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Involucrin modulates vitamin D receptor activity in the epidermis

  • Alina D. Schmidt
    Affiliations
    Division of Dermatology, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO, 63110 USA

    Center for Pharmacogenomics, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO, 63110 USA

    Center for the Study of Itch & Sensory Disorders, Washington University in St. Louis School of Medicine, St. Louis, MO 63110 USA
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  • Charlene Miciano
    Affiliations
    Division of Dermatology, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO, 63110 USA

    Center for Pharmacogenomics, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO, 63110 USA

    Center for the Study of Itch & Sensory Disorders, Washington University in St. Louis School of Medicine, St. Louis, MO 63110 USA
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  • Qi Zheng
    Affiliations
    Department of Dermatology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 USA
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  • Mary Elizabeth Mathyer
    Affiliations
    Division of Dermatology, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO, 63110 USA

    Center for Pharmacogenomics, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO, 63110 USA

    Center for the Study of Itch & Sensory Disorders, Washington University in St. Louis School of Medicine, St. Louis, MO 63110 USA
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  • Elizabeth A. Grice
    Affiliations
    Department of Dermatology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 USA
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  • Cristina de Guzman Strong
    Correspondence
    To Whom Correspondence Should Be Addressed: Cristina de Guzman Strong, PhD., Center for Cutaneous Biology and Immunology, Department of Dermatology, Immunology Program, Henry Ford Cancer Institute, Henry Ford Health, 1 Ford Place RM 4D46, Detroit, MI 48202 USA, Tel: (313) 876-7291, ,
    Affiliations
    Division of Dermatology, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO, 63110 USA

    Center for Pharmacogenomics, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO, 63110 USA

    Center for the Study of Itch & Sensory Disorders, Washington University in St. Louis School of Medicine, St. Louis, MO 63110 USA

    Center for Cutaneous Biology and Immunology, Department of Dermatology, Immunology Program, Henry Ford Cancer Institute, Henry Ford Health, Detroit, MI 48202 USA

    Department of Medicine, College of Human Medicine, Michigan State University, East Lansing, MI 48824
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Open AccessPublished:January 12, 2023DOI:https://doi.org/10.1016/j.jid.2022.12.009

      ABSTRACT

      Terminally differentiated keratinocytes are critical for epidermal function and surrounded by involucrin (IVL). Increased IVL expression is associated with a near selective sweep in European populations compared to African. This positive selection for increased IVL in the epidermis identifies human adaptation out-of-Africa. The functional significance is unclear. We hypothesize Ivl to modulate the environmentally sensitive Vitamin D receptor (Vdr) in the epidermis. We investigated Vdr activity in Ivl -/- and wild-type (WT) mice using vitamin D agonist (MC903) treatment and comprehensively determined the inflammatory response using single-cell RNA sequencing (scRNA-seq) and associated skin microbiome changes using 16S bacterial phylotyping. Vdr activity and target gene expression were reduced in Ivl -/- mouse skin, with decreased MC903-mediated skin inflammation and significant reductions in CD4+ T cells, basophils, macrophages, monocytes, and type II basal keratinocytes and increase in suprabasal keratinocytes. Coinciding with the dampened MC903-mediated inflammation, skin microbiota of Ivl -/- mice was more stable compared to WT mice, which exhibited a MC903-responsive increase in Bacteroidetes and decrease in Firmicutes. Together, our studies in Ivl -/- mice identify a functional role for Involucrin to positively impact Vdr activity and suggest an emerging IVL/VDR paradigm for adaptation in the human epidermis.

      Abbreviations:

      AF ((allele frequency)), CE ((cornified envelope)), DE-OTU ((differentially enriched operational taxonomic unit)), EDC ((Epidermal Differentiation Complex)), Envl ((envoplakin)), eQTL ((expression quantitative trait loci)), IVL ((involucrin)), OTU ((operational taxonomic unit)), Ppl ((periplakin)), (scRNA-seq) (single-cell RNA sequencing), UMAP ((uniform manifold approximation and projection)), VDR ((vitamin D receptor)), WT ((wild-type))

      INTRODUCTION

      The epidermis is the outermost tissue of the skin, providing crucial barrier function against the external environment (
      • Watt F.M.
      Mammalian skin cell biology: at the interface between laboratory and clinic.
      ,
      • Matsui T.
      • Amagai M.
      Dissecting the formation, structure and barrier function of the stratum corneum.
      ). It is composed of a hierarchical structure of proliferating and differentiated epidermal cells or keratinocytes that form the essential “brick” units of the skin barrier. The epidermis is also the primary site of vitamin D3 production (
      • Bikle D.
      • Christakos S.
      New aspects of vitamin D metabolism and action - addressing the skin as source and target.
      ). Keratinocytes are also known to elicit proper recruitment of immune cells in response to breaks in the skin barrier, a key defense mechanism to maintain skin homeostasis (
      • Niec R.E.
      • Rudensky A.Y.
      • Fuchs E.
      Inflammatory adaptation in barrier tissues.
      ). Keratinocytes as well respond to microbiota exposure with the induction of key epidermal differentiation gene expression (
      • Meisel J.S.
      • Sfyroera G.
      • Bartow-McKenney C.
      • Gimblet C.
      • Bugayev J.
      • Horwinski J.
      • et al.
      Commensal microbiota modulate gene expression in the skin.
      ) (
      • Uberoi A.
      • Bartow-McKenney C.
      • Zheng Q.
      • Flowers L.
      • Campbell A.
      • Knight S.A.B.
      • et al.
      Commensal microbiota regulates skin barrier function and repair via signaling through the aryl hydrocarbon receptor.
      ). Indeed, the biology of epidermal keratinocytes is complex and challenging and coupled with the need for these cells to quickly adapt amidst differing environments in order to ensure survival.
      Differentiated keratinocytes express many genes encoded within the Epidermal Differentiation Locus (EDC) critical for barrier function (
      • Volz A.
      • Korge B.P.
      • Compton J.G.
      • Ziegler A.
      • Steinert P.M.
      • Mischke D.
      Physical mapping of a functional cluster of epidermal differentiation genes on chromosome 1q21.
      ,
      • Mischke D.
      • Korge B.P.
      • Marenholz I.
      • Volz A.
      • Ziegler A.
      Genes encoding structural proteins of epidermal cornification and S100 calcium-binding proteins form a gene complex ("epidermal differentiation complex") on human chromosome 1q21.
      ,
      • Zhao X.P.
      • Elder J.T.
      Positional cloning of novel skin-specific genes from the human epidermal differentiation complex.
      ,
      • de Guzman Strong C.
      • Conlan S.
      • Deming C.B.
      • Cheng J.
      • Sears K.E.
      • Segre J.A.
      A milieu of regulatory elements in the epidermal differentiation complex syntenic block: implications for atopic dermatitis and psoriasis.
      ). One important EDC gene is involucrin (IVL), whose expression is a major marker for early epidermal differentiation (
      • Rice R.H.
      • Green H.
      Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions.
      ). IVL is a scaffolder for many cross-linked proteins that together form the mature cornified envelope (CE) surrounding the terminally differentiated keratinocyte (
      • Eckert R.L.
      • Green H.
      Structure and evolution of the human involucrin gene.
      ,
      • Steinert P.M.
      • Marekov L.N.
      The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal cornified cell envelope.
      ,
      • Robinson N.A.
      • LaCelle P.T.
      • Eckert R.L.
      Involucrin is a covalently crosslinked constituent of highly purified epidermal corneocytes: evidence for a common pattern of involucrin crosslinking in vivo and in vitro.
      ,
      • Nemes Z.
      • Marekov L.N.
      • Steinert P.M.
      Involucrin cross-linking by transglutaminase 1. Binding to membranes directs residue specificity.
      ,
      • Kajava A.V.
      alpha-Helical solenoid model for the human involucrin.
      ). Given the importance of CE formation for barrier function, it was surprising to find that Ivl knockout (-/-) mice exhibited no defects in the epidermal barrier and cornified envelope formation (
      • Djian P.
      • Easley K.
      • Green H.
      Targeted ablation of the murine involucrin gene.
      ). However, triple knockout mice for Ivl, envoplakin (Envl), and periplakin (Ppl) cornified envelope genes exhibited a delay in developmental barrier formation, dry, flaky skin, and persistent hyperkeratosis (
      • Sevilla L.M.
      • Nachat R.
      • Groot K.R.
      • Klement J.F.
      • Uitto J.
      • Djian P.
      • et al.
      Mice deficient in involucrin, envoplakin, and periplakin have a defective epidermal barrier.
      ). Together, these studies revealed that Ivl alone is dispensable for skin barrier development but, with coinciding Envl and Ppl deficiencies, is critical for epidermal barrier function.
      We recently discovered positive selection for a human IVL allele in populations of European ancestry (
      • Mathyer M.E.
      • Brettmann E.A.
      • Schmidt A.D.
      • Goodwin Z.A.
      • Oh I.Y.
      • Quiggle A.M.
      • et al.
      Selective sweep for an enhancer involucrin allele identifies skin barrier adaptation out of Africa.
      ). We identified a specific IVL haplotype that underwent a near selective sweep (>95% allele frequency [AF]) in contrast to that in Africa (32% AF). The European IVL haplotype consists of expression quantitative trait loci (eQTLs) associated with increased IVL gene expression in the skin in contrast to relatively lower IVL expression in the skin of African ancestry. To our knowledge, this is the first report of recent evolutionary adaptation in the human epidermis. Our finding suggests a selective benefit for increased IVL as humans migrated out of Africa. Here we revisit the role of involucrin for adaptation in the epidermis in response to the environment.
      The vitamin D receptor (Vdr) was recently determined to exhibit environmental sensitivity (
      • Romney A.L.T.
      • Davis E.M.
      • Corona M.M.
      • Wagner J.T.
      • Podrabsky J.E.
      Temperature-dependent vitamin D signaling regulates developmental trajectory associated with diapause in an annual killifish.
      ). This was discovered in killifish that exhibited differential downstream Vdr signaling when grown under different temperature conditions. Motivated by this novel discovery for Vdr as an environmentally sensitive receptor and its known expression in epidermal keratinocytes (
      • Stumpf W.E.
      • Sar M.
      • Reid F.A.
      • Tanaka Y.
      • DeLuca H.F.
      Target cells for 1,25-dihydroxyvitamin D3 in intestinal tract, stomach, kidney, skin, pituitary, and parathyroid.
      ), we reasoned VDR as a potential molecule that could link the levels of involucrin in the skin to environmental sensitivity and hence relevant to human epidermal adaptation. VDR is known to stimulate epidermal differentiation when activated by the vitamin D metabolite, 1,25-(OH)2D, produced by keratinocytes (
      • Hosomi J.
      • Hosoi J.
      • Abe E.
      • Suda T.
      • Kuroki T.
      Regulation of terminal differentiation of cultured mouse epidermal cells by 1 alpha,25-dihydroxyvitamin D3.
      ,
      • Bikle D.D.
      Vitamin D metabolism and function in the skin.
      ). Knockout mouse studies confirmed a requirement for Vdr for epidermal differentiation as Vdr null mice exhibited reduced levels of involucrin, filaggrin, and loricrin (
      • Xie Z.
      • Komuves L.
      • Yu Q.C.
      • Elalieh H.
      • Ng D.C.
      • Leary C.
      • et al.
      Lack of the vitamin D receptor is associated with reduced epidermal differentiation and hair follicle growth.
      ,
      • Bikle D.D.
      • Elalieh H.
      • Chang S.
      • Xie Z.
      • Sundberg J.P.
      Development and progression of alopecia in the vitamin D receptor null mouse.
      ), resulting in a defective permeability barrier (
      • Oda Y.
      • Uchida Y.
      • Moradian S.
      • Crumrine D.
      • Elias P.M.
      • Bikle D.D.
      Vitamin D receptor and coactivators SRC2 and 3 regulate epidermis-specific sphingolipid production and permeability barrier formation.
      ) and immune response (
      • Schauber J.
      • Dorschner R.A.
      • Coda A.B.
      • Buchau A.S.
      • Liu P.T.
      • Kiken D.
      • et al.
      Injury enhances TLR2 function and antimicrobial peptide expression through a vitamin D-dependent mechanism.
      ,
      • Muehleisen B.
      • Bikle D.D.
      • Aguilera C.
      • Burton D.W.
      • Sen G.L.
      • Deftos L.J.
      • et al.
      PTH/PTHrP and vitamin D control antimicrobial peptide expression and susceptibility to bacterial skin infection.
      ). Interestingly, VDR and vitamin D3 levels decrease upon IVL-marked terminal differentiation, suggesting IVL-associated negative feedback regulation for VDR (
      • Horiuchi N.
      • Clemens T.L.
      • Schiller A.L.
      • Holick M.F.
      Detection and developmental changes of the 1,25-(OH)2-D3 receptor concentration in mouse skin and intestine.
      ,
      • Pillai S.
      • Bikle D.D.
      • Elias P.M.
      1,25-Dihydroxyvitamin D production and receptor binding in human keratinocytes varies with differentiation.
      ). Moreover, VDR nuclear localization was found to be significantly reduced in the epidermis of black donor normal skin compared to that of white donors (
      • Hahn J.M.
      • Supp D.M.
      Abnormal expression of the vitamin D receptor in keloid scars.
      ) whose genotypes we have determined to be more commonly associated with relatively increased IVL (
      • Mathyer M.E.
      • Brettmann E.A.
      • Schmidt A.D.
      • Goodwin Z.A.
      • Oh I.Y.
      • Quiggle A.M.
      • et al.
      Selective sweep for an enhancer involucrin allele identifies skin barrier adaptation out of Africa.
      ). The findings using knockout mice and in vitro studies highlight a putative relationship between IVL and VDR. The more recent study using human diverse skin further suggests a role for higher IVL levels and its potential direct, downstream dosage effect on VDR to modulate epidermal function and underlie population-specific, human skin adaptation.
      We hypothesized a role for involucrin to modulate Vdr activity as a mode by which the epidermis adapts to the environment. To test this hypothesis, we investigated Vdr function in the epidermis of Ivl -/- mice with vitamin D agonist (MC903) exposure and determined the impact using single-cell RNA sequencing (scRNA-seq), flow cytometry, and 16S rRNA bacterial phylotyping to assess Vdr activated-skin inflammation and the anticipated, associated changes to the skin microbiome. Here we report decreased Vdr activity and Vitamin D-responsive gene expressions in Ivl -/- mice that exhibited reduced MC903-induced inflammation marked by scRNA-seq identification for decreased basophils, macrophages, monocytes, and CD4+ T cells and subsequent validation for decreased CD11b+ IgE+ basophils and CD4+ T cells and newly discovered increase in CD11b+ IgEhigh mast cells by flow cytometry. The decrease in Vdr-mediated inflammation in Ivl -/- mice coincided with no phylum level changes to the skin microbiome in contrast to WT that exhibited an MC903-responsive dysbiosis with increase in Bacteroidetes and decrease in Firmicutes. Together, our findings identify a functional role for Ivl to positively impact Vdr activity for the skin immune response in the epidermis.

