Cytokines In Keratinocytes Research Articles

Regulation of gingival epithelial cytokine response by bacterial cyclic dinucleotides

ABSTRACTBackground: Cyclic dinucleotides (cyclic di-guanosine monophosphate (c-di-GMP) and cyclic di-adenosine monophosphate (c-di-AMP)) and lipopolysaccharides (LPS) are pathogen-associated molecular patterns (PAMPs). Individual impacts of PAMPs on immune system have been evaluated, but simultaneous actions of multiple PAMPs have not been studied. Objective: Examination the effects of cyclic dinucleotides and Porphyromonas gingivalis LPS on gingival epithelial cytokine response. Methods: Human gingival keratinocytes (HMK) were incubated with 1, 10, and 100 µM concentrations of c-di-GMP and c-di-AMP, either in the presence or absence of P. gingivalis LPS. Intra- and extracellular levels of interleukin (IL)-1β, IL-8, IL-1Ra, monocyte chemoattractant protein (MCP)-1, and vascular endothelial growth factor (VEGF), were measured using the Luminex technique. Results: LPS decreased extracellular IL-8 levels, while the presence of c-di-AMP inhibited this effect. Incubating HMK cells with c-di-AMP (alone or with LPS) elevated the extracellular level of MCP-1. Extracellular VEGF level increased when cells were incubated with LPS and c-di-GMP together, or with c-di-AMP alone. LPS and c-di-AMP suppressed intracellular IL-1β levels. The c-di-AMP elevated intracellular levels of IL-1Ra. Conclusion: c-di-AMP and, to a lesser extent, c-di-GMP regulate keratinocyte cytokine response, either as an aggregator or as a suppressor of LPS, depending on the cytokine type.

Fas/FasL pathway and cytokines in keratinocytes in atopic dermatitis - Manipulation by the electromagnetic field.

BackgroundAtopic dermatitis (AD) is one of the most frequent skin diseases. Changes of the keratinocytes functionality play a major role in the development of AD. For example, activation of the Fas (CD95)/FasL (CD178) pathway in AD does not lead to extensive apoptosis in skin. Binding of the Fas receptor to its protein ligand—FasL, which are present on the (AD)-modified keratinocytes, should result in the sequential induction of cell death, but there is no evidence of extensive apoptosis of these cells. This suggests that non-apoptotic mechanism of Fas/FasL pathway is commonly encountered, although not examined in the case of AD, phenomenon. An electromagnetic field, which was used to influence cultured cells in this study, can modulate proliferation, apoptosis, differentiation, and metabolism in various cells.ObjectiveHere, we evaluate the possibility to manipulate the immune activation of AD keratinocytes and their response to the electromagnetic field, which was not tested before.MethodsKeratinocytes isolated from the skin of healthy subjects (n = 20) and patients with atopic dermatitis (n = 20) as well as HaCaT and PCS-200-010 cell were exposed to the 900 MHz electromagnetic field for 60 minutes. Cytometric analysis of viability, Fas/FasL, p-ERK, p-p38 and p-JNK expression and Luminex analysis of cytokine concentration were performed in two-time points: 4 and 24 hours after the exposition.ResultsThis research has shown upregulated Fas, FasL, p-ERK, p-p38, and p-JNK expression along with increased cytokine secretion (IL-1β, IL-4, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IL-31 and TNFα) by keratinocytes derived from the skin of patients with the AD when compared with healthy control. Exposure of keratinocyte cultures obtained from AD patients to EMF resulted in a decrease of 1β, IL-4, IL-10, IL-12, I L-13, IL-17, IL-31 and TNFα levels. Keratinocytes derived from the skin of AD patients are characterized by elevated Fas and FasL expression when compared to healthy control.ConclusionApoptotic and nonapoptotic activation of the Fas/FasL-dependent signaling pathway may play a significant role in the pathogenesis of AD, by adjusting the local cytokine and chemokine environment at the site of inflammation. Moreover, the electromagnetic field exhibits strong immunomodulatory effects on AD-modified keratinocytes.

