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Type 2 Inflammation Contributes to Skin Barrier Dysfunction in Atopic Dermatitis

Open AccessPublished:April 25, 2022DOI:https://doi.org/10.1016/j.xjidi.2022.100131
      Skin barrier dysfunction, a defining feature of atopic dermatitis (AD), arises from multiple interacting systems. In AD, skin inflammation is caused by host–environment interactions involving keratinocytes as well as tissue-resident immune cells such as type 2 innate lymphoid cells, basophils, mast cells, and T helper type 2 cells, which produce type 2 cytokines, including IL-4, IL-5, IL-13, and IL-31. Type 2 inflammation broadly impacts the expression of genes relevant for barrier function, such as intracellular structural proteins, extracellular lipids, and junctional proteins, and enhances Staphylococcus aureus skin colonization. Systemic anti‒type 2 inflammation therapies may improve dysfunctional skin barrier in AD.

      Abbreviations:

      AD (atopic dermatitis), AMP (antimicrobial peptide), CLDN (claudin), FFA (free fatty acid), hBD (human β-defensin), ILC2 (type 2 innate lymphoid cell), Jaki (Jak inhibitor), K (keratin), KC (keratinocyte), MMP (matrix metalloproteinase), NMF (natural moisturizing factor), PAR (protease-activated receptor), PDE-4 (phosphodiesterase-4), SC (stratum corneum), SG (stratum granulosum), TCI (topical calcineurin inhibitor), TCS (topical corticosteroid), TEWL (transepidermal water loss), Th (T helper), TJ (tight junction), TLR (toll-like receptor), TNF-α (tumor necrosis factor alpha), TYK (tyrosine kinase), ZO (zona occludens)

      Introduction

      Atopic dermatitis (AD) is a chronic pruritic inflammatory skin disease, whose pathogenesis is mediated by interactions between skin barrier impairment and an abnormal immune response featuring enhanced type 2 inflammation (Figure 1). Interactions between keratinocytes (KCs), innate immune cells (e.g., type 2 innate lymphoid cells [ILC2s], dendritic cells, mast cells, basophils, and eosinophils), adaptive immune cells (T and B cells), and an altered epidermal microbiome (with reduction of microbial diversity and predominance of Staphylococcus aureus) all contribute to AD pathogenesis (
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      Figure thumbnail gr1
      Figure 1Type 2 inflammation: primary immune cells, key cytokines and alarmins, exogenous targets, and representative diseases. AD is a predominantly type 2 inflammatory disease. Other type 2 allergic diseases include allergic rhinitis, asthma, CRSwNP, eosinophilic esophagitis, and food allergy. 1IL-25 is also known as IL-17E. AD, atopic dermatitis; CRSwNP, chronic rhinosinusitis with nasal polyps; ILC, innate lymphoid cell; TFH, T-follicular helper; Th, T helper.
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      Figure thumbnail gr2
      Figure 2IL-4 and IL-13 have overlapping but not identical functions; for example, both mediate inflammation and barrier dysfunction through immunologic and structural changes, both enhance pruritogenic pathways, and both promote Staphylococcus aureus colonization in AD. However, IL-4 but not IL-13 promotes the differentiation of Th cells from Th0 to Th2 cells (
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      ). AD, atopic dermatitis; DC, dendritic cell; ILC, innate lymphoid cell; Th, T helper.
      In this paper, we review the role of the components relevant to a functional skin barrier and highlight how skin barrier dysfunction promotes the development of type 2 inflammation and how type 2 inflammation, in turn, affects skin barrier dysfunction.

      The Skin Barrier

      The epidermis is the only epithelial surface with two barrier structures: the stratum corneum (SC), which is unique to the skin, and tight junctions (TJs), which are present in other epithelia as well (
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      ). Both the SC and the TJs limit penetration of and reaction to microbes, allergens/irritants, and toxins as well as prevent transepidermal water loss (TEWL).

      SC

      Corneocytes

      The SC, the outer layer of the epidermis, is composed of flattened, anucleated KCs (corneocytes) surrounded by a complex lipid-enriched extracellular matrix (Figure 3). Corneocytes are analogous to bricks and lipids to mortar in the original brick and mortar model of the SC (
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      ). This concept has since evolved into a dynamic model in which lipid composition and alignment of the SC allow for adaptation to external factors and are altered in diseases such as AD (
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      ).
      Figure thumbnail gr3
      Figure 3The key components of skin showing the differences between normal healthy skin (left side) and AD skin (right side), including the microbiome, corneocytes, antimicrobial peptides, lipids, NMFs, and tight junctions. In the brick and mortar model, the bricks represent corneocytes, and the mortar represents the extracellular lipids and other extracellular matrix components. In the confocal images, green staining represents ZO-1 and CLDN-1, and white represents cell nuclei. Confocal images from AD skin (on the right) show dramatically reduced green staining demonstrating reduced ZO-1 and CLDN-1, compared with normal skin (on the left). 1Confocal images were adapted with permission from
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      (http://creativecommons.org/licenses/by/4.0; modified), courtesy of Takaharu Okada at RIKEN IMS (Yokohama, Japan). 2Confocal images were provided with permission from
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      . AD, atopic dermatitis; CLDN, claudin; KLK, kallikrein; MMP, matrix metalloproteinase; NMF, natural moisturizing factor; SB, stratum basale; SC, stratum corneum; SG, stratum granulosum; SS, stratum spinosum; ZO, zona occludens.

