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IL-17 and IL-22 Promote Keratinocyte Stemness in the Germinative Compartment in Psoriasis

  • Anna-Karin Ekman
    Affiliations
    Ingrid Asp Psoriasis Research Center, Department of Clinical and Experimental Medicine, Division of Dermatology, Linköping University, Linköping, Sweden
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  • Cecilia Bivik Eding
    Affiliations
    Ingrid Asp Psoriasis Research Center, Department of Clinical and Experimental Medicine, Division of Dermatology, Linköping University, Linköping, Sweden
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  • Ingemar Rundquist
    Affiliations
    Ingrid Asp Psoriasis Research Center, Department of Clinical and Experimental Medicine, Division of Dermatology, Linköping University, Linköping, Sweden
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  • Charlotta Enerbäck
    Correspondence
    Correspondence: Charlotta Enerbäck, Department of Clinical and Experimental Medicine, Division of Dermatology, Linköping University, SE-581 85 Linköping, Sweden.
    Affiliations
    Ingrid Asp Psoriasis Research Center, Department of Clinical and Experimental Medicine, Division of Dermatology, Linköping University, Linköping, Sweden
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Open ArchivePublished:January 23, 2019DOI:https://doi.org/10.1016/j.jid.2019.01.014
      Psoriasis is an inflammatory skin disorder characterized by the hyperproliferation of basal epidermal cells. It is regarded as T-cell mediated, but the role of keratinocytes (KCs) in the disease pathogenesis has reemerged, with genetic studies identifying KC-associated genes. We applied flow cytometry on KCs from lesional and nonlesional epidermis to characterize the phenotype in the germinative compartment in psoriasis, and we observed an overall increase in the stemness markers CD29 (2.4-fold), CD44 (2.9-fold), CD49f (2.8-fold), and p63 (1.4-fold). We found a reduced percentage of cells positive for the early differentiation marker cytokeratin 10 and a greater fraction of CD29+ and involucrin+ cells in the psoriasis KCs than in nonlesional KCs. The up-regulation of stemness markers was more pronounced in the K10+ cells. Furthermore, the psoriasis cells were smaller, indicating increased proliferation. Treatment with IL-17 and IL-22 induced a similar expression pattern of an up-regulation of p63, CD44, and CD29 in normal KCs and increased the colony-forming efficiency and long-term proliferative capacity, reflecting increased stem cell-like characteristics in the KC population. These data suggest that IL-17 and IL-22 link the inflammatory response to the immature differentiation and epithelial regeneration by acting directly on KCs to promote cell stemness.

      Abbreviations:

      KC (keratinocyte), HEKn (neonatal human epidermal keratinocyte), siRNA (small interfering RNA), tumor necrosis factor-α (TNF-α)

      Introduction

      Psoriasis is widely regarded as an inflammatory T-cell–mediated disorder, where the hyperproliferation and disturbed maturation of the keratinocytes (KCs) are believed to be driven by T helper types 1- and 17-associated cytokines and chemokines (
      • Harden J.L.
      • Krueger J.G.
      • Bowcock A.M.
      The immunogenetics of psoriasis: a comprehensive review.
      ,
      • Lowes M.A.
      • Suarez-Farinas M.
      • Krueger J.G.
      Immunology of psoriasis.
      ). Genetic data support the role of a dysregulated immune system by showing a strong association with HLA-C, IL-12B, and IL23R. T helper type 17 cells and their downstream effector cytokines, IL-17 and IL-22, are central to pathogenesis, which is supported by the effectiveness of treatments targeting related pathways (
      • Harden J.L.
      • Krueger J.G.
      • Bowcock A.M.
      The immunogenetics of psoriasis: a comprehensive review.
      ,
      • Tsoi L.C.
      • Spain S.L.
      • Ellinghaus E.
      • Stuart P.E.
      • Capon F.
      • Knight J.
      • et al.
      Enhanced meta-analysis and replication studies identify five new psoriasis susceptibility loci.
      ). Although recent genetic advances suggest that both immune and epidermal components contribute to disease susceptibility, the role of KCs in the formation of psoriatic lesions has received less attention.
      The marked increase in proliferation and the disturbed differentiation of KCs in psoriasis (
      • Hawkes J.E.
      • Chan T.C.
      • Krueger J.G.
      Psoriasis pathogenesis and the development of novel targeted immune therapies.
      ) led to psoriasis long being regarded as a KC-driven disease (
      • Lowes M.A.
      • Suarez-Farinas M.
      • Krueger J.G.
      Immunology of psoriasis.
      ). The psoriatic epidermis manifests an incompletely formed granular layer and a stratum corneum where the KCs are not fully differentiated and still retain their nuclei (
      • Raychaudhuri S.K.
      • Maverakis E.
      • Raychaudhuri S.P.
      Diagnosis and classification of psoriasis.
      ). It also show an increased growth fraction of epidermal KCs and an approximately 30-fold increase in the production of cells compared with the normal epidermis (
      • Weinstein G.D.
      • McCullough J.L.
      • Ross P.A.
      Cell kinetic basis for pathophysiology of psoriasis.
      ). The mechanism of this enhanced proliferation is not fully understood, and no reduction in cell cycle time has been observed in plaques (
      • Castelijns F.A.
      • Gerritsen M.J.
      • van Erp P.E.
      • van de Kerkhof P.C.
      Cell-kinetic evidence for increased recruitment of cycling epidermal cells in psoriasis: the ratio of histone and Ki-67 antigen expression is constant.
      ,
      • van Ruissen F.
      • de Jongh G.J.
      • van Erp P.E.
      • Boezeman J.B.
      • Schalkwijk J.
      Cell kinetic characterization of cultured human keratinocytes from normal and psoriatic individuals.
      ). Epidermal KCs also display disturbed tissue homeostasis and dysregulated apoptosis, which may contribute to the thickened epidermal (
      • Eding C.B.
      • Enerback C.
      Involved and uninvolved psoriatic keratinocytes display a resistance to apoptosis that may contribute to epidermal thickness.
      ). Epidermal homeostasis is maintained through the activation of stem cells in the basal layer and the subsequent proliferation of early progenitor cells, often referred to as transient amplifying cells (
      • Ghadially R.
      25 years of epidermal stem cell research.
      ). In psoriasis, the phenotype of the proliferating epidermal KC subpopulation is still poorly defined. Here, using the best available markers of differentiation and stemness, we describe an overall more immature phenotype of psoriatic KCs. Furthermore, we found that IL-17 and IL-22 alter the expression of stem cell markers in KCs to a pattern that is reminiscent of that observed in psoriatic KCs.

