Epidermal equivalents of filaggrin null keratinocytes do not show impaired skin barrier function
Authors: Hanna Niehues, Joost Schalkwijk, Ivonne M.J.J. van Vlijmen-Willems, Diana Rodijk-Olthuis, Michelle M. van Rossum, Ewa Wladykowski, Johanna M. Brandner, Ellen H.J. van den Bogaard, Patrick L.J.M. Zeeuwen
Journal: Journal of Allergy and Clinical Immunology (2016)

The discovery that null alleles of the filaggrin (FLG) gene are a strong genetic risk factor for atopic dermatitis (AD) has caused a paradigm shift in understanding the etiology of this disease.1Palmer C.N. Irvine A.D. Terron-Kwiatkowski A. Zhao Y. Liao H. Lee S.P. et al.Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis.Nat Genet. 2006; 38: 441-446Crossref PubMed Scopus (2281) Google Scholar FLG-deficient mouse models showed barrier impairment and enhanced percutaneous allergen sensitization, exemplifying the potential functional consequences of insufficient FLG expression.2Kawasaki 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.J Allergy Clin Immunol. 2012; 129: 1538-1546.e6Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar Both in patients with AD carrying FLG mutations and in FLG-proficient patients, increased transepidermal water loss (TEWL) and skin permeability were noted in nonlesional skin, suggesting that skin barrier abnormalities are a general phenomenon in AD, not necessarily restricted to FLG mutations.3Jakasa I. Koster E.S. Calkoen F. McLean W.H. Campbell L.E. Bos J.D. et al.Skin barrier function in healthy subjects and patients with atopic dermatitis in relation to filaggrin loss-of-function mutations.J Invest Dermatol. 2011; 131: 540-542Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar Others reported a mild increase in TEWL in patients with ichthyosis vulgaris (IV) carrying 2 FLG null alleles, but failed to demonstrate increased TEWL in heterozygous carriers of an FLG null allele.4Perusquia-Ortiz A.M. Oji V. Sauerland M.C. Tarinski T. Zaraeva I. Seller N. et al.Complete filaggrin deficiency in ichthyosis vulgaris is associated with only moderate changes in epidermal permeability barrier function profile.J Eur Acad Dermatol Venereol. 2013; 27: 1552-1558Crossref PubMed Scopus (28) Google Scholar It could be argued that in vivo there may be confounding factors such as concomitant subclinical inflammation of the apparently normal skin that would obscure the effect of FLG deficiency on barrier function per se. For this reason, 3-dimensional (3D) skin models represent excellent models to investigate skin barrier function in a well-controlled setting. In vitro studies using various keratinocyte sources, 3D models, FLG gene knockdown approaches, and barrier assays have been published (summarized in Table E1 in this article's Online Repository at www.jacionline.org). Because of different experimental procedures, these studies appear contradictory and are difficult to interpret. None of them used genetically defined FLG null (FLG−/−) or FLG+/− keratinocytes, but all relied on gene knockdown approaches to lower FLG expression levels. In this study, we have used human epidermal equivalents (HEEs) generated from FLG null keratinocytes derived from patients with IV to study the effect on epidermal differentiation and barrier function, without confounding factors. We analyzed the outside-in and inside-out barrier of these equivalents using low molecular weight tracers as previously used in other studies. Remarkably, we did not observe altered skin barrier function in HEEs of completely FLG-deficient keratinocytes. All procedures for cell culture, cytokine stimulation, analysis of gene and protein expression, and epidermal barrier function were performed as described in this article's Methods section in the Online Repository at www.jacionline.org. Experiments were performed on fully differentiated HEEs expressing all markers of normal skin (see Fig E1 in this article's Online Repository at www.jacionline.org). For the FLG−/− HEEs, we used keratinocytes isolated