Biochemical and Biophysical Research Communications
Alternaria extract activates autophagy that induces IL-18 release from 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).
References (29)
- et al.
Alternaria-induced asthma
J. Allergy Clin. Immunol.
(2004) - et al.
The relationships among environmental allergen sensitization, allergen exposure, pulmonary function, and bronchial hyperresponsiveness in the childhood asthma management program
J. Allergy Clin. Immunol.
(1999) - et al.
Importance of IL-18-induced super Th1 cells for the development of allergic inflammation
Allergol. Int.
(2010) - et al.
IL-18 might reflect disease activity in mild and moderate asthma exacerbation
J. Allergy Clin. Immunol.
(2001) - et al.
The interleukin-1 family: back to the future
Immunity
(2013) - et al.
Inflammasome activation and IL-1beta and IL-18 processing during infection
Trends Immunol.
(2011) - et al.
Methods in mammalian autophagy research
Cell
(2010) - et al.
Autophagy intersections with conventional and unconventional secretion in tissue development, remodeling and inflammation
Trends Cell. Biol.
(2012) - et al.
Fatty acid synthase inhibitors cerulenin and C75 retard growth and induce caspase-dependent apoptosis in human melanoma A-375 cells
Biomed. Pharmacother.
(2007) - et al.
Genetic and histologic evidence for autophagy in asthma pathogenesis
J. Allergy Clin. Immunol.
(2012)
Clinical importance of Alternaria exposure in children
Am. J. Respir. Crit. Care Med.
Alternaria-induced release of IL-18 from damaged airway epithelial cells: an NF-kappaB dependent mechanism of Th2 differentiation?
PLoS One
Interleukin-18 in pulmonary inflammatory diseases
J. Interferon Cytokine Res.
Up-regulation of IL-18 in allergic rhinitis
Allergy
Cited by (27)
Silver birch pollen-derived microRNAs promote NF-κB-mediated inflammation in human lung cells
2021, Science of the Total EnvironmentCitation Excerpt :Autophagy triggered by air pollutants may suppress plane tree pollen-induced allergic inflammation in A549 cells (Shumin et al., 2021). Autophagy may also be involved in Alternaria extract-induced inflammation by stimulation of IL-18 production and A549 and NHBE cell damage (Murai et al., 2015) and in the pathophysiology of asthma (Poon et al., 2012). The presence of SBP-derived RNA molecules also induced the levels of 5-methylcytosine (m5C) RNA methyltransferases NSUN2 and NSUN5 (Bohnsack et al., 2019) in WI-38 cells.
Ambient particulate matter-associated autophagy alleviates pulmonary inflammation induced by Platanus pollen protein 3 (Pla3)
2021, Science of the Total EnvironmentCitation Excerpt :In this study, we demonstrated that SPPs could not only induce oxidative stress response (Figs. S4, 4) but also increase the level of TSLP in A549 cells (Figs. 5 and 6) and rats (Fig. 7). Although high levels of autophagy induce apoptosis, appropriate levels of autophagy could help to degrade damaged organelles in cells, such as mitochondria and peroxisomes (Seino et al., 2013), and played a negative role in the release of interleukins from airway epithelial cells exposed to allergens, thus the autophagy reduced Th2-type responses (Shi et al., 2012; Murai et al., 2015). In autophagy-deficient cells, damaged organelles, misfolded proteins and ROS would accumulate and resulted in inflammatory activation (Rao and Eissa, 2019).
Complex interplay between autophagy and oxidative stress in the development of pulmonary disease
2020, Redox BiologyCitation Excerpt :The activation of autophagy also correlates with the production of neutrophil extracellular traps (NETs) in neutrophils [339] and genetic polymorphisms of ATG5 and ATG7 contribute to neutrophilic airway inflammation in the pathogenesis of adult asthma [340]. Activation of autophagy in epithelial cell culture model systems exposed to allergens or other antigens, is also detrimental and promotes disease progression [341,342]. Genetic depletion of ATG5 and ATG14 or pharmacological inhibition of autophagy with 3-MA or bafilomycin A1 (Baf-A1) in epithelial cells treated with IL13 results in less mucus secretion and less CCL26 (eosinophil chemokine) production, demonstrating that autophagy is involved in the activation of the Th2 response in asthma [343].
Xenophagy in innate immunity: A battle between host and pathogen
2020, Developmental and Comparative ImmunologyCitation Excerpt :TLR engagement activates various defense mechanisms, such as phagosome maturation (Pauwels et al., 2017), within the macrophage (Maa and Leu, 2011) and engages in autophagy via the canonical or noncanonical autophagy pathway (Margaritopoulos et al., 2017). TLR1, 2, 3, 4, 5, and 7 can induce autophagosome formation during immune response (Murai et al., 2015; Li et al., 2019b; Zhu et al., 2017a; Zang et al., 2019; Singh et al., 2011; Molino et al., 2017). A canonical mechanistic study revealed that myeloid differentiation factor 88 (MyD88) and TRI, a TLR signaling complex component, interact with beclin 1 to facilitate autophagy induction and inhibit the interaction between beclin 1 and bcl-2 (Deretic, 2009).
The allergenic activity and clinical impact of individual IgE-antibody binding molecules from indoor allergen sources
2020, World Allergy Organization JournalCitation Excerpt :The increases in IL-5 and IL-13 that follow IL-33171 upregulation by Alternaria are independent of adaptive immunity, and regarded as an innate mechanism mediated by group 2 innate lymphoid cells (ILC2).172,173 A. alternata also activates autophagy in airway epithelial cells and induces them to secrete IL-18.174 Increased expression of cytokines (TSLP) and chemokines (CCL2) has been also reported upon exposure of lung epithelial cells to the extract of A. alternata via activation of Toll Like Receptor 2 (TLR-2) signaling.175