Jiping Zhao

IL-33 promotes airway remodeling and is a marker of asthma disease severity.

Abstract Objective: To investigate the function of interleukin-33 (IL-33) in the asthmatic airway remodeling and the relationship between IL-33 and asthma severity. Methods: IL-33 levels, sputum eosinophils percentage (EOS%), pulmonary function and total immunoglobulin (IgE) were measured for 45 patients with asthma and 40 non-allergic controls. Asthma severity was assessed. The expressions of IL-33 and reticular basement membrane (RBM) on bronchial biopsy specimens from eight asthma patients and eight non-allergic controls were observed after hematoxylin-eosin staining (HE) and immunohistochemical staining. In vitro experiments, real-time polymerase chain reactions and western blotting analysis were used to identify the specific effects of IL-33 administration. Results: Serum IL-33 levels in patients with asthma were higher than those in non-allergic controls. Moreover, in asthmatic patients, serum IL-33 levels were negatively correlated to forced expiratory volume in one second (FEV1, % predicted), and positively correlated to asthma severity. Increased expression of IL-33 and RBM thickening were observed on bronchial biopsy specimens obtained from patients with asthma. Serum IL-33 levels were positively correlated to basement membrane thickness. The production of fibronectin1 and type I collagen in human lung fibroblasts (HLF-1) increased at 24 h after IL-33 treatment in vitro. Pre-treatment with anti-ST2 antibody or fluticasone propionate (FP) suppressed the production of fibronectin1 and types I collagen induced by IL-33. Conclusions: IL-33 is a marker of asthma severity, and may contribute to airway remodeling in asthma by acting on human lung fibroblasts.

Type 2 innate lymphoid cells: A novel biomarker of eosinophilic airway inflammation in patients with mild to moderate asthma.

BACKGROUND Eosinophilic airway inflammation can predict the exacerbation of asthma, and we can improve the management of asthma by monitoring the eosinophilic airway inflammation. Although induced sputum and sputum eosinophil count is the gold standard test for diagnosing eosinophilic asthma, a more accessible and receptive method is needed for clinical practice. Type 2 innate lymphoid cells (ILC2) have recently been proposed to play a crucial role in eosinophilic inflammation and have been identified in peripheral blood from patients with asthma. OBJECTIVES We sought to identify simple and feasible biomarkers which can predict eosinophilic airway inflammation in asthmatic patients. METHODS Sputum was induced for the assessment of eosinophils in 150 asthmatic patients. In parallel, the proportion of ILC2s of peripheral blood lymphocytes (%ILC2), blood eosinophil counts, total immunoglobulin E (IgE), fractional exhaled nitric oxide (FeNO) and lung function tests were measured. 42 healthy donors served as controls. RESULTS 126 patients finished sputum induction and produced adequate sputum. The ILC2 level was significantly increased in eosinophilic asthmatic patients compared with non-eosinophilic asthmatic patients (0.117 ± 0.090versus0.035 ± 0.021, p < 0.001). A multiple regression model, including age, sex, BMI, blood eosinophil counts, FeNO, IgE and %ILC2, showed that %ILC2, blood eosinophil counts and FeNO were correlative factors of sputum eosinophil counts (p < 0.001, p = 0.037, p < 0.001, respectively) and %ILC2 was the most significant subset of airway eosinophilic inflammation (Estimate = 11.385). A receiver operating characteristic (ROC) analysis showed a sensitivity of 67.7% and a specificity of 95.3% for %ILC2 of 0.076 to distinguish eosinophilic asthmatic patients from non-eosinophilic asthmatic patients. CONCLUSION ILC2 is a surrogate marker of airway eosinophilic inflammation in patients with mild to moderate asthma and has great potential advantages for selecting the asthmatic patients most likely to benefit from therapeutics targeting Th2 inflammation.

Two single nucleotide polymorphisms in TSLP gene are associated with asthma susceptibility in Chinese Han population