from patients with IV (N = 5) which carry the 2 most frequent mutations of the FLG gene, leading to complete absence of FLG protein as verified by immunohistochemistry (see Fig E2 in this article's Online Repository at www.jacionline.org). Healthy volunteer keratinocytes (N = 6) were used as control (FLG+/+). We also considered the possibility that the FLG genotype may drive skin barrier impairment depending on a concomitant TH2-cytokine milieu. We therefore compared FLG−/− and FLG+/+ HEEs with and without stimulation by TH2 cytokines IL-4 and IL-13 during the entire air-liquid interface culture (Fig 1). Overall, despite the absence of keratohyalin granules and FLG protein in FLG−/− HEEs, we found no major differences between FLG−/− and FLG+/+ HEEs concerning general epidermal development, stratification, and morphology. The stratum corneum (SC) of FLG−/− HEEs was found to be marginally thinner but this was not significant (Fig 1; see Fig E3 in this article's Online Repository at www.jacionline.org). TH2 cytokines induced thickening of the viable epidermis (Fig E3), spongiosis, and apoptosis, as we have described previously.5van den Bogaard E.H. Bergboer J.G. Vonk-Bergers M. van Vlijmen-Willems I.M. Hato S.V. van der Valk P.G. et al.Coal tar induces AHR-dependent skin barrier repair in atopic dermatitis.J Clin Invest. 2013; 123: 917-927PubMed Google Scholar Differentiation proteins were strongly downregulated by TH2 cytokine treatment as reported before6Kim B.E. Leung D.Y. Boguniewicz M. Howell M.D. Loricrin and involucrin expression is down-regulated by Th2 cytokines through STAT-6.Clin Immunol. 2008; 126: 332-337Crossref PubMed Scopus (375) Google Scholar; however, expression levels were equal for both FLG genotypes (Fig 1, A). Quantitative PCR analysis revealed that TH2 cytokine stimulation caused a significant decrease in the expression of many genes that encode structural SC proteins (see Figs E4 and E5 in this article's Online Repository at www.jacionline.org). However, these were, with the exception of loricrin and caspase 14, not significantly different between the 2 FLG genotypes. Because tight junctions (TJs) are important in epidermal barrier formation, we analyzed the expression of TJ proteins of our HEEs. Occasionally, we observed a patchy expression of claudin-4 (Cldn-4) and occludin (Ocln) in FLG−/− HEEs compared with the continuous expression in FLG+/+ HEEs (Fig 1, B). A previous study in mice, however, reported that the epidermal TJ barrier is not affected by the loss of filaggrin.7Yokouchi M. Kubo A. Kawasaki H. Yoshida K. Ishii K. Furuse M. et al.Epidermal tight junction barrier function is altered by skin inflammation, but not by filaggrin-deficient stratum corneum.J Dermatol Sci. 2015; 77: 28-36Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar In vitro epidermal barrier function of the HEEs described above was assessed by outside-in and inside-out tracer penetration assays (Lucifer Yellow [LY] and EZ-Link Sulfo-NHS-LC-LC-Biotin). To validate the LY penetration assay for the detection of SC defects in our system, we used the detergent SDS as a positive control to compromise the HEE barrier capacity. We found LY penetration through the SC reaching the keratinocytes of the living epidermis following SDS treatment (see Fig E6 in this article's Online Repository at www.jacionline.org). Interestingly, under normal conditions as well as in a TH2 cytokine milieu, where expression of most structural proteins was strongly diminished, both SC of FLG+/+ and FLG−/− HEEs appeared impermeable for LY (Fig 1, C) and EZ-Link Sulfo-NHS-LC-LC-Biotin (see Fig E7 in this article's Online Repository at www.jacionline.org) when applied at the SC side of the HEE. The inside-out barrier with respect to water evaporation across the SC was measured by TEWL determination of FLG+/+ and FLG−/− HEEs. No differences were found between the groups (Fig 1, D). In addition, we used EZ-Link Sulfo-NHS-LC-LC-Biotin inside-out diffusion to investigate SC permeability