      Results

      Flow cytometric analysis of the germinative compartment shows an increased expression of stem cell markers in the psoriatic epidermis

      To characterize the phenotype in the germinative compartment in psoriasis, KCs from psoriatic lesional and nonlesional skin were isolated and analyzed with flow cytometry. Trypsin was used to dissociate the epidermal sheets, which enriched the immature and early differentiating cells of the epidermis, because trypsin does not dissociate terminally differentiated cells. To exclude doublet and cell debris, a gate was placed based on scatter properties. Within this population, cells were further gated on the lack of expression of CD45, a cell surface marker that is found on cells of hematopoietic origin. The derived KCs were analyzed based on their marker expression, evaluating the median fluorescence intensity in non-bimodal populations and further subgating the bimodal populations where positive and where negative cells could be clearly distinguished. An illustrative flowchart, as well as the strategy for singlet gating, scatter gating, and CD45 gating can be found in Figure 1a and b . The autofluorescence of unstained cells is shown in Supplementary Figure S1 online. The gates for the bimodal populations can be seen in Figure 2.
      Figure thumbnail gr1
      Figure 1KCs from psoriatic lesions display an immature phenotype and signs of proliferation. Events collected on a Beckman Coulter Gallios flow cytometer were gated to exclude doublets and cell debris. (a) A schematic of the gating strategy. (b) Gating to obtain the KC population, starting with the singlet gate to exclude doublets (top image), followed by the FSC/SSC gate (middle image) and the CD45 gate, where CD45+ cells were removed from the analysis by placing a KC gate on the CD45 cells (bottom image). KCs derived from psoriatic lesions and nonlesional skin were stained for the expression of CD29, CD44, CD49f, p63, and CD71. (c) The fluorescence overlay between the lesional (gray histograms) and nonlesional cells (transparent histograms), as well as the MFI levels. The x-axis in the histograms displays the fluorescence levels, and the y-axis displays the count. Brackets in the histograms show the fluorescence at which cells were considered positive for each marker. (d) The median FSC values as a measurement of cell size; n = 4–7. P ≤ 0.05, ∗∗P < 0.01. Fig., figure; FSC, forward scatter; KC, keratinocyte; MFI, median fluorescence intensity; SSC, side scatter.
      Figure thumbnail gr2
      Figure 2Keratinocyte subpopulations in psoriatic epidermis. The panels show the representative dot plots show the proportion of K10+/–, CD29+/–, and involucrin+/– populations in KCs derived from psoriatic lesions (lesional) and nonlesional skin (nonlesional). The dot plots display the fluorescence on the x-axis and the linear side scatter area (SS) on the y-axis. The graphs show the percentages of K10+, CD29+, and involucrin+ cells; n = 4–7. P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗P < 0.001. KC, keratinocyte.
      As expected, CD45 showed a higher percentage of leukocytes in the lesional psoriatic skin than in the control skin (2.3% ± 0.4% vs. control, 0.7% ± 0.1%; P < 0.05, data not shown). In the KC population that resulted from gating the CD45 cells, we found a higher expression of all the investigated stem cell markers (i.e., CD29, CD44, CD49f, and p63) in psoriatic KCs than in the KCs from nonlesional skin, with a 2.4-fold up-regulation of CD29, a 2.9-fold up-regulation of CD44, a 2.8-fold up-regulation of CD49f, and a 1.4-fold up-regulation of p63 (Figure 1c). We also found a tendency toward an up-regulation of the proliferation-associated marker CD71 in the psoriatic KCs (P = 0.08). The KCs derived from lesional psoriatic skin were markedly smaller (Figure 1d) than their nonlesional counterparts, indicative of an increase in proliferative capacity (
      • Barrandon Y.
      • Green H.
      Cell size as a determinant of the clone-forming ability of human keratinocytes.
      ).
      The markers K10, CD29, and involucrin displayed a bimodal expression patterns, with one positive and one negative cell population, which were altered in the KCs derived from psoriatic lesions. The most notable difference was the markedly smaller fraction of K10+ cells in the lesional cell fractions compared with the nonlesional fractions (51.3% ± 5.5% vs. control, 86.8% ± 1.8%, P < 0.001) (Figure 2). Furthermore, the cells derived from psoriatic lesions contained a larger fraction of CD29+ cells than the nonlesional cells (29.4% ± 5.5% vs. control, 9.1% ± 1.2%, P < 0.05) (Figure 2). The KCs from lesional psoriatic skin also contained a slightly larger proportion of involucrin+ cells than the corresponding nonlesional cells (71.3% ± 13.0% vs. control, 63.8% ± 13.1%, P < 0.05) (Figure 2).
      Because we observed a notable difference in the fraction of K10 and K10+ cells between psoriatic and nonpsoriatic KCs, we performed further gating on this marker (see Supplementary Figure S2 online). We found that psoriasis-derived K10+ cells displayed a more prominent up-regulation of the stem cell markers than the corresponding cells from nonlesional skin. The psoriatic K10+ population showed a 1.5-fold up-regulation of the expression of CD29, a 3.0-fold up-regulation of CD44, a 2.2-fold up-regulation of CD49f, and a 1.6-fold up-regulation of p63 (see Supplementary Figure S2a and b) compared with the nonlesional cells. The K10 population showed an up-regulation of only CD44 and involucrin (see Supplementary Figure S2a and c). The elevated levels of these markers suggest that, despite being in early differentiation, the psoriatic K10+ cells still retain an immature and proliferative phenotype that is not seen in their nonlesional skin counterparts. A detailed description of the specific marker expression in the K10+/– and CD29+/– populations is presented in Supplementary Figures S2 and S3 online, respectively. A detailed description of specific subpopulations for each marker can be found in Supplementary Table S1 online. The cell distribution obtained by the combined gating of K10 and CD29 is shown in Table 1, showing the altered frequency (% cells) of the populations.
      Table 1K10 and CD29 subpopulations and marker expression within K10 and CD29 subpopulations
      K10 and CD29 Subpopulations
      SubpopulationNonlesional (% cells), mean ± SEMPsoriasis (% cells), mean ± SEMP-Value
      K10+CD2983.0 ± 1.743.2 ± 4.1<0.0001
      K10CD29+5.3 ± 0.821.3 ± 5.00.02
      K10CD297.9 ± 2.027.4 ± 4.60.007
      K10+CD29+3.8 ± 0.98.1 ± 2.3n.s.
      Marker Expression in K10 and CD29 Subpopulations
      SubpopulationNonlesional (MFI), mean ± SEMPsoriasis (MFI), mean ± SEMP-Value
      K10+CD29
       CD446.24 ± 1.915.6 ± 2.3<0.01
       CD49f8.83 ± 1.316.4 ± 2.6n.s.
       CD710.75 ± 0.03.21 ± 1.2n.s.
       p635.13 ± 1.07.20 ± 0.9n.s.
       Involucrin102 ± 23.8104 ± 14.5n.s.
      K10CD29+
       CD442.8 ± 0.313.9 ± 2.4<0.05
       CD49f27.5 ± 3.750.3 ± 9.2n.s.
       CD711.02 ± 0.12.82 ± 0.70.05
       p637.18 ± 1.37.19 ± 1.0n.s.
       Involucrin56.8 ± 15.6110 ± 18.6n.s.
      K10CD29
       CD444.72 ± 2.59.43 ± 1.0n.s.
       CD49f4.75 ± 0.517.6 ± 4.0<0.05
       CD710.734 ± 0.11.90 ± 0.6n.s.
       p633.29 ± 0.75.38 ± 0.7<0.05
       Involucrin59.9 ± 21.479.3 ± 14.7n.s.
      K10+CD29+
       CD446.45 ± 1.524.5 ± 3.5<0.01
       CD49f20.7 ± 1.945.5 ± 10.8n.s.
       CD711.18 ± 0.15.21 ± 1.7n.s.
       p637.24 ± 1.19.79 ± 1.0<0.05
       Involucrin113 ± 35.2156 ± 7.2n.s.
      Abbreviations: MFI, median fluorescence intensity; n.s., not significant; SEM, standard error of the mean.
      CD44, which is an established stem cell marker in mammary epithelia (
      • Al-Hajj M.
      • Wicha M.S.
      • Benito-Hernandez A.
      • Morrison S.J.
      • Clarke M.F.
      Prospective identification of tumorigenic breast cancer cells.
      ,
      • Wright M.H.
      • Calcagno A.M.
      • Salcido C.D.
      • Carlson M.D.
      • Ambudkar S.V.
      • Varticovski L.
      Brca1 breast tumors contain distinct CD44+/CD24- and CD133+ cells with cancer stem cell characteristics.
      ), displayed an overall increase in the psoriasis cell population (Figure 1c). This up-regulation was pronounced even when cells were divided into K10+/– and CD29+/–populations (see Supplementary Figures S2 and S3). The altered expression of CD44 remained pronounced when keratinocytes were dual-gated for K10 and CD29, displaying an increase in the K10+CD29+, K10+CD29, and K10CD29+ populations (Table 1).
      We confirmed the flow cytometric findings using immunofluorescence staining on tissue sections of psoriatic skin and of control skin using antibodies directed against CD44, K10, p63, CD29, and involucrin (see Supplementary Figure S4 online). We also performed co-staining for the combination of K10 and CD44, as well as K10 and p63, to further visualize the localization of the markers (see Supplementary Figure S5 online). We were able to confirm the differential expression of these markers in psoriasis compared with control samples and describe their spatial distribution.
      CD44 was present in all layers of the epidermis. In psoriasis skin, the expression was particularly localized to the basal layers, with a gradually lower expression toward the skin surface, confirming an increased level of CD44 expression in the psoriatic epidermis, as well as a pronounced expression in the K10 cell layers. K10 expression was visible in all layers except the innermost. Psoriasis skin showed a homogenous expression in the outer layer, but more cells at the innermost layer lacked K10 expression. p63 was faintly expressed in the basal layers in the control skin, whereas in psoriasis, it formed a gradient toward the skin surface, with the strongest expression in the basal layer and no expression in the outmost layers. As expected and previously described, CD29 stained the basal layers in both psoriatic and control skin, but it was found in additional cell layers in the psoriatic epidermis, reflecting the increased percentage of CD29+ cells that we observed with flow cytometry. Although CD29 is also found in preadipocyte cells (
      • Festa E.
      • Fretz J.
      • Berry R.
      • Schmidt B.
      • Rodeheffer M.
      • Horowitz M.
      • et al.
      Adipocyte lineage cells contribute to the skin stem cell niche to drive hair cycling.
      ), the expression we observed was primarily localized to the epidermis. Involucrin was found in the outermost layer in psoriasis, as well as scattered through the epidermis, and it had a stronger expression in psoriatic skin than in control skin.

