Accepted Manuscript Epidermal equivalents of filaggrin null keratinocytes do not show impaired skin barrier function Hanna Niehues, MSc, Joost Schalkwijk, PhD, Ivonne M.J.J. van Vlijmen-Willems, BSc, Diana Rodijk-Olthuis, BSc, Michelle M. van Rossum, PhD, MD, Ewa Wladykowski, BSc, Johanna M. Brandner, PhD, Ellen H.J. van den Bogaard, PhD, Patrick L.J.M. Zeeuwen, PhD PII:
S0091-6749(16)31116-2
DOI:
10.1016/j.jaci.2016.09.016
Reference:
YMAI 12380
To appear in:
Journal of Allergy and Clinical Immunology
Received Date: 15 April 2016 Revised Date:
8 August 2016
Accepted Date: 7 September 2016
Please cite this article as: Niehues H, Schalkwijk J, van Vlijmen-Willems IMJJ, Rodijk-Olthuis D, van Rossum MM, Wladykowski E, Brandner JM, van den Bogaard EHJ, Zeeuwen PLJM, Epidermal equivalents of filaggrin null keratinocytes do not show impaired skin barrier function, Journal of Allergy and Clinical Immunology (2016), doi: 10.1016/j.jaci.2016.09.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Epidermal equivalents of filaggrin null keratinocytes do not show impaired skin barrier
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function
3 Authors:
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Hanna Niehues1, MSc, Joost Schalkwijk1, PhD, Ivonne M.J.J van Vlijmen-Willems1, BSc, Diana Rodijk-
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Olthuis1, BSc, Michelle M. van Rossum1, PhD, MD, Ewa Wladykowski2, BSc, Johanna M. Brandner2,
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PhD, Ellen H.J. van den Bogaard1,$, PhD and Patrick L.J.M. Zeeuwen1,$ , PhD
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Institute:
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Life Sciences, Nijmegen, The Netherlands; and 2Department of Dermatology and Venerology,
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University Hospital Hamburg-Eppendorf, Hamburg, Germany.
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These authors share senior authorship
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Department of Dermatology, Radboud university medical center, Radboud Institute for Molecular
Contact information of corresponding authors:
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Ellen van den Bogaard
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Adress: René Descartesdreef 1, 6525 GL Nijmegen, The Netherlands
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Phone number: +31 24 36 14093
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e-mail:
[email protected]
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Patrick Zeeuwen
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Adress: René Descartesdreef 1, 6525 GL Nijmegen, The Netherlands
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Phone number: +31 24 36 17245
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e-mail:
[email protected]
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Capsule summary:
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We investigated differentiation and barrier properties of epidermal equivalents derived from
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ichthyosis vulgaris keratinocytes that were homozygous for FLG null mutations. We found no effect
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on penetration of tracer molecules, compared with filaggrin proficient keratinocytes.
32 Keywords:
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filaggrin, ichthyosis vulgaris, atopic dermatitis, 3D epidermal equivalent, stratum corneum
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permeability
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36 Abbreviations:
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AD, atopic dermatitis; FLG, filaggrin; HE, hematoxylin and eosin; HEE, human epidermal equivalent;
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IL, interleukin; IV, ichthyosis vulgaris; LY, Lucifer Yellow; SC, stratum corneum; TEWL, trans epidermal
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water loss; TJ, tight junction; 3D, three dimensional
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To the Editor, The discovery that null alleles of the filaggrin (FLG) gene are a strong genetic risk factor for atopic
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dermatitis (AD) has caused a paradigm shift in understanding the etiology of this disease [1]. FLG
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deficient mouse models showed barrier impairment and enhanced percutaneous allergen
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sensitization, exemplifying the potential functional consequences of insufficient FLG expression [2].
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Both in AD patients carrying FLG mutations and in FLG proficient patients, increased transepidermal
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water loss (TEWL) and skin permeability was noted in non-lesional skin suggesting that skin barrier
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abnormalities are a general phenomenon in AD, not necessarily restricted to FLG mutations [3].
