Rosacea, a chronic inflammatory skin disorder primarily affecting the face, impacts a significant portion of the global population (1-3% prevalence). Characterized by erythema, telangiectasia, edema, papules, pustules, and flushing, rosacea is classified into four subtypes: erythematotelangiectatic (ETR), papulopustular (PPR), phymatous (PhR), and ocular rosacea. Pathogenesis involves skin barrier damage, dysregulated inflammation, neurovascular hyperactivity, glandular hyperplasia, and fibrosis. Current understanding of rosacea pathophysiology is limited by the lack of fully representative laboratory models and relies heavily on histomorphology, global gene analysis, and immunohistochemistry. Single-cell RNA sequencing (scRNA-seq) offers a powerful approach to define cellular composition, transcriptome dynamics, lineage tracing, and cell-cell communication in complex tissues with high resolution. This study aims to address the gap in knowledge by applying scRNA-seq to investigate the cellular and molecular mechanisms underlying rosacea development.

Existing research on rosacea pathophysiology has primarily focused on histomorphology, global gene expression profiling, and immunohistochemistry, offering limited insight into the cellular heterogeneity and dynamics within rosacea lesions. Studies have highlighted the involvement of damaged skin barrier function, dysregulated inflammatory responses, and neurovascular hyperactivity in rosacea pathogenesis. However, a detailed understanding of the cellular and molecular interactions driving these processes remains elusive. The role of specific cell types and their contributions to inflammation and vascular dysfunction in rosacea are not fully characterized. This lack of cellular resolution has hindered the development of targeted therapies for this common skin condition. The application of single-cell transcriptomics promises to overcome these limitations, providing an unprecedented level of detail into the complex cellular interplay involved in rosacea pathogenesis.

Skin biopsies were obtained from lesional and non-lesional facial skin of nine female rosacea patients (three ETR, three PPR, three PhR) and facial skin from three healthy female controls. Single-cell suspensions were prepared from the epidermis and dermis of these biopsies. scRNA-seq was performed using the Chromium Single Cell 3' Reagent Kits v3.1 (10x Genomics) and sequenced using an Illumina NovaSeq6000 System. Approximately 10,000 cells per sample were targeted for sequencing. Data processing involved quality control using Cell Ranger and Seurat (v4.3.0). Cells were clustered based on their gene expression profiles, and cell types were identified based on the expression of known marker genes. Subclustering analyses were performed to identify subpopulations within major cell types. Differentially expressed genes (DEGs) between lesional and non-lesional skin were identified using the FindMarkers function in Seurat. Gene Set Enrichment Analysis (GSEA) was used to identify enriched pathways within specific cell populations. Cell-cell communication analysis was performed using CellChat. To validate findings, a cathelicidin LL37-induced rosacea-like mouse model was used. Neutralizing antibodies against IFNγ, fibroblast depletion using Colla2^DTR mice, and PTGDS knockdown using siRNA were employed to investigate the functional roles of IFNγ signaling, fibroblasts, and PTGDS in rosacea development. Immunohistochemistry (IHC) and immunofluorescence (IF) were used to validate scRNA-seq findings. TEWL (transepidermal water loss) and ELISA for PGD2 were used to further assess the effects of the experimental manipulations.

The scRNA-seq analysis revealed a total of 11 major cell types in human facial skin, including keratinocytes, fibroblasts, Schwann cells, endothelial cells, vascular mural cells (vMCs), T cells, macrophages/dendritic cells (DCs), melanocytes, sweat gland cells, B cells, and mast cells. Significant alterations in the cellular composition were observed in rosacea skin compared to healthy controls. Notably, a subpopulation of keratinocytes exhibiting high expression of CD74 and upregulation of interferon (IFN)-related pathways was identified specifically in rosacea lesions. This subpopulation showed downregulation of skin barrier-related pathways. Neutralizing IFNγ alleviated rosacea-like phenotypes and skin barrier damage in a mouse model. Fibroblasts showed expansion of a pro-inflammatory subpopulation (C3+ fibroblasts) in papulopustular rosacea. These fibroblasts expressed high levels of chemokines (CCL19, CXCL1, CXCL2, CXCL12), contributing to inflammation. Schwann cells also showed expansion of a CD74+ subpopulation in papulopustular rosacea and upregulation of pro-inflammatory pathways. T cells, particularly Th1/Th17 cells and resident memory T cells (TRMs), were significantly increased in rosacea lesions. Macrophages showed increased expression of chemokines involved in T cell activation. Vascular mural cells showed activation of inflammatory pathways and impaired muscle contraction function. CellChat analysis showed that fibroblasts were the leading cell type producing outgoing signals to other cell types, highlighting their central role in coordinating the inflammatory response. Fibroblasts expressed high levels of PTGDS, leading to increased PGD2, a vasodilator. Depletion of fibroblasts or knockdown of PTGDS in mice prevented rosacea development. These findings suggest a critical role for fibroblasts in orchestrating both the inflammatory and vascular aspects of rosacea.

This study provides the first comprehensive single-cell transcriptomic atlas of rosacea skin lesions, identifying key cellular and molecular mechanisms contributing to this common skin disorder. The identification of a keratinocyte subpopulation with impaired barrier function and hyperactivated IFNγ signaling highlights a potential therapeutic target for rosacea. The pivotal role of fibroblasts in promoting inflammation and vasodilation through the production of chemokines and PGD2 respectively, offers further therapeutic opportunities. The study also reveals an expansion of Th1/Th17 cells and resident memory T cells (TRMs) in rosacea, providing new insights into the immune response. The findings suggest that fibroblasts act as central orchestrators of rosacea pathogenesis, integrating signals from other cell types and contributing to both inflammatory and vascular aspects of the disease. Targeting fibroblast activity may be a promising therapeutic strategy. Further research into the complex interactions between cell types and signaling pathways is crucial to advance the development of effective treatments.

This research demonstrates the power of single-cell transcriptomics in dissecting the complex cellular heterogeneity of rosacea. The study identified fibroblasts as a key determinant of rosacea pathogenesis, producing pro-inflammatory and vasodilatory signals that drive inflammation and vascular dysfunction. This understanding paves the way for targeted therapies focused on fibroblast modulation, offering new avenues for treating rosacea. Future work should focus on the specific molecular mechanisms driving fibroblast activation and exploring the possibility of developing targeted therapies against key molecules like PTGDS.

The study had a relatively small sample size, limiting the statistical power of certain analyses. Only female patients were included, potentially limiting the generalizability of the findings to males. The high proportion of keratinocytes in the healthy skin samples might have biased the estimation of the proportion of some cell types, particularly those present in smaller numbers, like fibroblasts. Further research is required to confirm the findings and to investigate potential sex-specific differences in rosacea pathogenesis.