Transcriptomic analysis of human skin wound healing and rejuvenation following ablative fractional laser treatment.

Ablative fractional laser treatment is considered the gold standard for skin rejuvenation. In order to understand how fractional laser works to rejuvenate skin, we performed microarray profiling on skin biopsies to identify temporal and dose-response changes in gene expression following fractional laser treatment. The backs of 14 women were treated with ablative fractional laser (Fraxel®) and 4 mm punch biopsies were collected from an untreated site and at the treated sites 1, 3, 7, 14, 21 and 28 days after the single treatment. In addition, in order to understand the effect that multiple fractional laser treatments have on skin rejuvenation, several sites were treated sequentially with either 1, 2, 3, or 4 treatments (with 28 days between treatments) followed by the collection of 4 mm punch biopsies. RNA was extracted from the biopsies, analyzed using Affymetrix U219 chips and gene expression was compared between untreated and treated sites. We observed dramatic changes in gene expression as early as 1 day after fractional laser treatment with changes remaining elevated even after 1 month. Analysis of individual genes demonstrated significant and time related changes in inflammatory, epidermal, and dermal genes, with dermal genes linked to extracellular matrix formation changing at later time points following fractional laser treatment. When comparing the age-related changes in skin gene expression to those induced by fractional laser, it was observed that fractional laser treatment reverses many of the changes in the aging gene expression. Finally, multiple fractional laser treatments, which cover different regions of a treatment area, resulted in a sustained or increased dermal remodeling response, with many genes either differentially regulated or continuously upregulated, supporting previous observations that maximal skin rejuvenation requires multiple fractional laser treatments. In conclusion, fractional laser treatment of human skin activates a number of biological processes involved in wound healing and tissue regeneration.

Combinations of peptides synergistically activate the regenerative capacity of skin cells in vitro.

OBJECTIVE: To explore synergistic effects related to skin regeneration, peptides with distinct biological mechanisms of action were evaluated in combination with different skin cell lines in the presence or absence of niacinamide (Nam). Furthermore, the synergistic responses of peptide combinations on global gene expression were compared with the changes that occur with fractional laser resurfacing treatment, a gold standard approach for skin rejuvenation, to further define optimal peptide combinations. METHODS: Microarray profiling was used to characterize the biological responses of peptide combinations (+/- Nam) relative to the individual components in epidermal keratinocyte and dermal fibroblast cell lines. Cellular functional assays were utilized to confirm the synergistic effects of peptide combinations. Bioinformatics approaches were used to link the synergistic effects of peptide combinations on gene expression to the transcriptomics of the skin rejuvenation response from fractional laser treatment. RESULTS: Microarray analysis of skin cells treated with peptide combinations revealed synergistic changes in gene expression compared with individual peptide controls. Bioinformatic analysis of synergy genes in keratinocytes revealed the activation of NRF2-mediated oxidative stress responses by a combination of Ac-PPYL, Pal-KTTKS and Nam. Additional analysis revealed direct downstream transcriptional targets of NRF2/ARE exhibiting synergistic regulation by this combination of materials, which was corroborated by a cellular reporter assay. NRF2-mediated oxidative stress response pathways were also found to be activated in the transcriptomics of the early skin rejuvenation response to fractional laser treatment, suggesting the importance of this biology in the early stages of tissue repair. Additionally, the second combination of peptides (pal-KT and Ac-PPYL) was found to synergistically restore cellular ATP levels that had been depleted due to the presence of ROS, indicating an additional mechanism, whereby peptide synergies may accelerate skin repair. CONCLUSION: Through combinatorial synergy studies, we have identified additional in vitro skin repair mechanisms beyond the previously described functions of individual peptides and correlated these to the transcriptomics of the skin rejuvenation response of fractional laser treatment. These findings suggest that specific peptides can act together, via complementary and synergistic mechanisms, to holistically enhance the regenerative capacity of in vitro skin cells.