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Others reported a mild increase of TEWL in ichthyosis vulgaris patients carrying two FLG null alleles,
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but failed to demonstrate increased TEWL in heterozygous carriers of a FLG null allele [4]. It could be
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argued that in vivo there may be confounding factors such as concomitant subclinical inflammation
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of the apparently normal skin that would obscure the effect of FLG deficiency on barrier function per
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se. For this reason, three dimensional (3D) skin models represent excellent models to investigate skin
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barrier function, in a well-controlled setting. In vitro studies using various keratinocyte sources, 3D
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models, FLG gene knockdown approaches and barrier assays have been published (summarized in
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Supplemental Table E1). Due to different experimental procedures these studies appear
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contradictory and are difficult to interpret. None of them used genetically defined FLG null (FLG-/-) or
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FLG+/- keratinocytes, but all relied on gene knockdown approaches to lower FLG expression levels. In
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this study we have used human epidermal equivalents (HEEs) generated from FLG null keratinocytes
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derived from ichthyosis vulgaris (IV) patients to study the effect on epidermal differentiation and
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barrier function, without confounding factors. We analyzed the outside-in and inside-out barrier of
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these equivalents using low molecular weight tracers as previously employed in other studies.
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Remarkably, we did not observe altered skin barrier function in HEEs of completely FLG deficient
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keratinocytes.
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All procedures for cell culture, cytokine stimulation, analysis of gene and protein expression and
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epidermal barrier function were performed as described in Supplemental Materials and Methods.
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Experiments were performed on fully differentiated HEEs expressing all markers of normal skin
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(Supplemental Figure E1). For the FLG-/- HEEs we used keratinocytes isolated from IV patients (N=5)
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which carry the two most frequent mutations of the FLG gene, leading to complete absence of FLG
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protein as verified by immunohistochemistry (Supplemental Figure E2). Healthy volunteer
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keratinocytes (N=6) were used as control (FLG+/+). We also considered the possibility that the FLG
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genotype may drive skin barrier impairment depending on a concomitant Th2-cytokine milieu. We
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therefore compared FLG-/- and FLG+/+ HEEs with and without stimulation by Th2-cytokines interleukin
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(IL)-4 and IL-13 during the entire air-liquid interface culture (Figure 1). Overall, despite the absence of
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ACCEPTED MANUSCRIPT keratohyalin granules and FLG protein in FLG-/- HEEs, we found no major differences between FLG-/-
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and FLG+/+ HEEs concerning general epidermal development, stratification and morphology. The
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stratum corneum of FLG-/- HEEs was found to be marginally thinner but this was not significant
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(Figure 1 and Supplemental Figure E3). Th2-cytokines induced thickening of the viable epidermis
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(Supplemental Figure E3) spongiosis and apoptosis, as we have described previously [5].
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Differentiation proteins were strongly downregulated by Th2 cytokine treatment as reported before
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[6], however, expression levels were equal for both FLG genotypes (Figure 1a). Quantitative PCR
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analysis revealed that Th2-cytokine stimulation caused a significant decrease in the expression of
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many genes that encode structural stratum corneum (SC) proteins (Supplemental Figure E4 and
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Supplemental Figure E5). However, these were, with the exception of loricrin and caspase 14, not
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significantly different between the two FLG genotypes. As tight junctions (TJs) are important in
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epidermal barrier formation, we analyzed expression of TJ proteins of our HEEs. Occasionally, we
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observed a patchy expression of claudin-4 (Cldn-4) and occludin (Ocln) in FLG-/- HEEs compared to the
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continuous expression in FLG+/+ HEEs (Figure 1b). A previous study in mice, however, reported that
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the epidermal tight junction barrier is not affected by the loss of filaggrin [7].
