Placenta growth factor augments airway hyperresponsiveness via leukotrienes and IL-13

Abstract

Airway hyperresponsiveness (AHR) affects 55%-77% of children with sickle cell disease (SCD) and occurs even in the absence of asthma. While asthma increases SCD morbidity and mortality, the mechanisms underlying the high AHR prevalence in a hemoglobinopathy remain unknown. We hypothesized that placenta growth factor (PlGF), an erythroblast-secreted factor that is elevated in SCD, mediates AHR. In allergen-exposed mice, loss of Plgf dampened AHR, reduced inflammation and eosinophilia, and decreased expression of the Th2 cytokine IL-13 and the leukotriene-synthesizing enzymes 5-lipoxygenase and leukotriene-C4-synthase. Plgf-/- mice treated with leukotrienes phenocopied the WT response to allergen exposure; conversely, anti-PlGF Ab administration in WT animals blunted the AHR. Notably, Th2-mediated STAT6 activation further increased PlGF expression from lung epithelium, eosinophils, and macrophages, creating a PlGF/leukotriene/Th2-response positive feedback loop. Similarly, we found that the Th2 response in asthma patients is associated with increased expression of PlGF and its downstream genes in respiratory epithelial cells. In an SCD mouse model, we observed increased AHR and higher leukotriene levels that were abrogated by anti-PlGF Ab or the 5-lipoxygenase inhibitor zileuton. Overall, our findings indicate that PlGF exacerbates AHR and uniquely links the leukotriene and Th2 pathways in asthma. These data also suggest that zileuton and anti-PlGF Ab could be promising therapies to reduce pulmonary morbidity in SCD.

Figure 1. PlGF deficiency blunts AHR.

(A) Experimental schema of induction of AHR in BALB/c mice. Mice were sensitized to HDM or saline i.p. and then exposed to HDM i.t. at the indicated time points; airway responsiveness was measured 48 hours after the last i.t. HDM exposure. (B) Airway responsiveness in Plgf^+/+ or Plgf^–/– mice was measured by airway pressure inflation after a challenge with acetylcholine. *P < 0.05 by 1-way ANOVA. (C) Airway responsiveness was also measured in response to increasing concentrations of methacholine using a forced oscillatory technique. HDM-treated Plgf^+/+ mice either injected with vehicle (Saline) or αPlGF. *P < 0.05 Plgf^+/+ HDM (vehicle) versus Plgf^–/– HDM; ^#P < 0.05 Plgf^+/+ HDM (vehicle) versus Plgf^+/+ HDM (αPlGF) by 2-way ANOVA. (D and E) Plasma IgE levels in Plgf^+/+ and Plgf^–/– measured in BALF after the last i.p. sensitization with HDM (D) or 48 hours after the last HDM airway exposure (E). *P < 0.05, **P < 0.01 by 1-way ANOVA. All data are presented as mean ± SEM; each symbol represents and individual mouse; n = 5 animals in the saline-treated groups and 6–10 animals in the HDM-treated groups. Results are from 1 experiment (B) and representative of 2 independent experiments (C–E).

Figure 2. PlGF deficiency reduces airway inflammation.

(A) Representative H&E lung sections of Plgf^+/+ and Plgf^–/– mice after i.p. sensitization and i.t. challenge with HDM depicting goblet cell hyperplasia (arrowheads) and smooth muscle proliferation (red arrows) in the peribronchiolar and perivascular regions, and inflammatory infiltrates in the alveolar sacs (black arrows). (B) Lung histological inflammation score of Plgf^–/– and Plgf^+/+ mice following HDM exposure; n = 7–10 mice per group. **P < 0.01 by Mann Whitney U test. Data are presented as mean ± SEM. Results are from 2 independent experiments.

Figure 3. PlGF deficiency reduces goblet cell metaplasia and eosinophil recruitment.

(A and B) MUC5AC staining (×20 and ×40 images shown) (A) and CLCA3 (×40) staining (B) of representative lung sections of HDM-exposed Plgf^+/+ and Plgf^–/– mice. (C) Differential cell counts in the BALF from saline- and HDM-exposed mice. *P < 0.05 by Mann Whitney U test; n = 4–5 animals per group. (D) Fold increase in blood eosinophils counts of Plgf^+/+ and Plgf^–/– mice following allergen exposure. **P < 0.01 by Mann Whitney U test; n = 3 animals in the saline-treated groups and 6–7 animals in the HDM-treated groups. All data are presented as mean ± SEM. Results are representative of 1 experiment (C and D).

Figure 4. PlGF mildly affects IL-13 BALF levels.

(A–C) IL-13, IL-4, and IL-5 levels in the BALF of Plgf^+/+ and Plgf^–/– mice 48 hours following the last allergen challenge. *P < 0.05 using Mann Whitney U test between Plgf^+/+ and Plgf^–/– groups of mice. Each symbol represents 1 mouse; mean ± SEM of IL-13, IL-5, and IL-4 levels are indicated by the horizontal line and error bars. Results are from 3 independent experiments.

Figure 5. STAT6 mediates IL-13–induced increases in PlGF production.

