Inhibition of PI3Kδ Enhances Poly I:C-Induced Antiviral Responses and Inhibits Replication of Human Metapneumovirus in Murine Lungs and Human Bronchial Epithelial Cells

Abstract

Viral infections of the airway can exacerbate respiratory diseases, such as asthma or chronic obstructive pulmonary disease (COPD), and accelerate disease progression. Phosphoinositide 3-kinase (PI3K)δ, a class 1A PI3K, has been studied as a potential target for achieving anti-oncogenic and anti-inflammatory effects. However, the role of PI3Kδ in antiviral responses is poorly understood. Using a synthetic double-stranded RNA poly I:C and a selective PI3Kδ inhibitor IC87114, we investigated the role of PI3Kδ signaling in poly I:C-induced expression of the T lymphocyte-inhibitory molecule programmed death 1 ligand 1 (PD-L1), inflammatory responses and antiviral interferon (IFN) responses. C57BL/6N mice were treated with IC87114 or vehicle by intratracheal (i.t.) instillation followed by i.t. administration of poly I:C. Poly I:C increased PD-L1 expression on epithelial cells, lymphocytes, macrophages, and neutrophils in the lungs and IC87114 suppressed poly I:C-induced PD-L1 expression on epithelial cells and neutrophils possibly via inhibition of the Akt/mTOR signaling pathway. IC87114 also attenuated poly I:C-induced increases in numbers of total cells, macrophages, neutrophils and lymphocytes, as well as levels of KC, IL-6 and MIP-1β in bronchoalveolar lavage fluid. Gene expression of IFNβ, IFNλ₂ and IFN-stimulated genes (ISGs) were upregulated in response to poly I:C and a further increase in gene expression was observed following IC87114 treatment. In addition, IC87114 enhanced poly I:C-induced phosphorylation of IRF3. We assessed the effects of IC87114 on human primary bronchial epithelial cells (PBECs). IC87114 decreased poly I:C-induced PD-L1 expression on PBECs and secretion of IL-6 and IL-8 into culture supernatants. IC87114 further enhanced poly I:C- induced increases in the concentrations of IFNβ and IFNλ_1/3 in culture supernatants as well as upregulated gene expression of ISGs in PBECs. Similar results were obtained in PBECs transfected with siRNA targeting the PIK3CD gene encoding PI3K p110δ, and stimulated with poly I:C. In human metapneumovirus (hMPV) infection of PBECs, IC87114 suppressed hMPV-induced PD-L1 expression and reduced viral replication without changing the production levels of IFNβ and IFNλ_1/3 in culture supernatants. These data suggest that IC87114 may promote virus elimination and clearance through PD-L1 downregulation and enhanced antiviral IFN responses, preventing prolonged lung inflammation, which exacerbates asthma and COPD.

Keywords: IC87114; bronchial epithelial cells; human metapneumovirus; interferon; phosphoinositide 3-kinase δ; poly I:C; programmed death 1 ligand 1.

Figure 1

Poly I:C-induced upregulation of PD-L1 on mouse lung cells. (A) Flow cytometry gating strategy to distinguish epithelial cells, lymphocytes, macrophages, and neutrophils in mouse lungs. Sorted and pelleted cells were stained with Diff-Quick and observed using an optical microscope. Scale bar, 20 μm. (B,C) Poly I:C or vehicle was administered intratracheally to mice and PD-L1 (B) or PD-1 (C) expression on lung cells was analyzed 24 h following administration using flow cytometry. Representative histograms are shown. SSC, side scatter; FSC, forward scatter; MFI, mean fluorescence intensity. All results are representative of at least three independent experiments. Data represent means ± SDs (n = 6–14 per group). *p < 0.01, **p < 0.001 by the Mann–Whitney U-test.

Figure 2

A PI3Kδ inhibitor attenuated poly I:C-induced upregulation of PD-L1 on mouse lung cells. (A,B) IC87114 was administered intratracheally (i.t.) to mice and PD-L1 (A) or PD-1 (B) expression on mouse lung cells was analyzed 24 h following treatment using flow cytometry. (C,D) IC87114 or vehicle was administered i.t. to mice followed by i.t. administration of poly I:C or vehicle. PD-L1 expression (C) on lung cells or cell viability (D) was analyzed 24 h following administration using flow cytometry. MFI, mean fluorescence intensity. All results are representative of at least three independent experiments. Data represent means ± SDs (n = 8–16 per group). *p < 0.05, **p < 0.01, ***p < 0.001 by the Mann–Whitney U-test or one-way ANOVA as appropriate.

Figure 3

A PI3Kδ inhibitor suppressed poly I:C-induced PD-L1 expression via inhibition of Akt/mTOR signaling pathway. (A) PBECs were pretreated with IC87114 or vehicle for 1 h, then stimulated with poly I:C. Cell lysates for RNA extraction were collected 6 h following stimulation and real-time quantitative reverse-transcriptase PCR was performed. Target gene expression levels were normalized to those of 18S rRNA. (B) BEAS-2B cells were pretreated with rapamycin or vehicle for 1 h, then stimulated with poly I:C. PD-L1 expression was analyzed 24 h following administration using flow cytometry. MFI, mean fluorescence intensity. (C) IC87114 or vehicle was administered intratracheally (i.t.) to mice followed by i.t. administration of poly I:C or vehicle. Lungs were collected 1 h following administration. Phosphorylated IκBα, total IκBα, phosphorylated Akt and total Akt were detected by western blotting and quantitated using densitometry. All results are representative of at least two independent experiments. Data represent means ± SDs (n = 4–6 per group). *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA.

