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Major differences between human atopic dermatitis and murine models, as determined by using global transcriptomic profiling

Published:October 01, 2016DOI:https://doi.org/10.1016/j.jaci.2016.08.029

      Background

      Atopic dermatitis (AD) is caused by a complex interplay between immune and barrier abnormalities. Murine models of AD are essential for preclinical assessments of new treatments. Although many models have been used to simulate AD, their transcriptomic profiles are not fully understood, and a comparison of these models with the human AD transcriptomic fingerprint is lacking.

      Objective

      We sought to evaluate the transcriptomic profiles of 6 common murine models and determine how they relate to human AD skin.

      Methods

      Transcriptomic profiling was performed by using microarrays and quantitative RT-PCR on biopsy specimens from NC/Nga, flaky tail, Flg-mutated, ovalbumin-challenged, oxazolone-challenged, and IL-23–injected mice. Gene expression data of patients with AD, psoriasis, and contact dermatitis were obtained from previous patient cohorts. Criteria of a fold change of 2 or greater and a false discovery rate of 0.05 or less were used for gene arrays.

      Results

      IL-23–injected, NC/Nga, and oxazolone-challenged mice show the largest homology with our human meta-analysis–derived AD transcriptome (37%, 18%, 17%, respectively). Similar to human AD, robust TH1, TH2, and also TH17 activation are seen in IL-23–injected and NC/Nga mice, with similar but weaker inflammation in ovalbumin-challenged mice. Oxazolone-challenged mice show a TH1-centered reaction, and flaky tail mice demonstrate a strong TH17 polarization. Flg-mutated mice display filaggrin downregulation without significant inflammation.

      Conclusion

      No single murine model fully captures all aspects of the AD profile; instead, each model reflects different immune or barrier disease aspects. Overall, among the 6 murine models, IL-23–injected mice best simulate human AD; still, the translational focus of the investigation should determine which model is most applicable.

      Key words

      Abbreviations used:

      AD (Atopic dermatitis), CD (Contact dermatitis), CLDN8 (Claudin 8), Ct (Threshold cycle), DEG (Differentially expressed gene), FCH (Fold change), FDR (False discovery rate), FLG (Filaggrin), LOR (Loricrin), MADAD (Meta-analysis–derived atopic dermatitis), OVA (Ovalbumin), OXA (Oxazolone), TLDA (TaqMan Low Density Array)
      Atopic dermatitis (AD) is the most common inflammatory skin disease
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      The Asian atopic dermatitis phenotype combines features of atopic dermatitis and psoriasis with increased TH17 polarization.
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      Although human testing is needed to elucidate the pathogenic contributions of various cytokines in patients with AD, murine AD models are essential for assessing new drugs at the preclinical level.
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      An ideal murine model should replicate both the epidermal barrier disruption and cutaneous inflammation of the human disease. Although many models have been used to simulate human AD, their transcriptomic profiles and relevance to the AD skin profile are not fully understood because a direct comparison between frequently used models and human AD is lacking. A number of spontaneous genetically engineered and inducible “AD-like” models have been described. These include NC/Nga,
      • Otsuka A.
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      • Egawa G.
      • Maekawa A.
      • Fujita T.
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      • et al.
      Possible new therapeutic strategy to regulate atopic dermatitis through upregulating filaggrin expression.
      flaky tail (Tmem79ma/ma Flgft/ft),
      • Saunders S.P.
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      • Porter R.M.
      • Cole C.
      • et al.
      Tmem79/Matt is the matted mouse gene and is a predisposing gene for atopic dermatitis in human subjects.
      • Moniaga C.S.
      • Egawa G.
      • Kawasaki H.
      • Hara-Chikuma M.
      • Honda T.
      • Tanizaki H.
      • et al.
      Flaky tail mouse denotes human atopic dermatitis in the steady state and by topical application with Dermatophagoides pteronyssinus extract.
      Flg-mutated (Flgft/ft),
      • Kawasaki H.
      • Nagao K.
      • Kubo A.
      • Hata T.
      • Shimizu A.
      • Mizuno H.
      • et al.
      Altered stratum corneum barrier and enhanced percutaneous immune responses in filaggrin-null mice.
      oxazolone (OXA)–challenged,
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      Characterization of a hapten-induced, murine model with multiple features of atopic dermatitis: structural, immunologic, and biochemical changes following single versus multiple oxazolone challenges.
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      • Homey B.
      • et al.
      Repeated epicutaneous exposures to ovalbumin progressively induce atopic dermatitis-like skin lesions in mice.
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      Furthermore, the IL-23–injected mouse model, which has been traditionally considered to resemble psoriasis
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      IL-23 stimulates epidermal hyperplasia via TNF and IL-20R2-dependent mechanisms with implications for psoriasis pathogenesis.
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      Genome-wide expression profiling of five mouse models identifies similarities and differences with human psoriasis.
      and exhibits the largest (25%) transcriptomic homology with human psoriasis fingerprinting among existing “psoriasis-like” models,
      • Suárez-Fariñas M.
      • Arbeit R.
      • Jiang W.
      • Ortenzio F.S.
      • Sullivan T.
      • Krueger J.G.
      Suppression of molecular inflammatory pathways by Toll-like receptor 7, 8, and 9 antagonists in a model of IL-23-induced skin inflammation.
      has also been found to display TH2 activation and simulate AD-like skin inflammation.
      • Bromley S.K.
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      • Ziegler S.F.
      • Luster A.D.
      IL-23 induces atopic dermatitis-like inflammation instead of psoriasis-like inflammation in CCR2-deficient mice.
      Although all murine AD-like models (with the exception of Flg-mutated mice)
      • Otsuka A.
      • Doi H.
      • Egawa G.
      • Maekawa A.
      • Fujita T.
      • Nakamizo S.
      • et al.
      Possible new therapeutic strategy to regulate atopic dermatitis through upregulating filaggrin expression.
      • Saunders S.P.
      • Goh C.S.
      • Brown S.J.
      • Palmer C.N.
      • Porter R.M.
      • Cole C.
      • et al.
      Tmem79/Matt is the matted mouse gene and is a predisposing gene for atopic dermatitis in human subjects.
      • Moniaga C.S.
      • Egawa G.
      • Kawasaki H.
      • Hara-Chikuma M.
      • Honda T.
      • Tanizaki H.
      • et al.
      Flaky tail mouse denotes human atopic dermatitis in the steady state and by topical application with Dermatophagoides pteronyssinus extract.
      • Kawasaki H.
      • Nagao K.
      • Kubo A.
      • Hata T.
      • Shimizu A.
      • Mizuno H.
      • et al.
      Altered stratum corneum barrier and enhanced percutaneous immune responses in filaggrin-null mice.
      • Man M.Q.
      • Hatano Y.
      • Lee S.H.
      • Man M.
      • Chang S.
      • Feingold K.R.
      • et al.
      Characterization of a hapten-induced, murine model with multiple features of atopic dermatitis: structural, immunologic, and biochemical changes following single versus multiple oxazolone challenges.
      • Wang G.
      • Savinko T.
      • Wolff H.
      • Dieu-Nosjean M.C.
      • Kemeny L.
      • Homey B.
      • et al.
      Repeated epicutaneous exposures to ovalbumin progressively induce atopic dermatitis-like skin lesions in mice.
      • Chan J.R.
      • Blumenschein W.
      • Murphy E.
      • Diveu C.
      • Wiekowski M.
      • Abbondanzo S.
      • et al.
      IL-23 stimulates epidermal hyperplasia via TNF and IL-20R2-dependent mechanisms with implications for psoriasis pathogenesis.
      are visibly inflamed, it is difficult to macroscopically differentiate AD-like dermatitis in mice from lesions mimicking contact dermatitis (CD) or psoriasis. For example, because flaky tail mice exhibit TH17-dominated skin inflammation,
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      this murine model might more closely simulate a psoriasis-like phenotype rather than AD-like fingerprinting.
      To evaluate the transcriptomic profiles of common AD models and determine how closely they resemble the intricate cytokine and epidermal abnormalities in AD skin, we profiled 5 common AD-like murine models, as well as the IL-23–injected model, which was previously considered to best mimic psoriasis. To be able to contrast all polar cytokine responses in human subjects, murine profiles were compared with transcriptomic profiles of AD and 2 other common inflammatory skin diseases, psoriasis and CD, that share clinical and tissue characteristics with AD.
      • Ewald D.
      • Malajian D.
      • Krueger J.
      • Workman C.
      • Wang T.
      • Tian S.
      • et al.
      Meta-analysis derived atopic dermatitis (MADAD) transcriptome defines a robust AD signature highlighting the involvement of atherosclerosis and lipid metabolism pathways.
      • Dhingra N.
      • Shemer A.
      • Correa da Rosa J.
      • Rozenblit M.
      • Fuentes-Duculan J.
      • Gittler J.K.
      • et al.
      Molecular profiling of contact dermatitis skin identifies allergen-dependent differences in immune response.
      • Tian S.
      • Krueger J.G.
      • Li K.
      • Jabbari A.
      • Brodmerkel C.
      • Lowes M.A.
      • et al.
      Meta-analysis derived (MAD) transcriptome of psoriasis defines the “core” pathogenesis of disease.
      We found that no single model fully captures all aspects of AD, but surprisingly, IL-23–injected mice show the most similar transcriptomic profile to human AD.