Keratinocyte Cytokine Networks Associated with Human Melanocytic Nevus Development

Melanocytes can group together in nevi, commonly thought to form because of intrinsic somatic mutations involving MAPK pathway activation. However, the role of the microenvironment, in particular keratinocytes, in nevogenesis is rarely studied. Melanocytes proliferate during the hair follicle growth phase and in some basal cell carcinomas, allowing us to construct keratinocyte gene expression clusters correlated with melanocyte activation. We asked whether such correlations are evident in the more subtle context of regulation of melanocyte behavior in normal skin. We considered genes which, when mutated in keratinocytes in mice, lead to nevogenesis. Across the human GTEx normal skin database, their expression was correlated with that of keratinocyte cytokines KITLG, HGF, FGF2, EDN1, and melanocyte markers. These cytokines have pleiotropic effects on melanocyte-specific and pigmentation genes and also influence mast cell gene expression. We show five classes of keratinocyte genes that, via germline genetic variation, influence melanocyte activity. These include genes involved in SHH signaling, structural keratins, ribosomal biogenesis, and stem cell governance. In agreement with the finding of KITLG linked to nevogenesis in human genome-wide association studies, we provide evidence that specific keratinocyte cytokines are components of networks that may drive or exacerbate nevus development.

Polyphyllin I Inhibits Propionibacterium acnes-Induced Inflammation In Vitro

Propionibacterium acnes (P. acnes) has been implicated in the progression of acne inflammation. Because current acne medications have various side effects, it is necessary to explore alternative medications possessing anti-inflammatory activity against P. acnes. We investigated the inhibitory effects of polyphyllin I (PPI) on P. acnes-induced inflammation in vitro. In this study, we examined the effects of PPI on the production of inflammatory cytokines in HaCaT keratinocytes treated with heat-killed P. acnes. These treated HaCaT keratinocytes showed increased expression of Toll-like receptor 2 (TLR2) and production of inflammatory cytokines. PPI significantly suppressed the secretion of inflammatory cytokines, including interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α, and the expression of TLR2 in P. acnes-treated cells. Moreover, we studied the influence of PPI on the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways in P. acnes-treated keratinocytes. PPI diminished the activation of NF-κB. Phosphorylated p38 levels were markedly increased after treatment with heat-killed P. acnes but were decreased after treatment with PPI, while the effect of PPI on ERK phosphorylation was not significant. Heat-killed P. acnes and PPI did not have any effect on JNK phosphorylation. Furthermore, we confirmed that NF-κB p65 inhibitor (BAY11-7082), p38 MAPK inhibitor (SB203580), and PPI blocked the expression of IL-8 in heat-killed P. acnes-treated cells. These results demonstrated that PPI has potential for development as a treatment for acne inflammation.

Fas/FasL pathway and cytokines in keratinocytes in atopic dermatitis – manipulation by the electromagnetic field.

Fas/FasL pathway and cytokines in keratinocytes in atopic dermatitis – manipulation by the electromagnetic field.

Fas/FasL pathway and cytokines in keratinocytes in atopic dermatitis – manipulation by the electromagnetic field.

Fas/FasL pathway and cytokines in keratinocytes in atopic dermatitis – manipulation by the electromagnetic field.

Pseudomonas aeruginosa flagellum is critical for invasion, cutaneous persistence and induction of inflammatory response of skin epidermis

ABSTRACT Pseudomonas aeruginosa, an opportunistic pathogen involved in skin and lung diseases, possesses numerous virulence factors, including type 2 and 3 secretion systems (T2SS and T3SS) and its flagellum, whose functions remain poorly known during cutaneous infection. Using isogenic mutants deleted from genes encoding each or all of these three virulence factors, we investigated their role in induction of inflammatory response and in tissue invasiveness in human primary keratinocytes and reconstructed epidermis. Our results showed that flagellum, but not T2SS and T3SS, is involved in induction of a large panel of cytokine, chemokine, and antimicrobial peptide (AMP) mRNA in the infected keratinocytes. Chemokine secretion and AMP tissular production were also dependent on the presence of the bacterial flagellum. This pro-inflammatory effect was significantly reduced in keratinocytes infected in presence of anti-toll-like receptor 5 (TLR5) neutralizing antibody. Bacterial invasion of human epidermis and persistence in a mouse model of sub-cutaneous infection were dependent on the P. aeruginosa flagellum. We demonstrated that flagellum constitutes the main virulence factor of P. aeruginosa involved not only in early induction of the epidermis inflammatory response but also in bacterial invasion and cutaneous persistence. P. aeruginosa is mainly sensed by TLR5 during the early innate immune response of human primary keratinocytes.

