Small interfering RNA targeting nerve growth factor alleviates allergic airway hyperresponsiveness

Abstract

Airway hyperresponsiveness is the hallmark of allergic asthma and caused by multiple factors. Nerve growth factor (NGF), a neurotrophin, is originally known for regulation of neural circuit development and function. Recent studies indicated that NGF contributes to airway hyperresponsiveness and pathogenesis of asthma. The objective of this study is to develop a small interfering RNA against NGF to attenuate airway hyperresponsiveness and further elucidate the underlying mechanism. In a murine model of allergic asthma, the ovalbumin-sensitized mice were intratracheally delivered small interfering RNA against NGF or administered an inhibitor targeting NGF receptor, tropomyosin-related kinase A, as a positive treatment control. In this study, knockdown NGF derived from pulmonary epithelium significantly reduced airway resistance in vivo. The levels of NGF, proinflammatory cytokines and infiltrated eosinophils in airway were decreased in small interfering RNA against NGF group but not in tropomyosin-related kinase A inhibitor and mock siRNA group. Furthermore, induction of neuropeptide (substance P) and airway innervation were mediated by NGF/tropomyosin-related kinase A pathway. These findings suggested that NGF targeting treatment holds the potential therapy for antigen-induced airway hyperresponsiveness via attenuation of airway innervation and inflammation in asthma.

Figure 1

The suppressive effects of siRNA against NGF (siNGF) in vitro. (a) The phenotypes of primary lung cells. (b) NGF inductions were assessed in primary lung culture in the absence or presence of different doses of TNF-α (0, 5, 10, and 20 ng/ml) at different time points (12, 24, and 48 hours). *P < 0.05, **P < 0.01, and ***P < 0.001 versus no TNF-α. (c) siNGF decreased NGF mRNA expression. Primary lung cells were infected by lentvirus for 48 hours and stimulated by TNF-α (20 ng/ml) for 6 hours. (d) The knockdown efficiency of siNGF. Primary lung cells were infected by lentvirus containing siNGF or mock siRNA (MOI: 10) for 48 hours and stimulated by TNF-α (20 ng/ml) for 48 hours. a*P < 0.05, a**P < 0.01, and a***P < 0.001 versus positive control. b*P < 0.05, b**P < 0.01, and b***P < 0.001 versus mock siRNA. Data are shown as mean ± SEM and representative of three independent experiments. MOI, multiplicity of infection; NGF, nerve growth factor; siRNA, small interfering RNA.

Figure 2

siNGF decreased OVA-specfic Abs and cytokine productions in BAL fluid. Lentivirus containing siNGF or mock siRNA (3 × 10⁶ infectious unit) was intratracheally delivered into the OVA-sensitized mice 3 days before the first challenge with OVA. The TrkA inhibitor was intranasally administered 3 hours before OVA challenge. After OVA challenges, sera and BAL fluid from the treated mice were collected and analyzed by ELISA assay. (a) The targeting location of lentivirus which was determined by immunofluorescent stain of anti-GFP expression After intratracheally delivering lentivirus containing GFP for 6 days, BAL cells were collected and lungs were isolated from treated mice following perfusion, fixation, and optimal cutting temperature compound embedding. BAL cells and lung were stained with anti-GFP antibody and secondary antibody conjugated DyLight-594. The sections were analyzed by fluorescence microscope. (b) OVA-specific antibody productions in sera. Data are presented as ELISA unit (E.U.). (c) NGF, IL-5 and eotaxin productions in BAL fluid. a*P < 0.05, a**P < 0.01, and a***P < 0.001 versus PC. b*P < 0.05, b**P < 0.01, and b***P < 0.001 versus mock siRNA. c*P < 0.05, c**P < 0.01, and c***P < 0.001 versus TrkA inhibitor. d***P < 0.001 versus siNGF. N = 6–8 per group. Data are shown as mean ± SEM and representative of five independent experiments. ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; siNGF, small interfering RNA against nerve growth factor; TrkA, tropomyosin-related kinase A. BAL, bronchoalveolar lavage.

