334 Impact of injury-induced inflammation on ex vivo skin culture as a preclinical model for atopic dermatitis

Abstract

Ex vivo skin culture holds promise as a preclinical model for understanding pathogenic mechanisms and testing therapeutics for skin diseases. This project studied the impact of ex vivo culture on the transcriptome profile and cytokine/chemokine production of nonlesional and lesional skin from adult atopic dermatitis (AD) patients (n=19) with moderate to severe disease. Punch biopsy (2mm) specimens were immediately processed for RNA (baseline) or cultured for 48 hours (37°C, 5% CO2, DMEM-10% FBS), followed by RNA isolation and supernatant collection. Transcriptome analyses were performed with HTA2.0 arrays (Affymetrix), and supernatants were analyzed with multiplex immunoassays. Microarray data analysis (t-SNE) revealed cultured samples clustered separately from baseline samples, which suggested culturing the skin had a dominant effect on gene expression and obscured AD transcriptome profiles. Relative to baseline samples, cultured AD skin exhibited increased inflammatory cytokine and chemokine transcripts. Proteins encoded by these transcripts were also detected in culture supernatants. These data demonstrated AD skin culture activates an inflammatory response distinct from that seen in vivo. Ex vivo culture of healthy donor skin also activated this response, which was determined to be interleukin-1 alpha (IL-1α)-dependent. In ex vivo skin culture, this IL-1α-induced inflammatory response obscured the ability of selected recombinant cytokines to induce known target genes. In conclusion, IL-1α activates a defined inflammatory response during ex vivo skin culture, which can obscure the native (in vivo) transcriptome/secretome of skin specimens. These results suggest including IL-1α neutralization as part of ex vivo skin culture systems would create a better preclinical skin disease model for evaluating therapeutic compounds.