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In vitro epidermal barrier function of the HEEs described above was assessed by outside-in and
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inside-out tracer penetration assays (Lucifer Yellow (LY) and EZ-Link™ Sulfo-NHS-LC-LC-Biotin). To
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validate the LY penetration assay for detection of SC defects in our system, we used the detergent
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sodium dodecyl sulfate (SDS) as a positive control, to compromise the HEE barrier capacity. We found
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LY penetration through the SC reaching the keratinocytes of the living epidermis following SDS
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treatment (Supplemental Figure E6). Interestingly, under normal conditions as well as in a Th2
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cytokine milieu, where expression of most structural proteins was strongly diminished, both stratum
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corneum of FLG+/+ and FLG-/- HEEs appeared impermeable for LY (Figure 1c) and EZ-Link™ Sulfo-NHS-
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LC-LC-Biotin (Supplemental Figure E7) when applied at the SC side of the HEE. The inside-out barrier
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with respect to water evaporation across the stratum corneum was measured by transepidermal
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water loss (TEWL) determination of FLG+/+ and FLG-/- HEEs. No differences were found between the
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groups (Figure 1d). In addition, we used EZ-Link™ Sulfo-NHS-LC-LC-Biotin inside-out diffusion to
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investigate stratum corneum permeability in HEEs of both genotypes (Figure 1e). Again, no
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differences were found.
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As sensitization against environmental antigens is a hallmark of AD, a leaky skin barrier has been
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postulated as the most plausible mechanism linking genetic alterations to disease phenotype.
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Conflicting outcomes of studies that have addressed this question during the last years
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(Supplemental Table E1) may be caused by differences in experimental setup or the lack of proper
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controls. Insufficient numbers of biological replicates, and off-target effects, a well known
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phenomenon in knockdown studies, may have played a role. Most of these studies obtained a
ACCEPTED MANUSCRIPT significant reduction of FLG mRNA or protein expression (70-90%), but none of them reached
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complete absence of expression. We used genetically defined keratinocytes, completely deficient of
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FLG protein, in a 3D epidermal equivalent model. This provided a unique possibility to study the role
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of FLG in skin barrier defects, reported for both IV and AD. Thereby, knockdown-derived off-target
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effects were excluded, while potential compensatory mechanisms by naturally occurring FLG
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deficiency were taken into account.
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In our study barrier function was tested by the polar solutes LY and biotin as these are commonly
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used in comparable studies. Although alteration of the permeability for these low molecular weight
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tracers was not observed, this does not completely rule out alterations of FLG-deficient epidermis
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with respect to permeability for environmental molecules with other biophysical properties such as
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microbial or airborne antigens, or fragments thereof. As microbiome alterations (e.g. Staphylococcus
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aureus colonization) may play a role in AD, the penetration of environmental stimuli may be the
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result of an interaction between microorganisms and genetic factors like FLG mutations, as
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suggested in a recent study [8]. Clearly, this is an interesting area for future research.
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Hanna Niehues1, MSc Joost Schalkwijk1 , PhD
Ivonne M.J.J van Vlijmen-Willems1, BSc
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Diana Rodijk-Olthuis1, BSc
Michelle M. van Rossum1, PhD, MD Ewa Wladykowski2, BSc Johanna M. Brandner2, PhD Ellen H.J. van den Bogaard1,$, PhD Patrick L.J.M. Zeeuwen1,$ , PhD
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1
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Life Sciences, Nijmegen, The Netherlands; and 2Department of Dermatology and Venerology,
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University Hospital Hamburg-Eppendorf, Hamburg, Germany.
Department of Dermatology, Radboud university medical center, Radboud Institute for Molecular
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Conflict of interest
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The authors state no conflict of interest.
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Acknowledgements
ACCEPTED MANUSCRIPT We thank the patients and volunteers that participated in this study, which is funded by a TOP grant
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(91211052) of The Netherlands Organization for Health Research and Development (ZonMw)
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awarded to Joost Schalkwijk. The funder had no role in study design, data collection and analysis,
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decision to publish, or preparation of the manuscript. The work of JMB was supported by the
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Deutsche Forschungsgemeinschaft (grant No Br-1982-4).
