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. Author manuscript; available in PMC: 2023 Feb 1.
Published in final edited form as: J Allergy Clin Immunol. 2021 Jul 13;149(2):708–717. doi: 10.1016/j.jaci.2021.07.003

Novel Role for Caspase Recruitment Domain Family Member 14 and its Genetic Variant rs11652075 in Skin Filaggrin Homeostasis

Stanley B DeVore 1,2, Mariana L Stevens 2, Hua He 3, Jocelyn M Biagini 1,2, John W Kroner 2, Lisa J Martin 1,3, Gurjit K Khurana Hershey 1,2,*
PMCID: PMC9119145  NIHMSID: NIHMS1727833  PMID: 34271060

Abstract

Background:

Low epidermal filaggrin (FLG) is a risk factor for atopic dermatitis (AD) and allergic co-morbidity. FLG mutations do not fully explain the variation in epidermal FLG levels, highlighting that other genetic loci may also regulate FLG expression.

Objective:

To identify genetic loci that regulate FLG expression and elucidate their functional and mechanistic consequences.

Methods:

A genome-wide association study (GWAS) of quantified skin FLG expression in lesional and baseline non(never)-lesional skin of children with AD in the Mechanisms of Progression of Atopic Dermatitis to Asthma in Children (MPAACH) cohort was conducted. CRISPR-Cas9 approaches were used to create isogenic human keratinocytes differing only at the identified variant rs11652075, and CARD14-deficient keratinocytes for subsequent mechanistic studies.

Results:

The GWAS identified the CARD14 rs11652075 variant to be associated with FLG expression in non(never)-lesional skin of children with AD. Rs11652075 is a CARD14 eQTL in human skin and primary human keratinocytes. The T variant destroys a functional CpG site resulting in reduced CpG methylation at this site (but not neighboring sites) in TT and CT compared to CC primary human keratinocytes and MPAACH children’s skin samples, and rs11652075 increases CARD14 expression in an allele-specific fashion. Further, studies in CRISPR-generated CC and TT isogenic keratinocytes, as well as CARD14-haplosufficient and deficient keratinocytes, reveal that IL-17A regulates FLG expression via CARD14, and that the underlying mechanisms are rs11652075 genotype-dependent.

Conclusion:

Our study identifies CARD14 as a novel regulator of FLG expression in the skin of children with AD. Further, CARD14 regulates skin FLG homeostasis in a rs11652075-dependent fashion.

Clinical Implication:

Our results highlight the CARD14 signaling pathway as a promising new target for therapeutic intervention in AD.

Keywords: Atopic Dermatitis, Filaggrin, CARD14, GWAS, Skin, Gene, Skin barrier

Capsule Summary:

Through a GWAS and subsequent functional analyses, the CARD14 variant rs11652075 was found to regulate FLG in non-lesional skin of children with AD, thus identifying a candidate pathway for novel therapeutics.

Introduction:

Atopic dermatitis (AD), or eczema, is a chronic, relapsing inflammatory skin disease affecting 15-30% of children globally and often precedes development of allergic asthma and other atopic diseases in the “atopic march” (1-3). A hallmark characteristic of AD is a dysfunctional skin barrier, which allows for epicutaneous penetration of environmental allergens and subsequent allergic sensitization (2). Loss-of-function mutations in the gene encoding filaggrin (FLG), a cornified envelope protein critical for epidermal barrier formation and function, are the most significant genetic risk factors for AD (4). However, low FLG expression levels are common in AD even in the absence of such mutations (4).

We recently found that low non(never)-lesional, but not lesional, skin FLG expression is associated with development of co-sensitization and moderate-severe AD (5), both key risk factors for future development of allergic AD co-morbidities (6). The importance of non-lesional skin FLG expression level in determining AD outcomes was similarly reported in another recent study (7). As such, there is a critical need to delineate factors that regulate FLG expression in non-lesional skin of patients with AD (2).

