Response Of Keratinocytes Research Articles

The mineralocorticoid receptor modulates timing and location of genomic binding by glucocorticoid receptor in response to synthetic glucocorticoids in keratinocytes.

Glucocorticoids (GCs) exert potent antiproliferative and anti-inflammatory properties, explaining their therapeutic efficacy for skin diseases. GCs act by binding to the GC receptor (GR) and the mineralocorticoid receptor (MR), co-expressed in classical and non-classical targets including keratinocytes. Using knockout mice, we previously demonstrated that GR and MR exert essential nonoverlapping functions in skin homeostasis. These closely related receptors may homo- or heterodimerize to regulate transcription, and theoretically bind identical GC-response elements (GRE). We assessed the contribution of MR to GR genomic binding and the transcriptional response to the synthetic GC dexamethasone (Dex) using control (CO) and MR knockout (MREKO ) keratinocytes. GR chromatin immunoprecipitation (ChIP)-seq identified peaks common and unique to both genotypes upon Dex treatment (1h). GREs, AP-1, TEAD, and p53 motifs were enriched in CO and MREKO peaks. However, GR genomic binding was 35% reduced in MREKO , with significantly decreased GRE enrichment, and reduced nuclear GR. Surface plasmon resonance determined steady state affinity constants, suggesting preferred dimer formation as MR-MR > GR-MR ~ GR-GR; however, kinetic studies demonstrated that GR-containing dimers had the longest lifetimes. Despite GR-binding differences, RNA-seq identified largely similar subsets of differentially expressed genes in both genotypes upon Dex treatment (3h). However, time-course experiments showed gene-dependent differences in the magnitude of expression, which correlated with earlier and more pronounced GR binding to GRE sites unique to CO including near Nr3c1. Our data show that endogenous MR has an impact on the kinetics and differential genomic binding of GR, affecting the time-course, specificity, and magnitude of GC transcriptional responses in keratinocytes.

Ionizing Radiation-Induced DNA Damage Response in Primary Melanocytes and Keratinocytes of Human Skin

Currently, our knowledge of how different cell types in a tissue microenvironment respond to low and high linear energy transfer (LET) radiation is highly restricted. In this study, a comparative analysis was performed on γ-ray-induced DNA damage and repair in primary human melanocytes and keratinocytes isolated from 3 donors. Our study demonstrates a modest interindividual variability in both melanocytes and keratinocytes in terms of both spontaneous and ionizing radiation (IR)-induced 53BP1 foci formation and persistence. Melanocytes, in general, showed a slightly elevated (1.66–2.79 folds more) 53BP1 foci induction relative to keratinocytes after exposure to different doses of γ-rays (0.1–2.5 Gy) radiation. To verify the influence of ATM kinase on IR-induced 53BP1 foci formation, melanocytes and keratinocytes were treated with a specific ATM kinase inhibitor (KU55993, 10 μM) for 1 h prior to radiation. ATM kinase inhibition resulted in the reduction of both spontaneous and IR-induced 53BP1 foci by 17–42% in both melanocytes and keratinocytes of all the 3 donors. Increased persistence of IR-induced 53BP1 foci number was observed in ATM-inhibited melanocytes and keratinocytes after different post exposure times (6 h and 24 h). Taken together, our study suggests that interindividual variations exist in the induction and repair of DNA double-strand breaks (DSBs) in melanocytes and keratinocytes and that ATM is crucial for an optimal DSB repair efficiency in both human skin cell types.

Neutrophil extracellular traps promote keratinocyte inflammation via AIM2 inflammasome and AIM2-XIAP in psoriasis.

The infiltration of neutrophils in the epidermis and the release of neutrophil extracellular traps (NETs) are important events in the pathogenesis of psoriasis, but the regulatory roles and internal mechanism of NETs in psoriasis are largely unknown. Here, we demonstrate that NETs can activate the absent-in-melanoma-2 (AIM2) inflammasome in keratinocytes through the p38-MAPK signalling pathway, and targeting NETs with CI-amidine in vivo reduces AIM2 expression and ameliorates imiquimod-induced psoriasis-like phenotype in mice. Notably, NETs-activated AIM2 in keratinocytes not only promotes IL-1β production through the classical inflammasome pathway but also promotes IFN-γ production via X-linked inhibitor of apoptosis protein (XIAP), thereby mediating the immune responses of keratinocytes. In conclusion, our study demonstrates that the NETs-AIM2 axis exerts multiple pro-inflammatory effects on keratinocytes and may serve as a potential target for psoriasis therapy.

