Research ArticleAsthma

DP2 antagonism reduces airway smooth muscle mass in asthma by decreasing eosinophilia and myofibroblast recruitment

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Science Translational Medicine  13 Feb 2019:
Vol. 11, Issue 479, eaao6451
DOI: 10.1126/scitranslmed.aao6451
  • Fig. 1 Increased ASM mass in asthma is reduced by fevipiprant.

    (A) Representative photomicrograph of a bronchial biopsy from a participant with severe asthma in the fevipiprant (DP2 antagonist) trial, showing increased ASM [brown-stained α-smooth muscle actin (α-SMA)], disrupted epithelium, and lamina propria. (B) ASM mass, as measured by percentage α-SMA–positive area, in bronchial biopsies from asthmatic subjects before treatment (Pre) and 12 weeks after treatment (Post) with fevipiprant (n = 14) or placebo (n = 13). A two-tailed paired t test was used for within group comparisons (P = 0.022 and P = 0.522), and a two-tailed unpaired t test was used to compare the difference in ASM mass observed after treatment with fevipiprant to that seen in the placebo group (P = 0.034).

  • Fig. 2 Computational model-based investigation of interactions between airway inflammation and ASM mass.

    (A) The mean time course from six simulations of the response to epithelial injury (50% denudation at time zero) over 180 days showing increased eosinophil numbers, ASM mass, and persistent epithelial damage in the model of airway remodeling in asthma versus resolution of the epithelial injury, eosinophil numbers, and persistently low ASM mass in the healthy control model (P < 0.01 for comparisons of each parameter over time between the patient model versus healthy control model, two-tailed unpaired t tests). (B) Predicted reduction in eosinophil number over 180 days (180d) after reduction in eosinophil recruitment or increase in eosinophil apoptosis in the computational model (n = 5 simulations). (C) Relative change in the ASM mass percentage at 180 days, predicted as a consequence of results in (B) (n = 5 simulations).

  • Fig. 3 ASM cells express functional DP2.

    (A) Representative photomicrograph of DP2 staining in bronchial biopsies from a subject with severe asthma (inset: isotype control). (B) Quantitative polymerase chain reaction cycle threshold (Ct) values for expression of ASM DP2 mRNA versus the 18S ribosomal RNA housekeeping gene RNA18S5 [mean DP2 Ct (95% CI), 27.9 (26.1 to 29.8); n = 7]. (C) Example histogram of DP2 expression (black trace) in ASM cells by flow cytometry versus isotype control antibody (gray trace); fold increase in geometric mean fluorescence intensity (GMFI) of anti-DP2 antibody/isotype control antibody (95% CI), 1.3 (1.2 to 1.4) [n = 15 donors, P < 0.001, two-tailed paired t test against isotype control]. (D) Representative photomicrographs (×20 magnification) showing ASM α-SMA expression (green, left; isotype control antibody, inset) and ASM DP2 expression (red, right; isotype control antibody, inset) by immunofluorescence staining. Nuclei are stained with 4′,6-diamidino-2-phenylindole (blue). (E) F-actin polymerization in primary human ASM cells (n = 9 donors) in response to DK-PGD2 treatment or Dulbecco’s modified Eagle’s medium (DMEM) containing 50% fetal bovine serum (FBS) as a positive control [geometric mean AUC DR of DK-PGD2 (95% CI), 46 (25 to 104) × 102; P = 0.01, one sample t test against a hypothetical value of zero]. (F) Intracellular calcium (Ca2+i) elevation in primary human ASM cells (n = 6 to 9 donors) in response to DK-PGD2 treatment or ionomycin (1.5 μg/ml) as a positive control [geometric mean AUC DR of DK-PGD2 (95% CI), 130 (78 to 230) × 103; P = 0.002, one sample t test against a hypothetical value of zero]. FITC, fluorescein isothiocyanate; PE, phycoerythrin. Data are plotted as means ± SEM. Two-tailed paired t tests were performed to compare each condition with its vehicle control; *P < 0.05, except FBS, where Wilcoxon matched-pairs signed-rank test was used, denoted by ^P < 0.05.

  • Fig. 4 A DP2 antagonist reduces ASM migration and recruitment of myofibroblasts and fibrocytes.

