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

DataSheet_1_IL-17 and IL-22 are pivotal cytokines to delay wound healing of S. aureus and P. aeruginosa infected skin.docx

IntroductionAlthough the presence of pathogens in skin wounds is known to delay the wound healing process, the mechanisms underlying this delay remain poorly understood. In the present study, we have investigated the regulatory role of proinflammatory cytokines on the healing kinetics of infected wounds.MethodsWe have developed a mouse model of cutaneous wound healing, with or without wound inoculation with Staphylococcus aureus and Pseudomonas aeruginosa, two major pathogens involved in cutaneous wound bacterial infections.ResultsAseptic excision in C57BL/6 mouse skin induced early expression of IL-1β, TNFα and Oncostatin M (OSM), without detectable expression of IL-22 and IL-17A/F. S. aureus and P. aeruginosa wound inoculation not only increased the expression of IL-1β and OSM, but also induced a strong cutaneous expression of IL-22, IL-17A and IL-17F, along with an increased number of infiltrating IL-17A and/or IL-22-producing γδ T cells. The same cytokine expression pattern was observed in infected human skin wounds. When compared to uninfected wounds, mouse skin infection delayed the wound healing process. Injection of IL-1α, TNFα, OSM, IL-22 and IL-17 together in the wound edges induced delayed wound healing similar to that induced by the bacterial infection. Wound healing experiments in infected Rag2KO mice (deficient in lymphocytes) showed a wound healing kinetic similar to uninfected Rag2KO mice or WT mice. Rag2KO infected-skin lesions expressed lower levels of IL-17 and IL-22 than WT, suggesting that the expression of these cytokines is mainly dependent on γδ T cells in this model. Wound healing was not delayed in infected IL-17R/IL-22KO, comparable to uninfected control mice. Injection of recombinant IL-22 and IL-17 in infected wound edges of Rag2KO mice re-establish the delayed kinetic of wound healing, as in infected WT mice.ConclusionThese results demonstrate the synergistic and specific effects of IL-22 and IL-17 induced by bacterial infection delay the wound healing process, regardless of the presence of bacteria per se. Therefore, these cytokines play an unexpected role in delayed skin wound healing.</p