IL-33 is clearly expressed in the airway of patients with asthma, but its role in asthma has not yet been fully understood. IL-17F is also involved in the pathogenesis of asthma. However, the regulatory mechanisms of IL-17F expression remain to be defined. To further indentify the role of IL-33 in asthma, we investigated the expression of IL-17F by IL-33 in bronchial epithelial cells and its signaling mechanisms.
Bronchial epithelial cells were stimulated with IL-33. The levels of IL-17F expression were analyzed using real-time PCR and ELISA. Next, the involvement of ST2, MAP kinases, and mitogen- and stress-activated protein kinase1 (MSK1) was determined by Western blot analyses. Various kinase inhibitors and anti-ST2 neutralizing Abs were added to the culture to identify the key signaling events leading to the expression of IL-17F, in conjunction with the use of short interfering RNAs (siRNAs) targeting MSK1.
IL-33 significantly induced IL-17F gene and protein expression. The receptor for IL-33, ST2, was expressed in bronchial epithelial cells. Among MAP kinases, IL-33 phosphorylated ERK1/2, but not p38MAPK and JNK. It was inhibited by the pretreatment of anti-ST2 neutralizing (blocking) Abs. MEK inhibitor significantly blocked IL-17F production. Moreover, IL-33 phosphorylated MSK1, and MEK inhibitor diminished its phosphorylation. Finally, MSK1 inhibitors and transfection of the siRNAs targeting MSK1 significantly blocked the IL-17F expression.
IL-33 induces IL-17F via ST2-ERK1/2-MSK1 signaling pathway in bronchial epithelial cells. These data suggest that the IL-33/IL-17F axis is involved in allergic airway inflammation and may be a novel therapeutic target.
IL-33 has been recently identified as a novel member of the IL-1 family and is a ligand for ST2 . The receptor for IL-33, ST2, is specifically expressed in Th2-type immune response cells such as eosinophils, mast cells, and Th2 cells, but not on Th1 cells [2-4]. Recent studies have reported that the IL-33/ST2 axis contributes to allergic airway inflammation. Administration of IL-33 into mice induces eosinophilia, airway hyperactivity, and goblet cell hyperplasia . Moreover, the administration of neutralizing antibodies against IL-33 or ST2 attenuates airway hyperactivity and eosinophilia [6, 7]. Increased IL-33 expression is observed in the airway of patients with asthma when compared with healthy subjects . In addition, a recent genome-wide association scan for sequence variants revealed that single-nucleotide polymorphisms in the IL-33 and ST2 genes are associated with asthma . Thus, IL-33 could be a potential therapeutic target for asthma. However, its role in asthma has not been fully understood.
The IL-17 family of cytokines contains six members (IL-17A–IL-17F) . We and other groups discovered human IL-17F [11-13]. The IL-17F gene is clearly expressed in BAL cells from patients with asthma . Moreover, we have demonstrated that a coding-region variant (H161R) of the IL17F gene is inversely associated with asthma and encodes an antagonist for the wild-type IL-17F [14, 15]. These findings suggest that IL-17F is one of the key cytokines deeply involved in the pathogenesis of asthma and is a valuable therapeutic target. IL-17F is expressed in activated CD4+ T cells, basophils, and mast cells, the three important cell types involved in asthma . Th17 cells selectively express IL-17A and IL-17F, but not IL-4 and INFγ, and play a crucial role in airway inflammation [16, 17]. However, we and other groups have demonstrated that IL-17F is mainly expressed in bronchial epithelium in the airway of asthmatics [18, 19], and its level in bronchial epithelium is correlated with the disease severity of asthma , while the mechanisms of epithelium-derived IL-17F expression in asthma are not yet understood. To further clarify the function of IL-33 and the regulation of IL-17F expression, the effects of IL-33 on the expression of IL-17F were investigated. In this study, we demonstrated, for the first time, that IL-33 is a potent inducer of IL-17F in bronchial epithelial cells via the activation of the ST2-ERK1/2-MSK1 signaling pathway.
Two different types of bronchial epithelial cells were used in this study: A bronchial epithelial cell line, BEAS-2B, and normal human bronchial epithelial cells (NHBEs) were cultured as previously described .
Analysis of IL-17F gene expression
Total RNA was extracted using RNeasy (Qiagen, Chatsworth, CA, USA) from 1 × 106 BEAS-2B cells and at 12 h after stimulation with 10, 100, and 200 ng/ml of IL-33 (R&D Systems, Minneapolis, MN, USA). cDNAs were synthesized from 500 ng of total RNA using the cDNA synthesis kit (TOYOBO, Tokyo, Japan), followed by real-time PCR. The sequences of primers were as follows: for IL-17F, forward, 5′-GAAGCTTGACATTGGCATCA-3′, reverse, 5′-GTGTAATTCCAGGGGGAGGT-3′; for G3PDH, forward, 5′-ACCACAGTCCATGCCATCAC-3′, reverse, 5′-TCCACCACCCTGTTGCTGTA-3′. Real-time PCR was carried out using a SYBR Green PCR kit (TOYOBO), gene-specific primers, and an ABI 7700 thermal cycler as previously described . The data were shown as fold induction relative to the control group. The values are expressed as mean ± SD (n = 6 experiments).
