Endoplasmic Reticulum Stress Responses in Pain

疼痛中的内质网应激反应

• 批准号: 10094265
• 负责人: Mario Danilo Boada
• 金额: $ 39.77万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-15 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10094265
• 关键词: Acetic Acids; Acute Pain; Address; Affect; Behavior; Behavioral; Bioinformatics; Biological Assay; Biology; Cells; Cluster Analysis; Communities; Development; Dinoprostone; Electrophysiology (science); Endoplasmic Reticulum; Exhibits; Foundations; Gene Expression; Genes; Genetic Transcription; Human; Hypersensitivity; Immune; Immunology; In Situ; Incidence; Individual; Infiltration; Inflammation; Inflammatory; Injury; Knockout Mice; Label; Leukocytes; Ligation; Medical; Modeling; Molecular; Mus; Nanotechnology; Nerve Degeneration; Neuroimmune; Neurons; Nociceptors; Operative Surgical Procedures; Outcome; Pain; Patients; Pattern; Pattern recognition receptor; Peripheral; Peripheral nerve injury; Pharmacology; Phenotype; Phosphotransferases; Positioning Attribute; Preventive; Process; Production; Prostaglandins; Protein Biosynthesis; Proteins; Quality of life; Recovery; Recovery of Function; Reflex action; Regulation; Resolution; Role; Signal Transduction; Social Impacts; Surgical incisions; Technology; Testing; Therapeutic; Tissues; Toll-like receptors; Tumor-infiltrating immune cells; XBP1 gene; arm; base; biological adaptation to stress; chronic inflammatory disease; chronic neuropathic pain; chronic pain; clinically relevant; conditional knockout; cytokine; economic impact; effective therapy; endoplasmic reticulum stress; genetic signature; genome-wide; immune activation; improved; in vivo; inflammatory pain; inhibitor/antagonist; injured; innovation; loss of function; misfolded protein; mouse model; nerve injury; neurobiotin; novel; painful neuropathy; partial recovery; prevent; programs; recruit; response; sciatic nerve; single-cell RNA sequencing; small molecule; tool; transcriptomics

Summary Peripheral nerve injury activates pattern recognition receptors (i.e. Toll-like receptors) in immune cells, thus triggering and maintaining inflammation, and ultimately determining the perpetuation of pain. Immune cell activation demands high levels of protein synthesis, folding and secretion, which are regulated by the endoplasmic reticulum (ER). An excessive demand in protein handling can evoke ER stress (accumulation of misfolded proteins) and consequently trigger robust activation of the unfolded protein response (UPR). IRE1α- XBP1 is the most evolutionarily conserved arm of the UPR and can be directly activated via Toll-like receptor engagement to promote the expression of pro-inflammatory factors. We have unveiled that conditional knockout (cKO) mice devoid of IRE1α/XBP1 in immune cells (Ern1/Xbp1f/f-Vav1cre), display decreased PGE2 production in vivo, reduced nociceptor responsiveness, and faster resolution of non-reflexive pain-related behaviors following paw incision. Similarly, these cKO mice exhibit improved recovery after partial sciatic nerve ligation (PSNL). Through unbiased genome-wide transcriptomic analyses we found that IRE1α-XBP1 signaling in leukocytes is critically required for the induction of prostanoids, cytokines and other novel factors such as Nupr1 (associated with chronic inflammatory diseases in humans). Of note, IRE1α-XBP1 overactivation has been correlated with painful or inflammatory conditions in humans. Therefore, we hypothesize that IRE1α-XBP1 signaling in leukocytes governs peripheral neuro-immune interactions and the development of chronic pain. Using cutting-edge experimental approaches, we will accomplish the following specific aims: 1) Determine how IRE1α-XBP1 signaling dictates the dynamics of immune cell infiltration and molecular changes that drive behavioral non-reflexive hypersensitivity following peripheral nerve injury. We postulate that the IRE1α-XBP1 arm operates as a key modulator of pro-algesic factors in immune cells, and that Nupr1 is a novel XBP1- dependent factor (using ChIP PCR) implicated in PSNL. 2) Establish how IRE1α-XBP1 activation governs individualized immune cellular reprogramming and how this drives the cross-talk with nociceptor afferents following peripheral nerve injury. Our hypothesis is that the reduced hypersensitivity observed in our cKO mice following PSNL is determined by discrete gene signatures in specific injury-infiltrating leukocyte subsets (using single cell RNA sequencing), and by the acquisition of a less responsive phenotype in nociceptors (via in vivo intracellular DRG recordings). 3) Define the therapeutic potential of inhibiting IRE1a to accelerate recovery from PSNL. We posit that pharmacological inhibition of IRE1α using MKC8866 (RNAase domain inhibitor) or KIRA6 (kinase domain inhibitor) will prevent or treat chronic neuropathic pain. Our team has expertise in pain biology and neuroimmune interactions (Romero-Sandoval), immunology and ER stress biology (Cubillos-Ruiz), in vivo electrophysiology (Boada), and scRNAseq and bioinformatics (Miller). Thus, we are uniquely positioned to test our innovative hypothesis and contribute to the development of novel non-narcotic treatments for chronic pain.