Abstract LB-355: Loss of keratinocyte desmoglein 1 occurs during melanoma development and alters crosstalk between keratinocytes and melanocytes

Abstract Ultraviolet (UV)-induced mutations in melanocytes are a well-established cause of melanoma. However, histologically normal “fields” of altered keratinocytes present in sun-exposed epidermis could also drive initiation and progression of melanoma. We previously demonstrated that UV radiation reduced expression of the keratinocyte desmosomal cadherin, desmoglein 1 (Dsg1), and that loss-of-function mutations in Dsg1 increased keratinocyte cytokine production in Severe dermatitis, multiple Allergies, and Metabolic wasting (SAM) syndrome. Based on these observations we hypothesized that loss of Dsg1 alters the microenvironment through increased cytokine signaling. Here, we show that Dsg1 expression is progressively reduced in regions surrounding human dysplastic nevi, melanoma in situ, and stage I/II melanomas. In contrast, keratinocyte E-cadherin was not significantly altered in early tumors. Dsg1 knockdown in keratinocytes in vitro increased phospho-EGFR, phospho-Stat3, and nuclear NFκB, while increasing keratinocyte mRNAs encoding IL-1α, IL-1β, IL-6 and IL-8 pro-inflammatory cytokines. Re-expressing silencing-resistant Dsg1 returned cytokine levels to control levels. To determine whether Dsg1 loss in keratinocytes elicits paracrine-mediated changes in melanocyte behavior, we treated normal primary melanocytes with conditioned media from Dsg1-silenced keratinocytes. Short-term (overnight) exposure to Dsg1-deficient conditioned media resulted in significantly increased melanocyte dendrite length, similar to morphology changes induced by UV exposure; however, long-term (7 day) exposure resulted in significantly shorter dendrites. Melanocytes exposed to Dsg1-deficient media increased melanin secretion and decreased melanin retention, a phenomenon associated with the process of transformation. Melanocytes incorporated into Dsg1-deficient 3D human skin equivalents exhibited aberrant behavior, including increased numbers and mis-localization to suprabasal layers. Based on these observations, we propose that under homeostatic conditions Dsg1 functions to maintain baseline levels of cytokine signaling, but that Dsg1 loss due to environmental stress disrupts keratinocyte:melanocyte communication through altered paracrine signaling to promote melanoma initiation. Citation Format: Christopher Arnette, Jodi L. Johnson, Hope E. Burks, Jennifer L. Koetsier, Kathleen J. Green. Loss of keratinocyte desmoglein 1 occurs during melanoma development and alters crosstalk between keratinocytes and melanocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-355.

The ARE-binding protein Tristetraprolin (TTP) is a novel target and mediator of calcineurin tumor suppressing function in the skin.

An increased incidence of skin inflammatory diseases is frequently observed in organtransplanted patients being treated with calcineurin inhibitor-based immunosuppressive agents. The mechanism of increased skin inflammation in this context has however not yet been clarified. Here we report an increased inflammation following inhibition of calcineurin signaling seen in both chemically induced mouse skin tumors and in tumors grafted from H-rasV12 expressing primary human keratinocytes (HKCs). Following UVB or TPA treatment, we specifically found that deletion of the calcineurin gene in mouse keratinocytes (MKCs) resulted in increased inflammation, and this was accompanied by the enhanced production of pro-inflammatory cytokines, such as TNFα, IL-8 and CXCL1. Furthermore, expression of the RNA-binding protein, tristetraprolin (TTP) was down-regulated in response to calcineurin inhibition, wherein TTP was shown to negatively regulate the production of pro-inflammatory cytokines in keratinocytes. The induction of TTP following TPA or UVB treatment was attenuated by calcineurin inhibition in keratinocytes, and correspondingly, disruption of calcineurin signaling down-regulated the amounts of TTP in both clinical and H-rasV12-transformed keratinocyte tumor models. Our results further demonstrated that calcineurin positively controls the stabilization of TTP in keratinocytes through a proteasome-dependent mechanism. Reducing the expression of TTP functionally promoted tumor growth of H-rasV12 expressing HKCs, while stabilizing TTP expression counteracted the tumor-promoting effects of calcineurin inhibition. Collectively these results suggest that calcineurin signaling, acting through TTP protein level stabilization, suppresses keratinocyte tumors by downregulating skin inflammation.

