Alternaria extract activates autophagy that induces IL-18 release from airway epithelial cells

https://doi.org/10.1016/j.bbrc.2015.05.076Get rights and content

Highlights

  • Alternaria activates autophagy that induces IL-18 release from epithelial cells.

  • This IL-18 release is independent of the canonical activation of the inflammasome.

  • Autophagy might be involved in the pathophysiology of Alternaria-associated asthma.

Abstract

Alternaria alternata is a major outdoor allergen that causes allergic airway diseases. Alternaria extract (ALT-E) has been shown to induce airway epithelial cells to release IL-18 and thereby initiate Th2-type responses. We investigated the underlying mechanisms involved in IL-18 release from ALT-E-stimulated airway epithelial cells. Normal human bronchial epithelial cells and A549 human lung adenocarcinoma cells were stimulated with ALT-E in the presence of different inhibitors of autophagy or caspases. IL-18 levels in culture supernatants were measured by ELISA. The numbers of autophagosomes, an LC3-I to LC3-II conversion, and p62 degradation were determined by immunofluorescence staining and immunoblotting. 3-methyladenine and bafilomycin, which inhibit the formation of preautophagosomal structures and autolysosomes, respectively, suppressed ALT-E-induced IL-18 release by cells, whereas caspase 1 and 8 inhibitors did not. ALT-E-stimulation increased autophagosome formation, LC-3 conversion, and p62 degradation in airway epithelial cells. LPS-stimulation induced the LC3 conversion in A549 cells, but did not induce IL-18 release or p62 degradation. Unlike LPS, ALT-E induced airway epithelial cells to release IL-18 via an autophagy dependent, caspase 1 and 8 independent pathway. Although autophagy has been shown to negatively regulate canonical inflammasome activity in TLR-stimulated macrophages, our data indicates that this process is an unconventional mechanism of IL-18 secretion by airway epithelial cells.

Introduction

A number of epidemiological studies have shown that the fungus Alternaria alternata is the source of a common allergen that causes allergic airway diseases, including bronchial asthma, and that sensitivity to this A. alternata allergen is associated with asthma severity, bronchial hyperresponsiveness, and fatal asthma exacerbations [1], [2], [3]. We recently demonstrated that an Alternaria extract (ALT-E) could induce airway epithelial cells to release interleukin (IL)-18 and thereby directly initiate Th2 differentiation by naive CD4+ T-cells [4]. Although originally discovered as a factor that induced IFN–γ production by Th1 cells, IL-18 also has the potential to induce IL-4 and IL-13 production by T cells, natural killer (NK) cells, NKT cells, mast cells, and basophils [5], [6]. Increased IL-18 expression and serum levels have been reported in humans with allergic airway diseases [7], [8]. A functional polymorphism of the IL-18 gene was also shown to be associated with the severity of bronchial asthma [9]. Taken together, these results suggest that IL-18 might play an important role in the pathophysiology of Alternaria-associated asthma.

IL-18 is a pro-inflammatory cytokine that belongs to the IL-1 family and is produced in a biologically inactive precursor form, pro-IL-18 [10]. Due to a lack of a signal peptide, as with IL-1β, mature IL-18 is secreted after pro-IL-18 is cleaved by the cysteine protease caspase 1. Caspase 1 activity is controlled by inflammasomes, which are multiprotein signaling complexes that detect microbial-derived molecular signatures or endogenous danger signals [11]. Several lines of evidence suggest that there is a non-canonical IL-1β and IL-18 activation pathway that involves caspase-8 and receptor interacting protein (RIP) kinases [12], [13]. Furthermore, in some inflammatory conditions, IL-1β and IL-18 processing might occur extracellularly due to the actions of a number of proteases, including neutrophil proteinase-3, mast cell chymase, matrix metalloproteinases, cathepsin G, and granzyme A [14], [15], [16].

Macroautophagy (often simply referred to as autophagy) is a cellular process that involves the recycling of cellular proteins and the removal of intracellular microorganisms through the lysosome machinery [17], [18]. Autophagy is a multistage process that comprises several steps: (1) formation of autophagosomes; (2) fusion of these autophagosomes with lysosomes to form autolysosomes; (3) degradation of autolysosome contents; and (4) utilization of these degradation products [19]. Autophagy is known to control inflammation by removing endogenous inflammasome agonists and by autophagic degradation of inflammasome components, which results in reduced inflammasome and caspase 1 activation [18], [20]. Additionally, autophagy-based unconventional secretion, or so-called autosecretion, has been shown to enable leaderless cytosolic proteins, such as IL-1β and IL-18, to be delivered extracellularly without entering the endoplasmic reticulum (ER)-to-Golgi secretory pathway [21], [22].

In this study, we investigated whether autophagy and inflammasome pathways were involved in IL-18 release from ALT-E-stimulated airway epithelial cells. We show that ALT-E-stimulation can induce airway epithelial cells to release IL-18 via an autophagy dependent and caspase 1 and 8 independent pathway.

Section snippets

Reagents

Caspase 1, 8 inhibitors and 3-methyladenine were purchased from Calbiochem (Gibbstown, NJ). C75, Bafilomycin A1, Pam3CSK4, MALP2, Poly (I:C), and LPS were from Sigma–Aldrich (St. Louis, MO). Flagellin, FSL-1, imiquimod, single stranded RNA40, and ODN 2006 were from InvivoGen (San Diego, CA).

Cell culture

Normal human bronchial epithelial (NHBE) cells were obtained from Cambrex Bio Science (Walkersville, MD) and A549 cells, a human lung adenocarcinoma line, were from the American Type Culture Collection

ALT-E-induced IL-18 release is independent of caspase 1 and 8 activation

We first investigated which cytokines were released from ALT-E stimulated NHBE cells by multiplex cytokine array analysis. Among the cytokines investigated, IL-18 levels in culture supernatants were dramatically increased after stimulation with ALT-E (Fig. 1A). We next examined whether conventional inflammasome pathways were involved in IL-18 release from airway epithelial cells in response to ALT-E stimulation using different inhibitors. As shown in Fig. 1B and C, 5–20 μM caspase-1 inhibitor

Discussion

In this study, we found that ALT-E-stimulation activated an autophagy-based unconventional secretion pathway in airway epithelial cells and thereby induced the extracellular release of IL-18 independent of caspase 1 and 8 activation.

A number of reports have shown negative effects of autophagy on IL-1β and IL-18 activation by inflammasomes and that caspase 1 had a dominant effect on degenerative autophagy [18], [20], [25]. In contrast to degenerative autophagy, a contribution of secretory

Acknowledgments

This study was supported by a Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research (MEXT-KAKENHI), Japan (NO. 25860850; HM), NAID (P01 AI062885-06A1; SS), the NHLBI Proteomic Center (N01HV00245; SS), NIEHS RO1 ES18948 (SS) and a Leon Bromberg Professorship (UTMB; SS).

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