摘要 周围神经损伤激活免疫细胞中的模式识别受体(即Toll样受体)，从而 引发和维持炎症，并最终决定疼痛的永久化。免疫细胞 激活需要高水平的蛋白质合成、折叠和分泌，这些都受 内质网(ER)。蛋白质处理的过度需求会引起内质网应激(内质网堆积 错误折叠的蛋白质)，从而触发未折叠蛋白反应(UPR)的强大激活。IRE1α- XBP1是UPR在进化上最保守的臂，可以通过Toll样受体直接激活 参与促进促炎因子的表达。我们已经揭晓了有条件的淘汰赛 (CKO)免疫细胞(Ern1/Xbp1f/f-Vav1cre)中缺乏IRE1α/XBP1的小鼠，表现为前列腺素E_2产生减少 在体内，伤害性感受器反应性降低，非反射性疼痛相关行为的解决速度更快 在爪子切开之后。同样，这些CKO小鼠在部分坐骨神经结扎后表现出更好的恢复 (PSNL)。通过无偏向全基因组转录分析，我们发现IRE1α-XBP1信号转导途径在 白细胞是诱导前列腺素、细胞因子和其他新的因子如Nupr1的关键 (与人类慢性炎症性疾病有关)。值得注意的是，IRE1α-XBP1过度激活 与人类的疼痛或炎症状况有关。因此，我们假设IRE1α-XBP1 白细胞中的信号调控外周神经免疫相互作用和慢性前列腺癌的发展 疼痛。利用前沿的实验方法，我们将实现以下具体目标：1)确定 IRE1α-XBP1信号如何决定免疫细胞渗透的动态和推动 周围神经损伤后的行为非反射性超敏反应。我们假设IRE1α-XBP1. ARM是免疫细胞中前痛觉因子的关键调节器，Nupr1是一种新的XBP1- 依赖因子(使用芯片聚合酶链式反应)与PSNL有关。2)确定IRE1α-XBP1激活是如何管理的 个体化免疫细胞重新编程及其如何驱动与伤害性感受器传入的串扰 在周围神经损伤后。我们的假设是在我们的CKO小鼠身上观察到的过敏性降低 随后的PSNL由特定的损伤浸润性白细胞亚群中的离散基因签名决定(使用 单细胞RNA测序)，以及在伤害性感受器中获得较不敏感的表型(通过体内 细胞内DRG记录)。3)确定抑制IRE1a加速康复的治疗潜力 PSNL.我们推测MKC8866(核糖核酸酶结构域抑制剂)或KIRA6对IRE1α的药理抑制作用 (激酶域抑制剂)将预防或治疗慢性神经病理性疼痛。我们的团队在疼痛生物学方面有专长 和神经免疫相互作用(Romero-Sandoval)，免疫学和内质网应激生物学(Cubillos-Ruiz)，活体 电生理学(BOADA)、scRNAseq和生物信息学(Miller)。因此，我们处于独特的地位，可以测试 我们的创新假设，并有助于开发新的非麻醉药物治疗慢性疼痛。