CHS-induced skin inflammation in K14-VEGF-A-tg mice differentially alters tissue cytokines, lymphatic drainage and DC migration at the DAY 2 and DAY 9 time point.

<p>(A) Schematic representation of the experiment: K14-VEGF-A-tg mice were grouped into a control (CTR) group, a DAY 2 and a DAY 9 group. On day -14 and day -7, mice from the DAY 9 group or the DAY 2 group, respectively, were sensitized by application of oxazolone (OXA) onto the belly and paws (indicated as OXA-SENS). 5 days later (day -9 or day -2), a CHS response was induced when challenging mice by topical oxazolone application onto the ears (indicated as OXA-CHALL). Ears of mice from all groups were analyzed on day 0. Notably, day 0 corresponded to 2 days (DAY 2 group) or 9 days (DAY 9 group) after challenge, i.e. the onset of inflammation (marked as a red bar). (<b>B</b>) The ear thickness was assessed as a measure of the ongoing inflammatory response. (<b>C-E</b>) ELISAs were performed on ear tissue protein extracts and the levels of the inflammatory cytokines (<b>C</b>) TNFα, (<b>D</b>) IFNγ and (<b>E</b>) IL-17 were measured. (<b>F,G</b>) To evaluate lymphatic drainage function, Evans Blue dye was injected into the ear skin of K14-VEGF-A-tg mice, and the dye content remaining in the ear was extracted and quantified 16 hours later. (<b>F</b>) Representative pictures taken immediately after Evans Blue injection. (<b>G</b>) Quantification revealed increased Evans Blue levels in the ears of mice with DAY 2- and DAY 9- inflamed ears, indicative of reduced drainage. (<b>H-J</b>) To study DC migration, FITC painting experiments were performed in the uninflamed (CTR), DAY 2- or DAY 9-inflamed ears. 18 hours after FITC application, mice were killed and single-cell suspensions of the ear draining auricular LNs were analyzed by FACS. (<b>H</b>) The percentage of I-A/I-E⁺CD11c⁺FITC⁺ cells in the dLN was significantly reduced in LNs draining DAY 2- but not DAY 9-inflamed ear skin. (<b>I</b>) Quantification of total cell numbers revealed that I-A/I-E⁺CD11c⁺FITC⁺ DCs were significantly increased in LNs draining DAY-9 but not DAY 2-inflamed ear skin. (<b>J</b>) FACS-based quantification or ear skin single –cell suspensions revealed that CD11c^hi DCs were significantly increased in DAY 2-and DAY 9-inflamed ear skin. Representative data from 1 out of 3 similar experiments (n = 5 mice/group) are shown. *p<0.05; **p<0.01; ***p<0.001.</p

DCs in LNs draining CHS-inflamed skin in WT mice display changes in their expression of co-stimulatory and MHC molecules.

<p>FACS analysis was performed on CD11c⁺ DCs present in auricular LNs draining DAY 2- and DAY 9-inflamed or uninflamed control (CTR) skin of WT mice. (<b>A–G</b>) Analysis of the expression levels of co-stimulatory and MHC molecules. The upper panel shows representative FACS plots. Blue line: CTR; green line: DAY 2; red line: Day 9; filled histogram: isotype control. To reduce complexity, only one out of three similar isotype control stainings is shown. The lower graph shows the Δ MFI (defined as the MFI of the specific staining – the MFI of the isotype control staining) values measured for each condition (n = 3-4 mice/group). (<b>A</b>) CD80, (<b>B</b>) CD86, (<b>C</b>) OX40L, (<b>D</b>) PD-L1, (<b>E</b>) CD40, (<b>F</b>) H-2K^d and (<b>G</b>) I-A^d. Representative data from 1 out of 2 similar experiments are shown. *p<0.05; **p<0.01; ***p<0.001.</p

Skin inflammation enhances the induction of a DTH response to OVA in WT mice.

