Caspase recruitment domain family member 14 (CARD14) and its variants are associated with both atopic dermatitis (AD) and psoriasis, but their mechanistic impact on skin barrier homeostasis is largely unknown. CARD14 is known to signal via NF-κB; however, CARD14-NF-κB signaling does not fully explain the heterogeneity of CARD14-driven disease. Here, we describe a direct interaction between CARD14 and MYC and show that CARD14 signals through MYC in keratinocytes to coordinate skin barrier homeostasis. CARD14 directly binds MYC and influences barrier formation in an MYC-dependent fashion, and this mechanism is undermined by disease-associated CARD14 variants. These studies establish a paradigm that CARD14 activation regulates skin barrier function by two distinct mechanisms, including activating NF-κB to bolster the antimicrobial (chemical) barrier and stimulating MYC to bolster the physical barrier. Finally, we show that CARD14-dependent MYC signaling occurs in other epithelia, expanding the impact of our findings beyond the skin.

BACKGROUND:Frequent asthma exacerbators, defined as those experiencing more than 1 hospitalization in a year for an asthma exacerbation, represent an important subgroup of individuals with asthma. However, this group remains poorly defined and understudied in children.OBJECTIVE:Our aim was to determine the molecular mechanisms underlying asthma pathogenesis and exacerbation frequency.METHODS:We performed RNA sequencing of upper airway cells from both frequent and nonfrequent exacerbators enrolled in the Ohio Pediatric Asthma Repository.RESULTS:Through molecular network analysis, we found that nonfrequent exacerbators display an increase in modules enriched for immune system processes, including type 2 inflammation and response to infection. In contrast, frequent exacerbators showed expression of modules enriched for nervous system processes, such as synaptic formation and axonal outgrowth.CONCLUSION:These data suggest that the upper airway of frequent exacerbators undergoes peripheral nervous system remodeling, representing a novel mechanism underlying pediatric asthma exacerbation.

Skin barrier disruptions are major risk factors for atopic dermatitis (AD) and atopic march progression. We recently found that the exonic CARD14 variant rs11652075 (R820W) is associated with low epidermal expression of the barrier protein filaggrin in children with AD, and that CARD14 regulates filaggrin in a genotype-dependent fashion. This suggests the variant negatively impacts barrier function. We thereby hypothesized that differential CARD14 signaling in variant keratinocytes confers risk for barrier dysfunction and AD. The transcriptomes of isogenic HaCaT human keratinocytes harboring homozygous wild-type (WT; CARD14C/C) or rs11652075 variant (CARD14T/T) alleles were compared by multiple bioinformatic approaches. Functional studies were conducted in the HaCaTs and in engineered skin substitutes (ESS) generated from primary keratinocytes and fibroblasts of each genotype. Gene enrichment and pathway analyses of differentially-expressed genes between CARD14C/C and CARD14T/T HaCaTs reveal that variant cells have attenuated signatures for MYC—a crucial regulator of skin barrier formation. Compared to WT, immunoblotting/staining confirms reduced total/nuclear MYC levels in variant HaCaTs and ESS that are attributable to elevated MYC degradation by autophagy. Co-immunoprecipitation and proximity ligation assays demonstrate CARD14 directly interacts with and regulates MYC. Finally, variant cells and ESS have reduced %Ki67+ populations and proliferation rates. Our data collectively identify CARD14 as a novel regulator of keratinocyte MYC and suggest that the variant disrupts this mechanism to reduce MYC levels and attenuate keratinocyte proliferation. This novel mechanism may significantly impact the formation of a functional skin barrier or, when disrupted, a suboptimal barrier that predisposes AD and the atopic march.

