Activation of toll‐like receptors 2, 3 or 5 induces matrix metalloproteinase‐1 and ‐9 expression with the involvement of MAPKs and NF‐κB in human epidermal keratinocytes

Please cite this paper as: Activation of toll‐like receptors 2, 3 or 5 induces matrix metalloproteinase‐1 and ‐9 expression with the involvement of MAPKs and NF‐κB in human epidermal keratinocytes. Experimental Dermatology 2010; 19 : e44–e49. Abstract: Toll‐like receptors (TLRs) on epidermal keratinocytes are the first line of defense against microbe invasion, and matrix metalloproteases (MMPs) regulate inflammation, cell migration and wound healing. In this study, we demonstrate that the mRNA and protein expressions of MMP‐1 and MMP‐9 in human epidermal keratinocytes are induced by ligands for TLR2, TLR3 and TLR5 [Pam3CSK4, Poly(I:C) and flagellin, respectively] in a dose‐dependent manner. We also found that the ligands for TLR2, TLR3 and TLR5 activate the MAP kinases, JNK and p38 MAPK, but not ERK1/2. Furthermore, treatment with the ligands for TLR2, TLR3 and TLR5 also induced the degradation of IκB‐α and activated the nuclear translocation of NF‐κB. MMP‐1 induction by the ligands for TLR2, TLR3 and TLR5 was inhibited by pretreatment with BAY11‐7082 (NF‐κB inhibitor) or SP600125 (JNK inhibitor), whereas MMP‐9 expression was inhibited by pretreatment with BAY11‐7082, SP600125 or SB203580. These findings demonstrate that the activation of TLR2, TLR3 or TLR5 induces the expression of MMP‐1 and MMP‐9 in human epidermal keratinocytes. In addition, NF‐κB or JNK mediated the MMP‐1 expression induced by TLR2, TLR3 and TLR5, whereas NF‐κB, JNK or p38 MAPK mediated the MMP‐9 expression induced by TLR2, TLR3 and TLR5.     

Increased expression of the histamine H4 receptor following differentiation and mediation of the H4 receptor on interleukin‐8 mRNA expression in HaCaT keratinocytes

Recent in vivo studies have demonstrated involvement of the histamine H4 receptor in pruritus and skin inflammation. We previously reported that an H4 receptor antagonist attenuated scratching behaviour and improved skin lesions in an experimental model of atopic dermatitis. We also reported the expression of the H4 receptor in human epidermal tissues. In this study, we investigated the expression of H4 receptor mRNA and the function of the receptor in a culture system that mimics in vivo inflammation on the HaCaT human keratinocyte cell line. Increased expression of the H4 receptor was observed in HaCaT cells following differentiation. Treatment of HaCaT cells with histamine and TNFα enhanced the mRNA expression of interleukin (IL)‐8. These increases in expression were significantly inhibited by the H4 receptor antagonist JNJ7777120. Our results indicate that IL‐8 mRNA expression might be enhanced by histamine and TNFα via H4 receptor stimulation in keratinocytes.     

IL‐17A synergistically enhances TLR3‐mediated IL‐36γ production by keratinocytes: A potential role in injury‐amplified psoriatic inflammation

Skin injury can trigger formation of new lesions in psoriasis (Koebner phenomenon). The mechanisms through which injury exacerbates psoriasis are unclear. During wound repair, epidermal keratinocytes are activated and produce abundant IL‐36γ, further promoting the skin inflammation. IL‐17A is the cornerstone cytokine in the pathogenesis of psoriasis. We sought to investigate the effects of IL‐17A on injury‐induced keratinocyte activation and IL‐36γ production. Here, we demonstrated that dsRNA released from necrotic keratinocytes induced the expression of IL‐36γ. Silencing of TLR3 by siRNA decreased the IL‐36γ induction by necrotic keratinocyte supernatant. Co‐stimulation with dsRNA and IL‐17A synergistically increased the expression of IL‐36γ and other proinflammatory mediators (CCL20, CXCL8, DEFB4 and LCN2) in keratinocytes. The synergistic effects were not dependent on TLR3 upregulation, TNF receptor signalling and mRNA stabilization. Co‐stimulation with dsRNA and IL‐17A resulted in an accumulation of IκBζ. The synergistic upregulation of IL‐36γ and proinflammatory mediators were inhibited by IκBζ siRNA. Co‐stimulation with IL‐17A and poly(I:C) markedly activated the p38 MAPK and NF‐κB pathway, compared with poly(I:C). Blockade of p38 MAPK and NF‐κB suppressed dsRNA/IL‐17A–mediated IκBζ and IL‐36γ induction. These findings demonstrated that IL‐17A synergistically enhanced the dsRNA‐mediated IL‐36γ production through a p38 MAPK‐, NF‐κB–, and IκBζ‐dependent mechanism.     