      Methods

       Murine models

      Transcriptomic profiling was performed with microarrays and quantitative RT-PCR (qRT-PCR) on biopsy specimens from 6 models, including NC/Nga, flaky tail, Flg-mutated, OVA-challenged, OXA-challenged, and IL-23–injected mice. Controls for murine models generally had similar genetic background, comprising the C57BL/6 (flaky tail, Flg-mutated, OVA, and IL-23), NC/Nga without mite infestation (NC/Nga), and BALB/c (OXA) laboratory strains. The samples of flaky tail, Flg-mutated, OVA-challenged, and NC/Nga mice were harvested at Kyoto University, Kyoto, Japan (Table I). Specimens from OXA-challenged mice were collected at Leo Pharma A/S (Ballerup, Denmark; Table I). The IL-23–injected mice gene array data were acquired from a previously published cohort (GSE50400),
      • Suárez-Fariñas M.
      • Arbeit R.
      • Jiang W.
      • Ortenzio F.S.
      • Sullivan T.
      • Krueger J.G.
      Suppression of molecular inflammatory pathways by Toll-like receptor 7, 8, and 9 antagonists in a model of IL-23-induced skin inflammation.
      and available RNA samples from these mice were used for qRT-PCR. See the Methods section in this article's Online Repository at www.jacionline.org for more details. The Institutional Animal Care and Use Committee of Kyoto University and Leo Pharma A/S approved all experiments.
      Table IMouse sample data
      SampleNo.AgeSexBiopsy locationSerum IgE level increaseDescriptionNo. of DEGs (all murine DEGs)
      Flaky tail526 wkFemaleEar+Flg and ma mutation226
      Flg mutated526 wkFemaleEarFlg mutation47
      C57BL/6526 wkFemaleEarControls for flaky tail and Flg-mutated mice
      NC/Nga (+mite)510 wkMaleEar+Mite exposure742
      NC/Nga (−mite)510 wkMaleEarNo mite exposure (controls for NC/Nga + mite)
      OVA challenged315 wkFemaleDorsal skin+C57BL/6 with OVA application373
      Control315 wkFemaleDorsal skinControls for OVA-challenged mice
      OXA challenged3∼10 wkFemaleEar+BALB/c with OXA application1004
      Acetone challenged3∼10 wkFemaleEarControls for OXA-challenged mice
      IL-23 injected56 wkFemaleDorsal skinNAC57BL/6 with IL-23 injections2753
      PBS injected56 wkFemaleDorsal skinNAControls for IL-23–injected mice
      NA, Not available.
      Representative images of each model are shown in Fig 1, A. Ear tissues from flaky tail, Flg-mutated, NC/Nga, and OXA-challenged mice were used, whereas dorsal skin was harvested from OVA-challenged and IL-23–injected mice. Flaky tail,
      • Moniaga C.S.
      • Egawa G.
      • Kawasaki H.
      • Hara-Chikuma M.
      • Honda T.
      • Tanizaki H.
      • et al.
      Flaky tail mouse denotes human atopic dermatitis in the steady state and by topical application with Dermatophagoides pteronyssinus extract.
      NC/Nga,
      • Matsuda H.
      • Watanabe N.
      • Geba G.P.
      • Sperl J.
      • Tsudzuki M.
      • Hiroi J.
      • et al.
      Development of atopic dermatitis-like skin lesion with IgE hyperproduction in NC/Nga mice.
      OVA-challenged,
      • Wang G.
      • Savinko T.
      • Wolff H.
      • Dieu-Nosjean M.C.
      • Kemeny L.
      • Homey B.
      • et al.
      Repeated epicutaneous exposures to ovalbumin progressively induce atopic dermatitis-like skin lesions in mice.
      and OXA-challenged
      • Man M.Q.
      • Hatano Y.
      • Lee S.H.
      • Man M.
      • Chang S.
      • Feingold K.R.
      • et al.
      Characterization of a hapten-induced, murine model with multiple features of atopic dermatitis: structural, immunologic, and biochemical changes following single versus multiple oxazolone challenges.
      models exhibit increased serum IgE levels. Flg-mutated mice show normal IgE levels,
      • Sasaki T.
      • Shiohama A.
      • Kubo A.
      • Kawasaki H.
      • Ishida-Yamamoto A.
      • Yamada T.
      • et al.
      A homozygous nonsense mutation in the gene for Tmem79, a component for the lamellar granule secretory system, produces spontaneous eczema in an experimental model of atopic dermatitis.
      and IgE data are unavailable for IL-23–injected mice (Table I).
      • Suárez-Fariñas M.
      • Arbeit R.
      • Jiang W.
      • Ortenzio F.S.
      • Sullivan T.
      • Krueger J.G.
      Suppression of molecular inflammatory pathways by Toll-like receptor 7, 8, and 9 antagonists in a model of IL-23-induced skin inflammation.
      Figure thumbnail gr1
      Fig 1A, Representative pictures of murine models. B, Venn diagram of overlapping unique HGNC symbol DEGs for 4 AD models. Flaky tail and Flg-mutated models are excluded because of their small numbers of DEGs. C, Proportion of MADAD DEGs represented in each model.
      • Ewald D.
      • Malajian D.
      • Krueger J.
      • Workman C.
      • Wang T.
      • Tian S.
      • et al.
      Meta-analysis derived atopic dermatitis (MADAD) transcriptome defines a robust AD signature highlighting the involvement of atherosclerosis and lipid metabolism pathways.
      IL-23–injected mice appear separated because these data are from our previous study (GSE50400).
      • Suárez-Fariñas M.
      • Arbeit R.
      • Jiang W.
      • Ortenzio F.S.
      • Sullivan T.
      • Krueger J.G.
      Suppression of molecular inflammatory pathways by Toll-like receptor 7, 8, and 9 antagonists in a model of IL-23-induced skin inflammation.
      D, Overlap between the top upregulated (red) and downregulated (dark blue) genes in each model and the MADAD transcriptome was estimated for rank N = 1 … 5000.
      • Lottaz C.
      • Yang X.
      • Scheid S.
      • Spang R.
      OrderedList—a bioconductor package for detecting similarity in ordered gene lists.
      The light blue line outlines the overlap expected by chance, such that any line above it indicates significant overlap.
      • Yang X.
      • Bentink S.
      • Scheid S.
      • Spang R.
      Similarities of ordered gene lists.
      HGNC, Human Genome Organization Gene Nomenclature Committee.

