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.