Discussion

In the present study, we found that poly (I:C) and IL-13 synergistically enhanced the production of CCL5 in bronchial epithelial cells. We regarded this experimental setup as a characteristic in vitromodel of airway disease, because CCL5 production is clinically observed in patients with repeated viral infections, eosinophilic asthma, and persistent Th2-type inflammation. The production of CCL5, induced by poly (I:C) plus IL-13, was regulated by the TLR3-IRF3-IFNAR/JAK1-PI3K pathway; this may be due to activation of the IL-13Rα2-PI3K pathways. This prompted our trial of ruxolitinib, a clinically available JAK1 inhibitor. We confirmed that ruxolitinib was a better inhibitor than FP for decreasing the synergistic production of CCL5 in vitro . Hence, ruxolitinib is a potential therapeutic agent for corticosteroid-resistant severe eosinophilic asthma with a persistent type-2 inflammation phenotype. This is the first observation of the potential role of ruxolitinib in this setting.
Exacerbation of asthma is strongly associated with various RNA virus infections, including rhinovirus, RSV, and EV (4, 22). Clinical and experimental data demonstrate that asthmatics have deficient immune responses to viruses and show higher viral loads and greater airway inflammation after viral infections than healthy subjects (25-27). The airway viral load in asthmatics correlates strongly with the severity of symptoms, hyperresponsiveness, and airflow limitations (26, 28), and is associated with airway eosinophilia. Furthermore, latent infection resulting from rhinovirus has been observed in 73% of stable asthmatics, in whom viral infection has been associated with eosinophilic lung infiltration and decreased lung function (29). In this study, we confirmed that poly (I:C), an RNA virus-related TLR3 ligand, stimulated CCL5 production in bronchial epithelial cells. Taken together, it can be stated that patients with asthma are highly susceptible to viral infections, and both active and latent virus infection enhance airway eosinophilia and increase disease severity.
Eosinophilic airway infiltration plays a major role in the pathogenesis of asthma. Airway eosinophilia is associated with recurring bronchial hyperresponsiveness and airflow limitations that account for the pathogenesis and the severity of asthma, respectively (30). Airway eosinophil counts in asthmatics are higher than in healthy subjects, and are further elevated after viral infection (26). Moreover, viral infections cause the release of IL-33 from bronchial epithelial cells, which enhances IL-13 and IL-5 production in Th0 cells and ILC-2s (26), providing a mechanism by which eosinophilic and Th2-type asthma commonly overlap. We have demonstrated that the virus-associated TLR3 ligand, along with either IL-13 or IL-4, enhanced CCL5 production in bronchial epithelial cells. Because a genome-wide association study and experiments using mouse asthma models have indicated that IL-13 contributes more strongly to the pathogenesis of arising asthma than does IL-4 (31, 32), we used IL-13 instead of IL-4 for the rest of the experiments. These findings demonstrate that viral infection in Th2-type asthma results in a prominent increase in airway CCL5 production, which offers a potential mechanism through which severe eosinophilic asthma develops.
CCL5 is a major eosinophil chemotactic molecule that exists in greater volumes in the lungs of asthmatics than in those of non-asthmatics (33, 34). Asthmatics show higher CCL5-mRNA expression in bronchial mucosal cells than in control non-asthmatic subjects (35). Experimental allergen challenge in asthmatics also causes an increase in the airway CCL5 level, which correlates positively with airway eosinophil counts (36). Airway CCL5 levels correlate positively with asthma severity, as measured by the percent predicted forced expiratory volume in 1 second (FEV1) (34). CCL5 levels are higher in severe asthmatics than in untreated mild asthmatics (37). Additionally, in a mice model, viral infection enhances airway CCL5 levels (38). Our in vitro study has demonstrated that poly (I:C) plus Th2-type cytokines synergistically stimulates CCL5 production in bronchial epithelial cells. The simulation of viral infection and Th2-type inflammation results in increased CCL5 production in bronchial epithelial cells, as is observed in humans. As such, we consider this experimental setup a representative in vitro model of severe eosinophilic asthma.
Multiple signal-transduction pathways are involved in virus-triggered CCL5 production in bronchial epithelial cells. ds-RNA binds to TLR3 and activates downstream NF-κB and IRF3 cascades, leading to CCL5 production (39). NF-κB directly activates a variety of genes associated with inflammation (40), whereas IRF3 induces gene expression of IFNs, which in turn leads to the expression of ISGs via receptors for type I IFN (41). Our data demonstrate that IRF3-knockdown strongly inhibited CCL5 production while Rel A-knockdown and BAY11-7082 failed to inhibit CCL5 production in BEAS-2B cells. These results show that the TLR3 ligand stimulated the BEAS-2B cells to produce CCL5 via IRF3 but not NF-κB.
Øvrevik et al. have also reported that Rel A-knockdown did not attenuate poly (I:C)-induced CCL5 production in BEAS-2B cells (42). However, other reports showed that poly (I:C) stimulated BEAS-2B cells to produce CCL5 via both the NF-κB and IRF3 pathways (43, 44). The reason for the discrepancy between our data and the existing literature remains unclear. There are two possible explanations for these differences: (1) we and Øvrevik et al.’s group used serum-free medium for culturing BEAS-2B cells, whereas others have used medium containing fetal bovine serum (42-44); and (2) the quality or purity of the poly (I:C) used may have differed between studies. The former is the more likely explanation, because the presence of fetal bovine serum leads to NF-κB activation (45). The latter is less likely, since poly (I:C) is a synthesized molecule. Nevertheless, our data agreed with previously demonstrated findings that show that the TLR3-IRF3 axis plays an important role in poly (I:C)-induced CCL5 production in bronchial epithelial cells.