      SC protein components

      Keratins have both structural and regulatory functions in the epidermis. The more than 20 different epithelial keratins form specific keratin pairs composed of type I (lower molecular weight and acidic) and type II (neutral basic) components (
      • Moll R.
      • Divo M.
      • Langbein L.
      The human keratins: biology and pathology.
      ;
      • Schweizer J.
      • Bowden P.E.
      • Coulombe P.A.
      • Langbein L.
      • Lane E.B.
      • Magin T.M.
      • et al.
      New consensus nomenclature for mammalian keratins.
      ;
      • Szeverenyi I.
      • Cassidy A.J.
      • Chung C.W.
      • Lee B.T.
      • Common J.E.
      • Ogg S.C.
      • et al.
      The human intermediate filament database: comprehensive information on a gene family involved in many human diseases.
      ). Keratin pairs crosslink with other keratin pairs to form keratin filaments, which interact with other proteins and the cell membrane to provide structural stability and flexibility to KCs (
      • Candi E.
      • Tarcsa E.
      • Digiovanna J.J.
      • Compton J.G.
      • Elias P.M.
      • Marekov L.N.
      • et al.
      A highly conserved lysine residue on the head domain of type II keratins is essential for the attachment of keratin intermediate filaments to the cornified cell envelope through isopeptide crosslinking by transglutaminases.
      ;
      • Lee C.H.
      • Coulombe P.A.
      Self-organization of keratin intermediate filaments into cross-linked networks.
      ;
      • 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.
      ). In the epidermis, keratin (K) 5 and K14 predominate in the stratum basale, whereas K1 and K10 predominate in the stratum spinosum and higher layers―the change from one pair type to another reflects KC differentiation (
      • Fuchs E.
      • Green H.
      Changes in keratin gene expression during terminal differentiation of the keratinocyte.
      ;
      • Moll R.
      • Franke W.W.
      • Schiller D.L.
      • Geiger B.
      • Krepler R.
      The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells.
      ;
      • Reichelt J.
      • Büssow H.
      • Grund C.
      • Magin T.M.
      Formation of a normal epidermis supported by increased stability of keratins 5 and 14 in keratin 10 null mice.
      ). In contrast, K6, K16, and K17 are associated with the repair of an injured epidermis and are upregulated in inflammatory disorders of the skin, such as AD and psoriasis (
      • Fu D.J.
      • Thomson C.
      • Lunny D.P.
      • Dopping-Hepenstal P.J.
      • McGrath J.A.
      • Smith F.J.D.
      • et al.
      Keratin 9 is required for the structural integrity and terminal differentiation of the palmoplantar epidermis.
      ;
      • Kumar V.
      • Bouameur J.E.
      • Bär J.
      • Rice R.H.
      • Hornig-Do H.T.
      • Roop D.R.
      • et al.
      A keratin scaffold regulates epidermal barrier formation, mitochondrial lipid composition, and activity.
      ;
      • Lessard J.C.
      • Piña-Paz S.
      • Rotty J.D.
      • Hickerson R.P.
      • Kaspar R.L.
      • Balmain A.
      • et al.
      Keratin 16 regulates innate immunity in response to epidermal barrier breach.
      ;
      • Leung D.Y.M.
      • Berdyshev E.
      • Goleva E.
      Cutaneous barrier dysfunction in allergic diseases.
      ;
      • Moll R.
      • Divo M.
      • Langbein L.
      The human keratins: biology and pathology.
      ;
      • Roth W.
      • Kumar V.
      • Beer H.D.
      • Richter M.
      • Wohlenberg C.
      • Reuter U.
      • et al.
      Keratin 1 maintains skin integrity and participates in an inflammatory network in skin through interleukin-18.
      ).
      KCs undergo cornification, marking their differentiation into corneocytes; the cells become compact owing to keratin crosslinking, and keratins and other proteins form a cornified envelope that lines the cell membrane (
      • Bowden P.E.
      • Quinlan R.A.
      • Breitkreutz D.
      • Fusenig N.E.
      Proteolytic modification of acidic and basic keratins during terminal differentiation of mouse and human epidermis.
      ;
      • Candi E.
      • Tarcsa E.
      • Digiovanna J.J.
      • Compton J.G.
      • Elias P.M.
      • Marekov L.N.
      • et al.
      A highly conserved lysine residue on the head domain of type II keratins is essential for the attachment of keratin intermediate filaments to the cornified cell envelope through isopeptide crosslinking by transglutaminases.
      ;
      • Eckert R.L.
      • Sturniolo M.T.
      • Broome A.M.
      • Ruse M.
      • Rorke E.A.
      Transglutaminase function in epidermis.
      ). During cornification, keratin filaments cross-link to FLG and other proteins lining the cell membrane (e.g., involucrin and loricrin) (
      • Candi E.
      • Tarcsa E.
      • Digiovanna J.J.
      • Compton J.G.
      • Elias P.M.
      • Marekov L.N.
      • et al.
      A highly conserved lysine residue on the head domain of type II keratins is essential for the attachment of keratin intermediate filaments to the cornified cell envelope through isopeptide crosslinking by transglutaminases.
      ;
      • Roth W.
      • Kumar V.
      • Beer H.D.
      • Richter M.
      • Wohlenberg C.
      • Reuter U.
      • et al.
      Keratin 1 maintains skin integrity and participates in an inflammatory network in skin through interleukin-18.
      ;
      • 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.
      ). Keratin filaments also connect to cell–cell adhesion structures, such as desmosomes, stabilizing connections between KCs (
      • Homberg M.
      • Magin T.M.
      Beyond expectations: novel insights into epidermal keratin function and regulation.
      ;
      • Kouklis P.D.
      • Hutton E.
      • Fuchs E.
      Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins.
      ;
      • Seltmann K.
      • Roth W.
      • Kröger C.
      • Loschke F.
      • Lederer M.
      • Hüttelmaier S.
      • et al.
      Keratins mediate localization of hemidesmosomes and repress cell motility.
      ). Under normal conditions, keratin-filled corneocytes swell and expand with exposure to water, which softens the keratin and allows the SC to bend and stretch (
      • Bouwstra J.A.
      • Groenink H.W.W.
      • Kempenaar J.A.
      • Romeijn S.G.
      • Ponec M.
      Water distribution and natural moisturizer factor content in human skin equivalents are regulated by environmental relative humidity.
      ,
      • Bouwstra J.A.
      • de Graaff A.
      • Gooris G.S.
      • Nijsse J.
      • Wiechers J.W.
      • van Aelst A.C.
      Water distribution and related morphology in human stratum corneum at different hydration levels.
      ).
      Keratins have multiple regulatory functions. For example, K1 downregulates the expression and secretion of the inflammatory cytokines IL-18, IL-33, and TSLP as well as damage-associated molecular patterns such as S100A8 and S100A9 (
      • Roth W.
      • Kumar V.
      • Beer H.D.
      • Richter M.
      • Wohlenberg C.
      • Reuter U.
      • et al.
      Keratin 1 maintains skin integrity and participates in an inflammatory network in skin through interleukin-18.
      ). K16 downregulates the expression of damage-associated molecular patterns and other inflammatory molecules involved in the innate immune response to skin barrier disruption (
      • Lessard J.C.
      • Piña-Paz S.
      • Rotty J.D.
      • Hickerson R.P.
      • Kaspar R.L.
      • Balmain A.
      • et al.
      Keratin 16 regulates innate immunity in response to epidermal barrier breach.
      ).
      Keratin expression is dysregulated in AD (
      • Guttman-Yassky E.
      • Bissonnette R.
      • Ungar B.
      • Suárez-Fariñas M.
      • Ardeleanu M.
      • Esaki H.
      • et al.
      Dupilumab progressively improves systemic and cutaneous abnormalities in patients with atopic dermatitis.
      ). K16 expression is increased in suprabasal epidermis in AD, corresponding with abnormal KC proliferation (
