PBECs were collected and cultured according to our previously described protocol (14). Briefly, PBECs were obtained during routine fibreoptic bronchoscopy from never-smoker patients with normal lung function and pulmonary nodules by performing bronchial brushings from healthy lobes without a pulmonary nodule (healthy PBECs). All bronchial brushings were obtained from the same anatomical region (bronchial generations 4–7). PBECs were also collected from patients with asthma and patients with COPD for use in some experiments. The characteristics of patients with asthma or COPD at bronchoscopy are shown in Table S1 . Unless otherwise stated, healthy PBECs were used in the experiments. The study protocol was approved by the Kyushu University Institutional Review Board for Clinical Research (29-170). All subjects provided written informed consent in accordance with the principles laid out in the Declaration of Helsinki.

Cells were cultured in flasks coated with collagen (Cell Applications, Inc., San Diego, CA, USA) containing supplemented bronchial epithelial growth medium (BEGM; Lonza, Basel, Swiss) at 37°C in 5% CO₂ and used within four passages. PBECs (approximately 80% confluency) were pretreated with 250 U/ml human IFNβ1a (PBL Assay Science, Piscataway, NJ, USA), 250 U/ml human IFNλ1 (PBL Assay Science), 10 μM IC87114 (BioVision, Milpitas, CA, USA), 10 μM AS604850 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) or vehicle for 1 h and then stimulated by the addition of 1 μg/mL poly I:C (Innaxon, Tewkesbury, United Kingdom) to the culture medium. In virus infection experiments, 10 μM IC87114 was added to cells for 1 h prior to hMPV infection, and IC87114 was maintained in medium after infection. PBECs were cultured in BEGM without hydrocortisone for at least 24 h prior to stimulation or infection.

PBECs were cultured to semi-confluence in 12-well plates. Cells were incubated in 100 μL of PBS containing 0.5% BSA containing PE anti-human PD-L1 (clone: MIH1; Invitrogen, Carlsbad, CA, USA) or PE anti-human PD-L2 (clone: MIH18; Invitrogen) at room temperature for 30 min in initial experiments. In some experiments, cells were incubated in 100 μL of PBS with 0.5% BSA containing APC anti-human PD-L1 (clone: MIH1; Invitrogen) and PE anti-human PD-L2 (clone: MIH18; Invitrogen) at room temperature for 30 min. Cells were thoroughly washed and analyzed using a BD FACSVerse flow cytometer with FACSuite software (Becton Dickinson, Franklin Lakes, NJ, USA). Ten thousand events were acquired in list mode with debris excluded by the forward scatter threshold.

For the analysis of cell viability, resuspended lung cells or PBECs were incubated with propidium iodide (PI), and non-viable cells (stained with PI) were counted in a flow cytometer with debris excluded by the forward scatter threshold.

PBECs cultured in 6-well plates (60%–80% confluence) were transiently transfected with 10 nM IFN regulatory factor 3 (IRF3) siRNA (Silencer^® Select Validated siRNA, s7507; Ambion, Life Technologies, Carlsbad, CA, USA), 10 nM PIK3CD siRNA (Silencer^® Select Validated siRNA, s10529; Ambion, Life Technologies) or 10 nM negative control (NC) siRNA (Silencer^® Select Negative Control siRNA, 4390843; Ambion, Life Technologies) using Lipofectamine^® RNAiMAX Reagent (Invitrogen) according to the manufacturer’s instructions. Lipofectamine^® RNAiMAX Reagent or siRNA were diluted in BEGM without hydrocortisone and antibiotics. Cells were used for experiments at 48 h after transfection.

