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

Abstract

Introduction: Although 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.

Methods: We 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.

Results: Aseptic 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.

Conclusion: These 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.

Keywords: Th17; cytokine; inflammation; pathogen; skin; wound healing.

Figure 1

Wound closure is delayed in infected skin excisional wounds.(A)Time course of wound closure expressed as the percentage of initial wound area at each time point, in the control wound (□) or wound infected with a mixture of S. aureus and P. aeruginosa (■). The data are expressed as the mean +/- SEM of 5 independent experiments, with at least 4 mice/group; ** p<0.01, ***p<0.001. (B) Representative photographs of the control and infected wounds at days 0, 2, 4, 6, 8, 10, 12 and 14 after injury..

Figure 2

IL-17 and IL-22 are produced by T cells in infected skin excisional wounds. (A) Cytokine mRNA expression into control or S. aureus and P. aeruginosa infected wounds were determined by RT-qPCR at day 2 and day 7. Each bar represents the mRNA fold increase over normal skin, in control-wounded skin (white) or infected wounded skin (black). The data correspond to the mean +/- SEM of 4 independent experiments; * p<0.05, ** p<0.01, ***p<0.001; (B) Five days after wound infection by the bacterial mixture (infected, I) or application of a control PBS solution (uninfected, C), an 8 cm² biopsy of back skin including 4 excisional wounds was collected and underwent mechanical and enzymatic treatment for cell dissociation before analysis by flow cytometry. (B, left) Representative gating strategy to analyze IL-17 and IL-22 expressions in CD3-positive T cells and TCRγδ expression among this IL-17⁺ and/or IL-22⁺, CD3⁺ T cell population. (B, right) Quantitative analysis of the number of IL-17-, IL-22- or IL-17/IL-22-positive T cells infiltrating infected or non-infected wounded skins. Data (mean +/- SEM) are representative of one out from three independent experiments including 5 mice per group. *p < 0.05. (C) IL-17A, IL-22, OSM, IL-1β and TNFα mRNA levels were determined by quantitative PCR in human wounds. Each bar represents the mRNA fold increase over normal human skin in uninfected (white) or infected wounded skin (black). The data correspond to the mean +/- SEM of 12 uninfected and 7 infected skin wound samples; * p<0.05, ** p<0.01, ***p<0.001. ns, not significant..

Figure 3

Combination of IL-17 and IL-22 are required for delayed closure in an infected skin wound. Time course of wound closure expressed as the percentage of initial wound area at each time point, (A) in the uninfected or infected wounds of WT mice (control □; infected ■) or IL-17R/IL-22KO mice (control ○; infected ●). (B) in the uninfected wound of WT mice injected with IL-17A (♦), IL-22 (▼). (C) in the uninfected wound of WT mice injected with IL-17A and IL-22 (▲), mix of IL-17A, OSM, TNFα, IL-22 and IL-1α (●) or PBS as a control (□). The data are expressed as the mean +/- SEM of 5 independent experiments, with at least 4 mice/group. * p<0.05, ** p<0.01, ***p<0.001..

Figure 4

Wound closure is not delayed in infected skin excisional wounds of Rag2KO mice, and exogenous IL-17A/F and IL-22 make up for this delay. Time course of wound closure expressed as the percentage of initial wound area at each time point, (A) in the uninfected (□) or infected (■) wounds of WT mice, infected Rag2KO mice (○) or infected Rag2KO mice with exogenous IL-17A and IL-22 (●). The data are expressed as the mean +/- SEM of 6 independent experiments, with at least 4 mice/group; ** p<0.01, ***p<0.001. ns, not significant. (B) Cytokine mRNA expression into control or infected wounds of Rag2KO mice was determined by RT-qPCR at day 2 and day 7. Each bar represents the mRNA fold increase over normal skin, in control-wounded skin (white) or infected wounded skin (black). The data correspond to the mean +/- SEM of 3 independent experiments; * p<0.05, ** p<0.01, ***p<0.001. ns, not significant. (C) BD3, S100A9, CXCL2, GR1 mRNA expression into control or infected wounds of WT and Rag2KO mice were determined by RT-qPCR at day 2 and day 7. Each bar represents the mRNA fold increase over normal skin in control-wounded WT mice skin (white), infected wounded WT mice skin (hatched), in control-wounded Rag2KO mice skin (dotted), infected wounded Rag2KO mice skin (black). The data correspond to the mean +/- SEM of 3 independent experiments; * p<0.05, ** p<0.01, ***p<0.001. ns, not significant. (D) The bacterial load after inoculation in skin wounds is unchanged between WT and Rag2KO mice. CFU determination for S. aureus and P. aeruginosa in wounded WT and Rag2KO mice skin 2 and 7 days after inoculation of S. aureus and P. aeruginosa. The data correspond to the mean +/- SEM of 3 independent experiments, ns, not significant.