      A psoriatic microenvironment of IL-17, IL-22, and tumor necrosis factor-α increases the expression levels of stem cell markers and alters the phenotypic behavior of KCs

      Because the psoriatic KCs displayed an overall immaturity, we hypothesized that stimuli present in the psoriatic microenvironment would be a contributing cause. Because of the strong T helper type 17 component in psoriasis (
      • Hawkes J.E.
      • Chan T.C.
      • Krueger J.G.
      Psoriasis pathogenesis and the development of novel targeted immune therapies.
      ), and because it has been suggested that IL-22 increases proliferation and delays differentiation (
      • Dudakov J.A.
      • Hanash A.M.
      • van den Brink M.R.
      Interleukin-22: immunobiology and pathology.
      ), we hypothesized that IL-17 and IL-22 would be key mediators in altering the KC phenotype in psoriasis. Indeed, treatment of cultured neonatal human epidermal KCs (HEKn) with a combination of IL-17, IL-22, and tumor necrosis factor (TNF)-α gave rise to an expression pattern similar to that observed in psoriatic KCs, with increases in CD44, p63, and CD29 and a decrease in K10. These changes were observed at both mRNA (Figure 3a) and protein (Figure 3b) levels. This was a synergistic effect, because TNF-α alone did not affect the levels of CD44 and p63 (see Supplementary Figure S6 online).
      Figure thumbnail gr3
      Figure 3Treatment with IL-17, IL-22, and TNF-α produces an in vivo-like phenotype. Cultured neonatal human epidermal KCs (HEKn) were treated with IL-17 (10 ng/ml), IL-22 (20 ng/ml), or a combination of either IL-17 or IL-22 with TNF-α (10 ng/ml) for 48 hours (quantitative PCR) or 72 h (immunocytochemistry). (a) The mRNA expression levels of CD44, p63, CD29, and K10 in HEKn cultured with IL-17, IL-22, IL-22 + TNF-α, IL-17 + TNF-α, or IL-17 + IL-22 + TNF-α. (b) The protein expression levels in HEKn cultured with IL-17 + IL-22 + TNF-α; n = 3–4. P ≤ 0.05. Scale bar = 50 μm. TNF, tumor necrosis factor.
      One characteristic feature of KC stem cells is a long-term proliferative potential (
      • Li A.
      • Simmons P.J.
      • Kaur P.
      Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype.
      ). Treatment of biopsy-derived KCs with IL-22 and IL-17 gave rise to a 3.9 and 6.3 times larger cell numbers, respectively, after 46 days of culture than for untreated cells (Figure 4a). When an equal number of cells were seeded in each passage, the proliferation increased for each passage, suggesting an increased proliferative rate as a function of long-term exposure to IL-17 and IL-22 (Figure 4b). Furthermore, cell sensitivity to the cytokines appeared to increase with time (see Supplementary Figure S7 online). There was no indication of increased differentiation or apoptosis in the untreated cells, which increased exponentially (Figure 4c). These findings suggest that IL-17 and IL-22 increase long-term proliferation in KCs. Like the reduced size observed in the lesion-derived psoriatic KCs, the size of the KCs decreased after treatment with IL-17 and IL-22 (see Supplementary Figure S8 online). We also found a large number of condensed nuclei in the treated cells, which is indicative of active cell proliferation (
      • Bazile F.
      • St-Pierre J.
      • D’Amours D.
      Three-step model for condensin activation during mitotic chromosome condensation.
      ).
      Figure thumbnail gr4
      Figure 4IL-17 and IL-22 promote proliferation and increase colony-forming efficiency in normal KCs. Early-passage KCs from normal skin biopsy samples were seeded and continually passaged upon 80% confluence. Every 3 days, the medium was changed to fresh medium containing IL-17 (5 ng/ml) or IL-22 (10 ng/ml). (a) The total cell yield was assessed after 46 days of culture. (b) The proliferation at each passage. (c) The total yield of cells after 46 days of cell culture in absolute numbers; n = 3–5. P ≤ 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. (d) Cultured HEKn were seeded at colony density and were treated with IL-17 (5 ng/ml) or IL-22 (10 ng/ml) for 10 days and subsequently stained with crystal violet (n = 6). (e) The colony formation of psoriatic KCs obtained from two psoriasis patients (psoriasis 1 and 2) and one nonpsoriasis control (normal). KC, keratinocyte.
      Furthermore, the colony-forming capacity (clonogenicity) is another indicator of stem cell-like characteristics of KCs (
      • Doucet Y.S.
      • Owens D.M.
      Isolation and functional assessment of cutaneous stem cells.
      ,
      • Moestrup K.S.
      • Andersen M.S.
      • Jensen K.B.
      Isolation and in vitro characterization of epidermal stem cells.
      ). The number of colonies is indicative of the growth potential of the cells, and the size of the colonies reflects their stage in terminal differentiation (
      • Barrandon Y.
      • Green H.
      Cell size as a determinant of the clone-forming ability of human keratinocytes.
      ). We found that HEKn treated with IL-17 or IL-22 showed a greater colony-forming capacity than untreated cells (Figure 4d), with an increase in both the number of colonies and the size of the colonies. Based on the flow cytometric analysis of psoriatic KCs, we anticipated that lesion-derived psoriatic KCs seeded at colony density would exhibit a colony-formation pattern comparable to that of undifferentiated KCs. When compared with normal KCs seeded at colony density for 10 days, psoriatic KCs formed a strikingly larger number of colonies, including colonies of larger size (Figure 4e).
      Next, we explored the molecular mechanisms by which IL-17 and IL-22 promote KC stemness. The principal mediators of IL-17 and IL-22 signaling are NF-κB and STAT3, respectively, whereas the MAPK pathway has been implicated in a tissue context-dependent manner (
      • Amatya N.
      • Garg A.V.
      • Gaffen S.L.
      IL-17 signaling: the yin and the yang.
      ,
      • Dudakov J.A.
      • Hanash A.M.
      • van den Brink M.R.
      Interleukin-22: immunobiology and pathology.
      ). We found that the selective MEK1 and MEK2 inhibitor U0126 (
      • Duncia J.V.
      • Santella 3rd, J.B.
      • Higley C.A.
      • Pitts W.J.
      • Wityak J.
      • Frietze W.E.
      • et al.
      MEK inhibitors: the chemistry and biological activity of U0126, its analogs, and cyclization products.
      ) and the NF-κB inhibitor caffeic acid phenethyl ester, prevented the up-regulation of CD44 (Figure 5a) and p63 mRNA (Figure 5b) in response to IL-17 and IL-22. The inhibition of the small GTPase RAC1, a regulator of the ERK pathway and a recently emerging mediator of psoriasis pathogenesis (
      • Winge M.C.
      • Ohyama B.
      • Dey C.N.
      • Boxer L.M.
      • Li W.
      • Ehsani-Chimeh N.
      • et al.
      RAC1 activation drives pathologic interactions between the epidermis and immune cells.
      ), reduced the CD44 and p63 mRNA expression levels (Figure 5a and b). The effects on the protein level were verified by flow cytometry and are presented in Figure 5c. These findings suggest that IL-17 and IL-22 induce CD44 and p63 through the RAC1/MEK/ERK/NF-κB pathway.
      Figure thumbnail gr5
      Figure 5NF-κB and MAPK mediate IL-17– and IL-22–dependent effects on KC stem cell markers. Cultured HEKn cells were pretreated for 1 hour with the inhibitors U0126 (20 μmol/L, MEK), caffeic acid phenethyl ester (20 μmol/L, NF-κB), SB203580 (20 μmol/L, p38 MAPK), Stattic (2 μmol/L, STAT3), or Rac1 inhibitor (50 μmol/L), before treatment with IL-17 (10 ng/ml) or IL-22 (20 ng/ml) in combination with TNF-α (10 ng/ml) for 48 hours. The mRNA expression of (a) CD44 and (b) p63 was related to untreated controls. (c) The reduced protein expression of CD44 after inhibitor treatment was confirmed using flow cytometry; n = 3–4, P < 0.05, ∗∗P < 0.01. KC, keratinocyte; TNF, tumor necrosis factor.
      CD44 has previously been shown to increase RAC1 upon binding to the CD44 ligands hyaluronan and streptococcal polysaccharide (
      • Bourguignon L.Y.
      Matrix hyaluronan-activated CD44 signaling promotes keratinocyte activities and improves abnormal epidermal functions.
      ). Because RAC1 has been implicated in psoriasis pathogenesis, we used small interfering RNA to silence CD44 and analyzed the expression of S100A7, S100A8, S100A9, and β-defensin 2, all of which are psoriasis-associated antimicrobial peptides regulated by IL-17 and IL-22. The basal level of S100A7, S100A8, and S100A9 were strongly suppressed in CD44 small interfering RNA (siRNA)–transfected cells compared with cells transfected with negative control siRNA. In response to stimulation by IL-17/TNF-α or IL-22/TNF-α, we detected a diminished up-regulation of the S100 proteins that reached statistical significance for S100A7 in response to IL-17/TNF-α (see Supplementary Figure S9 online).