Figure 3

siNGF decreased proinflammatory cytokines in lung. After sacrifice, lungs were isolated from the treated mice and homogenized by lysis buffer. Protein concentrations of lung homogenates were measured by bicinchoninic acid assay. NGF and cytokine concentrations were determined in 10 μg lung homogenate with ELISA kits. a*P < 0.05, a**P < 0.01, a***P < 0.001 versus positive control (PC). b*P < 0.05, b**P < 0.01, b***P < 0.001 versus mock siRNA. c*P < 0.05, c**P < 0.01, c***P < 0.001 versus TrkA inhibitor. d*P < 0.05, versus negative control. N = 6–8 per group. Data are shown as mean ± SEM and representative of five independent experiments. ELISA, enzyme-linked immunosorbent assay; NGF, nerve growth factor; TrkA, tropomyosin-related kinase A.

Figure 4

siNGF and TrkA inhibitor could reduce AHR. After OVA challenges, airway function of the treated mice was measured by (a) whole-body plethysmography or (b) invasive plethysmography. Results were expressed as the ratio of the baseline Penh value a or as the airway resistance value b. N = 6–8 per group. a*P < 0.05, a**P < 0.01, a***P < 0.001 versus PC. b*P < 0.05, b**P < 0.01, b***P < 0.001 versus mock siRNA. Data are shown as mean ± SEM and representative of five independent experiments. AHR, airway hyperresponsiveness; siNGF, small interfering RNA against nerve growth factor; siRNA, small interfering RNA; TrkA, tropomyosin-related kinase A.

Figure 5

TrkA inhibitor enhanced airway eosinophil infiltration. The cells from BAL fluid were stained with Liu staining after using cytospin. A total of 300 cells were counted under microscopy for the (a) cell population or (b) total cell number. N = 6–8 per group. a*P < 0.05, a**P < 0.01, a***P < 0.001 versus PC. b*P < 0.05, b**P < 0.01, b***P < 0.001 v.s mock siRNA. c*P < 0.05, c**P < 0.01, c***P < 0.001 versus TrkA inhibitor. Data are shown as mean ± SEM and representative of five independent experiments. siRNA, small interfering RNA; TrkA, tropomyosin-related kinase A. BAL, bronchoalveolar lavage.

Figure 6

siNGF decreased cell infiltration in lung. (a) Lung sections of the treated mice were prepared and stained with H&E staining for measurement of inflammatory cells around the airway. Bar = 100 µm (magnification: ×100). (b) The quantification of infiltration percentage in lung was analyzed by Image J software. a**P < 0.01 versus PC. b**P < 0.01 versus mock siRNA. c**P < 0.01 versus TrkA inhibitor. N = 6–8 per group. Data are shown as mean ± SEM and representative of five independent experiments. siNGF, small interfering RNA against nerve growth factor; siRNA, small interfering RNA; TrkA, tropomyosin-related kinase A.

Figure 7

siNGF reduced neuropeptide production and reduced airway innervation. (a) Lungs were isolated from the treated mice and homogenized by lysis buffer. Protein concentrations of lung homogenate were measured by bicinchoninic acid assay. Substance P concentrations were determined in 10 μg lung homogenate. (b) After OCT embedding, section and immunofluorescent staining, nerve fibers in lung sections were stained with nerve marker (PGP9.5). Bar = 100 µm. a*P < 0.05, a**P < 0.01, a***P < 0.001 versus positive control (PC). b*P < 0.05, b**P < 0.01, b***P < 0.001 versus mock siRNA. c*P < 0.05, c**P < 0.01, c***P < 0.001 versus TrkA inhibitor. N = 6–8 per group. Data are shown as mean ± SEM and representative of five independent experiments. siNGF, small interfering RNA against nerve growth factor; TrkA, tropomyosin-related kinase A.