Phenotyping acute and chronic atopic dermatitis-like lesions in Stat6VT mice identifies a role for IL-33 in disease pathogenesis

The Stat6VT mouse model of atopic dermatitis (AD) is induced by T-cell-specific expression of a constitutively active form of the protein signal transducer and activator of transcription 6 (STAT6). Although AD-like lesions are known to develop in Stat6VT mice, this study was designed to determine if these mice develop acute and chronic phases of disease similar to humans. To address this, AD-like lesions from Stat6VT mice were harvested at two different timepoints relative to their onset. Lesions harvested within 1 week after development were defined as acute lesions, and those present for 1 month or more were defined as chronic lesions. Acute and chronic AD-like lesions from Stat6VT mice exhibited histologic findings and cytokine expression patterns similar to acute and chronic AD lesions in humans. Further analysis revealed increased levels of interleukin (IL)-33 transcripts in AD-like lesions compared to Stat6VT nonlesional and wild-type skin controls. Immunofluorescence also revealed increased numbers of IL-33+ keratinocytes in Stat6VT lesional skin and localized IL-33+ keratinocytes to a keratin 5+ subset. Furthermore, AD-like disease was more severe in IL-33-deficient Stat6VT mice compared to IL-33-sufficient Stat6VT mice. These studies suggest that Stat6VT mice can serve as a model of acute and chronic AD and that IL-33 may attenuate inflammation in this system

STAT6-mediated keratitis and blepharitis: a novel murine model of ocular atopic dermatitis

PURPOSE: Atopic dermatitis (AD) is a common inflammatory disease that can affect the eye, resulting in ocular pathologies, including blepharitis, keratitis, and uveitis; however, the pathogenic mechanisms underlying the ocular manifestations of AD are not well understood. METHODS: In the present study, we characterized the ocular pathologies that develop in the Stat6VT mouse model of AD. We examined the cytokine profile of the eyelid lesions, measured the behavioral response, and documented the treatment response to topical steroids. RESULTS: Our results show that Stat6VT mice spontaneously developed blepharitis, keratitis, and uveitis similar to that observed in patients with AD. Histologic findings of allergic inflammation in affected eyelids in this model include the presence of a lymphocyte-predominant infiltrate and tissue eosinophilia in the dermis. Gene expression analysis of affected eyelid tissue by quantitative PCR revealed increased amounts of mRNAs for the Th2 cytokines IL-4, IL-5, and IL-13. In addition, increased eyelid scratching was seen in Stat6VT mice with blepharitis. Topical treatment with the corticosteroid clobetasol reduced eyelid inflammation, tissue eosinophilia, and Th2 cytokine expression. CONCLUSIONS: The development of AD-like ocular pathologies in this model supports the idea that in humans, AD-associated disease of the eye may be driven by Th2-mediated inflammation and demonstrates that the Stat6VT mouse may be a useful system in which to further investigate pathogenesis of and treatment strategies for blepharitis and other ocular diseases that develop in association with AD

Topical application of a platelet activating factor receptor agonist suppresses phorbol ester-induced acute and chronic inflammation and has cancer chemopreventive activity in mouse skin.

Platelet activating factor (PAF) has long been associated with acute edema and inflammatory responses. PAF acts by binding to a specific G-protein coupled receptor (PAF-R, Ptafr). However, the role of chronic PAF-R activation on sustained inflammatory responses has been largely ignored. We recently demonstrated that mice lacking the PAF-R (Ptafr-/- mice) exhibit increased cutaneous tumorigenesis in response to a two-stage chemical carcinogenesis protocol. Ptafr-/- mice also exhibited increased chronic inflammation in response to phorbol ester application. In this present study, we demonstrate that topical application of the non-hydrolysable PAF mimetic (carbamoyl-PAF (CPAF)), exerts a potent, dose-dependent, and short-lived edema response in WT mice, but not Ptafr -/- mice or mice deficient in c-Kit (c-KitW-sh/W-sh mice). Using an ear inflammation model, co-administration of topical CPAF treatment resulted in a paradoxical decrease in both acute ear thickness changes associated with a single PMA application, as well as the sustained inflammation associated with chronic repetitive PMA applications. Moreover, mice treated topically with CPAF also exhibited a significant reduction in chemical carcinogenesis. The ability of CPAF to suppress acute and chronic inflammatory changes in response to PMA application(s) was PAF-R dependent, as CPAF had no effect on basal or PMA-induced inflammation in Ptafr-/- mice. Moreover, c-Kit appears to be necessary for the anti-inflammatory effects of CPAF, as CPAF had no observable effect in c-KitW-sh/W-sh mice. These data provide additional evidence that PAF-R activation exerts complex immunomodulatory effects in a model of chronic inflammation that is relevant to neoplastic development