957 TNFα- and IL-17A-mediated induction of IL-36 cytokines in human keratinocytes

957 TNFα- and IL-17A-mediated induction of IL-36 cytokines in human keratinocytes

Circulating Cytokine/Chemokine Concentrations Respond to Ionizing Radiation Doses but not Radiation Dose Rates: Granulocyte-Colony Stimulating Factor and Interleukin-18.

Exposure to ionizing radiation is a crucial life-threatening factor in nuclear and radiological incidents. It is known that ionizing radiation affects cytokine/chemokine concentrations in the blood of B6D2F1 mice. It is not clear whether radiation dose rates would vary the physiological response. Therefore, in this study we utilized data from two experiments using B6D2F1 female mice exposed to six different dose rates ranging from low to high rates. In one experiment, mice received a total dose of 8 Gy (LD0/30) of 60Co gamma radiation at four dose rates: 0.04, 0.15, 0.30 and 0.47 Gy/min. Blood samples from mice were collected at 24 and 48 h postirradiation for cytokine/chemokine measurements, including interleukin (IL)-1β, IL-6, IL-10, keratinocyte cytokine (KC), IL-12p70, IL-15, IL-17A, IL-18, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage (GM)-CSF, macrophage (M)-CSF, monokine induced by gamma interferon (MIG), tumor necrosis factor (TNF)-α, fibroblast growth factor (FGF)-basic, vascular endothelial growth factor (VEGF) and platelet-derived growth factor basic (PDGF-bb). At 24 h after ionizing irradiation at dose rate of 0.04 Gy/min, significant increases were observed only in G-CSF and M-CSF ( P < 0.05). At 0.15 Gy/min, IL-10, IL-17A, G-CSF and GM-CSF concentrations were increased. At 0.3 Gy/min, IL-15, IL-18, G-CSF, GM-CSF, M-CSF, MCP-1, MIP-2, MIG, FGF-basic, VEGF and PDGF-bb were significantly elevated ( P < 0.05). At 0.47 Gy/min, IL-6, KC, IL-10, MCP-1, G-CSF, GM-CSF and M-CSF were significantly increased. At 48 h postirradiation, all cytokines/chemokines except MCP-1 returned to or were below their baselines, suggesting these increases are transient at LD0/30 irradiation. Of note, there is a limitation on day 2 because cytokines/chemokines are either at or below their baselines. Other parameters such as fms-like tyrosine kinase receptor-3 ligand (Flt-3 ligand) concentrations and lymphocyte counts, which have proven to be unaffected by radiation dose rates, can be used instead for assessing the radiation dose. However, in a separate radiation dose and time-course experiment, increases in IL-18 and G-CSF depended on the radiation doses but showed no significant differences between 0.58 and 1.94 Gy/min ( P > 0.05) at 3 and 6 Gy but not 12 Gy. G-CSF continued to increase up to day 7, whereas IL-18 increased on day 4 and remained above baseline level on day 7. Therefore, time after irradiation at different doses should be taken into consideration. To our knowledge, these results are the first to suggest that ionizing radiation, even at a very low-dose-rate (0.04 Gy/min), induces circulating G-CSF increases but not others for selected time points; radiation-induced increases in IL-18 at radiation dose rates between 0.15 and 1.94 Gy/min are also not in a radiation dose-rate-dependent manner. C-CSF, lymphocyte counts and circulating Flt-3 ligand should be explored further as possible biomarkers of radiation exposure at early time points. IL-18 is also worthy of further study as a potential biomarker at later time points.