PBECs cultured to semi-confluence in 6-well plates were lysed using Pierce^® RIPA Buffer (Thermo Fisher Scientific, San Diego, CA, USA) according to the manufacturer’s instructions. Protein samples (4 µg) were denatured, separated by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. Membranes were blocked with TBS-Tween (10 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 8.0) containing 5% skimmed milk for 1 h at room temperature. The membrane was then incubated with anti-PD-L1 (Cell Signaling Technology), anti-PD-L2 (Cell Signaling Technology), anti-the TANK-binding kinase-1 (TBK1) (Cell Signaling Technology, Beverly, MA, USA), anti-phospho-TBK1 (Ser172, Cell Signaling Technology), anti-IRF3 (Cell Signaling Technology), anti-phospho-IRF3 (Ser386, Cell Signaling Technology), anti-PI3 Kinase p110δ (Cell Signaling Technology) or anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) antibodies at 4°C overnight. Membranes were washed three times with TBS-Tween and then incubated with a horseradish peroxidase-conjugated secondary antibody for 60 min at room temperature. Membranes were washed three times with TBS-Tween, and specific bands were visualized using ImmunoStar^® LD (Wako, Osaka, Japan) according to the manufacturer’s instructions. All blots were imaged using the ChemiDoc™ XRS+ system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Densitometric analysis of band intensities was performed using Image J.

Total RNA was isolated from PBECs using TRI Reagent (Molecular Research Centre, Inc., Cincinnati, OH, USA). Reverse transcription was performed using Multiscribe Reverse Transcriptase (Invitrogen). Real-time quantitative RT-PCR analyses were performed once per sample using Fast Start SYBR Green Master (Roche, Mannheim, Germany) and a Thermal Cycler Dice Real Time System II (Takara, Shiga, Japan). The mRNA expression levels were calculated from the threshold cycle according to the ΔΔCt method. Target gene expression levels were normalized to the expression of 18S rRNA. Primer sequences are provided in Table S2 .

The CAN97-83 strain of hMPV was used; hMPV was propagated in Vero E6 cells (CRL-1586; ATCC, Manassas, VA, USA) and virus titer was determined by TCID₅₀ assay. In brief, virus was prepared in serum-free MEM (Gibco, Thermo Fisher Scientific, San Diego, CA, USA) containing 5 μg/ml trypsin (Gibco) by infecting cells in a 10-cm culture dish at a multiplicity of infection (MOI) of 0.05. After absorption for 2 h at 37°C, serum-free MEM containing 5 μg/ml trypsin was added. Serum-free medium containing 5 μg/ml trypsin was changed every other day during incubation and cells were incubated until a 70%–90% cytopathic effect was observed, usually within 7 days. Cells and culture supernatant were　collected and virus was obtained by freeze-thawing the cells. This strain of hMPV has been used in other studies (19), and we did not attempt further purification to avoid altering key properties of the virus (20).

Virus was quantified by immunostaining assay. Briefly, semi-confluent Vero E6 monolayers in a 24-well plate were infected with 200 μl of 10-fold serial dilutions. Trypsin (5 μg/ml) was added in serum-free MEM during infection. After absorption for 2 h at 37°C, 200 μl of MEM containing 5% FBS was added. After 6 days of cell culture, infected wells were identified by immunostaining with mouse anti-hMPV monoclonal primary antibody (MAB8510; EMD Millipore, Temecula, CA, USA) and horseradish peroxidase-labeled goat anti-mouse secondary antibody (Abcam, Cambridge, UK). Cells were visualized using DAB peroxidase substrate (Vector Laboratories, Inc., Burlingame, CA, USA) and the TCID₅₀/ml was determined using standard methods.

Control experiments using UV-inactivated hMPV were included to confirm that responses were a result of hMPV infection and not from factors remaining in the culture media following hMPV propagation. To UV-irradiate virus, hMPV was exposed to a short wavelength (254 nm) UV lamp at a distance of 5 cm for 15 min. PBECs (approximately 80% confluency) were washed with PBS and infected with hMPV in BEGM without hydrocortisone for 1 h at a MOI of 0.1. Cell monolayers were washed and then incubated in BEGM without hydrocortisone for 24–72 h. Standard biosecurity and institutional safety procedures were followed during this study.

Unless otherwise stated, data were expressed as means ± standard error (SEM). The Mann–Whitney U-test was used for comparisons between two groups. Comparisons of three or more groups were conducted using one-way analysis of variance (ANOVA) or two-way ANOVA followed by Tukey’s multiple comparisons test. Correlations were examined using Spearman correlation. All statistical analyses were conducted using GraphPad Prism 8 software (GraphPad Software, San Francisco, CA, USA). Differences were considered statistically significant at p<0.05.