OBJECTIF: Pour explorer les effets synergiques liés à la régénération cutanée, les peptides ayant des mécanismes d'action biologiques distincts ont été évalués en association dans différentes lignées cellulaires cutanées en présence ou en l'absence de niacinamide (Nam). De plus, les réponses synergiques des associations de peptides sur l'expression des gènes globale ont été comparées aux changements qui surviennent avec le traitement de resurfaçage au laser fractionné, une approche de référence pour le rajeunissement de la peau, afin de définir davantage les associations optimales de peptides. MÉTHODES: Le profilage de micro-réseau a été utilisé pour caractériser les réponses biologiques des combinaisons de peptides (+/-Nam) par rapport aux composants individuels dans les lignées cellulaires de kératinocytes épidermiques et de fibroblastes dermiques. Des tests fonctionnels cellulaires ont été réalisés pour confirmer les effets synergiques des associations de peptides. Des approches bio-informatiques ont été utilisées pour mettre en lien les effets synergiques des associations de peptides sur l'expression des gènes à la transcriptomique de la réponse de rajeunissement de la peau du traitement au laser fractionné. RÉSULTATS: L'analyse par micro-réseau des cellules cutanées traitées par des combinaisons de peptides a révélé des changements synergiques dans l'expression des gènes par rapport aux contrôles peptidiques individuels. L'analyse bio-informatique des gènes de synergie dans les kératinocytes a révélé une activation des réponses au stress oxydatif médiées par NRF2 par une association d'Ac-PPYL, de Pal-KTTKS et de Nam. Une analyse supplémentaire a révélé des cibles transcriptionnelles directes en aval de NRF2/ARE présentant une régulation synergique par cette combinaison de matériaux, qui a été corroborée par un test de gène rapporteur. Les voies de réponses au stress oxydatif médiées par NRF2 se sont également révélées activées dans la transcriptomique de la réponse précoce de rajeunissement cutané au traitement au laser fractionné, ce qui suggère l'importance de cette biologie dans les stades précoces de la réparation des tissus. De plus, une deuxième association de peptides (pal-KT et Ac-PPYL) s'est avérée restaurer de manière synergique les taux d'ATP cellulaire qui avaient été épuisés en raison de la présence de ROS, indiquant un mécanisme supplémentaire par lequel les synergies de peptides pourraient accélérer la réparation cutanée. CONCLUSION: Grâce à des études de synergie combinatoire, nous avons identifié des mécanismes de réparation cutanés in vitro supplémentaires au-delà des fonctions précédemment décrites des peptides individuels et les avons corrélés à la transcriptomique de la réponse de rajeunissement de la peau au traitement au laser fractionné. Ces résultats suggèrent que des peptides spécifiques peuvent agir ensemble, par le biais de mécanismes complémentaires et synergiques, pour améliorer de manière globale la capacité régénérative des cellules cutanées in vitro.

The vitamin A ester retinyl propionate has a unique metabolic profile and higher retinoid-related bioactivity over retinol and retinyl palmitate in human skin models.

Human skin is exposed daily to environmental stressors, which cause acute damage and inflammation. Over time, this leads to morphological and visual appearance changes associated with premature ageing. Topical vitamin A derivatives such as retinol (ROL), retinyl palmitate (RPalm) and retinyl propionate (RP) have been used to reverse these changes and improve the appearance of skin. This study investigated a stoichiometric comparison of these retinoids using in vitro and ex vivo skin models. Skin biopsies were treated topically to compare skin penetration and metabolism. Treated keratinocytes were evaluated for transcriptomics profiling and hyaluronic acid (HA) synthesis and treated 3D epidermal skin equivalents were stained for epidermal thickness, Ki67 and filaggrin. A retinoic acid receptor-alpha (RARα) reporter cell line was used to compare retinoid activation levels. Results from ex vivo skin found that RP and ROL have higher penetration levels compared with RPalm. RP is metabolized primarily into ROL in the viable epidermis and dermis whereas ROL is esterified into RPalm and metabolized into the inactive retinoid 14-hydroxy-4,14-retro-retinol (14-HRR). RP treatment yielded higher RARα activation and HA synthesis levels than ROL whereas RPalm had a null effect. In keratinocytes, RP and ROL stimulated similar gene expression patterns and pathway theme profiles. In conclusion, RP and ROL show a similar response directionality whereas RPalm response was inconsistent. Additionally, RP has a consistently higher magnitude of response compared with ROL or RPalm.

Metabolic dysfunction in human skin: Restoration of mitochondrial integrity and metabolic output by nicotinamide (niacinamide) in primary dermal fibroblasts from older aged donors.

