Abstract/Project Summary The effectiveness and mechanisms of stem cell-induced analgesia in treating dental pain is unknown. We demonstrate that i.v. injections of human Stem Cells of Apical Papilla (hSCAP) reverse apical periodontitis (infection of a tooth; AP)-induced mechanical allodynia. Moreover, hSCAP primed to periapical granulomas (peripheral sites of tooth infection) attenuate capsaicin (CAP)-evoked intracellular Ca2+ accumulation ([Ca2+]I) from trigeminal (TG) neurons and this effect is inhibited by anti-human macrophage migratory inhibitory factor (MIF)-antibody (Ab). Therefore, here, we will test the central hypothesis that apical periodontitis-induced nociceptive behaviors is reversed by hSCAP-derived release of MIF that directly inhibits TG neuronal activity. We propose the following aims to test the central hypothesis in male and female tissues: Specific Aim 1 will test the hypothesis that hSCAP-derived MIF inhibits TG neuronal activities in vitro and in vivo. We will use anti-human MIF-Ab to evaluate the effect of in vivo release of MIF by hSCAP on mechanical allodynia as well as spontaneous nociception using conditioned placed preference (CPP). Next, using CRISPR-Cas9, we will generate a hSCAP cell line with MIF knockout. This cell line will be used to test the hypothesis that in vitro and in vivo hSCAP-derived MIF inhibits TG neurons and AP-induced pain behaviors. Specific Aim 2 will test the hypothesis that neuronal CXCR4 and CD74 receptors mediate MIF-induced inhibition of TG neuronal activity in vitro and in vivo. MIF signals via the CXCR4 and CD74 receptors (21-23) that are expressed on mouse TG neurons (Fig. 11). We will evaluate the role of CXCR4 using AMD3100, a small molecule CXCR4 antagonist using in vitro Ca2+-imaging and in vivo behavioral assays. Next, we conditionally delete CXCR4 from TG sensory neurons to evaluate the function of CXCR4 receptors in vitro and in vivo. Next, will inject mice with AVV-shRNA against CD74 via an intraganglionic route to assess the effect of neuronal CD74 receptor knockdown on in vivo nociceptive behaviors and as well as in vitro Ca2+-imaging. Specific Aim 3 will determine the signaling pathway/s activated by MIF that contribute inhibition of TG neuronal activity. Signaling via CD74/CXCR4 receptor occurs via Gi/o proteins. We demonstrate that 1) pretreatment with pertussis toxin (PTx) completely reverses hSCAP-induced attenuation of [Ca2+]I (Fig. 15); and 2) MIF inhibits voltage gated Ca2+ currents (VGCaC; Fig 16) that is downstream of G activation. Using electrophysiological approaches, we will determine MIF signaling by determining Gi versus G signaling, type of VGCC inhibited, regulation of channels/receptors commonly known to mediate mechanical and spontaneous behaviors and finally study alternative Gi/o-mediated pathways commonly known post-MIF activation. These studies have high medical significance as they define a novel ligand-receptor system for treatin...