in HEEs of both genotypes (Fig 1, E). Again, no differences were found. As sensitization against environmental antigens is a hallmark of AD, a leaky skin barrier has been postulated as the most plausible mechanism linking genetic alterations to disease phenotype. Conflicting outcomes of studies that have addressed this question during the last years (Table E1) may be caused by differences in experimental setup or the lack of proper controls. Insufficient numbers of biological replicates, and off-target effects, a well-known phenomenon in knockdown studies, may have played a role. Most of these studies obtained a significant reduction in FLG mRNA or protein expression (70% to 90%), but none of them reached complete absence of expression. We used genetically defined keratinocytes, completely deficient of FLG protein, in a 3D epidermal equivalent model. This provided a unique possibility to study the role of FLG in skin barrier defects, reported for both IV and AD. Thereby, knockdown-derived off-target effects were excluded, while potential compensatory mechanisms by naturally occurring FLG deficiency were taken into account. In our study, barrier function was tested by the polar solutes LY and biotin as these are commonly used in comparable studies. Although alteration of the permeability for these low molecular weight tracers was not observed, this does not completely rule out alterations of FLG-deficient epidermis with respect to permeability for environmental molecules with other biophysical properties such as microbial or airborne antigens, or fragments thereof. As microbiome alterations (eg, Staphylococcus aureus colonization) may play a role in AD, the penetration of environmental stimuli may be the result of an interaction between microorganisms and genetic factors such as FLG mutations, as suggested in a recent study.8Jones A.L. Curran-Everett D. Leung D.Y. Food allergy is associated with Staphylococcus aureus colonization in children with atopic dermatitis.J Allergy Clin Immunol. 2016; 137: 1247-1248.e3Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar Clearly, this is an interesting area for future research. We thank the patients and volunteers who participated in this study. Primary human keratinocytes were obtained from four 4-mm diameter punch biopsies of genotyped healthy volunteers (FLG+/+) and patients with IV (FLG−/−), which were taken at the upper buttock by a dermatologist. All volunteers (aged between 25 and 65 years) signed informed consent for this study. Keratincoytes were isolated and expanded according to the Rheinwald-Green protocolE7Rheinwald J.G. Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.Cell. 1975; 6: 331-343Abstract Full Text PDF PubMed Scopus (3917) Google Scholar and stored in liquid nitrogen. FLG mutation analysis for the 2 most common mutations, R501X and 2282del4, was performed as described previously.E8van den Bogaard E.H. Bergboer J.G. Vonk-Bergers M. van Vlijmen-Willems I.M. Hato S.V. van der Valk P.G. et al.Coal tar induces AHR-dependent skin barrier repair in atopic dermatitis.J Clin Invest. 2013; 123: 917-927PubMed Google Scholar For the generation of HEEs, 300,000 primary human keratinocytes were seeded onto 0.4 μm pore size transwell filters (Thincerts, Greiner Bio-One GmbH, Frickenhausen, Germany). Before seeding, these transwells were incubated with 100 μg/mL rat-tail collagen (BD Biosciences, Breda, The Netherlands) diluted in sterile cold PBS at 4°C for 1 hour, or with 100 μg/mL fibrinogen, 10% FCS (HyClone), or 100 μg/mL poly-d-lysine at room temperature for 1 hour. Afterward, excessive coating solutions were carefully aspirated and filters were washed once with cold, sterile PBS. First, the primary keratinocytes were cultured in a submerged manner in proliferation medium (CnT-prime, CELLnTEC) for 2 days to form a homogeneously distributed monolayer. One day later, medium was changed to differentiation medium (60% 3D barrier, CELLnTEC complemented with 40% Dulbecco modified Eagle