O12 Environmental factors interact with FLG to affect cytokine expression in human primary keratinocytes
Authors: Johnston Luke , Etorban Mona , Becher Hannes , Elias Martina , Brown Sara 
Published: July 2023

Abstract
Atopic dermatitis (or eczema) is a highly inheritable, itchy inflammatory condition characterized by skin barrier dysfunction. Genome-wide association studies have identified &gt; 70 risk loci for eczema, of which loss-of-function mutations in FLG pose the strongest genetic risk. FLG encodes filaggrin, which contributes to many aspects of skin barrier function such as mechanical, biochemical, immunological and microbiological roles. In those with FLG loss-of-function mutations, exposure to environmental irritants and allergens has been associated with an increased prevalence of eczema, indicating a possible gene–environment interaction. Epidemiological studies have shown a higher prevalence of eczema in those with FLG mutations exposed to cat allergens, whereas childhood exposure to dogs may provide protection. Cigarette smoke exposure may affect eczema prevalence, but epidemiological studies have multiple confounding factors. Cigarette smoke signalling occurs through the aryl hydrocarbon receptor and results in an upregulation of FLG expression, suggesting possible protective effects. Studying environmental interactions with FLG genotype is challenging in vivo; therefore, we modelled the interaction in vitro using human primary keratinocytes. Primary keratinocytes were isolated from normal skin samples from donors undergoing routine surgery, with informed consent and the cells were cultured as monolayers. FLG was knocked down using small interfering RNA (siRNA) compared with a nontargeting control. Environmental exposures were modelled using clinical-grade cat or dog epithelium skin-prick test reagents, and standardized cigarette smoke condensate (CSC). These reagents were added at optimized concentrations to keratinocyte cultures overnight. Multiplex ELISAs and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) were performed on animal allergen-treated keratinocytes, and RT-qPCR and bulk RNA-Seq performed on CSC-treated samples. Exposure of keratinocytes to dog allergen stimulated an increase in multiple cytokines and chemokines of relevance to atopic inflammation, including tumour necrosis factor (TNF)-α, CCL2, CXCL8, interleukin (IL)-1α, IL-6, matrix metalloproteinase (MMP)9, CXCL1, IL-18 and granulocyte colony-stimulating factor (G-CSF). The combination of dog allergen and FLG knockdown showed a significant interaction: dog allergen and FLG knockdown together induced a greater increase in expression of TNF-α (n = 4; P = 0.016) and CCL2 (n = 3; P = 0.0009) than their additive effects. Cat allergen increased CXCL8, CXCL16, IL-6 and CXCL1 expression, and FLG knockdown showed interaction with cat allergen exposure to further increase CXCL8 expression (n = 4; P = 0.036). In FLG wild-type keratinocytes, CSC stimulated an increase in FLG expression (n = 6, mean 2.6-fold change; P = 0.03) and marked upregulation of CYP1A1, a marker of aryl hydrocarbon receptor signalling (n = 8, mean 71-fold change; P &lt; 0.0001). In contrast, FLG siRNA knockdown keratinocytes treated with CSC showed no significant increase in FLG expression and only a small, nonsignificant increase in CYP1A1 expression (n = 8; P = 0.084). Together, these findings provide experimental evidence of FLG–environment interactions in keratinocyte cytokine expression, illustrating the key role of innate immunity in this barrier cell type. Our experiments also provide important validation of findings from genetic epidemiological studies. Future work will investigate mechanistic pathways for potential therapeutic targets.