468 Preferentially Expressed Antigen in Melanoma Modulates Sensitivity to Retinoic Acid and Cell Cycle Control in Immortalized and Malignant Keratinocytes

Retinoids can be used to treat and prevent the formation of basal cell carcinoma (BCC) and squamous cell carcinomas (SCCs; notably cutaneous and oral SCC). Preferentially Expressed Antigen in Melanoma (PRAME), a gamete-specific repressor of retinoid-induced differentiation, is ectopically expressed in BCC and SCC tumors. However, the effects of PRAME on the development of these cancers and their sensitivity to retinoid therapies are unknown. The purpose of this study was to investigate PRAME’s influence on retinoid response and proliferation in benign human keratinocytes, BCC and SCC cells.

176 C10orf99/2610528A11Rik induces keratinocyte proinflammatory response and regulates lipid metabolism and barrier formation of the skin

The protective machinery of the skin opposes external agents by barrier and innate and adaptive immunity. The local interplay in the epithelial-immune microenvironment (EIME) contributes to the situation-specific protective/inflammatory responses, and its dysregulation generates inflammatory loops in atopic dermatitis and psoriasis. Keratinocytes are the key player in the interplay and inflammatory loops in the EIME. However, a common regulator of keratinocyte response in this machinery has not yet been identified. To address this issue, we analyzed skin specimens from four animal models for atopic dermatitis by microarray. We found that expression levels of one orphan gene, 2610528A11Rik, were up in all the models. Protein expression levels of the human ortholog, C10orf99, were much higher in the lesional skin of atopic dermatitis or psoriasis than in healthy skin. RNA-Seq analyses of normal human epidermal keratinocytes (NHEKs) transfected with C10orf99-expressing plasmids revealed their increased proinflammatory transcriptional response, and gene ontology analyses suggested the highest relation to “Lipid/cholesterol biosynthesis,” “Cadherin/cell adhesion,” and “Mitochondrion.” Furthermore, transcription factor (TF) enrichment analysis found that only one pathway activating SREBP1, a TF regulating sterol synthesis, was significantly impaired in C10orf99-expressing NHEKs. Moreover, treatment of 3D-cultured human epidermis with C10orf99 synthetic peptides during its stratification downregulated the expression levels of filaggrin and loricrin in a dose-dependent manner. Our results suggest that 2610528A11Rik/C10orf99 can be a master regulator that induces keratinocyte protective response and regulates barrier formation of the skin.

Low curcumin concentrations combined with blue light inhibits cutibacterium acnes biofilm-induced inflammatory response through suppressing MAPK and NF-κB in keratinocytes

BackgroundCurcumin has been employed as a photosensitizer agent during photodynamic therapy (PDT). Cutibacterium acnes (C. acnes) can cause an inflammatory response in human keratinocytes; however, no research has been conducted to determine whether curcumin and its photodynamic properties can prevent this inflammatory reaction. ObjectiveWe hypothesized that curcumin may control the C. acnes biofilm-induced inflammatory response in keratinocytes, either alone or in combination with blue light photodynamic therapy. MethodsFollowing C. acnes biofilm stimulation, human primary keratinocytes were treated with 20 μM curcumin solution alone or 5 μM curcumin with combined blue light irradiation. The amount of secreted protein was measured using an ELISA kit. The expression levels of Toll-like receptor 2 (TLR2) and its downstream proteins were determined using western blot. ResultsTreatment with 20 μM curcumin, but not 5 μM curcumin, reduced the inflammatory response to C. acnes biofilms in keratinocytes by blocking the TLR2/MAPK/NF-κB pathway. Interestingly, 5 μM curcumin combined with blue light also reduced the C. acnes biofilm-induced inflammation indicated above by blocking the TLR2/MAPK/NF-κB pathway. ConclusionCurcumin alone, in sufficient concentrations, or low-concentration curcumin with blue light had anti-inflammatory activity on keratinocytes stimulated by C. acnes biofilms through inhibition of MAPK and NF-κB signaling pathways by downregulating TLR2 expression.

