Journal: The Journal of Experimental Medicine

Article Title: IL-23 induced in keratinocytes by endogenous TLR4 ligands polarizes dendritic cells to drive IL-22 responses to skin immunization

doi: 10.1084/jem.20150376

Figure Lengend Snippet: IL-23 is essential for the IL-22 response to EC sensitization and for epidermal thickening and keratinocyte proliferation. (A and B) Serum IL-22 levels (A) and cytokine secretion OVA-stimulated skin DLN cells (B) in EC-sensitized Il23r −/− mice and WT controls. (C and D) Representative H&E staining and epidermal thickness (C) and representative immunohistochemical staining and numbers of Ki67 + cells in the epidermis (D) of skin from EC-sensitized Il23r −/− mice and WT controls. Bars: (C and D) 100 µm. (E) Cytokine secretion by skin DLN cells from EC-sensitized Il12p35 −/− mice and WT controls. Results in A represent 6 and 11 WT mice EC sensitized with saline and OVA, respectively, and 5 Il23r −/− mice EC sensitized with saline and OVA, respectively. Results in B–E represent five to six mice per group. Horizontal lines and vertical bars in A represent means and SEM, respectively. Columns and bars in B–E represent mean and SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant, two-tailed Student’s t test.

Article Snippet: Next, these cells were cultured in plate-bound antibodies against CD3 (145-2C11; 2 µg/ml; eBioscience) and soluble anti-CD28 (PV-1; 2 µg/ml; BioLegend), in the presence of IL-23 (40 ng/ml; R&D Systems), anti–IL-4 (11B11; 10 µg/ml; BioLegend), anti–IFN-γ (XMG1.2; 10 µg/ml; BioLegend), and anti–TGF-β1,2,3 (1D11; 10 µg/ml; R&D Systems) neutralizing antibodies to generate Th22 cells for 5 d. After extensive washings, 5 × 10 6 polarized T cells were adoptively transferred i.v. to naive WT or Il22 −/− recipients, which were challenged the same day by EC sensitization with OVA (100 µg/100 µl PBS).

Techniques: Staining, Immunohistochemical staining, Saline, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: IL-23 induced in keratinocytes by endogenous TLR4 ligands polarizes dendritic cells to drive IL-22 responses to skin immunization

doi: 10.1084/jem.20150376

Figure Lengend Snippet: 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.

Article Snippet: Next, these cells were cultured in plate-bound antibodies against CD3 (145-2C11; 2 µg/ml; eBioscience) and soluble anti-CD28 (PV-1; 2 µg/ml; BioLegend), in the presence of IL-23 (40 ng/ml; R&D Systems), anti–IL-4 (11B11; 10 µg/ml; BioLegend), anti–IFN-γ (XMG1.2; 10 µg/ml; BioLegend), and anti–TGF-β1,2,3 (1D11; 10 µg/ml; R&D Systems) neutralizing antibodies to generate Th22 cells for 5 d. After extensive washings, 5 × 10 6 polarized T cells were adoptively transferred i.v. to naive WT or Il22 −/− recipients, which were challenged the same day by EC sensitization with OVA (100 µg/100 µl PBS).

Techniques: Derivative Assay, Staining, Immunohistochemical staining, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: IL-23 induced in keratinocytes by endogenous TLR4 ligands polarizes dendritic cells to drive IL-22 responses to skin immunization

