Intelectin Is Essential For Allergen-induced IL-25, IL-33 And TSLP Expression In Asthma

Part I Intelectin is required for the airway hyperresponsiveness, eosinophilic inflammation, mucus metaplasia and the development of Th2 immune responses in allergic asthma modelsObjective:To investigate the role of intelectin (ITLN) in airway hyperresponsiveness (AHR), eosinophilic inflammation, mucus metaplasia and development of Th2 immune responses in allergic asthma models.Method:We used C57BL/6J mice to generate transgenic shRNA (ITLN KD) mice. Two asthma models, ovalbumin (OVA) asthma model and house dust mite (HDM) asthma model were used in our study. Pulmonary resistance in response to a range of concentrations of intravenous acetylcholine was measured. Cell counts for macrophages, eosinophils, lymphocytes and neutrophils in bronchoalveolar lavage fluid (BALF) were performed. In addition, mouse lungs were stained with hematoxylin and eosin (H&E) and the severity of peribronchial inflammation was scored. Lung sections were also stained with Periodic acid-Schiff (PAS) staining for detection of mucus. Five-?m-thick lung sections were used for immunohistochemistry with Muc5ac antibody to evaluated mucus. OVA-specific IgE in mouse serum were also detected by ELISA. We isolated total RNA from mouse lungs, and the transcript levels of Itlnl, Muc5ac,11-4,11-5,11-13 were determined by real-time PCR. Mouse lung and mediastinal lymph nodes were isolated, and the cells were stained with CD4 and IL-4 antibodies and analyzed on a FACSort flow cytometer.Results:Airway response to acetylcholine was the same in saline-challenged WT and ITLN KD mice. However, OVA-challenged ITLN KD mice were significantly protected from AHR when compared with WT mice. The numbers of total cells and eosinophils in BALF and the numbers of inflammatory cells around the conducting airways assessed by H&E staining were significantly lower (>50% reduction) in OVA -challenged ITLN KD mice when compared with WTmice. The numbers of periodic acid-schiff (PAS)-staining-positive, MUC5AC-staining-positive cells and the levels of Muc5ac transcripts were markedly reduced in OVA-challenged ITLN KD mice compared with WT mice. Serum levels of OVA-specific IgE were significantly decreased in OVA-challenged ITLN KD mice when compared to WT mice. We next analyzed the expression of theTh2 cytokines IL-4, IL-5, and IL-13 in mouse lung by real-time PCR and ELISA. OVA challenge induced the expression of IL-4, IL-5, and IL-13 in lung tissue and BALF of WT mice. However, the increase of these cytokines was significantly suppressed in OVA-challenged ITLN KD mice. We also investigated the role of ITLN in the pathogenesis of asthma using the HDM asthma model, since this allergen is more relevant to human asthma; the data were similar to our findings in OVA asthma model. We also found that percentages of Th2 cells (IL-4+CD4+) were increased in lung tissue and draining lymph node of OVA-challenged WT mice, but significantly decreased in OVA-challenged ITLN KD mice.Conclusion:ITLN is required for the development of AHR, airway inflammation, mucus overproduction and allergic response both in the OVA and HDM asthma models, and ITLN is also required for the differentiation of Th2 cells.Part II Intelectin is required for upregulation of the airway epithelial cytokines IL-25, IL-33, and TSLP after exposure to allergenObjective:To determine whether intelectin (ITLN) is required for allergen-induced IL-25, IL-33, and TSLP expression.Method:In mouse ovalbumin (OVA) and house dust mite (HDM) asthma models, IL-25, IL-33 and thymic stromal lymphopoietin (TSLP) in mouse lung homogenate were determined using ELISA. Later, Mice were intranasal aspiration of HDM for 3 consecutive days; Il-25,Il-33 and Tslp in mouse lungs were determined by real-time PCR. Human bronchial epithelial (BEAS-2B) cells were cultured in DMED/high glucose in 6-well plates. ITLN shRNA or scrambled shRNA plasmid were used to transfect BEAS-2B cells. Six hours later, the media were substituted with DMEM with or without HDM. At the indicated time points, cells were harvested for Real Time PCR or Western blot. EGFR inhibitor PD153035 and AG1478, and MEK inhibitor U0126 were added to the medium 1 h before HDM stimulation, and BEAS-2B cells were also harvested