      Discussion

      KC stem cells are located in the basal layer and display limited, slow division and the potential to self-renew (
      • Watt F.M.
      • Jensen K.B.
      Epidermal stem cell diversity and quiescence.
      ). The search for a molecular signature for stem cells in the epidermis began in the 1990s, when Jones and Watt showed that CD29 expression on the surface of epidermal KCs correlated with a high proliferative potential in vitro (
      • Jones P.H.
      • Watt F.M.
      Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.
      ). Although progress has been made in defining molecular markers for hair follicle epidermal stem cells residing in the bulge (
      • Blanpain C.
      • Fuchs E.
      Epidermal stem cells of the skin.
      ), stem cell markers for human interfollicular epidermis are less well defined (
      • Ghadially R.
      25 years of epidermal stem cell research.
      ,
      • Kretzschmar K.
      • Watt F.M.
      Markers of epidermal stem cell subpopulations in adult mammalian skin.
      ). Here, we used the putative stem cell markers CD29, CD44, CD49f, and p63 (
      • Ghadially R.
      25 years of epidermal stem cell research.
      ,
      • Moestrup K.S.
      • Andersen M.S.
      • Jensen K.B.
      Isolation and in vitro characterization of epidermal stem cells.
      ,
      • Pincelli C.
      • Marconi A.
      Keratinocyte stem cells: friends and foes.
      ) to characterize the differentiation state of psoriatic KCs in the germinative compartment. We also included the early differentiation, proliferation-associated CD71, and the early differentiation marker K10, as well as the established differentiation marker involucrin, which has been suggested to be aberrantly expressed in psoriasis (
      • Eckert R.L.
      • Yaffe M.B.
      • Crish J.F.
      • Murthy S.
      • Rorke E.A.
      • Welter J.F.
      Involucrin—structure and role in envelope assembly.
      ,
      • Ishida-Yamamoto A.
      • Iizuka H.
      Differences in involucrin immunolabeling within cornified cell envelopes in normal and psoriatic epidermis.
      ). With these markers of stemness and differentiation, we were able to identify higher levels of all the investigated stem cell markers in the psoriasis-derived KC fraction than in the fraction from normal skin. In addition, the psoriasis cell fraction contained a much larger population of K10 cells, as well as a larger proportion of CD29+ cells. The increased number of K10 cells is in line with findings indicating that several rows of suprabasal cells are negative for K10 transcripts or protein (
      • Bernerd F.
      • Magnaldo T.
      • Darmon M.
      Delayed onset of epidermal differentiation in psoriasis.
      ).
      A high KC expression of CD29 correlates with a high proliferative potential in vitro (
      • Jones P.H.
      • Watt F.M.
      Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.
      ). Upon KC commitment to terminal differentiation, CD29 is down-regulated (
      • Levy L.
      • Broad S.
      • Diekmann D.
      • Evans R.D.
      • Watt F.M.
      β1 integrins regulate keratinocyte adhesion and differentiation by distinct mechanisms.
      ). Previous attempts to identify the proliferating population in psoriasis have focused on sorting on the high CD29 expression (
      • Jones P.H.
      • Simons B.D.
      • Watt F.M.
      Sic transit gloria: farewell to the epidermal transit amplifying cell?.
      ), which identified a cell population with increased mRNA levels of CD49f and decreased levels of K10 mRNA (
      • Franssen M.E.
      • Zeeuwen P.L.
      • Vierwinden G.
      • van de Kerkhof P.C.
      • Schalkwijk J.
      • van Erp P.E.
      Phenotypical and functional differences in germinative subpopulations derived from normal and psoriatic epidermis.
      ). Similarly, in 1993, Bata-Csorgo et al. suggested that the proliferative population in the psoriatic epidermis is CD29+, with CD29+K10 cells as the primary hyperproliferative population in the psoriatic skin (
      • Bata-Csorgo Z.
      • Hammerberg C.
      • Voorhees J.J.
      • Cooper K.D.
      Flow cytometric identification of proliferative subpopulations within normal human epidermis and the localization of the primary hyperproliferative population in psoriasis.
      ).
      We observed a larger proportion of both K10CD29+ cells and K10CD29 cells in the psoriasis fraction than in the nonlesional fraction, as described in Table 1. This contrasts with the findings of
      • Bata-Csorgo Z.
      • Hammerberg C.
      • Voorhees J.J.
      • Cooper K.D.
      Flow cytometric identification of proliferative subpopulations within normal human epidermis and the localization of the primary hyperproliferative population in psoriasis.
      , who report the same relative proportion of K1/K10 cells in the normal and psoriatic CD29+ fraction (both 65.4% of CD29+ cells). The reason for this discrepancy is not clear, but it may relate to differences in protocols. The large fraction of K10CD29 cells indicates that many of the K10 cells in psoriasis may not be highly proliferative. This is supported by the elevated expression of involucrin that we observed in the K10 population. Instead, we found that the increase in stem cell markers was pronounced, especially in the K10+ fraction, which clearly suggests that K10+ cells are abnormal in psoriasis.
      The most pronounced increase in the investigated stem cell markers was the up-regulated CD44 expression in psoriasis-derived cells, which was persistent in all subpopulations, regardless of whether they were subdivided on K10 or CD29 expression. CD44 is firmly established as a stem cell marker in mammary epithelial cells (
      • Charafe-Jauffret E.
      • Ginestier C.
      • Birnbaum D.
      Breast cancer stem cells: tools and models to rely on.
      ), which have the same ectodermal origin as epidermal KCs. In breast cancer stem cells, the knockdown of CD44 induces cell differentiation (
      • Pham P.V.
      • Phan N.L.
      • Nguyen N.T.
      • Truong N.H.
      • Duong T.T.
      • Le D.V.
      • et al.
      Differentiation of breast cancer stem cells by knockdown of CD44: promising differentiation therapy.
      ). Furthermore,
      • Szabo A.Z.
      • Fong S.
      • Yue L.
      • Zhang K.
      • Strachan L.R.
      • Scalapino K.
      • et al.
      The CD44+ ALDH+ population of human keratinocytes is enriched for epidermal stem cells with long-term repopulating ability.
      proposed a CD44+ aldehyde dehydrogenase (i.e., ALDH+) epidermal KC population that is enriched in epidermal stem cells, with the CD44+ALDH+ cells displaying an ability for self-renewal, holoclone formation, and multipotency (
      • Szabo A.Z.
      • Fong S.
      • Yue L.
      • Zhang K.
      • Strachan L.R.
      • Scalapino K.
      • et al.
