Table 1.
Primer sequences for real time PCR
Fig 1.
Wound healing is delayed in the epidermis of IL-4 Tg mice.
a) Representative photomicrographs of wounds from days 0 to 21 after injury. Six 3mm full thickness excisional wounds were made on the dorsal skin of IL-4 Tg and WT C57BL/j mice. Bar = 3mm. b) Percent of wound closure. Similar results were obtained in another experiment. c) Photomicrographs of HE stained histologic sections of unwounded skin, days 3, 7, and 21 post-wounding. Bar = 200μm. d) Rate of wound re-epithelialization measured by histomorphometric analysis of tissue sections. e & f) Epithelial thickness and wound/scar thickness respectively, based on HE stained sections. * p<0.05, # p<0.01, ** p<0.001 compared to IL-4 Tg mice at the same time point, respectively. The number of mice used at each time point was 5.
Fig 2.
Injury induces stronger keratinocyte proliferation and more angiogenesis in IL-4 Tg mice than in WT mice.
Three mm full thickness excisional wounds were made on dorsal skin of IL-4 Tg and WT mice. a) Percent of Ki67 positive keratinocytes. b, c, and d) EGF, IGF-1, and FGF-7 (KGF-1) mRNA expression, respectively. e) Vessel density expressed as the percent CD31 positive area in wounds. f) VEGF mRNA expression. * p<0.05 and # p<0.01 compared to the other group at the same time point, respectively. NS: normal or unwounded skin. The number of mice used at each time point was 5.
Fig 3.
Injury induces robust inflammatory cell infiltration in wounds of IL-4 Tg mice.
Three mm full thickness excisional wounds were made on dorsal skin of IL-4 Tg and WT mice. At various time points, frozen sections were prepared for neutrophil (Gr-1), macrophage (CD68), CD3, and DETC staining. Positively stained cells in the wounds and wound margins were counted and the average number per 20x field was calculated. a and b) Time course of the number of neutrophils and macrophages, respectively. c and d) YM1 and Arg1 mRNA expression in wounds respectively. e and f) Time course of the number of dermal CD3+ lymphocytes and DETC, respectively. * p<0.05, # p<0.01, and ** p<0.001compared to WT at the same time point, respectively. NS: normal or unwounded skin. The number of mice used at each time point was 5.
Fig 4.
Injury boosts inflammatory cytokine expression in wounds of IL-4 Tg mice.
Three mm full thickness excisional wounds were harvested at various time points and total RNA was extracted. After reverse transcription, expression of cytokine mRNAs was semi-quantified using real time PCR: a) IL-1β, b) IL-6, c) TNF-α, d) IL-4, e) IL-10, f) IL-13, g) IFN-γ, h) IL-12, i) TGF-β1, j) CXCL-2, k) CCL-2, l) NLRP3, * p<0.05, # p<0.01, and @ p<0.001 compared to the other group at the same time point, respectively. NS: normal or unwounded skin. The number of mice used at each time point was 5.
Fig 5.
Collagen synthesis and deposition.
a) Representative microscopic images of Masson’s Trichrome staining of collagen at days 10, 14 and 21 post-wounding as well as unwounded skin. Blue: stained collagen. b) Corresponding enlarged inserts of a). c) Quantification of blue stained collagen by ImageJ. # p<0.01 and ** p<0.001 compared to IL-4 Tg mice. d) mRNA expression of collagen I and III. * p<0.05 compared to unwounded skin, # p<0.01 compared to IL-4 Tg mice. e) Microscopic images of picrosirious red staining of mature (collagen I, red/orange) and immature collagen (collagen III, green). f) Percent of mature and immature collagen based on picrosirious red staining. # p<0.01 compared to the other group. The number of mice used at each time point was 5.
Fig 6.
a) Wound breaking strength. * p<0.05, # p<0.01. b and c) LOX and MMP-9 mRNA expression, respectively. * p<0.05, # p<0.01 compared to the other group at the same time point. NS: normal or unwounded skin. The number of mice used at each time point was 5.
Fig 7.
Effect of IL-4 or a cocktail of IL-4 and inflammatory cytokines on migration of skin keratinocytes and fibroblasts in vitro.
Scratch wounds were made on monolayers of NHEK and NHDF after mitomycin C treatment to prevent cell proliferation. Cells were then treated with human recombinant IL-4, a cocktail of human recombinant IL-1β, IL-4, IL-6, and TNF-α, or human EGF for 24 hours. The defined areas were photographed at 0 and 24 hours post injury. The numbers of migrated cells in the wounds were counted. Data represent the averages of triplicate wells; similar results were obtained in a replicate experiment. a) NHEK, * p<0.05 between EGF treated group and other groups. b) NHDF, # p<0.001, between EGF treated group and other groups. * p<0.05 between IL-4 and cocktail treated groups.