Alterations in metabolism in skin are accelerated by environmental stressors such as solar radiation, leading to premature aging. The impact of aging on mitochondria is of interest given their critical role for metabolic output and the finding that environmental stressors cause lowered energy output, particularly in fibroblasts where damage accumulates. To better understand these metabolic changes with aging, we performed an in-depth profiling of the expression patterns of dermal genes in face, forearm, and buttock biopsies from females of 20-70 years of age that encode for all subunits comprising complexes I-V of the mitochondrial electron transport chain. This complements previous preliminary analyses of these changes. "Oxidative phosphorylation" was the top canonical pathway associated with aging in the face, and genes encoding for numerous subunits had decreased expression patterns with age. Investigations on fibroblasts from older aged donors also showed decreased gene expression of numerous subunits from complexes I-V, oxidative phosphorylation rates, spare respiratory capacity, and mitochondrial number and membrane potential compared to younger cells. Treatment of older fibroblasts with nicotinamide (Nam) restored these measures to younger cell levels. Nam increased complexes I, IV, and V activity and gene expression of representative subunits. Elevated mt-Keima staining suggests a possible mechanism of action for these restorative effects via mitophagy. Nam also improved mitochondrial number and membrane potential in younger fibroblasts. These findings show there are significant changes in mitochondrial functionality with aging and that Nam treatment can restore bioenergetic efficiency and capacity in older fibroblasts with an amplifying effect in younger cells.

A population-based gene expression signature of molecular clock phase from a single epidermal sample.

BACKGROUND: For circadian medicine to influence health, such as when to take a drug or undergo a procedure, a biomarker of molecular clock phase is required--one that is easily measured and generalizable across a broad population. It is not clear that any circadian biomarker yet satisfies these criteria. METHODS: We analyzed 24-h molecular rhythms in human dermis and epidermis at three distinct body sites, leveraging both longitudinal (n = 20) and population (n = 154) data. We applied cyclic ordering by periodic structure (CYCLOPS) to order the population samples where biopsy time was not recorded. With CYCLOPS-predicted phases, we used ZeitZeiger to discover potential biomarkers of clock phase. RESULTS: Circadian clock function was strongest in the epidermis, regardless of body site. We identified a 12-gene expression signature that reported molecular clock phase to within 3 h (mean error = 2.5 h) from a single sample of epidermis--the skin's most superficial layer. This set performed well across body sites, ages, sexes, and detection platforms. CONCLUSIONS: This research shows that the clock in epidermis is more robust than dermis regardless of body site. To encourage ongoing validation of this putative biomarker in diverse populations, diseases, and experimental designs, we developed SkinPhaser--a user-friendly app to test biomarker performance in datasets ( https://github.com/gangwug/SkinPhaser ).

Niacinamide mitigates SASP-related inflammation induced by environmental stressors in human epidermal keratinocytes and skin.

OBJECTIVE: To evaluate whether niacinamide (Nam) can mitigate production of inflammatory and senescence-related biomarkers induced by environmental stressors. METHODS: Human epidermal keratinocytes were exposed to UVB, urban dust, diesel exhaust and cigarette smoke extract and treated with Nam or vehicle control. Full thickness 3-D skin organotypic models were exposed to a combination of UVB and PM2.5 and treated with Nam or vehicle control. Quantitation of the SASP-related inflammatory mediators PGE2 , IL-6 and IL-8 was performed on cultured media. UVB-exposed keratinocytes treated with and without Nam were immunostained for the senescence biomarker Lamin B1 (LmnB1). Transcriptomics profiling of cigarette smoke extract effects on keratinocytes was performed. A double-blind, placebo-controlled clinical was conducted on 40 female panellists that were pretreated on back sites for two weeks with 5% Nam or vehicle and then exposed to 1.5 minimal erythemal dose (MED) solar-simulated radiation (SSR). Treated sites were compared with non-treated exposed sites for erythema and the skin surface IL-1αRA/IL-1α inflammatory biomarkers. RESULTS: Ultraviolet B induced synthesis of PGE2 , IL-8 and IL-6 and reduced LmnB1 levels in keratinocytes. Urban dust and diesel exhaust only stimulated synthesis of IL-8 whereas cigarette smoke extract only stimulated levels of PGE2 . In all exposures, treatment with Nam significantly mitigated synthesis of the inflammatory mediators and restored levels of UVB-reduced LmnB1. In the 3D skin equivalent model, Nam reduced IL-8 levels stimulated by a combination of topical PM2.5 and UV exposure. In a UV challenge clinical, pretreatment with 5% Nam reduced erythema and skin surface IL-1αRA/IL-1α inflammatory biomarkers that were induced by SSR. CONCLUSION: Since it is known that Nam has anti-inflammatory properties, we tested whether Nam can inhibit environmental stress-induced inflammation and senescence-associated secretory phenotype (SASP) biomarkers. We show Nam can reduce PGE2 , IL-6 and IL-8 levels induced by environmental stressors. Additionally, in vivo pretreatment with Nam can reduce UV-induced erythema and skin surface inflammatory biomarkers. These findings add to the body of evidence that Nam can mitigate the skin's inflammatory response elicited by environmental stressors. This supports Nam can potentially inhibit senescence and premature ageing and thereby maintain skin's functionality and appearance.

