Results
Poly (I:C) potentiates CCL5 production in human bronchial
epithelial
cells
Initially, we examined whether bronchial epithelial cells produced CCL5
when stimulated with poly (I:C), a ligand for TLR3. We found that poly
(I:C) enhanced CCL5 production in primary bronchial epithelial cells
(Fig. 1A). In BEAS-2B cells, poly (I:C) stimulated both CCL5 protein
release (Fig. 1B) and mRNA expression (Fig. 1C). For better
reproducibility we used BEAS-2B cells instead of primary bronchial cells
for all subsequent culture experiments. We also stimulated BEAS-2B cells
with CpG-ODN, another viral ligand for TLR9, but this treatment did not
augment cellular CCL5 production (Fig. 1D). These results demonstrated
that viral infection enhanced CCL5 production in bronchial epithelial
cells.
Poly (I:C)-induced CCL5 production is further enhanced by
the presence of Th2
cytokines
Next, we examined whether the presence of Th2-type cytokines further
stimulated poly (I:C)-induced CCL5 production in bronchial epithelial
cells. IL-13 enhanced poly (I:C)-induced CCL5 production (Fig. 1E) and
mRNA expression (Fig. 1F) in BEAS-2B cells. IL-4 also augmented poly
(I:C)-induced CCL5 production (Fig. 1G), even though neither IL-13 nor
IL-4 alone stimulated CCL5 production in BEAS-2B cells (Fig. 1E, G).
IL-33, IL-37, and CC16 in combination with poly (I:C) all failed to
stimulate CCL5 production (Supp. Fig. 1). We also examined whether IL-13
enhanced poly (I:C)-induced CXCL8 production, but it did not (Supp. Fig.
2). Together, these data show that poly (I:C) plus IL-13 or IL-4
synergistically induced CCL5 production in bronchial epithelial cells.
This experimental setup thus provides an in vitro model of severe
eosinophilic asthma associated with viral infection and persistent
type-2 inflammation.
Signal transduction mechanisms in BEAS-2B cells stimulated
with poly (I:C) and
IL-13
Using si-RNA techniques and inhibitors, we investigated the signal
transduction mechanisms that may be involved in CCL5 production of
BEAS-2B cells after stimulation with poly (I:C) and IL-13.
We first examined molecular signals for CCL5 production as induced by
poly (I:C) alone. TLR3- and IRF3-knockdown strongly inhibited poly
(I:C)-induced CCL5 production (Fig. 2A, B), whereas Rel A-knockdown and
NF-κB inhibitor (BAY11-7082) did not affect CCL5 production (Fig. 2C,
D). The neutralizing antibody against type I IFNs mixture inhibited poly
(I:C)-induced CCL5 production (Fig. 2E). Next, we assessed pathways that
were associated with the interferon receptor. We found that si-JAK1 and
the JAK1 inhibitor, ruxolitinib, attenuated poly (I:C)-induced CCL5
production (Fig. 2F, G). However, inhibitors for STAT1 and STAT3 did not
affect poly (I:C)-induced CCL5 production (Fig. 2H, I). These findings
suggest that canonical type I interferon receptor
(IFNAR)/JAK-STAT-associated pathways were not involved in the activation
of poly (I:C)-induced CCL5 production.
We subsequently assessed alternative pathways, including PI3K and
Erk1/2, and we found that PI3K, but not Erk1/2, is involved in poly
(I:C)-induced CCL5 production (Figs 2J, K). Therefore,
the TLR3-IRF3-IFNAR/JAK1-PI3K
cascade played an important role in poly (I:C)-induced production of
CCL5 in bronchial epithelial cells.
Next, we evaluated the role of IL-13 receptor-associated signals in
cells treated with both IL-13 and poly (I:C). Although the canonical
IL-13 receptor signal activated the IL-4Rα/IL-13Rα1/JAK1-STAT6 pathway,
STAT6-knockdown failed to inhibit the synergistic effect observed from
poly (I:C) and IL-13 (Fig. 3A), suggesting that an alternative pathway
was involved. Since IL-13 also activated the IL-13Rα2-PI3K-AKT pathway
(24), and we noted that the PI3K inhibitor attenuated CCL5 production
(Fig. 3B), it is likely that the IL-13Rα2-PI3K-AKT pathway is implicated
in bringing about the synergistic effect observed. IRF3- and JAK1-
knockdowns (Fig. 3C, D) and treatment with the JAK1/2 inhibitor
ruxolitinib (Fig. 3E) also attenuated CCL5 production, further
confirming that the TLR3-IRF3-IFNAR/JAK1-PI3K pathway is involved.
Together, these data demonstrate that IL-13 plus poly (I:C)
synergistically induced the production of CCL5 in BEAS-2B cells via the
TLR3-IRF3-IFNR/JAK1-PI3K-AKT and IL-13Rα2-PI3K pathways (Supp. Fig. 3).
Ruxolitinib is a potential therapeutic agent for severe
eosinophilic asthma.
Based on our data, the IFNAR/JAK1-PI3K pathway was a key regulator of
CCL5 production in our in vitro model of severe eosinophilic
asthma with persistent type-2 inflammation. Ruxolitinib, a JAK1
inhibitor, is already clinically available. Hence, we assessed the
therapeutic potential of ruxolitinib in comparison with FP, as measured
by the ability of both drugs to inhibit CCL5 production after induction
with both poly (I:C) and IL-13.
We first conducted a dose-response-inhibition experiment, followed by a
curve-fitting analysis. These experiments estimated that the maximal
inhibitory effects of ruxolitinib and FP against poly (I:C) induced-CCL5
production were 73.4% and 41.7% (Fig. 4A, B), respectively. We then
conducted a head-to-head comparison between ruxolitinib (10µM) and FP
(1µM). The selected concentrations of these drugs were based on the
maximal non-toxic concentrations as determined by preliminary
cell-toxicity experiments (Supp. Fig.4). We found that ruxolitinib
(10µM) was a stronger inhibitor of CCL5 production than FP (1µM) (Fig.
4C). The ruxolitinib activity in this in vitro model supports the
hypothesis that ruxolitinib is a better therapeutic option than FP for
managing eosinophilic asthma with type-2 inflammation.