       Human cohorts

      AD microarray data were obtained from our meta-analysis–derived atopic dermatitis (MADAD) transcriptome,
      • Ewald D.
      • Malajian D.
      • Krueger J.
      • Workman C.
      • Wang T.
      • Tian S.
      • et al.
      Meta-analysis derived atopic dermatitis (MADAD) transcriptome defines a robust AD signature highlighting the involvement of atherosclerosis and lipid metabolism pathways.
      which was comprised of 4 AD lesional versus nonlesional data sets with a total of 97 samples (41 paired lesional/nonlesional samples).
      This study also includes a previously published cohort of 25 patients with AD (9 female and 16 male patients; age, 23-73 years [median, 45 years]) with SCORAD scores from 33 to 77 (mean, 56.7; SD, 12.2) and serum eosinophil counts of 0.6% to 11.8% (median, 4.3%); among patients with AD, 14 (56%) of 25 had extrinsic AD (serum IgE level, 254-3000 kU/L; median, 1292.8 kU/L).
      • Noda S.
      • Suárez-Fariñas M.
      • Ungar B.
      • Kim S.J.
      • de Guzman Strong C.
      • Xu H.
      • et al.
      The Asian atopic dermatitis phenotype combines features of atopic dermatitis and psoriasis with increased TH17 polarization.
      • Khattri S.
      • Shemer A.
      • Rozenblit M.
      • Dhingra N.
      • Czarnowicki T.
      • Finney R.
      • et al.
      Cyclosporine in patients with atopic dermatitis modulates activated inflammatory pathways and reverses epidermal pathology.
      Ten previously published patients with moderate-to-severe psoriasis were included for comparisons (Psoriasis Area Severity Index score, 8.4-59.5; mean, 20.3; SD, 14.5).
      • Noda S.
      • Suárez-Fariñas M.
      • Ungar B.
      • Kim S.J.
      • de Guzman Strong C.
      • Xu H.
      • et al.
      The Asian atopic dermatitis phenotype combines features of atopic dermatitis and psoriasis with increased TH17 polarization.
      • Johnson-Huang L.M.
      • Suárez-Fariñas M.
      • Sullivan-Whalen M.
      • Gilleaudeau P.
      • Krueger J.G.
      • Lowes M.A.
      Effective narrow-band UVB radiation therapy suppresses the IL-23/IL-17 axis in normalized psoriasis plaques.
      Thirteen previously published patients with allergic CD (n = 10 with nickel allergy and n = 3 with fragrance allergy) were included.
      • Dhingra N.
      • Shemer A.
      • Correa da Rosa J.
      • Rozenblit M.
      • Fuentes-Duculan J.
      • Gittler J.K.
      • et al.
      Molecular profiling of contact dermatitis skin identifies allergen-dependent differences in immune response.
      Biopsy specimens from positive patch tests to these allergens were taken at 72 hours from patch test placement. All these subjects were European Americans. Patients' characteristics are summarized in Table E1 in this article's Online Repository at www.jacionline.org, and the entire list is available in Table E2 in this article's Online Repository at www.jacionline.org.

       qRT-PCR and gene arrays

      RNA was extracted with EZ-PCR Core Reagents (Life Technologies, Grand Island, NY). RNA was reverse transcribed to cDNA by using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, Calif). TaqMan Low Density Array (TLDA) cards (384-well plates preloaded with TaqMan assays) were used for qRT-PCR. For IFN-γ, IL-13, and IL-17A, which are frequently undetectable with TLDA, single TaqMan quantitative PCR was performed. All primers are listed in Table E3 in this article's Online Repository at www.jacionline.org. For IL-23–injected mice, PCR analyses were performed by using stored samples from a previous report.
      • Suárez-Fariñas M.
      • Arbeit R.
      • Jiang W.
      • Ortenzio F.S.
      • Sullivan T.
      • Krueger J.G.
      Suppression of molecular inflammatory pathways by Toll-like receptor 7, 8, and 9 antagonists in a model of IL-23-induced skin inflammation.
      Expression values (threshold cycle [Ct]) were normalized to Rplp0.
      Affymetrix Mouse Gene 2.1 ST Arrays (Affymetrix, Santa Clara, Calif) were used for flaky tail, Flg-mutated, OVA-challenged, and NC/Nga mice. Affymetrix Mouse Gene 1.0 ST Arrays were used for OXA-challenged mice, whereas the previously published IL-23–injected murine transcriptomic data were analyzed with the GeneChip Mouse Genome 430 2.0 Array (Affymetrix).
      The MADAD transcriptome combined all effect sizes for each probe set and identified a set of 595 differentially expressed genes (DEGs; 387 upregulated and 208 downregulated) by using a classical fold change (FCH) of 2 or greater and a false discovery rate (FDR) of 0.05 or less criteria.
      • Ewald D.
      • Malajian D.
      • Krueger J.
      • Workman C.
      • Wang T.
      • Tian S.
      • et al.
      Meta-analysis derived atopic dermatitis (MADAD) transcriptome defines a robust AD signature highlighting the involvement of atherosclerosis and lipid metabolism pathways.
      For qRT-PCR comparison, we used previously published data obtained from the abovementioned human cohorts. Because nonlesional AD skin is abnormal,
      • Suárez-Fariñas M.
      • Tintle S.J.
      • Shemer A.
      • Chiricozzi A.
      • Nograles K.
      • Cardinale I.
      • et al.
      Nonlesional atopic dermatitis skin is characterized by broad terminal differentiation defects and variable immune abnormalities.
      we also included a comparison of lesional AD to normal skin.

       Bioinformatics and statistical analyses

      Murine microarray data were preprocessed in R software by using the justRMA function of the affy package and normalized by using the normalize.quantiles.robust or normalize.quantiles.use.target (IL-23 and OXA models) function of the preprocessCore package.
      • Gautier L.
      • Cope L.
      • Bolstad B.M.
      • Irizarry R.A.
      affy—analysis of Affymetrix GeneChip data at the probe level.

      preprocessCore: a collection of pre-processing functions. Version R package version 1.30.0. Available at: https://www.bioconductor.org/packages/devel/bioc/manuals/preprocessCore/man/preprocessCore.pdf.

      Changes in expression profiles of each murine model and its controls were performed by using the linear model framework of limma package.
      • Ritchie M.E.
      • Phipson B.
      • Wu D.
      • Hu Y.
      • Law C.W.
      • Shi W.
      • et al.
      limma powers differential expression analyses for RNA-sequencing and microarray studies.
      P values from moderated t tests were adjusted for multiple testing by using the Benjamini-Hochberg procedure.
      Cross-species annotation from murine to human ensemble IDs was performed with the getLDS function biomart package.
      • Durinck S.
      • Moreau Y.
      • Kasprzyk A.
      • Davis S.
      • De Moor B.
      • Brazma A.
      • et al.
      BioMart and Bioconductor: a powerful link between biological databases and microarray data analysis.
      • Correa da Rosa J.
      • Malajian D.
      • Shemer A.
      • Rozenblit M.
      • Dhingra N.
      • Czarnowicki T.
      • et al.
      Patients with atopic dermatitis have attenuated and distinct contact hypersensitivity responses to common allergens in skin.
      • Esaki H.
      • Ewald D.A.
      • Ungar B.
      • Rozenblit M.
      • Zheng X.
      • Xu H.
      • et al.
      Identification of novel immune and barrier genes in atopic dermatitis by means of laser capture microdissection.
      • Suárez-Fariñas M.
      • Ungar B.
      • Correa da Rosa J.
      • Ewald D.A.
      • Rozenblit M.
      • Gonzalez J.
      • et al.
      RNA sequencing atopic dermatitis transcriptome profiling provides insights into novel disease mechanisms with potential therapeutic implications.
      Linear models were applied to analyze TLDA and qRT-PCR expression data based on Rplp0 normalized and negative transformed Ct (−dCt) values. Before applying these models to TLDA and qRT-PCR expression data but after normalization, we replaced nondetected −dCt values with 20% of the minimum dCt for each gene across all samples, while keeping it in the possible range of −40 to 40.

