Influence of scratching on skin profiles in chronic pruritus patients

慢性瘙痒是各种疾病的高发症状，给患者带来了沉重的痛苦负担。尽管如此，人们对潜在的途径知之甚少。这一点尤其适用于涉及抓挠的机制。抓挠缓解了瘙痒，但在CP中，它会导致皮肤完整性、解剖学和功能的损害。在PRUSEARCH和CP的第一个资助期，研究了抓挠对皮肤细胞和分子机制的影响-发现了分化的分枝模式和基因表达。在这个项目中，我们旨在深入分析由抓挠引起的表皮神经解剖改变，以及神经性和非神经性瘙痒标志物。我们将进一步研究慢性机械应激(相当于抓挠)对促进神经纤维改变和角质形成细胞代谢的因素的基因表达的影响，并确定基因表达的长期调节因素在维持瘙痒慢性化中的作用。我们将招募慢性结节性瘙痒(CNPG，SYN：结节性瘙痒；作为严重抓挠相关皮肤损害的模型)、特应性皮炎(AD；为了联系第一个资助期获得的数据)和相应的健康志愿者(共180名参与者)。使用(A)瘙痒、慢性划痕、(B)瘙痒、无划痕和(C)非瘙痒、正常皮肤的皮肤活检，我们将从形态上表征皮肤神经纤维的神经解剖和瘙痒标志物的表达。我们将分离角质形成细胞，在体外分析慢性机械应力对基因表达的影响。这将通过以神经可塑性相关基因为重点的信使核糖核酸测序来完成。不同区域的比较将使我们能够确定瘙痒和慢性抓挠对这些细胞的启动效应。神经细胞和角质形成细胞的共培养将用于第一个资助期内确定的候选基因的功能验证。我们将在培养基中使用尖峰或耗尽(分泌的蛋白质)或调节基因表达。为了在有划痕和未划痕的皮肤中获得功能关联，将进行局部电刺激以选择性地刺激伤害性感受器。最后，将为不同的CP实体以及慢性划痕定义DNA甲基化签名。通过分析相同的活检组织，可以确定DNA甲基化对基因表达谱的影响，从而维持时代性。总之，我们的目标是确定抓挠相关因素与形态变化、通过问卷获得的临床瘙痒特征和功能参数之间的因果关系。这些发现将通过定义与慢性抓挠相关的分子因素，并开启新的治疗策略，从而产生重要的翻译影响。

Chronic pruritus (CP) is a highly prevalent symptom of different diseases and constitutes a high burden of suffering for patients. Still, little is known about the underlying pathways. This holds especially true for the mechanisms involved in scratching. Scratching reliefs pruritus but, in CP, it can result in damage of the skin integrity, anatomy and function. The influence of scratching on the cutaneous cellular and molecular mechanisms have been investigated in the first funding period of PRUSEARCH and CP-differentiating branching pattern and gene expression have been found. In this project, we aim to analyze in-depth the epidermal neuroanatomical alterations, and neuronal and non-neuronal pruritic markers that are induced by scratching. We will further investigate the effect of chronic mechanical stress (as equivalent for scratching) on gene expression regarding factors contributing to nerve fiber alterations and metabolism of keratinocytes and we will define the role of long-term modifying factors of gene expression in maintaining chronicity of pruritus.We will recruit patients with chronic nodular prurigo (CNPG, syn: prurigo nodularis; as a model for severe scratch-related skin lesions), atopic dermatitis (AD; in order to connect to data obtained in the first funding period) and corresponding healthy volunteers (in total 180 participants). Using skin biopsies of (a) pruritic, chronically scratched, (b) pruritic, unscratched and (c) not-pruritic, normal skin, we will morphologically characterize the neuroanatomy of the cutaneous nerve fibers and expression of pruritogenic markers. We will isolate keratinocytes to analyze in vitro the effect of chronic mechanical stress on gene expression. This will be done by means of mRNA sequencing with focus on neuroplasticity-related genes. Comparison of the different areas will enable us to identify a priming effect of pruritus and chronic scratching on these cells. Co-culture of neuronal cells and the keratinocytes will be used for functional validation of candidate genes defined within the first funding period. We will use spiking or depletion in media (secreted proteins) or modulation of gene expression. To obtain a functional correlate in the scratched and unscratched skin, focal electrical stimulation will be performed for selective nociceptor stimulation. Finally, DNA methylation signatures will be defined for the different CP entities as well as for chronic scratching. Analyzing same biopsies will allow to define impact of DNA methylation on gene expression profiles and by this on maintaining chronicity.In sum, we aim to identify the causal relationships between the scratching-related factors and morphological changes, clinical pruritus characteristics obtained via questionnaires and functional parameters. The findings will have an important translational impact by defining molecular factors which are related to chronic scratching and which open novel therapeutically strategies.