Stimulation with IFNβ1a alone or poly I:C alone increased the expression of PD-L1 and PD-L2 on PBECs at 6 and 12 h following stimulation, respectively ( Figure 1A ). The combination treatment of IFNβ1a plus poly I:C upregulated the expression of PD-L1 and PD-L2 to higher levels compared with stimulation with IFNβ1a or poly I:C alone. Stimulation with IFNβ1a alone or the combination treatment of IFNβ1a plus poly I:C induced higher gene expression levels of IFN-stimulated gene (ISG) 56 than stimulation with poly I:C alone ( Figure 1B ). Similarly, IFNλ1 or poly I:C alone increased the expression of PD-L1 and PD-L2 on PBECs at 24 h after stimulation, and further upregulation of PD-L1 and PD-L2 was observed after the combination treatment of IFNλ1 plus poly I:C ( Figure S1A ). The cell viability of PBECs at 24 h after stimulation was not affected by treatment with IFNβ1a or IFNλ1 and/or poly I:C ( Figure S1B ).

Next, we assessed the effect of treatment with IFNβ1a and/or poly I:C on gene expression of PD-L1 and PD-L2. Treatment with IFNβ1a alone or poly I:C alone induced PD-L1 and PD-L2 gene expression 6 h following stimulation ( Figure 2A ). The combination treatment of IFNβ1a plus poly I:C further increased gene expression levels of PD-L1 and PD-L2 compared to treatment with IFNβ1a or poly I:C alone. IFNβ1a alone or poly I:C alone also increased the protein levels of PD-L1 and PD-L2 at 24 h following stimulation, and combination treatment enhanced both protein levels ( Figure 2B ).

We previously reported that the selective PI3Kδ inhibitor IC87114 attenuated poly I:C–induced PD-L1 expression on bronchial epithelial cells via inhibition of the Akt/mTOR signaling pathway (14). However, it has been unclear whether IC87114 affects poly I:C–induced PD-L2 expression. Here we analyzed the effects of IC87114 on the expression of PD-L1 and PD-L2 in PBECs. Although IC871114 did not affect poly I:C–induced gene expression levels of PD-L1, IC87114 markedly increased poly I:C–induced gene expression levels of PD-L2 at 12 h and 18 h following stimulation with poly I:C ( Figure 3A ). Consistent with our previous report, IC87114 suppressed poly I:C–induced PD-L1 expression at 24 h after poly I:C stimulation ( Figure 3B ). In contrast, IC87114 significantly enhanced poly I:C–induced PD-L2 expression. However, the combination treatment of IC87114 plus IFNβ1a did not additively enhanced gene expression levels of PD-L1 and PD-L2 ( Figure S2 ). PI3Kγ knockout mice were reported to be highly susceptible to lethality following infection with influenza A virus due to impaired antiviral IFNs responses (21). Hence, we also evaluated the effects of the selective PI3Kγ inhibitor AS604850 on the expression of PD-L1 and PD-L2. Unlike IC87114, AS604850 suppressed poly I:C–mediated gene and protein induction of both PD-L1 and PD-L2 expressions ( Figures 3C, D ). AS604850 also decreased poly I:C-induced gene expression levels of ISG56 ( Figure S3A ). Treatment with IC87114 or AS604850 with or without poly I:C did not affect the cell viability of PBECs ( Figure S3B ). These results indicate that inhibition of PI3Kδ signaling enhanced poly I:C–induced PD-L2 expression on PBECs, while inhibition of PI3Kγ signaling did not have this effect.

To confirm that inhibition of PI3Kδ signaling enhanced poly I:C–induced upregulation of PD-L2, we examined the effect of knockdown of the PIK3CD gene, which encodes PI3K p110δ, in PBECs. PI3K p110δ expression was suppressed in PBECs transfected with siPIK3CD at 48 h and 72 h following transfection compared with cells transfected with si-negative control (NC) ( Figure 4A ). PBECs transfected with siPIK3CD or siNC for 48 h were stimulated with poly I:C for 24 h, and PD-L2 expression was analyzed. Untreated cells transfected with siPIK3CD showed no changes in PD-L2 expression ( Figure 4B ). However, poly I:C–induced PD-L2 expression was enhanced on PBECs transfected with siPIK3CD compared with cells transfected with siNC ( Figure 4B ). Cell viability of PBECs was not affected by siRNA knockdown of PIK3CD and stimulation of poly I:C ( Figure 4C ).

