IL-22 derived from γδ T cells restricts Staphylococcus aureus infection of mechanically injured skin

Abstract

Background: Staphylococcus aureus is an opportunistic pathogen that colonizes the skin of patients with atopic dermatitis (AD) and aggravates their disease. Neutrophils and the cytokines IL-17A and IL-17F, which drive the expression of the neutrophil-attracting chemokines, are important for the clearance of S aureus infection. The cytokine IL-22 is often coproduced by IL-17-secreting cells. The levels of IL-22 are elevated in AD skin lesions.

Objective: We sought to determine the role of IL-22 in the clearance of S aureus infection of mouse skin subjected to tape stripping, a surrogate for scratching, a cardinal feature of AD.

Methods: S aureus was applied to the tape-stripped skin of wild-type and Il22^-/- mice. Bacterial burden was evaluated by enumerating colony-forming units. Quantitative PCR and ELISA were performed to quantify Il22 mRNA and IL-22 protein in mouse and human skin. Flow cytometry was used to enumerate neutrophils in the skin.

Results: Scratching the skin of healthy adults and tape stripping of mouse skin induced local expression of Il22 mRNA and IL-22 protein. Induction of Il22 expression by tape stripping was dependent on IL-23 and γδ T cells. Clearance of S aureus from tape-stripped skin was significantly impaired in Il22^-/- mice. Neutrophil infiltration and upregulation of expression of genes encoding the antimicrobial peptides antigen-6/urokinase-type plasminogen activator receptor related protein-1 and β-DEFENSIN 14 and the chemokine (C-X-C motif) ligand following tape stripping were significantly impaired in Il22^-/- mice.

Conclusions: These findings show that IL-22 is important for limiting the growth of S aureus on mechanically injured skin and caution that IL-23 and IL-22 blockade in patients with AD may enhance susceptibility to staphylococcal skin infection.

Keywords: AD; IL-22; S aureus; neutrophils.

Figure 1. Mechanical injury induces IL-22 expression in the skin

A. Il22 mRNA expression levels in the skin from WT (Balb/c) mice at 0, 3, 6, 12 and 24 hrs after tape-stripping, (n=3/group). The expression can be detected at 3 hrs, peaks at 6 hours and is undetectable (nd) at 24 hrs. B. IL-22 protein levels in the supernatants were quantitated after culturing the tape-stripped skin for 18hrs. C. Il22 mRNA expression in scratched versus unscratched human skin biopsies. One representative experiment out of two, with a total of 3 samples is shown. D. IL-22 protein levels quantitated by ELISA in supernatants from cultured scratched and control skin. Mean with SEM is shown *: p<0.05.

Figure 2. γδ T cells are the major IL-22 producing cells in response to mechanical skin injury

A. Il22 mRNA expression levels in the skin from unmanipulated WT (Balb/c), tape-stripped WT (Balb/c) mice, unmanipulated Rag2^-/- and tape-stripped Rag2^-/- mice (n=7/group). Il22 levels remain undetectable (nd) in unmanipulated skin. B. Il22 mRNA expression levels in the skin from unmanipulated WT (C57BL/6), tape-stripped WT mice, unmanipulated Tcrd^-/- and tape-stripped Tcrd^-/- mice (n=6, per group). C. Il22 mRNA expression levels quantitated in unmanipulated WT (C57BL/6), tape-stripped WT mice, unmanipulated Il23 (p19)^-/- and tape-stripped Il23 (p19)^-/- mice (n=3/ group). D. Il22 mRNA expression levels in unmanipulated WT (C57BL/6), tape-stripped WT mice, unmanipulated Rorc^-/- and tape-stripped Rorc^-/- mice (n=3/ group). Mean with SEM is shown *: p<0.05, **: p<0.01, ***: p<0.001

Figure 3. IL-22 limits cutaneous S. aureus infection

A. Schematic of S. aureus infection in tape-stripped mice is shown. B. Il22 mRNA expression determined at different times after tape stripping and after S. aureus infection (n=3/time-point). C. Representative pictures of WT and Il22^-/- mice, 5 days after S. aureus infection. D. Left, Bacterial colonies obtained from culture of skin homogenates from WT or Il22^-/- mice on Chrom-agar. Right, Bacterial CFU obtained from skin of WT or Il22^-/- mice (n=9 /group). Mean with SEM is shown *: p<0.05.

Figure 4. IL-22 is necessary for the early recruitment of neutrophils and the induction of Cxcl1, Cxcl2 and Cxcl3 expression

A. Schematic of S. aureus infection in neutrophil depleted mice. B. Left, Neutrophils, identified as CD11b⁺ Gr1^hi cells are shown in the tape stripped skin of isotype treated or anti-Ly6G treated mice. Right, Percentage of neutrophils in the two groups of mice (n=4/group). C. Bacterial CFUs enumerated from the skin biopsies of WT and neutrophil depleted mice. D. Left, Neutrophils quantified in the skin of WT or Il22^-/- mice in unmanipulated skin and 24 hrs after tape stripping, n=4/group. E. Neutrophil numbers in skin were quantitated in WT and Il22^-/- mice at indicated time points after tape stripping and following S. aureus infection (n=3/time-point). F. mRNA expression levels of chemokines Cxcl1, Cxcl2, Cxcl3 and Cxcl15 determined in unmanipulated and tape-stripped skin of WT or Il22^-/- mice, n=4/group. Mean with SEM is shown *: p<0.05, **: p<0.01, ***: p<0.001

Figure 5. γδ T cells are necessary for neutrophil recruitment and suppression of S. aureus infection in to tape-stripped skin of mice

A and B Percentages and numbers of neutrophils in unmanipulated and tape-stripped skin of Tcrd^-/- mice and WT controls, 24 hours after tape-stripping (n=3/group) *: p<0.05. C. Bacterial colonies obtained from homogenates of tape-stripped skin from Tcrd^-/- mice and WT controls cultured on Chrom-agar, 5 days after infection. D. Bacterial CFUs obtained from tape-stripped skin of Tcrd^-/- mice and WT controls (n=6/group). Mean with SEM are shown, p=0.058.

Figure 6. Reduced induction of Slurp1 and Defb14 anti-microbial peptides in WT and Il22^-/- mice upon tape stripping

A. mRNA expression levels of genes encoding antimicrobial peptides Camp and Defb1 from unmanipulated versus tape stripped WT and Il22^-/- mice. B. mRNA expression levels of genes encoding antimicrobial peptides Slurp1 and Defb14 from unmanipulated versus tape stripped WT and Il22^-/- mice. Mean with SEM is shown *: p<0.05, **: p<0.01, ***p<0.001, ****p<0.0001.