      The CD44+ ALDH+ population of human keratinocytes is enriched for epidermal stem cells with long-term repopulating ability.
      ). The inflammatory environment in the psoriatic plaque makes CD44 of even more interest, because this receptor can act as a co-receptor of inflammatory mediators like endothelial growth factor and can serve as an activator of MMP9 (
      • Misra S.
      • Hascall V.C.
      • Markwald R.R.
      • Ghatak S.
      Interactions between hyaluronan and its receptors (CD44, RHAMM) regulate the activities of inflammation and cancer.
      ).
      We hypothesized that the mechanism for driving the immature, stem cell-like features of psoriasis KCs is derived from the microenvironment in the psoriatic plaque. Both IL-17 and IL-22 are key cytokines in psoriasis. IL-22 contributes to many of the main features of psoriasis, such as acanthosis, loss of the granular layer, and hyperkeratosis, while also promoting KC differentiation, inducing antimicrobial peptide production and delaying differentiation (
      • 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.
      ,
      • Wolk K.
      • Haugen H.S.
      • Xu W.
      • Witte E.
      • Waggie K.
      • Anderson M.
      • et al.
      IL-22 and IL-20 are key mediators of the epidermal alterations in psoriasis while IL-17 and IFN-γ are not.
      ,
      • Wolk K.
      • Witte E.
      • Warszawska K.
      • Schulze-Tanzil G.
      • Witte K.
      • Philipp S.
      • et al.
      The Th17 cytokine IL-22 induces IL-20 production in keratinocytes: a novel immunological cascade with potential relevance in psoriasis.
      ). It has been suggested that IL-17 inhibits proliferation in a three-dimensional model of normal human skin (
      • Donetti E.
      • Cornaghi L.
      • Gualerzi A.
      • Baruffaldi Preis F.W.
      • Prignano F.
      An innovative three-dimensional model of normal human skin to study the proinflammatory psoriatic effects of tumor necrosis factor-alpha and interleukin-17.
      ) but does not affect terminal differentiation (
      • Donetti E.
      • Cornaghi L.
      • Gualerzi A.
      • Baruffaldi Preis F.W.
      • Prignano F.
      An innovative three-dimensional model of normal human skin to study the proinflammatory psoriatic effects of tumor necrosis factor-alpha and interleukin-17.
      ,
      • Wolk K.
      • Haugen H.S.
      • Xu W.
      • Witte E.
      • Waggie K.
      • Anderson M.
      • et al.
      IL-22 and IL-20 are key mediators of the epidermal alterations in psoriasis while IL-17 and IFN-γ are not.
      ). However, in the imiquimod-based mouse model of psoriasis, signaling through the IL-17 pathway reduces K10 levels, promotes KC hyperproliferation, and attenuates KC differentiation (
      • Ha H.L.
      • Wang H.
      • Pisitkun P.
      • Kim J.C.
      • Tassi I.
      • Tang W.
      • et al.
      IL-17 drives psoriatic inflammation via distinct, target cell-specific mechanisms.
      ). Furthermore, IL-17 increases wound healing kinetics in mice (
      • MacLeod A.S.
      • Hemmers S.
      • Garijo O.
      • Chabod M.
      • Mowen K.
      • Witherden D.A.
      • et al.
      Dendritic epidermal T cells regulate skin antimicrobial barrier function.
      ). The combination of IL-22, IL-17, and TNF-α with IL-1α and oncostatin M reduces the expression of the KC differentiation factors, K10, K1, desmoglein-1, desmocollin-1, and loricrin (
      • Rabeony H.
      • Petit-Paris I.
      • Garnier J.
      • Barrault C.
      • Pedretti N.
      • Guilloteau K.
      • et al.
      Inhibition of keratinocyte differentiation by the synergistic effect of IL-17A, IL-22, IL-1α, TNFα and oncostatin M.
      ).
      We found that IL-17 and IL-22, in combination with TNF-α, increased the expression of the investigated stem cell markers in normal KCs to a pattern similar to that observed in the entire psoriasis fraction. The increase in stem cell marker expression was accompanied by cellular characteristics typical of early progenitor cells, with an increased long-term proliferation ability and colony-forming efficiency. Large, abundant colonies were also seen in cultured treated KCs. Previous studies have shown that the cells that give rise to the largest colonies form holoclones, which express CD29 and p63 (
      • Jones P.H.
      • Watt F.M.
      Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.
      ,
      • Pellegrini G.
      • Dellambra E.
      • Golisano O.
      • Martinelli E.
      • Fantozzi I.
      • Bondanza S.
      • et al.
      p63 identifies keratinocyte stem cells.
      ). These cells also express high levels of CD49f and low levels of CD71 and display many stem cell features, including quiescence and long-term growth capacity (
      • Webb A.
      • Kaur P.
      Epidermal stem cells.
      ,
      • Webb A.
      • Li A.
      • Kaur P.
      Location and phenotype of human adult keratinocyte stem cells of the skin.
      ).
      The capacity of IL-17 and IL-22 to promote stemness, in a TNF-α microenvironment, was abolished upon treatment with inhibitors of the RAC1/MEK/ERK/NF-κB pathway. This pathway is of particular interest in psoriasis, because the expression of a constitutively active form of RAC1 in mice induces psoriasiform skin lesions with a transcriptional overlap with human psoriasis (
      • Winge M.C.
      • Ohyama B.
      • Dey C.N.
      • Boxer L.M.
      • Li W.
      • Ehsani-Chimeh N.
      • et al.
      RAC1 activation drives pathologic interactions between the epidermis and immune cells.
      ). RAC1 is also activated in response to the binding of streptococcal polysaccharide to the KC cell surface CD44 (
      • Cywes C.
      • Wessels M.R.
      Group A Streptococcus tissue invasion by CD44-mediated cell signalling.
      ), as well as the binding to its ligand hyaluronan (
      • Bourguignon L.Y.
      Matrix hyaluronan-activated CD44 signaling promotes keratinocyte activities and improves abnormal epidermal functions.
      ). We showed that CD44 mediates the expression of the IL-17/IL-22 downstream targets S100A7, S100A8, and S100A9, which is in line with these previous reports and suggests a role for CD44 in the regulation of these key psoriasis mediators. This suggests that RAC1 is involved both in CD44 regulation and mediating downstream effects of IL-17 and IL-22.
      In conclusion, we describe an overall more immature phenotype of psoriatic KCs. This immaturity is likely caused by the actions of the psoriatic key cytokines, IL-17 and IL-22, because these cytokines gave rise to a similar phenotype, induced proliferation, promoted a proliferation-like morphology, and enhanced the colony-forming capacity of normal KCs. The data suggest that IL-17 and IL-22 in the psoriatic microenvironment may act on KCs to promote proliferation and keep the KCs in an immature state.