Keratinocytes and immune cells in the epidermis are key drivers of inflammation in hidradenitis suppurativa providing a rationale for novel topical therapies

Hidradenitis suppurativa (HS) is a debilitating inflammatory skin disease characterized by painful nodules, drainage and scarring in skin folds. Injectable adalimumab is the only drug approved by the US Food and Drug Administration for the treatment of HS. Although systemic Janus kinase (JAK) inhibitors show promise, serious side-effects have been reported. There are no highly effective topical treatments for HS; furthermore, the contribution of epidermal keratinocytes to the intense inflammation has largely been unexplored. We investigated the role of keratinocytes and epidermal immune cells in HS inflammation at all Hurley stages of disease severity. We aimed to determine whether ruxolitinib can mitigate inflammation from keratinocytes and to develop a better understanding of how topical therapeutics might benefit patients with HS. We used skin samples from 87 patients with HS (Hurley stages I-III) and 39 healthy controls to compare keratinocyte- and immune cell-driven epidermal inflammation, in addition to the response of lesional HS keratinocytes to treatment with interferon (IFN)-γ and ruxolitinib. We used haematoxylin and eosin staining, immunohistochemistry, immunoblotting and quantitative reverse-transcription polymerase chain reaction assessments in whole skin, isolated epidermis, and cultured keratinocytes from healthy controls and both nonlesional and lesional HS skin to identify and define epidermal and keratinocyte-mediated inflammation in HS and how this may be targeted by therapeutics. HS lesional keratinocytes autonomously secreted high levels of chemokines, such as CCL2, CCL3 and CXCL3, which recruited neutrophils, CD8 T cells, and natural killer cells to the epidermis. Keratinocytes were the dominant source of tumour necrosis factor-α and interleukin (IL)-6 in HS lesions with little to no contribution from underlying dermal immune cells. In the presence of IFN-γ, which is dependent on immune cell infiltrate in vivo, keratinocytes expressed increased levels of additional cytokines including IL-1β, IL-12, IL-23 and IL-36γ. The JAK inhibitor ruxolitinib mitigated the expression of inflammatory cytokines and chemokines in HS lesional keratinocytes, thus providing a rationale for future study as a topical treatment for HS. This study demonstrates that keratinocytes actively recruit immune cells to HS epidermis and interactions between these cells drive a broad inflammatory profile in HS epidermis. Targeting epidermal inflammation in HS with novel topical formulations may be highly efficacious with reduced systemic side-effects.

Wound Healing Potential of an Oleoresin Essential Oil Chemotype from Canarium schweinfurthii Engl.

This study was conducted to investigate the chemical composition of essential oil (EO) extracted from an oleoresin of Canarium schweinfurthii widespread in the Gabonese tropical forest. A great variability in the chemical composition of EO was observed, among which a chemical profile rich in terpinolene and α-phellandrene (31.2 and 21.8%, respectively), was found and tested as a natural active ingredient for topical applications. After the evaluation of eye and skin irritancy and sensitization potentials of EO on in vitro and in chemico models, the in vitro modulating potential on a model of wound re-epithelialization was assessed. The terpinolene and α-phellandrene-rich chemotype have been proven to accelerate wound healing in a dose-dependent manner (concentration range from 1.8 to 9.0 μg/mL). In addition, the ability of this EO to modulate the pro-inflammatory response in human keratinocytes stimulated by UVB was observed in vitro by the reduction in levels of interleukin 6 (IL-6) and tumour necrosis factor-alpha (TNF-α), suggesting a possible implication during the inflammation phase of wound healing. Despite the high variability in EO composition, a method of solid-phase microextraction (SPME) of the oleoresin headspace is proposed for the in situ identification of the terpinolene and α-phellandrene-rich chemotype instead of conducting hydrodistillation. These results offer interesting perspectives for the development of innovative natural ingredients for the topical route, ingredients obtained in an eco-responsible and non-destructive way.

Platelet-activating factor and microvesicle particles as potential mediators for the toxicity associated with intoxicated thermal burn injury.

