Atopic dermatitis complicated by recurrent eczema herpeticum is characterized by multiple, concurrent epidermal inflammatory endotypes
A subgroup of atopic dermatitis (AD) patients suffers from recurrent, disseminated herpes simplex virus (HSV) skin infections, eczema herpeticum (EH). To determine transcriptional mechanisms of the skin and immune system pathobiology that underlies ADEH disease development, we performed RNA-sequencing analysis of non-lesional skin (epidermis, dermis) from AD patients with (ADEH+, n=15) and without (ADEH-, n=13) history of EH, and healthy controls (HC, n=15). We also performed RNA-sequencing on participants' plasmacytoid dendritic cells (pDCs) infected in vitro with HSV-1. ADEH+ patients exhibited dysregulated gene expression, limited in dermis (differentially expressed genes [DEGs]=14) and more widespread in epidermis (DEGs=129). ADEH+-upregulated epidermal DEGs were enriched in type 2 cytokine (T2) (IL4R, CCL22, CRLF2, IL7R), interferon (CXCL10, ICAM1, IFI44, IRF7), and IL-36? (IL36G) inflammatory gene pathways. All ADEH+ participants exhibited T2 and interferon endotypes and 87% were IL36G-high. In contrast, these endotypes were more variably expressed among ADEH- participants. ADEH+ skin also dysregulated epidermal differentiation complex (EDC) gene expression within LCE, S100, and SPRR families, which are involved in skin barrier function and antimicrobial activities. pDC transcriptional responses to HSV-1 infection were unaltered by ADEH status. Pathobiology underlying ADEH+ risk is associated with a unique, multi-faceted epidermal inflammation that accompanies dysregulation of EDC genes.  These findings will help direct future studies that define how these inflammatory patterns may drive risk of eczema herpeticum in AD. Overall design: Normal human embryonic keratinocytes (NHEK) from a single donor were purchased from ThermoFisher Scientific and maintained in EpiLife medium containing 0.06 mM CaCl2 and S7 supplement in 5% CO2 at 37°C. For NHEK differentiation, in triplicate, cells were cultured in EpiLife medium containing 1.3 mM CaCl2 for 3 days, then treated with 200 ng/ml of IL-36-gamma  (R&D systems) for 2 days. After treatment, the cells were harvested for RNA extraction using a Qiagen RNeasy Kit according to the manufacturer's instructions. Whole transcriptome libraries were constructed using the KAPA mRNA HyperPrep library kit (Roche Sequencing and Life Science, Kapa Biosystems, Wilmington, MA) from 250ng of total input RNA according to the manufacturers protocol. Barcoded libraries were pooled and sequenced using 150bp paired-end reads on the Illumina HiSeq 2500 system (Illumina, San Diego, CA). Raw sequencing reads were trimmed using skewer (v0.2.2) with the following parameters: end-quality=15, mean-quality=25, min=30. Read alignment to the human reference genome GRCh38 was performed using HISAT2 (v2.1.0) with default parameters. Gene quantification was performed using htseq-count (v0.9.1) and GRCh38 Ensemble v84 gene transcript model with the following parameters: stranded=reverse, a=20, mode=intersection-nonempty.