Ingenol mebutate 0.05% gel treatment causes rapid infiltration of T-cells, macrophages, and neutrophilic granulocytes.

<p>Biopsy specimens from all patients (<i>n</i> = 26) were subjected to histopathological evaluation. Skin tissues from the two treatment areas and all time points were assessed, and representative images are depicted. The panels depict hematoxylin & eosin staining as well as immunohistochemical staining for CD4⁺ T-lymphocytes, CD8⁺ T-lymphocytes, CD68⁺ macrophages/histiocytes, and myeloperoxidase (MPO⁺) neutrophils as indicated. The scale bar represents 100 μm.</p

Tumor-Preferential Induction of Immune Responses and Epidermal Cell Death in Actinic Keratoses by Ingenol Mebutate

<div><p>The rapid and strong clinical efficacy of the first-in-class, ingenol mebutate, against actinic keratosis (AK) has resulted in its recent approval. We conducted the first comprehensive analysis of the cellular and molecular mode of action of topical ingenol mebutate 0.05% gel in both AK and uninvolved skin of 26 patients in a phase I, single-center, open-label, within-patient comparison. As early as 1 day after application, ingenol mebutate induced profound epidermal cell death, along with a strong infiltrate of CD4⁺ and CD8⁺ T-cells, neutrophils, and macrophages. Endothelial ICAM-1 activation became evident after 2 days. The reaction pattern was significantly more pronounced in AK compared with uninvolved skin, suggesting a tumor-preferential mode of action. Extensive molecular analyses and transcriptomic profiling of mRNAs and microRNAs demonstrated alterations in gene clusters functionally associated with epidermal development, inflammation, innate immunity, and response to wounding. Ingenol mebutate reveals a unique mode of action linking directly to anti-tumoral effects.</p><p><b><i>Trial Registration</i>:</b> ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT01387711" target="_blank">NCT01387711</a></p></div

IL-9 induces angiogenesis in mice and tube formation in HDMEC.

<p>(<b>A</b>) Representative photomicrographs of VEGF and CD31 staining of paraffin-embedded sections of skin from WT or K5.hTGF-β1 transgenic mice injected intradermally for 4 days with 500 ng of IL-9 or vehicle (PBS) (Scale bar 200 µm). (<b>B,C</b>) Semi-quantitative scoring of VEGF and CD31 positivity (n = 4 mice per group). (<b>D</b>) <i>In vitro</i> angiogenesis assay (tube formation) was performed with human dermal micro vascular endothelial cells (HDMEC) in the presence or absence of IL-9 (scale bars 100 µm). (<b>E</b>) Bifurcations were counted as a measure of blood vessel formation. Data are from one of two independent experiments (n = 5 random fields in each sample). Error bars represent SEM. *, p)0.05; ***, p)0.001. (unpaired t-test). Similar results were obtained in three independent experiments.</p

Ingenol mebutate 0.05% gel activates cutaneous blood vessels and induces epidermal cell death.

<p>Biopsy specimens from both treatment areas of all patients (<i>n</i> = 26) and all time points were assessed immunohistochemically for expression of CD20⁺ B-lymphocytes, ICAM-1 (CD54), and CD1a⁺ cells. Cleaved caspase 3 and TdT-mediated dUTP-biotin nick end labeling (TUNEL) reactivity indicating apoptotic responses were detected in five of these patients (arrows indicate examples of TUNEL positive epidermal cells). The figure depicts representative slides from all stainings as indicated. The scale bar represents 100 μm.</p

Topical treatment with ingenol mebutate 0.05% gel affects expression of gene clusters relevant for inflammatory and wound healing responses, and induces a characteristic microRNA (miRNA) pattern.