      RESULTS

      Vdr-mediated inflammation is reduced in MC903-treated Ivl -/- mouse skin

      We investigated Vdr function in Ivl -/- and wild-type (WT) mice by way of MC903-mediated activation. Ivl -/- and WT mouse ear skin were treated daily for 12 days with MC903 that results in skin inflammation (
      • Li M.
      • Hener P.
      • Zhang Z.
      • Kato S.
      • Metzger D.
      • Chambon P.
      Topical vitamin D3 and low-calcemic analogs induce thymic stromal lymphopoietin in mouse keratinocytes and trigger an atopic dermatitis.
      ,
      • Kim B.S.
      • Siracusa M.C.
      • Saenz S.A.
      • Noti M.
      • Monticelli L.A.
      • Sonnenberg G.F.
      • et al.
      TSLP elicits IL-33-independent innate lymphoid cell responses to promote skin inflammation.
      ,
      • Moosbrugger-Martinz V.
      • Schmuth M.
      • Dubrac S.
      A Mouse Model for Atopic Dermatitis Using Topical Application of Vitamin D3 or of Its Analog MC903.
      ,
      • Walsh C.M.
      • Hill R.Z.
      • Schwendinger-Schreck J.
      • Deguine J.
      • Brock E.C.
      • Kucirek N.
      • et al.
      Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis.
      ). MC903-treated WT mice exhibited ear thickening and scaling compared to ethanol-treated ears (Figure 1a). By contrast, MC903-treated Ivl -/- mice exhibited less ear thickening and scaling. Histological findings further identified epidermal hyperplasia and hyperkeratosis observed in MC903-treated WT ear skin that was strikingly less evident in MC903-treated Ivl -/- and absent in ethanol-treated ear skin (Figure 1b). We assessed the longitudinal development of ear thickness over the 12-day treatment period and found an overall trend for decreased MC903-induced skin inflammation in Ivl -/- mice compared to WT (Figure 1c, Figure S1). Decreased inflammation in Ivl -/- mice was significant on days 6 and 7 and for each of days 9-12 (day 6, p = 0.044; day 7, p = 0.016; day 9, p = 0.039; day 10, p = 0.016; day 11, p = 0.007; and day 12, p = 0.004; one-way ANOVA with post-hoc Tukey HSD). Together, our findings for decreased skin inflammation in MC903-treated Ivl-/- adult mice identifies decreased Vdr activity in Ivl -/- epidermis.
      Figure thumbnail gr1
      Figure 1Reduced Vdr-mediated inflammation in MC903-treated Ivl -/- mouse skin. (a) MC903-treated Ivl -/- mouse ears displayed less scaling and thickness compared to WT. (b) Hematoxylin and eosin histology staining revealed epidermal hyperkeratosis and hyperplasia in WT ear skin vs. Ivl -/-. Scale bar = 50 μm. (c) Percent change in ear thickness (inflammation) was significantly reduced in MC903-treated Ivl -/- (n=10, orange) vs. WT mice (n=15, blue) at days 6 and 7 and days 9-12 vs. day 0. *p < 0.05, one-way ANOVA with post-hoc Tukey HSD. Error bars ±SEM. No percent change was observed in ethanol-treated control ears (Ivl -/-, n=10, dotted orange; WT, n=15, dotted blue). Vdr, vitamin D receptor; Ivl, Involucrin; WT, wild-type; ANOVA, ANalysis Of VAriance; HSD, Honestly Significant Difference.

      scRNA-seq identifies a decrease in Vitamin D-responsive gene expressions and decreased basophils, CD4+ T cells, macrophages, monocytes, and basal II keratinocytes and increased suprabasal keratinocytes in MC903-treated Ivl -/- skin