Thermal burn injuries (TBIs) in patients who are alcohol-intoxicated result in greater morbidity and mortality. The systemic toxicity found in human patients, which includes both immediate systemic cytokine generation with multiple organ failure and a delayed systemic immunosuppression, has previously been replicated in mouse models combining ethanol and localized TBI. Though considerable insights have been provided with these models, the exact mechanisms for these pathologic effects are unclear. In this review, we highlight the roles of the lipid mediator platelet-activating factor (PAF) and subcellular microvesicle particle (MVP) release in response to intoxicated thermal burn injury (ITBI) as effectors in the pathology. Particularly, MVP is released from keratinocytes in response to PAF receptor (PAFR) activation due to excess PAF produced by ITBI. These subcellular particles carry and thus protect the metabolically labile PAF which enable binding of this potent lipid mediator to several key sites. We hypothesize that PAF carried by MVP can bind to PAFR within the gut, activating myosin light chain kinase (MLCK). The subsequent gut barrier dysfunction in response to MLCK activation then allows bacteria to invade the lymphatic system and, eventually, the bloodstream, resulting in sepsis and resultant dysregulated inflammation in multiple organs. PAF in MVP also activate the skin mast cell PAFR resulting in migration of this key effector cell to the lymph nodes to induce immunosuppression. This review thus provides a mechanism and potential therapeutic approaches for the increased toxicity and immunosuppressive outcomes of TBI in the presence of acute ethanol exposure.

Biological response of epithelial and connective tissue cells to titanium surfaces with different ranges of roughness: An in-vitro study.

This study aimed to investigate how titanium (Ti) surface with different range roughness created by industrial machining influence the biological response of primary human gingival fibroblasts (HGFB) and keratinocytes (HGKC) in terms of cell proliferation and cytotoxicity. Four Ti surfaces of different roughness ranges were investigated: smooth (S: 0.08-0.1µm), minimally rough (MM: 0.3-0.5µm), moderately rough (MR: 1.2-1.4µm) and rough (R: 3.3-3.7µm). Discs topography and surface roughness were evaluated by scanning electron microscopy (SEM) and non-contact profilometer. Both cell lines were cultured, expanded, and maintained according to their supplier's protocols. Cell proliferation and cytotoxicity were evaluated at days 1, 3, 5, and 10 using cell viability and cytotoxicity colorimetric assays. Data were analysed via two-way ANOVA, one-way ANOVA and Tukey's post hoc test (p=0.05 for all tests). Both cell lines showed comparable initial proliferation activity of 70-86% for all the investigated roughnesses. HGKC showed better and higher proliferation % with S surface at all time points than all the other investigated surfaces which was significantly higher than MM at day 3 and higher than all the other investigated surfaces at day 5 and 10. On the other hand, HGFB exhibited the best proliferation with both MM and R surfaces with no significant differences from the other two surfaces (S and MR). Different surface roughnesses and exposure times showed significant effect on cell proliferation in both cell lines. Cytotoxicity for both cell lines was generally the highest on day 3, with the following order from highest to lowest: S (19.86%)>R>MR>MM for HGKC and MM (39.48%)>MR>S>R for HGFB. Different exposure times showed a significant effect on cell cytotoxicity in both cell lines and a significant effect of surface roughness in HGFB. All investigated roughness levels were sufficiently biologically compatible with cells representative of the major population of the soft tissue surrounding dental implants. However, the S surface was most cytotoxic to HGKC, while the MM surface was most cytotoxic to HGFB cells.

Two Gingival Cell Lines Response to Different Dental Implant Abutment Materials: An In Vitro Study.

Objectives: This study aimed to investigate the response of human gingival fibroblasts (HGFB) and human gingival keratinocytes (HGKC) towards different dental implant abutment materials. Methods: Five materials were investigated: (1) titanium (Ti), (2) titanium nitride (TiN), (3) cobalt-chromium (CoCr), (4) zirconia (ZrO2), and (5) modified polyether ether ketone (m-PEEK). Both cell lines were cultured, expanded, and seeded in accordance with the protocol of their supplier. Cell proliferation and cytotoxicity were evaluated at days 1, 3, 5, and 10 using colourimetric viability and cytotoxicity assays. Data were analysed via two-way ANOVA, one-way ANOVA, and Tukey’s post hoc test (p < 0.05 for all tests). Results: There was a statistically significant difference in cell proliferation of HGKC and HGFB cells in contact with different abutment materials at different time points, with no significant interaction between different materials. There was a significant effect on cell proliferation and cytotoxicity with different exposure times (p < 0.0001) for each material. Cell proliferation rates were comparable for both cell lines at the beginning of the study, however, HGFB showed higher proliferation rates for all materials at day 10 with better proliferation activities with ZrO and m-PEEK (40.27%) and (48.38%) respectively. HGKC showed significant interactions (p < 0.0001) in cytotoxicity between different materials. Conclusion: The present in vitro assessment investigated the biocompatibility of different abutment materials with soft tissue cells (HGFB and HGKC). The findings suggest that m-PEEK and TiN are biologically compatible materials with human cells that represent the soft tissue and can be considered as alternative implant abutment materials to Ti and ZrO2, especially when the aesthetic is of concern.

Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin-Induces Cell Cycle Arrest in a Glycogen Synthase Kinase (GSK)-3-Dependent Manner in Oral Keratinocytes.

Cytolethal distending toxins (Cdt) are produced by a diverse group of pathogens. One Cdt-producing organism, Aggregatibacter actinomycetemcomitans, plays a critical role in the pathogenesis of a unique form of periodontitis, formerly referred to as localized aggressive periodontitis. The active Cdt subunit, CdtB, is a potent phosphatidylinositol (PI) 3,4,5-triphosphate phosphatase capable of inducing PI-3-kinase signaling blockade, a requisite for Cdt-induced toxicity in lymphocytes. In this study, we extended our observations to include the oral keratinocyte response to AaCdt using cell lines and primary gingival keratinocytes. All three exhibited G2/M arrest when exposed to AaCdt toxin within 24 h. Toxin-treated cells exhibited reduced levels of pAkt and pGSK3β within 6 h. Pre-treatment with GSK3β kinase inhibitors, LY2090314, CHIR99021 and Tideglusib, abrogated Cdt-induced G2/M arrest. None of the oral epithelial cells exhibited evidence of apoptosis. Cells remained arrested in the G2/M phase for at least 72 h without evidence of DNA damage response activation (H2AX phosphorylation). Cdt-treated cells displayed increased phosphorylation of the cyclin dependent kinase 1 (CDK1); moreover, the GSK3 inhibitors blocked this increase and reduced total CDK1 levels. This study further clarifies the potential mechanism(s) contributing to Cdt toxicity and toxin-mediated pathogenesis.

Heat-clearing and blood-cooling decoction alleviates psoriasis by suppressing proliferation and inflammatory response of keratinocytes via EZH2/NF-κB

Psoriasis is a chronic recurrent dermatological disease that patients always suffer from different comorbidities. Chinese herbal medicine has been commonly used in the treatment of psoriasis for a long history. However, the mechanism operating between Qing-Ying Dizhuo Decoction (QYDD) (a heat-clearing and blood-cooling decoction) and psoriasis remains unclear. This paper aimed to study the role of QYDD in psoriasis and its underlying mechanism. HaCaT cells were induced by a mixture of IL-22, IL-17A, oncostatin M, TNF-α and IL-1α (M5) to construct the in vitro model of psoriasis. CCK-8 assay was utilized to assess the cell proliferation ability. The expressions of KRT1, KRT6, TNF-α, IL-1β, IL-6, IL-23, EZH2, IκBα, p65 and IKK were determined by qRT-PCR, western blot and ELISA. M5 induction increased levels of KRT6, IL-1β, IL-6, TNF-α and IL-23, enhanced the proliferation ability of HaCaT cells and decreased KRT1 expression, while QYDD treatment reversed these trends. Overexpression of EZH2 reversed the inhibitory effects of QYDD on cell inflammatory response and proliferation of M5-induced HaCaT cells. QYDD suppressed M5-activated NF-κB signaling pathway as evidenced by decreased levels of phosphorylated IKK and p65 and increased level of phosphorylated IκBα, while overexpression of EZH2 offset, in part, the regulation of the NF-κB pathway by QYDD. An NF-κB activator (betulinic acid) weakened the inhibitory effect of QYDD on the inflammatory response of HaCaT keratinocytes. QYDD appears to inactivate the NF-κB signaling pathway by inhibiting EZH2, thereby repressing the proliferation and suppressing inflammatory response of HaCaT cells and attenuating psoriasis.