<p>The profiles illustrated are heat-maps and 2-way hierarchical clusterings of deregulated genes of <b>(A)</b> the 95 most variable mRNAs, and <b>(B)</b> the 64 most variable miRNAs across samples (<i>n</i> = 24) for the first six patients in the study. Differentially expressed genes and miRNAs were identified by pair-wise comparison of the following groups: actinic keratosis day 0 (AK0) versus uninvolved skin day 0 (US0), AK2 versus AK0, US2 versus US0, and AK2 versus US2. The expression analysis included variance filtering (VAR >0.2), statistical significance test by analysis of variance (<i>P</i> < 0.01), expression cut-off (>2-fold), and the false-discovery-rate was controlled by the Benjamini-Hochberg procedure (q < 0.05). Microarray data can be found in the GEO repository (GSE63107). Arrows indicate mRNA genes and miRNAs that were selected for validation by quantitative polymerase chain reaction. The colors above the heat map indicate: US0 (dark blue, <i>n</i> = 6), US2 (light blue, <i>n</i> = 6), AK0 (green, <i>n</i> = 6), and AK2 (red, <i>n</i> = 6) samples, respectively. The red and green shades on the heatmap represent up- and down-regulated genes, respectively.</p

IL-9 enhances IL-17A production in human psoriasis.

<p>(<b>A</b>) Immunohistochemical staining of IL-9R in normal and lesional human psoriatic skin. (Scale bar 100 µm for normal skin and 50 µm for psoriatic skin). (<b>B</b>) Number of IL-9R positive cells in lesional skin from psoriatic patients or healthy skin from normal control subjects (n = 3 subjects per group). (<b>C</b>) Detection of IL-9 protein level in the culture supernatant of activated and cultured CD4+ T cells of healthy or psoriatic human subjects by ELISA (n = 4 subjects per group). (<b>D</b>) Detection of IL-17A in the culture supernatant of activated and cultured PBMC and CD4+ T cells of healthy or psoriatic human subjects as determined by ELISA. Cells were stimulated with IL-9 or left unstimulated (n = 3–4 subjects per group). (<b>E</b>) Dual ELIspot assay for IL-17 and IFN-γ of activated and cultured CD4+ T cells of psoriatic subjects in the presence or absence of IL-9. Number of CD4+ T cells secreting IL-17 or IFN-γ, or co-secreting IL-17 and IFN-γ were counted in samples from psoriatic patients (n = 4). (<b>F</b>) Detection of IL-17A in activated and cultured CD4+ T cells of psoriatic patients by ELISA. Cells were stimulated with different cytokine combinations (n = 4 subjects per group). Error bars represent SEM. *, p)0.05; **, p)0.01; ***, p)0.001. (unpaired t-test). ND, not detected.</p

Topical ingenol mebutate 0.05% gel activates cutaneous blood vessels, attracts B-cells, and induces epidermal cell death.

<p>Automated histomorphometric analyses on all samples from all patients (<i>n</i> = 26) were performed to quantitate expression of CD1a⁺ dendritic cells <b>(A)</b>, CD20⁺ B-cells <b>(B)</b>, and CD54 (ICAM-1) expressing cutaneous blood vessels and inflammatory cells within the dermis <b>(C)</b>. Expression of both CD20 and CD54 increased rapidly and strongly upon treatment with ingenol mebutate, and actinic keratosis (AK) lesions showed generally higher expression levels compared with uninvolved skin. <b>(D)</b> Dyskeratotic (dead) keratinocytes within the epidermis of all samples (<i>n</i> = 26 patients; five biopsy specimens each) were assessed using a scoring system ranging from 0 (absent, not depicted), 1 (few dyskeratotic cells), 2 (several dead cells) to 3 (numerous necrotic cells). No necrosis was evident in the dermis. TdT-mediated dUTP-biotin nick end labeling (TUNEL)⁺ apoptotic cells <b>(E)</b> and cleaved caspase 3 (CC3)⁺ nuclei <b>(F)</b> were determined in biopsies from five patients. A full statistical overview is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160096#pone.0160096.s007" target="_blank">S1 Table</a>.</p

Involvement of IL-9 in Th17-Associated Inflammation and Angiogenesis of Psoriasis

