ΔNp63 Controls a TLR3-Mediated Mechanism That Abundantly Provides Thymic Stromal Lymphopoietin in Atopic Dermatitis

<div><p>In the skin lesions of atopic dermatitis (AD), keratinocytes release large quantities of thymic stromal lymphopoietin (TSLP), causing unfavorable inflammation along with skin damage. Nevertheless, how TSLP influences keratinocytes themselves is still unknown. In this study, we showed that ΔNp63, a p53-homologue, predominantly expressed in keratinocytes regulated the receptor complex of TSLP, which determines susceptibility to self-derived TSLP. Expression of TSLP receptors in skin tissues and keratinocytes was assessed by immunohistochemistry and quantitative RT-PCR, and <i>in vitro</i> studies were also performed to examine the functional relevance of ΔNp63 in the expression of TSLP receptors and the constituting autocrine and/or paracrine pathway of TSLP under the condition of stimuli to innate receptors sensing cell damage. The results showed that normal keratinocytes in the upper epidermis preferentially expressed TSLP receptors and conversely lacked ΔNp63, which has an inhibitory effect on the expression of TSLP receptors. Interestingly, the epidermis of AD lesions was found to abundantly contain keratinocytes with low or undetectable levels of ΔNp63 (ΔNp63^lo/-). Moreover, in the absence of ΔNp63, keratinocytes readily presented TSLP and other cytokines by stimuli through Toll-like receptor 3 (TLR3). Together with the evidence that extrinsic TSLP itself augments TSLP production by keratinocytes without ΔNp63, the results indicate that ΔNp63^lo/- keratinocytes generate TSLP through a putative autocrine and/or paracrine pathway upon TLR3 stimulation within AD lesions, since moieties of damaged cells and pathogens stimulate TLR3.</p></div

Schematic diagram of findings in this study.

<p>AD lesions possess many ΔNp63^lo/- keratinocytes, which might be considered as a major source of TSLP. A potential TLR3 ligand such as damaged-cell-derived dsRNA suppresses the expression of ΔNp63, which eventually further upregulates TSLPR and IL-7Rα and augments the susceptibility to TSLP in ΔNp63^lo/- keratinocytes. Since TSLP itself can help ΔNp63^lo/- keratinocytes to produce endogenous TSLP under the condition of TLR3 stimulation, an autocrine and/or paracrine loop of TSLP are generated in ΔNp63^lo/- keratinocytes, leading to undesired inflammation of AD lesions.</p

TSLP induces signal transduction in ΔNp63-deficient HaCaT keratinocytes.

<p>Immunoblot analysis demonstrating phosphorylation of the p65 subunit of NF-κB at 24 hr after treatment with 10 µg/ml poly I:C and exogenous TSLP (10 ng/ml). The histogram shows the relative phospho- NF-κB expression normalized to NF-κB as determined by densitometric analysis. β-actin was used as a loading control. Data are representative of three independent experiments.</p

TSLP enhances inflammatory responses in ΔNp63-deficient keratinocytes.

<p>(<b>a, b</b>) Quantitative RT-PCR (<b>a</b>) and ELISA (<b>b</b>) demonstrate the level of endogenous TSLP at 24 hr (<b>a</b>) or 72 hr (<b>b</b>) after treatment with exogenous TSLP (1 or 10 ng/ml) under the condition of TLR3 stimulation (5 µg/ml poly I:C) in primary keratinocytes. (<b>c, d</b>) Quantitative RT-PCR (<b>c</b>) and ELISA (<b>d</b>) show the level of endogenous TSLP at 24 hr (<b>c</b>) or 72 hr (<b>d</b>) after stimulation with 10 µg/ml poly I:C and 10 ng/ml TSLP in siControl and sip63 HaCaT keratinocytes. (<b>e</b>) Quantitative RT-PCR confirming whether neutralization of TSLP inhibits the generation of TSLP under the condition of TLR3 stimulation (5 µg/ml poly I:C) in primary keratinocytes. One-way ANOVA followed by Tukey's multiple-comparison test. *<i>P</i><0.05, ***<i>P</i><0.005 and N.S., not significant. Data are representative of at least three independent experiments.</p

Unique expression profiles of ΔNp63 in AD epidermal lesions.

<p>(<b>a</b>) Immunohistochemical staining of ΔNp63 in normal skin and skin lesions of AD and SLE. Consecutive sections of each condition were stained with hematoxylin and eosin (HE). Bar  = 50 µm. (<b>b</b>) Level of ΔNp63 was classified into three categories depending on staining intensity, as indicated by ΔNp63^hi, ΔNp63^lo and ΔNp63⁻. The dot plot represents ratios of ΔNp63^lo/- keratinocytes in normal skin (n = 3) and AD lesions (n = 10). (<b>c</b>) Quantitative RT-PCR demonstrating the levels of TSLP and ΔNp63 mRNAs in normal skin and AD lesions. Student's <i>t</i> test. *<i>P</i><0.05 and ****<i>P</i><0.001. Data are representative of at least three independent experiments.</p

Expression of TSLPR and IL-7Rα in epidermal keratinocytes is controlled by ΔNp63.