      Materials and Methods

      Isolation of human epidermal KCs

      Skin punch biopsy samples (4 mm) were obtained from psoriasis patients at the local dermatology clinic. All the participating patients had been examined by a dermatologist. The patients were not receiving any systemic treatment. The psoriasis skin punch biopsy samples were obtained from an active, untreated, psoriatic lesion, and control skin was obtained from a nonlesional area from the same patient to minimize confounding variation. All the participants had given their written informed consent, and the study was approved by the Institutional Review Board of Linkoping University. The lesional and the nonlesional biopsy samples from each patient were simultaneously obtained, processed, stained, and analyzed, rendering a completely matched comparison. KC isolation was performed by placing the punch biopsy sample in 3.8% sterile ammonium thiocyanate (flow cytometry) for 30 minutes at room temperature or in dispase over night at 4 °C (culture). After incubation, the epidermis was removed and placed in trypsin, with mechanical disruption for 30 minutes at 37 °C.

      Staining and flow cytometry

      The KC suspension was filtered through a 70-μm mesh and fixed and permeabilized with the transcription factor buffer set (BD Biosciences, San Jose, CA). Fixation took place overnight at 4–8 °C, and antibody staining was performed on ice for 50 minutes. To avoid excessive spectral overlap and minimize the risk of compensation artifacts, cells were stained with either of two separate panels (see Supplementary Table S2 online). The following antibodies were used for analysis with flow cytometry: anti-CD29-brilliant violet 510, anti-CD44-PE.Cy7, anti-CD45-FITC, anti-CD49f-PerCP.Cy5.5, and anti-CD71- brilliant violet 421 (BD Biosciences) and anti-K10-APC and anti-involucrin-PE (Bio-Techne, Abingdon, UK). Anti-p63 (Bio-Techne) was conjugated using the Lynx rapid RPE conjugation kit (AbD Serotec, Kidlington, UK). Analysis was performed on a Gallios Flow Cytometer (Beckman Coulter, Bromma, Sweden). Between 2,000 and 10,000 events were collected. Data were analyzed using Kaluza Analysis Software (Beckman Coulter).