TLR3-induced CCL5 production is mediated by type I IFNs. IRF3 drives the expression of the gene encoding IFN and subsets of ISGs(46). Type I IFNs bind to the interferon-α/β receptor (IFNAR), which is composed of IFNAR1 and IFNAR2 (bearing JAK1 and TYK2, respectively), and activates multiple canonical pathways (i.e. STAT1-STAT2-IRF9, STAT1-STAT1, and STAT3-STAT3 mediated) (47). In addition, it has been also reported that IFNAR activates alternative cascades mediated by the PI3K-AKT and Erk 1/2 pathways (48), and another study has reported that type I IFN activates PI3K in JAK1-dependent manner (49). Our results have demonstrated that poly (I:C)-induced CCL5 production was attenuated by the neutralizing antibody against type I IFNs, JAK1-knockdown, the JAK1/2 inhibitor ruxolitinib, and the PI3K inhibitor LY294002, but not by inhibitors for STAT1, STAT3, or Erk 1/2. These findings indicate that poly (I:C)-induced CCL5 production is stimulated by TLR3-IRF3-IFNAR/JAK1-PI3K pathway but not by the canonical IFNAR/JAK-STAT pathway in BEAS-2B cells.
IL-13 and IL-4 both have augmented poly (I:C)-induced CCL5 production in BEAS-2B cells. The canonical receptor for IL-13 is a type II receptor complex that consists of IL-4Rα and IL-13Rα1 anchoring JAK1 and TYK2, respectively (50). The receptor for IL-4 receptor is a type I receptor complex that includes IL-4Rα and a common γ-chain that bears JAK1 and JAK3 (50), which also activates the PI3K pathway in JAK1-dependent manner (49, 51). The non-canonical IL-13 receptor is IL-13Rα2, which lacks JAKs and TYK2 (50) but activates the PI3K and Erk 1/2 pathways (52, 53).
Contrary to expected findings, IL-13 and poly (I:C)-induced CCL5 production was independent of STAT6 but was dependent on PI3K, indicating that the synergy of the two stimulating cytokines was possibly induced by the IL-13Rα2-PI3K pathway. This is consistent with a previous report whereby PI3K was found to bind to TLR3, which subsequently phosphorylated IRF3: an essential step for TLR3-IRF3-mediated gene induction (54). This signaling cascade can explain our observation that IL-13-augmented poly (I:C)-induced CCL5 production, while IL-13 alone did not stimulate CCL5 production. Moreover, the IFNAR/JAK1-PI3K pathway is also involved downstream of the TLR3-IRF3 pathway (49). Therefore, the JAK1-PI3K pathway is a key regulator for synergistic CCL5 production, which is a potential therapeutic target for severe asthma.
In our in vitro model of severe eosinophilic asthma that used IL-13 and poly (I:C), the JAK1-PI3K pathway played a pivotal role in CCL5 production. The JAK1 inhibitor, ruxolitinib, is already clinically available for treating myelofibrosis (21, 22). We demonstrated that ruxolitinib could more strongly decrease poly (I:C) and IL-13-induced CCL5 production compared with the corticosteroid, FP. These findings suggested that ruxolitinib may be used for treating severe asthma and is thus a potential therapeutic agent for corticosteroid-resistant severe eosinophilic asthma.
Despite recent advances in medication for bronchial asthma, approximately 10% of those diagnosed have uncontrolled symptoms (3). A common phenotype of severe asthma includes persistent type-2 inflammation, which is characterized by sputum eosinophilia, high doses of inhaled corticosteroids, severe airflow limitations, and airway hyperresponsiveness (3). We demonstrated, using an in vitromodel, that ruxolitinib is potentially beneficial for treating severe eosinophilic asthmatics who require high doses of inhaled corticosteroids. Previous studies have shown that JAK1 is involved in many signaling cascades of IFNs, growth factors, and cytokines (55). This suggests that ruxolitinib has a possible advantage, given its ability to inhibit multiple pathways, versus monoclonal antibody therapies that target only a single molecule. Furthermore, we demonstrated that ruxolitinib is a more effective inhibitor of CCL5 production than FP, which is also used in inhaled corticosteroids. This indicates that ruxolitinib inhalation therapy may have therapeutic potential, since it carries a lower risk of systemic toxicity than FP, although further research is required to confirm this.
Our study has some limitations. First, we used a cell line, BEAS-2B, in most experiments, although we initially used primary bronchial cells derived from healthy subjects and not asthmatic patients. As such, it is important to verify the efficacy of ruxolitinib in animal models of asthmatic disease. Second, although we found that the IL-13Rα2- PI3K pathway was implicated in the observed effects of poly (I:C) and IL-13, we did not investigate the impact of IL-13R α2 si-RNAs or inhibitors on this in vitro system. The effectiveness of ruxolitinib, however, in this cellular model of eosinophilic asthma is novel and encouraging and warrants additional exploration. In future we plan to conduct the following investigations: (1) evaluate the efficacy of ruxolitinib in a mouse asthma model; and (2) investigate the potential of ruxolitinib inhalation therapy, using intratracheal administration in animal models.
In conclusion, by treating BEAS-2B cells with poly (I:C) and IL-13, we developed an in vitro model of severe eosinophilic asthma with persistent type-2 inflammation, as evidenced by increased CCL5 production. The efficacy of the JAK1 inhibitor ruxolitinib in inhibiting CCL5 production in this in vitro model suggests that ruxolitinib has therapeutic potential in severe eosinophilic asthma, which requires high-dose inhaled corticosteroids. Further evaluation in animal models and clinical studies are necessary to confirm the suitability of ruxolitinib in patients.