contrast, during PDLC-MQ juxtacrine interactions, NLC-derived neuropeptides supported a regulatory profile, with reduced expression of CD68, CD80, and STAT1 and decreased production of TNF-α and IL-1β. Reciprocally, conditioned media from LPS-treated PDLC-MQ co-cultures modulated NLC signaling by increasing TRPV1 expression, while IL-10 present in the conditioned media suppressed TRPA1 expression, resulting in an overall CGRP-dominant and SP-suppressed neuropeptide profile. These findings identify a dynamic, bi-directional neuroimmune circuitry among neuron-like cells, macrophages, and periodontal ligament cells that differentially regulates inflammatory and regulatory signaling in a system relevant to Apical Periodontitis, providing mechanistic insight into neuroimmune coordination during periapical inflammation.