medium, Gibco, Life Technologies, Thermo Scientific, Bleiswijk, The Netherlands). On the third day, the transwell inserts were lifted to the air-liquid interface to induce differentiation and stratification of the epidermis. From now on, cells were cultured in differentiation medium until the end of the culture and they were refreshed with differentiation medium every other day. This HEE culture schedule is depicted in Fig E1. For TH2 cytokine stimulation, a mixture of 50 ng/mL IL-4 (Preprotech) and 50 ng/mL IL-13 (Preprotech, Rocky Hill, NJ) in HEE differentiation medium containing 0.05% BSA (Sigma-Aldrich, Zwijndrecht, The Netherlands) was added from day 1 of air-liquid interface culture until the end of the culture of the HEEs. LY (1 mM) (Sigma-Aldrich, Zwijndrecht, The Netherlands) or EZ-Link Sulfo-NHS-LC-LC-Biotin (Life Technologies) was added epicutaneously onto the HEE for 60 minutes, followed by formalin fixation and processing for histology. Sections (6 μm) were counterstained with 4′,6-diamidino-2-phenylindole (Boehringer Mannheim, Mannheim, Germany) and assessed with fluorescence microscopy for LY penetration or stained with an indirect immunoperoxidase technique (Vectastain, Vector Laboratories, Burlingame, Calif) for EZ-Link Sulfo-NHS-LC-LC-Biotin penetration. To show LY penetration on impaired skin barrier function in vitro, HEEs were topically treated with 0.5% SDS in PBS (Sigma-Aldrich) for 90 minutes followed by a 60-minute incubation with 1 mM LY as described above. Fully differentiated HEEs residing in the transwell were placed upside-down. EZ-Link Sulfo-NHS-LC-LC-Biotin (Life Technologies, Thermo Scientific, Bleiswijk, The Netherlands) in HEE differentiation medium was applied to the basal layer side of the HEE for 60 minutes, followed by formalin fixation and processing for histology. For visualization, 6-μm sections were stained with streptavidin Alexa Fluor 594 (Life Technologies, Thermo Scientific) and counterstained with 4′,6-diamidino-2-phenylindole (Boehringer Mannheim) and assessed with fluorescence microscopy dye penetration. Permeability barrier function was evaluated by measurement of TEWL with a condenser-chamber method (Aquaflux AF200, Biox Systems, London, United Kingdom). The HEEs, maintained in tissue culture exerts along with their medium, were first acclimatized to the ambient conditions of temperature and humidity for at least 30 minutes. The measurement head of the instrument was equipped with a stainless steel extension tube (5-mm bore diameter) to reach down into the transwell inserts. A gentle and controlled contact pressure of the tube onto the sample surface was achieved by mounting the measurement head on a purpose-designed probe holder with precise control of the vertical position of the probe. The same investigator performed all TEWL measurements. For each HEE, TEWL measurements were performed in triplicate and then averaged. HEEs were fixed in formalin and processed for routine histology. Paraffin sections (6 μm) were stained with antibodies using an indirect immunoperoxidase technique (Vectastain, Vector Laboratories, Burlingame, Calif) or immunofluorescence technique according to Gruber et al.E9Gruber R. Bornchen C. Rose K. Daubmann A. Volksdorf T. Wladykowski E. et al.Diverse regulation of claudin-1 and claudin-4 in atopic dermatitis.Am J Pathol. 2015; 185: 2777-2789Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar An overview of all used antibodies is presented in Table E2. RNA isolation, cDNA synthesis, and quantitative PCR analysis were performed as described earlier.E10Bergboer J.G. Tjabringa G.S. Kamsteeg M. van Vlijmen-Willems I.M. Rodijk-Olthuis D. Jansen P.A. et al.Psoriasis risk genes of the late cornified envelope-3 group are distinctly expressed compared with genes of other LCE groups.Am J Pathol. 