Children with atopic dermatitis (AD) have a higher risk for allergic sensitization than those without AD, but factors which predict persistent sensitization in children with AD remain uninvestigated. We sought to elucidate sensitization patterns in children with AD and to determine whether factors predict persistent allergen sensitization (PS) in children with AD as opposed to transient sensitization (TS) using machine learning. We defined longitudinal allergen sensitization profiles in 345 children with AD in the Mechanisms of Progression from Atopic Dermatitis to Asthma in Children (MPAACH) cohort. MPAACH is a pediatric AD cohort, recruited at 6-24 months of age, through hospital/community settings. We next determined which factors predict these sensitization patterns using random forest analysis. Children with AD were defined by 5 sensitization patterns: non-sensitized (NS), acquired (AS), transient (TS), persistent same (PS), and persistent different (PD). Most children were classified as NS (40.23%), followed by PS (24.19%), AS (15.16%), TS (14.58%), and PD (5.83%). No factors were predictive for NS vs AS. PD patterns were not evaluated further due to small sample size. The top 8 factors at the first visit that predicted PS vs TS were peanut, egg, and dog sensitizations, total allergen sensitization, lesional transepidermal water loss (TEWL), SCORAD (Scoring AD) assessment, and non-lesional maximum s100a8 and s100a9 mRNA expression. PS was predicted by having sensitization (skin prick test wheal ≥3mm diameter) to peanut, egg, or dog. In this cohort of young children with AD, allergic sensitization to peanut, egg, or dog was associated with persistent sensitization.

A disrupted skin barrier is a major risk factor for the development of atopic dermatitis (AD) and progression of the atopic march. We recently found that the CARD14 variant rs11652075 is associated with low epidermal expression of the barrier gene filaggrin (FLG) in children with AD, and that CARD14 regulates keratinocyte FLG expression in a rs11652075-dependent manner. Since CARD14 mediates signaling through multiple pathways, we hypothesized that differential signaling activity in keratinocytes harboring the variant allele confers risk to barrier dysfunction and AD.

Skin deficiency of kinesin family member 3A causes disrupted skin barrier function and promotes development of atopic dermatitis (AD). It is not known how well Kif3aK14∆/∆ mice approximate the human AD transcriptome. To determine the skin transcriptomic profile of Kif3aK14∆/∆ mice and compare it with other murine AD models and human AD, we performed RNA-seq of full-thickness skin and epidermis from 3- and 8-week-old Kif3aK14∆/∆ mice and compared the differentially expressed genes (DEGs) with transcriptomic datasets from mite-induced NC/Nga, flaky tail (Tmem79ma/ma Flgft/ft), and filaggrin-mutant (Flgft/ft) mice, as well as human AD transcriptome datasets including meta-analysis derived atopic dermatitis [MADAD] and the pediatric atopic dermatitis [PAD]. We then interrogated the Kif3aK14∆/∆ skin DEGs using the LINCS-L1000 database to identify potential novel drug targets for AD treatment. We identified 471 and 901 DEGs at 3 and 8 weeks of age, respectively, in the absence of Kif3a. Kif3aK14∆/∆ mice had 3.5-4.5 times more DEGs that overlapped with human AD DEGs compared to the flaky tail and Flgft/ft mice. Further, 55%, 85% and 75% of 8-week Kif3aK14∆/∆ DEGs overlapped with the MADAD and PAD non-lesional and lesional gene lists, respectively. Kif3aK14∆/∆ mice spontaneously develop a human AD-like gene signature, which better represents pediatric non-lesional skin compared to other mouse models including flaky tail, Flgft/ft and NC/Nga. Thus, Kif3aK14∆/∆ mice may model pediatric skin that is a precursor to the development of lesions and inflammation, and hence may be a useful model to study AD pathogenesis.

