Epidermal thymic stromal lymphopoietin predicts the development of atopic dermatitis during infancy

Abstract

To establish a primary prevention strategy for atopic dermatitis (AD), it is important to identify biomarkers that can predict the occurrence of AD. This study aimed to evaluate the expression level of epidermal proteins by using a tape stripping method to determine whether these proteins can be used as biomarkers predictive of AD development in infants. In this prospective birth cohort study, we followed 75 infants in a risk group and 12 in a control group for 2 years (Fig 1). The control group consisted of infants with both parents who had neither allergy nor immediate skin test reactivity to 8 common inhalant allergens (Dermatophagoides pteronyssinus, Dermatophagoides farinae, tree pollen mixture І & II, weed pollen mixture, grass pollen mixture, cat, and cockroach). The risk group was defined when at least 1 parent had both positive skin test response and a history of asthma or allergic rhinitis, or when at least 1 family member had AD. The diagnosis of AD was based on the Hanifin and Rajka criteria, and the severity was assessed using the SCORing Atopic Dermatitis (SCORAD) method. Transepidermal water loss (TEWL) was measured on the volar surface of the forearm at age 2 months by using a Tewameter TM300 (Courage & Khazaka, Köln, Germany). Tape stripping was done on the volar surface of the forearm at 2 months, and epidermal protein levels were measured by mass spectrometry as previously described to evaluate the expression of filaggrin, alpha enolase, corneodesmosin, fatty acid– binding protein, serpin B3, transglutaminase 3, and thymic stromal lymphopoietin (TSLP).1Broccardo C.J. Mahaffey S.B. Strand M. Reisdorph N.A. Leung D.Y. Peeling off the layers: skin taping and a novel proteomics approach to study atopic dermatitis.J Allergy Clin Immunol. 2009; 124 (e1-11): 1113-1115Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar Methods for mass spectrometry and statistics are described in this article's Online Repository at www.jacionline.org. This study was approved by the institutional review board (IRB) at Samsung Medical Center in Seoul (IRB no. 2011-11-072), and written informed consent was obtained from all the parents. The cumulative incidence of AD was higher in the risk group than in the control group at 24 months (64.8% vs 16.7%; P = .003) (see Fig E1 in this article's Online Repository at www.jacionline.org). Eczema symptoms at age 12 months occurred mostly on the face (72.7%), but clinical AD did not appear on the forearm where tape stripping was conducted. The mean SCORAD score in affected infants was 13.3 ± 5.8 and 12.9 ± 5.4 at age 12 and 24 months, respectively. There was no difference between the risk group and the control group in sex, birth weight, intrauterine period, birth type, mother's age, family's monthly income, and maternal education levels (see Table E1 in this article's Online Repository at www.jacionline.org). To estimate risk factors predicting AD development by the age of 24 months, a logistic regression model was used. A multivariable analysis was done by adjusting for TSLP and other variables with P value of less than .1 in a univariable analysis. When TSLP expression was dichotomized into low or high levels according to its median value, 0.83 pmol/mg skin (range, 0.32-1.60 pmol/mg skin), TSLP expression at 2 months was 5.3 times more likely to develop AD by age 24 months (95% CI, 1.3-21.4). When TSLP expression was added to family history, adjusted odds ratio (aOR) for AD development was higher in subjects with family history and high TSLP expression (aOR = 20.2; 95% CI, 1.5-272.3) than in those with family history alone (aOR = 12.6; 95% CI, 1.1-143.9; Fig 2). Male gender was also independently related to AD development (aOR = 5.5; 95% CI, 1.3-24.2). In contrast, other variables such as birth type, passive smoking, mold exposure during pregnancy, exclusive breast-feeding, TEWL on the nonlesional area of the forearm at 2 months, and filaggrin level at 2 months showed no statistical significance in a logistic regression model. The expression of alpha enolase, corneodesmosin, fatty acid–binding protein, serpin B3, and transglutaminase 3 at 2 months was not predictive of AD development (see Fig E2 in this article's Online Repository at www.jacionline.org). Taken together, our birth cohort study first demonstrated that epidermal TSLP expression at as early as age 2 months is linked with AD later in life. In particular, the predictive value for AD development was further increased by high TSLP expression in subjects with family history which is the strongest risk factor. It indicates that epidermal TSLP level may be used as an early predictor of AD development.2Sano Y. Masuda K. Tamagawa-Mineoka R. Matsunaka H. Murakami Y. Yamashita R. et al.Thymic stromal lymphopoietin expression is increased in the horny layer of patients with atopic dermatitis.Clin Exp Immunol. 2013; 171: 330-337Crossref PubMed Scopus (92) Google Scholar TSLP is expressed in the horny layer of stratum corneum, promotes TH2-type inflammation, and downregulates filaggrin expression like other cytokines such as IL-4, IL-13, IL-17, IL-22, IL-25, IL-31, and IL-33.2Sano Y. Masuda K. Tamagawa-Mineoka R. Matsunaka H. Murakami Y. Yamashita R. et al.Thymic stromal lymphopoietin expression is increased in the horny layer of patients with atopic dermatitis.Clin Exp Immunol. 