      Results

       Transcriptomic profiling of AD-like murine models

      Although transcriptomic fingerprinting has been thoroughly performed in AD skin,
      • Suárez-Fariñas M.
      • Dhingra N.
      • Gittler J.
      • Shemer A.
      • Cardinale I.
      • de Guzman Strong C.
      • et al.
      Intrinsic atopic dermatitis shows similar TH2 and higher TH17 immune activation compared with extrinsic atopic dermatitis.
      • Noda S.
      • Suárez-Fariñas M.
      • Ungar B.
      • Kim S.J.
      • de Guzman Strong C.
      • Xu H.
      • et al.
      The Asian atopic dermatitis phenotype combines features of atopic dermatitis and psoriasis with increased TH17 polarization.
      • Suárez-Fariñas M.
      • Tintle S.J.
      • Shemer A.
      • Chiricozzi A.
      • Nograles K.
      • Cardinale I.
      • et al.
      Nonlesional atopic dermatitis skin is characterized by broad terminal differentiation defects and variable immune abnormalities.
      • Esaki H.
      • Ewald D.A.
      • Ungar B.
      • Rozenblit M.
      • Zheng X.
      • Xu H.
      • et al.
      Identification of novel immune and barrier genes in atopic dermatitis by means of laser capture microdissection.
      • Suárez-Fariñas M.
      • Ungar B.
      • Correa da Rosa J.
      • Ewald D.A.
      • Rozenblit M.
      • Gonzalez J.
      • et al.
      RNA sequencing atopic dermatitis transcriptome profiling provides insights into novel disease mechanisms with potential therapeutic implications.
      • Gittler J.K.
      • Shemer A.
      • Suárez-Fariñas M.
      • Fuentes-Duculan J.
      • Gulewicz K.J.
      • Wang C.Q.
      • et al.
      Progressive activation of T(H)2/T(H)22 cytokines and selective epidermal proteins characterizes acute and chronic atopic dermatitis.
      • Quaranta M.
      • Knapp B.
      • Garzorz N.
      • Mattii M.
      • Pullabhatla V.
      • Pennino D.
      • et al.
      Intraindividual genome expression analysis reveals a specific molecular signature of psoriasis and eczema.
      a similar analysis is lacking for AD-like murine models. To establish the individual transcriptomic profiles of prominent AD-like models and their similarity to human disease, we profiled skin from 5 common AD-like models,
      • Moniaga C.S.
      • Egawa G.
      • Kawasaki H.
      • Hara-Chikuma M.
      • Honda T.
      • Tanizaki H.
      • et al.
      Flaky tail mouse denotes human atopic dermatitis in the steady state and by topical application with Dermatophagoides pteronyssinus extract.
      • Kawasaki H.
      • Nagao K.
      • Kubo A.
      • Hata T.
      • Shimizu A.
      • Mizuno H.
      • et al.
      Altered stratum corneum barrier and enhanced percutaneous immune responses in filaggrin-null mice.
      • Man M.Q.
      • Hatano Y.
      • Lee S.H.
      • Man M.
      • Chang S.
      • Feingold K.R.
      • et al.
      Characterization of a hapten-induced, murine model with multiple features of atopic dermatitis: structural, immunologic, and biochemical changes following single versus multiple oxazolone challenges.
      • Wang G.
      • Savinko T.
      • Wolff H.
      • Dieu-Nosjean M.C.
      • Kemeny L.
      • Homey B.
      • et al.
      Repeated epicutaneous exposures to ovalbumin progressively induce atopic dermatitis-like skin lesions in mice.
      • Matsuda H.
      • Watanabe N.
      • Geba G.P.
      • Sperl J.
      • Tsudzuki M.
      • Hiroi J.
      • et al.
      Development of atopic dermatitis-like skin lesion with IgE hyperproduction in NC/Nga mice.
      as well as from the IL-23–injected model, which is considered to best represent the IL-17/IL-23–centered inflammation in patients with psoriasis,
      • Suárez-Fariñas M.
      • Arbeit R.
      • Jiang W.
      • Ortenzio F.S.
      • Sullivan T.
      • Krueger J.G.
      Suppression of molecular inflammatory pathways by Toll-like receptor 7, 8, and 9 antagonists in a model of IL-23-induced skin inflammation.
      by using gene arrays (Table I). Criteria of an FCH of 2 or greater and an FDR of 0.05 or less were used to define DEGs between diseased murine skin and respective control skin. Lists of DEGs for each model are listed in Table E4 in this article's Online Repository at www.jacionline.org.
      A Venn diagram based on unique Human Genome Organisation Gene Nomenclature Committee symbol orthologs and proportional to DEGs illustrates the overlap between murine models (Fig 1, B). Flaky tail and Flg-mutated models are excluded from the diagram because they display the lowest numbers of DEGs (n = 226 and n = 47, respectively), despite being among the largest sample sizes (n = 5, Table I). Overall, IL-23–injected mice show the highest number of DEGs (n = 2753), followed by OXA-challenged (n = 1004), NC/Nga (n = 742), and OVA-challenged (n = 373) mice. Only 67 DEGs are shared among NC/Nga, OVA-challenged, OXA-challenged, and IL-23–injected mice, as represented in the center of the Venn diagram (Fig 1, B).

