S100A8 and S100A9 are messengers in the crosstalk between epidermis and dermis modulating a psoriatic milieu in human skin

S100A8 and S100A9 are members of the S100A8 protein family that exist as homodimers and heterodimers in neutrophils, monocytes, and macrophages. Recent studies have shown the pivotal roles of S100A8 and S100A9 in the propagation of inflammation and keratinocyte proliferation in psoriasis. We found significant up-regulation of S100A8 and S100A9 secretion from keratinocytes in psoriatic lesions. To mimic the in vivo secretory conditions of S100A8 and S100A9 from psoriatic epidermal keratinocytes, we used the culture medium (CM) of S100A8 and S100A8/A9 adenovirus-transduced keratinocytes to investigate the functions of S100A8 and S100A9. We detected increased levels of various pro-inflammatory cytokines in the CM, including IL-8 and TNF-α, which are involved in aggravating psoriatic skin lesions, and IL-6 and members of the CXCL family of pro-angiogenic cytokines. The CM increased immune cell migration and increased angiogenesis in human umbilical vein endothelial cells. In conclusion, we found that the upregulated production of S100A8 and S100A9 by psoriatic epidermal keratinocytes activated adjacent keratinocytes to produce several cytokines. Moreover, S100A8 and S100A9 themselves function as pro-angiogenic and chemotactic factors, generating a psoriatic milieu in skin.     

IL-22 is required for imiquimod-induced psoriasiform skin inflammation in mice

Psoriasis is a common chronic autoimmune skin disease of unknown cause that involves dysregulated interplay between immune cells and keratinocytes. IL-22 is a cytokine produced by the TH1, TH17, and TH22 subsets that are functionally implicated in the psoriatic pathology. We assessed the role of IL-22 in a mouse model where psoriasiform skin inflammation is triggered by topical application of the TLR7/8 agonist imiquimod. At the macroscopic level, scaly skin lesions induced by daily applications of imiquimod in wild-type mice were almost totally absent in IL-22-deficient mice or in mice treated with a blocking anti-IL-22 Ab. At the microscopic level, IL-22-deficient mice showed a dramatic decrease in the development of pustules and a partial decrease in acanthosis. At the molecular level, the absence or inhibition of IL-22 strongly decreased the expression of chemotactic factors such as CCL3 and CXCL3 and of biomarkers such as S100A8, S100A7, and keratin 14, which reflect the antimicrobial and hyperproliferative responses of keratinocytes. IL-22 also played a major role in neutrophil infiltration after imiquimod treatment. IL-23 was required for IL-22 production, and γδ TCR lymphocytes represented the major source of IL-22 in lymph nodes from imiquimod-treated mice. However, T cells were not absolutely required for IL-22 production because imiquimod-induced IL-22 expression in the skin is still preserved in Rag2(-/-) mice. Taken together, our data show that IL-22 is required for psoriasis-like lesions in the mouse imiquimod model and is produced by both T cells and innate immune cells.     

Induction of beta-defensin 3 in keratinocytes stimulated by bacterial lipopeptides through toll-like receptor 2

The epidermis, which covers the surface of all mammals, serves as a front line of defense against the invasion of pathogenic microbes and acts as a crucial site for innate immune responses. Various antimicrobial molecules are expressed not only on the surfaces of monocytes but also on epithelial cells. beta-Defensins, a family of antimicrobial peptides, are produced by several types of epithelial cells, including keratinocytes. However, the induction pathways for beta-defensins in keratinocytes are not fully understood. We hypothesized that bacterial components would trigger the expression of beta-defensins in keratinocytes through a toll-like receptor (TLR)-MyD88 signaling pathway that plays important roles in innate immunity. Production of TNF-alpha and IL-1 alpha following stimulation with lipopolysaccharide or bacterial lipopeptides was completely abolished in TLR2&TLR4-doubly deficient keratinocytes and in MyD88-deficient keratinocytes. Expression of murine beta-defensin was upregulated by bacterial lipopeptides in wild-type keratinocytes, while it was attenuated in TLR2-deficient keratinocytes. To evaluate the in vivo role of TLRs in keratinocytes, we inoculated Staphylococcus aureus into the tail skin from TLR2-deficient mice that had been grafted on the dorsal skin of syngeneic mice. The grafted skin from TLR2-deficient mice resulted in erosion. These studies strongly suggest that the TLR2-MyD88-dependent pathway in keratinocytes is essential for antimicrobial activity in vivo.     