Dual RNA-Sequencing and Liquid Chromatography–Mass Spectrometry Unveil Specific Insights on the Pathogenicity of Trichophyton mentagrophytes Complex

Trichophyton mentagrophytes is increasingly considered to be a public health hazard because it causes the most severe manifestations of dermatophytosis. In this study, we performed a series of studies to determine the pathogenicity of the T.mentagrophytes complex. We show that the T.mentagrophytes complex interacts with keratinocytes through pattern-recognition receptors‒MAPK/noncanonical NF-κB pathways and that the hyphal form of T.mentagrophytes is responsible for the increased inflammatory responses in keratinocytes. Moreover, SN-38 is likely a toxin of T.mentagrophytes that induces apoptosis in keratinocytes both invivo and invitro. Our results explain the severe pathogenicity and destructiveness of T.mentagrophytes observed in the clinic and pave the way for designing novel toxin-directed therapies to improve patient outcomes.

P05-03 Photobiomodulation-induced Nrf2 partially controls the inflammatory response in keratinocytes

P05-03 Photobiomodulation-induced Nrf2 partially controls the inflammatory response in keratinocytes

Supply Chain Disruptions during COVID-19 Pandemic Uncover Differences in Keratinocyte Culture Media

Various culture media are used to propagate keratinocytes (KCs) in vitro. The COVID-19 pandemic resulted in supply chain shortages necessitating substitutions to standard laboratory protocols, which resulted in many laboratories having to use culture media different from those they typically use. We screened available media on the KC line N/TERT2G and found that biological responses varied considerably across three culture media: KC serum-free media, KC growth medium 2, and defined media. We observed qualitative and quantitative differences in proliferation; KCs cultured in defined media had significantly lower proliferative capacity. KC differentiation was assessed by western blot for CLDN1, occludin, cytokeratin-10, and loricrin. Elevated expression of differentiation markers was observed in cells cultured in either KC growth medium 2 or defined media compared with those in cells cultured in KC serum-free media. KC barrier function was measured by transepithelial electrical resistance. KCs cultured in KC growth medium 2 and defined media developed significantly higher transepithelial electrical resistance than those cultured in KC serum-free media, and when treated with IL-4 and IL-13 or IL-17A, we observed variable responses. H&E staining on day 5 -post-differentiation showed greater epithelial thickness in KCs cultured in defined media and KC growth medium 2 than in those cultured in KC serum-free media. These findings show that the choice of culture media impacts the biological response of KCs in a manner that persists through differentiation in the same media.

Response of human gingival keratinocytes to hybrid CAD/CAM material eluates

ObjectivesThe aim of this study was to investigate the influence of hybrid CAD/CAM-blocks on immortalized human gingival keratinocytes (HGK). MethodsSamples of two different hybrid CAD/CAM materials [Lava™ Ultimate (3 M); VITA Enamic® (VITA Zahnfabrik)], a composite material [ceram.x® universal (Dentsply Sirona)] and a CAD/CAM ceramic [VITABLOCS® (VITA Zahnfabrik)] were stored in cell culture medium for 72 h to prepare eluates according to ISO-10993–12:2012. HGK were exposed to eluates for 6, 24 and 48 h. Cell monitoring was performed by RTCA iCELLigence™ system. The morphological changes were evaluated using phase contrast imaging. Specific biomarkers of apoptosis and terminal differentiation (Caspase-3, Involucrin) were analyzed semi quantitatively by indirect immunofluorescence (IIF). Protein levels and activation of MAP kinases ERK1/2 (p44/42) were quantified by Western blot. Data were statistically analyzed by unpaired t-test (p < 0.05). ResultsRegarding Vita Enamic® and Lava™ Ultimate, results of RTCA iCELLigence™ and Western blots showed no statistically significant differences (p > 0.05) compared to the negative control (HGK in native keratinocyte growth medium). No aberrant expression of Caspase-3 and Involucrin was detected in cells incubated with Vita® Enamic eluates Cells incubated with Lava™ Ultimate showed a higher expression of Involucrin after 24 h of incubation compared to the negative control. Statistically significant differences (p < 0.01) were found between cells incubated with ceram.x® universal and the negative control in RTCA iCELLigence™ assay and in quantitative measurements of Western blots after 6 h against phospho-p44/42 (p = 0.044). Increased expression of Caspase-3 and Involucrin were detected by IIF in cells after incubation with eluates of ceram.x® universal. SignificanceThe present data show no significant effect of hybrid materials on analyzed functions of cell behavior. A cytotoxic influence of ceram.x® universal eluates was observed in HGK in terms of a strong modulation of proliferation, morphology and protein expression.