Orf virus infection of human keratinocytes and dermal fibroblasts: Limited virus detection and interference with intercellular adhesion molecule‐1 up‐regulation

Orf virus (Parapoxvirus ovis, ORFV) is a dermatotropic virus causing pustular dermatitis in small ruminants and humans. We analysed isolated human primary keratinocytes (KC) and dermal fibroblasts (FB) for cell death and virus replication by infection with a patient‐derived ORFV isolate. ORFV infection was associated with rapid induction of cell death in KC allowing for considerable virus removal. Upon infection with ORFV, KC and FB harboured intracytoplasmic ORFV and showed viral protein presence; however, missing virus spread indicated an abortive infection. Upon ORFV exposure, KC but not FB secreted the pro‐inflammatory cytokine interleukin (IL)‐6. ORFV infection enhanced the frequency of KC expressing intercellular adhesion molecule (ICAM)‐1 which was independent of IL‐6. Interestingly, ORFV inhibited ICAM‐1 up‐regulation on infected but not on non‐infected KC. Even interferon‐γ, a potent inducer of ICAM‐1, up‐regulated ICAM‐1 only on non‐infected KC. Transfer of ORFV‐free supernatant from infected to non‐infected KC induced ICAM‐1 on non‐infected KC pointing to the involvement of soluble mediator(s). Similarly as in KC, in FB interference with ICAM‐1 up‐regulation by ORFV infection was also observed. In conclusion, we shed light on epidermal and dermal defense mechanisms to ORFV infection and point to a novel ICAM‐1‐related immune evasion mechanism of ORFV in human skin.     

The influence of temperature on 5‐aminolevulinic acid‐based photodynamic reaction in keratinocytes in vitro

Background/purpose: Based on the observation that increasing skin temperature could improve 5‐aminolevulinic acid (ALA) penetration and accumulation of protoporphyrin IX (PpIX) in the ALA‐based photodynamic therapy (PDT), this study was designed to investigate how temperature change varied the therapeutic effect of ALA‐based PDT in vitro. Methods: HaCat cells were cultured with or without ALA at various temperatures. ALA uptake and PpIX accumulation were analyzed before laser irradiation as the baseline. After irradiation, cell death and cytokine secretions in the media, including interleukin (IL)‐1α, tumor necrosis factor (TNF)‐α and basic fibroblast growth factor (bFGF) were assayed, and the morphological changes were recorded. Results: With increasing temperature, the amount of ALA uptake, intracellular PpIX concentration and cell death increased in both the PDT and the non‐PDT groups. Secretions of IL‐1α, TNF‐α and bFGF also increased and reached a peak around 44–47 °C and then declined at a higher temperature. This biphasic response might be due to protein thermolysis that occurs when cells reach beyond thermal tolerance. Conclusions: Elevating temperature could augment photodynamic reactions to a certain extent, but adverse effects occurred when cells were overheated.     

Interleukin‐15 inhibits the expression of differentiation markers induced by Ca2+ in keratinocytes

Interleukin (IL)‐15 is an important proinflammatory cytokine that can protect epidermal keratinocytes (KCs) from ultraviolet‐induced apoptosis and plays a role in the pathogenesis of psoriasis. However, the impact of IL‐15 on KC differentiation remains unknown. In this study, isolated human primary epidermal KCs were treated with various concentrations of IL‐15 for different times, and the expression of differentiation markers (keratin 1, involucrin and loricrin) and p53 as well as the activation of ERK, AKT and Notch induced by IL‐15 in the absence or presence of Ca²⁺ was detected by real‐time PCR and Western blot. The results showed that stimulation with Ca²⁺ alone increased the expression of KC differentiation markers and p53 and promoted the activation of Notch1. Pretreatment with IL‐15 resulted in a decrease in the Ca²⁺‐induced expression of KC differentiation markers and p53. Additionally, Ca²⁺ continually inhibited the phosphorylation of ERK1/2 and activated AKT, and IL‐15 reduced the effect of Ca²⁺ on ERK1/2 and AKT. FR180204, a specific inhibitor of ERK1/2 phosphorylation, slightly attenuated the effect of Ca²⁺ on the expression of differentiation markers and p53 and the activation of Notch1. In contrast, MK‐2206, an inhibitor of pAKT, strongly blocked the expression of the differentiation markers and p53 and the activation of Notch1. An anti‐IL‐15 antibody neutralized the effect of IL‐15 on KC differentiation. These results indicate that IL‐15 inhibits the Ca²⁺‐induced differentiation of KCs, mainly via the attenuation of Ca²⁺‐stimulated PI3K‐AKT signalling.     