We previously reported that IC87114 enhanced poly I:C–induced protein levels of IFNβ and IFNλ in bronchial epithelial cell culture supernatants (14). To investigate the mechanism of enhanced gene and protein expression of PD-L2 by IC87114 plus poly I:C, we examined the effects of IC87114 on the gene expression levels of poly I:C–induced antiviral IFNs and IRGs in PBECs. Stimulation with poly I:C increased gene expression levels of IFNβ and IFNλ at 3 h and 6 h following stimulation, respectively, as well as IRGs such as MxA and ISG56 at later time points ( Figure 5A ). Treatment with IC87114 together with poly I:C significantly enhanced gene expression levels of IFNs and IRGs compared with levels in cells treated with poly I:C alone.

Activation of TBK1/IRF3 pathway is essential to elicit antiviral IFN responses after the binding of viral dsRNA to toll-like receptor 3 (22). Thus, we examined the effects of IC87114 on TBK1 and IRF3 phosphorylation in PBECs by western blot analysis. The combined treatment of IC87114 plus poly I:C significantly increased the levels of phosphorylated TBK1 and IRF3 at 1 h and 2 h following stimulation, respectively, compared with levels in cells treated with poly I:C alone ( Figure 5B ). Total TBK1 and IRF3 levels were not affected by the combined treatment of IC87114 and poly I:C stimulation ( Figure S4 ).

Next, we assessed the effects of knockdown of the IRF3 gene, which encodes a key transcription factor in the poly I:C–induced antiviral IFNs response, on the expression of PD-L1 and PD-L2 in PBECs stimulated with poly I:C with or without IC87114. We confirmed decreased protein levels of IRF3 in PBECs at 48 h and 72 h following siIRF3 transfection ( Figure 6A ). PBECs transfected with siIRF3 or siNC for 48 h were stimulated with poly I:C alone or IC87114 plus poly I:C, and the expressions of PD-L1 and PD-L2 were analyzed 24 h following stimulation. We previously showed that the NF-κB pathway plays an essential role in poly I:C–induced upregulation of PD-L1 (23). Knockdown of the IRF3 gene did not attenuate poly I:C–induced PD-L1 and PD-L2 expression ( Figure 6B ). Regardless of transfection with siNC or siIRF3, IC87114 suppressed poly I:C–induced PD-L1 expression. Furthermore, the enhancement of poly I:C–induced PD-L2 expression by IC87114 was counteracted in PBECs transfected with siIRF3. Cell viability was not affected by stimulation of poly I:C alone or combined with IC87114 after siNC or siIRF3 transfection ( Figure S5 ).

The effects of IC87114 on poly I:C–induced PD-L1 and PD-L2 expressions were assessed using PBECs collected from patients with asthma or COPD. Similar to observations in healthy PBECs, treatment with IC87114 suppressed poly I:C–induced PD-L1 and increased poly I:C–induced PD-L2 in PBECs from patients with asthma ( Figure 7A ). In PBECs from patients with COPD, treatment with IC87114 increased poly I:C–induced PD-L2, but it did not affect poly I:C–induced PD-L1 ( Figure 7B ).

Finally, we investigated whether IC87114 affects virus-induced PD-L1 and PD-L2 expression in PBECs. Infection of PBECs with hMPV at a MOI of 0.1 induced the expression of PD-L1 and PD-L2 at 48 h and 72 h post-infection, and UV-irradiated hMPV did not increase PD-L1 or PD-L2 expression ( Figure 8A , S6 ). Similar to observations in PBECs treated with IC87114 plus poly I:C, hMPV-induced PD-L1 expression was suppressed in PBECs treated with IC87114 at 48 h post-infection, while IC87114 enhanced hMPV-induced PD-L2 expression ( Figure 8B ). There was a linear association between the gene expression of hMPV nucleocapsid protein (hMPV N) and hMPV-induced IFNβ, PD-L1, and PD-L2 in cells treated with or without IC87114 ( Figure 8C ). Treatment with IC87114 changed the slope of a regression line of IFNβ and PD-L2 larger, which indicates more efficient induction of IFNβ and PD-L2 gene responsive to hMPV infection in cells treated with IC87114.