       Murine models show differential profiles compared with human AD

      Each murine transcriptomic profile was compared with the MADAD transcriptome,
      • Ewald D.
      • Malajian D.
      • Krueger J.
      • Workman C.
      • Wang T.
      • Tian S.
      • et al.
      Meta-analysis derived atopic dermatitis (MADAD) transcriptome defines a robust AD signature highlighting the involvement of atherosclerosis and lipid metabolism pathways.
      a robust AD transcriptome across 4 independent cohorts, to compare how well the AD-like and IL-23–injected models represent human AD. The murine transcriptomes represent only 37%, 18%, 17%, and 11% of the human AD profile (MADAD) for IL-23–injected, NC/Nga, OXA-challenged, and OVA-challenged mice, respectively (Fig 1, C).
      • Ewald D.
      • Malajian D.
      • Krueger J.
      • Workman C.
      • Wang T.
      • Tian S.
      • et al.
      Meta-analysis derived atopic dermatitis (MADAD) transcriptome defines a robust AD signature highlighting the involvement of atherosclerosis and lipid metabolism pathways.
      • Suárez-Fariñas M.
      • Arbeit R.
      • Jiang W.
      • Ortenzio F.S.
      • Sullivan T.
      • Krueger J.G.
      Suppression of molecular inflammatory pathways by Toll-like receptor 7, 8, and 9 antagonists in a model of IL-23-induced skin inflammation.
      Only 4% of DEGs in flaky tail mice and 1% in Flg-mutated mice overlap with the MADAD transcriptome. The poor homology between human AD and the murine skin profiles is also illustrated as a heat map (see Fig E1 in this article's Online Repository at www.jacionline.org), showing relative expressions of MADAD genes within all murine transcriptomes. Fig E2 in this article's Online Repository at www.jacionline.org shows expression data by sample. Surprisingly, the transcriptome of IL-23–injected mice best represents the upregulated and downregulated components of the MADAD transcriptome, followed by NC/Nga, OXA-challenged, and OVA-challenged mice. Flg-mutated and flaky tail profiles are not well reflective of the MADAD transcriptome's increased or decreased elements. The MADAD DEGs with corresponding FCHs and FDRs are shown in Table E5 in this article's Online Repository at www.jacionline.org for each model.
      Because the choice of cutoffs and hybridization kits can influence intersections between DEGs of various transcriptomes,
      • Suárez-Fariñas M.
      • Lowes M.A.
      • Zaba L.C.
      • Krueger J.G.
      Evaluation of the psoriasis transcriptome across different studies by gene set enrichment analysis (GSEA).
      the MADAD transcriptome and transcriptomes of 6 murine models were also compared by using ranked gene lists instead of cutoffs, as previously described.
      • Swindell W.R.
      • Johnston A.
      • Carbajal S.
      • Han G.
      • Wohn C.
      • Lu J.
      • et al.
      Genome-wide expression profiling of five mouse models identifies similarities and differences with human psoriasis.
      • Suárez-Fariñas M.
      • Arbeit R.
      • Jiang W.
      • Ortenzio F.S.
      • Sullivan T.
      • Krueger J.G.
      Suppression of molecular inflammatory pathways by Toll-like receptor 7, 8, and 9 antagonists in a model of IL-23-induced skin inflammation.
      The 5000 highest upregulated and downregulated genes in each mouse transcriptome were identified and ranked according to FCHs, with lowest ranks assigned to the genes with highest regulations. We then analyzed the overlap of the MADAD transcriptome with ranked genes in each murine model. In Fig 1, D,
      • Lottaz C.
      • Yang X.
      • Scheid S.
      • Spang R.
      OrderedList—a bioconductor package for detecting similarity in ordered gene lists.
      • Yang X.
      • Bentink S.
      • Scheid S.
      • Spang R.
      Similarities of ordered gene lists.
      the red and blue lines correspond to the overlap between upregulated and downregulated genes, respectively, of murine models and the MADAD model at any given rank N, where N = 1, …, 5000. The light blue line outlines the level of overlap expected by chance (the 95% confidence region of hypergeometric distribution under the null hypothesis), such that any line above it indicates a significant level of overlap, and distances farther from it show a higher overlap.
      • Yang X.
      • Bentink S.
      • Scheid S.
      • Spang R.
      Similarities of ordered gene lists.
      For any given rank, IL-23–injected mice represent the highest and most significant overlap with the MADAD transcriptome and in decreasing order for OXA-challenged and NC/Nga mice. The OVA-challenged model has significant overlap with genes upregulated in human AD but does not capture the downregulated signature of human AD. The transcriptomes of flaky tail and Flg-mutated mice have poor overlap with human AD, which is consistent with the standard cutoff-based approaches (Fig 1, B and C).
      An enrichment analysis was also conducted by using Kyoto Encyclopedia of Genes and Genomes pathways to associate different mice profiles with relevant biological functions in human AD.
      • Rivals I.
      • Personnaz L.
      • Taing L.
      • Potier M.C.
      Enrichment or depletion of a GO category within a class of genes: which test?.
      • Zhang J.
      • Xing Z.
      • Ma M.
      • Wang N.
      • Cai Y.D.
      • Chen L.
      • et al.
      Gene ontology and KEGG enrichment analyses of genes related to age-related macular degeneration.
      Selected pathways significantly enriched in human AD and in murine models are shown in Fig 2, A, and Table E6 in this article's Online Repository at www.jacionline.org, with the black vertical line representing an FDR of 0.05. The genes represented in each pathway are listed in Table E7 in this article's Online Repository at www.jacionline.org. The most significantly enriched pathways in the MADAD transcriptome include peroxisome proliferator-activated receptor signaling, cytokine–cytokine receptor interaction, Janus kinase–signal transducer and activator of transcription signaling, T-cell receptor signaling, chemokine signaling, cell adhesion molecules, and adipocyte signaling pathways. IL-23–injected mice best simulate the inflammation and lipid pathways in human AD, with enrichments in some pathways also seen with NC/Nga and OXA-challenged mice. OVA-challenged mice only share the cytokine–cytokine receptor interaction, peroxisome proliferator-activated receptor signaling, and biosynthesis of unsaturated fatty acids pathways with the MADAD transcriptome. A single lipid pathway (biosynthesis of unsaturated fatty acids) is shared between Flg-mutated and flaky tail mice and the MADAD transcriptome, although this pathway is not significant for the MADAD transcriptome. Retinol metabolism, hedgehog signaling, and melanogenesis pathways are enriched in flaky tail mice but are not enriched in the MADAD transcriptome.
      Figure thumbnail gr2
      Fig 2A, Kyoto Encyclopedia of Genes and Genomes analysis in the respective MADAD and murine model transcriptomes. The black horizontal line indicates an FDR of 0.05. B, Heat map of the top 30 upregulated and downregulated MADAD DEGs with orthologs in murine models.
      • Ewald D.
      • Malajian D.
      • Krueger J.
      • Workman C.
      • Wang T.
      • Tian S.
      • et al.
      Meta-analysis derived atopic dermatitis (MADAD) transcriptome defines a robust AD signature highlighting the involvement of atherosclerosis and lipid metabolism pathways.
      Corresponding log2(FCH) values are shown. *P < .05, **P < .01, and ***P < .001. Coloring (blue, downregulated; red, upregulated) appears for an absolute log2(FCH) value of 1 or greater, with adjusted P values of less than .05. PPAR, Peroxisome proliferator-activated receptor.
      Fig 2, B,
      • Ewald D.
      • Malajian D.
      • Krueger J.
      • Workman C.
      • Wang T.
      • Tian S.
      • et al.
      Meta-analysis derived atopic dermatitis (MADAD) transcriptome defines a robust AD signature highlighting the involvement of atherosclerosis and lipid metabolism pathways.
      illustrates the top 30 upregulated and downregulated MADAD DEGs and their representation in each model (see Table E5). Again, IL-23–injected mice best represent not only the upregulated (eg, CCL18, S100A8, and keratin 16 [KRT16]) but also the downregulated DEGs (eg, claudin 8 [CLDN8]) in the MADAD transcriptome, whereas NC/Nga, OVA-challenged, and OXA-challenged mice mostly capture the upregulated but not the downregulated genes in the MADAD transcriptome. The flaky tail model only poorly represents key AD genes, and Flg-mutated mice entirely lack the top upregulated and downregulated human AD genes. The IL-23 model captures some lipid (elongation of very long-chain fatty acids [FEN1/Elo2, SUR4/Elo3, yeast]–like 3 [ELOVL3]), tight junction (CLDN8), and water channel (aquaporin 7) deficiencies, which were previously reported to characterize the barrier defects in patients with AD.
      • Ewald D.
      • Malajian D.
      • Krueger J.
      • Workman C.
      • Wang T.
      • Tian S.
      • et al.
      Meta-analysis derived atopic dermatitis (MADAD) transcriptome defines a robust AD signature highlighting the involvement of atherosclerosis and lipid metabolism pathways.
      • Esaki H.
      • Ewald D.A.
      • Ungar B.
      • Rozenblit M.
      • Zheng X.
      • Xu H.
      • et al.
      Identification of novel immune and barrier genes in atopic dermatitis by means of laser capture microdissection.
      • Westerberg R.
      • Tvrdik P.
      • Undén A.B.
      • Månsson J.E.
      • Norlén L.
      • Jakobsson A.
      • et al.
      Role for ELOVL3 and fatty acid chain length in development of hair and skin function.
      • Suárez-Fariñas M.
      • Gittler J.K.
      • Shemer A.
      • Cardinale I.
      • Krueger J.G.
      • Guttman-Yassky E.
      Residual genomic signature of atopic dermatitis despite clinical resolution with narrow-band UVB.

       Transcriptomic homology between mice and inflammatory skin diseases

      Because the signatures of the 6 murine models showed limited resemblance to human AD, we expanded our comparison to also include transcriptomic profiles of 2 other well-characterized polar inflammatory skin diseases: psoriasis and CD.
      • Noda S.
      • Suárez-Fariñas M.
      • Ungar B.
      • Kim S.J.
      • de Guzman Strong C.
      • Xu H.
      • et al.
      The Asian atopic dermatitis phenotype combines features of atopic dermatitis and psoriasis with increased TH17 polarization.
      • Khattri S.
      • Shemer A.
      • Rozenblit M.
      • Dhingra N.
      • Czarnowicki T.
      • Finney R.
      • et al.
      Cyclosporine in patients with atopic dermatitis modulates activated inflammatory pathways and reverses epidermal pathology.
      • Dhingra N.
      • Shemer A.
      • Correa da Rosa J.
      • Rozenblit M.
      • Fuentes-Duculan J.
      • Gittler J.K.
      • et al.
      Molecular profiling of contact dermatitis skin identifies allergen-dependent differences in immune response.
      • Johnson-Huang L.M.
      • Suárez-Fariñas M.
      • Sullivan-Whalen M.
      • Gilleaudeau P.
      • Krueger J.G.
      • Lowes M.A.
      Effective narrow-band UVB radiation therapy suppresses the IL-23/IL-17 axis in normalized psoriasis plaques.
      We then performed unsupervised clustering, with percentages reflecting robustness of each branch in the dendrogram (Fig 3, A).
      • Shimodaira H.
      An approximately unbiased test of phylogenetic tree selection.
      The resultant dendrogram establishes a tight cluster of inflammatory skin diseases, with clear separation from murine models. Flg-mutated and flaky tail mice cluster together and farther apart from other models. To elucidate the relative distances between individual models and human disease and to identify which murine model is closest to the human diseases, we performed a principal component analysis (Fig 3, B). The principal component analysis plot shows that murine models are separated from inflammatory skin diseases. IL-23–injected mice are closest to the AD profile (MADAD) on the PC1 axis, followed by CD (to fragrance) and surprisingly only then by psoriasis (Fig 3, B). Indeed, a principal variation component analysis also indicates that the organism explains 41.5% of the variations, with disease or mouse models accounting for only 12% (data not shown).
      Figure thumbnail gr3
      Fig 3Genome-wide neighboring analysis of the 8400 genes represented in human-murine orthologs. A, Dendrogram applying unsupervised clustering with the Pearson correlation distance and McQuitty agglomeration. Significant clusters have approximately unbiased (AU) P values of 95% or greater.
      • Shimodaira H.
      An approximately unbiased test of phylogenetic tree selection.
      B, Principal component analysis using log2(FCH) for the 8400 genes. PSO, Psoriasis.