      • Guttman-Yassky E.
      • Bissonnette R.
      • Ungar B.
      • Suárez-Fariñas M.
      • Ardeleanu M.
      • Esaki H.
      • et al.
      Dupilumab progressively improves systemic and cutaneous abnormalities in patients with atopic dermatitis.
      ;
      • Suárez-Fariñas M.
      • Tintle S.J.
      • Shemer A.
      • Chiricozzi A.
      • Nograles K.
      • Cardinale I.
      • et al.
      Nonlesional atopic dermatitis skin is characterized by broad terminal differentiation defects and variable immune abnormalities.
      ). In contrast, K1 and K10 expression is downregulated by IL-4 and IL-13 in AD lesional skin versus in healthy controls, which might contribute to the SC barrier defects seen in patients with AD and the release of proinflammatory and type 2–promoting alarmins (
      • Dai X.
      • Utsunomiya R.
      • Shiraishi K.
      • Mori H.
      • Muto J.
      • Murakami M.
      • et al.
      Nuclear IL-33 plays an important role in the suppression of FLG, LOR, keratin 1, and keratin 10 by IL-4 and IL-13 in human keratinocytes.
      ;
      • Imai Y.
      • Yasuda K.
      • Sakaguchi Y.
      • Haneda T.
      • Mizutani H.
      • Yoshimoto T.
      • et al.
      Skin-specific expression of IL-33 activates group 2 innate lymphoid cells and elicits atopic dermatitis–like inflammation in mice.
      ;
      • Totsuka A.
      • Omori-Miyake M.
      • Kawashima M.
      • Yagi J.
      • Tsunemi Y.
      Expression of keratin 1, keratin 10, desmoglein 1 and desmocollin 1 in the epidermis: possible downregulation by interleukin-4 and interleukin-13 in atopic dermatitis.
      ).
      FLG is a key structural protein in KCs. Its precursor pro-FLG is expressed in the stratum granulosum (SG) layer and is the major component of keratohyalin granules (
      • Presland R.B.
      • Kimball J.R.
      • Kautsky M.B.
      • Lewis S.P.
      • Lo C.Y.
      • Dale B.A.
      Evidence for specific proteolytic cleavage of the N-terminal domain of human profilaggrin during epidermal differentiation.
      ;
      • Resing K.A.
      • Thulin C.
      • Whiting K.
      • al-Alawi N.
      • Mostad S.
      Characterization of profilaggrin endoproteinase 1
      A regulated cytoplasmic endoproteinase of epidermis.
      ). During terminal differentiation, pro-FLG is dephosphorylated and cleaved to generate multiple FLG monomers (
      • Presland R.B.
      • Kimball J.R.
      • Kautsky M.B.
      • Lewis S.P.
      • Lo C.Y.
      • Dale B.A.
      Evidence for specific proteolytic cleavage of the N-terminal domain of human profilaggrin during epidermal differentiation.
      ;
      • Resing K.A.
      • Thulin C.
      • Whiting K.
      • al-Alawi N.
      • Mostad S.
      Characterization of profilaggrin endoproteinase 1
      A regulated cytoplasmic endoproteinase of epidermis.
      ;
      • Sandilands A.
      • Sutherland C.
      • Irvine A.D.
      • McLean W.H.I.
      Filaggrin in the frontline: role in skin barrier function and disease.
      ).
      FLG has multiple functions. It binds to keratin filaments in the KC cytoskeleton, forming an FLG–keratohyalin complex that cross-links to the cornified envelope, transforming KCs into arguably impervious corneocytes (i.e., “bricks”) (
      • Eckhart L.
      • Lippens S.
      • Tschachler E.
      • Declercq W.
      Cell death by cornification.
      ;
      • Presland R.B.
      • Kimball J.R.
      • Kautsky M.B.
      • Lewis S.P.
      • Lo C.Y.
      • Dale B.A.
      Evidence for specific proteolytic cleavage of the N-terminal domain of human profilaggrin during epidermal differentiation.
      ;
      • Sandilands A.
      • Sutherland C.
      • Irvine A.D.
      • McLean W.H.I.
      Filaggrin in the frontline: role in skin barrier function and disease.
      ). FLG degradation by the protease caspase-14 in outer SC layers produces natural moisturizing factors (NMFs) (see below) (
      • Hoste E.
      • Kemperman P.
      • Devos M.
      • Denecker G.
      • Kezic S.
      • Yau N.
      • et al.
      Caspase-14 is required for filaggrin degradation to natural moisturizing factors in the skin.
      ). FLG and NMFs are controlled in a finely balanced process of production, proteolysis, and inhibition that is crucial to skin barrier structure; hydration; and function, including pH regulation, microbial ecology, and possibly even UV protection (
      • Barker J.N.W.N.
      • Palmer C.N.
      • Zhao Y.
      • Liao H.
      • Hull P.R.
      • Lee S.P.
      • et al.
      Null mutations in the filaggrin gene (FLG) determine major susceptibility to early-onset atopic dermatitis that persists into adulthood.
      ;
      • Denecker G.
      • Hoste E.
      • Gilbert B.
      • Hochepied T.
      • Ovaere P.
      • Lippens S.
      • et al.
      Caspase-14 protects against epidermal UVB photodamage and water loss.
      ;
      • Kawasaki H.
      • Nagao K.
      • Kubo A.
      • Hata T.
      • Shimizu A.
      • Mizuno H.
      • et al.
      Altered stratum corneum barrier and enhanced percutaneous immune responses in filaggrin-null mice.
      ;
      • Palmer C.N.
      • Irvine A.D.
      • Terron-Kwiatkowski A.
      • Zhao Y.
      • Liao H.
      • Lee S.P.
      • et al.
      Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis.
      ;
      • Sandilands A.
      • Terron-Kwiatkowski A.
      • Hull P.R.
      • O'Regan G.M.
      • Clayton T.H.
      • Watson R.M.
      • et al.
      Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema.
      ;
      • Smith F.J.
      • Irvine A.D.
      • Terron-Kwiatkowski A.
      • Sandilands A.
      • Campbell L.E.
      • Zhao Y.
      • et al.
      Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris.
      ).
      Reduced expression and loss-of-function mutations of FLG are common in AD (
      • Barker J.N.W.N.
      • Palmer C.N.
      • Zhao Y.
      • Liao H.
      • Hull P.R.
      • Lee S.P.
      • et al.
      Null mutations in the filaggrin gene (FLG) determine major susceptibility to early-onset atopic dermatitis that persists into adulthood.
      ;
      • Baurecht H.
      • Irvine A.D.
      • Novak N.
      • Illig T.
      • Bühler B.
      • Ring J.
      • et al.
      Toward a major risk factor for atopic eczema: meta-analysis of filaggrin polymorphism data.
      ;
      • Nomura T.
      • Akiyama M.
      • Sandilands A.
      • Nemoto-Hasebe I.
      • Sakai K.
      • Nagasaki A.
      • et al.
      Specific filaggrin mutations cause ichthyosis vulgaris and are significantly associated with atopic dermatitis in Japan.
      ;
      • Palmer C.N.
      • Irvine A.D.
      • Terron-Kwiatkowski A.
      • Zhao Y.
      • Liao H.
      • Lee S.P.
      • et al.
      Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis.
      ;
      • Weidinger S.
      • O’Sullivan M.
      • Illig T.
      • Baurecht H.
      • Depner M.
      • Rodriguez E.
      • et al.
      Filaggrin mutations, atopic eczema, hay fever, and asthma in children.
      ,
      • Weidinger S.
      • Rodríguez E.
      • Stahl C.
      • Wagenpfeil S.
      • Klopp N.
      • Illig T.
      • et al.
      Filaggrin mutations strongly predispose to early-onset and extrinsic atopic dermatitis.
      ). Prevalence and types of FLG loss-of-function mutations vary among populations, with a very wide variation being reported for patients with AD (
      • Barker J.N.W.N.
      • Palmer C.N.
      • Zhao Y.
      • Liao H.
      • Hull P.R.
      • Lee S.P.
      • et al.
      Null mutations in the filaggrin gene (FLG) determine major susceptibility to early-onset atopic dermatitis that persists into adulthood.
      ;
      • Baurecht H.
      • Irvine A.D.
      • Novak N.
      • Illig T.
      • Bühler B.
      • Ring J.
      • et al.
      Toward a major risk factor for atopic eczema: meta-analysis of filaggrin polymorphism data.
      ;
      • Brown S.J.
      • McLean W.H.I.
      One remarkable molecule: filaggrin.
      ;
      • Nomura T.