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154 Figure legends
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Figure 1. Comparison of epidermal proliferation, differentiation and epidermal barrier between
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FLG+/+ and FLG-/- HEEs under normal or Th2-cytokine stimulated condition. (a) Morphological
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analysis and protein analysis by immunohistochemistry for markers of epidermal proliferation (Ki67)
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and differentiation (FLG, K10, IVL, LOR, LCE3 and TGM-1. (b) Immunofluorescence staining of HEE
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shows TJ proteins Cldn-4 (green) and Cldn-1 (red) at the left part and Ocln (green) at the right part
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combined with nuclear DAPI staining (blue). Amongst areas with normal TJ protein expression there
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are some areas with patchy or almost absent expression of Cldn-4 and Ocln in the FLG-/- HEEs; these
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are represented in the photographs. The arrows indicate the specific location of staining. (c) Outside-
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in stratum corneum permeability assay by LY penetration (green) combined with nuclear DAPI
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staining (blue). (d) TEWL analysis of unstimulated HEEs. (e) Inside-out stratum corneum permeability
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assay by EZ-Link™ Sulfo-NHS-LC-LC-Biotin penetration (red) combined with nuclear DAPI staining
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(blue). Pictures show combination of fluorescence and light microscopy images. White bars indicate
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stratum corneum (SC) layer. All scale bars = 100 µm. N=11 (N=6 FLG+/+ and N=5 FLG-/- HEEs).
169 References
171 172 173 174 175 176 177 178 179 180 181 182 183
1.
2. 3. 4.
5.
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Palmer, C.N., 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(4): p. 4416. Kawasaki, H., et al., Altered stratum corneum barrier and enhanced percutaneous immune responses in filaggrin-null mice. J Allergy Clin Immunol, 2012. 129(6): p. 1538-46 e6. Jakasa, I., 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(2): p. 540-2. Perusquia-Ortiz, A.M., 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(12): p. 1552-8. van den Bogaard, E.H., et al., Coal tar induces AHR-dependent skin barrier repair in atopic dermatitis. J Clin Invest, 2013. 123(2): p. 917-27.
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6. 7. 8.
Kim, B.E., et al., Loricrin and involucrin expression is down-regulated by Th2 cytokines through STAT-6. Clin Immunol, 2008. 126(3): p. 332-7. Yokouchi, 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(1): p. 28-36. Jones, A.L., D. Curran-Everett, and D.Y.M. Leung, Food allergy is associated with Staphylococcus aureus colonization in children with atoppic dermatitis. JACI, 2016. 137(4): p. 1247–1248.e3
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epidermal differentiation
Th2 control *
0.5
***
***
**
** **
***
0.0 LOR
IVL
HRNR LCE2A CASP14 TGM1
SC
FLG2
**
Th2 control
***
0.0
CLDN1
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fold difference
*
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fold difference
1.0
CLDN4
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Skin equivalent features
Barrier assay
siRNA + lipofectamine
- Less keratohyalin granules - Normal SC - Impaired lamellar bodies - 60% decrease in UCA - Increased UV sensitivity - Increased DNA damage - Normal keratin intermediate filament integrity
Lucifer Yellow penetration
Lanthanum perfusion
Increased paracellular permeability
- Intercellular and intracellular spongiosis
1% SDS for 15 min followed by testosterone and caffeine absorption
Increased IL-6 and IL-8 release and testosterone absorption
- Normal epidermal morphogenesis - Normal SC lipid organization and lipid composition - Less NMF - Normal skin surface pH - Altered lipid profile
Butyl-PABA
No differences in permeability
Testosterone and caffeine absorption
No differences in absorption (not shown)
Lucifer yellow penetration
Increased permeability
85% mRNA 95% protein
Gruber et al. 2011 [E2] (adapted from Mildner et al.)
Neonatal foreskin KC’s
siRNA + lipofectamine
Kuchler et al. 2011 [E3] (adapted from Mildner et al.)