BACKGROUND:Children with atopic dermatitis (AD) are often sensitized to food and aeroallergens, but sensitization patterns have not been analysed with biologic measures of disease pathogenicity.OBJECTIVE:We sought to define allergen sensitization grouping(s) using unbiased machine learning and determine their associations with skin filaggrin (FLG) and transepidermal water loss (TEWL) (assesses skin barrier integrity), S100A8 and S100A9 expression (assesses skin inflammation) and AD severity.METHODS:We studied 400 children with AD in the Mechanisms of Progression from Atopic Dermatitis to Asthma in Children (MPAACH) cohort to identify groupings of food and aeroallergen sensitizations. MPAACH is a paediatric AD cohort, aged 1-2, recruited through hospital/community settings between 2016 and 2018. We analysed these groupings' associations with AD biomarkers: skin FLG, S100A8 and S100A9 expression, total IgE, TEWL and AD severity.RESULTS:An unbiased machine learning approach revealed five allergen clusters. The most common cluster (N = 131), SPTPEP, had sensitization to peanut, egg and/or pets. Three low prevalence clusters, which included children with allergen sensitization other than peanut, egg or pets, were combined into SPTOther . SPTNEG included children with no sensitization(s). SPTPEP children had higher median non-lesional TEWL (16.9 g/m2 /h) and IgE (90 kU/L) compared with SPTOTHER (8.8 g/m2 /h and 24 kU/L; p = .01 and p < .001) and SPTNEG (9 g/m2 /h and 26 kU/L; p = .003 and p < .001). SPTPEP children had lower median lesional (0.70) and non-lesional (1.09) FLG expression compared with SPTOTHER (lesional: 0.9; p = .047, non-lesional: 1.78; p = .01) and SPTNEG (lesional: 1.47; p < .001, non-lesional: 2.21; p < .001). There were no differences among groupings in S100A8 or S100A9 expression.CONCLUSIONS AND CLINICAL RELEVANCE:In this largely clinic-based cohort of young children with AD, allergic sensitization to peanut, egg, cat or dog was associated with more severe disease and skin barrier function but not markers of cutaneous inflammation. These data need replicating in a population-based cohort but may have important implications for understanding the interaction between AD and allergic sensitization.

Children with atopic dermatitis (AD) often have allergen sensitizations. These sensitizations have not been analyzed using biologic measures of disease pathogenicity to define clinically relevant pediatric AD endotypes. We sought to define allergen sensitization grouping(s) and determine their associations with AD severity, transepidermal water loss (TEWL), and FLG expression to define clinically relevant endotypes. We studied 398 children with AD in the Mechanisms of Progression from Atopic Dermatitis to Asthma in Children (M-PAACH) cohort to identify groupings of aero- and food-allergen sensitizations. We then analyzed the associations between these groupings and AD severity, total IgE, TEWL, and skin FLG, S100A8, and S100A9 expression. An unbiased machine learning approach revealed five allergen sensitization clusters, which we used to define groups. The most common group (N=106), SPTPEP, had sensitization to peanut, egg and/or pets. SPTOther included children with allergen sensitization but not children within SPTPEP. SPTNEG included children with no sensitization(s). Regardless of sensitization load, SPTPEP children had higher SCORAD compared with SPTOTHER (p-value=0.007) or SPTNEG (p-value=3E-06), higher non-lesional TEWL compared with SPTOTHER (p=0.01) and SPTNEG (p=0.003), and decreased non-lesional FLG expression compared with SPTOTHER (p-value=0.01) and SPTNEG (p-value=0.0002). There was no difference in S100A8 or S100A9 expression between SPTPEP, SPTOTHER, and SPTNEG. The SPTPEP grouping represents a novel pediatric AD endotype that is independently associated with higher SCORAD, higher non-lesional TEWL, and decreased non-lesional FLG expression, which suggests a significant baseline barrier dysfunction and a common route of sensitization for these allergens.

Single nucleotide polymorphisms (SNPs) in the gene encoding kinesin family member 3A, KIF3A, have been associated with atopic dermatitis (AD), a chronic inflammatory skin disorder. We find that KIF3A SNP rs11740584 and rs2299007 risk alleles create cytosine-phosphate-guanine sites, which are highly methylated and result in lower KIF3A expression, and this methylation is associated with increased transepidermal water loss (TEWL) in risk allele carriers. Kif3aK14∆/∆ mice have increased TEWL, disrupted junctional proteins, and increased susceptibility to develop AD. Thus, KIF3A is required for skin barrier homeostasis whereby decreased KIF3A skin expression causes disrupted skin barrier function and promotes development of AD.