OBJECTIF: Évaluer si le niacinamide (Nam) peut atténuer la production de biomarqueurs inflammatoireset liés à la sénescence induits par les facteurs de stress environnementaux. MÉTHODES: Leskératinocytes épidermiques H uman ont été exposés aux UVB, à la poussière urbaine, aux gaz d'échappement diesel et à l'extrait de fumée de cigarette et traités avec nam ou contrôle de véhicule. Les modèles organotypic de peau 3D de pleine épaisseur ont été exposés à une combinaison d'UVB et de PM2.5 et traités avec nam ou commande de véhicule. La quantitation des médiateurs inflammatoires liés à la SASP PGE2 ,IL-6 et IL-8 a été réalisée sur des médias cultivés. Les kératinocytes exposés aux UVB traités avec et sans Nam étaient immunotachés pour le biomarqueur de sénescence Lamin B1 (LmnB1). Le profilage de transcriptomique des effets d'extrait de fumée de cigarette sur les kératinocytes a été exécuté. Un placebo contrôlé clinique à double insu a été menée sur 40 panélistes féminins qui ont été prétraités sur les sites arrière pendant deux semaines avec 5% Nam ou véhicule, puis exposés à 1,5 dose erythémique minimale (MED) rayonnement solaire simulé (SSR). Les sites traités ont été comparés à des sites exposés non traités pour l'érythème et la surface de la peau IL-1▫RA/IL-1▫ biomarqueurs inflammatoiress. RÉSULTATS: Synthèse induite par UVB des niveaux de PGE2, IL-8 et IL-6 et réduit de LmnB1 dans les kératinocytes. La poussière urbaine et les gaz d'échappement diesel n'ont stimulé que la synthèse de l'IL-8 alors que l'extrait de fumée de cigarette ne stimulait que les niveaux de PGE2 . Dans toutes les expositions, le traitement avec Nam a significativement atténué la synthèse des médiateurs inflammatoires et les niveaux restaurés de LmnB1 UVB-réduit. Dans le modèle équivalent de la peau 3D, Nam a réduit les niveaux d'IL-8 stimulés par une combinaison de PM combination of topical PM 2.5 topique et d'exposition aux UV. Dans un uv-défi clinique, prétraitement avec 5% Nam réduit érythème et la surface de la peau IL-1▫RA/IL-1▫ biomarqueurs inflammatoires qui ont été induits par SSR. CONCLUSION: Puisqu'il est connu que Nam a des propriétés anti-inflammatoires, nous avons testé si Nam peut inhiber l'inflammation induite par le stressenvironnementaltion et les biomarqueurs sécrétoires sécrétoires sécrétoires (SASP) associés à la sénescence. We montrent Nam peut réduire PGE2 ,IL-6, et IL-8 niveaux induits par les facteurs de stress environnementaux. En outre, le prétraitement in vivo avec Nam peut réduire l'érythème induit par les UV et les biomarqueurs inflammatoires de surface de la peau. Ces résultats ajoutent à l'oody bde la preuve que Nam peut atténuer la réponse inflammatoire de la peau provoquée par lesfacteurs de stress environnementaux. Cela soutient Nam peut potentiellement inhiber la sénescence et le vieillissement prématuré et ainsi maintenir la fonctionnalité de la peau et l'apparence.

Transcriptomic Analysis Links Eosinophilic Esophagitis and Atopic Dermatitis.