Herein we utilized the Mechanisms of Progression of Atopic Dermatitis to Asthma in Children (MPAACH) early life cohort of children with AD (5) to perform a GWAS to identify genetic variants associated with epidermal FLG levels and identified the common rs11652075 missense variant of the Caspase Activation and Recruitment Domain Family Member 14 (CARD14) gene as a novel genetic locus associated with reduced epidermal FLG expression in non-lesional skin. We found the variant destroys a CpG site and induces allelic CARD14 expression in keratinocytes, suggesting the variant is a CARD14 expression quantitative trait locus (eQTL). Finally, we show IL-17A, which activates CARD14 signaling in keratinocytes (8), suppresses FLG through CARD14 in a rs11652075 genotype-dependent manner. Taken together, our work identifies the CARD14 gene and its variant rs11652075 as regulators of FLG mRNA levels within skin.

Methods

Study Design and Approval.

The GWAS utilized the MPAACH cohort, whose recruitment details, exclusion and inclusion criteria have been detailed previously (5) and are summarized in the Online Methods. The MPAACH study was approved by the Cincinnati Children’s Hospital Medical Center Institutional Review Board under protocol number 2016-5842, and informed consent was provided by all subjects.

Genome Wide Association Study.

To test for the association between FLG levels and single nucleotide polymorphisms (SNPs), linear regression models of log-transformed (to address normality) non-lesional FLG were performed in PLINK (Version 1.07). Age, sex and race were included as co-variates. For the lesional analysis, non-lesional FLG was an additional covariate to account for baseline differences. Multiple testing correction and several quality control metrics were employed during the analysis. Details of FLG measurement and statistical methodology are available in the Online Repository.

Other Methods:

Please see the Online Methods and Table E1 for a thorough description of all methods and materials.

Results:

The CARD14 rs11652075 variant is associated with reduced non-lesional FLG levels in the epidermis of children with AD

The importance of non-lesional skin FLG expression level in determining AD outcomes has been reported by our group and others (5,7). To elucidate genetic variants that are associated with skin FLG expression, we performed a GWAS on MPAACH participants using non-lesional or lesional FLG mRNA levels as a continuous outcome variable. Skin FLG mRNA levels were quantified from mRNA isolated from skin tape strips taken from lesional (site of active or historical lesion) and non(never)-lesional (no current lesion and no history of a lesion at this site and >10 cm from a lesional site) sites from each MPAACH participant at the enrollment visit as previously described (5). The first 240 participants enrolled in MPAACH who passed genotyping QC and had data available for non-lesional skin FLG expression level were included in this study. Previous studies have validated the reproducibility and reliability of the FLG expression data (5), and that the skin tape strips sample stratum corneum keratinocytes (9). The characteristics of the genotyped cohort compared to the non-genotyped cohort and the overall cohort are detailed in Table E2. There was a small but significant difference in race.

Analyses of non-lesional FLG levels identified 2 SNPs, rs56344002 (p=3.8x10−7) and rs11652075 (p=4.2x10−7), that surpassed the multiple-testing correction significance threshold of 4.2x10−7 based on an LD-adjusted Bonferroni correction in order to account for correlation between variables and more appropriately control type I error than traditional Bonferroni correction, which assumes independence (10) (Figure 1A, Table E3). Rs56344002 is an intron variant located in NEUROTRIMIN (NTM), and rs11652075 is a missense variant in CARD14. Imputation of SNPs in the CARD14 and NTM genes reveals that there are several SNPs in moderate to strong LD with rs11652075 and rs56344002 that exhibit p-values <10−4, further supporting the significance of these two variants (Figure E1) in non-lesional skin. Genetic association of lesional FLG levels identified no SNPs that surpassed the multiple testing threshold (Figure 1A; Table E4). Race-stratified analyses support similar effects in the black and non-black populations (Tables E3 and E4).

Figure 1. GWAS of FLG expression, and CARD14 is highly expressed in epithelial tissues, where rs11652075 is an eQTL.

Figure 1.

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A. Manhattan plot of the GWAS of non-lesional and lesional FLG in the MPAACH cohort (N = 240). The horizontal dashed red line indicates the preset threshold of p = 4.2×10−7 B. Schematic of the location of rs11652075 within the CARD14 gene and the nucleotide change caused by the rs11652075. C. Tissue-specific expression of the CARD14 gene in median transcripts per million according to the Genomic Tissue Expression (GTEx) database. D. The tissue-specific normalized effect size (NES) of rs11652075 on CARD14 expression according to the GTEx database.

