Effects of inhaled corticosteroids on the expression of TNF family molecules in murine model of allergic asthma

<p><b>Background:</b> The tumor necrosis factor superfamily member LIGHT (the official gene symbol approved by NCBI Gene Database), an inflammatory factor secreted by T cells after allergen exposure, recently discovered to play crucial roles in asthmatic airway remodeling. However, it is unclear whether LIGHT could be controlled by inhaled corticosteroids, a key component of asthma management. This study was to investigate the effects and potential mechanisms of inhaled budesonide on the expressions of LIGHT and its receptors (LTβR and HVEM) of lung tissues in ovalbumin-sensitized mice. <b>Materials and Methods:</b> Thirty-three BALB/c mice were randomly divided into the control, asthma model, and budesonide treatment groups (11 in each group). Mice were sensitized and challenged by OVA to develop mouse model of chronic asthma, and treated with aerosolized budesonide before OVA challenge. Bronchoalveolar lavage fluid (BALF) and lungs were obtained after the final OVA challenge. Protein and mRNA Levels of LIGHT, LTβR, and HVEM in the lungs were investigated by immunohistochemistry, image analysis, and real-time PCR. Expressions of IL-6 and IFN-γ in BALF were measured by ELISA. <b>Results:</b> Inhaled budesonide significantly reduced protein and mRNA levels of lung LIGHT, LTβR, and HVEM in asthmatic mice. Correspondingly, the number of eosinophils and neutrophils and IL-6 levels in BALF after budesonide treatment were found to be decreased, whereas the IFN-γ levels in BALF were increased. Moreover, the expressions of LIGHT and HVEM mRNA showed positive correlation with IL-6 levels in the treatment group. <b>Conclusions:</b> Inhaled budesonide can down-regulate the expressions of LIGHT, LTβR, and HVEM in the lungs of asthmatic mice, and LIGHT/LTβR/HVEM interactions may be a potentially key target for asthma treatment.</p

Copy number loss of HRH4 in GCs.

<p>(A) Real-time PCR assay was carried out as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031207#s2" target="_blank">Materials and Methods</a> section, and the results were obtained from indicated group of samples. Boxplots of relative copy number of HRH4 mRNA measured with Real-Time PCR analysis showing median; box: 25th–75th percentile; bars: largest and smallest values within 1.5 box lengths; little cross: outliers. mRNA expression level of HRH4 in groups with deleted (n = 23) or unaltered (n = 108) DNA copies. (B) Representative figure of FISH analysis using chromosome 18q specific alpha satellite DNA probe and chromosome 18q11 specific probe for HRH4 gene. I. Nucleus of ANT tissue with two signals for each of green and orange, showing no deletion of chromosome 18q or HRH4 gene. II. Nucleus of GC tissue with 0–2 signals for green and 0–2 signals for orange, indicating relative deletion in chromosome 18q or HRH4 gene.</p

HRH4 activation induced growth arrest in gastric carcinoma cell lines.

<p>(A) Mock-AGS and H4R-AGS cells were treated with 10⁻⁵ M histamine, CB or CB accompanied by HRH4 antagonist (JNJ7777120) pretreatment, and cell-cycle distributions were determined by propidium iodide flow cytometry analysis. Each value is the mean±s.e. of triplicate data representative for three independent experiments. *p<0.05 and **p<0.01 vs. Control, H4R-AGS cells without any treatment. (B) Colony-formation assay. 5×10³ Mock-AGS and H4R-AGS cells were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031207#pone-0031207-g003" target="_blank">Fig. 3A</a> and maintained in G418 for 14 days, and the colonies were stained with Giemsa. The bar graph shows the absolute colony (≥50 cells) number±s.e. in duplicate experiments. *p<0.05 and **p<0.01 vs. Control, H4R-AGS cells without any treatment. (C) Mock-AGS and H4R-AGS cells were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031207#pone-0031207-g003" target="_blank">Fig. 3A</a>, WST-1 (Roche) assay measuring the activity of mitochondrial dehydrogenases was performed following the manufacturer's instruction at 0-, 1-, 2-, 3- ,4- ,5- day time points. Each value is the means.e. of triplicate data representative for three independent experiments. Error bars represent standard deviation of the mean.</p

Expressions of cell cycle proteins regulated by HRH4.

