IL-1β upregulates Muc5ac expression via NF-κB-induced HIF-1α in asthma

Highlights

• IL-1β is related to Muc5ac in asthma.

• IL-1β increased Muc5ac expression.

• HIF-1α is essential to IL-1β-induced Muc5ac expression in asthma.

• HIF-1α could bind to Muc5ac promoter.

• NF-κB is responsible for IL-1β-upregulated HIF-1α in asthma.

Abstract

The manifest and important feature in respiratory diseases, including asthma and COPD (chronic obstructive pulmonary disease), is the increased numbers and hypersecretion of goblet cells and overexpression of mucins, especially Muc5ac. Many proinflammatory cytokines play important roles in goblet cell metaplasia and overproduction of Muc5ac. However, the effect of IL-1β on Muc5ac expression in asthma remains unknown. Here, we detected the correlation between IL-1β and Muc5ac in asthma patients and further explored the mechanism of IL-1β-induced Muc5ac overexpression. Our results showed that Muc5ac and IL-1β were up-regulated in 41 patients with asthma and that Muc5ac overexpression was related with IL-1β in asthma (R² = 0.668, p ≪ 0.001). Furthermore, the correlation between IL-1β and Muc5ac is higher in severe group than that in moderate group. In vitro experiments with normal human bronchial epithelial cells (NHBECs) showed that IL-1β up-regulated Muc5ac expression in NHBEC in a time- and dosage-dependent manner. Hypoxia-induced HIF-1α was responsible for Muc5ac expression mediated by IL-1β. Knocking down HIF-1α by siRNA decreased Muc5ac expression under hypoxia even in IL-1β-treated NHBEC cells. Luciferase reporter assay showed that HIF-1α enhanced Muc5ac promoter activity in HEK293T cells. HIF-1α could specifically bind to the promoter of Muc5ac by EMSA. The correlation among IL-1β, HIF-1α and Muc5ac was observed in patients with asthma. Mechanically, NF-κB activation was essential to IL-1β-induced HIF-1α upregulation via the canonical pathway of NF-κB. The level of nuclear p65, a subunit of NF-κB, was obviously increased in NHBEC cells under IL-1β treatment. IL-1β did not change either HIF-1α or Muc5ac expression when inhibiting NF-κB signaling with Bay11-7082, an inhibitor of NF-κB. Collectively, we concluded that IL-1β up-regulated Muc5ac expression via NF-κB-induced HIF-1α in asthma and provided a potential therapeutic target for asthma.

Introduction

The hallmarks of the airways in many chronic respiratory diseases, such as chronic bronchitis, bronchiectasis and bronchial asthma, are involved in goblet cell hyperplasia and metaplasia, which are associated with mucus hypersecretion. Hypersecretion from numerous goblet cells is regarded as an important contribution to mucus plugging and airway obstruction [1]. Excessive mucus in the airways plays a crucial role in the increase in the frequency and duration of infection, even in the increase in morbidity and mortality in respiratory diseases [2]. Gel-forming mucins include MUC2, MUC5AC, MUC5B, MUC19, and so on. Among these, MUC5AC is the common and major respiratory mucin in goblet cell [3]. The source of mucus is different between the large airways and the small airways. In the large airways, mucus is secreted by goblet cells and submucosal glands, whereas in the small airways mucus is only produced by the goblet cell. Goblet cells can rapidly produce mucus under certain stimuli in the manner of exocytosis, and then form a mucus layer in the airways. Muc5ac overexpression has been observed both in the large and small ariways and plays an important role in the development of asthma. Many proinflammatory cytokines, such as IL-13 and IL-4, up-regulate Muc5ac in lung tissue with asthma via activating transcription 6 (STAT6) [4]. These Th2 cytokines induce goblet cell metaplasia/hyperplasia via STAT6, which in turn results in an increase of Muc5ac mRNA and protein levels as expressed by the new airway goblet cells.

Interleukin-1 (IL-1), first described as lymphocyte-activating cytokine, was discovered in the 1970s as a potent inflammatory cytokine [5], [6]. IL-1 family was consisted of at least two members. Among them, IL-1β plays an important role in severe inflammatory diseases, such as the rare cryopyrin-associated periodic syndromes (CAPS). The treatment of anti-IL-1β can completely control the progression of most of these diseases [7], [8], [9]. Increasing evidences have indicated that IL-1 is an important regulator in airway hyperresponsiveness and the cytokine production related to Th2 cells [10]. Recently, several studies reported that IL-1-neutralizing drugs reduced airway hyperreactivity and decreased Th2-related cytokines in animal asthma models [11], [12]. In addition, IL-1β mediates MUC5AC gene expression through CREB and/or NF-κB [13], [14]. However, the microenvironment of lung tissue in asthma is complicated. Thus, whether IL-1β could regulate Muc5ac expression through other mechanism such as hypoxia-induced HIF-1α in asthma remain largely unknown.

The balance of oxygen supply and demand is required to normal tissue development. However, the balance can be disturbed by numerous physiological and pathophysiological conditions. Under the condition of the reduction in oxygen tension, some tissues usually trigger signaling events and up-regulate several genes to allow for short- and long-term adaptation to hypoxia. The up-regulation of the hypoxia-inducible factor (HIF) family is often initiated under hypoxia, and consequently regulates the transcription of other genes. Although hypoxia can induce HIF expression, HIF-1α accumulation and transcription can also be triggered under normoxic and inflammatory conditions [15]. Indeed, cytokine-induced up-regulation of HIF-1 demonstrates that HIF-1 is an important line between inflammation and cancer [16], [17].

In this study, we evaluated the effect of HIF-1α on Muc5ac expression in the presence or absence of IL-1β. Our results suggested that HIF-1α could enhance Muc5ac overexpression in NHBECs under IL-1β treatment. Muc5ac was triggered through HIF-1α binding to Muc5ac promoter. Mechanically, we found that IL-1β activated NF-κB signaling and then promoted HIF-1α expression.