Effect of CPAF treatment on ear thickness changes over an 18 day course of thrice weekly PMA applications in WT and <i>Ptafr</i>-/- mice.

<p><i>4A. Topical CPAF treatment suppresses PMA-induced changes in ear thickness in WT mice.</i> CPAF (6 nmoles) alone or PMA +/- CPAF were applied to WT mouse ears thrice weekly for 18 days. Skin thickness measurements were taken at time 0 and just prior to each reagent application. After subtraction of the time 0 ear thickness, ear thickness changes were plotted as the mean and SEM (n = 4–5 mice per group). PMA relative to PMA+CPAF (^a), PMA+CPAF relative to CPAF(^b); *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001; 2-tailed <i>t</i>-test. <i>4B. Topical CPAF treatment is ineffective in altering PMA-induced ear thickness changes in Ptafr-/- mice. Ptafr</i>-/- mice were treated and assessed as in 4A above. For the sake of comparison, the data for the PMA + CPAF treatment in WT mice is included. (mean and SEM; n = 4–5 mice per group). WT mice treated with PMA + CPAF exhibit a significant decrease in ear thickness measurements relative to <i>Ptafr</i>-/- mice treated with PMA + CPAF (*, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001; 2-tailed <i>t</i>-test). <i>4C. Topical CPAF treatment suppresses PMA-induced skin thickness increases in dorsal epidermis following 18 days of treatment.</i> The dorsal epidermis of SKH-1 mice was treated thrice weekly with vehicle, CPAF, PMA, or PMA + CPAF. Doses of PMA and CPAF were the same as that used for the tumorigenesis studies in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111608#pone-0111608-g003" target="_blank">Fig 3</a>. ***, <i>p</i><0.05 relative to PMA treated skin; 2-tailed <i>t</i>-test (n = 3 per group).</p

Dose and time related acute ear inflammation changes in response to topical CPAF.

<p><i>1A</i>. <i>Topical CPAF dose-dependently induces rapid inflammatory responses as measured by ear thickness measurements.</i> One ear of WT and <i>Ptafr</i> (-/-) were treated with one of three doses of CPAF (20 µl of a 0.1, 0.3, and 1.0 mM solution for a total treatment dose of 2, 6, or 20 nmole CPAF per ear). The contralateral ear was treated with acetone alone (VEH). Ear thickness was measured prior to treatment and 2 hours after treatment. After the pretreatment ear thickness values were subtracted, the mean and SEM were plotted (n = 4 for 20 nmole and n = 8 for 2 & 6 nmole CPAF & VEH treated mouse ears). <i>1B</i>. <i>Topical CPAF treatment induces a rapid, but transient increase in inflammation as measured by ear thickness changes.</i> One ear of wildtype (WT) and <i>Ptafr</i> (-/-) mice was treated with 20 µl of CPAF (20 nmoles of a 0.1 mM solution in acetone) and 20 µl of acetone (VEH) was applied to the contralateral ear. Ear thickness was measured just prior to reagent application and at 1, 2, 4, and 8 hours after application. Results represent the mean and SEM (n = 4 mice) after subtracting the pretreatment ear thickness. CPAF induced a significant increase in ear thickness in WT mice relative to the WT+VEH treated ears. *, <i>p</i><0.05; **, <i>p</i><0.01; ***; <i>p</i><0.001; 2-tailed <i>t</i>-test.</p

CPAF induces transient ear thickness changes are blocked in c-Kit^W-sh/W-sh mast cell deficient mice.

