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 干细胞诱导的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）可以逆转根尖牙周炎 （牙齿感染; AP）引起的机械性异常性疼痛。此外，hSCAP引发根尖周肉芽肿， （牙齿感染的外周部位）减弱辣椒素（CAP）诱导的细胞内Ca 2+积聚（[Ca 2 +]I） 从三叉神经（TG）神经元，这种作用是抑制抗人巨噬细胞迁移抑制因子 （MIF）-antibody（Ab）.因此，在这里，我们将测试的核心假设，根尖牙周炎诱导 伤害感受行为被hSCAP衍生的直接抑制TG神经元活性的MIF释放逆转。 我们提出了以下目标，以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神经元活性的假设 和体内。我们将使用抗人MIF-Ab来评估通过hSCAP在体内释放MIF对 机械性异常性疼痛以及使用条件性位置偏爱（CPP）的自发性伤害感受。接下来， 使用CRISPR-Cas9，我们将产生具有MIF敲除的hSCAP细胞系。该细胞系将用于测试 体外和体内hSCAP衍生的MIF抑制TG神经元和AP诱导的疼痛的假设 行为。 具体目标2将检验神经元CXCR 4和CD 74受体介导MIF诱导的细胞凋亡的假设。 在体外和体内抑制TG神经元活性。通过CXCR 4和CD 74受体的MIF信号 （21-23），其在小鼠TG神经元上表达（图11）。我们将评估CXCR 4的作用， AMD 3100，一种小分子CXCR 4拮抗剂，使用体外Ca 2+成像和体内行为测定。 接下来，我们从TG感觉神经元中有条件地删除CXCR 4以评估CXCR 4受体的功能 在体外和体内。接下来，将通过神经节内途径向小鼠注射针对CD 74的AVV-shRNA， 评估神经元CD 74受体敲低对体内伤害性行为的影响，以及 体外钙离子显像。 特异性目的3将确定由MIF激活的有助于抑制TG的信号通路 神经元活动经由CD 74/CXCR 4受体的信号传导经由Gl/o蛋白发生。我们证明：（1） 用百日咳毒素（PTx）预处理完全逆转hSCAP诱导的[Ca 2 +] i衰减（图15）; 和2）MIF抑制G β激活下游的电压门控Ca 2+电流（VGCaC;图16）。使用 电生理学方法，我们将通过确定G β i与G β i信号传导来确定MIF信号传导， VGCC类型的抑制，通常已知的通道/受体的调节介导机械和 自发行为，并最终研究MIF后常见的G i/o介导的替代途径 activation. 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，用于治疗 感染引起的疼痛，可能导致新的非阿片类镇痛药。重要的是， 啮齿类动物模型中的人hSCAP促进了机制研究并增加了翻译意义。 相关性：这些研究旨在研究用于牙痛的新型非阿片类镇痛药。