for real-time PCR. For phospholyted EGFR, total EGFR, phospholyted ERK and total ERK Western blotting, rabbit monoclonal phospho-EGFR (Tyr1068), EGFR, phospho-p44/42 MAPK/ERK1/2 (Thr202/Tyr204) and MAPK/ERK1/2 antibody from Cell Signaling Technology were used.Results:OVA and HDM challenge each increased lung IL-25, IL-33 and TSLP protein in wild type (WT) mice, but these increases were significantly suppressed in transgenic ITLN shRNA (ITLN KD) mice. In the model of 3 consecutive daily intranasal challenges with HDM, we found that HDM induced the expression of ITLN1, IL-25, IL-33 and TSLP at 2 and 6h after the last HDM exposure in the lung of WT mice. However, HDM-induced upregulation of ITLN 1, IL-25, IL-33 and TSLP were markedly suppressed in ITLN KD mice. We examined the expression of ITLN1, IL-25, IL-33 and TSLP expression in BEAS-2B cells after exposure to HDM for 0,2,6,24 h. HDM induced ITLN1, IL-25, IL-33 and TSLP expression in BEAS-2B cells at 2 and 6h. However, HDM-induced increases in these cytokines were suppressed when ITLN1 expression was silenced by RNA interference. Pretreatment of BEAS-2B cells with galactose also inhibited HDM-induced IL-25, IL-33, and TSLP mRNA expression. When BEAS-2B cells pretreated with EGFR inhibitors AG1478 and PDl53035 and the MAPK/ERK kinase (MEK) inhibitor U0126, we found that these inhibitors each blocked HDM-induced IL-25, IL-33, and TSLP expression. Furthermore, HDM stimulation increased EGFR and MAPK/ERK phosphorylation at 2h and 4h, respectively, and ITLN1 shRNA transfection inhibited HDM-induced EGFR and MAPK/ERK phosphorylation.Conclusion:ITLN is necessary for allergen-induced IL-25, IL-33, and TSLP expression in human airway epithelial cells. The activation of EGFR and MAPK/ERK mediates HDM-induced IL-25, IL-33, and TSLP expression, and that ITLN1 expression is required for HDM-induced EGFR and MAPK/ERK activation.Part ? The expression of intelectin in airway of subjects with asthmaObjective:Determined the mRNA and protein level of intelectin (ITLN) in airway brushings and endobronchial biopsies, to find the association between ITLN and eosinophilic airway inflammation.Method:We recruited 23 healthy control subjects and 48 subjects with asthma. All subjects were recruited from Tongji Hospital and were Chinese. For each subject, we recorded demographic information, collected blood and induced sputum samples. The spirometry were performed, FEV1 (percentage predicted) and the dosage of methacholine provoking a 20% fall of in forced expiratory volume in the first second (FEV1 PD20)< 2.5mg and/or >12% increase in FEVI following inhalation of 200?g salbutamol were recorded. The number of eosinophil in peripheral blood and induced sputum samples were also collected. We brushed 10 sites within the subsegmental bronchi of right middle and lower lobes (10 gentle upward and downward strokes per site). The total RNA from bronchial epithelial brushings was isolated and the expression of ITLN was determined by Real Time PCR. Since ITLN is secreted, plasma ITLN1 levels were also measured by ELISA sets. We used forceps to biopsy left lower lobe carinae and fixed samples in polyoxymethylene. We stained 2 ?m sections with hematoxylin and eosin (H&E) or intelectin antibody for endobronchial. Furthermore, the analysis of the association between epithelial ITLN1 transcript levels and eosinophils in induced sputum were also conducted.Results:Subjects with asthma had lower FEV1 and methacholine PD20 and higher serum IgE levels, blood and induced sputum eosinophil numbers than healthy control subjects. ITLN immunostaining in airway biopsy samples revealed that ITLN-positive cells were mainly airway mucous cells and submucosal gland cells. ITLN1 transcript level in bronchial epithelial brushings and plasma ITLN1 levels were markedly increased in subjects with asthma when compared with healthy controls. Furthermore, epithelial JTLN1 transcript levels were significantly correlated with the levels of eosinophils in induced sputum.Conclusion:ITLN is mainly expressing in airway mucous cells and submucosal gland cells. The expression level of ITLN was markedly higher in asthma patients when compared with the healthy controls. Epithelial ITLN level was also significantly correlated with eosinophilic airway inflammation.