<p>WT and c-Kit^W-sh/W-sh mice were treated with vehicle (VEH) alone on one ear, and 20 ml of 0.3 mM CPAF (6 nmole) on the contralateral ear. Ear thickness was measured both prior to and 2 hrs after reagent application. After subtraction of the ear thickness at time 0, the mean and SEM were plotted (n = 5 for WT mice, n = 4 for Kit^W-sh/W-sh mice). *, <i>p</i><0.05; 2-tailed <i>t</i>-test.</p

Topical application of CPAF suppresses DMBA/PMA-induced tumorigenesis.

<p><i>3A. Topical treatment with CPAF suppresses DMBA/PMA-induced tumor multiplicity.</i> SKH-1 mice were treated once with DMBA+/- CPAF, then with PMA or PMA+CPAF for 25 weeks. Durable tumors were counted on a weekly basis. Tumor multiplicity (Avg tumor number per mouse was plotted at each week. Results represent the mean and SEM for n = 19–20 mice/group. <i>p</i><0.05 for weeks 9–12,14–25; <i>p</i><0.01 for week 13; Mann-Whitney U test. <i>3B. Topical CPAF delayed tumor incidence in mice treated with DMBA/PMA.</i> The percent of mice remaining tumor free over the 25 week chemical tumorigenesis study were plotted using a survival curve. The treatment with CPAF resulted in a significant change in the tumor incidence, with a median time until first tumor occurrence of 8 weeks for DMBA/PMA treated and 9 weeks for DMBA/PMA+CPAF treated mice. *, <i>p</i><0.05; Log-rank (Mantel-Cox) Test. <i>3C</i>. <i>Topical CPAF treatment results in a smaller number of large tumors (≥3 mm in greatest diameter) after 25 weeks of treatment.</i> Tumor size distribution was plotted as the number of tumors in each size distribution for each treatment group. Histopathologic exam showed no significant difference in the rates of papilloma and SCC formation between the treatment groups (not shown). ***, <i>p</i> = 0.0001 Fisher's exact test. <i>3D</i>. <i>DMBA/PMA-induced MPO activity is suppressed by CPAF.</i> After the mice were euthanized following 25 weeks of DMBA/PMA +/- CPAF treatment, tumor free areas of skin were excised and MPO activity was assessed in tissue lysates. After normalization to total protein, MPO activity was plotted as the mean and SEM (n = 5–9 mice per group). **, <i>p</i><0.05; ***, <i>p</i><0.001; 2-tailed <i>t</i>-test.</p

Phenotyping acute and chronic atopic dermatitis-like lesions in Stat6VT mice identifies a role for IL-33 in disease pathogenesis

The Stat6VT mouse model of atopic dermatitis (AD) is induced by T-cell-specific expression of a constitutively active form of the protein signal transducer and activator of transcription 6 (STAT6). Although AD-like lesions are known to develop in Stat6VT mice, this study was designed to determine if these mice develop acute and chronic phases of disease similar to humans. To address this, AD-like lesions from Stat6VT mice were harvested at two different timepoints relative to their onset. Lesions harvested within 1 week after development were defined as acute lesions, and those present for 1 month or more were defined as chronic lesions. Acute and chronic AD-like lesions from Stat6VT mice exhibited histologic findings and cytokine expression patterns similar to acute and chronic AD lesions in humans. Further analysis revealed increased levels of interleukin (IL)-33 transcripts in AD-like lesions compared to Stat6VT nonlesional and wild-type skin controls. Immunofluorescence also revealed increased numbers of IL-33+ keratinocytes in Stat6VT lesional skin and localized IL-33+ keratinocytes to a keratin 5+ subset. Furthermore, AD-like disease was more severe in IL-33-deficient Stat6VT mice compared to IL-33-sufficient Stat6VT mice. These studies suggest that Stat6VT mice can serve as a model of acute and chronic AD and that IL-33 may attenuate inflammation in this system