doi: 10.1084/jem.20150376

Figure Lengend Snippet: Tape stripping induces IL-23 expression in keratinocytes of epidermis. (A) Il23p19 mRNA expression in ear skin of BALB/c mice at 0, 6, and 24 h after tape stripping. Results are expressed as fold increase relative to nontape-stripped skin. (B) IL-23 release by explants of skin minced with scissors and cultured for 24 h with or without 10 µg/ml CHX. (C) Expression of Il23p19 mRNA (left) and Keratin 5 mRNA (right) in dermis and epidermis isolated from ear skin 6 h after tape stripping. Results are expressed as fold increase relative to epidermis of nontape-stripped skin. (D) Representative immunohistochemical staining for IL-23 in the skin. Staining with an isotype-matched irrelevant antibody (Ctrl IgG) was used as a negative control. Bars, 100 µm. (E) Representative FACS analysis of CD11c + Langerin + cells in ears from Langerin-eGFP-DTR mice treated by i.v. administration of DT or saline 24 h before sacrifice and qPCR analysis of Il23p19 mRNA expression 6 h after tape stripping the ear skin of Langerin-eGFP-DTR mice that were administered DT or saline 24 h before tape stripping. Results are expressed as fold increase relative to nontape-stripped skin of saline-treated Langerin-eGFP-DTR mice. Results represent at least 5 mice for each group in A–C and E and 3 mice in D. Columns and bars represent mean and SEM. *, P < 0.05; **, P < 0.01; ns, not significant, two-tailed Student’s t test.

Article Snippet: Next, these cells were cultured in plate-bound antibodies against CD3 (145-2C11; 2 µg/ml; eBioscience) and soluble anti-CD28 (PV-1; 2 µg/ml; BioLegend), in the presence of IL-23 (40 ng/ml; R&D Systems), anti–IL-4 (11B11; 10 µg/ml; BioLegend), anti–IFN-γ (XMG1.2; 10 µg/ml; BioLegend), and anti–TGF-β1,2,3 (1D11; 10 µg/ml; R&D Systems) neutralizing antibodies to generate Th22 cells for 5 d. After extensive washings, 5 × 10 6 polarized T cells were adoptively transferred i.v. to naive WT or Il22 −/− recipients, which were challenged the same day by EC sensitization with OVA (100 µg/100 µl PBS).

Techniques: Stripping Membranes, Expressing, Cell Culture, Isolation, Immunohistochemical staining, Staining, Negative Control, Saline, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: IL-23 induced in keratinocytes by endogenous TLR4 ligands polarizes dendritic cells to drive IL-22 responses to skin immunization

doi: 10.1084/jem.20150376

Figure Lengend Snippet: 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-γ 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 OVA 323-339 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.

Article Snippet: Next, these cells were cultured in plate-bound antibodies against CD3 (145-2C11; 2 µg/ml; eBioscience) and soluble anti-CD28 (PV-1; 2 µg/ml; BioLegend), in the presence of IL-23 (40 ng/ml; R&D Systems), anti–IL-4 (11B11; 10 µg/ml; BioLegend), anti–IFN-γ (XMG1.2; 10 µg/ml; BioLegend), and anti–TGF-β1,2,3 (1D11; 10 µg/ml; R&D Systems) neutralizing antibodies to generate Th22 cells for 5 d. After extensive washings, 5 × 10 6 polarized T cells were adoptively transferred i.v. to naive WT or Il22 −/− recipients, which were challenged the same day by EC sensitization with OVA (100 µg/100 µl PBS).

Techniques: Expressing, Cell Culture, Isolation, Derivative Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: IL-23 induced in keratinocytes by endogenous TLR4 ligands polarizes dendritic cells to drive IL-22 responses to skin immunization

doi: 10.1084/jem.20150376

Figure Lengend Snippet: 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-Cre Tg/0 Il23r flox/flox mice and Il23r flox/flox controls. (E) Cytokine secretion by CD4 + OT-II T cells cultured with DCs isolated from the skin DLN of Cd11c-Cre Tg/0 Il23r flox/flox mice and Il23r flox/flox controls 24 h after tape striping and OVA application. (F) Cytokine secretion by OVA-stimulated skin DLN cells from Cd11c-Cre Tg/0 Il23r flox/flox mice and Il23r flox/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-Cre Tg/0 Il23r flox/flox mice and Il23r flox/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.

