Pathogenic Mechanism And Single Cell Transcriptome Atlas In Xerosis Induced Itch

Itch is an unpleasant skin sensation that elicits the desire of reflex to scratch.According to the duration of symptoms,itch can be defined as acute itch and chronic itch.Acute itch lasts less than six weeks and acute itch lasts more than six weeks.Chronic itch has high morbidity rate and relapsing pathogenic condition.Because of massive skin lesions and intense itch,patients always suffer from comorbidities like depression and sleep problem which substantially impairs life quality.Xerosis（dry skin）itch is one of the most common chronic itch diseases,which is characterized by dry,desquamated,cracked and itchy skin.It has complex pathogenic mechanisms,involving various cell types like keratinocytes,immune cells,neurons and glias and lacking of specific clinical drugs.The pathogenic hypothesis of xerosis itch includes increased oxidative stress,over-expressed ion channels,itchy nerve endings sensitization and elevated release of proinflammatory cytokines.TRPM2 is a calcium permeable nonselective cation channel and main oxidative stress sensor.It expresses in key organs of itch conduction like skin,dorsal root ganglion,spinal cord and brain.Whether TRPM2 may participate in oxidative stress related xerosis itch conduction?Our research found that globally knock out TRPM2 didn’t affect histamine,compound 48/80,chloroquine,β-alanine and BAM8-22 induced itch,but specially reduced oxidative stress induced itch.The pathogenic mechanisms of atopic dermatitis and xerosis are closely related to increased oxidative stress.Therefore,we further investigated whether TRPM2 mediated above chronic itch diseases.Globally knock out TRPM2 didn’t affect atopic dermatitis induced itch,but specially reduced xerosis induced itch.Among xerosis patients,getting dressed or slightly touched can increase the itch sensation and such phenomenon is called touch evoked itch or alloknesis.We also found that globally knock out TRPM2 reduced xerosis induced alloknesis.Dorsal root ganglion and keratinocyte are closely related to xerosis itch.Previous studies have shown that DRG TRPM2 were activated by warmth,mediating 23-38℃environmental temperature sensation.Therefore,we firstly investigated whether DRG TRPM2 mediated xerosis itch.Although immunofluorescence staining showed that TRPM2 mainly expressed in small and medium size DRG neurons,but specially knock out DRG TRPM2 didn’t affect xerosis itch,indicating DRG TRPM2 didn’t participate in the pathogenic mechanisms of xerosis itch.Then,we further investigated whether keratinocyte TRPM2 mediated xerosis itch.Immunofluorescence staining and in situ hybrization showed that TRPM2 expressed in both mouse and human keratinocyte.Moreover,calcium imaging and electrophysiology showed that TRPM2 functioned in both mouse and human keratinocyte.Specially knock out keratinocyte TRPM2 significantly relieved xerosis itch,indicating keratinocyte TRPM2 mediated xerosis itch conduction.Knockout TRPM2 or pharmacological inhibit TRPM2 reduced oxidative stress induced calcium influx both in mouse and human keratinocyte,indicating ROS-TRPM2-Ca²⁺pathway mediated xerosis itch conduction.Overall,our research firstly confirmed that TRPM2 functionally expressed in mouse as well as human keratinocyte and the ROS-TRPM2-Ca²⁺pathway of keratinocyte regulated xerosis itch.Our result provided a new target for the treatment of xerosis itch.The barrier function of skin depends on the structural integrity and orderly metabolism of epidermal layer.When free water and lipid are lacking in epidermis,epidermal skin will be dry,cracked and itchy.Chronic xerosis（dry skin）itch is very common clinical symptom and its pathogenic mechanism has not been clearly studied.In order to further explore the pathogenic mechanism of xerosis itch,Application of acetone/ether mixture followed by water（AEW）was used to establish classic mouse model of xerosis itch.Single cell transcriptome sequencing was performed in the skin tissues of normal and xerosis mice.Totally,we analyzed 11846 skin cells of normal mice and 7137 skin cells of xerosis mice.We used Principal Component Analysis（PCA）,t-Distributed Stochastic Neighbor Embedding（t-SNE）and Find Clusters function in Seurat to cluster skin cells in 0.1 resolution.The cells were divided into ten groups.The Find All Markers function in Seurat and Wilcoxon Rank Sum Test was used to identify marker genes in each cell groups and certain cell types were annotated according to other reports in literature.Finally,we annotated skin cells as five categories: epithelial cells,fibroblasts,monocytes,adipocytes,and endothelial cells.Further comparison of the percentages of each cell types showed that xerosis altered the proportions of epithelial cells,fibroblasts,monocytes and endothelial cells.Differential analysis of gene expression profiles between normal mice and xerosis mice was performed using the Find Markers function in Seurat.Gene Ontology（GO）enrichment analysis showed that: the expression of genes involved in wound healing,proliferation,development and differentiation of epithelial cells,ribosome organization and assembly were significantly different in epithelial cells;the expression of genes involved in extracellular matrix organization,extracellular structure organization,cell-substrate adhesion and amoeba-type cell migration were significantly different in fibroblasts;the expression of genes involved in positive regulation of cytokine production,positive regulation of cell activation,lymphocyte migration and cell chemotaxis were significantly different in monocytes and the expression of genes involved in amoeba-type cell migration,actin filament organization,regulation of vasculature development and regulation of angiogenesis were significantly different in endothelial cells.GO enrichment analysis only classified and described the differentially expressed genes in skin cells between normal mice and xerosis mice in terms of molecular function,biological process and cell composition.Numerous studies have shown that orderly cell-cell communication is essential for maintaining normal physiological function of skin and abnormal cell-cell can lead to skin diseases such as melanoma,eczema,psoriasis and so on.There are still few reports on the cell-cell communication patterns in xerosis itch and our study used the newly published Cell Chat tool to further analyze the communication differences between normal and xerosis skin cells.Cell Chat analysis revealed that macrophage migration inhibitory factor（MIF）signaling pathway had the highest communication probability in xerosis mice.Xerosis significantly up-regulated Interleukin 1（IL1）and Angiopoietin（ANGPT）signaling pathways and down-regulated VISFATIN and Interleukin 6（IL6）signaling pathways.In the future,we will further verify the function of the above significantly changed secretory signaling pathways.In conclusion,our research firstly performed skin single cell transcriptome atlas of chronic xerosis itch mouse model and further analysis by Cell Chat revealed that MIF,IL1,ANGPT,VISFATIN and IL6 may be the key signaling pathways in the pathogenic mechanisms of xerosis.Our research will provide a new insight for exploring the pathogenesis of xerosis itch.