2013; 171: 330-337Crossref PubMed Scopus (92) Google Scholar, 3Kim J.H. Bae H.C. Ko N.Y. Lee S.H. Jeong S.H. Lee H. et al.Thymic stromal lymphopoietin downregulates filaggrin expression by signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK) phosphorylation in keratinocytes.J Allergy Clin Immunol. 2015; 136: 205-208.e9Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 4Thyssen J.P. Kezic S. Causes of epidermal filaggrin reduction and their role in the pathogenesis of atopic dermatitis.J Allergy Clin Immunol. 2014; 134: 792-799Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 5Seltmann J. Roesner L.M. von Hesler F.W. Wittmann M. Werfel T. IL-33 impacts on the skin barrier by downregulating the expression of filaggrin.J Allergy Clin Immunol. 2015; 135: 1659-1661.e4Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar A recent study showed that the long isoform of TSLP exerts proinflammatory activities, whereas the short isoform plays an anti-inflammatory role.6Fornasa G. Tsilingiri K. Caprioli F. Botti F. Mapelli M. Meller S. et al.Dichotomy of short and long thymic stromal lymphopoietin isoforms in inflammatory disorders of the bowel and skin.J Allergy Clin Immunol. 2015; 136: 413-422Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar Stratum corneum TSLP level is reduced by moisturizer application in AD patients,2Sano Y. Masuda K. Tamagawa-Mineoka R. Matsunaka H. Murakami Y. Yamashita R. et al.Thymic stromal lymphopoietin expression is increased in the horny layer of patients with atopic dermatitis.Clin Exp Immunol. 2013; 171: 330-337Crossref PubMed Scopus (92) Google Scholar and it may explain how AD development is prevented by the application of moisturizer to neonates.7Horimukai K. Morita K. Narita M. Kondo M. Kitazawa H. Nozaki M. et al.Application of moisturizer to neonates prevents development of atopic dermatitis.J Allergy Clin Immunol. 2014; 134: 824-830.e6Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar Although TSLP is an important biomarker in the Korean cohort, it is noteworthy that having a group of biomarkers, rather than 1 biomarker, is important because it may be different according to races and AD phenotypes.8Leung D.Y. Guttman-Yassky E. Deciphering the complexities of atopic dermatitis: shifting paradigms in treatment approaches.J Allergy Clin Immunol. 2014; 134: 769-779Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar In an Irish birth cohort study, increased TEWL was found before the occurrence of AD and the authors suggested that measurement of TEWL at ages 2 days and 2 months was predictive of AD development at 1 year.9Kelleher M. Dunn-Galvin A. Hourihane J.O. Murray D. Campbell L.E. McLean W.H. et al.Skin barrier dysfunction measured by transepidermal water loss at 2 days and 2 months predates and predicts atopic dermatitis at 1 year.J Allergy Clin Immunol. 2015; 135: 930-935.e1Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar However, we failed to find an association between TEWL at age 2 months and later development of AD in our Korean cohort. This may be explained by the extent of inflammation in the skin. Interestingly, the mean SCORAD score in the Irish study at 6 and 12 months was 21.54 ± 16.29 and 18.56 ± 14.92, respectively, showing that the Irish infants had more severe AD than did ours. It is plausible that subclinical skin inflammation of the Irish study population at age 2 months could impair the skin barrier function as opposed to our cohort, although clinical AD was not apparent in both studies. Another possibility is that the pathobiology of AD is different in Korea versus Ireland.10Noda 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.J Allergy Clin Immunol. 2015; 136: 1254-1264Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar Indeed, levels of TH17- and TH22-related cytokines have been found to be higher in Asian than in European American AD skin biopsies. In conclusion, epidermal TSLP protein expression may serve as an early biomarker to predict the development of infantile AD. To facilitate AD prevention studies, additional skin tape stripping studies are needed to identify biomarkers that predict infants at risk of developing AD in different racial groups. We tried to select the study population according to very strict criteria. We excluded parents with asthma or allergic rhinitis from the risk group unless they were sensitized to common inhalant allergens as well. Subjects with a history of AD were also excluded if eczema symptoms were not found on site. Likewise, all parents with asymptomatic sensitization were excluded from the control group even though they had never presented with asthma or allergic rhinitis symptoms. Finally, we excluded 279 subjects out of 388 after initial screening, and successfully followed 75 infants in the risk group and 12 in the control group for 2 years. Patients were directed to avoid showering on the day of the collection. A total of 20 consecutive D-Squame tape strips (22 mm diameter, CuDerm, Dallas, Tex) were applied