<p>(A) Schematic representation of the experiment: WT mice were grouped into a control (CTR) group, a DAY 2 and a DAY 9 group. On day -14 and day -7, mice from the DAY 9 group or the DAY 2 group, respectively, were sensitized by application of oxazolone (OXA) onto the belly and paws (indicated as OXA-SENS). 5 days later (day -9 or day -2) a CHS response was induced, by challenging mice from the DAY 9 or DAY 2 groups with oxazolone on the right ear (indicated as OXA-CHALL). In mice belonging to the DAY 9 group, OXA was repeatedly applied onto the right ear on day -7 and day -4, to maintain the inflammatory response. On day 0, corresponding to 2 days (DAY 2 group) and 9 days (DAY 9 group) after the onset of inflammation (marked as a red bar), a DTH experiment was initiated in mice from all treatment groups. To this end, all mice were immunized by OVA injection into the CHS-inflamed or control right ear (indicated as OVA-SENS). On day 7, a DTH response to OVA was induced by OVA injection into the left ear (indicated as OVA-CHALL). The strength of the immune response in the left ear was analyzed two days later (day 9). (<b>B</b>) The ear swelling response in the left ear was significantly stronger when mice had been immunized by OVA injection into the inflamed, right ear. The Δ ear thickness is shown, defined as the difference between the ear thickness measured at baseline and the ear thickness measured two days after DTH induction. CFA group: As a positive control group, some untreated mice were immunized by injection of OVA plus CFA into the uninflamed right ear (R) on day 0, followed by a regular OVA challenge into the left ear (L) on day 7 (as described in 3B). (<b>C–F</b>) FACS analysis was performed on single-cell suspensions of the DTH-challenged, left ear to quantify leukocyte infiltration. Quantification of (<b>C</b>) total leukocytes (CD45⁺), (<b>D</b>) neutrophils (CD11b⁺Gr1⁺), (<b>E</b>) CD4⁺CD3⁺CD45⁺ T cells and (<b>F</b>) CD8⁺CD3⁺CD45⁺ T cells. (<b>G</b>) Quantification of OVA-specific total IgG in the serum of mice. Representative data from 1 out of 3 similar experiments (n = 6–7 mice/group) are shown. *p<0.05; **p<0.01.</p

Skin inflammation enhances the induction of a DTH response to OVA in K14-VEGF-A-tg mice.

<p>(A) Schematic representation of the experiment: K14-VEGF-A-tg mice were grouped into a control (CTR) group, a DAY 2 and a DAY 9 group. On day -14 and day -7, mice from the DAY 9 group or the DAY 2 group, respectively, were sensitized by application of oxazolone (OXA) onto the belly and paws (indicated as OXA-SENS). 5 days later (day -9 or day -2), a CHS response was induced by challenging mice with oxazolone on the right ear (indicated as OXA-CHALL). On day 0, corresponding to 2 days (DAY 2 group) or 9 days (DAY 9 group) after the onset of inflammation (marked as a red bar), a DTH experiment was initiated in mice from all treatment groups. To this end, all mice were immunized by OVA injection into the CHS-inflamed or control right ear (indicated as OVA-SENS). On day 7, a DTH response to OVA was induced by OVA injection into the left ear (indicated as OVA-CHALL). The strength of the immune response in the left ear was analyzed two days later (day 9). (<b>B</b>) The ear swelling response in the left ear was significantly stronger in mice that had been immunized by OVA injection into the inflamed, right ear. The Δ ear thickness is shown, defined as the difference between the ear thickness measured at baseline and the ear thickness measured two days after DTH induction. CPG group: As a positive control group, some untreated control mice were immunized by injection of OVA plus CpG into their uninflamed right ears (R) on day 0, followed by a regular OVA challenge into the left ear (L) on day 7 (as described in 2A). (<b>C–F</b>) FACS analysis was performed on single-cell suspensions of the DTH-challenged left ear (L) to quantify leukocyte infiltration. Quantification of (<b>C</b>) total leukocytes (CD45⁺), (<b>D</b>) neutrophils (CD11b⁺.Gr1⁺), (<b>E</b>) CD4⁺CD3⁺CD45⁺ T cells and (<b>F</b>) CD8⁺CD3⁺CD45⁺ T cells. Representative data from 1 out of 3 similar experiments (n = 5 mice/group) are shown. *p<0.05; **p<0.01; ***p<0.001.</p

DCs isolated from LNs draining CHS-inflamed skin of WT mice are more potent inducers of T-cell activation in vitro.

<p>CD11c⁺ DCs were FACS-sorted from the auricular LNs of WT BALB/c mice with uninflamed (CTR) or DAY 2- or DAY 9-inflamed ear skin. Subsequently, CFSE-labeled TCR transgenic CD4⁺ T cells isolated from DO11.10 mice were incubated for 3 days with DCs and OVA peptide at a T cell: DC ratio of 5:1. CFSE dilution assays revealed that DCs isolated from LNs draining DAY 2- and DAY 9-inflamed skin were significantly more potent in inducing T cell proliferation than DCs isolated from control LNs. (<b>A</b>) Representative FACS plots showing CFSE dilution in dividing T cells. Numbers indicate the percentage of gated cells. (<b>B</b>) Quantification of the percentage of proliferating T cells. Significantly higher levels of (<b>C</b>) IFNγ, (<b>D</b>) IL-17 and (<b>E</b>) IL-4 were quantified in the supernatant of co-cultures containing DCs isolated from LNs draining DAY 2- or DAY 9-inflamed skin. Representative data from 1 out of 2 similar experiments (n = 3) per condition are shown. *p<0.05.</p

Blockade of IL-12/23-p40 reduces the induction of adaptive immune responses towards OVA in WT mice with DAY 9-inflamed ears.