Background: Eosinophilic esophagitis (EoE) is commonly associated with concomitant atopic diseases including atopic dermatitis (AD) and allergic airway (AA) diseases including asthma. Despite this link and the shared pathologic features across these three disorders, detailed analyses of the unifying molecular pathways are lacking. Objectives: We sought to investigate the mRNA expression profile overlap between EoE, AA, and AD and to identify the involvement of interleukin 13 (IL-13) in modulating gene expression. Methods: Whole-genome mRNA expression analyses were performed on tissue specimens (biopsies or nasal brushes) from patients with EoE, AD, and AA, and IL-13-stimulated primary human epithelial cells from the esophagus, the skin, and the airways. Results: By human disease expression profiles, EoE evidenced a significantly higher overlap (p = 0.0006) with AD (181 transcripts; 10%) than with AA (124 transcripts, 7%). Only 18 genes were found to be commonly dysregulated among the three diseases; these included filaggrin, histamine receptor H1, claudin 1, cathepsin C, plasminogen activator urokinase receptor, and suppressor of cytokine signaling 3. Ontogenetic analysis revealed a common immune/inflammatory response among the three diseases and a different epithelial response (epidermal cell differentiation) between EoE and AA. The overlap between the IL-13-stimulated epithelial cell transcriptome and the respective disease transcriptome was 22, 9, and 5% in EoE, AD, and AA, respectively, indicating a greater involvement of the IL-13 pathway in EoE than AA (p = 0.0007) or AD (p = 0.02). Conclusion: EoE, AD, and AA share a common set of disease-specific transcripts, highlighting common molecular etiology. Their comparative analysis indicates relatively closer relationships between EoE and AD, particularly centered around IL-13-driven pathways. Therefore, these findings provide an increased rationale for shared therapeutic strategies.

Population-level rhythms in human skin with implications for circadian medicine.

Skin is the largest organ in the body and serves important barrier, regulatory, and sensory functions. The epidermal layer shows rhythmic physiological responses to daily environmental variation (e.g., DNA repair). We investigated the role of the circadian clock in the transcriptional regulation of epidermis using a hybrid experimental design, in which a limited set of human subjects (n = 20) were sampled throughout the 24-h cycle and a larger population (n = 219) were sampled once. We found a robust circadian oscillator in human epidermis at the population level using pairwise correlations of clock and clock-associated genes in 298 epidermis samples. We then used CYCLOPS to reconstruct the temporal order of all samples, and identified hundreds of rhythmically expressed genes at the population level in human epidermis. We compared these results with published time-series skin data from mice and found a strong concordance in circadian phase across species for both transcripts and pathways. Furthermore, like blood, epidermis is readily accessible and a potential source of biomarkers. Using ZeitZeiger, we identified a biomarker set for human epidermis that is capable of reporting circadian phase to within 3 hours from a single sample. In summary, we show rhythms in human epidermis that persist at the population scale and describe a path to develop robust single-sample circadian biomarkers.

Phenotypic Characterization of Eosinophilic Esophagitis in a Large Multicenter Patient Population from the Consortium for Food Allergy Research.

BACKGROUND: Eosinophilic esophagitis (EoE) is increasingly common, but data on phenotypic aspects are still incomplete. OBJECTIVES: To describe the clinical, endoscopic, and histopathologic features of a large number of children and adults with EoE across the United States. METHODS: This was a multisite single visit registry enrolling subjects aged 6 months to 65 years with EoE. Participants provided responses regarding their medical history, with verification of the diagnosis and history by the study teams. RESULTS: A total of 705 subjects were analyzed (median [interquartile range] age at enrollment 11.2 [6.7-17.7] years, 68.2% male, 87.9% whites). Of these, 67 subjects had concurrent gastrointestinal eosinophilia, with gastric mucosa most common. An age- and race-dependent time gap was present between symptom onset and time of diagnosis (adults and whites with longer gap). Food allergy and atopic dermatitis were associated with a decrease in this gap. Symptoms varied with age (more dysphagia and food impaction in adults) and with race (more vomiting in non-whites). Esophageal rings and strictures at diagnosis were more common in adults, although esophageal eosinophilia was comparable among age groups. Concomitant allergic disease (91%), infectious/immunologic disorders (44%), neurodevelopmental disorders (30%), and failure to thrive (21%) were common. Depression/anxiety increased with age. EoE was reported in 3% of parents and 4.5% of siblings. CONCLUSIONS: Gastrointestinal eosinophilia is present in approximately 10% of patients with EoE; the symptom-diagnosis time gap is influenced by age, race, food allergy, and atopic dermatitis; symptoms vary with race; concurrent infectious/immunologic disorders and mental health disorders are common; and the level of esophageal eosinophils is comparable in patients with and without fibrostenotic features.

