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

神经性瘙痒导致生活质量的严重损害。在第一个资助期，我们招募了245名患有多神经病变（PNP）的患者：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，其特征是在电C纤维刺激和机械痛觉过敏时具有高疼痛评分。在皮肤活组织检查中，我们注意到瘙痒患者真皮神经纤维的明显丢失，并伴有剩余C纤维的强分支。通过rna测序（RNA-seq）和定量PCR，我们发现仅瘙痒患者皮肤活检样本中IFI44L的表达增加。我们证明了免疫组化方法在皮下雪旺细胞（SC）类型定位中的可行性。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特征。值得注意的是，IFI44L是microRNA let-7d的直接靶点，我们之前发现let-7d在慢性疼痛患者的皮肤中增加，而let-7抑制SCs中netrin的表达。鉴于let-7家族成员在SC中强烈表达，我们假设SC中表达let-7- ifi44l -netrin通路成员可能介导PNP患者亚组的瘙痒和疼痛。因此，我们将研究瘙痒和疼痛中的SC形态和蛋白质表达，旨在更深入地了解定义明确的亚组患者皮肤中的SC相关通路。将招募疼痛或瘙痒的PNP患者，并通过临床测量仔细评估。为了进行比较，我们将纳入肱桡瘙痒患者，并从合作项目#5中获得牛皮癣患者的皮肤样本。然后，我们将在C纤维刺激下识别和表征瘙痒、疼痛和高疼痛敏感性的PNP患者群体，旨在了解他们的心理物理特征，并选择同质样本用于高通量转录组学技术。接下来，寡核苷酸标记抗体多重snRNA-seq将允许我们分析从大量临床队列中获得的冷冻保存皮肤活检样本中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皮肤sc的转录组谱，并定义稳态和反应性sc的分子特征。多重RNA原位作图将使我们能够将SC亚型的反应性水平映射回潜在的皮肤微环境。综上所述，结合单细胞和空间转录组学工具，对来自神经性疼痛和瘙痒患者的高质量冷冻皮肤活检样本进行研究，将使我们能够为未来的介入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，鉴定与瘙痒或疼痛相关的新SC特征将有助于更好地理解它们在轴突病理和临床表型中的作用。

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.