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

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 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

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 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