Analysis of IL-17F protein expression
Cell supernatants in BEAS-2B cells and NHBEs were harvested from cultures in the absence or presence of 10, 100, or 200 ng/ml of IL-33 at 4, 12, 24, or 48 h after stimulation. IL-17F protein levels in the supernatants were determined with ELISA. ELISAs were developed using anti-human IL-17F antibody (R&D Systems), and human recombinant IL-17F protein (R&D Systems) was used for IL-17F protein standard curve. ELISAs were prepared by coating the bottom of a 96-well plate with 100 µl of 0.88 µg/ml capture antibody. Then, 100 µl of the supernatants was incubated, and 100 µl of 100 ng/ml detection antibody was added after washing with PBST, followed by the addition of TMB substrate (KPL, Gaithersburg, MD, USA) after washing with PBST. The values are expressed as mean ± SD (n = 6 experiments). The minimum concentration detected by this method was 20.0 pg/ml. IL-17A protein levels in the supernatants were determined with a commercially available ELISA kit (R&D Systems), according to the manufacturer's instruction. The minimum concentration detected by this method was 15.0 pg/mL. The values are expressed as mean ± SD (n = 6 experiments).
Involvement of ST2 in the expression of IL-17F
For analysis of the expression of ST2, the total cellular extracts (1 × 106 cell equivalents/lane) of BEAS-2B cells, NHBEs, and a recombinant human ST2 protein (Sino Biological Inc. Beijing, China) as positive control were subjected to 5–20% Tris–glycine gel electrophoresis (DRC, Tokyo, Japan), followed by transfer onto polyvinylidene difluoride membranes (Bio-Rad, Tokyo, Japan) as previously described . The antibodies (Ab) used were mouse anti-ST2 Ab and mouse IgG1 Abs for isotype control (R&D Systems). BEAS-2B cells were preincubated with different doses of these Abs as indicated for 1 h, followed by stimulation of the cells with medium or IL-33 for 24 h. IL-17F protein levels in supernatants were measured by ELISA as described above. The values are expressed as mean ± SD (n = 6 experiments).
Detection of MAP kinases and MSK1
For analysis of activation of MAP kinases and MSK1, the cells were treated with IL-33 (100 ng/ml) and in some cases with or without treatment with the MEK1/2 inhibitor PD98059, anti-ST2 neutralizing Abs, or a vehicle control (0.1%DMSO) for 1 h. Following treatment, Western blotting analysis was performed as described above. The Abs used were anti-ERK1/2 Ab, anti-phospho-ERK1/2 Ab, anti-phospho-JNK Ab, anti-phospho-p38MAPK Ab, anti-phospho-MSK1 Ab (Cell Signaling Technology, Danvers, MA, USA), and anti-MSK1 Ab (Santa Cruz Biotechnology, Santa Cruz, CA). A panel of kinase protein controls was included and run in parallel. Two controls included phosphorylated JNK and p38MAPK control cellular extracts (Cell Signaling Technology).
Effect of MAP kinase and MSK1 inhibitors on the expression of IL-17F
For analysis of involvement of the ERK1/2-MSK1 pathway, BEAS-2B cells were treated in the presence or absence of the following kinase inhibitors at varying doses: MEK1/2 inhibitor, PD98059; p38MAPK inhibitor, SB202190; JNK inhibitor, SP600125; MSK1 inhibitors, H89 and Ro-31-8220 (Calbiochem, San Diego, CA); and a vehicle control, 0.1%DMSO for 1 h before treatment with IL-33 (100 ng/ml). The supernatants were harvested at 24 h after stimulation for analyses with ELISA. IL-17F protein levels in the supernatants were determined as described above. These values are expressed as mean ± SD (n = 6 experiments).
Effect of knockdown of MSK1 with siRNA
Predesigned siRNAs for MSK1 (Santa Cruz Biotechnology) and control siRNAs (Ambion, Tokyo, Japan) were used. The siRNA transfection into BEAS-2B cells was performed according to the manufacturer's instruction. The supernatants were then harvested at 24 h after stimulation with 100 ng/ml of IL-33 and subjected to analysis by ELISA, respectively (each n = 6 experiments). IL-17F protein levels in the supernatants are expressed as mean ± SD.