       qRT-PCR highlights significant differences in polar immune activation among murine models

      qRT-PCR was performed on a wide array of hallmark human AD inflammatory and barrier genes that are often less than detection levels in arrays and share murine orthologs to validate and extend the microarray findings to polar cytokines. mRNA expressions were also compared with those obtained in our AD and psoriasis cohorts and, for selected available genes, also CD (to nickel and fragrance) populations (Fig 4).
      • Dhingra N.
      • Shemer A.
      • Correa da Rosa J.
      • Rozenblit M.
      • Fuentes-Duculan J.
      • Gittler J.K.
      • et al.
      Molecular profiling of contact dermatitis skin identifies allergen-dependent differences in immune response.
      Overall, similar patterns are seen in qRT-PCR and gene arrays. IL-23–injected, NC/Nga, OXA-challenged, and OVA-challenged mice show large increases in innate, TH2, and TH1 cytokine pathways (IL-1β, IL-13, and IFN-γ), as well as increased KRT16 (marking epidermal hyperplasia),
      • Hodak E.
      • Gottlieb A.B.
      • Anzilotti M.
      • Krueger J.G.
      The insulin-like growth factor 1 receptor is expressed by epithelial cells with proliferative potential in human epidermis and skin appendages: correlation of increased expression with epidermal hyperplasia.
      and the OXA-challenged model has the highest activation of the TH2 cytokine IL-13. Only IL-23–injected and NC/Nga mice reflect all diverse cytokine pathways, including IL-17A activation. The downregulation of terminal differentiation genes (filaggrin [FLG] and loricrin [LOR]) that characterizes AD is best captured by Flg-mutated and flaky tail models and to some extent by IL-23–injected, OVA, and OXA models but is missing in NC/Nga mice.
      Figure thumbnail gr4
      Fig 4Heat map of qRT-PCR–derived mRNA expression differences. CD data are shown separated because data from only 13 genes were available.
      • Dhingra N.
      • Shemer A.
      • Correa da Rosa J.
      • Rozenblit M.
      • Fuentes-Duculan J.
      • Gittler J.K.
      • et al.
      Molecular profiling of contact dermatitis skin identifies allergen-dependent differences in immune response.
      Corresponding log2(FCH) values are shown. +P < .1, *P < .05, **P < .01, and ***P < .001. Dendrograms represent unsupervised clustering with Euclidean distance and McQuitty agglomeration. LS, Lesional; N/A, not available; NL, nonlesional; PSO, psoriasis.

