Research ArticleANTIVIRAL IMMUNITY

PGD2/DP2 receptor activation promotes severe viral bronchiolitis by suppressing IFN-λ production

See allHide authors and affiliations

Science Translational Medicine  09 May 2018:
Vol. 10, Issue 440, eaao0052
DOI: 10.1126/scitranslmed.aao0052
  • Fig. 1 Severe bronchiolitis induced by PVM/CRE coexposure promotes PGD2.

    (A) Study design. Neonatal mice were infected with PVM [1 plaque-forming unit (PFU)] at 7 days old and/or exposed to CRE 3 days later. End points were assessed 2 hours later at 5, 7, or 10 dpi. To induce asthma-like pathology, mice were reinfected with PVM and exposed to CRE as shown, and end points were assessed at 66 dpi. h-PGDS expression in (B) peribronchial leukocytes or in (C and D) AECs (arrows indicate positive AECs), analyzed by two-way analysis of variance (ANOVA). (E) Neonates were infected with PVM and/or treated with low-dose exogenous IL-33. h-PGDS in AECs was assessed at 7 dpi. (F) PGD2 in the lung. Data are presented as mean ± SEM or as box-and-whisker plots to show quartiles (boxes) and range (whiskers) and are representative of two experiments (n = 5 to 8 mice per group per experiment), analyzed by one-way ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with vehicle-treated mice. #P < 0.05 and ###P < 0.001 compared with PVM/CRE mice.

  • Fig. 2 RSV infection promotes PGD2.

    Pediatric human bronchial epithelial cells isolated from healthy children were uninfected (UI) or infected with RSV (MOI, 1). (A) h-PGDS (red) and nuclei (blue) in uninfected (left) or 48 hours after RSV infection (right) in hAECs. (B) h-PGDS expression was quantified as a percentage of hAECs. (C) PGD2 was detected in cell supernatants. Box-and-whisker plots show quartiles (boxes) and range (whiskers), and data are representative of three experiments (n = 4 individual hAEC samples per group in duplicate), analyzed by one-way ANOVA. (D) Details of bronchiolitis patients. (E) Nasal swabs were collected from healthy infants or those admitted to the hospital with RSV bronchiolitis, and PGD2 was measured. n = 7 to 14 infants per group, analyzed by Mann-Whitney test. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 3 DP2 antagonism promotes antiviral immunity in PVM/CRE mice.

    (A) Study design. Mice were inoculated at 7 days old with PVM and then exposed to CRE 3 dpi (intranasal). Some mice were treated daily with a DP2 antagonist (AM156) from 3 to 9 dpi (oral gavage). End points were assessed at 5, 7, and 10 dpi. (B) Weight gain. (C) Viral load in AECs detected by immunohistochemistry, or (D) whole-lung viral copies assessed by quantitative polymerase chain reaction (qPCR). (E) IFN-α, IFN-λ (IL-28A/B), and IFN-γ expression in bronchoalveolar lavage fluid (BALF) and IL-12p40 expression in lung were assessed at 5 dpi (top) and 7 dpi (bottom). (F) Interferon regulatory factor 7 (Irf7) gene and (G) Caspase-3 (Casp3) gene expression in whole lung was assessed by qPCR. (H) Mice were inoculated with PVM ± CRE and treated daily with AM156. Some mice were treated with anti–IL-28A (intraperitoneally) at 4 dpi. (I) Viral load in AECs or (J) viral copies. (K) Irf7 and (L) Casp3 expression. Data are presented as mean ± SEM or as box-and-whisker plots to show quartiles (boxes) and range (whiskers) and are representative of two experiments (n = 5 to 10 mice per group), analyzed by one-way or two-way ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with PVM-alone mice or as indicated. #P < 0.05 and ###P < 0.001 compared with PVM/CRE mice. Dashed lines represent uninfected controls.

  • Fig. 4 DP2 antagonism ameliorates type 2 inflammation in PVM/CRE mice.

