DataSheet_1_Dermal fibroblasts are the key sensors of aseptic skin inflammation through interleukin 1 release by lesioned keratinocytes.docx

IL-1 plays a crucial role in triggering sterile inflammation following tissue injury. Although most studies associate IL-1 release by injured cells to the recruitment of neutrophils for tissue repair, the inflammatory cascade involves several molecular and cellular actors whose role remains to be specified. In the present study, we identified dermal fibroblasts among the IL-1R1-expressing skin cells as key sensors of IL-1 released by injured keratinocytes. After in vitro stimulation by recombinant cytokines or protein extracts of lysed keratinocytes containing high concentrations of IL-1, we show that dermal fibroblasts are by far the most IL-1-responsive cells compared to keratinocytes, melanocytes and endothelial cells. Fibroblasts have the property to respond to very low concentrations of IL-1 (from 10 fg/ml), even in the presence of 100-fold higher concentrations of IL-1RA, by increasing their expression of chemokines such as IL-8 for neutrophil recruitment. The capacity of IL-1-stimulated fibroblasts to attract neutrophils has been demonstrated both in vitro using cell migration assay and in vivo using a model of superficial epidermal lesion in IL-1R1-deficient mice which harbored reduced expression of inflammatory mediators and neutrophil skin infiltration. Together, our results shed a light on dermal fibroblasts as key relay cells in the chain of sterile inflammation induced after epidermal lesion.</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

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

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

Exogenous IL-22 promotes cell survival of GBM cell lines.

<p>U87MG (A) and U118MG (B) cell lines were incubated for 24 to 48 h in basal culture medium (FCS 10%, +) and in serum-free medium (FCS 10%,-) in the presence of exogenous IL-22 (+) or without IL-22 (-). Cell proliferation was determined using BrdU cell proliferation assay. The data are represented as histograms of proliferating cells in relative units ± SEM of three independent experiments. *, a value of <i>p</i> < 0.05; when compared with respective control without exogenous IL-22. (C, D) Apoptotic ratios of soluble nucleosomes were detected by ELISA cell death for U87MG (C) and U118MG (D) cell lines induced after 48 h of incubation with Fas activation alone (Fas-L, +) or in combination with 20 ng/mL of recombinant IL-22 (IL-22, +). Histograms mean ratio of apoptotic cells ± SEM of three independent experiments. *, <i>p</i> < 0.05; ***, <i>p</i> < 0.001; when compared with respective control. (E) The antiapoptotic factor Bcl-xL expression was assessed by western blotting (in reference to actin) in total cellular protein extracted from cells treated or not with Fas-L, with or without recombinant IL-22 for 48 h.</p

Expression of IL-22, IL-22R1 and IL-10R2 in GBM cell lines.

<p>(A-C) Quantitative RT-PCR analysis of IL-22 and its two receptors (IL-22R1, IL-10R2) in total RNA extracted from U87MG and U118MG cell lines. Positive controls were the human epidermal keratinocytes (NHEK) for IL-22R1 (A) and IL-10R2 (B) and the human psoriatic skin biopsies (PSO) for IL-22 expression (C). Target gene expression was normalized to the housekeeping GAPDH mRNA. (D, E) Detection of IL-22R1 (D) and IL-10R2 (E) proteins assessed by western blot analysis in the two studied cell lines. Positive control was the colorectal cancer cell line HT29 for both IL-22R1 and IL-10R2 expression. Actin was used as a loading protein control. (F) Confocal microscopy analysis of IL-22R1 and IL-10R2 labeled with specific antibodies and revealed with Alexa fluor-488 conjugated fluorescent antibodies (green) in U87MG and U118MG cell lines. Nuclei were counter stained with the blue-fluorescent DNA stain DAPI. Scale bars, 10μm.</p

IL-22 reduces ERK1/2 phosphorylation in GBM cell lines.

<p>(A, B) The expression of P-ERK1/2 and the total amount of ERK1/2 were analyzed by western blotting in total cellular protein extracted from U87MG (A) and U118MG (B) cells treated with IL-22 for the indicated times. Thirty mg of protein lysates was analyzed for P-ERK1/2 and total ERK1/2. The density of each P-ERK1/2 band was corrected for variance in loading, using the density of the corresponding total ERK1/2. The expression level was evaluated as the ratio of phosphorylated ERK1/2 protein densities between control (0 min) and treated cells. A representative results of three independent experiments. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001; when compared with control. (C, D) Effect of U0126 on proliferation of GBM cells. BrdU cell proliferation assays of U87MG (C) and U118MG (D) cells treated for 24 h in serum-free medium with vehicle (non-treated; NT) or with 0.5 and 5 μM of U0126. The data are represented as histograms of proliferating cells in relative units. Error bars indicate ± SEM. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01.</p

IL-22 enhances Akt phosphorylation in GBM cell lines.

<p>(A) The expression of phosphorylated Akt and the total amount of Akt were analyzed by western blotting for U87MG along a 3 h treatment with recombinant IL-22. Thirty mg of protein lysates was analyzed for P-Akt (Ser473) and total Akt by western blot analysis. The density of each P-Akt band was corrected for variance in loading, using the density of the corresponding total Akt. The expression level was evaluated as the ratio of phosphorylated Akt protein densities between control (0 min) and treated cells. A representative results of three independent experiments. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; when compared with control. (B) Western blot analysis of cellular protein extracted from U87MG cells pretreated with increasing concentrations of LY294002 for 2 h, then treated or not with recombinant IL-22 for 20 min. Thirty mg of protein lysates was analyzed for P-Akt (Ser473) and total Akt. Actin was used as a loading protein control.</p

IL-22 activates the STAT3 signaling pathway in GBM cell lines.

<p>(A, B) The ability of IL-22 to activate signaling pathway in GBM cells was assessed using antibodies specific to STAT3 and Phospho-STAT3 (P-STAT3). U87MG (A) and U118MG (B) cells were stimulated with IL-22 and harvested at indicated times. Thirty mg of protein lysates was analyzed for P-STAT3 (Tyr705) and total STAT3 by western blot analysis. The density of each P-STAT3 band was corrected for variance in loading, using the density of the corresponding total STAT3. The expression level was evaluated as the ratio of phosphorylated STAT3 protein densities between control (0 min) and treated cells. Histograms are means ± SEM of three independent experiments. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001; when compared with control.</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