      • Akiyama M.
      • Sandilands A.
      • Nemoto-Hasebe I.
      • Sakai K.
      • Nagasaki A.
      • et al.
      Specific filaggrin mutations cause ichthyosis vulgaris and are significantly associated with atopic dermatitis in Japan.
      ;
      • Palmer C.N.
      • Irvine A.D.
      • Terron-Kwiatkowski A.
      • Zhao Y.
      • Liao H.
      • Lee S.P.
      • et al.
      Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis.
      ;
      • Weidinger S.
      • O’Sullivan M.
      • Illig T.
      • Baurecht H.
      • Depner M.
      • Rodriguez E.
      • et al.
      Filaggrin mutations, atopic eczema, hay fever, and asthma in children.
      ,
      • Weidinger S.
      • Rodríguez E.
      • Stahl C.
      • Wagenpfeil S.
      • Klopp N.
      • Illig T.
      • et al.
      Filaggrin mutations strongly predispose to early-onset and extrinsic atopic dermatitis.
      ). FLG loss-of-function mutations are associated with more severe AD (
      • Brown S.J.
      • McLean W.H.I.
      One remarkable molecule: filaggrin.
      ,
      • Brown S.J.
      • McLean W.H.
      Eczema genetics: current state of knowledge and future goals.
      ;
      • Brown S.J.
      • Sandilands A.
      • Zhao Y.
      • Liao H.
      • Relton C.L.
      • Meggitt S.J.
      • et al.
      Prevalent and low-frequency null mutations in the filaggrin gene are associated with early-onset and persistent atopic eczema.
      ;
      • Margolis D.J.
      • Apter A.J.
      • Gupta J.
      • Hoffstad O.
      • Papadopoulos M.
      • Campbell L.E.
      • et al.
      The persistence of atopic dermatitis and filaggrin (FLG) mutations in a US longitudinal cohort.
      ;
      • Weidinger S.
      • Rodríguez E.
      • Stahl C.
      • Wagenpfeil S.
      • Klopp N.
      • Illig T.
      • et al.
      Filaggrin mutations strongly predispose to early-onset and extrinsic atopic dermatitis.
      ), earlier onset of AD, greater risk of allergen sensitization and other atopic disorders (
      • Brown S.J.
      • Asai Y.
      • Cordell H.J.
      • Campbell L.E.
      • Zhao Y.
      • Liao H.
      • et al.
      Loss-of-function variants in the filaggrin gene are a significant risk factor for peanut allergy.
      ;
      • Palmer C.N.
      • Ismail T.
      • Lee S.P.
      • Terron-Kwiatkowski A.
      • Zhao Y.
      • Liao H.
      • et al.
      Filaggrin null mutations are associated with increased asthma severity in children and young adults.
      ), and higher incidence of eczema herpeticum (
      • Gao P.S.
      • Rafaels N.M.
      • Hand T.
      • Murray T.
      • Boguniewicz M.
      • Hata T.
      • et al.
      Filaggrin mutations that confer risk of atopic dermatitis confer greater risk for eczema herpeticum.
      ). FLG loss-of-function mutations are also associated with mild AD, but the association is weaker than that seen for severe disease (
      • Brown S.J.
      • Relton C.L.
      • Liao H.
      • Zhao Y.
      • Sandilands A.
      • Wilson I.J.
      • et al.
      Filaggrin null mutations and childhood atopic eczema: a population-based case-control study.
      ).
      Notably, FLG expression may be reduced in patients with AD without FLG mutations. Type 2 inflammatory mediators, including IL-4, IL-13, IL-31, IL-33, and TSLP, reduce FLG expression (
      • Howell M.D.
      • Kim B.E.
      • Gao P.
      • Grant A.V.
      • Boguniewicz M.
      • DeBenedetto A.
      • et al.
      Cytokine modulation of atopic dermatitis filaggrin skin expression.
      ,
      • Howell M.D.
      • Kim B.E.
      • Gao P.
      • Grant A.V.
      • Boguniewicz M.
      • Debenedetto A.
      • et al.
      Cytokine modulation of atopic dermatitis filaggrin skin expression.
      ;
      • Kim J.H.
      • Bae H.C.
      • Ko N.Y.
      • Lee S.H.
      • Jeong S.H.
      • Lee H.
      • et al.
      Thymic stromal lymphopoietin downregulates filaggrin expression by signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK) phosphorylation in keratinocytes.
      ;
      • Sehra S.
      • Yao Y.
      • Howell M.D.
      • Nguyen E.T.
      • Kansas G.S.
      • Leung D.Y.M.
      • et al.
      IL-4 regulates skin homeostasis and the predisposition toward allergic skin inflammation.
      ;
      • Seltmann J.
      • Roesner L.M.
      • von Hesler F.W.
      • Wittmann M.
      • Werfel T.
      IL-33 impacts on the skin barrier by downregulating the expression of filaggrin.
      ). This has also been observed in skin inflammation mediated by Th17 (IL-17), Th22 (IL-22), and Th1 (IL-1α, IL-1β, and TNF-α) (
      • Archer N.K.
      • Jo J.H.
      • Lee S.K.
      • Kim D.
      • Smith B.
      • Ortines R.V.
      • et al.
      Injury, dysbiosis, and filaggrin deficiency drive skin inflammation through keratinocyte IL-1α release.
      ;
      • Boniface K.
      • Bernard F.X.
      • Garcia M.
      • Gurney A.L.
      • Lecron J.C.
      • Morel F.
      IL-22 inhibits epidermal differentiation and induces proinflammatory gene expression and migration of human keratinocytes.
      ;
      • Danso M.O.
      • van Drongelen V.
      • Mulder A.
      • van Esch J.
      • Scott H.
      • van Smeden J.
      • et al.
      TNF-α and Th2 cytokines induce atopic dermatitis-like features on epidermal differentiation proteins and stratum corneum lipids in human skin equivalents.
      ;
      • Gutowska-Owsiak D.
      • Schaupp A.L.
      • Salimi M.
      • Selvakumar T.A.
      • McPherson T.
      • Taylor S.
      • et al.
      IL-17 downregulates filaggrin and affects keratinocyte expression of genes associated with cellular adhesion.
      ,
      • Gutowska-Owsiak D.
      • Schaupp A.L.
      • Salimi M.
      • Taylor S.
      • Ogg G.S.
      Interleukin-22 downregulates filaggrin expression and affects expression of profilaggrin processing enzymes.
      ;
      • Kezic S.
      • O'Regan G.M.
      • Lutter R.
      • Jakasa I.
      • Koster E.S.
      • Saunders S.
      • et al.
      Filaggrin loss-of-function mutations are associated with enhanced expression of IL-1 cytokines in the stratum corneum of patients with atopic dermatitis and in a murine model of filaggrin deficiency.
      ;
      • Oyoshi M.K.
      • Murphy G.F.
      • Geha R.S.
      Filaggrin-deficient mice exhibit Th17-dominated skin inflammation and permissiveness to epicutaneous sensitization with protein antigen.
      ;
      • Tan Q.
      • Yang H.
      • Liu E.
      • Wang H.
      P38/ERK MAPK signaling pathways are involved in the regulation of filaggrin and involucrin by IL-17.
      ). Repetitive scratching, detergent use, low humidity, exogenous or endogenous proteases, air pollution, and topical and oral corticosteroids can also reduce FLG expression (
      • Danby S.G.
      • Chittock J.
      • Brown K.
      • Albenali L.H.
      • Cork M.J.
      The effect of tacrolimus compared with betamethasone valerate on the skin barrier in volunteers with quiescent atopic dermatitis.
      ;
      • Goleva E.
      • Berdyshev E.
      • Leung D.Y.M.
      Epithelial barrier repair and prevention of allergy.
      ;
      • Sheu H.M.
      • Lee J.Y.
      • Chai C.Y.
      • Kuo K.W.
      Depletion of stratum corneum intercellular lipid lamellae and barrier function abnormalities after long-term topical corticosteroids.
      ,
      • Sheu H.M.
      • Tai C.L.
      • Kuo K.W.
      • Yu H.S.
      • Chai C.Y.
      Modulation of epidermal terminal differentiation in patients after long-term topical corticosteroids.
      ;
      • Thyssen J.P.
      • Kezic S.
      Causes of epidermal filaggrin reduction and their role in the pathogenesis of atopic dermatitis.
      ). FLG has multiple repeats (typically 10‒12) within the locus (
      • Brown S.J.
      • Kroboth K.
      • Sandilands A.
      • Campbell L.E.
      • Pohler E.
      • Kezic S.
      • et al.
      Intragenic copy number variation within filaggrin contributes to the risk of atopic dermatitis with a dose-dependent effect.
