Transcriptomic patterns of dermal Schwann cell reactivity in neuropathic itch and pain

神经性瘙痒症导致生活质量的显著损害。在第一个资助期间，我们招募了245名多发性神经病（PNP）患者：24名报告瘙痒为唯一症状，58名报告瘙痒和疼痛。我们定义了一个PNP亚组，其特征在于电刺激C纤维和机械痛觉过敏后的高疼痛评级。在皮肤活组织检查中，我们注意到瘙痒患者的真皮神经纤维明显丢失，而剩余的C纤维分支很强。通过RNA测序（RNA-seq）和定量PCR，我们发现IFI 44 L在来自仅瘙痒患者的皮肤活检样品中的表达增加。我们证明了免疫组化法定位表皮下雪旺细胞（SC）类型的可行性。利用单核RNA-seq（snRNA-seq），我们可以表征SC亚型并解码其亚型特异性转录组签名。值得注意的是，IFI 44 L是microRNA let-7 d的直接靶点，我们先前发现let-7 d在慢性疼痛患者的皮肤中增加，let-7抑制SC中的netrin表达。鉴于let-7家族成员在SC中强烈表达，我们假设let-7-IFI 44 L-netrin途径成员的SC表达可能介导PNP患者亚组中的瘙痒和疼痛。因此，我们将研究瘙痒和疼痛中的SC形态和蛋白表达，旨在更深入地了解明确定义的患者亚组皮肤中的SC相关途径。将招募疼痛或瘙痒的PNP患者，并通过临床测量进行仔细评估。为了进行比较，我们将纳入肱桡瘙痒症患者，并接受来自合作项目#5的银屑病患者的皮肤样本。然后，我们将确定和表征组的PNP患者的瘙痒，疼痛和高疼痛敏感性后，C纤维刺激，旨在了解他们的心理物理特征，并在选择同质样品用于高通量转录组技术。接下来，带有多核苷酸标签的抗体多重snRNA-seq将使我们能够分析从大型临床队列中获得的冷冻保存的皮肤活检样本的细胞类型特异性基因表达。通过生物信息学分析，我们将能够解码不同临床实体之间的皮肤SC的转录组学谱，并定义稳态和反应性SC的分子特征。多重RNA原位定位将使我们能够将SC亚型反应性水平映射到底层皮肤微环境。总之，使用来自具有神经性疼痛和瘙痒的良好特征的患者的高质量冷冻皮肤活检样本与单细胞和空间转录组学工具相结合，将使我们能够为未来的干预性研究确定新的细胞类型特异性靶点和生物标志物。我们假设，新的SC特性相对于瘙痒或疼痛的鉴定将有助于更好地了解其在轴突病理和临床表型相关的作用。

Neuropathic pruritus leads to significant impairment of life quality. During the first funding period, we recruited 245 patients with polyneuropathies (PNP): 24 reporting itch as only symptom and 58 reporting itch and pain. We defined a PNP subgroup characterized by high pain ratings upon electrical C fiber stimulation and mechanical hyperalgesia. In skin biopsies, we noted a pronounced loss of dermal nerve fibers paralleled by a strong branching of remaining C fibers in patients with itch. By RNA-sequencing (RNA-seq) and quantitative PCR, we found increased expression of IFI44L in skin biopsy samples from patients with itch only. We showed feasibility of subepidermal Schwann cell (SC) type mapping by immunohistochemistry. Utilizing single-nucleus RNA-seq (snRNA-seq), we could characterize SC subtypes and decode their subtype-specific transcriptomic signatures. Notably, IFI44L is a direct target of the microRNA let-7d, which we previously found to be increased in skin from patients with chronic pain, and let-7 represses netrin expression in SCs. Given that let-7 family members are strongly expressed in SCs, we postulate that SC expression of let-7-IFI44L-netrin pathway members might mediate itch and pain in a subgroup of PNP patients. Thus, we will investigate SC morphology and protein expression in itch and pain, aiming at a deeper understanding of SC related pathways in the skin of well-defined subgroups of patients. PNP patients with either pain or itch, will be recruited and carefully assessed by clinical measurements. For comparison, we will include patients with brachioradial pruritus and receive skin samples of patients with psoriasis from collaborating project #5. We will then identify and characterize groups of PNP patients with itch, pain and high pain sensitivity upon C fiber stimulation aiming at understanding their psychophysical characteristics and at selecting homogenous samples to be used in high-throughput transcriptomic technologies. Next, oligonucleotide-tagged antibody multiplex snRNA-seq will allow us to analyze cell type-specific gene expression from cryo-preserved skin biopsy samples obtained from large clinical cohorts. By bioinformatic analysis, we will be able to decode the transcriptomic profile of cutaneous SCs between different clinical entities and define molecular traits of homeostatic and reactive SCs. Multiplex RNA in situ mapping will enable us to map back levels of SC subtype reactivity to the underlying skin microenvironment. In summary, work with high-quality frozen skin biopsy samples from well-characterized patients with neuropathic pain and itch in combination with single-cell and spatial transcriptomic tools will allow us to identify novel cell-type specific targets and biomarkers for future interventional studies. We hypothesize that identification of novel SC characteristics relative to itch or pain will help better understand their role in relation to axon pathology and the clinical phenotype.