A purinergic P2Y6 receptor agonist prodrug modulates airway inflammation, remodeling, and hyperreactivity in a mouse model of asthma

Figures & data

Figure 1 Pulmonary function mechanics are improved in GC021109-treated mice.

Notes: Acute MCh-induced pulmonary function response curves in 10-week-old mice after 6 weeks of treatment with saline, GC021109, HDM (80 µg), or HDM + GC021109 at either 10 or 100 µg/kg weight/dose. Pulmonary resistance in the (A) GC021109 (10 µg/kg weight/dose) treatment group and (B) GC021109 (100 µg/kg weight/dose) treatment group. Pulmonary compliance in the (C) GC021109 (10 µg/kg weight/dose) treatment group and (D) GC021109 (100 µg/kg weight/dose) treatment group. Pulmonary elastance in the (E) GC021109 (10 µg/kg weight/dose) treatment group and (F) GC021109 (100 µg/kg weight/dose) treatment group. The mice treated with HDM alone exhibited significant changes in pulmonary resistance, compliance, and elastance curves in a pattern consistent with asthma. The combination of HDM and GC021109 reversed these changes. The effect appeared stronger with the 100 µg/kg weight/dose GC021109 dose. GC021109 given with saline was not different from saline alone. Treatment groups: 1 – saline, 2 – saline + GC021109, 3 – HDM + GC021109, 4 – HDM. Values are mean ± SEM of N=4–5 mice per group. *P<0.05, saline control compared to HDM; ^@P<0.05, HDM + GC021109 compared to HDM; ^$P<0.01, HDM + GC021109 compared to HDM; ^‡P<0.01, saline control compared to HDM; ^▼P<0.005, HDM + GC021109 compared to HDM; ^#P<0.005, saline control compared to HDM.
Abbreviations: HDM, house dust mite; MCh, methacholine; SEM, standard error of means.

Figure 2 GC021109 prevents HDM-induced peribronchiolar and perivascular inflammatory cell infiltration.

Notes: Representative photomicrographs of hematoxylin/eosin-stained lung (5-µm sections) from mice treated 6 weeks with either saline, HDM, saline + GC021109 (100 µg/kg weight/dose), or HDM + GC021109 (100 µg/kg weight/dose). Arrows point to inflammatory cell infiltration in the peribronchiolar and perivascular space. Inflammatory cells were prominently increased by HDM treatment. This response was absent in mice treated concomitantly with HDM and GC021109. *Vascular lumen, **bronchiolar lumen. Bar represents 50 µm.
Abbreviation: HDM, house dust mite.

Figure 3 Increases in lung lavage content of inflammatory cells and protein after HDM exposure are normalized by the addition of GC021109.

Notes: (A) Total inflammatory cell numbers in lung lavage fluid from mice after exposure to saline (solid green), HDM (green striped), GC021109 (10 µg/kg weight/dose – solid red, 100 µg/kg weight/dose – solid black), and HDM + GC021109 (10 µg/kg weight/dose – striped red, 100 µg/kg weight/dose – striped black). Counts were performed using a hemocytometer. (B) Cumulative differential cell counts in lung lavage from saline-treated, HDM-treated, and HDM + GC021109 (100 µg/kg weight/dose)-treated mice. (C) Protein concentration in lung lavage fluid. Conditions were the same as in (A). Data represent mean ± SEM; N=5 mice per group. *P<0.01, compared to control; ^{^}P<0.01, compared to HDM; ^‡P<0.001, compared to HDM.

Abbreviations: HDM, house dust mite; LL, lung lavage; SEM, standard error of means.

Figure 4 Asthma-related cytokines in lung lavage fluid are increased after HDM exposure but reduced by the addition of GC021109 in a dose-dependent pattern.

Notes: Cytokines were measured in lung lavage fluid by multiplex cytokine immunoassay. The figure shows the levels of IL-4, IL-5, G-CSF, and IL-12 in lavage fluid from mice treated with saline, GC021109, HDM, and HDM + GC021109 (10 and 100 µg/kg weight/dose). GC021109 alone did not alter the lung lavage cytokine levels as compared to saline, although IL-12 levels in the saline group appeared elevated. IL-4, IL-5, and G-CSF were strongly increased in HDM-treated mice, while IL-12 remained high. Concomitant treatment with HDM and GC021109 (10 or 100 µg/kg weight/dose) produced a dose-dependent reduction in all 4 cytokines. Data represent mean ± SEM; N=5 mice per group. *P<0.05, compared to control; ^{^}P<0.05, compared to HDM; ^@P<0.10, compared to HDM.
Abbreviations: HDM, house dust mite; SEM, standard error of means; IL, interleukin; G-CSF, granulocyte colony stimulating factor.