ABSTRACT Background: Asthma is a chronic inflammatory disease of the airway that is mediated by T-helper 2(TH2) cells. Thymic stromal lymphopoietin (TSLP) can aggravate asthmatic lung inflammation by activating dendritic cells (DCs) to promote TH2 differentiation. TSLP promoter polymorphisms are associated with susceptibility to bronchial asthma in Japanese population. We sought to determine whether single nucleotide polymorphisms (SNPs) in TSLP gene are associated with asthma in Chinese Han population. Objective: To analyze the polymorphism of the two SNPs Rs2289276 and Rs2289278 in TSLP gene and to evaluate the association between the two SNPs and asthma susceptibility in Chinese Han population by using case-control study. Methods: five hundred and thirty one asthmatic patients and 540 age–sex matched normal controls were collected and DNA were extracted from peripheral blood, then the genotypes of SNPs Rs2289276 and Rs2289278 in TSLP gene were detected with polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP), genotype and allele frequencies were calculated and analyzed with Chi-square test. Results: Frequencies of CC/CT/TT genotypes at Rs2289276 site were 0.4706/0.4392/0.0902 in the asthmatic patients and 0.5604/0.3800/0.0595 in the healthy controls. Frequencies of CC/CG/GG genotypes at Rs2289278 site were 0.6502/0.2966/0.0532 in the asthmatic patients and 0.5795/0.3428/0.0777 in the healthy controls. The genotype and allele frequencies of the two SNPs in asthma patients were significantly different from those in the healthy controls. Rs2289278 C allele was correlated with decreased FEV1: FVC (P ≤ .05). Conclusions: TSLP variants are significantly associated with bronchial asthma. TSLP might be a new therapeutic target molecule for asthma.

Central role of cellular senescence in TSLP-induced airway remodeling in asthma.

Background Airway remodeling is a repair process that occurs after injury resulting in increased airway hyper-responsiveness in asthma. Thymic stromal lymphopoietin (TSLP), a vital cytokine, plays a critical role in orchestrating, perpetuating and amplifying the inflammatory response in asthma. TSLP is also a critical factor in airway remodeling in asthma. Objectives To examine the role of TSLP-induced cellular senescence in airway remodeling of asthma in vitro and in vivo. Methods Cellular senescence and airway remodeling were examined in lung specimens from patients with asthma using immunohischemical analysis. Both small molecule and shRNA approaches that target the senescent signaling pathways were used to explore the role of cellular senescence in TSLP-induced airway remodeling in vitro. Senescence-Associated β-galactosidase (SA-β-Gal) staining, and BrdU assays were used to detect cellular senescence. In addition, the Stat3-targeted inhibitor, WP1066, was evaluated in an asthma mouse model to determine if inhibiting cellular senescence influences airway remodeling in asthma. Results Activation of cellular senescence as evidenced by checkpoint activation and cell cycle arrest was detected in airway epithelia samples from patients with asthma. Furthermore, TSLP-induced cellular senescence was required for airway remodeling in vitro. In addition, a mouse asthma model indicates that inhibiting cellular senescence blocks airway remodeling and relieves airway resistance. Conclusion TSLP stimulation can induce cellular senescence during airway remodeling in asthma. Inhibiting the signaling pathways of cellular senescence overcomes TSLP-induced airway remodeling.

Thymic stromal lymphopoietin promotes asthmatic airway remodelling in human lung fibroblast cells through STAT3 signalling pathway

This study aimed to identify the role and regulation of thymic stromal lymphopoietin (TSLP) in asthmatic airway remodelling. To identify the expression of TSLP, α smooth muscle actin (α‐SMA) and collagen I in bronchial tissues, bronchial biopsy specimens were collected from patients with asthma and healthy controls and stained with specific antibodies, respectively. To characterize the signalling pathways regulated by TSLP, we silenced or overexpressed TSLP in human lung fibroblast (HLF‐1) cells by shRNA approaches or transfection and detected the expression of TSLP receptor (TSLPR) by enzyme‐linked immunosorbent assay and Western blot analysis. In TSLP signalling pathway, the protein expression of total signal transducer and activator of transcription 3 (STAT3), STAT5, the phosphorylation of STAT3 (pSTAT3) and STAT5 (pSTAT5), TSLP, α‐SMA and collagen I were also detected by Western blotting. In addition, the α‐SMA, collagen I and mRNA expression were determined by real‐time reverse‐transcription. To further confirm the TSLP‐STAT3 signalling pathway in HLF‐1 cells, we inhibited STAT3 activity by targeted small molecules and then detected TSLP‐induced expression of α‐SMA and collagen I in both mRNA and protein levels by quantitative real‐time reverse‐transcription and Western blotting, respectively. First, overexpression of TSLP, α‐SMA and collagen I was detected in epithelium collected from patients with asthma. Second, STAT3 activity and the expression of α‐SMA and collagen I were controlled, regulated by TSLP. Specifically, the pSTAT3, α‐SMA and collagen I were induced by the introduction of TSLP in HLF‐1 cells, and the repression of α‐SMA and collagen I was detected after TSLP silencing. Third, no changes of pSTAT5 were found in the presence of the STAT3 inhibitor, and TSLP‐induced α‐SMA and collagen I upregulation is in a STAT3 dependent manner. If we inhibit STAT3 activity by STAT3 targeted small molecules, TSLP‐induced α‐SMA and collagen I upregulation cannot be detected. The functions of TSLP in asthmatic airway remodelling were performed through STAT3 signalling pathway. Copyright