Analysis of gene expression profiles of multiple skin diseases identifies a conserved signature of disrupted homeostasis.

Triggers of skin disease pathogenesis vary, but events associated with the elicitation of a lesion share many features in common. Our objective was to examine gene expression patterns in skin disease to develop a molecular signature of disruption of cutaneous homeostasis. Gene expression data from common inflammatory skin diseases (eg psoriasis, atopic dermatitis, seborrhoeic dermatitis and acne) and a novel statistical algorithm were used to define a unifying molecular signature referred to as the "unhealthy skin signature" (USS). Using a pattern-matching algorithm, analysis of public data repositories revealed that the USS is found in diverse epithelial diseases. Studies of milder disruptions of epidermal homeostasis have also shown that these conditions converge, to varying degrees, on the USS and that the degree of convergence is related directly to the severity of homeostatic disruption. The USS contains genes that had no prior published association with skin, but that play important roles in many different disease processes, supporting the importance of the USS to homeostasis. Finally, we show through pattern matching that the USS can be used to discover new potential dermatologic therapeutics. The USS provides a new means to further interrogate epithelial homeostasis and potentially develop novel therapeutics with efficacy across a spectrum of skin conditions.

Whole-exome sequencing uncovers oxidoreductases DHTKD1 and OGDHL as linkers between mitochondrial dysfunction and eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is an allergic inflammatory esophageal disorder with a complex underlying genetic etiology often associated with other comorbidities. Using whole-exome sequencing (WES) of 63 patients with EoE and 60 unaffected family members and family-based trio analysis, we sought to uncover rare coding variants. WES analysis identified 5 rare, damaging variants in dehydrogenase E1 and transketolase domain-containing 1 (DHTKD1). Rare variant burden analysis revealed an overabundance of putative, potentially damaging DHTKD1 mutations in EoE (P = 0.01). Interestingly, we also identified 7 variants in the DHTKD1 homolog oxoglutarate dehydrogenase-like (OGDHL). Using shRNA-transduced esophageal epithelial cells and/or patient fibroblasts, we further showed that disruption of normal DHTKD1 or OGDHL expression blunts mitochondrial function. Finally, we demonstrated that the loss of DHTKD1 expression increased ROS production and induced the expression of viperin, a gene previously shown to be involved in production of Th2 cytokines in T cells. Viperin had increased expression in esophageal biopsies of EoE patients compared with control individuals and was upregulated by IL-13 in esophageal epithelial cells. These data identify a series of rare genetic variants implicating DHTKD1 and OGDHL in the genetic etiology of EoE and underscore a potential pathogenic role for mitochondrial dysfunction in EoE.

Profound loss of esophageal tissue differentiation in patients with eosinophilic esophagitis.

BACKGROUND: A key question in the allergy field is to understand how tissue-specific disease is manifested. Eosinophilic esophagitis (EoE) is an emerging tissue-specific allergic disease with an unclear pathogenesis. OBJECTIVE: Herein we tested the hypothesis that a defect in tissue-specific esophageal genes is an integral part of EoE pathogenesis. METHODS: We interrogated the pattern of expression of esophagus-specific signature genes derived from the Human Protein Atlas in the EoE transcriptome and in EPC2 esophageal epithelial cells. Western blotting and immunofluorescence were used for evaluating expression of esophageal proteins in biopsy specimens from control subjects and patients with active EoE. Whole-exome sequencing was performed to identify mutations in esophagus-specific genes. RESULTS: We found that approximately 39% of the esophagus-specific transcripts were altered in patients with EoE, with approximately 90% being downregulated. The majority of transcriptional changes observed in esophagus-specific genes were reproduced in vitro in esophageal epithelial cells differentiated in the presence of IL-13. Functional enrichment analysis revealed keratinization and differentiation as the most affected biological processes and identified IL-1 cytokines and serine peptidase inhibitors as the most dysregulated esophagus-specific protein families in patients with EoE. Accordingly, biopsy specimens from patients with EoE evidenced a profound loss of tissue differentiation, decreased expression of keratin 4 (KRT4) and cornulin (CRNN), and increased expression of KRT5 and KRT14. Whole-exome sequencing of 33 unrelated patients with EoE revealed 39 rare mutations in 18 esophagus-specific differentially expressed genes. CONCLUSIONS: A tissue-centered analysis has revealed a profound loss of esophageal tissue differentiation (identity) as an integral and specific part of the pathophysiology of EoE and implicated protease- and IL-1-related activities as putative central pathways in disease pathogenesis.