Antibacterial and Anti-inflammatory Activity of Extracts and Major Constituents Derived from Stachytarpheta indica Linn. Leaves and Their Potential Implications for Wound Healing.

Wounds of various types continue to have a severe socioeconomic impact on the cost of health care. Globally, there has been increased interest surrounding the identification of bioactive compounds that promote or modulate the wound healing process. Stachytarpheta indica Linn. is traditionally used to heal wounds and relieve inflammation; however, the theorised pharmacological properties have not yet been scientifically validated. In this study, dried and ground plant leaves were extracted with water and methanol, which were then subjected to various analyses. The antimicrobial activity of the plant extracts and isolated compounds was determined using well diffusion assays, while the minimum inhibitory concentrations were determined with a colorimetric assay. Morphological changes of human keratinocytes in response to plant extracts were observed with differential interference contrast microscope imaging. Cell viability, proliferation, and migratory effects post-treatment with the plant extracts were also evaluated via colorimetric cytotoxicity assays and a real-time cell analyser protocol. Anti-inflammatory effects of plant extracts and isolated compounds were evaluated by flow cytometry and cyclooxygenase and lipoxygenase enzyme inhibition assays. Three active compounds, i.e. ipolamiide, verbascoside and iso-verbascoside, were isolated from S. indica leaves. Verbascoside demonstrated broad-range antibacterial activity and imposed strong inhibition at 9.77μg/mL against Staphylococci spp. S. indica extracts (0.1-0.2mg/mL) were shown to improve human keratinocyte proliferation up to 60% and induce morphological changes by producing cytoplasmic projections at concentrations higher than 0.4mg/mL. Plant extracts (6.25-100μg/mL) and individual compounds (3.125-50μg/mL) elicited strong anti-inflammatory effects by suppressing the expression of interleukin-8 and inhibiting cyclooxygenase-1 and 5-lipoxygenase enzymes. Collectively, these results indicate that plant extracts and isolated compounds derived from S. indica have the potential to inhibit bacterial growth, promote tissue regeneration and reduce inflammation, hence, potentially providing the basis for a novel therapeutic for the treatment of wounds.

827 Psoriasis exacerbation by obesity reflects reduced adiponectin regulation of PPAR-γ/Th17 pathway activation and is reversal by adiponectin receptor agonism

Although obesity-related deficiency of adipokines is known to contribute to increased psoriasis severity and decreased clinical response to interventions, the mechanism by which it exacerbates psoriasis is poorly defined. We hypothesized that adipokines suppress the activation of innate immune responses in keratinocytes and skin-resident Th17/γδ T cells. In preliminary studies, we confirmed that diet-induced obese (DIO) mice had significantly lower serum adiponectin levels than lean mice when subjected to topical imiquimod (IMQ) treatments to induce the psoriasis-like disease, supporting the use of this model for studies.

011 A new immunocompetent skin model recapitulates molecular features of psoriasis

Psoriasis is a chronic inflammatory skin disease involving interactions between keratinocytes and both the innate and adaptive immune systems. The IL-17/IL-23 axis is described to drive the release of inflammatory and immune factors in keratinocytes (cytokines, antimicrobial peptides, chimiokines), which sit at the heart of an inflammation amplification loop. To this date, there are very few immunocompetent models recapitulating as close as possible psoriatic skin. In this context, the aim of this work was to develop a 3D model of psoriatic lesional skin summarizing biological features of the pathology. As the dialogue between Th17 lymphocytes (LTh17) and keratinocytes plays a central role in psoriasis pathogenesis, a model taking into account these two biological actors was developed. A reconstructed epidermis (RE) was co-cultured with differentiated LTh17. RE morphology as well as proliferation/differentiation imbalance and inflammatory immune response were explored. As it is reported in psoriatic lesions, results showed that IL-17 family cytokines were increased. Histological analysis demonstrated that the psoriatic mimic epidermis exhibited morphological changes. Moreover, mechanisms of proliferation and differentiation appeared profoundly altered while a high keratinocyte inflammatory response characteristic of psoriasis was observed. In conclusion, we have succeeded in designing a 3D in vitro model reproducing molecular alterations of psoriatic skin. The co-culture of the RE with differentiated LTh17 led to a typical inflammatory response in keratinocytes, thus proving the successful communication of the two biological compartments. This physiological relevant model is suitable for the assessment of natural therapeutic molecules dedicated to psoriasis treatment.