      We next determined the cell types that underlie the Vdr-mediated inflammatory response at a single-cell resolution using scRNA-seq. Single-cell suspensions from MC903-treated ear skin from Ivl -/- and WT mice were obtained for scRNA-seq (10X Genomics). A total of 25,799 single cells were sequenced and analyzed from both Ivl -/- and WT mice (n=2 for each genotype). Seurat4.0 clustering analyses identified 20 cell populations in MC903-treated ear skin (Figure 2a ). Keratinocytes comprise a majority of the populations with 13 distinct keratinocyte clusters that were identified in both genotypes (Figure 2b). Additionally, five immune cell types were found including basophils, macrophages, monocytes, natural killer T cells, and CD4+ T cells, with the remaining two populations comprised of fibroblasts and melanocytes. We confirmed annotation of the cell clusters using gene markers specific for each cell population (
      • Joost S.
      • Zeisel A.
      • Jacob T.
      • Sun X.
      • La Manno G.
      • Lonnerberg P.
      • et al.
      Single-Cell Transcriptomics Reveals that Differentiation and Spatial Signatures Shape Epidermal and Hair Follicle Heterogeneity.
      ) (Tables S1-2).
      Figure thumbnail gr2
      Figure 2scRNA-seq identifies differential expressions for Vitamin D-responsive genes and decreases in basophils, macrophages, CD4+ T cells, monocytes, and basal II keratinocytes and an increase in suprabasal keratinocytes in MC903-treated Ivl -/- skin. (a) UMAP of scRNA-seq-identified cell types in both MC903-treated WT and Ivl -/- skin (n=2/genotype). (b) Individual UMAPs of cell types in MC903-treated WT (left) and Ivl -/- (right) skin. (c) Vitamin D-responsive Vdr, Cyp24a1, Snw1, Pdia3, and Rxra genes are differentially expressed between MC903-treated WT and Ivl -/- skin and ranked by number of clusters with significance and by number found in keratinocyte clusters (Wilcoxon signed-rank test, *adj. p <0.05, see also Table S3). (d) Bar plots (proportions of cells) for Ivl -/- (orange) and WT (blue). Basophils, macrophages, CD4+ T cells, monocytes, and basal II keratinocytes were decreased. Suprabasal keratinocytes were increased in Ivl -/- vs. WT (chi-squared goodness of fit, *p < 0.05). (e) Predominant IL-4 and IL-6 expressions in basophils that were reduced in MC903-treated Ivl-/- vs. WT skin. Ivl, Involucrin; WT, wild-type; UMAP, Uniform Manifold Approximation and Projection.
      Figure thumbnail gr3
      Figure 3CD4+ T cells and CD11b+ IgE+ basophils are reduced and CD11b+ IgEhigh mast cells are increased in MC903-treated Ivl -/- mouse skin. (a) Flow cytometry pseudocolor dot plots for CD45+ gated cell types shown in representative MC903-treated WT and Ivl -/- ear skin (day 12). (b) Bar graphs of cell type mean percentages are also shown. CD4+ T cells and CD11b+ IgE+ basophils in MC903-treated Ivl -/- ear skin were significantly decreased. CD11b+ IgEhigh mast cells were also significantly increased in MC903-treated Ivl -/- ear skin. (CD4+ T cells, *p < 0.04; basophils, p < 0.05; mast cells, *p < 0.03, one-sided t-test). No significant differences were found for both monocytes and macrophages (n=5/genotype). Error bars ±SEM. Ivl, Involucrin; WT, wild type.
      We next investigated the impact of MC903 treatment on Vitamin D-responsive gene expression in Ivl -/- mice that exhibited a dampened Vdr-mediated inflammatory response. Fourteen Vdr-regulated genes in GSEA (GOBP_RESPONSE_TO_VITAMIN_D; MM6812) were expressed in at least one scRNA-seq cell cluster for either MC903-treated Ivl -/- or WT skin (Figure 2c, Figure S2). Of these, ten genes were differentially expressed in at least one scRNA-seq cell cluster in MC903-treated Ivl -/- vs. WT skin (adj p <0.05; Table S3). A majority of these gene expression differences in Ivl -/- skin were decreased (38 out of 41 clusters) and were found primarily in the keratinocyte clusters (17 out of 38 decreased clusters). Vdr, Cyp24a1, Snw1, Pdia3, and Rxra were the top 5 differentially expressed genes that were observed in at least 3 clusters (Figure 2c). Vdr, Snw1, and Pdia3 were all decreased in basal cycling (0), suprabasal cycling (3), and suprabasal infundibular (6) keratinocytes in MC903-treated Ivl -/- mice. More importantly, Vdr was the top gene that was found to have the most number of keratinocyte clusters exhibiting differential expression, a total of 9 that were all decreased in Ivl -/- mice. By contrast, Vdr-regulated Cyp24a1 was increased in several Ivl -/- keratinocyte clusters and melanocytes. Cyp24a1 encodes 24-hydroxylase that breaks down active Vitamin D (
      • Makin G.
      • Lohnes D.
      • Byford V.
      • Ray R.
      • Jones G.
      Target cell metabolism of 1,25-dihydroxyvitamin D3 to calcitroic acid. Evidence for a pathway in kidney and bone involving 24-oxidation.
      ,
      • Reddy G.S.
      • Tserng K.Y.
      Calcitroic acid, end product of renal metabolism of 1,25-dihydroxyvitamin D3 through C-24 oxidation pathway.
      ,
      • Schlingmann K.P.
      • Kaufmann M.
      • Weber S.
      • Irwin A.
      • Goos C.
      • John U.
      • et al.
      Mutations in CYP24A1 and idiopathic infantile hypercalcemia.
      ). The finding for increased Cyp24a1 in MC903-treated Ivl -/- skin suggests a compensatory mechanism in response to MC903 and likely non-Vdr regulated given the decreased Vdr expression in these mice. Together, our findings identifies significant decreases in Vdr and Vitamin D-responsive genes that underlie the dampened MC903-mediated inflammation in Ivl -/- mice.
      We further determined if there was a difference in the proportions between each scRNA-seq population in MC903-treated Ivl -/- mice compared to WT. The proportions for each of five cell populations were significantly reduced in MC903-treated Ivl -/- mice: basophils, macrophages, basal IIb keratinocytes, CD4+ T cells, and monocytes (Figure 2d, chi-squared goodness of fit, p < 0.00001, p < 0.00001, p < 0.00001, p < 0.0455, and p < 0.00014, respectively). However, suprabasal keratinocytes were significantly increased in MC903-treated Ivl-/- ear skin (Figure 2d, chi-squared goodness of fit, p < 0.028) and concomitant with a decrease in basal IIb keratinocytes. The finding suggests a higher turnover in basal IIb keratinocytes and downstream effect for the increased number of suprabasal keratinocytes. The reduction in basophils led us to further investigate the significance of IL-4 and IL-6 that are also known key drivers for MC903-induced skin inflammation (
      • Kim B.S.
      • Wang K.
      • Siracusa M.C.
      • Saenz S.A.
      • Brestoff J.R.
      • Monticelli L.A.
      • et al.
      Basophils promote innate lymphoid cell responses in inflamed skin.
      ,
      • Hussain M.
      • Borcard L.
      • Walsh K.P.
      • Pena Rodriguez M.
      • Mueller C.
      • Kim B.S.
      • et al.
      Basophil-derived IL-4 promotes epicutaneous antigen sensitization concomitant with the development of food allergy.
      ,
      • Walsh C.M.
      • Hill R.Z.
      • Schwendinger-Schreck J.
      • Deguine J.
      • Brock E.C.
      • Kucirek N.
      • et al.
      Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis.
      ). Our scRNA-seq analysis resolved predominant IL-4 and IL-6 expressions in basophils as shown in MC903-treated mice and for which basophil numbers were significantly decreased in the Ivl -/- mice (Figure 2de). In summary, scRNA-seq resolves the single cell architecture of the dampened MC903-induced inflammation in Ivl -/- mice identifying reduced basophils, macrophages, CD4+ T cells, monocytes, and a notable keratinocyte response (basal IIb keratinocyte depletion with increased suprabasal cells) and further highlights skin immune cellular interconnectivity.

      Flow cytometry validates decreased CD4+ T cells and basophils and reveals increased mast cells in MC903-treated Ivl -/- mouse skin

      We sought to validate the scRNA-seq-identified immune cell types that were reduced in MC903-treated Ivl -/- compared to WT mice by flow cytometry. CD4+ T cells, CD11c+ dermal dendritic cells, granulocytes, monocytes, eosinophils, and CD8+ T cells were previously determined to be increased based on immunofluorescent and histological findings in MC903-induced inflamed skin (
      • Li M.
      • Hener P.
      • Zhang Z.
      • Kato S.
      • Metzger D.
      • Chambon P.
      Topical vitamin D3 and low-calcemic analogs induce thymic stromal lymphopoietin in mouse keratinocytes and trigger an atopic dermatitis.
      ). However, of the CD45+ leukocytes in the MC903-treated skin, we found no significant differences in dendritic cells (CD11c+), eosinophils (CD11b+, Ly6g-), neutrophils (CD11b+, Ly6g+) and B cells (CD19+) between Ivl -/- and WT mice (Figure S3). We also did not find significant differences in monocytes (CD11b+, F4/80-, Ly6g-) and macrophages (CD11b+, F4/80+, Ly6g-) (Figure 3ab). However, both CD4+ T cells and CD11b+ IgE+ basophils were significantly reduced in Ivl -/- compared to WT treated ear skin (Figure 3ab, one-sided t-test, p < 0.04, p < 0.05) respectively. Interestingly, we also observed an increase in a distinct population of CD11b+ IgEhigh cells in MC903-treated Ivl -/- skin, representative of mast cells that was significantly increased compared to MC903-treated WT ear skin (one-sided t-test, p < 0.03) (Figure 3ab). Together, our flow cytometry results confirm our scRNA-seq findings for decreased basophil and CD4+ T cell infiltrates yet with an additional discovery for increased CD11b+ IgEhigh mast cells in MC903-treated Ivl -/- mice.