S100A7, Jab1, and p27kip1 expression in psoriasis and S100A7 CRISPR-activated human keratinocyte cell line

Psoriasis, a chronic immune-mediated inflammatory skin disease, is characterized by dysregulated keratinocyte proliferation. The EF-hand calcium binding protein S100A7 has been found to be overexpressed in psoriatic keratinocytes. It is know that S100A7 may interact with Jab1, a cofactor that stabilizes c-Jun. Jab1 is known to downregulate the expression of the cell cycle inhibitor p27^Kip1 in some cancer models. In this study, we aimed to investigate the possible interaction between S100A7 and Jab1 and the downstream effects on p27 ^Kip1 expression in normal human keratinocyte cells transfected with S100A7 CRISPR activation plasmid and in archival psoriatic skin samples. Our results showed that the upregulated S100A7 colocalizes with Jab1 at the nuclear level in transfected cells and psoriatic skin samples. We also showed a differential protein expression of Jab1 between cytoplasmic and nuclear compartments, thus suggesting Jab1 translocation from nucleus to cytoplasm. p27 ^Kip1 protein expression patterns would imply a translocation from nucleus and a subsequent degradation of this protein. The upregulation of S1007 and its interaction with Jab1 would contribute to the p27 ^Kip1-dependent impaired proliferation that characterizes psoriatic skin.     

Interleukin-1 regulates keratinocyte expression of T cell targeting chemokines through interleukin-1 receptor associated kinase-1 (IRAK1) dependent and independent pathways

IL-1 is a potent pro-inflammatory cytokine that activates intracellular signaling cascades some of which may involve IL-1 receptor associated kinase-1 (IRAK1). Psoriasis is a T cell dependent chronic inflammatory condition of the skin of unknown cause. IL-1 has been implicated in psoriasis pathology, but the mechanism has not been elucidated. Interestingly, expression of IRAK1 is elevated in psoriatic skin. To identify a potential link between IL-1, keratinocytes and T cells in skin inflammation we employed pathway-focused microarrays to evaluate IL-1 dependent gene expression in keratinocytes. Several candidate mRNAs encoding known T cell chemoattractants were identified in primary keratinocytes and the stable keratinocyte cell line HaCaT. CCL5 and CCL20 mRNA and protein levels were confirmed up-regulated by IL-1 in concentration and time-dependent manners. Furthermore IL-1 synergized with IFN-γ and TNF-α. Expression of CXCL9, CXCL10 and CXCL11 mRNAs was also increased in response to IL-1, but protein could only be detected in medium from cells treated with IFN-γ alone or in combination with IL-1. Over-expression of IRAK1 led to increased constitutive and cytokine induced production of CCL5 and CCL20. Inhibition of IRAK1 activity through RNAi or expression of a dominant negative mutant blocked production of CCL5 and CCL20 but had no effect upon the IL-1 enhancement of IFN-γ induced CXCL9, CXCL10 and CXCL11 production. In conclusion IL-1 regulates T cell targeting chemokine production in keratinocytes through IRAK1 dependent and independent pathways. These pathways may contribute to acute and chronic skin inflammation.     