on the volar surface of the right forearm at age 2 months. On application of the first tape disc, 4 marks were placed around the disc with a pen so that subsequent discs could be applied to the same location. Each tape disc was placed adhesive side up in its own 6-well plate and then frozen at −80°C. Proteins were extracted by using a buffer composed of 0.01% 3-(3-[1,1-bisalkyloxyethyl] pyridin-1-yl)propane-1-sulfonate (Protein Discovery, Knoxville, Tenn) in 50 mmol/L ammonium bicarbonate with 1 × HALT protease inhibitors, EDTA-free (Thermo Fisher Scientific, Rockford, Ill), and 50 mmol/L dithiothreitol (Bio-Rad, Hercules, Calif) and incubated on a rocker at room temperature for 1 hour. Extraction buffer was pooled from tape disc, transferred into polypropylene 1.5-mL microcentrifuge tubes, lyophilized, and stored at −80°C. Proteins were precipitated in 300 mL ice-cold precipitation buffer consisting of 0.1% formic acid in 80:20 methanol:water (VWR, West Chester, Pa, and Thermo Fisher Scientific, respectively). Samples were incubated at −20°C and vortexed for 30 seconds every 10 minutes for 1 hour, and then centrifuged at 18,000g at 4°C for 20 minutes. The supernatant was removed, and the protein pellet was resuspended in 8 mol/L urea (Sigma Ultra, St Louis, Mo) in 100 mmol/L TRIS HCl (pH 8.5; Thermo Fisher Scientific). Proteins were reduced with 5 mmol/L tris[2-carboxyethyl]phosphine (Thermo Fisher Scientific) for 20 minutes and alkylated with 500 mmol/L iodoacetamide (Bio-Rad, Hercules, Calif) for 15 minutes. Urea was diluted to 2 mol/L with 100 mmol/L TRIS HCl (pH 8.5), and samples were incubated at 37°C overnight with 100 ng trypsin (Trypsin Gold; Promega, Madison, Wis). Samples were then cleaned by using PepClean C-18 spin columns (Thermo Fisher Scientific) following the manufacturer's protocol. All reagents were of the highest grade available for mass spectrometry. Mass spectrometry was carried out as previously described.E1Broccardo C.J. Mahaffey S.B. Strand M. Reisdorph N.A. Leung D.Y. Peeling off the layers: skin taping and a novel proteomics approach to study atopic dermatitis.J Allergy Clin Immunol. 2009; 124 (e1-11): 1113-1115Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar Samples were run in triplicate on an Agilent 1200 series HPLC (Agilent Technologies, Santa Clara, Calif) and Agilent ETD ion trap (model 6340) mass spectrometer with an HPLC chip to evaluate the expression of filaggrin, alpha enolase, corneodesmosin, fatty acid–binding protein, serpin B3, transglutaminase 3, and TSLP. Data were analyzed using SPSS for Windows (version 23.0, SPSS, Chicago, Ill). The Fisher exact test was applied to determine differences in the proportions of sex, delivery mode, parental history of allergic diseases, parental atopy, monthly income, maternal education levels, and the incidence of AD between the risk group and the control group. The Mann-Whitney U test was used to analyze differences in birth weights, intrauterine periods, and maternal ages between the risk group and the control group. To estimate risk factors predicting AD development by the age of 24 months, univariable and multivariable logistic regression analyses were used. In a univariable analysis, candidate variables for adjustment included epidermal protein levels, sex, parental history of allergic diseases, delivery mode, maternal education levels, move to a new house during pregnancy, mold exposure during pregnancy, exclusive breast-feeding during the first 4 months of life, and TEWL at age 2 months. TSLP and epidermal protein levels were dichotomized according to their median values. In a multivariable regression analysis, TSLP and other variables with a P value of less than .1 in univariable analysis were selected and adjusted for each other. The combined effect of TSLP and family history was also evaluated using a multivariable logistic regression. A P value of less than .05 was considered to be significant.Fig E2Univariable analysis for epidermal proteins influencing AD development by age 24 months. OR, Odds ratio.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Characteristics of study participantsCharacteristicRisk group (n = 75)Control group (n = 12)P valueSex: male43 (57.3)8 (66.7).754Birth weight (kg)3.2 ± 0.43.1 ± 0.5.434Intrauterine period (wk)39.5 ± 6.139.4 ± 6.3.696Mother's age at child's birth (y)32.1 ± 2.832.8 ± 3.3.555Birth type.746 Vaginal delivery53 (70.7)8 (66.7) Cesarean section22 (29.3)4 (33.3)Paternal history of allergic diseases Atopic dermatitis19 (25.3)0 (0).041 Allergic rhinitis25 (33.3)0 (0).012 Asthma5 (6.7)0 (0).467Maternal history of allergic diseases Atopic dermatitis18 (24.0)0 (0)<.001 Allergic rhinitis46 (63.0)0 (0)<.001 Asthma3 (4.0)0 (0).637Paternal atopy∗Positive skin prick test result to common allergens.46 (63.0)0 (0)<.001Maternal atopy∗Positive skin prick test result to common allergens.50 (66.7)0 (0)<.001Monthly income (US $).971 ≤17999 (12.0)1 (8.3) 1800-359936 (48.0)6 (50.0) 3600-539922 (29.3)4 (33.3) ≥54008 (10.7)1 (8.3)Maternal education.546 College4 (5.3)0 (0) High school or less71 (94.7)12 (100.0)Values are n (%) or mean ± SD.∗ Positive skin prick test result to common allergens. Open table in a new tab Values are n (%) or mean ± SD.