    All experiments were carried out in serum-free media. (A) Data for the wound closure after 24 hours of ASM cells that had been grown in monolayers and then wounded by scratching with a pipette tip, followed by incubation with different treatments for 24 hours are shown: DP2 agonist DK-PGD2 (n = 4 to 5 donors), the DP2 antagonists fevipiprant (n = 8 donors), CAY10471 (n = 6 to 8 donors), and OC000459 (n = 7 donors) or DMEM culture medium containing 10% FBS as a positive control. Two-tailed paired t tests were performed to compare each condition with its vehicle control; *P < 0.05 versus vehicle control. Data are expressed as means ± SEM. (B) Representative photographs of ASM monolayers wounded by scratching with a pipette tip after 24 hours [vehicle control for fevipiprant (500 nM), and DMEM containing 10% FBS control (top); vehicle control for CAY10471 (100 nM) and OC000459 (100 nM) (bottom)]; black lines represent the wound edge at 0 hours. (C) PGD2 release by unwounded and wounded ASM cells after 24 hours; P = 0.02, Wilcoxon matched-pairs signed-rank test (n = 10 donors). Data are expressed as means ± SEM. (D) Representative flow cytometry traces of isotype control antibodies (gray traces) versus α-SMA expression [left, black trace; mean percentage fibrocyte population positive for α-SMA expression (95% CI), 97% (93 to 100%); n = 4 donors] and DP2 expression [right, black trace; GMFI fold difference DP2 antibody/isotype control antibody (95% CI), 1.6 (1.3 to 2); P = 0.0064, two-tailed paired t test against isotype control antibody (n = 6 donors)] by fibrocytes. (E) Correlation between change in myofibroblast number in the lamina propria and absolute change in ASM mass as a percentage of the total biopsy area [fevipiprant, black circle (n = 14); placebo, black triangle (n = 13)]; Spearman (95% CI) r = 0.347 (−0.050 to 0.649), P = 0.076. (F) Correlation between change in fibrocyte number in the lamina propria and absolute change in ASM mass as a percentage of the total biopsy area [fevipiprant, black circle (n = 12); placebo, black triangle (n = 13)]; Spearman (95% CI) r = 0.538 (0.169 to 0.774), P = 0.006. (G) Predicted relative reduction in percent ASM mass at 180 days in the computational model as a consequence of reduced myofibroblast recruitment (30 to 50%) in combination with a 40% reduction in eosinophil recruitment in the computational model (n = 5 simulations).

  • Table 1 Agents, rules, and model.

    The computational airway model rule set, parameters, and which parameters were altered to observe airway remodeling. N/A, not applicable.

    CategoryActivityRulesParametersBaseline valueComments
    EpithelialMigrationDedifferentiated epithelial
    cells migrate to repair the
    damaged epithelium and
    then undergo proliferation.
    Migration rate5 μm/hourMigration rate was consistent with in vitro
    studies (4143).
    Migration delay5 hoursDedifferentiated cells migrate after 5 hours,
    doubled to 10 hours to represent slowly
    recovering epithelium.
    DifferentiationCells at the edge
    dedifferentiate into a
    flattened phenotype.
    Dedifferentiation
    probability
    50%A 50% probability per iteration was applied to
    determine whether an epithelial cell
    dedifferentiates. This was altered to 5% per
    iteration for the defective epithelium.
    Delay in
    proliferation
    2 to 8 hoursEdge cells require 2 to 8 hours to flatten out (41);
    this was increased by three times (6 to 24 hours)
    for slow epithelial recovery.
    ProliferationDedifferentiated epithelial
    cells proliferate after their
    migration to the opposite
    edge of a compromised
    epithelium.
    Ciliated:goblet
    ratio
    0.7:0.3 and
    0.1:0.9
    The number of goblet cells increase by two to
    three times in severe asthma (4447) given the
    inflammation status in disease. The probability of
    differentiating to a ciliated:goblet cell fate
    was either 0.7:0.3 (less inflamed) or 0.1:0.9
    (more inflamed).
    ApoptosisThis feature was not
    included in the baseline
    model.
    Auto-denudationN/ANo apoptosis rate was applied to the baseline
    model. In “mild” shedding every 25 hours, cells
    underwent apoptosis with 50% probability,
    “moderate” apoptosis with the probability of 1% at
    each iteration, “extreme” 10% at each iteration.
    InflammatoryCell migration
    and activation
    Inflammatory cells migrate
    into the airway in response
    to epithelial denudation,
    and their activation
    promotes eosinophil
    recruitment.
    Frequency of
    recruitment
    120 hoursInflammatory cells were added every 120 hours to
    the model with recruitment frequency increased
    to 60 and 50 hours in disease.
    Inflammatory
    cells recruited
    30Inflammatory cell number was chosen to reflect
    previous reports (11), and in disease, this was
    increased by 1.5 to 2 times (48).
    Eosinophil
    recruitment
    45Eosinophil recruitment was triggered by increased
    inflammatory cell number (45), based on
    previous reports (11), and in disease, this
    threshold was halved.
    DegranulationInflammatory cell activation
    promotes cell recruitment
    and fibroblast
    differentiation.
    Inflammatory
    cytokine release
    25/75/105 hoursThe “universal” inflammatory cell was assigned
    150 hours life, releasing cytokines for 25 hours (or
    75 and 105 hours for epithelial integrity of <55 and
    30%, respectively). In disease, inflammatory cell
    life was reduced to 65, 40, and 25 hours (releasing
    cytokines for 85, 110, and 125 hours, respectively).
    ApoptosisInflammatory cells promote
    eosinophil survival.
    Eosinophil life in
    the airway
    10/30/45 hoursEosinophils survival is ~10 hours within a normal
    airway (49). This was increased to 30 and
    45 hours for epithelial integrity of <55 and 30%,
    respectively. In disease, the life of eosinophils
    was doubled to 20, 60, and 90 hours.
    MesenchymalDifferentiationInflammatory cells promote
    increased ASM mass.
    ASM activation25 cellsThe threshold number of inflammatory cells above
    which myofibroblast-ASM differentiation is induced
    (11), which was reduced by 50% in disease.
    DifferentiationInflammatory cells promote
    fibroblast-myofibroblast
    differentiation.
    (Myo)fibroblast
    differentiation
    probability
    30%Increased fibroblast differentiation or myofibroblast
    recruitment in the presence of activated
    inflammatory cells increased to 50% in disease.
    ProliferationFibroblast proliferation
    increased by inflammatory
    and epithelial cells.
    Fibroblast
    growth rate
    132 iterationsAnimal models indicate that lung fibroblasts divide
    every 5.5 days (132 iterations in model), which
    reduces to 2 days (48 iterations in model) in the
    animal model of asthma (50).
    ApoptosisMyofibroblast survival
    supported by activated
    inflammatory cells.
    Myofibroblast
    apoptosis
    5 cellsThe threshold number of inflammatory cells below
    which myofibroblast apoptosis was induced,
    which was reduced to two in disease.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/479/eaao6451/DC1

