Supplementary file 1_Integration of bulk and single-cell transcriptomics with spatial analysis identifies candidate hub genes and inferred regulatory programs in atopic dermatitis.docx

Regulon activity was computationally inferred. GSE197023 comprised 20 spatial transcriptomic samples, including 7 lesional AD, 7 non-lesional AD, and 6 healthy-control samples, and was analyzed using cell-population deconvolution. CCR7 immunoreactivity was assessed by immunohistochemistry in independent healthy-control and lesional AD skin samples (n = 5 per group) analyze.</p>Results<p>WGCNA identified disease-severity-associated gene modules enriched in immune and cell-cycle-related processes.

Nominal-P-value-based integration with differential expression results identified seven exploratory candidate hub genes (GZMB, CCR7, GPR183, MMP12, IL7R, RGS1, and KLHDC7B) associated with immune- cell abundance patterns. FDR-adjusted sensitivity analysis supported MMP12 and RGS1 in the lesional-versus-non-lesional comparison, whereas none of the seven genes reached nominal or FDR-adjusted significance in the paired treatment analysis.

Single-cell reference mapping showed cell-type-associated expression of GZMB in cytotoxic T-cell populations and CCR7 and MMP12 in dendritic-cell subsets. Computational regulon analysis identified candidate cell-type-associated regulatory signals and differences in inferred regulon activity across treatment-state groups. Spatial deconvolution indicated predominantly dermal distributions of inferred immune-cell populations.

Immunohistochemical assessment showed higher relative CCR7 immunoreactivity in lesional AD skin than in healthy-control skin.</p>Conclusion<p>These cross-modal analyses linked candidate molecular signals todendritic-cell- and T- cell-associated patterns in AD. These findings are exploratory and hypothesis-generating and do not establish validated biomarkers, causal regulatory mechanisms, or predictors of treatment response.

Independent cohort replication and functional and cell-type-specific validation are required.</p>