CARD14 is highly expressed in the skin, where the rs11652075 T allele is a candidate tissue-specific CARD14 eQTL

Rs11652075 is a C>T transition variant within exon 20 of the CARD14 gene (Figure 1B). CARD14 is a scaffolding protein that, upon activation, nucleates the assembly of B-cell lymphoma/leukemia 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) into the CARD14-BCL10-MALT1 (CBM) complex which induces nuclear factor kappa B (NFkB) and mitogen activated protein kinase (MAPK) signaling (11-14). Notably, NFkB and MAPK signaling have been previously implicated in FLG regulation (15-23), but this has not been examined in the context of CARD14 signaling. CARD14 variants, including rs11652075, are also associated with psoriasis, a distinct inflammatory skin disease that shares several characteristics with early-onset pediatric AD (11,24,25). Thus, we prioritized the rs11652075 variant for biologic investigation.

Publicly available Genotype-Tissue Expression (GTEx) data reveal that CARD14 expression is highest in epithelial tissues including sun-exposed and non-sun-exposed skin and esophageal mucosa (>15 transcripts per million (TPM)), whereas its expression is low (<0.6 TPM) in nearby tissues including the esophageal muscularis, fibroblasts, and subcutaneous adipose (Figure 1C), consistent with previous data (12). Furthermore, the normalized effect size (NES) of the rs11652075 T allele demonstrates that the T allele is associated with higher CARD14 expression in non-sun-exposed skin and the esophageal mucosa (Figure 1D). Conversely, the T allele is associated with reduced CARD14 expression in several tissues, including the esophageal muscularis, fibroblasts, and subcutaneous adipose. These findings indicate that CARD14 is highly expressed in the skin, and rs11652075 is a candidate tissue-specific CARD14 eQTL.

The rs11652075 variant destroys a cytosine-phosphate-guanine (CpG) site within the CARD14 gene

To determine why the rs11652075 T allele is associated with increased skin CARD14 expression (Figure 1D), we examined the variant’s genomic context and found that the cytosine affected by rs11652075 is within a putative CpG site (Figure 2A). CpG cytosines can be enzymatically methylated to epigenetically alter chromatin conformation and gene transcription (26). Given that CpG polymorphisms are associated with epigenetic and transcriptional changes (27-29), we next examined methylation of the rs11652075 CpG site in primary keratinocytes from individuals of each genotype to determine if it is a functional CpG that is destroyed by the C>T transition. Primary keratinocyte donor characteristics are detailed in Table E5. Pyrosequencing of bisulfite-converted DNA revealed that the rs11652075 CpG is 86.5% methylated in homozygous C allele (CC) keratinocytes, but that this methylation is decreased to 39.2% and 8.5% in heterozygous (CT) and homozygous T allele (TT) keratinocytes, respectively (Figure 2B). Importantly, pyrosequencing of bisulfite-converted DNA isolated from skin tape strips taken from MPAACH children exhibited similar methylation trends, with CC subjects exhibiting 94.4% methylation at the rs11652075 CpG, but only 53.9% and 9.9% methylation in CT and TT subjects, respectively (Figure 2C). Four neighboring CpGs downstream of rs11652075 exhibited only minor methylation changes in primary keratinocytes and no detectable changes in human skin, indicating that the effects of rs11652075 on methylation are specific to the rs11652075 CpG (Figure E2A and Figure E2B). These data demonstrate that the rs11652075 CpG is functional but is destroyed by the C>T transition. This finding suggests epigenetic mechanisms may contribute to the differential CARD14 expression observed in the GTEx data.

Figure 2. The rs11652075 T-allele abrogates methylation of CARD14 at the rs11652075 CpG and increases CARD14 expression in primary human keratinocytes.

Figure 2.

