IFNγ and cytosolic dsDNA regulate inflammatory caspase-5 and IL-1β release by epidermal keratinocytes.

<p>A-B, Regulation of IL-1β and caspase-5 in Th1/Th17 cytokine-stimulated keratinocytes analyzed by RTqPCR and normalized to β-actin. Data represent mean + SEM, *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001, Student’s <i>t</i> test, n = 9. C, Keratinocytes stimulated with IFNγ, transfected with dsDNA [Poly(dA:dT)] ± DNase and the DNA-dependent IL-1β release was analyzed by ELISA. D, Representative immunoblotting of corresponding supernatants analyzed for DNA-dependent activation of caspase-5 (exposure times; caspase-5, 30s; cleaved caspase-5, 30min) compared to loading control (Ponceau staining), three independent experiments. E, Keratinocytes stimulated with IFNγ, transfected with dsDNA and indicated siRNA and the caspase-5 dependent IL-1β release was analyzed by ELISA. C, E, Data represent mean + SEM, **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001, Student’s <i>t</i> test, n = 3–6.</p

The antimicrobial peptide psoriasin (S100A7) mediates cytokine-dependent caspase regulation and IL-1β release by epidermal keratinocytes.

<p>A, E, Keratinocytes stimulated with IFNγ, IL-17A, transfected with dsDNA and indicated siRNA, and the psoriasin-dependent IL-1β release was analyzed by ELISA. Data represent mean + SEM, **, <i>p</i> < 0.01, Student’s <i>t</i> test, n = 7–8. B, D, Regulation of IL-1β, caspase-1, caspase-5 in cytokine-stimulated keratinocytes, transfected with psoriasin-targeting siRNA was analyzed by RTqPCR and normalized to β-actin. Data represent mean + SEM, *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001 determined by Student’s <i>t</i> test, n = 9. C, Induction of psoriasin in cytokine-stimulated keratinocytes analyzed by RTqPCR and normalized to β-actin. Data represent mean + SEM, *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001 determined by ANOVA, n = 9.</p

IL-17A amplifies caspase-5 induction and controls NLRP1-mediated IL-1β release by epidermal keratinocytes.

<p>A-D, Regulation of IL-1β, caspase-1, caspase-5, NLRP1in IFNγ- and IL-17A -stimulated keratinocytes analyzed by RTqPCR and normalized to β-actin. Data represent mean + SEM of three independent experiments performed in triplicates *, p < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001 determined by ANOVA. E, dsDNA-transfected keratinocytes stimulated with IFNγ, IL-17A, and the cytokine-dependent IL-1β release was analyzed by ELISA. Data represent mean + SEM *, p < 0.05; *** p< 0.001 determined by ANOVA, n = 6. F, IFNγ and IL17A-treated keratinocytes, transfected with dsDNA and with siRNA targeting NLRP1 (N1) or non-coding siRNA (Co), and the supernatant was analyzed for NLRP1-dependent activation of caspase-5 (exposure time 20s; active p10, p20 subunits; exposure time, 120s) and caspase-1 (exposure time 35s). Corresponding protein levels were quantified by densitometry, n = 3 and compared to Ponceau staining (loading control). G, Keratinocytes stimulated with IFNγ, IL-17A transfected with dsDNA and indicated siRNA, and the NLRP1 inflammasome-dependent IL-1β release was analyzed by ELISA. Data represent mean + SEM, ***, p < 0.001 determined by ANOVA, n = 6.</p

Inflammatory caspase-5 is increased and active in psoriatic skin.

<p>A-C, Increased expression of IL-1β, caspase-1, caspase-5 in healthy (H) and psoriatic (P) skin analyzed by RTqPCR and normalized to PBGD. Data represent mean + SEM **, <i>p</i> < 0.01, Student’s <i>t</i> test (n = 6–8). D, Immunofluorescent staining of caspase-5 in healthy skin which was enhanced in psoriasis (n = 3), scale bar = 50μm. Representative control section stained with secondary antibody (ab) only and DAPI. E, Representative immunoblot analysis of healthy and psoriatic skin stained for caspase-5, indicated are pro-caspase-5 and active caspase-5 in psoriasis compared to healthy skin versus β-actin (n = 3).</p

Vitamin D interferes with IL-1β release by epidermal keratinocytes and suppresses caspase-5 expression in epidermal keratinocytes in psoriasis.

<p>A, dsDNA-transfected keratinocytes stimulated with IFNγ, IL-17A, and the vitamin D-dependent IL-1β release was analyzed by ELISA, n = 4. B, Keratinocytes stimulated with IFNγ, IL-17A and vitamin D-dependent caspase-5 levels were detected by immunoblotting, n = 3. C, Vitamin D-dependent regulation of caspase-5 in keratinocytes stimulated with IFNγ, IL-17A and analyzed by RTqPCR and normalized to β-actin. Data represent mean + SEM *, p < 0.05 determined by Student’s <i>t</i> test, n = 9. D, Representative immunofluorescent staining of caspase-5 in psoriatic skin was reduced after calcipotriol treatment, scale bar = 50 μm. Skin sections of three psoriatic patients were examined. E, F, Representative caspase-5 immunoblotting of caspase-5 levels in skin lysates from psoriatic patients reduced after calcipotriol treatment, protein levels were quantified by densitometry versus β-actin. Data represent mean + SEM, *, <i>p</i> < 0.05 determined by Student’s <i>t</i> test. Skin lysates of three psoriatic patients were examined. G, H, Regulation of caspase-5 and IL-1β levels in skin lysates from psoriatic patients after calcipotriol treatment as analyzed by RTqPCR and normalized to PBGD. Data represent mean + SEM *, <i>p</i> < 0.05; **, <i>p</i> < 0.01 determined by Student’s <i>t</i> test, n = 3.</p

Additional file 2: Table S1. of A hypofractionated radiation regimen avoids the lymphopenia associated with neoadjuvant chemoradiation therapy of borderline resectable and locally advanced pancreatic adenocarcinoma

Phenotyping of peripheral blood immune cells. (DOCX 16 kb