      ). Copy number variants are associated with AD in some but not all populations. For example, in a cohort study in Ireland, reduced copy numbers were more frequent in patients with AD than in normal controls (
      • Brown S.J.
      • Kroboth K.
      • Sandilands A.
      • Campbell L.E.
      • Pohler E.
      • Kezic S.
      • et al.
      Intragenic copy number variation within filaggrin contributes to the risk of atopic dermatitis with a dose-dependent effect.
      ), whereas studies in other populations did not find any association between copy number variation and the risk of AD (
      • Fernandez K.
      • Asad S.
      • Taylan F.
      • Wahlgren C.F.
      • Bilcha K.D.
      • Nordenskjöld M.
      • et al.
      Intragenic copy number variation in the filaggrin gene in Ethiopian patients with atopic dermatitis.
      ;
      • Fulton R.L.
      • Margolis D.J.
      • Sockler P.G.
      • Mitra N.
      • Wong X.F.C.C.
      • Common J.E.
      No association of filaggrin copy number variation and atopic dermatitis risk in White and Black Americans.
      ).
      FLG deficiency is associated with reductions in SC structure, hydration, antimicrobial function, and epithelial buffering capacity in AD and increases in skin pH, percutaneous absorption, and protease activity (
      • Brauweiler A.M.
      • Bin L.
      • Kim B.E.
      • Oyoshi M.K.
      • Geha R.S.
      • Goleva E.
      • et al.
      Filaggrin-dependent secretion of sphingomyelinase protects against staphylococcal α-toxin-induced keratinocyte death.
      ;
      • Flohr C.
      • England K.
      • Radulovic S.
      • McLean W.H.I.
      • Campbel L.E.
      • Barker J.
      • et al.
      Filaggrin loss-of-function mutations are associated with early-onset eczema, eczema severity and transepidermal water loss at 3 months of age.
      ;
      • Kawasaki H.
      • Nagao K.
      • Kubo A.
      • Hata T.
      • Shimizu A.
      • Mizuno H.
      • et al.
      Altered stratum corneum barrier and enhanced percutaneous immune responses in filaggrin-null mice.
      ;
      • Kezic S.
      • Kemperman P.M.J.H.
      • Koster E.S.
      • de Jongh C.M.
      • Thio H.B.
      • Campbell L.E.
      • et al.
      Loss-of-function mutations in the filaggrin gene lead to reduced level of natural moisturizing factor in the stratum corneum.
      ;
      • Thyssen J.P.
      • Kezic S.
      Causes of epidermal filaggrin reduction and their role in the pathogenesis of atopic dermatitis.
      ;
      • Vávrová K.
      • Henkes D.
      • Strüver K.
      • Sochorová M.
      • Školová B.
      • Witting M.Y.
      • et al.
      Filaggrin deficiency leads to impaired lipid profile and altered acidification pathways in a 3D skin construct.
      ). FLG-knockdown KCs have reduced levels of K10, TJ proteins (zona occludens [ZO]-1, claudin [CLDN]-1, and occludin), and human β-defensin (hBD)-2; and increased cysteine proteases, which can degrade TJ proteins (
      • Hönzke S.
      • Wallmeyer L.
      • Ostrowski A.
      • Radbruch M.
      • Mundhenk L.
      • Schäfer-Korting M.
      • et al.
      Influence of Th2 cytokines on the cornified envelope, tight junction proteins, and β-defensins in filaggrin-deficient skin equivalents.
      ;
      • Wang X.W.
      • Wang J.J.
      • Gutowska-Owsiak D.
      • Salimi M.
      • Selvakumar T.A.
      • Gwela A.
      • et al.
      Deficiency of filaggrin regulates endogenous cysteine protease activity, leading to impaired skin barrier function.
      ). Reduction in FLG expression reduces the levels of FLG metabolites such as NMFs. This results in an increase in SC pH, which activates serine proteases (
      • Elias P.M.
      • Hatano Y.
      • Williams M.L.
      Basis for the barrier abnormality in atopic dermatitis: outside-inside-outside pathogenic mechanisms.
      ;
      • Goleva E.
      • Berdyshev E.
      • Leung D.Y.M.
      Epithelial barrier repair and prevention of allergy.
      ;
      • Wang X.W.
      • Wang J.J.
      • Gutowska-Owsiak D.
      • Salimi M.
      • Selvakumar T.A.
      • Gwela A.
      • et al.
      Deficiency of filaggrin regulates endogenous cysteine protease activity, leading to impaired skin barrier function.
      ) and induces the expression of the proinflammatory cytokines, IL-1α, IL-1β, and TSLP (
      • Hönzke S.
      • Wallmeyer L.
      • Ostrowski A.
      • Radbruch M.
      • Mundhenk L.
      • Schäfer-Korting M.
      • et al.
      Influence of Th2 cytokines on the cornified envelope, tight junction proteins, and β-defensins in filaggrin-deficient skin equivalents.
      ;
      • Kezic S.
      • O'Regan G.M.
      • Lutter R.
      • Jakasa I.
      • Koster E.S.
      • Saunders S.
      • et al.
      Filaggrin loss-of-function mutations are associated with enhanced expression of IL-1 cytokines in the stratum corneum of patients with atopic dermatitis and in a murine model of filaggrin deficiency.
      ;
      • Nylander-Lundqvist E.
      • Egelrud T.
      Formation of active IL-1 beta from pro-IL-1 beta catalyzed by stratum corneum chymotryptic enzyme in vitro.
      ;
      • Wood L.C.
      • Elias P.M.
      • Calhoun C.
      • Tsai J.C.
      • Grunfeld C.
      • Feingold K.R.
      Barrier disruption stimulates interleukin-1 alpha expression and release from a pre-formed pool in murine epidermis.
      ). Reduced FLG expression is also linked to increased levels of arachidonic acid and its metabolite 12-hydroxy-eicosatetraenoic acid in KCs, leading to increased inflammation and impairing late epidermal differentiation (
      • Blunder S.
      • Rühl R.
      • Moosbrugger-Martinz V.
      • Krimmel C.
      • Geisler A.
      • Zhu H.
      • et al.
      Alterations in epidermal eicosanoid metabolism contribute to inflammation and impaired late differentiation in FLG-mutated atopic dermatitis.
      ).
      The manifestations of FLG-deficient skin are much more dramatic when combined with the biological actions of IL-4 and IL-13. For example, in an in vitro study, IL-4 and IL-13 stimulation induced spongiosis and increased epidermal thickening, skin pH, and permeability in both normal and FLG-deficient skin equivalents (
      • Hönzke S.
      • Wallmeyer L.
      • Ostrowski A.
      • Radbruch M.
      • Mundhenk L.
      • Schäfer-Korting M.
      • et al.
      Influence of Th2 cytokines on the cornified envelope, tight junction proteins, and β-defensins in filaggrin-deficient skin equivalents.
      ). However, in FLG-deficient equivalents, IL-4 and IL-13 decreased the levels of skin barrier proteins (e.g., involucrin and loricrin), TJ proteins (e.g., occludin), and hBD-2 and increased basal layer proliferation rates and TSLP levels to a greater extent than in normal skin equivalents. This suggests that the combination of type 2 immunity and FLG deficiency may promote AD development more than either alone.
      NMFs are composed of FLG degradation products (i.e., free amino acids, urocanic acid, and pyrrolidine carboxylic acid), urea, and lactate derived from sweat. Under normal conditions, the decrease in hydration from middle to outer SC levels promotes FLG detachment from the corneocyte envelope and degradation, forming NMFs (
      • Rawlings A.V.
      • Matts P.J.
      Stratum corneum moisturization at the molecular level: an update in relation to the dry skin cycle.
      ;
      • Sandilands A.
      • Sutherland C.
      • Irvine A.D.
      • McLean W.H.I.
      Filaggrin in the frontline: role in skin barrier function and disease.
      ).
      NMFs retain moisture, contributing to barrier function by promoting epidermal hydration through osmotic gradients that allow the movement of water into the corneocytes (
      • Björklund S.
      • Andersson J.M.
      • Pham Q.D.
      • Nowacka A.
      • Topgaard D.
      • Sparr E.
      Stratum corneum molecular mobility in the presence of natural moisturizers.
      ;
      • Kezic S.
      • Kemperman P.M.J.H.
      • Koster E.S.
      • de Jongh C.M.
      • Thio H.B.