(A) HDM sensitization further increases PlGF production in Plgf^+/+ mice compared with control (saline-treated) mice; n = 3 animals in the saline-treated group and 5–9 animals in the HDM-treated group. **P < 0.01 by Mann-Whitney test. (B) Relative Plgf mRNA expression in the lungs of Stat6^+/+ and Stat6^–/– mice after HDM or saline exposure. ***P < 0.001 by Mann-Whitney U test; n = 6–8 mice per group. (C) Expression of PlGF protein in airway, alveolar epithelial cells, and alveolar macrophages in WT mice (top left panel), as compared with Plgf^–/– mice (top right panel); ×20 images. A magnified view (×40) showing increased PlGF expression in alveolar epithelial cells and macrophages in WT mice following Th2-driven STAT6 activation (bottom left panel), compared with PlGF expression in Stat6^–/– (bottom middle panel) and Plgf^–/– (bottom right panel) alveolar epithelial cells and macrophages. All data are presented as mean ± SEM. Results are representative of 2 independent experiments.

Figure 6. PlGF activates the LT pathway.

(A) The relative Alox5 mRNA expression in the lungs of Plgf^+/+ and Plgf^–/– BALB/c mice 48 hours after the last i.t. HDM exposure. *P < 0.05, **P < 0.01 by ANOVA (Bonferroni). (B) IHC showing 5-LO staining in lungs of saline and HDM-exposed Plgf^+/+ and Plgf^–/– BALB/c mice; magnification ×50. (C) CysLT levels in Plgf^+/+ and Plgf^–/– BALB/c mice measured in urine collected 24 hours after the last HDM exposure. (D) Cysltr1 mRNA expression in the lungs of HDM-exposed Plgf^+/+ and Plgf^–/– BALB/c mice. *P < 0.05 by Mann Whitney U test. All data are presented as mean ± SEM. Results are representative of 1 experiment (A, B, and D) and from 2 independent experiments (C).

Figure 7. Expression of PLGF mRNA and its downstream target genes is increased in respiratory epithelial cells of patients with asthma.

(A) Relative quantification of PLGF mRNA expression in nasal epithelial cells of human subjects with no asthma (n = 9) and those with asthma (n = 18). **P < 0.01 by Mann Whitney U test. All data are presented as mean ± SEM. (B) Heat map of PlGF pathway genes in the microarray profile of some of the subjects shown in A (n = 4 normal, and n = 8 asthmatic). The normalized expression intensity is indicated by the color code: low (blue), medium (yellow to orange), and high (red). *P < 0.05 by unpaired t test. The fold increase is shown as mean ± SEM in parenthesis. Results are from of 2 experiments (A) and 1 experiment (B).

Figure 8. PlGF and cytokine levels in chimeric SCD mice.

(A) Experimental schema showing generation of chimeric HbS (Chi-HbS) and BL/6 (Chi-BL/6) mice by BM transplantation. (B) Plasma PlGF levels of HDM- and saline-exposed Chi-HbS and Chi-BL/6 mice. *P < 0.05 by Mann Whitney U test; n = 17–19 animals per group. (C–F) Eotaxin, IL-13, IL-4, and IL-5 levels in the BALF of saline- and HDM-exposed chimeric mice. All data are presented as mean ± SEM; each symbol represents 1 mouse. Results are from 3 independent experiments (B) or representative of 1 experiment (C and D).

Figure 9. PlGF mediates AHR seen in SCD mice through a LT-dependent pathway.

(A) Airway responsiveness to methacholine of saline- or HDM-exposed Chi-BL/6 and Chi-HbS mice. *P < 0.05, HDM-exposed Chi-BL/6 versus HDM-treated Chi-HbS group, 2-way ANOVA. (B) Airway responsiveness to methacholine of HDM-exposed Chi-BL/6 and Chi-HbS mice treated with vehicle control, αPlGF, or zileuton. ***P < 0.001, HDM-exposed Chi-HbS (vehicle) versus HDM-exposed Chi-HbS (αPlGF); ^###P < 0.001, HDM-exposed Chi-HbS (vehicle) versus HDM-exposed Chi-HbS (zileuton); ^##P < 0.01, HDM-treated Chi-HbS (vehicle) versus Chi-BL/6 by 2-way ANOVA. n = 6–8 animals per group. (C) Urine cysteinyl LTs of HDM-exposed Chi-BL/6 and Chi-HbS mice treated with vehicle control, αPlGF, or zileuton. *P < 0.05, ***P < 0.001 by 1-way ANOVA; n = 5–6 animals per group. All data are presented as mean ± SEM. Results are from 2 independent experiments.

Figure 10. Schematic showing the mechanism of PlGF-induced AHR and the positive feedback loop between IL-13 and PlGF.

PlGF is secreted by erythroid progenitors from the BM in response to hypoxia and erythropoietin (Epo) in SCD. PlGF stimulates the expression of 5-LO, which generates CysLT, and increases CysLTR1 expression. Sickle hematopoietic cells additionally increase LTC4S expression, which further contributes to CysLT generation. LT and IL-13 are key effectors of AHR. PlGF blockade or 5-LO inhibition reduces AHR. IL-13 levels activate its downstream effector molecule, STAT6, which in turn further augments PlGF production from pulmonary airway epithelial cells, eosinophils, and alveolar macrophages. The latter explains the elevated PlGF levels in allergen-challenged WT mice and in patients with asthma.