TIMP3 is a CLOCK-dependent diurnal gene that inhibits the expression of UVB-induced inflammatory cytokines in human keratinocytes.

As the outermost physical barrier of an organism, the skin is diurnally exposed to UV radiation (UVR). Recent studies have revealed that the skin exhibits a circadian rhythm in various functions, and this oscillation is disturbed and reset via a strong environmental cue, the UVR. However, a molecular link between circadian perturbation by UVR and UVR-induced cellular responses has not been investigated. We identified tissue inhibitor of metalloproteinase (TIMP)-3 as a novel circadian locomotor output cycles kaput (CLOCK)–dependent diurnal gene by using a CLOCK-knockdown strategy in human keratinocytes. Among dozens of identified transcripts down-regulated by CLOCK knockdown, TIMP3 displayed a rhythmic expression in a CLOCK-dependent manner, in which the expression of matrix metalloproteinase (MMP)-1 and inflammatory cytokines, such as TNF-α, chemokine (C-X-C motif) ligand (CXCL)-1, and IL-8, were inversely regulated. Upon UVB exposure, the expression of CLOCK and TIMP3 was down-regulated, which led to an up-regulation of secretion of MMP1 and TNF-α proteins and in the transcription of CXCL1 and IL-8 via CCAAT-enhancer binding protein (C/EBP)-α. UVB-induced TNF-α secretion increased further or decreased by knockdown or overexpression of TIMP3, respectively, as well as by CLOCK. As a novel CLOCK-dependent diurnal gene, TIMP3 inhibits the expression of inflammatory cytokines that are up-regulated by UV irradiation in human keratinocytes. Thus, our work suggests a molecular link between circadian perturbation by UVR and UVR-induced inflammation.—Park, S., Kim, K., Bae, I.-H., Lee, S. H., Jung, J., Lee, T. R., Cho, E.-G. TIMP3 is a CLOCK-dependent diurnal gene that inhibits the expression of UVB-induced inflammatory cytokines in human keratinocytes.

Rottlerin as a therapeutic approach in psoriasis: Evidence from in vitro and in vivo studies

Rottlerin is a natural polyphenolic compound that was initially indicated as a PKCδ inhibitor. However, it was recently revealed that it may target a number of molecules and have biological effects on various cell types and is considered as a possible agent for tumor and cell proliferative diseases. Psoriasis is a chronic inflammatory cutaneous disorder with undefined etiology and is characterized by abnormal cellular proliferation, angiogenesis, and inflammation. Therefore, this paper investigates the regulatory effects of rottlerin on normal human epidermal keratinocytes (NHEKs) and imiquimod (IMQ)-induced psoriasiform (IPI) lesions. In vitro results showed that rottlerin inhibited cell proliferation in NHEKs through growth arrest and NFκB inhibition. It may also induce apoptosis in an autophagy-dependent pathway. We found that rottlerin inhibited human microvascular endothelial cells tube formation on matrigel. Rottlerin also decreased the cell senescence of keratinocytes and intracellular ROS generation, which indicated its antioxidant effect. We also showed that rottlerin affects the expression of keratinocyte proliferation biomarkers. In 12-O-tetradecanoylphorbol13-acetate (TPA)–induced keratinocytes, rottlerin significantly inhibited the expression of the induced pro-inflammatory cytokines in keratinocytes. An animal experiment provided the corresponding evidence based on this evidence in vitro, by using IPI model, we found that rottlerin could relieve the psoriasiform of BALB/c mice by inhibiting keratinocyte proliferation, inflammatory cell infiltration, and vascular proliferation. In conclusion, our results suggest that rottlerin may prove useful in the development of therapeutic agents against psoriasis. However, the deep mechanism still requires further study.