Interferon Regulatory Factor 6 Has a Protective Role in the Host Response to Endotoxic Shock

Interferon Regulatory Factor (IRF) 6, a member of the IRF family, is essential for epidermal and orofacial embryonic development. Irf6 is strongly expressed in keratinocytes, in which it regulates epidermal proliferation, differentiation, and migration. A recent role for Irf6 in Toll-like receptor 2-dependent chemokine gene expression was also reported in an epithelial cell line. However, a function for Irf6 in innate immune cells was not previously reported. In the present study, we investigated the expression and function of Irf6 in bone marrow-derived neutrophils and macrophages. We show here, using a conditional knockout of Irf6 in lysosymeM expressing cells, that Irf6 is required for resistance to LPS-induced endotoxic shock. In addition, Irf6-deficient bone marrow-derived neutrophils exhibited increased chemotactic index and velocity compared with wild-type cells in vitro. TLR4-specific KC and IL6 secretions were upregulated in Irf6-deficient bone marrow-derived macrophages in vitro. These cells also exhibited an increased level of phosphorylated IkBa. Collectively, our findings suggest a role for Irf6 in the resistance to endotoxic shock due to NFk-B-mediated alteration of cytokine production.     

S100A8/A9, a key mediator for positive feedback growth stimulation of normal human keratinocytes

S100A8 and S100A9 are known to be up-regulated in hyperproliferative and psoriatic epidermis, but their function in epidermal keratinocytes remains largely unknown. Here we show that (1) S100A8 and S100A9 are secreted by cultured normal human keratinocytes (NHK) in a cytokine-dependent manner, (2) when applied to NHK, recombinant S100A8/A9 (a 1:1 mixture of S100A8 and S100A9) induced expression of a number of cytokine genes such as IL-8/CXCL8, CXCL1, CXCL2, CXCL3, CCL20, IL-6, and TNFalpha that are known to be up-regulated in psoriatic epidermis, (3) the S100A8/A9-induced cytokines in turn enhanced production and secretion of S100A8 and S100A9 by NHK, and (4) S100A8 and S100A8/A9 stimulated the growth of NHK at a concentration as low as 1 ng/ml. These results indicate the presence of a positive feedback loop for growth stimulation involving S100A8/A9 and cytokines in human epidermal keratinocytes, implicating the relevance of the positive feedback loop to the etiology of hyperproliferative skin diseases, including psoriasis.     

Reduced TRPC channel expression in psoriatic keratinocytes is associated with impaired differentiation and enhanced proliferation

Psoriasis is a characteristic inflammatory and scaly skin condition with typical histopathological features including increased proliferation and hampered differentiation of keratinocytes. The activation of innate and adaptive inflammatory cellular immune responses is considered to be the main trigger factor of the epidermal changes in psoriatic skin. However, the molecular players that are involved in enhanced proliferation and impaired differentiation of psoriatic keratinocytes are only partly understood. One important factor that regulates differentiation on the cellular level is Ca(2+). In normal epidermis, a Ca(2+) gradient exists that is disturbed in psoriatic plaques, favoring impaired keratinocyte proliferation. Several TRPC channels such as TRPC1, TRPC4, or TRPC6 are key proteins in the regulation of high [Ca(2+)](ex) induced differentiation. Here, we investigated if TRPC channel function is impaired in psoriasis using calcium imaging, RT-PCR, western blot analysis and immunohistochemical staining of skin biopsies. We demonstrated substantial defects in Ca(2+) influx in psoriatic keratinocytes in response to high extracellular Ca(2+) levels, associated with a downregulation of all TRPC channels investigated, including TRPC6 channels. As TRPC6 channel activation can partially overcome this Ca(2+) entry defect, specific TRPC channel activators may be potential new drug candidates for the topical treatment of psoriasis.     

Hyperproliferation of normally quiescent keratinocytes in non-lesional psoriatic skin due to high calcium concentration (an organotypic culture model)