<p>Mock-AGS and H4R-AGS were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031207#pone-0031207-g003" target="_blank">Fig. 3A</a>, and total cell lysates were examined by western blotting.</p

CNVs of HRH4 in GC tissues and matched adjacent normal tissues (ANTs).

<p>CNVs of HRH4 in GC tissues and matched adjacent normal tissues (ANTs).</p

Decreased H4R expression in GCs.

<p>(A) Representative blots of HRH4 expression in normal mucosa and gastric tumor tissues. α-tubulin was used as a stable endogenous control. (B) The histogram shows the anlysis of the results from the immunoblottings. The relative expression value of HRH4 protein (normalized by α-tubulin) in GCs is expressed as an average ratio±s.e. of tumor dose to matched adjacent normal tissue dose. *p<0.01 vs. Control, ANT. (C) Real-time PCR assay was carried out as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031207#s2" target="_blank">Materials and Methods</a> section, boxplots of relative HRH4 mRNA(HRH4/GAPDH) measured with real-time PCR analysis showing median; box: 25th–75th percentile; bars: largest and smallest values within 1.5 box lengths; little cross: outliers. The results were obtained from 3 reactions in each sample. (D) Representative immunofluorescent microscope analysis of paired samples of GC tissue and adjacent normal tissue using anti human HRH4 monoclonal antibody (red). Nuclei were stain with DAPI (blue). Sample I: gastric cancer (GC); sample II: adjacent normal control (ANT). Arrows point to region of positive staining.</p

Comparison of CNVs of HRH4 gene between adjacent normal tissues (ANT) and healthy normal controls (HNC) ^* from peripheral blood.

<p>*ANT represents adjacent normal tissues; HNC represents healthy normal controls</p

FISH results in 10 GC/ANT pairs.

<p>FISH results in 10 GC/ANT pairs.</p

DataSheet_2_OPN promotes pro-inflammatory cytokine expression via ERK/JNK pathway and M1 macrophage polarization in Rosacea.docx

Rosacea is a chronic inflammatory dermatosis that involves dysregulation of innate and adaptive immune systems. Osteopontin (OPN) is a phosphorylated glycoprotein produced by a broad range of immune cells such as macrophages, keratinocytes, and T cells. However, the role of OPN in rosacea remains to be elucidated. In this study, it was found that OPN expression was significantly upregulated in rosacea patients and LL37-induced rosacea-like skin inflammation. Transcriptome sequencing results indicated that OPN regulated pro-inflammatory cytokines and promoted macrophage polarization towards M1 phenotype in rosacea-like skin inflammation. In vitro, it was demonstrated that intracellular OPN (iOPN) promoted LL37-induced IL1B production through ERK1/2 and JNK pathways in keratinocytes. Moreover, secreted OPN (sOPN) played an important role in keratinocyte-macrophage crosstalk. In conclusion, sOPN and iOPN were identified as key regulators of the innate immune system and played different roles in the pathogenesis of rosacea.</p

DataSheet_1_OPN promotes pro-inflammatory cytokine expression via ERK/JNK pathway and M1 macrophage polarization in Rosacea.xls

Rosacea is a chronic inflammatory dermatosis that involves dysregulation of innate and adaptive immune systems. Osteopontin (OPN) is a phosphorylated glycoprotein produced by a broad range of immune cells such as macrophages, keratinocytes, and T cells. However, the role of OPN in rosacea remains to be elucidated. In this study, it was found that OPN expression was significantly upregulated in rosacea patients and LL37-induced rosacea-like skin inflammation. Transcriptome sequencing results indicated that OPN regulated pro-inflammatory cytokines and promoted macrophage polarization towards M1 phenotype in rosacea-like skin inflammation. In vitro, it was demonstrated that intracellular OPN (iOPN) promoted LL37-induced IL1B production through ERK1/2 and JNK pathways in keratinocytes. Moreover, secreted OPN (sOPN) played an important role in keratinocyte-macrophage crosstalk. In conclusion, sOPN and iOPN were identified as key regulators of the innate immune system and played different roles in the pathogenesis of rosacea.</p