Keratinocyte Sonic Hedgehog Upregulation Drives the Development of Giant Congenital Nevi via Paracrine Endothelin-1 Secretion

Giant congenital nevi are associated with clinical complications such as neurocutaneous melanosis and melanoma. Virtually nothing is known about why some individuals develop these lesions. We previously identified the sonic hedgehog (Shh) pathway regulator Cdon as a candidate nevus modifier gene. Here we validate this by studying Cdon knockout mice, and go on to establishing the mechanism by which Shh exacerbates nevogenesis. Cdon knockout mice develop blue nevi without the need for somatic melanocyte oncogenic mutation. In a mouse model carrying melanocyte NRASQ61K, we found that strain backgrounds that carry genetic variants that cause increased keratinocyte Shh pathway activity, as measured by Gli1 and Gli2 expression, develop giant congenital nevi. Shh components are also active adjacent to human congenital nevi. Mechanistically, this exacerbation of nevogenesis is driven via the release of the melanocyte mitogen endothelin-1 from keratinocytes. We then suppressed nevus development in mice using Shh and endothelin antagonists. Our work suggests an aspect of nevus development whereby keratinocyte cytokines such as endothelin-1 can exacerbate nevogenesis, and provides potential therapeutic approaches for giant congenital nevi. Furthermore, it highlights the notion that germline genetic variation, in addition to somatic melanocyte mutation, can strongly influence the histopathological features of melanocytic nevi.

Staphylococcal Biofilms in Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disorder that is a major public health burden worldwide. AD lesions are often colonized by Staphylococcus aureus and Staphylococcus epidermidis. An important aspect of Staphylococcus spp. is their propensity to form biofilms, adhesive surface-attached colonies that become highly resistant to antibiotics and immune responses, and recent studies have found that clinical isolates colonizing AD skin are often biofilm-positive. Biofilm formation results in complex bacterial communities that have unique effects on keratinocytes and host immunity. This review will summarize recent studies exploring the role of staphyloccocal biofilms in atopic dermatitis and the implications for treatment. Recent studies suggest an important role for biofilms in the pathogenesis of numerous dermatologic diseases including AD. S. aureus biofilms have been found to colonize the eccrine ducts of AD skin, and these biofilms influence secretion of keratinocyte cytokines and trigger differentiation and apoptosis of keratinocytes. These activities may act to disrupt barrier function and promote disease pathogenesis as well as allergen sensitization. Formation of biofilm is a successful strategy that protects the bacteria from environmental danger, antibiotics, and phagocytosis, enabling chronic persistence in the host. An increasing number of S. aureus skin isolates are resistant to conventional antibiotics, and staphylococcal biofilm communities are prevalent on the skin of individuals with AD. Staphylococcal colonization of the skin impacts skin barrier function and plays multiple important roles in AD pathogenesis.

Crystallinity of Double-Stranded RNA-Antimicrobial Peptide Complexes Modulates Toll-Like Receptor 3-Mediated Inflammation.

Double-stranded RNA (dsRNA) induces production of pro-inflammatory cytokines in normal human epidermal keratinocytes (NHEK) by specific binding to endosomal Toll-like receptor-3 (TLR3). Recently, it has been shown that hyperactivation of TLR3 in psoriatic keratinocytes by dsRNA can occur in the presence of human antimicrobial peptide (AMP) LL37. Here, we combine synchrotron X-ray scattering, microscopy, computer simulations, and measurements of NHEK cytokine production to elucidate a previously unanticipated form of specific molecular pattern recognition. LL37 and similar α-helical AMPs can form pro-inflammatory nanocrystalline complexes with dsRNA that are recognized by TLR3 differently than dsRNA alone. dsRNA complexes that activate IL-6 production in NHEK and those that do not are both able to enter cells and co-localize with TLR3. However, the crystallinity of these AMP-dsRNA complexes, specifically the geometric spacing between parallel dsRNA and the repeat number of ordered dsRNA, strongly influences the level of TLR3 activation. Crystalline complexes that present dsRNA at a spacing that matches with the steric size of TLR3 can recruit and engage multiple TLR3 receptors, driving receptor clustering and immune amplification, whereas crystalline complexes that exhibit poor steric matching do not. Reverse-transcription quantitative PCR of IL-6 during siRNA knockdown of TLR3 confirms that cytokine production is due to TLR3: High levels of IL-6 transcription are observed for sterically matched complexes without TLR3 knockdown, whereas such activity is abrogated with TLR3 knockdown.