      • Campbell L.E.
      • et al.
      Loss-of-function mutations in the filaggrin gene lead to reduced level of natural moisturizing factor in the stratum corneum.
      ). NMFs maintain and buffer the acidic pH of the SC, which may reduce colonization by pathogenic bacteria (
      • Kezic S.
      • Kemperman P.M.J.H.
      • Koster E.S.
      • de Jongh C.M.
      • Thio H.B.
      • Campbell L.E.
      • et al.
      Loss-of-function mutations in the filaggrin gene lead to reduced level of natural moisturizing factor in the stratum corneum.
      ;
      • Krien P.M.
      • Kermici M.
      Evidence for the existence of a self-regulated enzymatic process within the human stratum corneum – an unexpected role for urocanic acid.
      ;
      • Miajlovic H.
      • Fallon P.G.
      • Irvine A.D.
      • Foster T.J.
      Effect of filaggrin breakdown products on growth of and protein expression by Staphylococcus aureus.
      ). NMFs also promote epidermal maturation and desquamation (
      • Kezic S.
      • O’Regan G.M.
      • Yau N.
      • Sandilands A.
      • Chen H.
      • Campbell L.E.
      • et al.
      Levels of filaggrin degradation products are influenced by both filaggrin genotype and atopic dermatitis severity.
      ). Decreased SC NMF levels are associated with dry skin and skin diseases such as ichthyosis vulgaris and AD. IL-4 and IL-13 reduce FLG levels and sweat secretion, which thereby affect NMF composition and function (
      • Howell M.D.
      • Kim B.E.
      • Gao P.
      • Grant A.V.
      • Boguniewicz M.
      • DeBenedetto A.
      • et al.
      Cytokine modulation of atopic dermatitis filaggrin skin expression.
      ,
      • Howell M.D.
      • Kim B.E.
      • Gao P.
      • Grant A.V.
      • Boguniewicz M.
      • Debenedetto A.
      • et al.
      Cytokine modulation of atopic dermatitis filaggrin skin expression.
      ;
      • Sehra S.
      • Yao Y.
      • Howell M.D.
      • Nguyen E.T.
      • Kansas G.S.
      • Leung D.Y.M.
      • et al.
      IL-4 regulates skin homeostasis and the predisposition toward allergic skin inflammation.
      ).
      Loricrin and involucrin are key structural proteins of the cornified envelope that anchor keratin filaments, providing mechanical strength and flexibility to the corneocytes (
      • Candi E.
      • Tarcsa E.
      • Digiovanna J.J.
      • Compton J.G.
      • Elias P.M.
      • Marekov L.N.
      • et al.
      A highly conserved lysine residue on the head domain of type II keratins is essential for the attachment of keratin intermediate filaments to the cornified cell envelope through isopeptide crosslinking by transglutaminases.
      ;
      • Roth W.
      • Kumar V.
      • Beer H.D.
      • Richter M.
      • Wohlenberg C.
      • Reuter U.
      • et al.
      Keratin 1 maintains skin integrity and participates in an inflammatory network in skin through interleukin-18.
      ;
      • 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.
      ). Both loricrin and involucrin are highly insoluble in late-stage KC differentiation, resulting from disulfide and transglutaminase cross-linking within the molecules and to other proteins in the cell envelope in corneocytes (
      • Hohl D.
      • Mehrel T.
      • Lichti U.
      • Turner M.L.
      • Roop D.R.
      • Steinert P.M.
      Characterization of human loricrin. Structure and function of a new class of epidermal cell envelope proteins.
      ;
      • 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.
      ;
      • 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.
      ). Loricrin is more prominent toward the cytoplasmic surface of the envelope, whereas involucrin is localized proximate to the lipid portion of the envelope (
      • Jarnik M.
      • de Viragh P.A.
      • Schärer E.
      • Bundman D.
      • Simon M.N.
      • Roop D.R.
      • et al.
      Quasi-normal cornified cell envelopes in loricrin knockout mice imply the existence of a loricrin backup system.
      ;
      • 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.
      ).
      IL-4 and IL-13 downregulate loricrin and involucrin expression in KCs (
      • Kim B.E.
      • Howell M.D.
      • Guttman-Yassky E.
      • Gilleaudeau P.M.
      • Cardinale I.R.
      • Boguniewicz M.
      • et al.
      TNF-α downregulates filaggrin and loricrin through c-Jun N-terminal kinase: role for TNF-α antagonists to improve skin barrier.
      ,
      • Kim B.E.
      • Leung D.Y.M.
      • Boguniewicz M.
      • Howell M.D.
      Loricrin and involucrin expression is down-regulated by Th2 cytokines through STAT-6.
      ), which may account for the reduced levels observed in AD. TNF-α reduces loricrin and involucrin expression, which also explains their reduced levels in psoriasis (
      • Kim B.E.
      • Howell M.D.
      • Guttman-Yassky E.
      • Gilleaudeau P.M.
      • Cardinale I.R.
      • Boguniewicz M.
      • et al.
      TNF-α downregulates filaggrin and loricrin through c-Jun N-terminal kinase: role for TNF-α antagonists to improve skin barrier.
      ,
      • Kim B.E.
      • Leung D.Y.M.
      • Boguniewicz M.
      • Howell M.D.
      Loricrin and involucrin expression is down-regulated by Th2 cytokines through STAT-6.
      ). Interestingly, silencing FLG expression in normal human KCs reduced involucrin expression but upregulated the expression of loricrin and IL-2, IL-4, IL-5, and IL-13 (
      • Dang N.N.
      • Pang S.G.
      • Song H.Y.
      • An L.G.
      • Ma X.L.
      Filaggrin silencing by shRNA directly impairs the skin barrier function of normal human epidermal keratinocytes and then induces an immune response.
      ).
      Proteases have multiple roles in the SC, mediated by both their direct proteolytic activity and through protease-activated receptors (PARs) (Figure 3). They influence SC cohesion, degrade corneodesmosome proteins (desmogleins and desmocollins) during homeostatic desquamation, regulate lipid synthesis by degrading enzymes that process extracellular lipids, and reduce lipid secretion into the extracellular matrix by stimulating the type 2 plasminogen receptor (
      • Borgoño C.A.
      • Michael I.P.
      • Komatsu N.
      • Jayakumar A.
      • Kapadia R.
      • Clayman G.L.
      • et al.
      A potential role for multiple tissue kallikrein serine proteases in epidermal desquamation.
      ;
      • Brattsand M.
      • Egelrud T.
      Purification, molecular cloning, and expression of a human stratum corneum trypsin-like serine protease with possible function in desquamation.
      ;
      • Caubet C.
      • Jonca N.
      • Brattsand M.
      • Guerrin M.
      • Bernard D.
      • Schmidt R.
      • et al.
      Degradation of corneodesmosome proteins by two serine proteases of the kallikrein family, SCTE/KLK5/hK5 and SCCE/KLK7/hK7.
      ;
      • Hachem J.P.
      • Houben E.
      • Crumrine D.
      • Man M.Q.
      • Schurer N.
      • Roelandt T.
      • et al.
      Serine protease signaling of epidermal permeability barrier homeostasis.
      ,
      • Hachem J.P.
      • Man M.Q.
      • Crumrine D.
      • Uchida Y.
      • Brown B.E.
      • Rogiers V.
      • et al.
      Sustained serine proteases activity by prolonged increase in pH leads to degradation of lipid processing enzymes and profound alterations of barrier function and stratum corneum integrity.
      ;
      • Sales K.U.
      • Masedunskas A.
      • Bey A.L.
      • Rasmussen A.L.
      • Weigert R.
      • List K.
      • et al.
      Matriptase initiates activation of epidermal pro-kallikrein and disease onset in a mouse model of netherton syndrome.
      ;
      • Watkinson A.
      Stratum corneum thiol protease (SCTP): a novel cysteine protease of late epidermal differentiation.
      ).
      Serine protease activity is increased in both lesional and nonlesional AD skin (
      • Komatsu N.
      • Saijoh K.
      • Kuk C.
      • Liu A.C.
      • Khan S.
      • Shirasaki F.
      • et al.
      Human tissue kallikrein expression in the stratum corneum and serum of atopic dermatitis patients.