2011; 178: 1470-1477Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar All primers were designed and used as described previously.E11Zeeuwen P.L. de Jongh G.J. Rodijk-Olthuis D. Kamsteeg M. Verhoosel R.M. van Rossum M.M. et al.Genetically programmed differences in epidermal host defense between psoriasis and atopic dermatitis patients.PLoS One. 2008; 3: e2301Crossref PubMed Scopus (42) Google Scholar Target gene expression was normalized to the expression of the house keeping gene human acidic ribosomal phosphoprotein P0 (RPLP0). The ΔΔCt method was used to calculate relative mRNA expression levelsE12Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (124358) Google Scholar (see Table E3 for primer sequences). Quantitative PCR data were analyzed using 2-way ANOVA to test the effect of both FLG genotype and TH2 cytokine stimulation on mRNA expression. For this, we used the ΔCt values of n = 11 HEEs (n = 6 FLG+/+, n = 5 FLG−/−).Fig E2Filaggrin null-mutation. A, H&E staining of a 4-mm skin biopsy punch of one of the patients with IV from whom keratinocytes were isolated for this study. General morphology and stratification of the epidermis is normal, except for absence of the FLG-containing keratohyalin granules that are normally present in the stratum granulosum. B, FLG protein staining is negative, revealing complete loss of the FLG expression in this patient. Scale bar = 100 μm. C, Keratinocytes from patients with IV used in this study carry the 2 most frequently occurring mutations R501X and 2282del4 located on the first repeat of the FLG gene. D, FLG mRNA expression analysis of FLG−/− and FLG+/+ epidermal equivalents. EDC, Epidermal differentiation complex; H&E, hematoxylin and eosin. Bars represent mean (N = 2 for both groups).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Epidermal thickness of FLG+/+ and FLG−/− HEEs. Thickness of the viable epidermal layers (stratum basale, stratum spinosum, stratum granulosum) and the entire epidermal thickness were measured in unstimulated (control) and TH2-cytokine-stimulated HEEs. Measurements were taken using AxioVison software. Two-way ANOVA was performed to determine significant changes in epidermal thickness.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4mRNA expression changes on TH2 stimulation of HEEs. HEEs of 11 primary keratinocyte donors (n = 6 FLG+/+, n = 5 FLG−/−) were stimulated with TH2 cytokines IL-4 and IL-13 during air-liquid interface culture. Epidermal differentiation and TJ genes were significantly downregulated on TH2 stimulation. Two-way ANOVA statistical analysis was performed to test the influence of the TH2 stimulation. Data are represented as mean ± SEM. *P < .05, **P < .001, and ***P < .0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E5mRNA expression data of FLG+/+ and FLG−/− HEEs. Relative mRNA expression levels of genes concerning epidermal differentiation (A) and TJs (B). The figures present quantitative PCR expression data of HEEs of 11 primary keratinocyte donors (n = 6 FLG+/+, n = 5 FLG−/−) unstimulated (control) or with TH2 stimulation. The house keeping gene ribosomal phosphoprotein P0 (RPLP0) was used for normalization. Two-way ANOVA was performed to determine significant changes in expression levels between FLG+/+ and FLG−/− HEEs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E5mRNA expression data of FLG+/+ and FLG−/− HEEs. Relative mRNA expression levels of genes concerning epidermal differentiation (A) and TJs (B). The figures present quantitative PCR expression data of HEEs of 11 primary keratinocyte donors (n = 6 FLG+/+, n = 5 FLG−/−) unstimulated (control) or with TH2 stimulation. The house keeping gene ribosomal phosphoprotein P0 (RPLP0) was used for normalization. Two-way ANOVA was performed to determine significant changes in expression levels between FLG+/+ and FLG−/− HEEs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E6SDS application disrupts the epidermal barrier in vitro. A, Representative H&E staining of an HEE after 90 minutes topical application of 0.5% SDS to disrupt the epidermal barrier. B, Immunofluorescence detection of LY (green), which was incubated for 60 minutes after SDS application, in combination