Neonatal foreskin KC’s
>90% (not shown) siRNA + HiPerFect
Van Drongelen et al. 2013 [E4]
N/TERT immortalized KC cell line
Vavrova et al. 2014 [E5] (adapted from Mildner et al.) Pendaries et al. 2014 [E6]
82% mRNA
Neonatal foreskin KC’s
Adult epidermal KC’s
shRNA + electroporation 85% mRNA 80% protein siRNA + lipofectamine 75% mRNA shRNA + lentivirus
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- Less KC layers - Reduced SC thickness - Less keratohyalin granules - Impaired SC formation - Reduction in proliferation - Normal skin surface pH - Decreased NMF levels - Increased UV sensitivity - Altered epidermal differentiation - Activation pro-caspase 14 inhibition
Barrier permeability Increased permeability
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Mildner et al. 2010 [E1]
Keratinocyte source Neonatal foreskin KC’s
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Supplemental Table E1: Filaggrin knockdown studies using in vitro 3D skin models
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Abbreviations: Butyl-PABA, butyl 4-aminobenzoate; KC, keratinocyte; mRNA, messenger RNA; NMF, natural moisturizing factor; N/TERT, normal human keratinocytes immortalized by stable human telomerase transfection; SC, stratum corneum; shRNA, small hairpin RNA (stable transduction); siRNA, small interfering RNA (transient transfection); UCA, urocanic acid; UV, ultra violet radiation
ACCEPTED MANUSCRIPT Supplemental Table E2. Antibodies for immunohistochemistry and immunofluorescence (grey) Antibody clone, manufacturer
Dilution
Cytokeratin-10
DE-K10, Euro Diagnostics
1:100
Filaggrin
NCL-Filaggrin, Novocastra
1:200
Loricrin
PRB145P, Covance
1:2000
Transglutaminase-1
H-87, Santa Cruz
Involucrin
MON-150, generated by our group [E13]
1:20
Late cornified envelope 3
Generated by our group [E14]
1:5000
4',6-diamidino-2-phenylindole
Boehringer Mannheim
Streptavidin Alexa Fluor 594
Life Technologies
Claudin-1
JAY.8, Thermo Fisher Scientific
1:1500
Claudin-4
3E2C1, Thermo Fisher Scientific
1:700
Occludin
N19, Santa Cruz
1:250
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1:3000 1:200
ACCEPTED MANUSCRIPT Supplemental Table E3. Primers used for qPCR. HUGO alias gene name
forward primer 5’-3’
reverse primer 3’-5’
E*
CASP14
Caspase 14
acaaaccatcccaacatacac
gatatgtcctttcctcttcgt
2,12
CLDN1
Claudin 1
ccagtcaatgccaggtacga
ttggatagggccttggtgtt
1,78
CLDN4
Claudin 4
gctgtaaacaggtttgggca
tcagaggggatcagtctcca
1,85
FLG
Filaggrin
acttcactgagtttcttctgatggtatt
tccagacttgagggtctttttctg
1,89
FLG2
Filaggrin 2
accaggttcacttaaacttgca
atgacatccactgtgtctggatc
1,96
HRNR
Hornerin
tacaaggcgtcatcactgtcatc
IVL
Involucrin
acttatttcgggtccgctaggt
SC
atctggatcgtttggattcttcag
2,12
gagacatgtagagggacagagtcaag
1,93
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ggacctgtcccagagtgatg
gatccaggatgggctcttg
2,10
2A LOR
Loricrin
aggttaagacatgaaggatttgcaa
ggcaccgatgggcttagag
2,08
OCLN
Occludin
attggtcaccgagggagga
taaaccaatctgctgcgtccta
1,86
caccattgaaatcctgagtgatgt
tgaccagcccaaaggagaag
2,02
cccccgcaatgagatctaca
atcctcatggtccacgtacaca
1,99
hARP, 60S acidic ribosomal protein P0 TGM1
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RPLP0
Transglutaminase 1
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*E is efficiency as fold increase in fluorescence per PCR cycle
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