Filaggrin knockdown and Toll-like receptor 3 (TLR3) stimulation enhanced the production of thymic stromal lymphopoietin (TSLP) from epidermal layers

Keratinocytes constitute the first-line barrier against exogenous antigens and contain Toll-like receptors (TLRs), which function as pattern-recognition molecules to activate antimicrobial innate immune responses. In an effort to ascertain whether or not filaggrin (filament-aggregating protein) expression affected the TLR-mediated responses of keratinocytes, we transfected filaggrin siRNA into HaCaT human keratinocyte cells and determined that thymic stromal lymphopoietin (TSLP) and IL-6 secretion were increased by poly(I:C) stimulus. Additionally, TSLP expression is increased in filaggrin knockdown as well as TLR3 stimulation in reconstituted human epidermal layers. Therefore, the findings of this study show that reduced filaggrin levels may influence innate immune responses via TLR stimuli and may contribute to the pathogenesis of inflammatory skin disease via TSLP expression.     

SIG1459: A novel phytyl‐cysteine derived TLR2 modulator with in vitro and clinical anti‐acne activity

Cutibacterium (formerly Propionibacterium acnes) is a major contributor to the pathogenesis of acne. C. acnes initiates an innate immune response in keratinocytes via recognition and activation of toll‐like receptor‐2 (TLR2), a key step in comedogenesis. Tetramethyl‐hexadecenyl‐cysteine‐formylprolinate (SIG1459), a novel anti‐acne isoprenylcysteine (IPC) small molecule, is shown in this study to have direct antibacterial activity and inhibit TLR2 inflammatory signalling. In vitro antibacterial activity of SIG1459 against C. acnes was established demonstrating minimal inhibitory concentration (MIC = 8.5 μmol\L), minimal bactericidal concentration (MBC = 16.1 μmol\L) and minimal biofilm eradication concentration (MBEC = 12.5 μmol\L). To assess SIG1459's anti‐inflammatory activity, human keratinocytes were exposed to C. acnes and different TLR2 ligands (peptidoglycan, FSL‐1, Pam3CSK4) that induce pro‐inflammatory cytokine IL‐8 and IL‐1α production. Results demonstrate SIG1459 inhibits TLR2‐induced IL‐8 release from TLR2/TLR2 (IC₅₀ = 0.086 μmol\L), TLR2/6 (IC₅₀ = 0.209 μmol\L) and IL‐1α from TLR2/TLR2 (IC₅₀ = 0.050 μmol\L). To assess the safety and in vivo anti‐acne activity of SIG1459, a vehicle controlled clinical study was conducted applying 1% SIG1459 topically (n = 35 subjects) in a head‐to‐head comparison against 3% BPO (n = 15 subjects). Utilizing the Investigator Global Assessment scale for acne as primary endpoint, results demonstrate 1% SIG1459 significantly outperformed 3% BPO over 8 weeks, resulting in 79% improvement as compared to 56% for BPO. Additionally, 1% SIG1459 was well tolerated. Thus, SIG1459 and phytyl IPC compounds represent a novel anti‐acne technology that provides a safe dual modulating benefit by killing C. acnes and reducing the inflammation it triggers via TLR2 signalling.     