In vitro model for studying esophageal epithelial differentiation and allergic inflammatory responses identifies keratin involvement in eosinophilic esophagitis.

Epithelial differentiation is an essential physiological process that imparts mechanical strength and barrier function to squamous epithelia. Perturbation of this process can give rise to numerous human diseases, such as atopic dermatitis, in which antigenic stimuli can penetrate the weakened epithelial barrier to initiate the allergic inflammatory cascade. We recently described a simplified air-liquid interface (ALI) culture system that facilitates the study of differentiated squamous epithelia in vitro. Herein, we use RNA sequencing to define the genome-wide transcriptional changes that occur within the ALI system during epithelial differentiation and in response to allergic inflammation. We identified 2,191 and 781 genes that were significantly altered upon epithelial differentiation or dysregulated in the presence of interleukin 13 (IL-13), respectively. Notably, 286 genes that were modified by IL-13 in the ALI system overlapped with the gene signature present within the inflamed esophageal tissue from patients with eosinophilic esophagitis (EoE), an allergic inflammatory disorder of the esophagus that is characterized by elevated IL-13 levels, altered epithelial differentiation, and pro-inflammatory gene expression. Pathway analysis of these overlapping genes indicated enrichment in keratin genes; for example, the gene encoding keratin 78, an uncharacterized type II keratin, was upregulated during epithelial differentiation (45-fold) yet downregulated in response to IL-13 and in inflamed esophageal tissue from patients. Thus, our findings delineate an in vitro experimental system that models epithelial differentiation that is dynamically regulated by IL-13. Using this system and analyses of patient tissues, we identify an altered expression profile of novel keratin differentiation markers in response to IL-13 and disease activity, substantiating the potential of this combined approach to identify relevant molecular processes that contribute to human allergic inflammatory disease.

TNF-related apoptosis-inducing ligand (TRAIL) regulates midline-1, thymic stromal lymphopoietin, inflammation, and remodeling in experimental eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is an inflammatory disorder of the esophagus defined by eosinophil infiltration and tissue remodeling with resulting symptoms of esophageal dysfunction. TNF-related apoptosis-inducing ligand (TRAIL) promotes inflammation through upregulation of the E3 ubiquitin-ligase midline-1 (MID1), which binds to and deactivates the catalytic subunit of protein phosphatase 2Ac, resulting in increased nuclear factor κB activation. OBJECTIVE: We sought to elucidate the role of TRAIL in EoE. METHODS: We used Aspergillus fumigatus to induce EoE in TRAIL-sufficient (wild-type) and TRAIL-deficient (TRAIL(-/-)) mice and targeted MID1 in the esophagus with small interfering RNA. We also treated mice with recombinant thymic stromal lymphopoietin (TSLP) and TRAIL. RESULTS: TRAIL deficiency and MID1 silencing with small interfering RNA reduced esophageal eosinophil and mast cell numbers and protected against esophageal circumference enlargement, muscularis externa thickening, and collagen deposition. MID1 expression and nuclear factor κB activation were reduced in TRAIL(-/-) mice, whereas protein phosphatase 2Ac levels were increased compared with those seen in wild-type control mice. This was associated with reduced expression of CCL24, CCL11, CCL20, IL-5, IL-13, IL-25, TGFB, and TSLP. Treatment with TSLP reconstituted hallmark features of EoE in TRAIL(-/-) mice and recombinant TRAIL induced esophageal TSLP expression in vivo in the absence of allergen. Post hoc analysis of gene array data demonstrated significant upregulation of TRAIL and MID1 in a cohort of children with EoE compared with that seen in controls. CONCLUSION: TRAIL regulates MID1 and TSLP, inflammation, fibrosis, smooth muscle hypertrophy, and expression of inflammatory effector chemokines and cytokines in experimental EoE.