    Mice were treated as per Fig. 3A. (A) ILC2s in the lung. (B) Eosinophils in the BALF. (C) PGD2 expression in the lung and (D) IL-33 expression in the BALF. ILC2s were isolated from PVM/CRE mice and cultured with PGD2 ± DP2 antagonist (AM156) or IFN-λ. (E) IL-5 and (F) IL-13 expression was measured in the supernatant. (G) Airway smooth muscle (ASM) mass in vivo expressed as √area/perimeter of the basement membrane (Pbm). (H) ASM cells were isolated and cultured from neonatal mice and stimulated, and proliferation was measured using V450 dye dilution. Data are presented as mean ± SEM. (I) ASM cells were stimulated with PGD2, DP1-specific, or DP2-specific agonists, and proliferation was measured. (J) PVM/CRE-infected mice were treated with the DP2 antagonist as per Fig. 3A and then exposed to PVM and CRE in later life (as per Fig. 1A) to induce asthma-like pathologies. Eosinophils in the BALF. (K) Muc5a expression as a percentage of AECs. (L) ASM mass. (M) Airway resistance (Rn) in response to increasing doses of methacholine (MCh). Data are presented as mean ± SEM or as box-and-whisker plots to show quartiles (boxes) and range (whiskers) and are representative of two experiments (n = 4 to 8 mice per group or four to five replicates in vitro), analyzed by Mann-Whitney test or one-way or two-way ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001. Dashed lines represent uninfected controls.

  • Fig. 5 DP2 but not DP1 antagonism prevents severe bronchiolitis in PVM/CRE mice.

    (A) Study design. Mice were inoculated at 7 days old with PVM and then exposed to CRE 3 dpi (intranasal). Some mice were treated daily from 3 dpi with a DP2 antagonist (AM156), a DP1 antagonist (MK-0524), or both antagonists from 3 to 9 dpi (oral gavage). End points were assessed at 5, 7, and 10 dpi. (B) Viral load in AECs detected by immunohistochemistry or (C) whole-lung viral copies assessed by qPCR. (D) IFN-λ (IL-28A/B) expression in the BALF at 7 dpi. (E) Irf7 and (F) Casp3 gene expression in whole lung assessed by qPCR. (G) Eosinophils in the BALF at 10 dpi. (H) ILC2s in the lung at 10 dpi. (I) ASM mass expressed as √area/perimeter of the basement membrane at 10 dpi (Pbm). Data are presented as mean ± SEM or as box-and-whisker plots to show quartiles (boxes) and range (whiskers) and are representative of two experiments (n = 4 to 8 mice per group), analyzed by one-way or two-way ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001. Dashed lines represent uninfected controls.

  • Fig. 6 DP1 activation promotes IFN-λ to limit viral load and type 2 inflammation.

    (A) Study design. All mice were inoculated at 7 days old with PVM. PVM/CRE-coexposed mice were exposed to CRE at 3 dpi and received no other treatment. Mice that received PGD2, a DP1 agonist (BW245C), or a DP2 agonist [15(R)-15-methyl-PGD2] were treated daily from 3 to 9 dpi. End points were assessed at 5, 7, and 10 dpi. (B) Viral load in AECs detected by immunohistochemistry or (C) whole-lung viral copies assessed by qPCR. (D) IFN-λ (IL-28A/B) expression in the BALF at 5 and 7 dpi. (E) Irf7 gene and (F) Casp3 gene expression in whole lung assessed by qPCR. (G) Cleaved caspase-3 expression (green) and nuclei (blue). (H) ILC2s in the lung at 10 dpi. (I) Eosinophils in the BALF at 10 dpi. (J) ASM mass expressed as √area/perimeter of the basement membrane at 10 dpi. (K) ILC2s were cultured with IL-33 ± DP1 agonist (BW245C). ILC2 proliferation was measured by dilution of carboxyfluorescein diacetate succinimidyl ester (CFSE). (L) IL-5 and (M) IL-13 secretion measured in the supernatant. Box-and-whisker plots show quartiles (boxes) and range (whiskers), and data are representative of two experiments (n = 4 to 8 mice per group or five replicates in vitro), analyzed by one-way or two-way ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to PVM-alone mice or as indicated. Dashed lines represent uninfected controls.

  • Fig. 7 DP2 antagonism or DP1 activation of AECs promotes IFN-λ expression to alleviate viral burden.