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A. Schematic of the location of the rs11652075 CpG site and other nearby CpGs. B-C. The percent methylation of the rs11652075 CpG site in (B) primary human donor keratinocytes and (C) human skin from MPAACH subjects of each rs11652075 genotype (CC, CT, TT). Data were analyzed using a one-way ANOVA with Tukey’s post-hoc tests. Primary keratinocytes: N = 3 (CT, TT) or 4 (CC) donors per genotype. MPAACH skin tapes: N = 5 (TT) or 7 (CC, CT) subjects per genotype. D. Schematic of the allele-specific qPCR method. E. The relative expression of CARD14 from the T allele versus C allele determined by allele-specific qPCR in primary keratinocytes from 3 separate donors heterozygous for rs11652075. Data were analyzed using a one-sample two-tailed t-test on the expression ratio using a reference value of 1. N = 6 (donors 894, 929) or 7 (donor 904) per donor. All data are represented as mean ± SD.

The rs11652075 T allele is expressed more efficiently than the C allele in keratinocytes

Since the rs11652075 T allele is associated with both increased CARD14 expression in skin (Figure 1D) and altered CARD14 methylation in primary keratinocytes and skin (Figure 2B and Figure 2C), we hypothesized that the transcriptional efficiency of CARD14 is greater from the T allele compared to the C allele. Since the variant is exonic we utilized allele-specific qPCR (Figure 2D) to calculate the ratio of transcripts expressed from the two rs11652075 alleles (a T:C ratio) in primary keratinocytes derived from three heterozygous (CT) individuals (Table E5). Expression was consistently higher from the T allele with the T:C ratio from three separate donors (894, 904, and 929) being 1.5, 1.34, and 1.49, respectively (Figure 2E). These results, which corroborate the GTEx data (Figure 1D), suggest that the rs11652075 T allele is expressed more efficiently than the C allele in keratinocytes and may thus be a keratinocyte CARD14 eQTL.

CARD14 mediates IL-17A-induced FLG suppression in a rs11652075 genotype-dependent fashion.

Since our GWAS identified the rs11652075 CARD14 variant to be associated with reduced non-lesional FLG, we investigated whether CARD14 signaling directly mediates FLG suppression. The cytokine IL-17A triggers CARD14 signaling in keratinocytes via its receptor IL-17R (8), which is constitutively expressed on keratinocytes (30). Interestingly, IL-17A is known to reduce keratinocyte FLG expression (15,16,31), and altered type 17 responses are implicated in AD pathogenesis—especially pediatric AD, which is the focus of the present study (25,32-37). For these studies we utilized HaCaT human keratinocytes, which are homozygous wild-type (CC) at rs11652075 (CARD14C/C, Figure E3), cultured in high-calcium medium (1.9 mM) to promote a more differentiated state. We treated HaCaT human keratinocytes with IL-17A for 24 hours with and without mepazine, a compound that disrupts CARD14 signaling by inhibiting MALT1 enzymatic activity (38,39). IL-17A treatment reduced FLG by 20.0%; however, mepazine co-treatment failed to rescue FLG expression (Figure 3A), suggesting that IL-17A-induced FLG suppression is not mediated by CARD14 signaling through MALT1 enzymatic activity in CARD14C/C HaCaT keratinocytes. The CARD14 target genes CCL20 and CXCL8 (encoding IL-8) (13,40) were both induced by IL-17A, but CXCL8 induction was prevented by mepazine co-treatment and a similar, but not significant, trend was observed for CCL20 in the CARD14C/C HaCaT keratinocytes (Figure 3A). However, as these trends were observed in CARD14C/C HaCaT keratinocytes that lacked the rs11652075 T allele, we were prompted to consider if the mechanisms underlying IL-17A-induced FLG suppression were rs11652075 genotype-dependent.

Figure 3. IL-17A-induced FLG suppression is rescued by the MALT1 inhibitor mepazine in a rs11652075 genotype-dependent manner.

Figure 3.

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A. The fold change expression of FLG, CCL20 and CXCL8 in CARD14C/C HaCaT keratinocytes upon IL-17A ± mepazine treatment. N = 6. B. Same as (A) but in primary keratinocytes of each rs11652075 genotype. N = 5. C. Fold change expression of FLG, CCL20 and CXCL8 in CRISPR-Cas9-generated CARD14T/T HaCaT keratinocytes isogenic to the CC HaCaT keratinocytes in panel A except at rs11652075. N = 6. All data were analyzed on non-normalized ddCt values using repeated-measures two-way ANOVAs with Tukey’s post-hoc tests. Data are shown normalized to control and are represented as mean ± SD. Symbols designate values from individual replicates. ns = not significant, NT = non-treated, M = mepazine.