      Cells for culture and culture conditions

      The culture conditions for the KCs are described in the Supplementary Materials online. Treatment with IL-17 (10 ng/ml) or IL-22 (20 ng/ml) alone, or in combination with TNF-α (10 ng/ml), was performed for 48 hours (quantitative PCR) or 72 hours (immunocytochemistry). For inhibitor experiments, the cells were pretreated for 1 hour with caffeic acid phenethyl ester (20 μmol/L), Stattic (2 μmol/L), SB203580 (20 μmol/L), and U0126 (20 μmol/L) (all from Abcam, Cambridge, UK) or the RAC1 inhibitor (50 μmol/L) (Calbiochem, San Diego, CA) before the addition of cytokines or vehicle DMSO. To down-regulate CD44 expression, HEKn were transfected with 50 nmol/L CD44 siRNA (Qiagen, Hilden, Germany) 8 hours before the addition of IL-17 and IL-22 in combination with TNF-α for 48 hours. AllStars Negative control siRNA (Qiagen) served as control. Transfection was performed using FlexiTube premix siRNA (Qiagen).

      RNA extraction, cDNA synthesis, and quantitative PCR

      Cells were lysed in RLT buffer (Qiagen), and RNA was extracted using the RNeasy mini kit (Qiagen). cDNA was synthesized using the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Fermentas, Vilnius, Lithuania). Quantitative PCR was performed using SYBR green for the expression analyses of CD44, p63, CD29, and K10 and using predesigned TaqMan gene expression assays for the analyses of S100A7, S100A8, and S100A9 (Applied Biosystems, Foster City, CA), on a real-time 7500 HT system (Applied Biosystems). The primer sequences are listed in Supplementary Table S3 online. Expression data were normalized to RPLP0 using the comparative Ct (2–ΔΔCt) method.

      Immunofluorescence

      HEKn and skin punch biopsy samples from healthy control and lesional psoriasis skin were fixed, paraffin embedded, and stained for CD44, p63, K10, CD29 and involucrin with a standard protocol. A detailed description of the staining process can be found in the Supplementary Materials. Alexa Fluor 488 and 555 (both from Molecular Probes, Eugene, OR) were used as secondary antibodies.
      Negative controls were obtained by omitting the primary antibody and displayed no staining. Fluorescence intensity was analyzed with Image J software, version 1.51j (National Institutes of Health, Bethesda, MD).

      Colony-forming efficiency assay and long-term proliferation

      Colony-forming efficiency and proliferation assays were performed on KCs isolated from skin biopsy samples and HEKn. We cultured the cells without a feeder layer, as described by
      • Doucet Y.S.
      • Owens D.M.
      Isolation and functional assessment of cutaneous stem cells.
      . Briefly, second-passage KCs were seeded and treated with IL-17 (5 ng/ml) or IL-22 (10 ng/mL). After 10 days, colonies were fixed and stained with 0.05% crystal violet. The cells in the proliferation assay were cultured for 46 days and counted upon passaging. A detailed description of the culture process can be found in the Supplementary Materials.

      Statistics

      Differences between groups were analyzed using Student t test or the Mann-Whitney U test. A P-value of less than or equal to 0.05 was considered significant. The data are presented as the mean ± standard error of the mean. Statistical comparisons were performed in GraphPad Prism, version 6.0 (GraphPad Software, San Diego, CA).

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      This research was funded by the Ingrid Asp Foundation, the Welander Foundation, the Swedish Psoriasis Association, and the Medical Research Council.

      Supplementary Material

      Cells for culture and culture conditions

      KCs isolated from skin biopsy samples were cultured in KC serum-free medium with l-glutamine supplemented with 25 μg/ml bovine pituitary extract and 1 ng/ml epidermal growth factor (Gibco, Gaithersburg, MD). HEKn (Cascade Biologics, Life Technologies, Carlsbad, CA) were cultured in complete EpiLife medium supplemented with 1% EpiLife defined growth supplement, CaCl2 (0.06 μmol/L) (all from Cascade Biologics, Paisley, UK). All media contained 1% amphotericin B (Gibco) and 1% penicillin/streptomycin (Lonza, Verviers, Belgium).

      Immunofluorescence

      HEKn were fixed in 4% formaldehyde and permeabilized with 0.1% saponin, followed by incubation with primary antibody overnight at 4 °C and subsequent incubation with secondary Alexa Fluor 488 conjugated antibody (Molecular Probes, Eugene, OR).
      Skin punch biopsy samples were obtained from healthy control skin and lesional psoriasis skin and were fixed and paraffin embedded. The sections were deparaffinized in Histolab-clear (Histolab Products, Gothenburg, Sweden) and rehydrated in ethanol. Heat-induced antigen retrieval was performed in citrate (pH = 6) antigen retrieval buffer (DAKO, Glostrup, Denmark); 5% bovine serum albumin was used as blocking agent. Incubation with primary antibody was carried out overnight at 4 °C followed by 1-hour incubation with Alexa Fluor 555 conjugated secondary antibody. DAPI nuclear counterstain was performed.
      The following primary antibodies were used: CD44 (BD Biosciences), p63 (Abcam), K10 (Epitomics, Burlingame, CA), CD29 (Novus Biologicals, Littleton, CO), and involucrin (Novus Biologicals) and secondary Alexa Fluor 488 and Alexa Fluor 555 conjugated antibodies (Molecular Probes, Eugene, OR).