      MC903-treated WT skin exhibit increased Bacteroidetes and decreased Firmicutes whereas Ivl -/- untreated skin exhibit increased Firmicutes (Streptococcus and Aerococcus) and decreased Bacteroidetes (Muribaculaceae) OTUs

      We next examined the impact of Vdr-mediated inflammation on Ivl -/- and WT skin microbiomes. Specific microbiome changes (dysbiosis) are sufficient to drive skin inflammation as observed in barrier-impaired, ADAM17-deficient mice (
      • Kobayashi T.
      • Glatz M.
      • Horiuchi K.
      • Kawasaki H.
      • Akiyama H.
      • Kaplan D.H.
      • et al.
      Dysbiosis and Staphylococcus aureus Colonization Drives Inflammation in Atopic Dermatitis.
      ). However, whether Vdr-mediated inflammation drives microbial changes per se is an underexplored area of investigation. The dampened Vdr activity and decreased Vdr and Vitamin D-responsive gene expression in Ivl -/- skin also provides us with the opportunity to more directly address the microbial changes specific to Vdr activity that are active in WT and compromised in Ivl -/- skin. As microbiota exposure induces host expression of involucrin in the skin (
      • Meisel J.S.
      • Sfyroera G.
      • Bartow-McKenney C.
      • Gimblet C.
      • Bugayev J.
      • Horwinski J.
      • et al.
      Commensal microbiota modulate gene expression in the skin.
      ,
      • Uberoi A.
      • Bartow-McKenney C.
      • Zheng Q.
      • Flowers L.
      • Campbell A.
      • Knight S.A.B.
      • et al.
      Commensal microbiota regulates skin barrier function and repair via signaling through the aryl hydrocarbon receptor.
      ), we further explore the significance of involucrin for shaping microbial community structure.
      To profile skin microbiota dynamics, we used 16S rRNA sequencing of the V1-V3 hypervariable region to identify bacterial taxa from ear swab collections from 12-week-old mice before and after MC903 treatment. 16S microbiome sequencing reads were individually phylotyped and subsequently clustered into phylogeny-based operational taxonomic units (OTUs) (
      • Zheng Q.
      • Bartow-McKenney C.
      • Meisel J.S.
      • Grice E.A.
      HmmUFOtu: An HMM and phylogenetic placement based ultra-fast taxonomic assignment and OTU picking tool for microbiome amplicon sequencing studies.
      ). We first assessed the beta (inter-sample) diversity with respect to MC903 treatment in Ivl -/- and WT skin and observed that clustering between microbial communities was significantly associated with both genotypes (WT or Ivl -/-) and MC903 treatment (before or after) (Figure 4a, p < 0.005, PERMANOVA test) (GreenGenes v13.8 (
      • DeSantis T.Z.
      • Hugenholtz P.
      • Larsen N.
      • Rojas M.
      • Brodie E.L.
      • Keller K.
      • et al.
      Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB.
      )). Closer examination of the microbial phyla on untreated WT skin revealed a composition dominated by the phylum Firmicutes and phylum Bacteroidetes consistent with previous reports in WT murine ear skin microbiota (
      • Ren D.
      • Gong S.
      • Shu J.
      • Zhu J.
      • Rong F.
      • Zhang Z.
      • et al.
      Mixed Lactobacillus plantarum Strains Inhibit Staphylococcus aureus Induced Inflammation and Ameliorate Intestinal Microflora in Mice.
      ,

      Moskovicz V, Ben-El R, Horev G, Mizrahi B. Skin microbiota dynamics following B. subtilis formulation challenge: an in vivo study in mice. BMC Microbiol 2021;21(1):231.

      ) (Figure 4b). By contrast, we found reductions in Bacteroidetes, Actinobacteria, and Verrucomicrobia and an increase in Firmicutes in Ivl -/- untreated skin compared to WT (Figure 4b). We next compared the alpha diversity (intra-sample) of untreated WT and Ivl -/- skin by calculating the Shannon Index, which takes into account both richness and evenness of operational taxonomic units (OTUs) observed in a sample. We detected a significant depletion in the alpha diversity of untreated Ivl -/- skin compared to WT (p < 0.001, t-test) (Figure 4c).
      Figure thumbnail gr4
      Figure 4MC903-treated WT skin show increased Bacteroidetes and decreased Firmicutes whereas Ivl -/- untreated skin exhibit increased Streptococcus and Aerococcus (Firmicutes) and decreased Muribaculaceae (Bacteroidetes) taxa. (a) Clustering between microbial communities based on unweighted UniFrac distance (beta diversity) was significantly associated with both genotype (WT or Ivl -/-) and MC903 treatment (before or after). Axis 1 (26.1% variance) and Axis 2 (16.3% variance) (OTU_dist ∼ study_group * TimePoint, p < 0.005). (b) Relative abundance of top phylum-level OTUs in untreated WT and Ivl -/- skin (WT and Ivl -/-, each n=3). (c) Decreased alpha diversity (Shannon index) in untreated Ivl -/- vs. WT skin (***p < 0.01). (d) Relative abundance of top phylum-level OTUs in MC903-treated WT and Ivl -/- skin. (e) Decreased alpha diversity in MC903-treated Ivl -/- vs. WT skin (*p < 0.05). (f) Alpha diversity is significantly reduced in MC903-treated WT skin (*p < 0.05) but not in MC903-treated Ivl -/- skin. DE-OTU MA plots of (g) untreated Ivl -/- vs. WT skin; Streptococcus, Aerococcus, Lachnospiraceae, and Muris (phylum Firmicutes) comprise a majority of top 15 significantly upregulated DE-OTUs and Muribaculaceae (phylum Bacteroidetes) represent the most common top 15 significantly downregulated DE-OTUs and (h) MC903-treated Ivl -/- vs. WT skin; Streptococcus and Aerococcus are also found in a majority of top 15 significantly upregulated DE-OTUs with Muribaculaceae in a majority of the top 15 significantly downregulated DE-OTUs. All MA plots shown with FDR < 0.1, log2FC>=1. Ivl, Involucrin; WT, wild-type; DE-OTUs, differentially expressed operational taxonomic units; FDR, false discovery rate; log2FC, log-fold change.
      We next determined the longitudinal effect of MC903 treatment on the skin microbiota. MC903 treatment in WT skin resulted in an increase in phylum Bacteroidetes and a decrease in phylum Firmicutes compared to untreated skin (Figure 4d). In contrast, MC903 treatment in Ivl -/- skin did not change the phylum composition of the skin microbiota compared to untreated Ivl -/- skin (Figure 4d). Yet alpha diversity in MC903-treated Ivl -/- skin was significantly reduced compared to MC903-treated WT skin and correlates with the dampened skin inflammation in Ivl -/- treated skin compared to WT treated skin (p < 0.05) (Figure 4e). Relative to untreated skin, alpha diversity in WT mice was significantly reduced after MC903 treatment (Figure 4f, p < 0.05, t-test). In contrast, alpha diversity in Ivl -/- mice did not change after MC903 treatment, albeit with a slight increase compared to baseline skin (Figure 4f). Together, our results reveal potential dysbiosis in Ivl -/- baseline skin marked by a decrease in Bacteroidetes, Actinobacteria, and Verrucomicrobia and an increase in Firmicutes phyla. These changes were also consistent with a significant decrease in alpha diversity that persisted even upon MC903 treatment. We further identified dysbiosis in our longitudinal study of WT skin upon MC903 treatment marked by an increase in Bacteroidetes, a decrease in Firmicutes, and a significant reduction in alpha diversity.
      We next determined the differentially enriched OTUs (DE-OTUs) that underlie the microbiota differences observed in Ivl -/- skin and upon MC903 treatment in WT skin. We compared the abundance of each phylogeny-based OTU per mouse group before and after treatment (
      • Love M.I.
      • Huber W.
      • Anders S.
      Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.
      ). In untreated skin, we identified 122 upregulated and 28 downregulated differentially enriched OTUs (DE-OTUs) in Ivl -/- skin compared to WT (Figure 4g, Table S4). A majority of the top 15 upregulated OTUs in Ivl -/- untreated skin (66%) were comprised of phylum Firmicutes (Aerococcus, Streptococcus, Lachnospiraceae, and Muris). By contrast, the most common taxa of the top 15 downregulated OTUs in Ivl -/- untreated skin were Muribaculaceae (Bacteroidetes phylum) (47%) and formerly known as S24-7 (
      • Ormerod K.L.
      • Wood D.L.
      • Lachner N.
      • Gellatly S.L.
      • Daly J.N.
      • Parsons J.D.
      • et al.
      Genomic characterization of the uncultured Bacteroidales family S24-7 inhabiting the guts of homeothermic animals.
      ). In MC903-treated skin, we identified a total of 115 upregulated and 52 downregulated DE-OTUs (Figure 4h, Table S5) in Ivl -/- skin vs. WT. Similar to untreated skin, a majority (66%) of the top 15 upregulated OTUs in MC903-treated Ivl -/- skin were comprised of members from the phylum Firmicutes (Aerococcus and Streptococcus). Again, similar to untreated skin, a majority (60%) of the downregulated OTUs were Bacteroidetes (Muribaculaceae).
      In summary, our results suggest changes to the skin microbiota in Ivl -/- skin that were stable even after MC903 treatment. Our longitudinal study further identifies alterations to the microbiota in WT and not in Ivl -/- skin with decreased Vdr-mediated inflammation. This supports the hypothesis that Vdr-mediated inflammation can drive dysbiosis. As housing conditions can influence the skin microbiota, future studies using isolated cages and additional single-housed mice can further bolster these findings and provide insight into the cause and effect relationship between host and microbiota.