    Materials and Methods

    Fig. S1. The virtual airway at baseline.

    Fig. S2. Model parameters and agent interactions.

    Fig. S3. Flow cytometric analysis of ASM cells.

    Fig. S4. DK-PGD2, fevipiprant, CAY10471, and OC000459 had no effect on ASM cell number, apoptosis, or necrosis after 24 hours.

    Fig. S5. DK-PGD2, fevipiprant, and CAY10471 had no effect on ASM proliferation after 72 hours.

    Fig. S6. DK-PGD2, fevipiprant, and CAY10471 had no effect on basal or BK-induced ASM contraction.

    Fig. S7. Conceptual summary.

    Table S1. Description of agents used in computational model.

    Table S2. Alterations made to epithelial parameters in the computational model.

    Table S3. Alterations made to mesenchymal parameters in the computational model.

    Table S4. Alterations made to inflammatory parameters in the computational model.

    Table S5. Clinical characteristics of subjects that provided additional bronchial biopsies for primary ASM cultures.

    Table S6. Analysis of expression of genes involved in PGD2 biosynthesis and metabolism in ASM cells.

    Table S7. Output of computations simulating pathological airway remodeling.

    Data file S1. Data values for individual experiments.

    References (5170)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. The virtual airway at baseline.
    • Fig. S2. Model parameters and agent interactions.
    • Fig. S3. Flow cytometric analysis of ASM cells.
    • Fig. S4. DK-PGD2, fevipiprant, CAY10471, and OC000459 had no effect on ASM cell number, apoptosis, or necrosis after 24 hours.
    • Fig. S5. DK-PGD2, fevipiprant, and CAY10471 had no effect on ASM proliferation after 72 hours.
    • Fig. S6. DK-PGD2, fevipiprant, and CAY10471 had no effect on basal or BK-induced ASM contraction.
    • Fig. S7. Conceptual summary.
    • Table S1. Description of agents used in computational model.
    • Table S2. Alterations made to epithelial parameters in the computational model.
    • Table S3. Alterations made to mesenchymal parameters in the computational model.
    • Table S4. Alterations made to inflammatory parameters in the computational model.
    • Table S5. Clinical characteristics of subjects that provided additional bronchial biopsies for primary ASM cultures.
    • Table S6. Analysis of expression of genes involved in PGD2 biosynthesis and metabolism in ASM cells.
    • Table S7. Output of computations simulating pathological airway remodeling.
    • References (5170)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Data values for individual experiments.

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