Calcium plays an important role in the regulation of different functions of keratinocytes. In the present work we studied the effect of different extracellular calcium concentrations (0.01 mM-2.0 mM) on the proliferation and differentiation of human keratinocytes in normal human and non-lesional psoriatic skin. Using explant culture model, the proliferative and differentiated subsets of keratinocytes were detected by specific antibodies related to cell proliferation [beta-1 integrin (CD29), proliferating cell antigen (Ki67), proliferating cell nuclear antigen (PCNA)] and differentiation [differentiated cell cytokeratins (K1/K10) and differentiating cell antigen (lectin Ulex europaius agglutinin, UEA-1)]. After 4 days of culturing at high Ca2+ (2.0 mM) we observed marked hyperproliferation among the normally quiescent keratinocytes of non-lesional psoriatic skin. In normal uncultured and cultured skin and in uncultured and two-day-cultured non-lesional psoriatic skin both at normal (1.2 mM) and at high (2.0 mM) Ca2+ concentration only one layer of basal CD29+/Ki67+/K1/K10-/UEA-1- cell was observed. In sections from non-lesional psoriatic skin cultured for 4 days in the presence of high Ca2+ (2.0 mM) this cell population has expanded from at least three layers above the basement membrane. This expanded cell population of the 4-day high Ca2+ cultured non-lesional skin showed clear PCNA positive staining on frozen sections with the strongest positivity among the most basal localized cells. These data suggest that (i) extracellular Ca2+ concentration can influence the proliferation of basal ("stem") keratinocytes, (ii) the proliferative response to high Ca2+ concentration of psoriatic non-lesional basal keratinocytes differs from that of normal basal keratinocytes, (iv) changes in the extracellular Ca2+ milieu might play a role in the induction of the hyperproliferative psoriatic lesion.     

In vitro and in situ expression of IL-23 by keratinocytes in healthy skin and psoriasis lesions: enhanced expression in psoriatic skin

Keratinocytes contribute to cutaneous immune responses through the expression of cytokines. We investigated whether human keratinocytes can express IL-23, a newly defined IFN-gamma-inducing cytokine composed of a unique p19 subunit and a p40 subunit shared with IL-12. Cultured keratinocytes from normal and lesional psoriatic skin were found to express constitutively mRNA for both subunits of IL-23. Low but significant levels of the heterodimeric IL-23 protein could be detected in cell lysates and supernatants from stimulated keratinocytes by immunoblotting and ELISA. Functional analysis showed that these low levels of keratinocyte-derived IL-23 were sufficient to enhance the IFN-gamma production by memory T cells. Immunostaining of skin sections confirmed expression of both subunits of IL-23 by keratinocytes in situ and also revealed expression of this cytokine in the dermal compartment. IL-23 expression was significantly higher in psoriatic lesional skin, compared with normal and psoriatic nonlesional skin. The immunostained preparations of cultured cells and IL-23 levels in culture supernatants did not show any difference between normal and psoriatic keratinocytes indicating no intrinsic aberration of IL-23 expression in keratinocytes from psoriatic skin. Double staining of cytospin preparations demonstrated that IL-23 p19 is also expressed by epidermal Langerhans cells, dermal dendritic cells, and macrophages. Psoriasis is a chronic inflammatory skin disease mediated by IFN-gamma-expressing type 1 memory T cells. As IL-23 is important to activate memory T cells to produce IFN-gamma, its augmented expression of IL-23 by keratinocytes and cutaneous APC may contribute to the perpetuation of the inflammation process in this disease.     

MiRNA expression in psoriatic skin: reciprocal regulation of hsa-miR-99a and IGF-1R

Background

Psoriasis is a complex disease at the cellular, genomic and genetic levels. The role of microRNAs in skin development was shown in a keratinocyte-specific Dicer knockout mouse model. Considering that two main characteristics of psoriasis are keratinocytes hyperproliferation and abnormal skin differentiation, we hypothesized that aberrant microRNA expression contributes to the psoriatic phenotype. Here, we describe the differential expression of miRNAs in psoriatic involved and uninvolved skin as compared to normal skin, revealing an additional aspect of this complex disorder.