      DISCUSSION

      Our study identifies a functional role for Ivl to positively regulate Vdr activity in the epidermis. Here we find that Vdr activation and several Vitamin D-responsive genes including Vdr are reduced in MC903-treated Ivl-/- mice resulting in a dampened Vdr-mediated inflammatory response. The response is characterized by a significant reduction in CD4+ T cells, decreases in basophils, monocytes, macrophages, and basal II keratinocytes, and a concomitant increase in suprabasal keratinocytes. Indeed, MC903 is an analog of the natural VDR ligand, 1,25-(OH)2D, yet its topical application also stimulates VDR activity, resulting in skin inflammation albeit at a lower calcemic activity (
      • Carlberg C.
      Molecular basis of the selective activity of vitamin D analogues.
      ). We further validated our scRNA-seq findings for increased CD4+ T cells and basophils in MC903-treated WT skin but also discovered an increase in mast cells in MC903-treated Ivl -/- skin. Although flow cytometry did not fully recapitulate the scRNA-seq findings for monocytes and macrophages, not all monocytes and macrophage clusters as determined by scRNA-seq directly correlate with cell surface marker expression (
      • Sanin D.E.
      • Ge Y.
      • Marinkovic E.
      • Kabat A.M.
      • Castoldi A.
      • Caputa G.
      • et al.
      A common framework of monocyte-derived macrophage activation.
      ). Indeed, recent scRNA-seq studies have determined that multiple subsets of monocytes and their derived macrophages exist and each marked by dynamic heterogeneity in its transcriptional programs (
      • Villani A.C.
      • Satija R.
      • Reynolds G.
      • Sarkizova S.
      • Shekhar K.
      • Fletcher J.
      • et al.
      Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors.
      ,
      • Zilionis R.
      • Engblom C.
      • Pfirschke C.
      • Savova V.
      • Zemmour D.
      • Saatcioglu H.D.
      • et al.
      Single-Cell Transcriptomics of Human and Mouse Lung Cancers Reveals Conserved Myeloid Populations across Individuals and Species.
      ,
      • Sanin D.E.
      • Ge Y.
      • Marinkovic E.
      • Kabat A.M.
      • Castoldi A.
      • Caputa G.
      • et al.
      A common framework of monocyte-derived macrophage activation.
      ). This also creates even more challenges to further resolve these subset(s) and ascribe function. Nevertheless, increased monocytes have been reported in several MC903 studies (
      • Li M.
      • Hener P.
      • Zhang Z.
      • Kato S.
      • Metzger D.
      • Chambon P.
      Topical vitamin D3 and low-calcemic analogs induce thymic stromal lymphopoietin in mouse keratinocytes and trigger an atopic dermatitis.
      ,
      • Kim B.S.
      • Siracusa M.C.
      • Saenz S.A.
      • Noti M.
      • Monticelli L.A.
      • Sonnenberg G.F.
      • et al.
      TSLP elicits IL-33-independent innate lymphoid cell responses to promote skin inflammation.
      ,
      • Walsh C.M.
      • Hill R.Z.
      • Schwendinger-Schreck J.
      • Deguine J.
      • Brock E.C.
      • Kucirek N.
      • et al.
      Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis.
      ). Future studies are needed to further resolve the monocyte subsets impacted in the context of MC903-mediated inflammation and other inflammatory conditions. Additionally, Ivl -/- skin also exhibited microbial dysbiosis with increases in Firmicutes phyla, Streptococcus and Aerococcus, and a decrease in Bacteroidetes Muribaculaceae that was sustained even after MC903 treatment. Together, our results identify a functional role for involucrin to facilitate Vdr signaling in the epidermis for skin immune crosstalk and cellular composition as well as for impacting the microbial community structure.
      Previous work in vitro suggested a potential negative feedback for IVL on VDR (
      • Horiuchi N.
      • Clemens T.L.
      • Schiller A.L.
      • Holick M.F.
      Detection and developmental changes of the 1,25-(OH)2-D3 receptor concentration in mouse skin and intestine.
      ) (
      • Pillai S.
      • Bikle D.D.
      • Elias P.M.
      1,25-Dihydroxyvitamin D production and receptor binding in human keratinocytes varies with differentiation.
      ). VDR and Vitamin D3 levels concomitantly decreased and coincided with the onset of IVL-marked terminal differentiation as determined in cultured keratinocytes. However, our in vivo work in mice challenges this notion and rather establishes that the presence of involucrin instead positively regulates epidermal VDR activity. This is further supported by our scRNA-seq findings in Ivl -/- keratinocyte clusters for decreased Vdr and Snw1, Pdia3, and Rxra that co-regulate with Vdr (
      • Quack M.
      • Carlberg C.
      The impact of functional vitamin D(3) receptor conformations on DNA-dependent vitamin D(3) signaling.
      ,
      • Zhang C.
      • Dowd D.R.
      • Staal A.
      • Gu C.
      • Lian J.B.
      • van Wijnen A.J.
      • et al.
      Nuclear coactivator-62 kDa/Ski-interacting protein is a nuclear matrix-associated coactivator that may couple vitamin D receptor-mediated transcription and RNA splicing.
      ,
      • Chen J.
      • Doroudi M.
      • Cheung J.
      • Grozier A.L.
      • Schwartz Z.
      • Boyan B.D.
      Plasma membrane Pdia3 and VDR interact to elicit rapid responses to 1alpha,25(OH)(2)D(3).
      ).
      The increase in mast cells observed in MC903-treated Ivl -/- mice is notable despite the dampened skin inflammation yet observed dysbiosis. Mast cells are long-lived (up to 30 days) in contrast to short-lived basophils (up to 3 days) and hence are likely to reflect a more sustained cellular response to an environmental trigger (
      • Igawa S.
      • Di Nardo A.
      Skin microbiome and mast cells.
      ). Interestingly, it was recently discovered that germ-free mice have immature mast cells (
      • Wang Z.
      • Mascarenhas N.
      • Eckmann L.
      • Miyamoto Y.
      • Sun X.
      • Kawakami T.
      • et al.
      Skin microbiome promotes mast cell maturation by triggering stem cell factor production in keratinocytes.
      ). Skin microbiota reconstitution in these mice facilitated normal mast cell maturation thus identifying a functional role for microbiota in shaping mast cell differentiation. We speculate, based on these findings, that the observed mast cell increase in MC903-treated Ivl -/- mice may likely arise from prior skin dysbiosis and warrants future investigations.
      Additionally, our findings for potential dysbiosis in Ivl-deficient mouse skin suggests a requirement for involucrin to maintain microbial homeostasis in comparison to its reference WT isogenic C57Bl/6 mouse strain. We further identify dysbiosis associated with Vdr-mediated inflammation in WT skin. Indeed, additional studies are needed to more completely exclude the possibility of cage effects in comparing Ivl-deficient to WT skin microbiota at baseline yet we included the collection and analysis of negative and positive control specimens to minimize this concern. With our longitudinal study design, we were able to capture the changes in the microbiome due to treatment. Even with the observed microbiota changes, the microbiome of Ivl-/- skin does not appear to give rise to pathogenic infection even after MC903 treatment and also over time as we did not observe any overt infections in MC903-treated mice and also in adult Ivl-/- mice up to 2 years of age in pathogen-free housing conditions (data not shown). An additional interpretation is that the presence of involucrin may promote a niche-specific microbiome with enrichment for specific members of the Firmicutes, Proteobacteria, and Bacteroidetes phyla at the expense of the Bacteriodetes phylum, Muribaculaceae, and distinct members of the Firmicutes and Verrumocrobia phyla.
      Until now, our current understanding of human skin evolution has been illuminated with the discovery of skin pigmentation as an adaptation to the degree of UVB sun exposure (
      • Jablonski N.G.
      • Chaplin G.
      Colloquium paper: human skin pigmentation as an adaptation to UV radiation.
      ). Our current research expands our understanding of skin evolution with the identification of IVL-mediated adaptation for the epidermis. Our previous work identified recent positive selection for increased IVL in European populations (
      • Mathyer M.E.
      • Brettmann E.A.
      • Schmidt A.D.
      • Goodwin Z.A.
      • Oh I.Y.
      • Quiggle A.M.
      • et al.
      Selective sweep for an enhancer involucrin allele identifies skin barrier adaptation out of Africa.
      ) suggesting a functional role for IVL dosage that we addressed in this paper. Indeed, earlier studies have highlighted genetic innovation for IVL with the expansion of glutamine-rich tandem repeats across mammalian and primate clades and the more recent emergence of the “late” repeat domain that arose only in humans and continues to expand in repeat number across human populations (
      • Eckert R.L.
      • Green H.
      Structure and evolution of the human involucrin gene.
      ,
      • Djian P.
      • Green H.
      Vectorial expansion of the involucrin gene and the relatedness of the hominoids.
      ,
      • Teumer J.
      • Green H.
      Divergent evolution of part of the involucrin gene in the hominoids: unique intragenic duplications in the gorilla and human.
      ,
      • Tseng H.
      • Green H.
      The involucrin gene of the owl monkey: origin of the early region.
      ). In conclusion, our findings highlight an exciting paradigm for involucrin dosage to modulate vitamin D receptor function that affects epidermal crosstalk and modulation of the skin immune system.

      MATERIALS AND METHODS

      Mice

      WT and Ivl -/- C57BL/6 mice were group-housed in pathogen-free, barrier conditions and approved by the Division of Comparative Medicine Animal Studies Committee (Washington University in St. Louis School of Medicine) and in accordance with the NIH Guide for the Care and Use of Laboratory Animals. MC903 treatment (1 nmol) was administered daily to the same ear (ethanol control on the opposite ear) in 11-14 week-old mice for 12 days. Ear thickness was measured daily with a Peacock dial thickness gauge caliper (Ozaki MFG, Japan).