Figure 5 GC021109 reduces HDM-induced increases in peribronchial and perivascular α-SMA.

Notes: (A) Representative photomicrographs of α-SMA immunostaining in lungs from mice exposed to saline, GC021109 (10 and 100 µg/kg weight/dose), HDM, and HDM + GC021109 (10 and 100 µg/kg weight/dose). Arrows point to α-SMA immunostaining around bronchioles (orange) and blood vessels (blue). Increased α-SMA expression was observed in HDM-exposed lungs compared to controls but not in HDM + GC021109 (both doses). Bar =50 µm. (B) Quantification of the relative area of α-SMA immunopositivity around bronchioles and arteries, expressed as a percentage of the total area of the peri-luminal muscle layer. Area measurements were made using ImageJ (National Institutes of Health). HDM treatment significantly increased, and HDM + GC021109 (100 µg/kg weight/dose) prevented increase in α-SMA expression. Data represent mean ± SEM; N=4 lungs (3 measurements each). ^‡P<0.0001, compared to control; ^{^}P<0.005, compared to HDM.

Abbreviations: HDM, house dust mite; SEM, standard error of means; α-SMA, alpha smooth muscle actin.

Figure 6 Peribronchial thickness identified by micro-CT scan is increased by HDM and ameliorated by HDM + GC021109.

Notes: Mice treated with saline, HDM, or HDM + GC021109 (100 µg/kg weight/dose) underwent micro-CT scanning to image pulmonary airways. Images were captured at peak inspiration. Peribronchial thickness at the level of the third-generation bronchi on coronal images was measured in both the coronal and corresponding transverse axial planes. Only bronchi whose shape indicated parallel (coronal) or perpendicular (sagittal, i.e. transverse axis) orientation to the image plane were measured. Data represent mean ± SEM; N=3 mice (1–2 measurements per lung). ^@P<0.10, compared to saline of same image orientation; *P<0.05, compared to HDM of same image orientation; ^{^}P<0.005, compared to saline of same image orientation; ^‡P<0.005, compared to HDM of same image orientation.
Abbreviations: HDM, house dust mite; SEM, standard error of means; CT, computed tomography.

Figure S1 LLF fluid cell differential counts noted by a hemocytometer after Wright’s staining from mice treated with saline,

Notes: GC021109 (10 and 100 µg/kg weight/dose), HDM, or HDM + GC021109 (10 and 100 µg/kg weight/dose). Black bars: lymphocytes; green bars: macrophages; red bars: eosinophils. Data are expressed as % total white blood cells counted, and shown as mean ± SEM; N=5 mice.

Abbreviations: HDM, house dust mite; SEM, standard error of means; LLF, lung lavage fluid.

Figure S2 Additional selected cytokines significantly increased in LLF fluid by HDM and normalized by the addition of GC021109.

Notes: These results are from the same animals and were obtained in the multiplex assay explained in the legend of Figure 4. They are shown as supplementary data as cytokines of interest in allergen-induced asthma that were markedly increased by HDM sensitization but the response to added GC021109 with HDM sensitization did not demonstrate a clear dose–response relationship. Nonetheless, these data do illustrate the tendency of added GC021109 to profoundly reduce the levels of specific cytokines in HDM mice. Specific treatment conditions are labeled along the X axis. The Y axis represents picograms/mL of IL-6, IL-17, LIF, and CXCL1, respectively. Data represent mean ± SEM; N=3–9; *P<0.05, compared to the saline controls; ^▼P<0.01, compared to saline controls; ^‡P<0.005, compared to saline controls; ^@P<0.10, compared to HDM: ^{^}P<0.05, compared to HDM. The HDM + GC021109 condition in the IL-6 panel had very low identical measurements; thus, no standard error or statistical test results are shown.

Abbreviations: HDM, house dust mite; SEM, standard error of means; LLF, lung lavage fluid; IL, interleukin; LIF, leukemia inhibitory factor; CXCL1, chemokine (C-X-C) motif ligand 1.

Figure S3 Representative micro-CT images of mice from the control, HDM, and HDM + GC021109 treatment groups.

Notes: Coronal (top) and transverse axis (bottom) images are shown for each condition. The horizontal line in the coronal images represents the level at which the corresponding transverse axis images were viewed and is the level of the third-generation bronchi. Arrows point to peribronchial airway thickness in third-generation airways.

Abbreviations: HDM, house dust mite; CT, computed tomography.