<p>(<b>a</b>) Immunofluorescence labeling confocal microscopy to localize TSLPR and IL-7Rα in the area of epidermis where ΔNp63 was not detected. Bar  = 20 µm. (<b>b</b>, <b>c</b>) Quantitative RT-PCR (<b>b</b>) and immunoblot analysis (<b>c</b>) against ΔNp63, TSLPR, IL-7Rα and TSLP in control siRNA (siControl) and p63-specific siRNA (sip63)-transfected HaCaT keratinocytes. β-actin was used as a loading control. The cells were harvested for assays at 72 hr after siRNA transfection. Student's <i>t</i> test. **<i>P</i><0.01 and N.S., not significant. (<b>d</b>) Immunofluorescence labeling fluorescence microscopy to localize TSLPR in siControl and sip63 HaCaT keratinocytes. The cells were fixed at 72 hr after siRNA introduction. DAPI, 4′,6-diamidino-2-phenylindole. Bar  = 50 µm. Data are representative of at least three independent experiments.</p

TLR3 stimulation alters the expression levels of AD-related molecules in primary keratinocytes.

<p>(<b>a</b>) Quantitative RT-PCR demonstrating the levels of ΔNp63, p73, TSLP, TSLPR and IL-7Rα mRNAs at 24 hr after treatment with 25 µg/ml poly I:C. One-way ANOVA followed by Tukey's multiple-comparison test. ***<i>P</i><0.005 and ****<i>P</i><0.001 versus control. (<b>b</b>) Immunoblot analysis showing the level of ΔNp63 at different time course after treatment with 25 µg/ml poly I:C. (<b>c</b>) Phase-contrast microscopy demonstrating the morphological changes of primary keratinocytes at 24 hr after 25 µg/ml poly I:C treatment. Bar  = 50 µm. Data are representative of three independent experiments.</p

Cholesterol accumulation in the livers of <i>Sik3</i>^−/− mice after feeding with a high-cholesterol diet.

<p>(A) Male mice were fed a 2% cholesterol diet for 4 months (12–30 weeks) and then sacrificed (n = 5). (B) HE staining of the liver (sets at the <i>upper</i> and <i>lower left</i>). The arrows indicate eosin-negative foci which with autofluorescence (<i>lower right</i>: red, and nuclei are blue (DAPI)). The magnification is the same in each set. (C) Cholesterol and TG levels in the liver and serum were measured (n = 5). *** indicates <i>p</i><0.001. Means and SEM are shown. (D) FPLC analysis of serum lipids. (E) Serum levels of alanine aminotransferase (ALT) were monitored at the indicated time points. * and ** indicate <i>p</i><0.05 and <i>p</i><0.01, respectively. (F) Quantitative polymerase chain reaction analysis of inflammatory molecules in the liver.</p

Gene expression profile in the liver.

<p>(A) One-year-old male mice (n = 5) were fasted for 4 h, and the liver mRNA levels were measured using quantitative polymerase chain reaction (qPCR). Red and blue indicate the up- and down-regulated genes in <i>Sik3</i>^−/− mice, respectively. +, fold increase; -, fold decrease. The threshold is set at <i>p = </i>0.1. The values marked with an asterisk (*) were obtained using PCR-array kits (n = 3). The abbreviations for the genes and the PCR primers used are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037803#pone.0037803.s007" target="_blank">Table S2</a>. Mit, mitochondria; TCA, tricarboxylic acid cycle. (B) Intracellular signaling molecules and their activation status in the liver were examined by western blot analysis. (C) Immunohistochemical analysis of SIK3 substrates (CRTC2 and HDAC5) in the liver.</p

<i>Sik3</i>^−/− mice are less tolerant to a cholic acid (CA)-containing diet.

<p>(A) Mice (n = 6, but <i>Sik3</i>^−/− mouse died before 1 month) were fed a diet supplemented with 0.25% cholic acid for 1 month (12–16 weeks) and then sacrificed. (B) HE staining of the liver (<i>left</i>), BSEP staining (<i>right</i>: BSEP is green and nuclei are blue (DAPI)). The magnification is the same in each set. (C) Photographs of gallbladders (scale, 1 mm). (D) The color of bile juice and bile sand in the gallbladder. (E) HE staining of the gallbladder. The magnification is the same in both panels. (F) The levels of bile acid (BA), cholesterol (Chol), and phospholipids (PL) in bile juice from the gallbladder were measured. * and ** indicate <i>p</i><0.05 and <0.01, respectively. Means and SEM are shown. (G) Serum BA and alanine aminotransferase (ALT) levels were monitored at the indicated periods. All ALT data points are <i>p</i><0.001, except day 0. (H) Serum alkaline phosphatase (ALP) and total bilirubin (T-Bil) levels were measured. (I) Cholesterol and TG levels in the liver and serum were measured. *** indicates <i>p</i><0.001. (J) FPLC analysis of serum lipids.</p