To address this possibility, we stimulated primary human keratinocytes isolated from human donors of each rs11652075 genotype (Figure E3) with IL-17A ± mepazine and measured FLG, CCL20, and CXCL8 expression. The CC keratinocytes exhibited a trend similar to the CARD14C/C HaCaT keratinocytes: IL-17A reduced FLG by 60.4% and was unaffected by mepazine co-treatment. In CT and TT keratinocytes, IL-17A also suppressed FLG by 45.1% and 50.3% respectively; however, their response to mepazine co-treatment was notably different than CC keratinocytes: mepazine co-treatment not just rescued FLG, but actually induced FLG expression in CT and TT keratinocytes by 2.61- and 1.55-fold, respectively (Figure 3B). Furthermore, IL-17A-dependent CCL20 and CXCL8 induction in CT and TT keratinocytes was more effectively blunted by mepazine; this was most evident in the TT keratinocytes. These data support a rs11652075 genotype-dependent mechanism regulating FLG downstream of IL-17A.

Since primary keratinocytes are derived from heterogeneic individuals, we cannot discount that variants distinct from rs11652075 contribute to the differential IL-17A response. We therefore used CRISPR-Cas9 to knock-in the rs11652075 T allele into the CC HaCaT keratinocytes, thus generating homozygous T allele (CARD14T/T) HaCaT cells that are isogenic to CARD14C/C HaCaT cells (Figure E3). IL-17A treatment of CARD14T/T HaCaT keratinocytes resulted in a 33.9% reduction in FLG. However, similar to CT and TT primary keratinocytes but in contrast to CARD14C/C HaCaT cells and CC primary keratinocytes, mepazine co-treatment rescued FLG to above baseline, inducing its expression 1.21-fold (Figure 3C). Taken together, these results reveal that CARD14 signaling mediates IL-17A-induced FLG suppression in a rs11652075 genotype-dependent manner.

CARD14 knockout abrogates FLG suppression in HaCaT keratinocytes

To confirm that CARD14-mediated signaling is specifically involved in FLG regulation we used CRISPR-Cas9 to sequentially knockout both CARD14 alleles in CARD14C/C (also designated as CARD14+/+) HaCaT keratinocytes, thereby creating both CARD14 haploinsufficient (CARD14+/−) and deficient (CARD14−/−) HaCaT lines (Figure E4). Since endogenous CARD14 protein is undetectable even in CARD14+/+ HaCaT keratinocyte lysates, we confirmed knockdown efficacy by incorporating the CRISPR-Cas9-induced edits into CARD14-FLAG expression plasmids (Figure E5A), which failed to express CARD14 protein when transfected into HaCaT keratinocytes (Figure E5B). We observed a stepwise increase in baseline FLG expression with CARD14 allele loss, with haploinsufficient and deficient HaCaT strains exhibiting 2.24- and 3.81-fold higher FLG relative to wild-type. Whereas IL-17A reduced FLG levels by 24.3% in CARD14+/+ HaCaT keratinocytes, this effect was blunted to 19.4% in CARD14+/− HaCaT keratinocytes, and no significant reduction was detected in CARD14−/− HaCaT keratinocytes (Figure 4A). To evaluate the effects of CARD14 signaling on FLG expression independent of a cell-surface receptor (i.e. IL-17R), we treated CARD14+/+ HaCaT keratinocytes with phorbol 12-myristate 13-acetate (PMA) and ionomycin with and without mepazine pre-treatment (40). PMA/ionomycin (PI) alone suppressed FLG by 67.0%, but mepazine rescued expression by 50.0% (Figure 4B). However, as observed with IL-17A, the use of CARD14+/− HaCaT keratinocytes blunted PI-induced FLG suppression to an only 38.7% reduction, and no significant change was detected in CARD14−/− HaCaT cells (Figure 4C). Taken together, these data confirm that CARD14 has a functional role in regulating the FLG gene in keratinocytes.

Figure 4. CARD14 deficiency increases baseline FLG levels and rescues FLG suppression induced by IL-17A and PMA/ionomycin (PI).