      Flow Cytometry

      MC903-treated ear tissue was dissociated (0.25% Trypsin/EDTA) at 37 °C (750 rpm) for 90 mins and Fc receptor was blocked. Antibodies are listed in Supplementary Material. Gates were based on the specificity of cell surface marker vs. isotype control staining with dead cell exclusions (Hoechst or 7AAD positive) via BD FACSAria II and FlowJo10.6.2 analysis.

      scRNA-seq

      Treated ears were dissociated in trypsin at 37°C (750 rpm) for 90 minutes and strained prior to two rounds of centrifugation (300 rcf) for 7 mins at 4°C. A total of 20,000 live cells (Hoechst-) per sample were captured by flow cytometry (AriaII) and submitted for 10X Genomics library preparation and sequencing. Unfiltered feature-barcode matrix per sample from 10X Cell Ranger was obtained for each Ivl -/- and WT replicate (n=2 per genotype). Analysis was performed with the R programming environment (v4.1.0) and Seurat (v4.0.3) (
      • Hao Y.
      • Hao S.
      • Andersen-Nissen E.
      • Mauck 3rd, W.M.
      • Zheng S.
      • Butler A.
      • et al.
      Integrated analysis of multimodal single-cell data.
      ). Cells were excluded for any of the following criteria: cell of low quality (< 700 genes), cell “doublets” (>8000 genes), and dead/stressed cell (> 60% mitochondrial gene expression). Ivl -/- single cells were down-sampled to the WT populations (8135 cells per genotype). All data was scaled and normalized with the centered-log ratios parameter, followed by principal component analysis (10 principal components), and batch correction and integration using Harmony (
      • Korsunsky I.
      • Millard N.
      • Fan J.
      • Slowikowski K.
      • Zhang F.
      • Wei K.
      • et al.
      Fast, sensitive and accurate integration of single-cell data with Harmony.
      ). UMAP was determined by FindNeighbors using dimensions 1 through 10 as input parameter and FindClusters with a resolution of 0.8 for cell cluster discovery using the Louvain algorithm. Marker genes were identified separately for each genotyped samples using FindAllMarkers function. Cell clusters were determined by comparing reference datasets in (
      • Joost S.
      • Zeisel A.
      • Jacob T.
      • Sun X.
      • La Manno G.
      • Lonnerberg P.
      • et al.
      Single-Cell Transcriptomics Reveals that Differentiation and Spatial Signatures Shape Epidermal and Hair Follicle Heterogeneity.
      ) and My Geneset on Immunological Genome Project (immgen.org). Analysis of vitamin D responsive genes involved the comparisons of gene expression means for genes listed in GSEA Mouse Gene Set: GOBP_CELLULAR_RESPONSE_TO_VITAMIN_D (MM9698) (
      • Subramanian A.
      • Tamayo P.
      • Mootha V.K.
      • Mukherjee S.
      • Ebert B.L.
      • Gillette M.A.
      • et al.
      Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.
      ) (
      • Liberzon A.
      • Birger C.
      • Thorvaldsdottir H.
      • Ghandi M.
      • Mesirov J.P.
      • Tamayo P.
      The Molecular Signatures Database (MSigDB) hallmark gene set collection.
      ).

      Microbiome

      Skin microbiota was collected using pre-moistened sterile swabs on adult mouse ear skin (co-housed littermates by genotype) prior to and after 12 days of MC903 treatment (n=3, WT and Ivl -/- each). Air swabs from each cage were obtained as negative controls. Bacterial swabs were subject to bacterial genome isolation and submitted for 16S rRNA microbiome sequencing (V1-V3 regions) on Illumina Miseq. A total of ∼720,000 reads were obtained with mean of 30k and median of 29k per sample. Quality control was performed with AlignerBoost v1.8.3 (
      • Zheng Q.
      • Grice E.A.
      AlignerBoost: A Generalized Software Toolkit for Boosting Next-Gen Sequencing Mapping Accuracy Using a Bayesian-Based Mapping Quality Framework.
      ) and demultiplexed with Flexbarv3.5 (
      • Roehr J.T.
      • Dieterich C.
      • Reinert K.
      Flexbar 3.0 - SIMD and multicore parallelization.
      ). Chloroplast and mitochondrial reads that mapped to the taxonomy class Chloroplast or family mitochondria were removed from the analysis. Phylogenetic assignment was obtained using HmmUFOtuv1.4.2 (
      • Zheng Q.
      • Bartow-McKenney C.
      • Meisel J.S.
      • Grice E.A.
      HmmUFOtu: An HMM and phylogenetic placement based ultra-fast taxonomic assignment and OTU picking tool for microbiome amplicon sequencing studies.
      )with the GreenGene97% OTU database (release 13.8) database and pseudo-node on (all OTUs as leaf nodes). Statistics were calculated using R package phyloseq v1.32.0 (
      • McMurdie P.J.
      • Holmes S.
      phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data.
      )and DEseq2v1.28.1 (
      • Love M.I.
      • Huber W.
      • Anders S.
      Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.
      ). To identify differentially enriched OTUs (DE-OTUs), 16s microbiome data were converted to DESeq2 objects and ran using a negative binomial linear model as ∼ study_group*time_point. Thresholds for significant DE-OTUs were FDR < 0.1 and absolute log2FC >= 1.

      DATA AVAILABILITY STATEMENT

      Datasets related to this article can be found at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA821259/, hosted at NCBI SRA, BioProject accession PRJNA821259 and at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA786852/, hosted at NCBI SRA, BioProject accession PRJNA786852.

      CONFLICT OF INTEREST STATEMENT

      C.dG.S. and Erin A. Brettmann are listed as inventors on utility patent application 63/090,801 submitted by Washington University in St. Louis for the compositions and methods of involucrin for treating skin diseases, disorders, or conditions. C.dG.S. is the founder of Evoly Skin, LLC that is developing new technologies for skin barrier health.

      ACKNOWLEDGEMENTS

      We thank Lloyd Miller for helpful discussion, Pascaline Akitani, Dorjan Brinja, and Erica Lantelme at the Flow Cytometry and Fluorescence Activated Cell Sorting Core, Jennifer Ponce and Mirhanda Allen at the Genome Technology Access Core (GTAC) at the McDonnell Genome Institute for scRNA-seq library prep, sequencing, and preliminary analyses, Jordan Harris in the Grice lab for DNA extraction, Simon Knight in the Grice lab for microbiota genomic isolation, Penn CHOP Microbiome Core for library prep and sequencing, and John Edwards for bioinformatics assistance. Support for this work was provided by the Society of Investigative Dermatology Sun Pharma Post-doctoral Award (M.E.M.), NIH/NHGRI T32 HG000045 (A.D.S.), NIH/NHGRI R25HG006687 (Opportunities in Genomics Research training, C.M.), NIH/NCRR/NCI P30CA91842 (GTAC), NIH/NIAMS P30AR69589 (Penn Skin Biology and Diseases Resource-based Center), and NIH/NIAMS R01AR065523, R56AR075427, and R01AR079888 (C.dG.S.) funds. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
      AUTHOR CONTRIBUTIONS
      Conceptualization: CdGS; Data Curation: ADS, CM, QZ, CdGS; Formal Analysis: ADS, CM, QZ, EAG, CdGS; Funding Acquisition: EAG, CdGS; Investigation: ADS, CM, QZ, MEM, EAG, CdGS; Methodology: ADS, CM, QZ, MEM, EAG, CdGS; Project Administration: EAG, CdGS; Resources: EAG, CdGS; Software: ADS, CM, QZ; Supervision: EAG, CdGS; Validation: ADS, CdGS; Visualization: ADS, CM, QZ, CdGS; Writing – Original Draft Preparation: ADS, MEM, CdGS; Writing – Review and Editing – ADS, CM, QZ, MEM, EAG, CdGS.