The statistical significance of differences was determined by analysis of variance (anova). The values are expressed as mean ± SD from independent experiments. Any difference with P values <0.05 was considered significant. When anova indicated a significant difference, the Scheffe F-test was used to determine the difference between groups.
IL-33 induces the expression of IL-17F
First, the levels of IL-17F gene expression were analyzed by real-time PCR. IL-33 significantly induced IL-17F gene expression in a dose-dependent manner when compared with control (Fig. 1A). IL-17F proteins were weakly detected in untreated cells, but protein levels in supernatants were significantly increased and peaked at 24-h time point in both bronchial epithelial cell types, BEAS-2B cells and NHBEs (Fig. 1B). These cells showed similar kinetics. Although IL-17A has high homology with IL-17F, IL-17A was not induced by IL-33 24 h after stimulation (Fig. 1C).
Involvement of ST2 in the expression of IL-17F by IL-33
Bronchial epithelial cells constitutively expressed the receptor for IL-33, ST2 (Fig. 2A). Moreover, pretreatment of the cells with anti-ST2 neutralizing Abs significantly abolished IL-33-induced IL-17F protein expression in a dose-dependent manner (Fig.2B). In contrast, isotype control Abs had no significant effect on IL-17F expression.
Activation of MAP kinases by IL-33
Whereas no activation of p38MAPK and JNK kinases was seen at any time points (Fig. 3A) even after a 120-min stimulation of the cells with IL-33, Western blotting analysis revealed that the phosphorylation of ERK1/2 reached a maximum at 20 min and returned to baseline levels by 120 min in BEAS-2B cells (Fig. 3A). Pretreatment of the cells with anti-ST2 neutralizing Abs diminished the activation of IL-33-induced ERK1/2 (Fig. 3B). In addition, preincubation of the cells with MEK1/2 inhibitor, PD98059, diminished the activation of ERK1/2 in BEAS-2B cells, and preincubation of DMSO did not affect the phosphorylation of ERK1/2 (Fig. 3C).
Pretreatment of the cells for 1 h with a MEK1/2 inhibitor, PD98059, significantly decreased the levels of IL-33-induced IL-17F expression in BEAS-2B cells, while 1-h pretreatment of the cells with vehicle alone (DMSO) did not affect IL-17F expression. In addition, the protein levels of IL-17F were unchanged in IL-33-treated cells in the presence of varying doses of p38MAPK inhibitor, SB202190, and JNK inhibitor, SP600125 (Fig. 4).
Activation of MSK1 by IL-33
Transient phosphorylation of MSK1 was observed upon stimulation of the cells with IL-33, reaching the maximum at 30 min after stimulation (Fig. 5A). To establish the interrelationship between ERK1/2 and MSK1, the cells were treated with MEK1/2 inhibitor, PD98059, before the stimulation with IL-33. Pretreatment of PD98059 diminished the activation of MSK1 induced by IL-33 (Fig. 5B).
Effect of MSK1 inhibition on IL-33-induced IL-17F expression
Pretreatment with MSK1 inhibitors, Ro-31-8220 and H89, significantly blocked IL-33-induced IL-17F expression (Fig. 6A). Finally, to further confirm whether MSK1 plays a role in IL-33-induced IL-17F expression, total MSK1 expression was diminished in the cells by transfecting with siRNA targeting MSK1 (Fig. 6B). As shown in Fig. 6C, its expression induced by IL-33 was significantly inhibited in cells transfected with siRNA targeting MSK1, while no significant difference was seen in wild-type cells and cells transfected with a control siRNA.
In this study, we demonstrated that a new cytokine, IL-33, significantly induces expression of IL-17F, but not IL-17A, in bronchial epithelial cells through the activation of ST2-ERK1/2-MSK1 signaling pathway. These findings suggest that IL-33 is a potent inducer of IL-17F and is involved in the pathogenesis of allergic airway inflammation via the induction of IL-17F.
IL-33 plays a central role in allergic airway inflammation. IL-33 is able to induce airway hyper-responsiveness and goblet cell hyperplasia . The blockade of IL-33 inhibited allergen-induced airway eosinophils and mucus hypersecretion . The expression of IL-33 is significantly increased within the airways of subjects with severe asthma when compared with those with mild asthma . However, its function has not been fully clarified. Here, we found that IL-33 is able to induce IL-17F expression. IL-33 is produced by several lung structural cells including bronchial epithelial cells [1, 21]. Recent study demonstrated that bronchial epithelial cells are able to induce IL-33 by an airborne allergen such as Alternaria alternata . This suggests that bronchial epithelial cells play a crucial role for allergic airway inflammation, at least partially, via the induction of IL-33 and IL-17F expression.
