Synergistic activity of proinflammatory cytokines on inhibition of KDM expression by NHEK.

<p>NHEK were cultured in the presence or absence of 10/ml of IL-1α, IL-17A, IL-22, OSM and TNFα alone or in combination for 24 h. Quantitative RT-PCR analysis was carried out on total RNA from 4 independent NHEK cultures. mRNA expression levels for cytokeratin 10 (CK10), cytokeratin 1 (CK1), desmoglein 1 (DSG1), desmocollin 1 (DSC1), fatty acid binding protein 5 (FABP5), calmodulin-like skin protein (CLSP), loricrin (LOR) and filaggrin (FLG) were normalized using GAPDH housekeeping gene and expressed as the fold decrease under unstimulated cultures. (A) Comparison of the activity of IL-1α, IL-17A, IL-22, OSM and TNFα alone or in combination (M5) on expression of keratinocyte differentiation markers. (B) Comparison of the activity of mix of 4 cytokines versus mix of 5 cytokines (M5) on expression of keratinocyte differentiation markers. All data are represented as mean and SEM of 4 independent experiments. One-way ANOVA with a Dunnett post-test were used for statistical evaluation and p values were as follows: *p<0.05, **p<0.01, ***p<0.001.</p

Synergistic activity of proinflammatory cytokines on inhibition of KDM expression by Reconstituted Human Epidermis.

<p>RHE have been cultured for 10-water interface using an appropriate differentiation medium and then with or without recombinant IL-1α, IL-17A, IL-22, OSM or TNFα alone or in combination during 24 h for mRNA quantification. Quantitative RT-PCR analysis was carried out and expression levels for KDM were normalized using GAPDH housekeeping gene and expressed as the fold to unstimulated control cultures. Data are mean and SEM of one experiment representative of two. One-way ANOVA with a Dunnett post-test were used for statistical evaluation and p values were as follows: *p<0.05, **p<0.01, ***p<0.001.</p

Activities of proinflammatory cytokines on the differentiation of Reconstituted Human Epidermis.

<p>(A) RHE have been cultured for 10 days at the air-water interface using an appropriate differentiation medium and then with or without recombinant IL-1α, IL-17A, IL-22, OSM or TNFα alone or in combination during 72 h for immunohistological analysis. RHE were fixed, embedded in paraffin and 4 µm vertical sections were stained with Hematoxylin and Eosin (HE) or with anti-CK10, anti-LOR, anti-FLG, anti-IVL or anti-S100A7 mAbs. Results are from one experiment representative of two. (B) RHE have been cultured for 10 days at the air-water interface using an appropriate differentiation medium and then with or without recombinant IL-1α, IL-17A, IL-22, OSM and TNFα (3 ng/ml), with or without JAKs inhibitor (10 µM) during 72 h. RHE were fixed, embedded in paraffin and 4 µm vertical sections were stained with Hematoxylin and Eosin. Results are from one experiment representative of three.</p

Skin and liver expression of A-SAA in a mouse model of psoriasiform dermatitis.

<p>C57BL/6 mice were treated daily during six days with imiquimod 5% cream (IMQ), with Vaseline (VAS) or were not treated (NT). (A) SAA1/2 and (B) SAA3 mRNA expression was determined by RT-qPCR. All data represent mean ± SEM relative expression to <i>GAPDH</i>. Statistical comparisons were performed using t test (*p<0.05; **p<0.01; ns, non-significant).</p

A-SAA production by NHEK stimulated with human IL-1α, IL-17A, IL-22, OSM, TNF-α, alone or in combination.

<p>(A) A-SAA mRNA expression and (B) protein secretion by NHEK stimulated with M5 were analyzed at different time-periods. (C) A-SAA mRNA expression and (D) protein secretion were determined 40 hours after cytokine activation. (E) A-SAA mRNA expression and (F) protein secretion by NHEK 40 hours after stimulation with four cytokines by sequentially subtracting either recombinant IL-1α, IL-17A, IL-22, OSM or TNF-α from M5. A-SAA mRNA and protein were quantified by RT-qPCR in NHEK and ELISA in supernatants, respectively. Data represent the mean ± SEM of three experiments with duplicates. Statistical comparisons were performed using 2way ANOVA test or t test (*p<0.05; **p<0.01; ***p<0.001). (G) Compared to resting control, (H) intracellular A-SAA staining was detected by immunofluorescence in the cytoplasm of NHEK stimulated with M5 in the presence of brefeldin A.</p

A-SAA mRNA expression in the skin and the serum of psoriatic and control patients.

<p>A-SAA mRNA expression in (A) the skin and (B) the serum of psoriatic patients is compared to healthy controls. Positive correlations of (C) serum A-SAA and CRP protein levels and (D) A-SAA mRNA expression in the skin and A-SAA protein concentrations in the serum of psoriatic patients. A-SAA mRNA levels from psoriatic skins are increased with (E) psoriasis severity, as evaluated by PASI, (F) disease duration, (G) cigarette smoking and (H) metabolic syndrome, respectively. A-SAA mRNA expression from 37 psoriatic skins was compared to 28 healthy skins and quantified by RT-qPCR. A-SAA protein concentrations in 17 psoriatic sera were determined by immunonephelemetry and compared to those of 11 healthy sera. Values are expressed as mean ± SEM. Statistical comparisons were performed using t test or Spearman rank correlation test (*p<0.05; **p<0.01; ***p<0.0001; ns, non-significant).</p

Expression of proinflammatory mediators by A-SAA-stimulated NHEK and in synergy with IL-17A.

<p>(A) NHEK were incubated with A-SAA (10 μg/ml) for 24 hours and the expression of TNF-α, S100A7, S100A8, hBD2, CCL20 and A-SAA was determined by RT-qPCR. (B) IL-17A (10 ng/ml) had a synergistic effect with rA-SAA. After A-SAA and IL-17A costimulation, mRNA expression of S100A7, hBD2 and A-SAA were further increased compared to A-SAA or IL-17A alone. Three independent experiments with duplicates were performed. Values are expressed as mean ± SEM fold change above unstimulated NHEK. Statistical comparisons were performed using t test (*p<0.05; **p<0.01; ***p<0.001).</p

Characteristics of psoriatic patients.

<p>Data are expressed as Mean ± SD or percentage (%). Abbreviations: PASI (Psoriasis Area and Severity Index), BMI (Body Mass Index).</p

A-SAA and IL-17A expression in skin samples from atopic dermatitis and psoriasis patients.

<p>Cutaneous expression of (A) A-SAA and (B) IL-17A mRNA in freshly isolated skin samples, measured by RT-qPCR. All data are represented as mean ± SEM relative expression to <i>GAPDH</i>. Statistical comparisons were performed using t test (*p<0.05; ***p<0.0001; ns, non-significant).</p