      Supplementary Material

      SUPPLEMENTARY MATERIAL

      SUPPLEMENTARY FIGURE LEGENDS
      Figure thumbnail fx1
      Figure S1Ivl -/- males and females exhibit similar trends to each other and for decreased MC903-treated skin inflammation compared to WT males and females. Ivl -/- males and females show relatively similar inflammatory responses and also for decreased inflammation in comparison to WT males and females. Percent change in ear thickness was significantly reduced in MC903-treated Ivl -/- males (n=5) compared to treated WT males (n=9) at day 12 and was also significantly reduced in MC903-treated Ivl -/- females (n=5) compared to treated WT females (n=6) at days 7, 11, and 12. *p< 0.05, one-way ANOVA with post-hoc Tukey HSD. Error bars ±SEM.
      Figure thumbnail fx2
      Figure S2scRNA-seq identifies differential expressions for Vitamin D-responsive genes in scRNA-seq cell clusters in MC903-treated Ivl -/- vs. WT skin.
      Figure thumbnail fx3
      Figure S3Ivl -/- mice exhibit similar MC903-treated immune infiltrate responses as WT mice. No significant differences were found for dendritic cells (CD11c+), B cells (CD19+), neutrophils (CD11b+, Ly6g+), and eosinophils (CD11b+, Ly6g-) in MC903-treated Ivl -/- mice (n=5) compared to WT (n=5) after 12 days of daily MC903 treatment (one-sided t-test).
      SUPPLEMENTARY METHODS
      The following antibodies were used for flow cytometry: Fc block (CD16/CD32 [93, #14-0161-82], 1:400; eBioscience, San Diego, CA), CD4-PE (Gk1.5, 1:400; Biolegend, San Diego, CA), CD8-APC (53-6.7, 1:400; Biolegend, San Diego, CA), CD19-APC-Cy7 (1D3, 1:400; BD Pharmingen, San Jose, CA), CD11b-FITC (M1/70, 101205, 1:400; Biolegend, San Diego, CA), CD11c-PE-cyanin5 (N418, 1:400; eBioscience, San Diego, CA), Ly6g- PE-Cy7 (A8, 560601, 1:400; BD Biosciences, San Diego, CA), F4/80-PE (BM8, 12-4801-80, 1:400; Invitrogen, Waltham, MA), IgE-BV421 (R35-72, 564207, 1:200; BD Horizon, San Jose, CA), and CD45-APC (30-F11, 561018, 1:400; BD Pharmingen, San Jose, CA) (Eberle et al., 2019) (Signaling, 2021). The following antibodies were used for flow cytometry isotype controls: FITC Rat IgG2b (A95-1, 556923, 1:400; BD Biosciences, San Diego, CA), PE-Cy™7 Rat IgG2a (R35-95, 557855, 1:400; BD Biosciences, San Diego, CA), PE Rat IgG2a (R35-95, 551799, 1:400; BD Biosciences, San Diego, CA), APC-Cy™7 Rat IgM (R4-22, 560571, 1:200; BD Biosciences, San Diego, CA), BV421 Rat IgG1 (R3-34, 562868, 1:200; BD Biosciences, San Diego, CA), and APC Rat IgG2b (A95-1, 553991, 1:400; BD Biosciences, San Diego, CA).
      SUPPLEMENTARY REFERENCES
      Eberle JU, Radtke D, Nimmerjahn F, Voehringer D. Eosinophils Mediate Basophil-Dependent Allergic Skin Inflammation in Mice. J Invest Dermatol 2019;139(9):1957-65 e2.
      Signaling C. Mouse Immune Cell Marker Guide. https://wwwcellsignalcom/pathways/immune-cell-markers-mouse 2021.

      REFERENCES

        • Bikle D.
        • Christakos S.
        New aspects of vitamin D metabolism and action - addressing the skin as source and target.
        Nat Rev Endocrinol. 2020; 16: 234-252
        • Bikle D.D.
        Vitamin D metabolism and function in the skin.
        Mol Cell Endocrinol. 2011; 347: 80-89
        • Bikle D.D.
        • Elalieh H.
        • Chang S.
        • Xie Z.
        • Sundberg J.P.
        Development and progression of alopecia in the vitamin D receptor null mouse.
        J Cell Physiol. 2006; 207: 340-353
        • Carlberg C.
        Molecular basis of the selective activity of vitamin D analogues.
        J Cell Biochem. 2003; 88: 274-281
        • Chen J.
        • Doroudi M.
        • Cheung J.
        • Grozier A.L.
        • Schwartz Z.
        • Boyan B.D.
        Plasma membrane Pdia3 and VDR interact to elicit rapid responses to 1alpha,25(OH)(2)D(3).
        Cell Signal. 2013; 25: 2362-2373
        • de Guzman Strong C.
        • Conlan S.
        • Deming C.B.
        • Cheng J.
        • Sears K.E.
        • Segre J.A.
        A milieu of regulatory elements in the epidermal differentiation complex syntenic block: implications for atopic dermatitis and psoriasis.
        Hum Mol Genet. 2010; 19: 1453-1460
        • DeSantis T.Z.
        • Hugenholtz P.
        • Larsen N.
        • Rojas M.
        • Brodie E.L.
        • Keller K.
        • et al.
        Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB.
        Appl Environ Microbiol. 2006; 72: 5069-5072
        • Djian P.
        • Easley K.
        • Green H.
        Targeted ablation of the murine involucrin gene.
        J Cell Biol. 2000; 151: 381-388
        • Djian P.
        • Green H.
        Vectorial expansion of the involucrin gene and the relatedness of the hominoids.
        Proc Natl Acad Sci U S A. 1989; 86: 8447-8451
        • Eckert R.L.
        • Green H.
        Structure and evolution of the human involucrin gene.
        Cell. 1986; 46: 583-589
        • Hahn J.M.
        • Supp D.M.
        Abnormal expression of the vitamin D receptor in keloid scars.
        Burns. 2017; 43: 1506-1515
        • Hao Y.
        • Hao S.
        • Andersen-Nissen E.
        • Mauck 3rd, W.M.
        • Zheng S.
        • Butler A.
        • et al.
        Integrated analysis of multimodal single-cell data.
        Cell. 2021; 184: 3573-3587 e29
        • Horiuchi N.
        • Clemens T.L.
        • Schiller A.L.
        • Holick M.F.
        Detection and developmental changes of the 1,25-(OH)2-D3 receptor concentration in mouse skin and intestine.
        J Invest Dermatol. 1985; 84: 461-464
        • Hosomi J.
        • Hosoi J.
        • Abe E.
        • Suda T.
        • Kuroki T.
        Regulation of terminal differentiation of cultured mouse epidermal cells by 1 alpha,25-dihydroxyvitamin D3.
        Endocrinology. 1983; 113: 1950-1957
        • Hussain M.
        • Borcard L.
        • Walsh K.P.
        • Pena Rodriguez M.
        • Mueller C.
        • Kim B.S.
        • et al.
        Basophil-derived IL-4 promotes epicutaneous antigen sensitization concomitant with the development of food allergy.
        J Allergy Clin Immunol. 2018; 141: 223-234 e5
        • Igawa S.
        • Di Nardo A.
        Skin microbiome and mast cells.
        Transl Res. 2017; 184: 68-76
        • Jablonski N.G.
        • Chaplin G.
        Colloquium paper: human skin pigmentation as an adaptation to UV radiation.
        Proc Natl Acad Sci U S A. 2010; 107: 8962-8968
        • Joost S.
        • Zeisel A.
        • Jacob T.
        • Sun X.
        • La Manno G.
        • Lonnerberg P.
        • et al.
        Single-Cell Transcriptomics Reveals that Differentiation and Spatial Signatures Shape Epidermal and Hair Follicle Heterogeneity.
        Cell Syst. 2016; 3: 221-237 e9
        • Kajava A.V.
        alpha-Helical solenoid model for the human involucrin.
        FEBS Lett. 2000; 473: 127-131
        • Kim B.S.
        • Siracusa M.C.
        • Saenz S.A.
        • Noti M.
        • Monticelli L.A.
        • Sonnenberg G.F.
        • et al.
        TSLP elicits IL-33-independent innate lymphoid cell responses to promote skin inflammation.
        Sci Transl Med. 2013; 5 (170ra16)
        • Kim B.S.
        • Wang K.
        • Siracusa M.C.
        • Saenz S.A.
        • Brestoff J.R.
        • Monticelli L.A.
        • et al.
        Basophils promote innate lymphoid cell responses in inflamed skin.
        J Immunol. 2014; 193: 3717-3725
        • Kobayashi T.
        • Glatz M.
        • Horiuchi K.
        • Kawasaki H.
        • Akiyama H.
        • Kaplan D.H.
        • et al.
        Dysbiosis and Staphylococcus aureus Colonization Drives Inflammation in Atopic Dermatitis.
        Immunity. 2015; 42: 756-766
        • Korsunsky I.
        • Millard N.
        • Fan J.
        • Slowikowski K.
        • Zhang F.
        • Wei K.
        • et al.
        Fast, sensitive and accurate integration of single-cell data with Harmony.
        Nat Methods. 2019; 16: 1289-1296
        • Li M.
        • Hener P.
        • Zhang Z.
        • Kato S.
        • Metzger D.
        • Chambon P.
        Topical vitamin D3 and low-calcemic analogs induce thymic stromal lymphopoietin in mouse keratinocytes and trigger an atopic dermatitis.
        Proc Natl Acad Sci U S A. 2006; 103: 11736-11741
        • Liberzon A.
        • Birger C.
        • Thorvaldsdottir H.
        • Ghandi M.
        • Mesirov J.P.
        • Tamayo P.
        The Molecular Signatures Database (MSigDB) hallmark gene set collection.
        Cell Syst. 2015; 1: 417-425
        • Love M.I.
        • Huber W.
        • Anders S.
        Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.
        Genome Biol. 2014; 15: 550
        • Makin G.
        • Lohnes D.
        • Byford V.
        • Ray R.
        • Jones G.
        Target cell metabolism of 1,25-dihydroxyvitamin D3 to calcitroic acid. Evidence for a pathway in kidney and bone involving 24-oxidation.
        Biochem J. 1989; 262: 173-180
        • Mathyer M.E.
        • Brettmann E.A.
        • Schmidt A.D.
        • Goodwin Z.A.
        • Oh I.Y.
        • Quiggle A.M.
        • et al.
        Selective sweep for an enhancer involucrin allele identifies skin barrier adaptation out of Africa.
        Nat Commun. 2021; 12: 2557
        • Matsui T.
        • Amagai M.
        Dissecting the formation, structure and barrier function of the stratum corneum.
        Int Immunol. 2015; 27: 269-280
        • McMurdie P.J.
        • Holmes S.
        phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data.
        PLoS One. 2013; 8e61217
        • Meisel J.S.
        • Sfyroera G.
        • Bartow-McKenney C.
        • Gimblet C.
        • Bugayev J.
        • Horwinski J.
        • et al.
        Commensal microbiota modulate gene expression in the skin.
        Microbiome. 2018; 6: 20
        • Mischke D.
        • Korge B.P.
        • Marenholz I.
        • Volz A.
        • Ziegler A.
        Genes encoding structural proteins of epidermal cornification and S100 calcium-binding proteins form a gene complex ("epidermal differentiation complex") on human chromosome 1q21.
        J Invest Dermatol. 1996; 106: 989-992
        • Moosbrugger-Martinz V.
        • Schmuth M.
        • Dubrac S.
        A Mouse Model for Atopic Dermatitis Using Topical Application of Vitamin D3 or of Its Analog MC903.
        Methods Mol Biol. 2017; 1559: 91-106
      1. Moskovicz V, Ben-El R, Horev G, Mizrahi B. Skin microbiota dynamics following B. subtilis formulation challenge: an in vivo study in mice. BMC Microbiol 2021;21(1):231.

