Fig 2: TLR4 is essential for the induction of IL-23 expression in mouse skin by tape stripping and for the IL-22 response to EC immunization. (A–D) Il23p19 mRNA expression 6 h after tape stripping of the ear skin of WT mice housed under GF versus specific pathogen–free (SPF) conditions (A); Myd88−/− and Trif−/− mice and WT controls (B); Il1r1−/−, Il18r−/−, Il1rl1−/−, and Il1r1−/− mice treated with anti-ST2 and anti–IL-18–neutralizing mAbs or control IgG (C); Tlr2−/−, Tlr5−/−, Unc93b−/−, and TLR4 mutant (mut.), C3HeJ mice, and WT controls (D). (E) S100a8 and S100a9 mRNA expression 6 h after tape stripping of ear skin. (F) Il23p19 mRNA expression 3 h after i.d. injection of low MW HA or rS100a8 in the back skin of TLR4 mutant mice and WT controls. (G) Il23p19 mRNA expression in Pam212 cells stimulated for 6 h with low MW HA with or without preincubation with the indicated inhibitors of NFKβ (BAY-11-7082), p38 (SB 203580), ERK (U-0126), or JNK (SP600125). Results in A–F are derived from at least five mice for each group. Results in G represent four independent experiments. Columns and bars in A–F and horizontal and vertical bars in G represent mean and SEM. *, P < 0.05; **, P < 0.01; ns, not significant, two-tailed Student’s t test.

Fig 3: IL-23 in mechanically injured skin polarizes migratory skin DCs to drive an IL-22 response by inducing endogenous IL-23 expression. (A) Cytokine secretion of IL-22 and IL-17A by CD4+ OT-II T cells cultured with DCs isolated from skin DLN of WT or Il23r-/- mice and pretreated for 24 h with skin explant–conditioned medium (SCM) harvested from cultured explants of tape-stripped (+) or with medium (-) as control. (B) Cytokine secretion of IL-22 and IL-17A by CD4+ OT-II T cells cultured with DCs isolated from skin DLN of WT mice and pretreated for 24 h with conditioned medium harvested from cultured epidermal layer explants of tape-stripped (+) or unmanipulated (-) skin from WT and Il23p19-/- mice. (C) Cytokine secretion by CD4+ OT-II T cells cultured with DCs isolated from the skin DLN of WT or Il23r-/- mice 24 h after tape striping and OVA application. No exogenous OVA were added to the cultures. (D) FACS analysis of IL-23R expression by DCs from the skin of Cd11c-CreTg/0Il23rflox/flox mice and Il23rflox/flox controls. (E) Cytokine secretion by CD4+ OT-II T cells cultured with DCs isolated from the skin DLN of Cd11c-CreTg/0Il23rflox/flox mice and Il23rflox/flox controls 24 h after tape striping and OVA application. (F) Cytokine secretion by OVA-stimulated skin DLN cells from Cd11c-CreTg/0Il23rflox/flox mice and Il23rflox/flox controls that were EC sensitized with OVA for 7 wk. (G) Representative H&E staining and epidermal thickness of skin in OVA EC-sensitized Cd11c-CreTg/0Il23rflox/flox mice and Il23rflox/flox controls. Bar, 100 µm. Results in A-G represent four to six mice for each group. Columns and bars represent mean and SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant, two-tailed Student’s t test.

Fig 4: IL-23 derived from both nonhematopoietic and hematopoietic cells is important for the IL-22 response to EC sensitization. (A and B) Cytokine secretion by OVA-stimulated skin DLN cells from WT?p40-/-, p40-/- ?WT, and WT?WT control chimeras. (A and B) Representative H&E staining of the skin and epidermal thickness (C and E) and representative immunohistochemical staining of the skin and numbers of Ki67+ cells in the epidermis (D and F) in EC-sensitized WT?p40-/-, p40-/-?WT, and WT?WT control chimeras. Bars: (C–F) 100 µm. Results in A–F represent five to six mice per group. Columns and bars in A–F represent mean and SEM. *, P < 0.05; **, P < 0.01; ns, not significant, two-tailed Student’s t test.

Fig 5: IL-23 polarizes DCs from skin DLN to drive IL-22 production by naive CD4+ T cells by inducing them to express endogenous IL-23 expression. (A) qPCR analysis of Il23r mRNA expression by CD11c+ DCs from spleen, skin and skin DLN. Results as expressed relative to splenic DCs. (B) Representative FACS analysis of IL-23R expression by CD11c+MHCII+/high DCs and percentage of IL-23R+CD11c+MHCII+/high DCs in spleen, skin, and skin DLN from WT mice. (C) IL-22 secretion by CD4+ OT-II cells cultured with DCs isolated from spleen, skin, and skin DLN of WT mice pretreated with rIL-23 or medium for 24 h. (D) IL-17A, IL-13, and IFN-? secretion by CD4+ OT-II T cells cultured with rIL-23 primed DCs isolated from the skin DLN of WT mice. (E) Expression of Il23p19 mRNA by skin DLN DCs from WT mice cultured for 3 h with rIL-23. (F) IL-23 secretion by DCs from skin DLN of WT mice after preincubation with rIL23 for 16 h, followed by extensive washing and culture for the indicated times. (G) Effect of IL-23 neutralizing antibody or its isotype control on the ability of IL-23 primed and washed DCs isolated from skin DLN to drive IL-22 production by naive CD4+ OTII T cells. (H) IL-22 production by naive CD4+ OTII T cells stimulated with OVA323-339 peptide and IL-23 primed DCs derived from the skin DLN of WT or Il23p19-/- mice. Results in A–H represent DCs obtained from four to six mice for each group. Columns and bars represent mean and SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant, two-tailed Student’s t test.