<p>(A) Schematic representation of the experiment: WT mice were grouped into a control (CTR): IgG group, a DAY 9: IgG or a DAY 9: αp40 group. On day -14 mice in the DAY 9: IgG and DAY 9:αp40 groups were sensitized by application of oxazolone (OXA) onto the belly and paws (indicated as OXA-SENS). 5 days later (day -9) a CHS response was induced, by challenging mice with oxazolone on the right ear (indicated as OXA-CHALL). OXA was repeatedly applied onto the right ear on day -7 and day -4, to maintain the inflammatory response. On day -1 the DAY 9:αp40 group was treated with an IL-12/23-p40 blocking antibody (αp40). Mice in the DAY 9: IgG and the CTR: IgG groups were treated with an IgG control antibody (IgG). On day 0 a DTH experiment was initiated in mice from all treatment groups. To this end, all mice were immunized by OVA injection into the CHS-inflamed or control right ear (indicated as OVA-SENS). On day 10, a DTH response to OVA was induced by OVA injection into the left ear (indicated as OVA-CHALL). The strength of the immune response in the left ear was analyzed two days later (day 12). (B) The ear swelling response in the left ear was significantly weaker when mice had been treated with the anti-IL-12/23p40 antibody compared to mice treated with the corresponding isotype control. The Δ Ear thickness is shown, which is defined as the difference between the ear thickness measured at baseline and the ear thickness measured two days after DTH induction. FACS analysis was performed on single-cell suspensions of the DTH-challenged, left ear to quantify leukocyte infiltration. Quantification of (C) total leukocytes (CD45⁺), (D) neutrophils (CD11b⁺Gr1⁺ cells), (E) CD4⁺CD3⁺CD45⁺ T cells and (F) CD8⁺CD3⁺CD45⁺ T cells. (G) Quantification of serum OVA-specific IgG titer. Data from 2 pooled experiments (n = 12–14 mice/group) are shown. *p<0.05; **p<0.01; ***p<0.001.</p

Nrf2 links epidermal barrier function with antioxidant defense.

The skin provides an efficient permeability barrier and protects from microbial invasion and oxidative stress. Here, we show that these essential functions are linked through the Nrf2 transcription factor. To test the hypothesis that activation of Nrf2 provides skin protection under stress conditions, we determined the consequences of pharmacological or genetic activation of Nrf2 in keratinocytes. Surprisingly, mice with enhanced Nrf2 activity in keratinocytes developed epidermal thickening, hyperkeratosis and inflammation resembling lamellar ichthyosis. This resulted from upregulation of the cornified envelope proteins small proline-rich proteins (Sprr) 2d and 2h and of secretory leukocyte peptidase inhibitor (Slpi), which we identified as novel Nrf2 targets in keratinocytes. Since Sprrs are potent scavengers of reactive oxygen species and since Slpi has antimicrobial activities, their upregulation contributes to Nrf2's protective function. However, it also caused corneocyte fragility and impaired desquamation, followed by alterations in the epidermal lipid barrier, inflammation and overexpression of mitogens that induced keratinocyte hyperproliferation. These results identify an unexpected role of Nrf2 in epidermal barrier function, which needs to be considered for pharmacological use of Nrf2 activators

AutoTube: a novel software for the automated morphometric analysis of vascular networks in tissues

Due to their involvement in many physiologic and pathologic processes, there is a great interest in identifying new molecular pathways that mediate the formation and function of blood and lymphatic vessels. Vascular research increasingly involves the image-based analysis and quantification of vessel networks in tissue whole-mounts or of tube-like structures formed by cultured endothelial cells in vitro. While both types of experiments deliver important mechanistic insights into (lymph)angiogenic processes, the manual analysis and quantification of such experiments are typically labour-intensive and affected by inter-experimenter variability. To bypass these problems, we developed AutoTube, a new software that quantifies parameters like the area covered by vessels, vessel width, skeleton length and branching or crossing points of vascular networks in tissues and in in vitro assays. AutoTube is freely downloadable, comprises an intuitive graphical user interface and helps to perform otherwise highly time-consuming image analyses in a rapid, automated and reproducible manner. By analysing lymphatic and blood vascular networks in whole-mounts prepared from different tissues or from gene-targeted mice with known vascular abnormalities, we demonstrate the ability of AutoTube to determine vascular parameters in close agreement to the manual analyses and to identify statistically significant differences in vascular morphology in tissues and in vascular networks formed in in vitro assays