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齿疼痛的有效性和机制尚不清楚。 我们证明了i.v.对顶乳头（HSCAP）反向根尖牙周炎的人类干细胞注射 （牙齿感染； AP）诱导的机械性异常。此外，HSCAP启动至根尖的肉芽肿 （牙齿感染的外围部位）衰减辣椒素（CAP）诱发的细胞内Ca2+积累（[CA2+] I） 抗人类巨噬细胞迁移抑制因子抑制三叉神经元（TG）神经元（TG）神经元 （MIF） - 抗体（AB）。因此，在这里，我们将测试中心牙周炎诱导的中心假设 HSCAP衍生的MIF释放直接抑制TG神经元活性的MIF释放可以逆转伤害性行为。 我们提出以下旨在检验男性和女性时间的中心假设： 特定目标1将检验以下假设：HSCAP衍生的MIF在体外抑制TG神经元活性 和体内。我们将使用抗人Mif-AB来评估HSCAP对MIF释放的效果 使用条件放置偏好（CPP）的机械异常性疾病以及赞助的nocetion。下一个， 使用CRISPR-CAS9，我们将与MIF敲除生成HSCAP单元线。该单元线将用于测试 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。 特定目标2将检验以下假设：神经元CXCR4和CD74受体介导MIF诱导的假设 在体外和体内抑制TG神经元活性。 MIF信号通过CXCR4和CD74受体 （21-23）在小鼠TG神经元上表达（图11）。我们将使用CXCR4的角色使用 AMD3100，一种使用体外CA2+成像和体内行为测定的小分子CXCR4拮抗剂。 接下来，我们从TG感觉神经元中有条件删除CXCR4来评估CXCR4受体的功能 体外和体内。下一 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。 特定的目标3将确定由MIF激活的信号传导途径，从而抑制TG 神经元活性。通过CD74/CXCR4受体通过GI/O蛋白进行信号传导。我们证明了1） 用百日咳毒素（PTX）预处理完全逆转了[Ca2+] i的HSCAP诱导的衰减（图15）； 2）MIF抑制G激活下游的电压门控Ca2+电流（VGCAC;图16）。使用 电生理方法，我们将通过确定GI与G信号传导来确定MIF信号传导， 抑制VGCC的类型，调节通道/受体通常已知会介导机械和 赞助行为，最后研究替代性GI/O介导的途径，通常已知的MIF 激活。 这些研究具有很高的医学意义，因为它们定义了一种用于治疗的新型配体受体系统 感染引起的疼痛，可能导致新的非阿片类镇痛药。重要的是，联合使用的 啮齿动物模型中的人类HSCAP促进了机械研究并增加了翻译意义。 相关性：这些研究旨在研究新的非阿片类镇痛药以解决牙齿疼痛。