S‐allyl cysteine inhibits TNF‐α‐induced inflammation in HaCaT keratinocytes by inhibition of NF‐ κB‐dependent gene expression via sustained ERK activation

Tumor necrosis factor‐α (TNF‐α)‐induced keratinocyte inflammation plays a key role in the pathogenesis of multiple inflammatory skin diseases. Here we investigated the anti‐inflammatory effect of S‐allyl cysteine (SAC) on TNF‐α‐induced HaCaT keratinocyte cells and the mechanism behind its anti‐inflammatory potential. SAC was found to inhibit TNF‐α‐stimulated cytokine expression. Further, SAC was found to inhibit TNF‐α‐induced activation of p38, JNK and NF‐κB pathways. Interestingly, SAC was found to differentially regulate ERK MAP kinase in cells. TNF‐α‐induced transient ERK activation and SAC treatment resulted in sustained ERK activation both in the presence and absence of TNF‐α. Additionally, SAC failed to inhibit the TNF‐α‐induced expression of the pro‐inflammatory cytokines TNF‐α and IL‐1β when cells were treated with the MEK inhibitor PD98059, suggesting that the anti‐inflammatory effect of SAC is via sustained activation of the ERK pathway. Since ERK activation has been reported to negatively regulate NF‐κB‐driven gene expression and we find that SAC activates ERK and negatively regulates NF‐κB, we investigated whether there existed any crosstalk between the ERK and the NF‐κB pathways. NF‐κB‐dependent reporter assay, visualization of the nuclear translocation of NF‐κB‐p65 subunit and determination of the cellular levels of I‐κB, the inhibitor of NF‐κB, revealed that SAC inhibited TNF‐α‐induced NF‐κB activation, and PD98059 treatment reversed this effect. These results collectively suggest that SAC inhibits TNF‐α‐induced inflammation in HaCaT cells via a combined effect entailing the inhibition of the p38 and the JNK pathways and NF‐κB pathway via the sustained activation of ERK.     

E‐FABP induces differentiation in normal human keratinocytes and modulates the differentiation process in psoriatic keratinocytes in vitro

Epidermal fatty acid‐binding protein (E‐FABP) is a lipid carrier, originally discovered in human epidermis. We show that E‐FABP is almost exclusively expressed in postmitotic (PM) keratinocytes, corresponding to its localization in the highest suprabasal layers, while it is barely expressed in keratinocyte stem cells (KSC) and transit amplifying (TA) keratinocytes. Transfection of normal human keratinocytes with recombinant (r) E‐FABP induces overexpression of K10 and involucrin. On the other hand, E‐FABP inhibition by siRNA downregulates K10 and involucrin expression in normal keratinocytes through NF‐κB and JNK signalling pathways. E‐FABP is highly expressed in psoriatic epidermis, and it is mainly localized in stratum spinosum. Psoriatic PM keratinocytes overexpress E‐FABP as compared to the same population in normal epidermis. E‐FABP inhibition in psoriatic keratinocytes markedly reduces differentiation, while it upregulates psoriatic markers such as survivin and K16. However, under high‐calcium conditions, E‐FABP silencing downregulates K10 and involucrin, while survivin and K16 expression is completely abolished. These data strongly indicate that E‐FABP plays an important role in keratinocyte differentiation. Moreover, E‐FABP modulates differentiation in psoriatic keratinocytes.     

Activation of Toll‐like receptors alters the microRNA expression profile of keratinocytes

Keratinocytes recognize invading pathogens by various receptors, among them Toll‐like receptors (TLRs), and provide the first line of defense in skin immunity. The role of microRNAs in this important defense mechanism has not been explored yet. Our aim was to identify microRNAs involved in the innate immune response of keratinocytes. MicroRNA expression profiling revealed that the TLR2 ligand zymosan, the TLR3 ligand poly(I:C) or the TLR5 ligand flagellin significantly altered the microRNA expression in keratinocytes. The regulation of microRNAs was concentration‐dependent and it could be neutralized by siRNAs specific for TLR2, TLR3 and TLR5, respectively, confirming the specificity of the TLR response. Interestingly, one microRNA, miR‐146a, was strongly induced by all studied TLR ligands, while other microRNAs were regulated in a TLR‐ or time point‐specific manner. These findings suggest an important role for microRNAs in the innate immune response of keratinocytes and provide a basis for further investigations.     