      Discussion

      Over the past decades, many murine AD-like models,
      • Moniaga C.S.
      • Egawa G.
      • Kawasaki H.
      • Hara-Chikuma M.
      • Honda T.
      • Tanizaki H.
      • et al.
      Flaky tail mouse denotes human atopic dermatitis in the steady state and by topical application with Dermatophagoides pteronyssinus extract.
      • Kawasaki H.
      • Nagao K.
      • Kubo A.
      • Hata T.
      • Shimizu A.
      • Mizuno H.
      • et al.
      Altered stratum corneum barrier and enhanced percutaneous immune responses in filaggrin-null mice.
      • Man M.Q.
      • Hatano Y.
      • Lee S.H.
      • Man M.
      • Chang S.
      • Feingold K.R.
      • et al.
      Characterization of a hapten-induced, murine model with multiple features of atopic dermatitis: structural, immunologic, and biochemical changes following single versus multiple oxazolone challenges.
      • Wang G.
      • Savinko T.
      • Wolff H.
      • Dieu-Nosjean M.C.
      • Kemeny L.
      • Homey B.
      • et al.
      Repeated epicutaneous exposures to ovalbumin progressively induce atopic dermatitis-like skin lesions in mice.
      • Matsuda H.
      • Watanabe N.
      • Geba G.P.
      • Sperl J.
      • Tsudzuki M.
      • Hiroi J.
      • et al.
      Development of atopic dermatitis-like skin lesion with IgE hyperproduction in NC/Nga mice.
      including humanized AD,
      • Carretero M.
      • Guerrero-Aspizua S.
      • Illera N.
      • Galvez V.
      • Navarro M.
      • García-García F.
      • et al.
      Differential features between chronic skin inflammatory diseases revealed in skin-humanized psoriasis and atopic dermatitis mouse models.
      topical vitamin D analogue application,
      • Turner M.J.
      • Dasilva-Arnold S.C.
      • Yi Q.
      • Mehrotra P.
      • Kaplan M.H.
      • Travers J.B.
      Topical application of a vitamin D analogue exacerbates atopic dermatitis and induces the atopic dermatitis-like phenotype in Stat6VT mice.
      • Li M.
      • Hener P.
      • Zhang Z.
      • Kato S.
      • Metzger D.
      • Chambon P.
      Topical vitamin D3 and low-calcemic analogs induce thymic stromal lymphopoietin in mouse keratinocytes and trigger an atopic dermatitis.
      or allergen/staphylococcal enterotoxin B models,
      • Turner M.J.
      • Dasilva-Arnold S.C.
      • Yi Q.
      • Mehrotra P.
      • Kaplan M.H.
      • Travers J.B.
      Topical application of a vitamin D analogue exacerbates atopic dermatitis and induces the atopic dermatitis-like phenotype in Stat6VT mice.
      • Li M.
      • Hener P.
      • Zhang Z.
      • Kato S.
      • Metzger D.
      • Chambon P.
      Topical vitamin D3 and low-calcemic analogs induce thymic stromal lymphopoietin in mouse keratinocytes and trigger an atopic dermatitis.
      • Ando T.
      • Matsumoto K.
      • Namiranian S.
      • Yamashita H.
      • Glatthorn H.
      • Kimura M.
      • et al.
      Mast cells are required for full expression of allergen/SEB-induced skin inflammation.
      have been developed as tools for understanding AD pathogenesis and as preclinical models. The most complete view of the human AD profile is based on transcriptomic profiling of skin lesions.
      • Suárez-Fariñas M.
      • Dhingra N.
      • Gittler J.
      • Shemer A.
      • Cardinale I.
      • de Guzman Strong C.
      • et al.
      Intrinsic atopic dermatitis shows similar TH2 and higher TH17 immune activation compared with extrinsic atopic dermatitis.
      • Noda S.
      • Suárez-Fariñas M.
      • Ungar B.
      • Kim S.J.
      • de Guzman Strong C.
      • Xu H.
      • et al.
      The Asian atopic dermatitis phenotype combines features of atopic dermatitis and psoriasis with increased TH17 polarization.
      • Ewald D.
      • Malajian D.
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      Meta-analysis derived atopic dermatitis (MADAD) transcriptome defines a robust AD signature highlighting the involvement of atherosclerosis and lipid metabolism pathways.
      Key AD features include prominent activation of inflammatory pathways and large defects in epidermal differentiation, tight junctions, and lipid profiles.
      • Suárez-Fariñas M.
      • Tintle S.J.
      • Shemer A.
      • Chiricozzi A.
      • Nograles K.
      • Cardinale I.
      • et al.
      Nonlesional atopic dermatitis skin is characterized by broad terminal differentiation defects and variable immune abnormalities.
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      • Rozenblit M.
      • Zheng X.
      • Xu H.
      • et al.
      Identification of novel immune and barrier genes in atopic dermatitis by means of laser capture microdissection.
      • Suárez-Fariñas M.
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      RNA sequencing atopic dermatitis transcriptome profiling provides insights into novel disease mechanisms with potential therapeutic implications.
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      • Kuo I.H.
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      • et al.
      Activation of epidermal toll-like receptor 2 enhances tight junction function: implications for atopic dermatitis and skin barrier repair.
      • De Benedetto A.
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      • Georas S.N.
      • Cheadle C.
      • et al.
      Tight junction defects in patients with atopic dermatitis.
      Unlike psoriasis, which is largely TH17/IL-23 polarized, AD shows more diverse cytokine expression, with TH2 but also TH22 pathway activation,
      • Noda S.
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      • Guttman-Yassky E.
      The translational revolution and use of biologics in patients with inflammatory skin diseases.
      with possible roles also suggested for the IL-23/TH17 axis, particularly in specific AD populations, such as Asian patients with AD.
      • Noda S.
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      • Ungar B.
      • Kim S.J.
      • de Guzman Strong C.
      • Xu H.
      • et al.
      The Asian atopic dermatitis phenotype combines features of atopic dermatitis and psoriasis with increased TH17 polarization.
      Global transcriptomic profiling of individual murine models is essential for understanding their distinct cytokine and barrier alterations and how well they resemble human AD fingerprinting compared with profiles of other common polar inflammatory skin diseases.
      • Swindell W.R.
      • Johnston A.
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      • et al.
      Genome-wide expression profiling of five mouse models identifies similarities and differences with human psoriasis.
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      • Krueger J.G.
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      This study is the first to evaluate the fingerprinting of commonly used AD-like (and a representative psoriasis-like) murine models and their relevance to human AD but also to psoriasis and CD.
      A proper model of human AD skin should appropriately represent its 2 main features: relevant cytokine networks and epidermal pathology. However, our data suggest that no single murine model is able to accurately simulate both aspects of AD. Our data separate the models into 2 clusters. One group includes flaky tail and Flg-mutated mice, which are least reminiscent of human AD, particularly of its immune skewing. Flaky tail mice show a TH17-dominated polarization, and Flg-mutated mice demonstrate no apparent clinical or tissue inflammation, which is quite different from the human AD profile. The other group, including IL-23–injected, NC/Nga, OXA-challenged, and OVA-challenged mice, show a broad range of polar immune activation. With the exception of the IL-23–injected model, which simulates some barrier features of human AD, such as reductions in tight junctions (CLDN8), lipids, and LOR, other models in this group mostly do not reflect the epidermal abnormalities of AD.
      Surprisingly, among all evaluated models, IL-23–injected mice show the largest overlap with human AD skin, which is unexpected because this model has been traditionally used for psoriasis.
      • Hedrick M.N.
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      • Shirakawa A.K.
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      • et al.
      CCR6 is required for IL-23-induced psoriasis-like inflammation in mice.
      This model demonstrates remarkable activation of innate immunity; the TH1, TH2, and TH17/IL-23 axes; and some epidermal alterations. Still, IL-23–injected mice have increased neutrophil counts and only sparse eosinophils and mast cells,
      • Suárez-Fariñas M.
      • Arbeit R.
      • Jiang W.
      • Ortenzio F.S.
      • Sullivan T.
      • Krueger J.G.
      Suppression of molecular inflammatory pathways by Toll-like receptor 7, 8, and 9 antagonists in a model of IL-23-induced skin inflammation.
      • Bromley S.K.
      • Larson R.P.
      • Ziegler S.F.
      • Luster A.D.
      IL-23 induces atopic dermatitis-like inflammation instead of psoriasis-like inflammation in CCR2-deficient mice.
      which can be found in human patients with AD.
      • Groneberg D.A.
      • Bester C.
      • Grützkau A.
      • Serowka F.
      • Fischer A.
      • Henz B.M.
      • et al.
      Mast cells and vasculature in atopic dermatitis—potential stimulus of neoangiogenesis.
      • Simon D.
      • Braathen L.R.
      • Simon H.U.
      Eosinophils and atopic dermatitis.
      Furthermore, FLG downregulation, which represents a hallmark of AD, is absent in this model. This model thus replicates the TH2 activation of AD, as well as the TH17/TH22 signature of psoriasis and some AD subtypes. Although IgE levels have not been evaluated in our IL-23 model,
      • Suárez-Fariñas M.
      • Arbeit R.
      • Jiang W.
      • Ortenzio F.S.
      • Sullivan T.
      • Krueger J.G.
      Suppression of molecular inflammatory pathways by Toll-like receptor 7, 8, and 9 antagonists in a model of IL-23-induced skin inflammation.
      • Bromley S.K.
      • Larson R.P.
      • Ziegler S.F.
      • Luster A.D.
      IL-23 induces atopic dermatitis-like inflammation instead of psoriasis-like inflammation in CCR2-deficient mice.
      IgE responses take at least several weeks to develop.
      • Wang G.
      • Savinko T.
      • Wolff H.
      • Dieu-Nosjean M.C.
      • Kemeny L.
      • Homey B.
      • et al.
      Repeated epicutaneous exposures to ovalbumin progressively induce atopic dermatitis-like skin lesions in mice.
      Perhaps, unlike other models, which have more chronic T-cell activation and are able to induce IgE responses over time, the IL-23 model is a short-term skin inflammation model (profile is created within 7 days).
      Although the NC/Nga model does not reflect the various barrier defects of human AD, including decreases in differentiation, tight junctions, and lipids (ie, FLG, LOR, CLDN8, and ELOVL3),
      • Ewald D.
      • Malajian D.
      • Krueger J.
      • Workman C.
      • Wang T.
      • Tian S.
      • et al.
      Meta-analysis derived atopic dermatitis (MADAD) transcriptome defines a robust AD signature highlighting the involvement of atherosclerosis and lipid metabolism pathways.
      • Suárez-Fariñas M.
      • Tintle S.J.
      • Shemer A.
      • Chiricozzi A.
      • Nograles K.
      • Cardinale I.
      • et al.
      Nonlesional atopic dermatitis skin is characterized by broad terminal differentiation defects and variable immune abnormalities.
      • Esaki H.
      • Ewald D.A.
      • Ungar B.
      • Rozenblit M.
      • Zheng X.
      • Xu H.
      • et al.
      Identification of novel immune and barrier genes in atopic dermatitis by means of laser capture microdissection.
      • Suárez-Fariñas M.
      • Ungar B.
      • Correa da Rosa J.
      • Ewald D.A.
      • Rozenblit M.
      • Gonzalez J.
      • et al.
      RNA sequencing atopic dermatitis transcriptome profiling provides insights into novel disease mechanisms with potential therapeutic implications.