<div><p>It is thought that a Th1/Th17-weighted immune response plays a predominant role in the pathogenesis of psoriasis. Our findings now indicate a link between IL-9, a Th2 and Th9 cytokine, and Th17 pathway in psoriasis. In K5.hTGF-β1 transgenic mice, exhibiting a psoriasis-like phenotype, we found increased IL-9R and IL-9 expression in the skin and intradermal IL-9 injection induced Th17-related inflammation. IL-9 also promoted angiogenesis and VEGF and CD31 overexpression in mice <em>in vivo</em> and increased tube formation of human endothelial cells <em>in vitro</em>. Injecting anti-IL-9 antibody into K5.hTGF-β1 transgenic mice not only diminished inflammation (including skin infiltration by T cells, monocytes/macrophages, and mast cells) and angiogenesis but also delayed the psoriasis-like skin phenotype. Notably, injection of anti-psoriatic acting anti-IL-17 antibody reduced skin IL-9 mRNA and serum IL-9 protein levels in K5.hTGF-β1 transgenic mice and prevented IL-9-induced epidermal hyperplasia and inflammation of the skin of wild type mice. In addition, we observed that IL-9R expression in lesional skin from psoriasis patients was markedly higher than in healthy skin from control subjects. Moreover, IL-9 significantly enhanced IL-17A production by cultured human peripheral blood mononuclear cells or CD4+ T cells, especially in psoriasis patients. Thus, IL-9 may play a role in the development of psoriatic lesions through Th17-associated inflammation and angiogenesis.</p> </div

IL-9 accelerates psoriasis-like inflammation in K5.hTGF-β1 transgenic mice.

<p>(<b>A</b>) Representative photomicrographs of immunohistochemical staining of IL-9 and IL-9R in the dorsal skin of K5.hTGF-β1 transgenic mice (Scale bar 200 µm for WT and 50 and 100 µm for K5.hTGF-β1 epidermis and dermis, respectively). (<b>B</b>) Real time PCR analysis for IL-9 in the dorsal skin of WT and K5.hTGF-β1 transgenic mice (n = 7 mice per group). (<b>C</b>) K5.hTGF-β1 transgenic mice were injected intradermally for 4 days with 500 ng of IL-9 or vehicle (PBS) and skin samples were collected 24 hours after the last IL-9 injection (C,D). Representative photomicrographs of HE-stained paraffin-embedded skin sections (Scale bar 200 µm). Histological quantification of mean epidermal thickness (n = 5 mice per group). (<b>D</b>) Dermal infiltration by CD3+ T cells, CD68+ monocytes/macrophages, and mast cells in the dorsal skin of WT mice (n = 5 mice per group). Data shown represent mean numbers of cells per ×200 microscopic field. Error bars represent SEM. *, p)0.05 (unpaired t-test). Similar results were obtained in two independent experiments.</p

Inflammatory skin reactions induced by ingenol mebutate 0.05% gel are more pronounced in actinic keratosis (AK)-lesioned areas compared with uninvolved skin.

<p><b>(A)</b> CONSORT study diagram. This was a phase I, single-center, open-label, within-patient comparison trial to explore the biological effects of ingenol mebutate gel applied once daily for 2 consecutive days in patients with actinic keratosis (AK) in a 25-cm² area on the extremities and a 25-cm² area of uninvolved-skin (US) on the inner upper arm. <b>(B)</b> Typical skin reactions during the course of the trial in AK treatment areas of three representative patients at the dorsum of the hand (upper panel) (<i>n</i> = 26) and uninvolved skin of the inner upper arm (lower panel) (<i>n</i> = 26) at day 0 (baseline), day 1 (after one treatment application), and day 2 (after two treatment applications) as well as during follow-up at days 8 and 29. <b>(B)</b> The composite local skin response (LSR) score is the sum of six individual LSR scores including erythema, flaking/scaling, crusting, swelling, vesiculation/pustulation, and erosion/ulceration, which range from 0 to 4, with higher numbers indicating more severe reactions. This was calculated at each study visit for each patient, with a theoretical maximum composite score of 24. Patients were assessed on days 0, 1, 2, 8, and 29. No unexpected signs of local or systemic toxicity were noted. Illustrated are averages of the LSR; error bars indicate standard error of the mean.</p