Thymic stromal lymphopoietin promotes asthmatic airway remodeling in human lung fibroblast cells through stat3 signaling pathway

This study aimed to identify the role and regulation of thymic stromal lymphopoietin (TSLP) in asthmatic airway remodelling. To identify the expression of TSLP, α smooth muscle actin (α‐SMA) and collagen I in bronchial tissues, bronchial biopsy specimens were collected from patients with asthma and healthy controls and stained with specific antibodies, respectively. To characterize the signalling pathways regulated by TSLP, we silenced or overexpressed TSLP in human lung fibroblast (HLF‐1) cells by shRNA approaches or transfection and detected the expression of TSLP receptor (TSLPR) by enzyme‐linked immunosorbent assay and Western blot analysis. In TSLP signalling pathway, the protein expression of total signal transducer and activator of transcription 3 (STAT3), STAT5, the phosphorylation of STAT3 (pSTAT3) and STAT5 (pSTAT5), TSLP, α‐SMA and collagen I were also detected by Western blotting. In addition, the α‐SMA, collagen I and mRNA expression were determined by real‐time reverse‐transcription. To further confirm the TSLP‐STAT3 signalling pathway in HLF‐1 cells, we inhibited STAT3 activity by targeted small molecules and then detected TSLP‐induced expression of α‐SMA and collagen I in both mRNA and protein levels by quantitative real‐time reverse‐transcription and Western blotting, respectively. First, overexpression of TSLP, α‐SMA and collagen I was detected in epithelium collected from patients with asthma. Second, STAT3 activity and the expression of α‐SMA and collagen I were controlled, regulated by TSLP. Specifically, the pSTAT3, α‐SMA and collagen I were induced by the introduction of TSLP in HLF‐1 cells, and the repression of α‐SMA and collagen I was detected after TSLP silencing. Third, no changes of pSTAT5 were found in the presence of the STAT3 inhibitor, and TSLP‐induced α‐SMA and collagen I upregulation is in a STAT3 dependent manner. If we inhibit STAT3 activity by STAT3 targeted small molecules, TSLP‐induced α‐SMA and collagen I upregulation cannot be detected. The functions of TSLP in asthmatic airway remodelling were performed through STAT3 signalling pathway. Copyright

Regulatory mechanism of pyrrolidine dithiocarbamate is mediated by nuclear factor‑κB and inhibits neutrophil accumulation in ARDS mice

The aim of the present study was to investigate the regulatory mechanism of nuclear factor (NF)-κB on polymorphonuclear neutrophil (PMN) accumulation and the inflammatory response in lung tissues with acute respiratory distress syndrome (ARDS), as well as the therapeutic effect of pyrrolidine dithiocarbamate (PDTC). Mouse models of ARDS were established by intraperitoneal injection of lipopolysaccharide (LPS). BALB/c mice were divided into control, LPS and PDTC + LPS groups. The expression of PMN adhesion molecules, CD11b/CD18 and intercellular adhesion molecule-1 (ICAM-1), were detected by immunohistochemistry, while the protein expression levels of NF-κB p65 in the lung tissue were analyzed by western blot analysis. In addition, flow cytometry was used to investigate the apoptosis rate of PMNs in the bronchoalveolar fluid, and the expression levels of interleukin (IL)-1β, IL-8 and tumor necrosis factor (TNF)-α and myeloperoxidase (MPO) activity were also determined. Following an intraperitoneal injection of LPS, alveolar septum rupture, pulmonary interstitial hyperemia and PMN infiltration in the alveolar was observed. The protein expression of p65 in the pulmonary cytoplasm decreased, while the expression of p65 in the nucleus increased. The levels of IL-8, IL-1β and TNF-α increased and the high expression status was maintained for 24 h. As the time increased, CD11b/CD18 and ICAM-1 expression increased, as well as MPO activity, while the apoptosis of PMNs was delayed. Compared with the LPS group, the expression of p65 in the pulmonary cytoplasm and the PMN apoptosis rate increased following PDTC intervention, while the expression of p65 in the nucleus decreased, as well as the expression levels of the cytokines and MPO activity. Therefore, PDTC reduced the production of inflammatory cytokines via the NF-κB pathway, which reduced the activation of PMNs in the lung tissue and promoted PMN apoptosis.

