Ell-dependent inflammation [9]. CCR2 is expressed on innate cells as well as activated Th17 cells, and so we examined whether CCR2 deficiency reduced IL-22 production in IL-23-injected ears. However, real-time RT-PCR analysis did not 548-04-9 reveal any significant difference in the expression of IL-22 in the ears of WT and CCR22/2 mice either early (day 6) or late (day 12) following the initiation of intradermal IL-23 injections (Figure 5). Recent studies demonstrate that IL-22-producing T cells are present in both psoriatic and chronic atopic dermatitis lesions [48,49]. IL-22 induces keratinocyte proliferation and epidermal hyperplasia, and so may contribute to the epidermal thickening that we observe in both IL-23-injected WT and CCR22/2 skin.Figure 1. IL-23 AVP custom synthesis injection induces increased inflammation in CCR22/2 mice compared to WT mice. Ears of CCR22/2 and WT mice were injected every other day with 20 mL PBS alone or containing 500 ng IL-23. Ear thickness was measured one day following each injection. Data are from at least 13 mice/group total in three separate experiments. *p,0.01 versus all other groups. doi:10.1371/journal.pone.0058196.gIL-23 Induces Th2 Inflammation in CCR22/2 MiceFigure 2. Eosinophils and mast cells accumulate in ears of IL-23-injected CCR22/2 mice. (A) H E-stained sections of ears from IL-23injected WT and CCR22/2 mice at day 12. a, acanthosis; h, hyperkeratosis; p, parakeratosis; o, orthokeratosis; d, dermal inflammatory infiltrate; s; spongiosis; m, intracorneal microabscess. Enlargements of the boxed areas within the WT IL-23 or CCR22/2 IL-23 image are displayed below the corresponding photo. Black arrows indicate eosinophils. Green arrows indicate neutrophils. (B) Toluidine blue-stained sections of ear from IL-23injected WT and CCR22/2 mice at day 12. Arrows indicate mast cells. doi:10.1371/journal.pone.0058196.gIL-23 Induces Th2 Inflammation in CCR22/2 MiceFigure 3. Increased epidermal thickness and accumulation of eosinophils and mast cells in ears of 
IL-23-injected CCR22/2 compared to WT mice. (A) Average epidermal thickness was measured on 1407003 H E-stained sections. *p,0.02. Data are from two independent experiments with three-four mice/group. (B) Ears were digested with collagenase and recovered leukocytes were analyzed by flow cytometry to determine numbers of inflammatory dendritic cells (CD11c+ CD11b+ Ly6c+) within ears of IL-23 injected WT and CCR22/2 mice. *p,0.05. Data are from one experiment with three mice/group. Average percent neutrophils (C) and eosinophils 
(D) among leukocytes in H E sections of ears from IL23-injected WT and CCR22/2 mice at day 12. (E) Average percent mast cells among leukocytes in toluidine blue sections of ears from IL-23-injected WT and CCR22/2 mice at day 12. Slides were imaged (x200, original magnification) and numbers of eosinophils or neutrophils among leukocytes were counted. Data are from four mice/genotype with at least three fields counted per mouse. *p,0.05; **p,0.005. doi:10.1371/journal.pone.0058196.gCutaneous Expression of TSLP and IL-4 is Increased in CCR22/2 MicePrevious studies of inflammatory responses in CCR22/2 mice have observed increased Th2 cytokine production, with a corresponding decrease in Th1 cytokine expression. Although we did not detect a significant difference in the expression of IL-22 in the IL-23-injected ears of WT and CCR22/2 mice, we reasoned that a decrease in other Th17 or in Th1 cytokines, or an increase in Th2 cytokines might explain the increase.Ell-dependent inflammation [9]. CCR2 is expressed on innate cells as well as activated Th17 cells, and so we examined whether CCR2 deficiency reduced IL-22 production in IL-23-injected ears. However, real-time RT-PCR analysis did not reveal any significant difference in the expression of IL-22 in the ears of WT and CCR22/2 mice either early (day 6) or late (day 12) following the initiation of intradermal IL-23 injections (Figure 5). Recent studies demonstrate that IL-22-producing T cells are present in both psoriatic and chronic atopic dermatitis lesions [48,49]. IL-22 induces keratinocyte proliferation and epidermal hyperplasia, and so may contribute to the epidermal thickening that we observe in both IL-23-injected WT and CCR22/2 skin.Figure 1. IL-23 injection induces increased inflammation in CCR22/2 mice compared to WT mice. Ears of CCR22/2 and WT mice were injected every other day with 20 mL PBS alone or containing 500 ng IL-23. Ear thickness was measured one day following each injection. Data are from at least 13 mice/group total in three separate experiments. *p,0.01 versus all other groups. doi:10.1371/journal.pone.0058196.gIL-23 Induces Th2 Inflammation in CCR22/2 MiceFigure 2. Eosinophils and mast cells accumulate in ears of IL-23-injected CCR22/2 mice. (A) H E-stained sections of ears from IL-23injected WT and CCR22/2 mice at day 12. a, acanthosis; h, hyperkeratosis; p, parakeratosis; o, orthokeratosis; d, dermal inflammatory infiltrate; s; spongiosis; m, intracorneal microabscess. Enlargements of the boxed areas within the WT IL-23 or CCR22/2 IL-23 image are displayed below the corresponding photo. Black arrows indicate eosinophils. Green arrows indicate neutrophils. (B) Toluidine blue-stained sections of ear from IL-23injected WT and CCR22/2 mice at day 12. Arrows indicate mast cells. doi:10.1371/journal.pone.0058196.gIL-23 Induces Th2 Inflammation in CCR22/2 MiceFigure 3. Increased epidermal thickness and accumulation of eosinophils and mast cells in ears of IL-23-injected CCR22/2 compared to WT mice. (A) Average epidermal thickness was measured on 1407003 H E-stained sections. *p,0.02. Data are from two independent experiments with three-four mice/group. (B) Ears were digested with collagenase and recovered leukocytes were analyzed by flow cytometry to determine numbers of inflammatory dendritic cells (CD11c+ CD11b+ Ly6c+) within ears of IL-23 injected WT and CCR22/2 mice. *p,0.05. Data are from one experiment with three mice/group. Average percent neutrophils (C) and eosinophils (D) among leukocytes in H E sections of ears from IL23-injected WT and CCR22/2 mice at day 12. (E) Average percent mast cells among leukocytes in toluidine blue sections of ears from IL-23-injected WT and CCR22/2 mice at day 12. Slides were imaged (x200, original magnification) and numbers of eosinophils or neutrophils among leukocytes were counted. Data are from four mice/genotype with at least three fields counted per mouse. *p,0.05; **p,0.005. doi:10.1371/journal.pone.0058196.gCutaneous Expression of TSLP and IL-4 is Increased in CCR22/2 MicePrevious studies of inflammatory responses in CCR22/2 mice have observed increased Th2 cytokine production, with a corresponding decrease in Th1 cytokine expression. Although we did not detect a significant difference in the expression of IL-22 in the IL-23-injected ears of WT and CCR22/2 mice, we reasoned that a decrease in other Th17 or in Th1 cytokines, or an increase in Th2 cytokines might explain the increase.