Genome-wide association analysis of eosinophilic esophagitis provides insight into the tissue specificity of this allergic disease.

Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder associated with allergic hypersensitivity to food. We interrogated >1.5 million genetic variants in EoE cases of European ancestry and subsequently in a multi-site cohort with local and out-of-study control subjects. In addition to replicating association of the 5q22 locus (meta-analysis P=1.9×10(-16)), we identified an association at 2p23 spanning CAPN14 (P=2.5×10(-10)). CAPN14 was specifically expressed in the esophagus, was dynamically upregulated as a function of disease activity and genetic haplotype and after exposure of epithelial cells to interleukin (IL)-13, and was located in an epigenetic hotspot modified by IL-13. Genes neighboring the top 208 EoE-associated sequence variants were enriched for esophageal expression, and multiple loci for allergic sensitization were associated with EoE susceptibility (4.8×10(-2)

Genetic and epigenetic underpinnings of eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a complex, polygenic disorder caused by genetic predisposition and environmental exposures. Because of the recent emergence of EoE as a bona fide global health concern, a paucity of available therapeutic and diagnostic options exists. However, rapid progress has been made in an effort to rectify this lack and to improve understanding of the factors that cause EoE. This article highlights key advances in elucidating the genetic (and epigenetic) components involved in EoE.

Genetics of eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a complex genetic disorder characterized by eosinophilic inflammation within the esophagus. Multiple epidemiological studies estimate the prevalence of EoE is 4 in 10,000, with a higher disease prevalence in individuals of European ancestry and in males, highlighting a genetic etiology of the disease. EoE has often been noted to occur in multiple family members, particularly siblings, in a non-Mendelian pattern, indicating the heritable component of EoE is likely complex in nature. Although EoE is a newly diagnosed disorder involving a complex polygenic etiology, much progress has been made towards identifying the molecular pathways contributing to the disease pathogenesis and the genetic variants associated with disease susceptibility using a variety of approaches (genome-wide and candidate gene) as well as study designs (case-control and family-based cohorts). Here, we discuss the major scientific findings that have shaped the current molecular and genetic landscape of EoE as well as the major obstacles in the discovery of disease causal variants in complex disorders.

Intestinal CCL11 and eosinophilic inflammation is regulated by myeloid cell-specific RelA/p65 in mice.

In inflammatory bowel diseases (IBDs), particularly ulcerative colitis, intestinal macrophages (MΦs), eosinophils, and the eosinophil-selective chemokine CCL11, have been associated with disease pathogenesis. MΦs, a source of CCL11, have been reported to be of a mixed classical (NF-κB-mediated) and alternatively activated (STAT-6-mediated) phenotype. The importance of NF-κB and STAT-6 pathways to the intestinal MΦ/CCL11 response and eosinophilic inflammation in the histopathology of experimental colitis is not yet understood. Our gene array analyses demonstrated elevated STAT-6- and NF-κB-dependent genes in pediatric ulcerative colitis colonic biopsies. Dextran sodium sulfate (DSS) exposure induced STAT-6 and NF-κB activation in mouse intestinal F4/80(+)CD11b(+)Ly6C(hi) (inflammatory) MΦs. DSS-induced CCL11 expression, eosinophilic inflammation, and histopathology were attenuated in RelA/p65(Δmye) mice, but not in the absence of STAT-6. Deletion of p65 in myeloid cells did not affect inflammatory MΦ recruitment or alter apoptosis, but did attenuate LPS-induced cytokine production (IL-6) and Ccl11 expression in purified F4/80(+)CD11b(+)Ly6C(hi) inflammatory MΦs. Molecular and cellular analyses revealed a link between expression of calprotectin (S100a8/S100a9), Ccl11 expression, and eosinophil numbers in the DSS-treated colon. In vitro studies of bone marrow-derived MΦs showed calprotectin-induced CCL11 production via a p65-dependent mechanism. Our results indicate that myeloid cell-specific NF-κB-dependent pathways play an unexpected role in CCL11 expression and maintenance of eosinophilic inflammation in experimental colitis. These data indicate that targeting myeloid cells and NF-κB-dependent pathways may be of therapeutic benefit for the treatment of eosinophilic inflammation and histopathology in IBD.