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.