Article Snippet: Next, these cells were cultured in plate-bound antibodies against CD3 (145-2C11; 2 µg/ml; eBioscience) and soluble anti-CD28 (PV-1; 2 µg/ml; BioLegend), in the presence of IL-23 (40 ng/ml; R&D Systems), anti–IL-4 (11B11; 10 µg/ml; BioLegend), anti–IFN-γ (XMG1.2; 10 µg/ml; BioLegend), and anti–TGF-β1,2,3 (1D11; 10 µg/ml; R&D Systems) neutralizing antibodies to generate Th22 cells for 5 d. After extensive washings, 5 × 10 6 polarized T cells were adoptively transferred i.v. to naive WT or Il22 −/− recipients, which were challenged the same day by EC sensitization with OVA (100 µg/100 µl PBS).

Techniques: Expressing, Cell Culture, Isolation, Staining, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: IL-23 induced in keratinocytes by endogenous TLR4 ligands polarizes dendritic cells to drive IL-22 responses to skin immunization

doi: 10.1084/jem.20150376

Figure Lengend Snippet: 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.

Article Snippet: Next, these cells were cultured in plate-bound antibodies against CD3 (145-2C11; 2 µg/ml; eBioscience) and soluble anti-CD28 (PV-1; 2 µg/ml; BioLegend), in the presence of IL-23 (40 ng/ml; R&D Systems), anti–IL-4 (11B11; 10 µg/ml; BioLegend), anti–IFN-γ (XMG1.2; 10 µg/ml; BioLegend), and anti–TGF-β1,2,3 (1D11; 10 µg/ml; R&D Systems) neutralizing antibodies to generate Th22 cells for 5 d. After extensive washings, 5 × 10 6 polarized T cells were adoptively transferred i.v. to naive WT or Il22 −/− recipients, which were challenged the same day by EC sensitization with OVA (100 µg/100 µl PBS).

Techniques: Expressing, Stripping Membranes, Mutagenesis, Injection, Derivative Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: IL-23 induced in keratinocytes by endogenous TLR4 ligands polarizes dendritic cells to drive IL-22 responses to skin immunization

doi: 10.1084/jem.20150376

Figure Lengend Snippet: IL-23 is released in human skin by scratching and polarizes human skin DCs to drive an IL-22 response. (A) IL-23 release by explants of unmanipulated human skin and skin obtained 6 h after scratching from the same individual. (B) FACS analysis of IL-23R expression by adult human blood DCs, and foreskin dermal DCs and epidermal LCs. (C) Secretion of IL-22, by allogeneic human naive CD4 + cells stimulated with anti-CD3– and anti-CD28–coated beads in the presence of rIL-23 pretreated and washed LCs from the skin or the blood of normal subjects. (D) Secretion of IL-13, IFN-γ, and IL-17A by allogeneic human naive CD4 + cells stimulated with anti-CD3– + anti-CD28–coated beads in the presence of rIL-23 primed LCs from human skin. Results in A, C, and D represent three independent experiments. Results in B represent two independent experiments each, including two to three subjects. Columns and bars represent mean and SEM. *, P < 0.05; ns, not significant, two-tailed Student’s t test.

Article Snippet: Next, these cells were cultured in plate-bound antibodies against CD3 (145-2C11; 2 µg/ml; eBioscience) and soluble anti-CD28 (PV-1; 2 µg/ml; BioLegend), in the presence of IL-23 (40 ng/ml; R&D Systems), anti–IL-4 (11B11; 10 µg/ml; BioLegend), anti–IFN-γ (XMG1.2; 10 µg/ml; BioLegend), and anti–TGF-β1,2,3 (1D11; 10 µg/ml; R&D Systems) neutralizing antibodies to generate Th22 cells for 5 d. After extensive washings, 5 × 10 6 polarized T cells were adoptively transferred i.v. to naive WT or Il22 −/− recipients, which were challenged the same day by EC sensitization with OVA (100 µg/100 µl PBS).

Techniques: Expressing, Two Tailed Test