The signaling pathway of IL-33 has not yet been fully understood. IL-33 exerts its cytokine activity through the specific receptor, ST2 . Although the expression of ST2 has been a useful marker to characterize Th2 cells, we and other groups have demonstrated that bronchial epithelial cells also express ST2 [23, 24]. In the upstream signaling pathway, IL-33 activates the ERK1/2 pathway in bronchial epithelial cells . IL-33 signal mediates ERK1/2 via ST2, as pretreatment of the cells with anti-ST2 Abs diminished IL-33-induced ERK1/2 activation, and significantly inhibited IL-17F expression. This suggests that the ST2-ERK1/2 pathway is a central one for IL-33 in bronchial epithelial cells. In contrast, little information has been available regarding its downstream signaling pathway. Here, we identified that MSK1 is a novel signaling molecule of IL-33. MSK1 is located downstream of the ERK1/2 cascade, as a MEK inhibitor blocked the phosphorylation of MSK1. Moreover, the activation of MSK1 is essential for IL-17F expression by IL-33, as MSK1 inhibitors and siRNA targeting MSK1 blocked its expression. This signaling pathway might be a potential pharmacological target in the IL-33-mediated airway inflammation. However, all inhibitors and siRNAs used in this study did not completely abrogate IL-33-induced IL-17F expression. This suggests the potential involvement of other signaling pathways. Further study is needed to clarify a novel signaling molecule of IL-33.
We found that IL-17F is clearly elicited by IL-33. IL-17F is involved in the pathogenesis of asthma, and upregulated expression of IL-17F is seen in BAL cells from asthmatics following segmental allergen challenge . Similarly, IL-17F is clearly upregulated in the mouse model of asthma . Moreover, immunocytochemistry showed that IL-17F is expressed in both bronchial epithelium and inflammatory infiltrates in the airway of asthmatics [18, 19]. We have also reported that IL-17F is a candidate gene for asthma susceptibility [14, 15]. IL-17F exerts multiple functions in airway inflammation. In vitro, IL-17F is able to secrete a number of cytokines and chemokines in several cell types. We have shown that IL-17F induces various cytokines and chemokines in bronchial epithelial cells [11, 20, 27-30]. Moreover, IL-17F acts on eosinophils and lung structural cells such as vein endothelial cells and fibroblasts to induce several cytokines and chemokines [12, 13, 31]. These cell types may play pivotal roles in asthma in response to IL-17F. In the mouse model of asthma, IL-17F enhances allergic airway inflammation such as goblet cell hyperplasia and increasing airway hyper-reactivity . IL-17F shows the highest homology with IL-17A among the IL-17 family . In this study, we found different expression of IL-17A and IL-17F, despite their high homology. IL-17A was not induced by IL-33 in bronchial epithelial cells. Consistent with this, it is reported that IL-33 is not able to produce IL-17A in bone narrow-derived basophils and mast cells . Moreover, their expression pattern in asthmatic airway is not identical . Unlike IL-17F, IL-17A was not expressed in airway epithelium. However, little is known about the differences in the signaling pathways for the expression of IL-17A and IL-17F. Additional work is needed to determine their regulatory mechanisms.
Th17 cells are implicated in the pathogenesis of airway diseases, and Th17 cells have been observed in bronchial tissues taken from patients with severe asthma . However, Th17 cells may not be the major cell source of IL-17F in airway diseases . Indeed, IL-17F is produced by many cell types such as basophils, mast cells, monocytes, memory CD4+ T cells, NKT cells, and NKT cells [11, 13, 35]. Here, we have identified a novel cell source, bronchial epithelial cells, for IL-17F. Interestingly, IL-17F immunoreactivity in airway epithelial cells is correlated with the disease severity of asthma . However, the inducer and regulatory mechanisms of IL-17F expression in airway inflammation are unclear. The current study has demonstrated, for the first time, that IL-33 is a novel inducer, and epithelial IL-17F expression is mediated by ST2-ERK1/2-MSK1 signaling pathway. Hence, the potential involvement of IL-33 in the airway inflammatory process is likely mediated, in part, through the induction of IL-17F. The IL-33/IL-17F axis might be especially important in the pathophysiologic events of airway inflammation.
In conclusion, this study has revealed a new cytokine interactive network, wherein IL-33 induces IL-17F through the activation of the ST2-ERK1/2-MSK1 signaling pathway. The respective roles of IL-33 and IL-17F in airway inflammation have been suggested, and our study provides evidence for a functional linkage between these 2 cytokines, further strengthening their role in the regulation of allergic airway inflammation.
We thank Beverly Plunkett, Hideaki Watanabe, and Miho Kawaguchi for their excellent technical assistance. This work was supported by Grants-in-Aid for Scientific Research (C) 23591455 and the Alumni Award of Showa University School of Medicine. S. K. Huang was supported, in part, by National Institute of Health (AI-052468) and National Health Research Institutes.
Conflict of interest
The authors have no financial conflicts of interest.