      Colony-forming efficiency assay and long-term proliferation

      Second-passage KCs were seeded at 500 KCs/well in 12-well plates for the colony formation assay and 10,000 KCs/well in 6-well plates for the proliferation assay. The day after seeding, the cells were treated with IL-17 (5 ng/ml) or IL-22 (10 ng/ml). Every 3 days, the medium was changed, and fresh cytokines were added. After 10 days, the colonies were fixed in 4% formaldehyde and stained with 0.05% crystal violet. The cells in the proliferation assay were continually passaged when 80% confluency was reached. At each passage the number of cells in each well was determined, and the cells were replated at 10,000 cells per well, irrespective of cell yield. The cumulative number of cells derived since the first plating of 10,000 cells was also determined at each passage. The total cell yield after 46 days of culture was calculated under the assumption that all the cells from the previous passages had been replated.
      Figure thumbnail fx1
      Supplementary Figure S1Autofluorescence of unstained KCs. Histograms demonstrating the autofluorescence of unstained KCs in each fluorescence channel. The x-axis demonstrates the fluorescence, the y-axis demonstrates the count.
      Figure thumbnail fx2
      Supplementary Figure S2A detailed description of the K10+/– population. (a) Dot plots with the gating strategy to determine the positive and negative population. (b) The median fluorescence intensity of each respective marker and the percentage of cells positive for each marker in the K10+ population. (c) Displays the median fluorescence intensity (MFI) of each respective marker and the percentage of cells positive for each marker in the K10 population; n = 4–7. P ≤ 0.05, ∗∗P < 0.01.
      Figure thumbnail fx3
      Supplementary Figure S3A detailed description of the CD29+/– population. (a) Dot plots with the gating strategy to determine the positive and negative population. (b) Median fluorescence intensity (MFI) of each respective marker and the percentage of cells positive for each marker in the CD29+ population. (c) The median fluorescence intensity of each respective marker and the percentage cells positive for each marker in the CD29 population; n = 4–7. P ≤ 0.05.
      Figure thumbnail fx4
      Supplementary Figure S4Immunofluorescence describing the localization of CD44, p63, K10, CD29, and involucrin in psoriasis and control skin. Markers are stained red; bottom pictures include DAPI counterstaining of cell nuclei (blue). Photos are representative of three independent experiments. Scale bars = 50 μm.
      Figure thumbnail fx5
      Supplementary Figure S5Immunofluorescence describing the co-localization of CD44 and p63 with K10. Double immunofluorescence staining of (a) CD44 (green staining) and K10 (red staining) or (b) p63 (green staining) and K10 (red staining) of control and psoriasis skin. The rightmost column provides the merged stainings. Photos are representative of three experiments.
      Figure thumbnail fx6
      Supplementary Figure S6mRNA expression analysis of CD44 and p63 following TNF-α treatment. mRNA expression analysis of CD44 and p63 in HEKn treated with (gray) or without (white) tumor necrosis factor-α (10 ng/ml). n = 3. ns, non-significant.
      Figure thumbnail fx7
      Supplementary Figure S7Comparison of the sensitivity of normal KCs to cytokine treatment after passage 1 and passage 5. For details on experimental procedure, see . ∗∗P ≤ 0.01.
      Figure thumbnail fx8
      Supplementary Figure S8Representative images showing cell and nuclear size of cultured neonatal human epidermal KCs (HEKn) after treatment with IL-17 (5 ng/ml) or IL-22 (10 ng/ml). Arrows indicate examples of cells with a reduced size following IL-22 or IL-17 treatment. Scale bar = 500 μm.
      Figure thumbnail fx9
      Supplementary Figure S9The mRNA expression of S100 proteins following siRNA-mediated silencing of CD44. Quantitative PCR analyses of the mRNA expression of CD44, S100A7, S100A8, and S100A9 in CD44 siRNA-transfected HEKn treated with IL-22 + TNF-α or IL-17 + TNF-α. Gene expression was compared with untreated negative control siRNA-transfected cells; n = 4, P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001, ∗∗∗∗P ≤ 0.0001. C, control; Neg, negative; siRNA, small interfering RNA; TNF, tumor necrosis factor.
      Supplementary Table S1Marker expression in keratinocyte subpopulations
      Subpopulation% Positive CellsP-ValueMFIP-Value
      NonlesionalPsoriasisNonlesionalPsoriasis
      p63+ cells93.3 ± 1.996.3 ± 2.2<0.055.2 ± 0.96.8 ± 0.9<0.05
       K10 expression in p63+ cells90.2 ± 1.949.8 ± 6.8<0.01371.3 ± 179.080.6 ± 50.0n.s.
       CD29 expression in p63+ cells6.3 ± 1.626 ± 9.3n.s.1.4 ± 0.12.8 ± 0.4<0.05
       CD49f expression in p63+ cells93.4 ± 1.697.7 ± 0.7n.s.9.3 ± 1.322.7 ± 3.3<0.05
       CD71 expression in p63+ cells13.2 ± 4.669.7 ± 9.6<0.0010.8 ± 0.02.6 ± 0.8n.s.
      CD49f+ cells91.5 ± 1.996.3 ± 1.39n.s.9.0 ± 1.322.1 ± 3.5<0.05
       K10 expression in CD49f+ cells90.0 ± 1.549.4 ± 6.5<0.001366.3 ± 179.072.5 ± 43.6n.s.
       CD29 expression in CD49f+ cells6.6 ± 1.726.4 ± 9.5n.s.1.4 ± 0.12.87 ± 0.4<0.05
       CD71 expression in CD49f+ cells13.6 ± 4.870.0 ± 10.0<0.0010.8 ± 0.02.8 ± 0.8<0.05
       p63 expression in CD49f+ cells95.3 ± 1.997.7 ± 1.6<0.055.3 ± 1.06.9 ± 0.9<0.05
      CD71+ cells13.0 ± 4.668.0 ± 10.0<0.010.8 ± 0.02.6 ± 0.8n.s.
       K10 expression in CD71+ cells71.6 ± 8.052.6 ± 6.7n.s.330.0 ± 18389.2 ± 52.5n.s.
       CD29 expression in CD71+ cells14.2 ± 5.428.5 ± 9.8n.s.2.5 ± 0.93.1 ± 0.4n.s.
       CD49f expression in CD71+ cells96.3 ± 1.299.2 ± 0.3n.s.20.6 ± 6.026.3 ± 3.4n.s.
       p63 expression in CD71+ cells95.4 ± 1.899.2 ± 0.4n.s.8.4 ± 2.27.7 ± 1.2n.s.
      Abbreviations: MFI, median fluorescent intensity; n.s., not significant.
      Supplementary Table S2Antibody panels
      Panel 1Panel 2
      CD45-FITCCD45-FITC
      K10-APCK10-APC
      CD29-BV510CD29-BV510
      involucrin-PEp63-PE
      CD44-PE.Cy7CD49f-PerCP.Cy5.5
      CD71-BV421
      Supplementary Table S3Primer sequences
      GenePrimer Sequence
      CD29
       Forward5′-CCTACTTCTGCACGATGTGATG-3′
       Reverse5′-CCTTTGCTACGGTTGGTTACATT-3′
      CD44
       Forward5′-CTGCCGCTTTGCAGGTGTA-3′
       Reverse5′-CATTGTGGGCAAGGTGCTATT-3′
      K10
       Forward5′-GGGCTAAACAGCATCACCATGT-3′
       Reverse5′-GCGGGAGGAAGAGTAGTGCTT-3′
      p63
       Forward5′-TCTCTTTCCCACCCCGAGAT-3′
       Reverse5′-CGGCGAGCATCCATGTC-3′
      RPLP0
       Forward5′-ACTGTGCCAGCCCAGAACA-3′
       Reverse5′-AGCCTGGAAAAAGGAGGTCTTC-3′

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