Evaluation of the Role of Macrophage Migratory Inhibitory Factor (MIF) in mediating Stem Cell Analgesia in a Model of Orofacial Pain

评估巨噬细胞迁移抑制因子（MIF）在口面部疼痛模型中介导干细胞镇痛的作用

• 批准号: 10585412
• 负责人: Nikita Ruparel
• 金额: $ 46.38万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2027-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10585412
• 关键词: AMD3100; Absence of pain sensation; Acetaminophen; Afferent Neurons; Animals; Antibodies; Apical; Attenuated; Behavior; Behavioral Assay; CRISPR/Cas technology; CXCR4 Receptors; CXCR4 gene; Capsaicin; Cell Line; Coculture Techniques; Data; Degenerative polyarthritis; Disease Progression; Effectiveness; Electrophysiology (science); Endodontics; Evaluation; Female; Fostering; Goals; Home; Homing; Human; Image; In Vitro; Infection; Injections; Knock-out; Knowledge; Ligands; Macrophage; Mechanics; Mediating; Medical; Mesenchymal Stem Cells; Migraine; Modeling; Mus; Neurons; Nociception; Non-Steroidal Anti-Inflammatory Agents; Opioid; Orofacial Pain; Pain; Pathway interactions; Patients; Periapical Granuloma; Periapical Periodontitis; Peripheral; Persistent pain; Pertussis Toxin; Proteins; Regulation; Research; Rodent Model; Role; Route; Sex Differences; Signal Pathway; Signal Transduction; Site; System; Testing; Therapeutic; Tissues; Tooth structure; Toothache; Trigeminal System; Up-Regulation; antagonist; antinociception; attenuation; comparison control; conditioned place preference; experience; human stem cells; in vitro activity; in vivo; knock-down; male; mechanical allodynia; microbial; mouse model; non-opioid analgesic; novel; orofacial; pain behavior; painful neuropathy; peripheral pain; programs; public health relevance; receptor; secondary analysis; side effect; small hairpin RNA; small molecule; stem cells; tongue papilla; voltage

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 Gi/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 Gi versus G signaling, type of VGCC inhibited, regulation of channels/receptors commonly known to mediate mechanical and spontaneous behaviors and finally study alternative Gi/o-mediated pathways commonly known post-MIF activation. These studies have high medical significance as they define a novel ligand-receptor system for treating infection-induced pain, possibly leading to new non-opioid analgesics. Importantly, the combined use of human hSCAP in rodent models foster mechanistic research and increases translational significance. RELEVANCE: These studies aim at studying new class of non-opioid analgesics for dental pain.

摘要/项目摘要 干细胞诱导镇痛治疗牙痛的有效性和机制尚不清楚。 我们证明，静脉注射人类根尖乳头干细胞 (hSCAP) 可逆转根尖周炎 （牙齿感染；AP）引起的机械异常性疼痛此外，hSCAP 引发根尖肉芽肿。 （牙齿感染的周围部位）减弱辣椒素 (CAP) 引起的细胞内 Ca2+ 积累 ([Ca2+]I) 来自三叉神经 (TG) 神经元，这种作用被抗人巨噬细胞迁移抑制因子抑制 (MIF)-抗体(Ab) 因此，在这里，我们将检验根尖周炎诱发的中心假设。 hSCAP 衍生的 MIF 释放直接抑制 TG 神经元活性，从而逆转伤害感受行为。 我们提出以下目标来检验男性和女性组织中的中心假设： 具体目标 1 将检验 hSCAP 衍生的 MIF 在体外抑制 TG 神经元活动的假设 我们将使用抗人 MIF-Ab 来评估 hSCAP 体内释放 MIF 的效果。 机械性异常性疼痛以及使用条件性位置偏好（CPP）的自发性伤害感受 使用 CRISPR-Cas9，我们将生成具有 MIF 敲除的 hSCAP 细胞系，该细胞系将用于测试。 假设体外和体内 hSCAP 衍生的 MIF 抑制 TG 神经元和 AP 诱导的疼痛 行为。 具体目标 2 将检验神经元 CXCR4 和 CD74 受体介导 MIF 诱导的假设 通过 CXCR4 和 CD74 受体抑制 TG 神经元活性。 (21-23) 在小鼠 TG 神经元上表达（图 11）。我们将使用 CXCR4 来评估其作用。 AMD3100，一种小分子 CXCR4 拮抗剂，采用体外 Ca2+ 成像和体内行为测定。 接下来，我们有条件地从TG感觉神经元中删除CXCR4来评估CXCR4受体的功能 接下来，将通过神经节内途径向小鼠注射针对 CD74 的 AVV-shRNA。 评估神经元 CD74 受体敲低对体内伤害性行为的影响以及 体外 Ca2+ 成像。 具体目标 3 将确定 MIF 激活的信号通路，从而抑制 TG 通过 CD74/CXCR4 受体的神经元活动通过 Gi/o 蛋白发生。 用百日咳毒素（PTx）预处理完全逆转hSCAP诱导的[Ca2+]I减弱（图15）； 2) MIF 抑制 G 激活下游的电压门控 Ca2+ 电流（VGCaC；图 16）。 电生理学方法，我们将通过确定 Gi 与 G 信号传导来确定 MIF 信号传导， VGCC 类型受到抑​​制，通道/受体的调节通常已知介导机械和 自发行为，并最终研究 MIF 后众所周知的替代 Gi/o 介导的途径 激活。 这些研究具有很高的医学意义，因为它们定义了一种新的配体-受体系统来治疗 感染引起的疼痛，可能导致新的非阿片类镇痛药的联合使用。 啮齿动物模型中的人类 hSCAP 促进了机制研究并增加了转化意义。 相关性：这些研究旨在研究新型非阿片类镇痛药治疗牙痛。