Methodology/Principal Findings

Expression arrays were used to compare microRNA expression in normal skin versus psoriatic involved and uninvolved skin. Fourteen differentially expressed microRNAs were identified, including hsa-miR-99a, hsa-miR-150, hsa-miR-423 and hsa-miR-197. The expression of these microRNAs was reevaluated by qPCR. IGF-1R, which is involved in skin development and the pathogenesis of psoriasis, is a predicted target of hsa-miR-99a. In an in situ hybridization assay, we found that IGF-1R and miR-99a are reciprocally expressed in the epidermis. Using a reporter assay, we found that IGF-1R is targeted by hsa-miR-99a. Moreover, over expression of miR-99a in primary keratinocytes down-regulates the expression of the endogenous IGF-1R protein. Over expression of miR-99a also inhibits keratinocyte proliferation and increases Keratin 10 expression. These findings suggest that overexpression of hsa-miR-99a in keratinocytes drives them towards differentiation. In primary keratinocytes grown in high Ca⁺⁺, miR-99a expression increases over time. Finally, we found that IGF1 increases the expression of miR-99a.

Conclusions/Significance

We identified several microRNAs that are expressed differentially in normal and psoriatic skin. One of these miRNAs is miR-99a that regulates the expression of IGF-1R. Moreover, miR-99a seems to play a role in the differentiation of keratinocytes. We suggest that miR-99a is one of the regulators of the IGF-1R signaling pathway in keratinocytes. Activation of IGF1 signaling results in elevation of miR-99a which represses the expression of IGF-1R.     

Bi-compartmental communication contributes to the opposite proliferative behavior of Notch1-deficient hair follicle and epidermal keratinocytes

Notch1-deficient epidermal keratinocytes become progressively hyperplastic and eventually produce tumors. By contrast, Notch1-deficient hair matrix keratinocytes have lower mitotic rates, resulting in smaller follicles with fewer cells. In addition, the ratio of melanocytes to keratinocytes is greatly reduced in hair follicles. Investigation into the underlying mechanism for these phenotypes revealed significant changes in the Kit, Tgfbeta and insulin-like growth factor (IGF) signaling pathways, which have not been previously shown to be downstream of Notch signaling. The level of Kitl (Scf) mRNA produced by Notch1-deficient follicular keratinocytes was reduced when compared with wild type, resulting in a decline in melanocyte population. Tgfbeta ligands were elevated in Notch1-deficient keratinocytes, which correlated with elevated expression of several targets, including the diffusible IGF antagonist Igfbp3 in the dermal papilla. Diffusible stromal targets remained elevated in the absence of epithelial Tgfbeta receptors, consistent with paracrine Tgfbeta signaling. Overexpression of Igf1 in the keratinocyte reversed the phenotype, as expected if Notch1 loss altered the IGF/insulin-like growth factor binding protein (IGFBP) balance. Conversely, epidermal keratinocytes contained less stromal Igfbp4 and might thus be primed to experience an increase in IGF signaling as animals age. These results suggest that Notch1 participates in a bi-compartmental signaling network that controls homeostasis, follicular proliferation rates and melanocyte population within the skin.     

Transforming growth factor-beta 1 localization in normal and psoriatic epidermal keratinocytes in situ.

Transforming growth factor-beta 1 (TGF beta 1) is a potent inhibitor of epithelial cell proliferation and its effects on growth and differentiation have been extensively characterized in cultured keratinocytes. We used two TGF beta 1-specific polyclonal antibodies (anti-LC and anti-CC) to determine the presence of TGF beta 1 peptide in keratinocytes in sections of normal human skin in situ and in both plaque and nonplaque skin from individuals with psoriasis. In contrast to the differentiation phenotype expressed by keratinocytes in normal epidermis, keratinocytes in the psoriatic plaque exhibit a hyperproliferative/regenerative differentiation phenotype. Anti-TGF beta 1 staining was observed primarily in the epidermis. Anti-LC TGF beta 1 antibody stained nonproliferating, differentiated suprabasal keratinocytes intracellularly in normal skin but did not stain psoriatic plaques from five of seven patients. In contrast, anti-CC TGF beta 1 antibody stained suprabasal keratinocytes extracellularly in psoriatic plaques, but did not stain normal skin. Both anti-LC and anti-CC stained suprabasal keratinocytes intracellularly in nonplaque psoriatic skin. Thus, the conformation or structure of TGF beta 1 and its localization vary in keratinocytes with distinct differentiation phenotypes suggesting that TGF beta 1 is a potential modulator of keratinocyte differentiation in vivo. Selective association of TGF beta 1 with nonproliferating keratinocytes in the suprabasal layers of the epidermis and its exclusion from the proliferating keratinocytes in the basal layer suggest that it may be a physiological regulator of keratinocyte proliferation. In addition, the intracellular localization of TGF beta 1 peptide in both normal and psoriatic keratinocytes suggests that it is constitutively synthesized by epidermal keratinocytes in vivo.     