FIZZ1 promotes airway remodeling through the PI3K/Akt signaling pathway in asthma

Found in inflammatory zone 1 (FIZZ1) plays a vital role in pulmonary inflammation and angiogenesis. In addition, FIZZ1 plays a role in the early stages of airway remodeling in asthma by increasing the expression of α smooth muscle actin (α-SMA) and type I collagen. However, the role of FIZZ1 in the airway remodeling of asthma remains unclear. In the present study, FIZZ1 was identified to be upregulated in ovalbumin (OVA)-induced asthmatic mice, along with phosphorylated protein kinase B (Akt). Following FIZZ1 recombinant protein co-culture in the murine lung epithelial-cell line, Akt phosphorylation was upregulated, however, following transfection with FIZZ1-small hairpin RNA, the phosphorylation levels were decreased. The variation in α-SMA and type I collagen expression levels was consistent with the Akt phosphorylation levels. Intratracheal administration of LY294002 and Akt inhibitor IV to the asthmatic mice was capable of reducing airway inflammation, downregulating the expression of α-SMA, type I collagen and fibronectin-1 and increasing the expression of E-cadherin. In conclusion, the present study demonstrated that FIZZ1 promoted airway remodeling in asthma via the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Blocking the PI3K/Akt signaling pathway may attenuate the early stages of airway remodeling induced by OVA by regulating the abnormal process of epithelial-mesenchymal transition.

Autophagy plays a role in FSTL1-induced epithelial mesenchymal transition and airway remodeling in asthma

Asthma is a chronic disease related to airway hyperresponsiveness and airway remodeling. Airway remodeling is the important reason of refractory asthma and is associated with differentiation of airway epithelia into myofibroblasts via epithelial-mesenchymal transition (EMT) to increase the process of subepithelial fibrosis. There is growing evidence that autophagy modulates remodeling. However, the underlying molecular mechanisms of these effects are still unclear. In this study, we hypothesized that Follistatin-like 1 (FSTL1) promotes EMT and airway remodeling by intensifying autophagy. With the use of transmission electron microscopy (TEM), double-membrane autophagosomes were detected in the airways of patients and mice. More autophagosomes were in patients with asthma and OVA-challenged mice compared with healthy controls. The expression of FSTL1 and beclin-1 was upregulated in the airways of patients with asthma and OVA-challenged mice, accompanied by airway EMT and remodeling. In OVA-challenged Fstl1+/- mice, the degree of airway remodeling and autophagy was decreased compared with control mice. The effects of FSTL1 on autophagy and EMT were also tested in 16HBE cells in vitro. Additionally, inhibition of autophagy by using LY-294002 and siRNA-ATG5 reduced the FSTL1-induced EMT in 16HBE cells, as measured by E-cadherin, N-cadherin, and vimentin expression. In line herewith, administration of LY-294002 reduced the expression of autophagy, EMT, and airway remodeling markers in FSTL1-challenged WT mice. Taken together, our study suggests that FSTL1 may induce EMT and airway remodeling by activating autophagy. These findings may provide novel avenues for therapeutic research targeting the autophagy and FSTL1 pathway, which may be beneficial to patients with refractory asthma.

IL-25 Involved in Airway Inflammation of OVA-Induced Asthmatic Mice and the Inhibitory Effect of Glucocorticoid

Objective: To study the expression of IL-25 in airway inflammation of OVA-induced asthmatic mice and the effects of glucocorticoid. Methods: The asthmatic mice were induced by ovalbumin (OVA) sensitization and challenge. This model was confirmed by detecting airway responsiveness, serum levels of IgE and lung histopathology. We also detected the inflammatory cells by cell counting plate and cytokines by ELISA, respectively. We also detected the expression of IL-25 by quantitative real-time PCR and immunohistochemistry. Results: Compared with the mice in control group, the asthmatic mice showed elevated airway responsiveness and high serum levels of IgE. We also detected more inflammatory cells and high level of inflammatory cytokines in mice of OVA group. The expression of IL-25 was also significantly increased in both mRNA and protein levels in OVA group. Furthermore, treating mice with glucocorticoid could inhibit inflammatory cells and cytokines infiltration and reduce IL-25 expression. Conclusion: IL-25 level is increased in OVA-induced asthmatic airway inflammation and glucocorticoid is capable of inhibiting IL-25 expression as well as other Th2 cytokines, thus presents a promising strategy for the treatment of asthma.