        • Muehleisen B.
        • Bikle D.D.
        • Aguilera C.
        • Burton D.W.
        • Sen G.L.
        • Deftos L.J.
        • et al.
        PTH/PTHrP and vitamin D control antimicrobial peptide expression and susceptibility to bacterial skin infection.
        Sci Transl Med. 2012; 4: 135ra66
        • Nemes Z.
        • Marekov L.N.
        • Steinert P.M.
        Involucrin cross-linking by transglutaminase 1. Binding to membranes directs residue specificity.
        J Biol Chem. 1999; 274: 11013-11021
        • Niec R.E.
        • Rudensky A.Y.
        • Fuchs E.
        Inflammatory adaptation in barrier tissues.
        Cell. 2021; 184: 3361-3375
        • Oda Y.
        • Uchida Y.
        • Moradian S.
        • Crumrine D.
        • Elias P.M.
        • Bikle D.D.
        Vitamin D receptor and coactivators SRC2 and 3 regulate epidermis-specific sphingolipid production and permeability barrier formation.
        J Invest Dermatol. 2009; 129: 1367-1378
        • Ormerod K.L.
        • Wood D.L.
        • Lachner N.
        • Gellatly S.L.
        • Daly J.N.
        • Parsons J.D.
        • et al.
        Genomic characterization of the uncultured Bacteroidales family S24-7 inhabiting the guts of homeothermic animals.
        Microbiome. 2016; 4: 36
        • Pillai S.
        • Bikle D.D.
        • Elias P.M.
        1,25-Dihydroxyvitamin D production and receptor binding in human keratinocytes varies with differentiation.
        J Biol Chem. 1988; 263: 5390-5395
        • Quack M.
        • Carlberg C.
        The impact of functional vitamin D(3) receptor conformations on DNA-dependent vitamin D(3) signaling.
        Mol Pharmacol. 2000; 57: 375-384
        • Reddy G.S.
        • Tserng K.Y.
        Calcitroic acid, end product of renal metabolism of 1,25-dihydroxyvitamin D3 through C-24 oxidation pathway.
        Biochemistry. 1989; 28: 1763-1769
        • Ren D.
        • Gong S.
        • Shu J.
        • Zhu J.
        • Rong F.
        • Zhang Z.
        • et al.
        Mixed Lactobacillus plantarum Strains Inhibit Staphylococcus aureus Induced Inflammation and Ameliorate Intestinal Microflora in Mice.
        Biomed Res Int. 2017; 20177476467
        • Rice R.H.
        • Green H.
        Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions.
        Cell. 1979; 18: 681-694
        • Robinson N.A.
        • LaCelle P.T.
        • Eckert R.L.
        Involucrin is a covalently crosslinked constituent of highly purified epidermal corneocytes: evidence for a common pattern of involucrin crosslinking in vivo and in vitro.
        J Invest Dermatol. 1996; 107: 101-107
        • Roehr J.T.
        • Dieterich C.
        • Reinert K.
        Flexbar 3.0 - SIMD and multicore parallelization.
        Bioinformatics. 2017; 33: 2941-2942
        • Romney A.L.T.
        • Davis E.M.
        • Corona M.M.
        • Wagner J.T.
        • Podrabsky J.E.
        Temperature-dependent vitamin D signaling regulates developmental trajectory associated with diapause in an annual killifish.
        Proc Natl Acad Sci U S A. 2018; 115: 12763-12768
        • Sanin D.E.
        • Ge Y.
        • Marinkovic E.
        • Kabat A.M.
        • Castoldi A.
        • Caputa G.
        • et al.
        A common framework of monocyte-derived macrophage activation.
        Sci Immunol. 2022; 7eabl7482
        • Schauber J.
        • Dorschner R.A.
        • Coda A.B.
        • Buchau A.S.
        • Liu P.T.
        • Kiken D.
        • et al.
        Injury enhances TLR2 function and antimicrobial peptide expression through a vitamin D-dependent mechanism.
        J Clin Invest. 2007; 117: 803-811
        • Schlingmann K.P.
        • Kaufmann M.
        • Weber S.
        • Irwin A.
        • Goos C.
        • John U.
        • et al.
        Mutations in CYP24A1 and idiopathic infantile hypercalcemia.
        N Engl J Med. 2011; 365: 410-421
        • Sevilla L.M.
        • Nachat R.
        • Groot K.R.
        • Klement J.F.
        • Uitto J.
        • Djian P.
        • et al.
        Mice deficient in involucrin, envoplakin, and periplakin have a defective epidermal barrier.
        J Cell Biol. 2007; 179: 1599-1612
        • Steinert P.M.
        • Marekov L.N.
        The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal cornified cell envelope.
        J Biol Chem. 1995; 270: 17702-17711
        • Stumpf W.E.
        • Sar M.
        • Reid F.A.
        • Tanaka Y.
        • DeLuca H.F.
        Target cells for 1,25-dihydroxyvitamin D3 in intestinal tract, stomach, kidney, skin, pituitary, and parathyroid.
        Science. 1979; 206: 1188-1190
        • Subramanian A.
        • Tamayo P.
        • Mootha V.K.
        • Mukherjee S.
        • Ebert B.L.
        • Gillette M.A.
        • et al.
        Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.
        Proc Natl Acad Sci U S A. 2005; 102: 15545-15550
        • Teumer J.
        • Green H.
        Divergent evolution of part of the involucrin gene in the hominoids: unique intragenic duplications in the gorilla and human.
        Proc Natl Acad Sci U S A. 1989; 86: 1283-1286
        • Tseng H.
        • Green H.
        The involucrin gene of the owl monkey: origin of the early region.
        Mol Biol Evol. 1989; 6: 460-468
        • Uberoi A.
        • Bartow-McKenney C.
        • Zheng Q.
        • Flowers L.
        • Campbell A.
        • Knight S.A.B.
        • et al.
        Commensal microbiota regulates skin barrier function and repair via signaling through the aryl hydrocarbon receptor.
        Cell Host Microbe. 2021; 29: 1235-12348 e8
        • Villani A.C.
        • Satija R.
        • Reynolds G.
        • Sarkizova S.
        • Shekhar K.
        • Fletcher J.
        • et al.
        Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors.
        Science. 2017; 356
        • Volz A.
        • Korge B.P.
        • Compton J.G.
        • Ziegler A.
        • Steinert P.M.
        • Mischke D.
        Physical mapping of a functional cluster of epidermal differentiation genes on chromosome 1q21.
        Genomics. 1993; 18: 92-99
        • Walsh C.M.
        • Hill R.Z.
        • Schwendinger-Schreck J.
        • Deguine J.
        • Brock E.C.
        • Kucirek N.
        • et al.
        Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis.
        Elife. 2019; 8
        • Wang Z.
        • Mascarenhas N.
        • Eckmann L.
        • Miyamoto Y.
        • Sun X.
        • Kawakami T.
        • et al.
        Skin microbiome promotes mast cell maturation by triggering stem cell factor production in keratinocytes.
        J Allergy Clin Immunol. 2017; 139: 1205-12016 e6
        • Watt F.M.
        Mammalian skin cell biology: at the interface between laboratory and clinic.
        Science. 2014; 346: 937-940
        • Xie Z.
        • Komuves L.
        • Yu Q.C.
        • Elalieh H.
        • Ng D.C.
        • Leary C.
        • et al.
        Lack of the vitamin D receptor is associated with reduced epidermal differentiation and hair follicle growth.
        J Invest Dermatol. 2002; 118: 11-16
        • Zhang C.
        • Dowd D.R.
        • Staal A.
        • Gu C.
        • Lian J.B.
        • van Wijnen A.J.
        • et al.
        Nuclear coactivator-62 kDa/Ski-interacting protein is a nuclear matrix-associated coactivator that may couple vitamin D receptor-mediated transcription and RNA splicing.
        J Biol Chem. 2003; 278: 35325-35336
        • Zhao X.P.
        • Elder J.T.
        Positional cloning of novel skin-specific genes from the human epidermal differentiation complex.
        Genomics. 1997; 45: 250-258
        • Zheng Q.
        • Bartow-McKenney C.
        • Meisel J.S.
        • Grice E.A.
        HmmUFOtu: An HMM and phylogenetic placement based ultra-fast taxonomic assignment and OTU picking tool for microbiome amplicon sequencing studies.
        Genome Biol. 2018; 19: 82
        • Zheng Q.
        • Grice E.A.
        AlignerBoost: A Generalized Software Toolkit for Boosting Next-Gen Sequencing Mapping Accuracy Using a Bayesian-Based Mapping Quality Framework.
        PLoS Comput Biol. 2016; 12e1005096
        • Zilionis R.
        • Engblom C.
        • Pfirschke C.
        • Savova V.
        • Zemmour D.
        • Saatcioglu H.D.
        • et al.
        Single-Cell Transcriptomics of Human and Mouse Lung Cancers Reveals Conserved Myeloid Populations across Individuals and Species.
        Immunity. 2019; 50: 1317-1334 e10