Abstract 4010: Tonggyu-tang, a traditional Korean medicine, suppresses inflammation, potential implications in tumor microenvironment

Abstract The critical roles of inflammation in the development of cancer have long been appreciated. A growing body of evidence supports the notion that infiltrates of inflammatory cells into tumor microenvironment influence the tumor progression by providing bioactive molecules including pro-inflammatory cytokines. Importantly, the increased number of mast cells within tumor microenvironment has been associated with a poor survival in cancer patients. Moreover, keratinocyte inflammation is known to be crucial for skin tumor development. The use of natural products to reduce inflammation in tumor microenvironment is gaining an interest, because of their reduced toxicity toward normal cells. In this study, we tested the effects of Tonggyu-tang (TGT) which is composed of 14 different herbal extracts on the activity of mast cells. We found that TGT significantly reduced the expression and production of inflammatory cytokines such as IL-4, IL-6, IL-8, and TNF-α in PMA and ionomycin- stimulated HMC-1 (human mast cell line-1). In an attempt to determine molecular mechanism underlying the inhibitory effects of TGT on cytokine expression, we revealed that TGT suppressed MAPK signaling pathway including ERK, p38, and JNK as well as NF-κB pathway, which are known to regulate inflammatory cytokine expression. Similar results were obtained from the LPS-stimulated HaCaT cells, immortalized human keratinocytes. Taken together, our results demonstrate that TGT suppresses inflammation by inhibiting the expression of pro-inflammatory cytokine in both mast cells and keratinocytes, thereby potentially leading to inhibition of tumor progression. Citation Format: Hyoin Kim, Seong-Gyu Ko, Yong Cheol Shin, Ji Hye Kim, Hye-Sook Seo, Tai Young Kim, Se Hyang Hong, Kangwook Lee, Jin Mo Ku, Myeong-Sun Kim, Yu-Jeong Choi, Soo-yeon Kang, Chunhoo Cheon, Youme Ko, Huang Ching Wen, Yui Sasaki, Sohyeon Kang. Tonggyu-tang, a traditional Korean medicine, suppresses inflammation, potential implications in tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4010. doi:10.1158/1538-7445.AM2017-4010

Anthralin modulates the expression pattern of cytokeratins and antimicrobial peptides by psoriatic keratinocytes

BackgroundPsoriasis is an inflammatory skin disease with aberrant keratinocyte proliferation, presumably as a result of immune cell activation. Th17 cytokines like IL-17A and IL-22 are critically implicated in epidermal thickening, altered keratinocyte differentiation and production of innate factors such as antimicrobial peptides. Psoriasis treatment options include modern targeted therapies using anti-cytokine antibodies and traditional non-targeted treatments like anthralin (dithranol). While the mode of action of anti-cytokine antibodies is defined, the effects of topical anthralin on psoriatic skin are not fully understood. ObjectiveThis study aims to unravel the direct effects of anthralin on keratinocyte proliferation, differentiation and production of psoriasis-associated factors. MethodsWe tested the effects of anthralin on cell proliferation, cytokeratin expression and changes in the expression of antimicrobial peptides using primary keratinocytes and 3D psoriasis tissue models with and without stimulation of the psoriasis-promoting cytokines IL-17A and IL-22. Moreover, we compared the findings derived from monolayer and multilayer cultures to data derived from lesional skin of patients with psoriasis before and under treatment with anthralin. ResultsOur study shows that anthralin directly induces cell apoptosis in vitro in monolayer cultures but not in 3D psoriasis tissue models treated with IL-17A and IL-22. Yet, keratinocyte proliferation as determined by Ki-67 staining is impaired by anthralin in vivo. In lesional skin but not in 3D psoriasis tissue models anthralin rapidly normalizes cytokeratin (CK)16 expression. Furthermore, anthralin directly inhibits DEFB4 expression in vitro and in vivo, while other antimicrobial peptides and cytokines studied like IL-6 and IL-8 are regulated differently in vitro and in vivo. ConclusionsOur results show that anthralin directly regulates DEFB4A expression. However, its beneficial effects on psoriasis cannot be explained by direct effects on keratinocyte differentiation or cytokine expression.