Figure 4.

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A. Relative expression of FLG, CCL20 and CXCL8 at baseline and with IL-17A treatment in CARD14+/+, CARD14+/−, and CARD14−/− CRISPR-generated HaCaT keratinocytes isogenic except for rs11652075. N = 5. B. Fold change expression in wild-type CARD14C/C HaCaT keratinocytes upon 8 hours of PI ± mepazine. N = 5. C. Same as (A) but with PI treatment. N = 4. All data were analyzed on non-normalized ddCt values using repeated-measures two-way ANOVAs with Tukey’s post-hoc tests. Data are shown normalized to control and are represented as mean ± SD. Symbols designate values from individual replicates. ns = not significant; NT = non-treated; PI = PMA/ionomycin.

Discussion:

Herein, we conducted the first GWAS of skin FLG expression as an eQTL and identified CARD14 as an important regulator of FLG expression in non-lesional skin. No variants were significantly associated with FLG levels in lesional skin, possibly due to the effects of local inflammation on FLG expression overpowering genetic effects (41). Further, we demonstrate that the CARD14 rs11652075 variant is functional: the variant destroys a CpG and disrupts its normally high level of methylation, and CARD14 is transcribed more efficiently from the risk (T) allele than reference (C) allele. Finally, we show that CARD14 signaling regulates FLG downstream of IL-17A, and that the underlying mechanisms are rs11652075-dependent. Together, these experiments suggest a model in which the rs11652075 T allele increases CARD14 levels and activity resulting in reduced FLG expression, contributing to the skin barrier defect observed in AD (Figure 5).

Figure 5. A model for the mechanistic contribution of CARD14 rs11652075 to reduced FLG expression in non-lesional skin.

Figure 5.

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The T allele prevents methylation at the rs11652075 CpG site and increases CARD14 expression in heterozygous and homozygous keratinocytes. Since CARD14 regulates FLG expression, altered CARD14 levels and genotype-dependent signaling mechanisms contribute to suppressed FLG that may promote to a disrupted barrier phenotype in non-lesional skin. Me = methyl group.

Our study is the first unbiased genetic association study to link CARD14 and rs11652075 to skin barrier function and AD. This is likely due to our analytic approach, which aimed to identify variants associated with a specific quantitative AD risk factor (2) (FLG levels) rather than with AD presence versus absence (42). Only one previous study has examined CARD14 in the AD context, which associated two CARD14 loss-of-function variants with severe AD (43). However, the effects of these variants on FLG expression were not evaluated.

Our data clearly demonstrate that rs11652075 affects CARD14 methylation and expression. However, we cannot exclude other potential mechanisms that contribute to the cumulative impact of rs11652075 on AD. In addition to transcriptional changes, rs11652075 is also a missense variant (R820W) within the full-length CARD14 protein (24,44) and is thus likely to induce protein-level alterations. This is the focus of ongoing experiments.

Our study is the first to show that CARD14 signaling directly regulates FLG expression. Upon CARD14 activation and CBM assembly, MALT1 activates NFkB and major MAPK pathways (JNK, ERK and p38), and proteolytically inactivates enzymes that attenuate these pathways, such as A20 (11-14). Low A20 levels have been detected in the skin of human AD patients, and epidermal A20 deletion induces NFkB/MAPK activity and exacerbates experimental AD in mice (45). NFkB signaling is a known contributor to AD pathogenesis (46-48) and can suppress FLG (21). While JNK signaling induces FLG (18,19,22), conflicting reports exist regarding the impact of ERK and p38 on FLG expression (16,20,23). Notably, ERK and p38 suppress FLG in response to IL-17A (16), making them the likely pathways mediating FLG suppression downstream of CARD14. Furthermore, differential activation of these pathways may underly the rs11652075-dependent effects of CARD14 signaling on FLG regulation. Although FLG is expressed in monolayer, future experiments addressing these underlying mechanisms will utilize three-dimensional models (e.g., air-liquid interface and epidermal organoid models) that more optimally recapitulate FLG expression than monolayer culture, which is a limitation of the present study.