      ;
      • Voegeli R.
      • Rawlings A.V.
      • Breternitz M.
      • Doppler S.
      • Schreier T.
      • Fluhr J.W.
      Increased stratum corneum serine protease activity in acute eczematous atopic skin.
      ). Increased serine protease activity compromises barrier function by increasing the degradation of corneodesmosomes and extracellular lipid-processing enzymes, reducing ceramide production (a characteristic abnormality of AD) (
      • Borgoño C.A.
      • Michael I.P.
      • Komatsu N.
      • Jayakumar A.
      • Kapadia R.
      • Clayman G.L.
      • et al.
      A potential role for multiple tissue kallikrein serine proteases in epidermal desquamation.
      ;
      • Di Nardo A.
      • Wertz P.
      • Giannetti A.
      • Seidenari S.
      Ceramide and cholesterol composition of the skin of patients with atopic dermatitis.
      ;
      • Hachem J.P.
      • Man M.Q.
      • Crumrine D.
      • Uchida Y.
      • Brown B.E.
      • Rogiers V.
      • et al.
      Sustained serine proteases activity by prolonged increase in pH leads to degradation of lipid processing enzymes and profound alterations of barrier function and stratum corneum integrity.
      ,
      • Hachem J.P.
      • Crumrine D.
      • Fluhr J.
      • Brown B.E.
      • Feingold K.R.
      • Elias P.M.
      pH directly regulates epidermal permeability barrier homeostasis, and stratum corneum integrity/cohesion.
      ;
      • Imokawa G.
      • Abe A.
      • Jin K.
      • Higaki Y.
      • Kawashima M.
      • Hidano A.
      Decreased level of ceramides in stratum corneum of atopic dermatitis: an etiologic factor in atopic dry skin?.
      ). Serine proteases and cysteine proteases activate the PAR2 receptor, which regulates the secretion of lamellar bodies and cornification (
      • Demerjian M.
      • Hachem J.P.
      • Tschachler E.
      • Denecker G.
      • Declercq W.
      • Vandenabeele P.
      • et al.
      Acute modulations in permeability barrier function regulate epidermal cornification: role of caspase-14 and the protease-activated receptor type 2.
      ;
      • Hachem J.P.
      • Houben E.
      • Crumrine D.
      • Man M.Q.
      • Schurer N.
      • Roelandt T.
      • et al.
      Serine protease signaling of epidermal permeability barrier homeostasis.
      ), and is linked to increased inflammation, itch, and epidermal barrier disruption (
      • Briot A.
      • Deraison C.
      • Lacroix M.
      • Bonnart C.
      • Robin A.
      • Besson C.
      • et al.
      Kallikrein 5 induces atopic dermatitis-like lesions through PAR2-mediated thymic stromal lymphopoietin expression in netherton syndrome.
      ;
      • Wilson S.R.
      • Thé L.
      • Batia L.M.
      • Beattie K.
      • Katibah G.E.
      • McClain S.P.
      • et al.
      The epithelial cell-derived atopic dermatitis cytokine TSLP activates neurons to induce itch.
      ). Both endogenous and exogenous proteases (e.g., from allergens, such as cockroach and dust mites, or from bacteria, such as S. aureus alpha-toxin) activate PAR2 (
      • Ebeling C.
      • Lam T.
      • Gordon J.R.
      • Hollenberg M.D.
      • Vliagoftis H.
      Proteinase-activated receptor-2 promotes allergic sensitization to an inhaled antigen through a TNF-mediated pathway.
      ;
      • Hachem J.P.
      • Houben E.
      • Crumrine D.
      • Man M.Q.
      • Schurer N.
      • Roelandt T.
      • et al.
      Serine protease signaling of epidermal permeability barrier homeostasis.
      ;
      • Jeong S.K.
      • Kim H.J.
      • Youm J.K.
      • Ahn S.K.
      • Choi E.H.
      • Sohn M.H.
      • et al.
      Mite and cockroach allergens activate protease-activated receptor 2 and delay epidermal permeability barrier recovery.
      ;
      • Kato T.
      • Takai T.
      • Fujimura T.
      • Matsuoka H.
      • Ogawa T.
      • Murayama K.
      • et al.
      Mite serine protease activates protease-activated receptor-2 and induces cytokine release in human keratinocytes.
      ). PAR2 activation reduces the expression of TJ proteins (occludin, CLDN-1, and ZO-1) and impairs TJ function, as assessed by reduced transepithelial electrical resistance (owing to a diminished barrier to ions) and increased permeability to small proteins (
      • Nadeau P.
      • Henehan M.
      • de Benedetto A.
      Activation of protease-activated receptor 2 leads to impairment of keratinocyte tight junction integrity.
      ). Thus both allergens and cutaneous dysbiosis may promote skin barrier disruption in AD through PAR2-mediated mechanisms. Finally, PAR2 agonists also increase the expression of IL-4 and IL-13 by mast cells, whereas PAR2 inhibition blocks IL-4 and IL-13 expression, decreases skin thickening, and suppresses itching in AD models (
      • Barr T.P.
      • Garzia C.
      • Guha S.
      • Fletcher E.K.
      • Nguyen N.
      • Wieschhaus A.J.
      • et al.
      PAR2 pepducin-based suppression of inflammation and itch in atopic dermatitis models.
      ). Of interest, Netherton syndrome, a monogenic AD-like syndrome characterized by the loss of serine protease inhibition due to a mutation in SPINK5 (which codes for the protease inhibitor LEKTI), is associated with kallikrein 5‒mediated PAR2 activation resulting in the production of the pro–type 2 cytokine TSLP by KCs (
      • Briot A.
      • Lacroix M.
      • Robin A.
      • Steinhoff M.
      • Deraison C.
      • Hovnanian A.
      Par2 inactivation inhibits early production of TSLP, but not cutaneous inflammation, in Netherton syndrome adult mouse model.
      ).
      Matrix metalloproteinases (MMPs), which affect tissue remodeling and inflammatory cell migration into the epidermis, may also play an important role in AD pathogenesis (
      • Groneberg D.A.
      • Bester C.
      • Grützkau A.
      • Serowka F.
      • Fischer A.
      • Henz B.M.
      • et al.
      Mast cells and vasculature in atopic dermatitis—potential stimulus of neoangiogenesis.
      ;
      • Harper J.I.
      • Godwin H.
      • Green A.
      • Wilkes L.E.
      • Holden N.J.
      • Moffatt M.
      • et al.
      A study of matrix metalloproteinase expression and activity in atopic dermatitis using a novel skin wash sampling assay for functional biomarker analysis.
      ;
      • Purwar R.
      • Kraus M.
      • Werfel T.
      • Wittmann M.
      Modulation of keratinocyte-derived MMP-9 by IL-13: a possible role for the pathogenesis of epidermal inflammation.
      ). MMP activity was 10‒24 times greater in saline wash samples from AD lesional skin than in healthy controls, which do not normally express MMPs (
      • Harper J.I.
      • Godwin H.
      • Green A.
      • Wilkes L.E.
      • Holden N.J.
      • Moffatt M.
      • et al.
      A study of matrix metalloproteinase expression and activity in atopic dermatitis using a novel skin wash sampling assay for functional biomarker analysis.
      ). IL-13 induces MMP-9 expression in KCs, and expression of both MMP-9 and IL-13 is increased in acute AD lesions (
      • Purwar R.
      • Kraus M.
      • Werfel T.
      • Wittmann M.
      Modulation of keratinocyte-derived MMP-9 by IL-13: a possible role for the pathogenesis of epidermal inflammation.
      ). MMP-12, which induces inflammatory cell aggregation, is also upregulated in lesional and nonlesional AD skin (
      • Brunner P.M.
      • Suárez-Fariñas M.
      • He H.
      • Malik K.
      • Wen H.C.
      • Gonzalez J.
      • et al.
      The atopic dermatitis blood signature is characterized by increases in inflammatory and cardiovascular risk proteins.
      ;
      • Pavel A.B.
      • Zhou L.
      • Diaz A.
      • Ungar B.
      • Dan J.
      • He H.
      • et al.
      The proteomic skin profile of moderate-to-severe atopic dermatitis patients shows an inflammatory signature.
      ;
      • Zhu J.
      • Wang Z.
      • Chen F.
      Association of key genes and pathways with atopic dermatitis by bioinformatics analysis.
      ).

