Figure 2: Hyperglycaemia inhibits IL-33 to decrease REG3A in diabetes.

(a) IL-17 production by ELISA in skin extracts taken from 2 mm skin surrounding the wound edges of normal and T1D mice at indicated times (n=4). (b) Immunoblot of REG3A in NHEKs stimulated by different doses of rhIL-33 for 12 h. (c) RegIIIγ and IL-33 production by ELISA in skin extracts taken as in a (n=4). (d,e) Immunohistochemical analyses of IL-33 in day-3 skin wounds of normal and T1D mice (d) or skin wounds of normal and diabetic patients taken as in Fig. 1b (e). Long scale bars represent 200 μm while short scale bars represent 50 μm. Black rectangles designate region of × 400 magnification shown in inset. (f) Immunoblot of RegIIIγ in the skin wounds of T1D mice treated with PBS or rmIL-33. The first two samples were day-3 skin wounds of normal and T1D mice. The other three samples were from the skin wounds of T1D mice treated with rmIL-33 a day before wounding (−1) or 1 or 3 days post wounding. (g) Wound healing of normal and T1D mice treated with (n=7) or without rmIL-33 (n=6). (h) IL-33 production in NHEKs induced by different doses of rhIL-17 for 24 h. (i) The production of IL-33 and RegIIIγ in day-3 skin wounds of wild-type (WT) and Il17^−/− mice. (j) REG3A production in NHEKs induced by 200 ng ml⁻¹ rhIL-17 before and after IL-33 was silenced. (k) Immunoblot of RegIIIγ in skin wounds of WT and Il17^−/− mice treated with PBS or 2 μg rmIL-33. (l) IL-33 production induced by 200 ng ml⁻¹ rhIL-17 in NHEKs exposed to 20 mM glucose or mannitol for 24 h. (m) Immunofluorescent staining of IL-33 in NHEKs treated as in l. Scale bars represent 25 μm. (n) IL-33 production in NHEKs treated with 200 ng ml⁻¹ rhIL-17 in the presence or absence of 20 mM glucose before and after AGE was inhibited by 2 mM aminoguanidine. The abbreviations used here are IL-33 shRNA (IL-33 sh), glucose (Glu), mannitol (Man), aminoguanidine (AG). *P<0.05, **P<0.01 and ***P<0.001. P values were determined by two-way analysis of variance (ANOVA). Data are means±s.e.m. and representative of two to three independent experiments.