There is a growing appreciation for the role of IL-17A in AD (32-37). Elevated IL-17A levels have been observed in both the lesional and non-lesional skin of pediatric AD patients (25,37,49,50), and is thought to contribute to AD pathogenesis by promoting the differentiation of pro-allergic T-helper 2 cells (34) and by suppressing FLG in keratinocytes (15,16,31). While it is known that IL-17A stimulates CARD14 signaling (8), our study shows that CARD14 is necessary for IL-17A-induced FLG suppression. Furthermore, the effects of IL-17A on FLG appear to be rs11652075-dependent, with the T allele being associated with both reduced FLG levels and improved FLG rescue upon CBM inhibition. Our findings thus clearly demonstrate that CARD14 signaling is one mechanism by which IL-17A modulates keratinocyte FLG expression and contributes to AD pathogenesis.

IL-17A and the IL-23/IL-17A axis are also critical in the development of psoriasis and pityriasis rubra pilaris (PRP), two inflammatory skin diseases which, notably, are associated with CARD14 mutations (24,51-54). Despite being distinct diseases, early-onset pediatric AD and psoriasis share similar inflammatory environments (25). An unsupervised clustering analysis revealed that the molecular profile of pediatric non-lesional AD is more similar to that of adult psoriatic skin than adult AD skin, including elevated IL-17A, CCL20 and IL-8 (37). Multiple reports have noted PRP patients with gain-of-function CARD14 mutations who exhibit several AD-like features, including eczematous lesions, elevated total and allergen-specific IgE, and near absence of epidermal FLG (55,56). Aberrant CARD14 signaling may thus be the nexus between the pathogeneses of these distinct inflammatory skin conditions. Interestingly, the C allele of the rs11652075 variant has been linked to both psoriasis and PRP by several groups (54,57-67), though the underlying mechanisms are unclear. Further research is warranted to define the role of CARD14 in the pathogenesis of AD versus that of other inflammatory skin diseases, and how its role is influenced or conditioned by the presence of the rs11652075 C versus T allele.

Our findings highlight a novel role of CARD14 and the rs11652075 variant in the regulation of FLG in keratinocytes. Low epidermal FLG disrupts the skin barrier and is a significant risk factor for AD development (2,4,5). Since the T allele of rs11652075 is common (a 47.68% global minor allele frequency (68)), rs11652075 may contribute to reduced FLG levels and compromised barrier function in many individuals, underscoring the high impact of our studies. Our findings thus reveal the potential for targeting CARD14 signaling in the prevention or treatment of AD, particularly in CT and TT individuals.

Supplementary Material

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Acknowledgements:

We thank the participating families and staff on the MPAACH study. We also thank Dr. Dorothy Supp and Jennifer Hahn, M.S. for their assistance and for generously providing the primary human keratinocytes utilized in this study.

Funding:

This work was supported by U19AI70235 (G.K.K.H., J.M.B., and L.J.M.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Abbreviations:

AD

Atopic Dermatitis

BCL10

B-cell lymphoma/leukemia 10

CARD14

Caspase recruitment domain family member 14

Cas9

CRISPR associated protein 9

CBM

CARD14-BCL10-MALT1

CCL20

C-C motif chemokine ligand 20

CpG

Cytosine-phosphate-guanine

CRISPR-Cas9

Clustered regularly interspaced short palindromic repeats

CXCL8

C-X-C motif chemokine ligand 8

eQTL

Expression quantitative trail locus

ERK

Extracellular signal-regulated kinase

FLG

Filaggrin

GWAS

Genome-wide association study

GTEx

Genotype-Tissue Expression

IL

Interleukin

JNK

c-Jun N-terminal kinase

LD

Linkage disequilibrium

MALT

Mucosa-associated lymphoid tissue lymphoma translocation protein 1

MAPK

Mitogen activated protein kinase

MPAACH

Mechanisms of Progression of Atopic Dermatitis to Asthma in Children

NES

Normalized effect size

NFkB

Nuclear factor kappa B

PI

PMA/ionomycin

PMA

Phorbol 12-myristate 13-acetate

PRP

Pityriasis rubra pilaris

qPCR

Quantitative polymerase chain reaction

SNP

Single nucleotide polymorphism

TPM

Transcripts per million

Footnotes

Competing interests: The authors declare no conflicts of interest.

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