      • Gittler J.K.
      • Shemer A.
      • Suárez-Fariñas M.
      • Fuentes-Duculan J.
      • Gulewicz K.J.
      • Wang C.Q.
      • et al.
      Progressive activation of T(H)2/T(H)22 cytokines and selective epidermal proteins characterizes acute and chronic atopic dermatitis.
      it clearly shows the diverse immune polarizations in human AD, including innate, TH1, TH2, and TH17 pathways. Thus NC/Nga mice might be useful to investigate the complex cytokine interactions but not the barrier aberrations of patients with AD. OVA-challenged mice also replicate activation of inflammatory pathways in patients with AD, although to a lesser extent than IL-23–injected and NC/Nga models.
      OXA-challenged mice demonstrate high TH1 and marginal TH2 inductions, with only limited barrier defects (LOR). IL-17A induction is absent in these mice, but a few downstream mediators (S100A8 and lipocalin 2 [LCN2]) are upregulated.
      Flaky tail mice show selective TH17 activation, as previously reported.
      • Oyoshi M.K.
      • Murphy G.F.
      • Geha R.S.
      Filaggrin-deficient mice exhibit TH17-dominated skin inflammation and permissiveness to epicutaneous sensitization with protein antigen.
      • Bonefeld C.M.
      • Petersen T.H.
      • Bandier J.
      • Agerbeck C.
      • Linneberg A.
      • Ross-Hansen K.
      • et al.
      Epidermal filaggrin deficiency mediates increased systemic T-helper 17 immune response.
      Although the TH17 axis is a central driver of psoriasis, some role of this pathway in patients with AD, including induction of the TH2 axis, has recently been suggested.
      • Noda S.
      • Suárez-Fariñas M.
      • Ungar B.
      • Kim S.J.
      • de Guzman Strong C.
      • Xu H.
      • et al.
      The Asian atopic dermatitis phenotype combines features of atopic dermatitis and psoriasis with increased TH17 polarization.
      • Koga C.
      • Kabashima K.
      • Shiraishi N.
      • Kobayashi M.
      • Tokura Y.
      Possible pathogenic role of Th17 cells for atopic dermatitis.
      • Nakajima S.
      • Kitoh A.
      • Egawa G.
      • Natsuaki Y.
      • Nakamizo S.
      • Moniaga C.S.
      • et al.
      IL-17A as an inducer for Th2 immune responses in murine atopic dermatitis models.
      Thus although flaky tail mice replicate some TH17 activation and reductions in FLG and LOR seen in patients with AD, data obtained from these mice should be applied cautiously to human AD because this model lacks TH2 activation, which is the hallmark of the AD immune response.
      Flg-mutated mice are the only murine model showing significant downregulation of FLG, similar to reductions observed in human AD.
      • Thyssen J.P.
      • Kezic S.
      Causes of epidermal filaggrin reduction and their role in the pathogenesis of atopic dermatitis.
      • Strid J.
      • McLean W.H.
      • Irvine A.D.
      Too much, too little or just enough: a goldilocks effect for il-13 and skin barrier regulation?.
      • Guttman-Yassky E.
      • Nograles K.E.
      • Krueger J.G.
      Contrasting pathogenesis of atopic dermatitis and psoriasis—part I: clinical and pathologic concepts.
      However, Flg-mutated mice lack both clinical and transcriptomic skin inflammation
      • Kawasaki H.
      • Nagao K.
      • Kubo A.
      • Hata T.
      • Shimizu A.
      • Mizuno H.
      • et al.
      Altered stratum corneum barrier and enhanced percutaneous immune responses in filaggrin-null mice.
      and additional barrier abnormalities that are seen in patients with AD, suggesting its limited use as a model to replicate the inflammation and global barrier defects in AD. A summary of clinical and laboratory characteristics of all analyzed models is provided in Table II.
      • Otsuka A.
      • Doi H.
      • Egawa G.
      • Maekawa A.
      • Fujita T.
      • Nakamizo S.
      • et al.
      Possible new therapeutic strategy to regulate atopic dermatitis through upregulating filaggrin expression.
      • Moniaga C.S.
      • Egawa G.
      • Kawasaki H.
      • Hara-Chikuma M.
      • Honda T.
      • Tanizaki H.
      • et al.
      Flaky tail mouse denotes human atopic dermatitis in the steady state and by topical application with Dermatophagoides pteronyssinus extract.
      • Kawasaki H.
      • Nagao K.
      • Kubo A.
      • Hata T.
      • Shimizu A.
      • Mizuno H.
      • et al.
      Altered stratum corneum barrier and enhanced percutaneous immune responses in filaggrin-null mice.
      • Man M.Q.
      • Hatano Y.
      • Lee S.H.
      • Man M.
      • Chang S.
      • Feingold K.R.
      • et al.
      Characterization of a hapten-induced, murine model with multiple features of atopic dermatitis: structural, immunologic, and biochemical changes following single versus multiple oxazolone challenges.
      • Wang G.
      • Savinko T.
      • Wolff H.
      • Dieu-Nosjean M.C.
      • Kemeny L.
      • Homey B.
      • et al.
      Repeated epicutaneous exposures to ovalbumin progressively induce atopic dermatitis-like skin lesions in mice.
      • Chan J.R.
      • Blumenschein W.
      • Murphy E.
      • Diveu C.
      • Wiekowski M.
      • Abbondanzo S.
      • et al.
      IL-23 stimulates epidermal hyperplasia via TNF and IL-20R2-dependent mechanisms with implications for psoriasis pathogenesis.
      • Bromley S.K.
      • Larson R.P.
      • Ziegler S.F.
      • Luster A.D.
      IL-23 induces atopic dermatitis-like inflammation instead of psoriasis-like inflammation in CCR2-deficient mice.
      Table IISummary of clinical and histologic features of mouse models
      ClinicalHistology
      Visible inflammationTEWL increaseSC water content reductionPruritic activitySerum IgE increaseEpidermal hyperplasiaParakeratosisT cellDC
      Flaky tail
      • Otsuka A.
      • Doi H.
      • Egawa G.
      • Maekawa A.
      • Fujita T.
      • Nakamizo S.
      • et al.
      Possible new therapeutic strategy to regulate atopic dermatitis through upregulating filaggrin expression.
      • Moniaga C.S.
      • Egawa G.
      • Kawasaki H.
      • Hara-Chikuma M.
      • Honda T.
      • Tanizaki H.
      • et al.
      Flaky tail mouse denotes human atopic dermatitis in the steady state and by topical application with Dermatophagoides pteronyssinus extract.
      +++++++++
      Flg mutated
      • Kawasaki H.
      • Nagao K.
      • Kubo A.
      • Hata T.
      • Shimizu A.
      • Mizuno H.
      • et al.
      Altered stratum corneum barrier and enhanced percutaneous immune responses in filaggrin-null mice.
      NC/Nga
      • Otsuka A.
      • Doi H.
      • Egawa G.
      • Maekawa A.
      • Fujita T.
      • Nakamizo S.
      • et al.
      Possible new therapeutic strategy to regulate atopic dermatitis through upregulating filaggrin expression.
      +++++++++
      Ova
      • Suárez-Fariñas M.
      • Dhingra N.
      • Gittler J.
      • Shemer A.
      • Cardinale I.
      • de Guzman Strong C.
      • et al.
      Intrinsic atopic dermatitis shows similar TH2 and higher TH17 immune activation compared with extrinsic atopic dermatitis.
      +++NA+++++
      OXA
      • Man M.Q.
      • Hatano Y.
      • Lee S.H.
      • Man M.
      • Chang S.
      • Feingold K.R.
      • et al.
      Characterization of a hapten-induced, murine model with multiple features of atopic dermatitis: structural, immunologic, and biochemical changes following single versus multiple oxazolone challenges.
      +++++++++
      IL-23 injected
      • Chan J.R.
      • Blumenschein W.
      • Murphy E.
      • Diveu C.
      • Wiekowski M.
      • Abbondanzo S.
      • et al.
      IL-23 stimulates epidermal hyperplasia via TNF and IL-20R2-dependent mechanisms with implications for psoriasis pathogenesis.
      • Bromley S.K.
      • Larson R.P.
      • Ziegler S.F.
      • Luster A.D.
      IL-23 induces atopic dermatitis-like inflammation instead of psoriasis-like inflammation in CCR2-deficient mice.
      +NANANANA++++
      DC, Dendritic cell; NA, not available; SC, stratum corneum; TEWL, transepidermal water loss.
      Human inflammatory skin diseases share a number of features but maintain distinct differences in tissue structure and immune activation. Although downregulation of differentiation genes clearly differentiates AD from the other 2 inflammatory skin diseases, other immune and epidermal features are shared among the 3 conditions. These characteristics include increased epidermal hyperplasia and activation of TH1, TH17, and TH22 pathways, although, as expected, TH2 polarization is minimal in patients with psoriasis compared with that seen in patients with AD.
      • Noda S.
      • Suárez-Fariñas M.
      • Ungar B.
      • Kim S.J.
      • de Guzman Strong C.
      • Xu H.
      • et al.
      The Asian atopic dermatitis phenotype combines features of atopic dermatitis and psoriasis with increased TH17 polarization.
      Our study has several limitations. First, only mRNA and not protein levels were measured, and assessments were limited to a single time point. Second, we mainly used B6 mice, although BALB/c mice might be more appropriate in certain cases. Third, we did not perform functional in vivo tests (ie, transepidermal water loss, corneal water content, and pruritus measures).
      • Otsuka A.
      • Doi H.
      • Egawa G.
      • Maekawa A.
      • Fujita T.
      • Nakamizo S.
      • et al.
      Possible new therapeutic strategy to regulate atopic dermatitis through upregulating filaggrin expression.
      • Man M.Q.
      • Hatano Y.
      • Lee S.H.
      • Man M.
      • Chang S.
      • Feingold K.R.
      • et al.
      Characterization of a hapten-induced, murine model with multiple features of atopic dermatitis: structural, immunologic, and biochemical changes following single versus multiple oxazolone challenges.
      The biopsy specimens were taken from different locations and at different ages in mice, possibly affecting gene expression.
      • Swindell W.R.
      • Johnston A.
      • Sun L.
      • Xing X.
      • Fisher G.J.
      • Bulyk M.L.
      • et al.
      Meta-profiles of gene expression during aging: limited similarities between mouse and human and an unexpectedly decreased inflammatory signature.
      Finally, comparisons with different AD endotypes were not possible here and should be addressed in future larger cohort studies of various ages and races.
      Overall, no single murine model fully captures all immune and barrier aspects of human AD skin, and phenotypic differences between AD and murine models are significant, regardless of which model is chosen. Nevertheless, of the models studied, IL-23–injected mice appear to best replicate the human AD profile. IL-23–injected and NC/Nga mice demonstrate a wide array of inflammatory axes, including TH2 activation, and are best suited to assess this AD-centric axis and how it interacts with other activated cytokines in patients with AD. OXA-challenged and OVA-challenged mice, despite showing remarkable inflammation, are not necessarily AD specific. IL-23–injected, OXA-challenged, and OVA-challenged models have the advantage that inducing agents can be used on different mouse backgrounds, including existing knockouts of specific genes, to dissect cutaneous inflammation. Flaky tail mice do not capture any key features of human AD. Flg-mutated mice are best only to represent the FLG downregulation in patients with AD but not other AD characteristics. Thus the choice of which murine model to use depends on the translational focus of the investigator, but caution should be exercised when translating murine data to human inflammatory skin diseases.
      Clinical implications
      When testing new drugs for AD, murine models can be used to study barrier or immune features, but human trials are needed to determine effects on the actual disease profile.

      Supplementary data

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