with DAPI staining for cell nuclei (blue). DAPI, 4′-6-Diamidino-2-phenylindole, dihydrochloride. H&E, Hematoxylin and eosin. Scale bar = 100 μm. N = 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E7Normal barrier function of FLG−/− HEEs. A, H&E staining of FLG+/+ and FLG−/− HEEs used for barrier permeability assays after 8 days of air-liquid interface culture. B, Staining of biotin, which was incubated apically on the HEEs for 60 minutes, shows equal epidermal barrier function in both genotypes as the dye remained on top of the SC, not reaching the viable cell layers of the epidermis. C, Lucifer yellow dye (green) was incubated apically on the HEEs for 60 minutes. The fluorescent dye remains on top of the stratum corneum only, revealing equal barrier function in both genotypes. Nuclei were stained with DAPI (blue). H&E, Hematoxylin and eosin. DAPI, 49-6-Diamidino-2-phenylindole, dihydrochloride. Scale bar = 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Filaggrin knockdown studies using in vitro 3D skin modelsAuthorsKeratinocyte sourceKnockdownSkin equivalent featuresBarrier assayBarrier permeabilityMildner et al 2010E1Mildner M. Jin J. Eckhart L. Kezic S. Gruber F. Barresi C. et al.Knockdown of filaggrin impairs diffusion barrier function and increases UV sensitivity in a human skin model.J Invest Dermatol. 2010; 130: 2286-2294Abstract Full Text Full Text PDF PubMed Scopus (222) Google ScholarNeonatal foreskin KCssiRNA + lipofectamine85% mRNA95% proteinLess keratohyalin granulesNormal SCImpaired lamellar bodies60% decrease in UCAIncreased UV sensitivityIncreased DNA damageLY penetrationIncreased permeabilityGruber et al. 2011E2Gruber R. Elias P.M. Crumrine D. Lin T.K. Brandner J.M. Hachem J.P. et al.Filaggrin genotype in ichthyosis vulgaris predicts abnormalities in epidermal structure and function.Am J Pathol. 2011; 178: 2252-2263Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar (adapted from Mildner et alE1Mildner M. Jin J. Eckhart L. Kezic S. Gruber F. Barresi C. et al.Knockdown of filaggrin impairs diffusion barrier function and increases UV sensitivity in a human skin model.J Invest Dermatol. 2010; 130: 2286-2294Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar)Neonatal foreskin KCssiRNA + lipofectamine>90% (not shown)Normal keratin intermediate filament integrityLanthanum perfusionIncreased paracellular permeabilityKuchler et al 2011E3Kuchler S. Henkes D. Eckl K.M. Ackermann K. Plendl J. Korting H.C. et al.Hallmarks of atopic skin mimicked in vitro by means of a skin disease model based on FLG knock-down.Altern Lab Anim. 2011; 39: 471-480PubMed Google Scholar (adapted from Mildner et alE1Mildner M. Jin J. Eckhart L. Kezic S. Gruber F. Barresi C. et al.Knockdown of filaggrin impairs diffusion barrier function and increases UV sensitivity in a human skin model.J Invest Dermatol. 2010; 130: 2286-2294Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar)Neonatal foreskin KCssiRNA + HiPerFect82% mRNAIntercellular and intracellular spongiosis1% SDS for 15 min followed by testosterone and caffeine absorptionIncreased IL-6 and IL-8 release and testosterone absorptionvan Drongelen et al 2013E4van Drongelen V. Alloul-Ramdhani M. Danso M.O. Mieremet A. Mulder A. van Smeden J. et al.Knock-down of filaggrin does not affect lipid organization and composition in stratum corneum of reconstructed human skin equivalents.Exp Dermatol. 2013; 22: 807-812Crossref PubMed Scopus (40) Google ScholarN/TERT immortalized KC cell lineshRNA + electroporation85% mRNA80% proteinNormal epidermal morphogenesisNormal SC lipid organization and lipid compositionButyl-PABANo differences in permeabilityVavrova et al 2014E5Vavrova K. Henkes D. Struver K. Sochorova M. Skolova B. Witting M.Y. et al.Filaggrin deficiency leads to impaired lipid profile and altered acidification pathways in a 3D skin construct.J Invest Dermatol. 