Interleukin‐17A affects extracellular vesicles release and cargo in human keratinocytes

Psoriasis is a chronic inflammatory systemic disease caused by deregulation of the interleukin‐23/‐17 axis that allows the activation of Th17 lymphocytes and the reprogramming of keratinocytes proliferative response, thereby inducing the secretion of cyto‐/chemokines and antimicrobial peptides. Beside cell‐to‐cell contacts and release of cytokines, hormones and second messengers, cells communicate each other through the release of extracellular vesicles containing DNA, RNA, microRNAs and proteins. It has been reported the alteration of extracellular vesicles trafficking in several diseases, but there is scarce evidence of the involvement of extracellular vesicles trafficking in the pathogenesis of psoriasis. The main goal of the study was to characterize the release, the cargo content and the capacity to transfer bioactive molecules of extracellular vesicles produced by keratinocytes following recombinant IL‐17A treatment if compared to untreated keratinocytes. A combined approach of standard ultracentrifugation, RNA isolation and real‐time RT‐PCR techniques was used to characterize extracellular vesicles cargo. Flow cytometry was used to quantitatively and qualitatively analyse extracellular vesicles and to evaluate cell‐to‐cell extracellular vesicles transfer. We report that the treatment of human keratinocytes with IL‐17A significantly modifies the extracellular vesicles cargo and release. Vesicles from IL‐17A‐treated cells display a specific pattern of mRNA which is undid by IL‐17A neutralization. Extracellular vesicles are taken up by acceptor cells irrespective of their content but only those derived from IL‐17A‐treated cells enable recipient cells to express psoriasis‐associated mRNA. The results imply a role of extracellular vesicles in amplifying the pro‐inflammatory cascade induced in keratinocyte by pro‐psoriatic cytokines.     

Psoriatic keratinocytes are resistant to tumor necrosis factor alpha's induction of mRNA for the NMDA‐R2C subunit

Psoriatic individuals demonstrate accelerated healing and the Koebner phenomenon, suggesting that psoriatic proliferation of keratinocytes is not inhibited appropriately after skin injury. Serial analysis of gene expression in TNFα‐exposed keratinocytes shows the greatest alteration in expression of NMDA‐R2C. Expression of the NMDA receptor is altered in diseased skin containing TNFα, and TNFα plays a prominent role in psoriasis. An abnormality in induction of NMDA‐R2C by TNFα in psoriatic keratinocytes may explain their lack of growth inhibition. We compared the capacity of TNFα to induce expression of NMDA‐R2C in normal and psoriatic (involved and uninvolved) keratinocytes in vitro. After 72 h of incubation with TNFα, normal keratinocytes demonstrated a significant induction of NMDA‐R2C mRNA compared with control cultures, whereas psoriatic keratinocytes showed no induction. In an in vitro model of wounding (scratches on monolayers), TNFα inhibited migration/proliferation of keratinocytes only at the edge of NMDA‐R2C expressing wounded monolayers of normal keratinocytes.     

Z‐ligustilide ameliorated ultraviolet B‐induced oxidative stress and inflammatory cytokine production in human keratinocytes through upregulation of Nrf2/HO‐1 and suppression of NF‐κB pathway

Ultraviolet B (UVB), a harmful environmental factor, is responsible for a variety of skin disorders including skin inflammation through reactive oxygen species (ROS) and inflammatory mediator production. Here, we investigated the effect of Z‐ligustilide (Z‐lig), an active ingredient isolated from the medicinal plants Cnidium officinale and Angelica acutiloba, on UVB‐induced ROS generation and inflammatory mediator production in normal human epidermal keratinocytes (NHEKs) as well as its underlying mechanisms. Z‐lig significantly rescued UVB‐induced NHEKs damage in a dosage‐dependent manner. Pretreatment of NHEKs with Z‐lig inhibited UVB‐induced ROS production in NHEKs. Both silencing the nuclear factor E2‐related factor 2 (Nrf2) and the supplement of tin protoporphyrin IX (SnPP), a haeme oxygenase‐1 (HO‐1) inhibitor, cancelled the inhibitory effect of Z‐lig on UVB‐induced ROS upregulation in NHEKs. Moreover, pretreatment of NHEKs with Z‐lig reduced UVB‐induced nuclear factor kappa B (NF‐κB)‐dependent inflammatory mediators (IL‐6, IL‐8 and MCP‐1) production at both mRNA and protein level. In the presence of Z‐lig, UVB‐induced NF‐κB subunit p65 nuclear translocation was abolished, and the IκBα degradation was suppressed. Taken together, these findings suggest that Z‐lig can suppress UVB‐induced ROS generation through Nrf2/HO‐1 upregulation and inflammation by suppressing the NF‐κB pathway, suggesting that Z‐lig may be beneficial in protecting skin from UVB exposure.