      SC lipid components

      Skin barrier lipids are localized in the extracellular matrix surrounding corneocytes and are secreted from lamellar bodies before cornification (Figure 3) (
      • Elias P.M.
      • Cullander C.
      • Mauro T.
      • Rassner U.
      • Kömüves L.
      • Brown B.E.
      • et al.
      The secretory granular cell: the outermost granular cell as a specialized secretory cell.
      ;
      • Menon G.K.
      • Feingold K.R.
      • Elias P.M.
      Lamellar body secretory response to barrier disruption.
      ). By weight, these lipids include approximately 47% ceramides, 24% cholesterol, 18% cholesterol esters, and 11% free fatty acids (FFAs) (
      • Ohno Y.
      • Nakamichi S.
      • Ohkuni A.
      • Kamiyama N.
      • Naoe A.
      • Tsujimura H.
      • et al.
      Essential role of the cytochrome P450 CYP4F22 in the production of acylceramide, the key lipid for skin permeability barrier formation.
      ;
      • Rawlings A.V.
      • Matts P.J.
      Stratum corneum moisturization at the molecular level: an update in relation to the dry skin cycle.
      ;
      • van Smeden J.
      • Bouwstra J.A.
      Stratum corneum lipids: their role for the skin barrier function in healthy subjects and atopic dermatitis patients.
      ). The SC contains several types of ceramides, many of which have very long fatty acid chains and are highly hydrophobic (
      • Berdyshev E.
      • Goleva E.
      • Bronova I.
      • Dyjack N.
      • Rios C.
      • Jung J.
      • et al.
      Lipid abnormalities in atopic skin are driven by type 2 cytokines.
      ;
      • Rawlings A.V.
      • Matts P.J.
      Stratum corneum moisturization at the molecular level: an update in relation to the dry skin cycle.
      ;
      • van Smeden J.
      • Bouwstra J.A.
      Stratum corneum lipids: their role for the skin barrier function in healthy subjects and atopic dermatitis patients.
      ).
      Lipids form densely packed layers in the central portion of the SC, becoming less densely packed and more gel-like closer to the surface (
      • Brancaleon L.
      • Bamberg M.P.
      • Sakamaki T.
      • Kollias N.
      Attenuated total reflection–Fourier transform infrared spectroscopy as a possible method to investigate biophysical parameters of stratum corneum in vivo.
      ;
      • Pilgram G.S.K.
      • Engelsma-van Pelt A.M.
      • Bouwstra J.A.
      • Koerten H.K.
      Electron diffraction provides new information on human stratum corneum lipid organization studied in relation to depth and temperature.
      ). Alterations of this packing pattern, resulting from altered lipid composition and lipid-chain shortening, are thought to contribute significantly to skin barrier impairment in AD (
      • Berdyshev E.
      • Goleva E.
      • Bronova I.
      • Dyjack N.
      • Rios C.
      • Jung J.
      • et al.
      Lipid abnormalities in atopic skin are driven by type 2 cytokines.
      ;
      • Pilgram G.S.
      • Vissers D.C.
      • van der Meulen H.
      • Pavel S.
      • Lavrijsen S.P.
      • Bouwstra J.A.
      • et al.
      Aberrant lipid organization in stratum corneum of patients with atopic dermatitis and lamellar ichthyosis.
      ;
      • van Smeden J.
      • Bouwstra J.A.
      Stratum corneum lipids: their role for the skin barrier function in healthy subjects and atopic dermatitis patients.
      ;
      • van Smeden J.
      • Janssens M.
      • Kaye E.C.J.
      • Caspers P.J.
      • Lavrijsen A.P.
      • Vreeken R.J.
      • et al.
      The importance of free fatty acid chain length for the skin barrier function in atopic eczema patients.
      ). Fatty acid chains are lengthened by elongases (
      • Ewald D.A.
      • Malajian D.
      • Krueger J.G.
      • Workman C.T.
      • Wang T.
      • Tian S.
      • et al.
      Meta-analysis derived atopic dermatitis (MADAD) transcriptome defines a robust AD signature highlighting the involvement of atherosclerosis and lipid metabolism pathways.
      ;
      • van Smeden J.
      • Bouwstra J.A.
      Stratum corneum lipids: their role for the skin barrier function in healthy subjects and atopic dermatitis patients.
      ). The expression of the elongases ELOVL1, ELOVL3, and ELOVL6 is reduced in lesional AD skin, resulting in shortened fatty acid chains and increased skin barrier permeability (
      • Berdyshev E.
      • Goleva E.
      • Bronova I.
      • Dyjack N.
      • Rios C.
      • Jung J.
      • et al.
      Lipid abnormalities in atopic skin are driven by type 2 cytokines.
      ;
      • Danso M.
      • Boiten W.
      • van Drongelen V.
      • Gmelig Meijling K.
      • Gooris G.
      • El Ghalbzouri A.
      • et al.
      Altered expression of epidermal lipid bio-synthesis enzymes in atopic dermatitis skin is accompanied by changes in stratum corneum lipid composition.
      ). The higher proportion of short fatty acids correlates with changes in lipid organization and skin barrier function and is associated with AD severity (
      • Janssens M.
      • van Smeden J.
      • Gooris G.S.
      • Bras W.
      • Portale G.
      • Caspers P.J.
      • et al.
      Increase in short-chain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients.
      ;
      • Li S.
      • Villarreal M.
      • Stewart S.
      • Choi J.
      • Ganguli-Indra G.
      • Babineau D.C.
      • et al.
      Altered composition of epidermal lipids correlates with Staphylococcus aureus colonization status in atopic dermatitis.
      ;
      • van Smeden J.
      • Janssens M.
      • Kaye E.C.J.
      • Caspers P.J.
      • Lavrijsen A.P.
      • Vreeken R.J.
      • et al.
      The importance of free fatty acid chain length for the skin barrier function in atopic eczema patients.
      ). IL-4 and IL-13 inhibit KCs expression of ELOVL1, ELOVL3, and ELOVL6 (
      • Berdyshev E.
      • Goleva E.
      • Bronova I.
      • Dyjack N.
      • Rios C.
      • Jung J.
      • et al.
      Lipid abnormalities in atopic skin are driven by type 2 cytokines.
      ;
      • Danso M.
      • Boiten W.
      • van Drongelen V.
      • Gmelig Meijling K.
      • Gooris G.
      • El Ghalbzouri A.
      • et al.
      Altered expression of epidermal lipid bio-synthesis enzymes in atopic dermatitis skin is accompanied by changes in stratum corneum lipid composition.
      ), and IL-4 inhibits ceramide synthesis (
      • Hatano Y.
      • Terashi H.
      • Arakawa S.
      • Katagiri K.
      Interleukin-4 suppresses the enhancement of ceramide synthesis and cutaneous permeability barrier functions induced by tumor necrosis factor-alpha and interferon-gamma in human epidermis.
      ).

      Sweat

      Sweat is an important component of the skin barrier. Sweat forms a protective layer on the SC surface, contributing to thermoregulation, moisturizing the skin surface, and regulating water retention (
      • Murota H.
      • Matsui S.
      • Ono E.
      • Kijima A.
      • Kikuta J.
      • Ishii M.
      • et al.
      Sweat, the driving force behind normal skin: an emerging perspective on functional biology and regulatory mechanisms.
      ). In addition to water, electrolytes, lactate, basic nitrogenous compounds (e.g., urea, ammonia), amino acids, and proteins (
      • Hiragun T.
      • Hiragun M.
      • Ishii K.
      • Kan T.
      • Hide M.
      Sweat allergy: extrinsic or intrinsic?.
      ), sweat contains antimicrobial peptides (AMPs) (e.g., dermcidin and cathelicidin [see below]) and secretory IgA, which protect against infection (
      • Imayama S.
      • Shimozono Y.
      • Hoashi M.
      • Yasumoto S.
      • Ohta S.
      • Yoneyama K.
      • et al.
      Reduced secretion of IgA to skin surface of patients with atopic dermatitis.
      ;
      • Metze D.
      • Kersten A.
      • Jurecka W.
      • Gebhart W.
      Immunoglobulins coat microorganisms of skin surface: a comparative immunohistochemical and ultrastructural study of cutaneous and oral microbial symbionts.
      ;
      • Murakami M.
      • Ohtake T.
      • Dorschner R.A.
      • Schittek B.
      • Garbe C.
      • Gallo R.L.
      Cathelicidin anti-microbial peptide expression in sweat, an innate defense system for the skin.
      ). TJs prevent sweat ducts from leaking sweat contents into the dermis. CLDN-3 is the most prevalent TJ protein that regulates sweat gland permeability (
      • Yamaga K.
      • Murota H.
      • Tamura A.
      • Miyata H.
      • Ohmi M.
      • Kikuta J.
      • et al.
      Claudin-3 loss causes leakage of sweat from the sweat gland to contribute to the pathogenesis of atopic dermatitis.
      ).
      Patients with AD may have an impaired ability to sweat (
      • Takahashi A.
      • Murota H.
      • Matsui S.
      • Kijima A.
      • Kitaba S.
      • Lee J.B.
      • et al.
      Decreased sudomotor function is involved in the formation of atopic eczema in the cubital fossa.
      ;
      • Yamaga K.
      • Murota H.
      • Tamura A.
      • Miyata H.
      • Ohmi M.
      • Kikuta J.
      • et al.
      Claudin-3 loss causes leakage of sweat from the sweat gland to contribute to the pathogenesis of atopic dermatitis.
      ), causing sweat leakage into the dermis leading to local inflammation and reducing the beneficial effects of sweating (
      • Shiohara T.
      • Doi T.
      • Hayakawa J.
      Defective sweating responses in atopic dermatitis.
      ;
      • Takahashi A.
      • Murota H.
      • Matsui S.
      • Kijima A.
      • Kitaba S.
      • Lee J.B.
      • et al.
      Decreased sudomotor function is involved in the formation of atopic eczema in the cubital fossa.
      ). Skin dryness and increased susceptibility to infection caused by decreased sweating at the skin surface may also worsen AD symptoms (
      • Murota H.
      • Yamaga K.
      • Ono E.
      • Katayama I.
      Sweat in the pathogenesis of atopic dermatitis.
      ;
      • Shimoda-Komatsu Y.
      • Sato Y.
      • Yamazaki Y.
      • Takahashi R.
      • Shiohara T.
      A novel method to assess the potential role of sweating abnormalities in the pathogenesis of atopic dermatitis.
      ). Dermal sweat leakage is due to reduced CLDN-3 expression in AD sweat ducts (
      • Yamaga K.
      • Murota H.
      • Tamura A.
      • Miyata H.
      • Ohmi M.
      • Kikuta J.
      • et al.
      Claudin-3 loss causes leakage of sweat from the sweat gland to contribute to the pathogenesis of atopic dermatitis.
      ). AD exacerbation may also result from sweat allergy―an IgE-mediated hypersensitivity to sweat, particularly to fungal (Malassezia) protein antigens commonly found in sweat (
      • Hiragun T.
      • Ishii K.
      • Hiragun M.
      • Suzuki H.
      • Kan T.
      • Mihara S.
      • et al.
      Fungal protein MGL_1304 in sweat is an allergen for atopic dermatitis patients.
      ;
      • Takahagi S.
      • Tanaka A.
      • Hide M.
      Sweat allergy.
      ). Sweat glands also express IL-13 receptors, suggesting a role for type 2 inflammation in sweat regulation (
      • Akaiwa M.
      • Yu B.
      • Umeshita-Suyama R.
      • Terada N.
      • Suto H.
      • Koga T.
      • et al.
      Localization of human interleukin 13 receptor in non-haematopoietic cells.