    (A) hAECs were preincubated with 50 nM DP2 antagonist (AM156) before infection with RSV (MOI, 1). Viral copies were assessed by qPCR at 24 and 48 hours after infection. (B) IL28A gene expression and (C) IFN-λ (IL-28A) protein expression. (D) IRF7 and (E) CASP3 gene expression. (F) hAECs or mouse AECs (mAECs) were infected with RSV or PVM (MOI, 1), respectively. Cells were preincubated with 100 nM DP1 agonist (BW245C). Viral copies were assessed by qPCR at 24 and 48 hours after RSV infection. (G) PVM viral copies and (H) G protein expression in mAECs differentiated at the air-liquid interface. (I) IL28A gene expression in hAECs (left) and mAECs (right). (J) IFN-λ (hIL-28A or mIL-28A/B) protein expression. (K) IRF7 gene expression. (L) CASP3 gene expression. Box-and-whisker plots show quartiles (boxes) and range (whiskers), and data are representative of three experiments (n = 6 individual patients or mice in duplicate), analyzed by Wilcoxon or Mann-Whitney test. *P < 0.05, **P < 0.01, and ***P < 0.001. Dashed lines represent uninfected controls.

  • Fig. 8 DP1 activation of AECs promotes IFN-λ expression.

    (A) hAECs were preincubated with anti–IL-28A before infection (MOI, 1) ± DP1 agonist. Viral copies and (B) IRF7 gene and (C) CASP3 gene expression were assessed by qPCR at 48 hours. (D) hAECs were treated with polyI:C ± DP1 agonist. IL28A gene expression, (E) IFN-λ (IL-28A) protein expression, and (F) IRF7 and (G) CASP3 gene expression. Box-and-whisker plots show quartiles (boxes) and range (whiskers), and data are representative of three experiments (n = 6 individual patients per mice in duplicate), analyzed by Wilcoxon or Mann-Whitney test. *P < 0.05. Dashed lines represent uninfected controls.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/440/eaao0052/DC1

    Materials and Methods

    Fig. S1. h-PGDS expression.

    Fig. S2. Effect of DP2 antagonism on antiviral immune cells.

    Fig. S3. Effect of DP2 antagonism on type 2 inflammation.

    Fig. S4. Effect of DP2 antagonism is not mediated by IL-13.

    Fig. S5. Effect of DP1 and/or DP2 antagonism on antiviral cytokines.

    Fig. S6. Effect of DP1 and/or DP2 agonism on antiviral cytokines.

    Fig. S7. Effect of anti-TH2, DP1-alone or RSV+DP2 agonism in hAECs.

    Fig. S8. A simplified schematic of the proposed mechanisms by which PGD2 suppresses or promotes antiviral immunity.

    Table S1. hAEC donor characteristics.

    Table S2. Oligonucleotide sequences used in this study.

    Table S3. Primary data from this study.

    References (6365)

  • Supplementary Material for:

    PGD2/DP2 receptor activation promotes severe viral bronchiolitis by suppressing IFN-λ production

    Rhiannon B. Werder, Jason P. Lynch, Jennifer C. Simpson, Vivian Zhang, Nick H. Hodge, Matthew Poh, Elizabeth Forbes-Blom, Christina Kulis, Mark L. Smythe, John W. Upham, Kirsten Spann, Mark L. Everard, Simon Phipps*

    *Corresponding author. Email: simon.phipps{at}qimrberghofer.edu.au

    Published 9 May 2018, Sci. Transl. Med. 10, eaao0052 (2018)
    DOI: 10.1126/scitranslmed.aao0052

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. h-PGDS expression.
    • Fig. S2. Effect of DP2 antagonism on antiviral immune cells.
    • Fig. S3. Effect of DP2 antagonism on type 2 inflammation.
    • Fig. S4. Effect of DP2 antagonism is not mediated by IL-13.
    • Fig. S5. Effect of DP1 and/or DP2 antagonism on antiviral cytokines.
    • Fig. S6. Effect of DP1 and/or DP2 agonism on antiviral cytokines.
    • Fig. S7. Effect of anti-TH2, DP1-alone or RSV+DP2 agonism in hAECs.
    • Fig. S8. A simplified schematic of the proposed mechanisms by which PGD2 suppresses or promotes antiviral immunity.
    • Table S1. hAEC donor characteristics.
    • Table S2. Oligonucleotide sequences used in this study.
    • Legend for table S3
    • References (6365)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S3 (Microsoft Excel format). Primary data from this study.

Navigate This Article