Figure 4

A PI3Kδ inhibitor suppressed poly I:C-induced influx of inflammatory cells and increases in pro-inflammatory chemokines and cytokines in BALF. IC87114 or vehicle was administered intratracheally (i.t.) to mice followed by i.t. administration of poly I:C or vehicle. BALF were collected 24 h following administration. (A) Cells were enumerated in BALF. (B) Pro-inflammatory cytokine and chemokine levels in BALF were measured by ELISA. The dotted line shows the lower limit of detection. ^#Levels in all samples were below the detection limit. All results are representative of at least three independent experiments. Data represent means ± SDs (n = 6–12 per group). *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA.

Figure 5

A PI3Kδ inhibitor enhanced poly I:C-induced IFN responses in mouse lung. IC87114 or vehicle was administered intratracheally (i.t.) to mice followed by i.t. administration of poly I:C or vehicle. Lungs were collected at 6 (A) or 24 h (B) post-administration and RNA was extracted. Real-time quantitative reverse-transcriptase PCR was performed and target gene expression levels were normalized to those of GAPDH. Data represent means ± SDs (n = 6–12 per group). All results are representative of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA.

Figure 6

A PI3Kδ inhibitor enhanced poly I:C-induced phosphorylation of IRF3 in mouse lung. IC87114 or vehicle was administered intratracheally (i.t.) to mice followed by i.t. administration of poly I:C or vehicle. Lungs were collected 3 h following administration. Phosphorylated IRF3 and total IRF3 were detected by western blotting and quantitated using densitometry. All results are representative of at least two independent experiments. Data represent means ± SDs (n = 4–5 per group). *p < 0.05, **p < 0.001 by one-way ANOVA.

Figure 7

Effects of PI3Kδ inhibitor on poly I:C-induced upregulation of PD-L1, production of cytokines and chemokines, and IFN responses in PBECs. PBECs were pretreated with IC87114 or vehicle for 1 h, then stimulated with poly I:C. Cell lysates for RNA extraction or cells and culture supernatants were collected 6 or 24 h following administration, respectively. (A,B) PD-L1 expression (A) on PBECs or cell viability (B) was analyzed using flow cytometry. Representative histograms are shown. MFI, mean fluorescence intensity. (C) Pro-inflammatory cytokine and chemokine levels in supernatants were measured by ELISA. (D) IFN levels in supernatants were measured by ELISA. The dotted line shows the lower limit of detection. ^#Levels in all samples were below the detection limit. (E) RNA was extracted and real-time quantitative reverse-transcriptase PCR was performed. Target gene expression levels were normalized to those of 18S rRNA. All results are representative of at least two independent experiments. Data represent means ± SDs (n=6 per group) of three replicates from a minimum of two independent donors. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA.

Figure 8

Effects of siRNA targeting the PIK3CD gene on poly I:C-induced upregulation of PD-L1, production of cytokines and IFN responses in PBECs. (A) Representative western blots showing PI3K p110δ in PBECs treated with PIK3CD siRNA or negative control (NC) siRNA for 48 or 72 h. Band intensity was quantitated using densitometry. (B–D) PBECs were transfected with PIK3CD siRNA or NC siRNA for 48 h, then stimulated with poly I:C or vehicle for 24h. (B) PD-L1 expression on PBECs was analyzed using flow cytometry. MFI, mean fluorescence intensity. (C) IL-6 levels in supernatants were measured by ELISA. (D) IFN levels in supernatants were measured by ELISA. The dotted line shows the lower limit of detection. ^#Levels in all samples were below the detection limit. (E) Viable cells (PI negative) were identified using flow cytometry. All results are representative of at least two independent experiments. Data represent means ± SDs (n = 6–9 per group) of three replicates from a minimum of two independent donors. *p < 0.05, **p < 0.01, ***p < 0.001 by one- or two-way ANOVA as appropriate.

Figure 9

A PI3Kδ inhibitor attenuated hMPV-induced upregulation of PD-L1 on PBECs. (A) PBECs were infected with hMPV (MOI 0.1) and PD-L1 expression was analyzed at the indicated times using flow cytometry. (B) IC87114 or vehicle was added prior to and after hMPV infection (MOI 0.1). PD-L1 expression was analyzed at 48 hpi using flow cytometry. MFI, mean fluorescence intensity. All results are representative of at least two independent experiments. Data represent means ± SDs (n = 6 per group) of three replicates from a minimum of two independent donors. *p < 0.05, **p < 0.01, ***p < 0.001 by one- or two-way ANOVA as appropriate.

Figure 10

A PI3Kδ inhibitor reduced replication of hMPV in PBECs without changing the production level of IFNs in supernatants. IC87114 or vehicle was added prior to and after hMPV (MOI 0.1) (A,B) or hMPV-GFP (MOI 0.1) (C) infection. (A) Cell culture supernatants were collected at 48 hpi and IFN levels in supernatants were measured by ELISA. The dotted line shows the lower limit of detection. ^#Levels in all samples were below the detection limit. (B) Cell lysates for RNA extraction were collected at 24, 36, and 48 hpi and real-time quantitative reverse-transcriptase PCR was performed. Target gene expression levels were normalized to those of 18S rRNA. (C) Images of hMPV-GFP-infected cells at 72 hpi were obtained using fluorescence microscopy (10× objective lens) and the number of cells infected with hMPV-GFP (4× objective lens) was counted. Scale bar, 200 μm. All results are representative of at least two independent experiments. Data represent means ± SDs (n = 6 per group) of three replicates from a minimum of two independent donors. *p < 0.05, **p < 0.01, ***p < 0.001 by one- or two-way ANOVA and spearman correlation as appropriate.