MiR-146b Probably Assists miRNA-146a in the Suppression of Keratinocyte Proliferation and Inflammatory Responses in Psoriasis

miR-146a inhibits inflammatory responses in human keratinocytes and in different mouse models of skin inflammation. Little is known about the role of miR-146b in the skin. In this study, we confirmed the increased expression of miR-146a and miR-146b (miR-146a/b) in the lesional skin of patients with psoriasis. The expression of miR-146a was approximately twofold higher than that of miR-146b in healthy human skin, and it was more strongly induced by stimulation of proinflammatory cytokines in keratinocytes and fibroblasts. miR-146a/b target genes regulating inflammatory responses or proliferation were altered in the skin of patients with psoriasis, among which FERMT1 was verified as a direct target of miR-146a. In silico analysis of genome-wide data from >4,000 psoriasis cases and >8,000 controls confirmed a moderate association between psoriasis and genetic variants in the miR-146a encoding gene. Transfection of miR-146a/b suppressed and inhibition enhanced keratinocyte proliferation and the expression of psoriasis-related target genes. Enhanced expression of miR-146a/b-influenced genes was detected in cultured keratinocytes from miR-146a-/- and skin fibroblasts from miR-146a-/- and miR-146b-/- mice stimulated with psoriasis-associated cytokines as compared with wild-type mice. Our results indicate that besides miR-146a, miR-146b is expressed and might be capable of modulation of inflammatory responses and keratinocyte proliferation in psoriatic skin.

Echinacea purpurea-derived alkylamides exhibit potent anti-inflammatory effects and alleviate clinical symptoms of atopic eczema

BackgroundAtopic eczema (AE) is a chronic inflammatory and pruritic skin disease. There is still an unmet need for topical anti-inflammatory and anti-pruritic substances exhibiting an excellent safety profile. The endocannabinoid system is known to regulate various aspects of cutaneous barrier and immune functions, thus targeting it may be a valid approach for alleviating the symptoms of AE. ObjectiveTo assess the putative efficacy of Echinacea purpurea-derived alkylamides (Ec. extract) activating cannabinoid (CB)-2 receptors in exerting anti-inflammatory effects and alleviating symptoms of AE. MethodsIn vitro anti-inflammatory efficiency was investigated by monitoring the effects of Ec. extract on poly-(I:C)-induced pro-inflammatory cytokine expression (Q-PCR) and release (ELISA) of HaCaT keratinocytes. Irritancy and sensitization potential (assessed by Human Repeat Insult Patch Test; Clinical trial 1); clinical efficiency in alleviating symptoms of AE (Clinical trial 2) as well as effects on human skin structure and lipid content (Clinical trial 3 followed by transmission electron microscopy and HPTLC) were investigated in randomized double blind clinical trials. ResultsEc. extract significantly reduced mRNA expression as well as release of poly-(I:C)-induced pro-inflammatory cytokines (IL-6 and IL-8) in keratinocytes. Thus, not surprisingly, the well-tolerated (Clinical trial 1) Ec. extract-based cream reduced local SCORAD statistically significantly, not only compared to baseline, but also compared to the comparator (Clinical trial 2). Of great importance, besides the in vitro anti-inflammatory effects, administration of the Ec. extract-based cream also resulted in significantly higher levels of overall epidermal lipids, ceramide EOS (ω-esterified fatty acid+sphingosine sphingoid base), and cholesterol at Day 15 compared to baseline as well as significantly greater numbers of intercellular lipid lamellae in the intercellular space (Clinical trial 3). ConclusionThe investigated Ec. extract shows great potential in alleviating cutaneous symptoms of AE, and by exerting remarkable anti-inflammatory actions and restoring the epidermal lipid barrier, it will be very likely a well-tolerated, powerful novel ingredient for the adjuvant therapy of AE.