EGFR-driven up-regulation of decoy receptor 3 in keratinocytes contributes to the pathogenesis of psoriasis

Decoy receptor 3 (DcR3) is a soluble receptor of Fas ligand (FasL), LIGHT (TNFSF14) and TNF-like molecule 1A (TL1A) and plays pleiotropic roles in many inflammatory and autoimmune disorders and malignant diseases. In cutaneous biology, DcR3 is expressed in primary human epidermal keratinocytes and is upregulated in skin lesions in psoriasis, which is characterized by chronic inflammation and angiogenesis. However, the regulatory mechanisms of DcR3 over-expression in skin lesions of psoriasis are unknown. Here, we demonstrate that DcR3 can be detected in both dermal blood vessels and epidermal layers of psoriatic skin lesions. Analysis of serum samples showed that DcR3 was elevated, but FasL was downregulated in psoriatic patients compared with normal individuals. Additional cell studies revealed a central role of epidermal growth factor receptor (EGFR) in controlling the basal expression of DcR3 in keratinocytes. Activation of EGFR by epidermal growth factor (EGF) and transforming growth factor (TGF)-α strikingly upregulated DcR3 production. TNF-α?enhanced DcR3 expression in both keratinocytes and endothelial cells compared with various inflammatory cytokines involved in psoriasis. Additionally, TNF-α-enhanced DcR3 expression in keratinocytes was inhibited when EGFR was knocked down or EGFR inhibitor was used. The NF-κB pathway was critically involved in the molecular mechanisms underlying the action of EGFR and inflammatory cytokines. Collectively, the novel regulatory mechanisms of DcR3 expression in psoriasis, particularly in keratinocytes and endothelial cells, provides new insight into the pathogenesis of psoriasis and may also contribute to the understanding of other diseases that involve DcR3 overexpression.     

Signal transductions induced by bone morphogenetic protein-2 and transforming growth factor-beta in normal human osteoblastic cells

Transforming growth factor beta (TGF-beta) activates Ras/MAPK signaling in many cell types. Because TGF-beta and BMP-2 exert similar effects, we examined if this signaling is stimulated by both factors and analyzed the relationship between this signaling and the Smads in osteoblasts. BMP-2 and TGF-beta stimulated Ras, MAPK, and AP-1 activities. The DNA binding activities of c-Fos, FosB/Delta FosB, Fra-1, Fra-2, and JunB were up-regulated whereas JunD activity was decreased. c-Fos, FosB/Delta FosB, and JunB were associated with Smad4. The stimulation of AP-1 by BMP-2 and TGF-beta was dependent on Smad signaling, and anti-Smad4 antibody interfered with AP-1 activity. Thus, BMP-2 and TGF-beta activate both Ras/MAPK/AP-1 and Smad signaling in osteoblasts with Smads modulating AP-1 activity. To determine the roles of MAPK in BMP-2 and TGF-beta function, we analyzed the effect of ERK and p38 inhibitors on the regulation of bone matrix protein expression and JunB and JunD levels by these two factors. ERK and p38 mediated TGF-beta suppression of osteocalcin and JunD as well as stimulation of JunB. p38 was essential in BMP-2 up-regulation of type I collagen, fibronectin, osteopontin, osteocalcin, and alkaline phosphatase activity whereas ERK mediated BMP-2 stimulation of fibronectin and osteopontin. Thus, ERK and p38 differentially mediate TGF-beta and BMP-2 function in osteoblasts.