2014; 134: 746-753Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar (adapted from Mildner et alE1Mildner M. Jin J. Eckhart L. Kezic S. Gruber F. Barresi C. et al.Knockdown of filaggrin impairs diffusion barrier function and increases UV sensitivity in a human skin model.J Invest Dermatol. 2010; 130: 2286-2294Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar)Neonatal foreskin KCssiRNA + lipofectamine75% mRNALess NMFNormal skin surface pHAltered lipid profileTestosterone and caffeine absorptionNo differences in absorption (not shown)Pendaries et al 2014E6Pendaries V. Malaiss J. Pellerin L. Le Lamer M. Nachat R. Kezic S. et al.Knockdown of filaggrin in a three-dimensional reconstructed human epidermis impairs keratinocyte differentiation.J Invest Dermatol. 2014; 134: 2938-2946Abstract Full Text Full Text PDF PubMed Scopus (101) Google ScholarAdult epidermal KCsshRNA + lentivirus90% mRNA90% proteinLess KC layersReduced SC thicknessLess keratohyalin granulesImpaired SC formationReduction in proliferationNormal skin surface pHDecreased NMF levelsIncreased UV sensitivityAltered epidermal differentiationActivation pro-caspase 14 inhibitionLY penetrationIncreased permeabilityButyl-PABA, Butyl 4-aminobenzoate; KC, keratinocyte; NMF, natural moisturizing factor; N/TERT, normal human keratinocytes immortalized by stable human telomerase transfection; shRNA, small hairpin RNA (stable transduction); siRNA, small interfering RNA (transient transfection); UCA, urocanic acid; UV, ultraviolet radiation. Open table in a new tab Table E2Antibodies for immunohistochemistry and immunofluorescence (boldface)Target proteinAntibody clone, manufacturerDilutionCytokeratin-10DE-K10, Euro Diagnostics1:100FilaggrinNCL-Filaggrin, Novocastra1:200LoricrinPRB145P, Covance1:2000Transglutaminase-1H-87, Santa Cruz1:100InvolucrinMON-150, generated by our groupE13van Duijnhoven J.L. Schalkwijk J. Kranenborg M.H. van Vlijmen-Willems I.M. Groeneveld A. van Erp P.E. et al.MON-150, a versatile monoclonal antibody against involucrin: characterization and applications.Arch Dermatol Res. 1992; 284: 167-172Crossref PubMed Scopus (28) Google Scholar1:20Late cornified envelope 3Generated by our groupE14Niehues H. van Vlijmen-Willems I.M. Bergboer J.G. Kersten F.F. Narita M. Hendriks W.J. et al.Late cornified envelope (LCE) proteins: distinct expression patterns of LCE2 and LCE3 members suggest non-redundant roles in human epidermis and other epithelia.Br J Dermatol. 2015; 174: 795-802Crossref Scopus (13) Google Scholar1:50004′,6-Diamidino-2-phenylindoleBoehringer Mannheim1:3000Streptavidin Alexa Fluor 594Life Technologies1:200Claudin-1JAY.8, Thermo Fisher Scientific1:1500Claudin-43E2C1, Thermo Fisher Scientific1:700OccludinN19, Santa Cruz1:250 Open table in a new tab Table E3Primers used for quantitative PCRHUGO symbolAlias gene nameForward primer 5′-3′Reverse primer 3′-5′E∗E is efficiency as fold increase in fluorescence per PCR cycle.CASP14Caspase 14acaaaccatcccaacatacacgatatgtcctttcctcttcgt2.12CLDN1Claudin 1ccagtcaatgccaggtacgattggatagggccttggtgtt1.78CLDN4Claudin 4gctgtaaacaggtttgggcatcagaggggatcagtctcca1.85FLGFilaggrinacttcactgagtttcttctgatggtatttccagacttgagggtctttttctg1.89FLG2Filaggrin 2accaggttcacttaaacttgcaatgacatccactgtgtctggatc1.96HRNRHornerintacaaggcgtcatcactgtcatcatctggatcgtttggattcttcag2.12IVLInvolucrinacttatttcgggtccgctaggtgagacatgtagagggacagagtcaag1.93LCE2ALate cornified envelope 2Aggacctgtcccagagtgatggatccaggatgggctcttg2.10LORLoricrinaggttaagacatgaaggatttgcaaggcaccgatgggcttagag2.08OCLNOccludinattggtcaccgagggaggataaaccaatctgctgcgtccta1.86RPLP0hARP, 60S acidic ribosomal protein P0caccattgaaatcctgagtgatgttgaccagcccaaaggagaag2.02TGM1Transglutaminase 1cccccgcaatgagatctacaatcctcatggtccacgtacaca1.99∗ E is efficiency as fold increase in fluorescence per PCR cycle. Open table in a new tab Butyl-PABA, Butyl 4-aminobenzoate; KC, keratinocyte; NMF, natural moisturizing factor; N/TERT, normal human keratinocytes immortalized by stable human telomerase transfection; shRNA, small hairpin RNA (stable transduction); siRNA, small interfering RNA (transient transfection); UCA, urocanic acid; UV, ultraviolet radiation.