Figure 4.

NGF effect on TRPV₁-mediated Ca²⁺ influx in children’s HBE. A: NGF protein expression measured by Western blot in primary HBE cells from children without (1, 2, 5, 11) and with asthma (12–15) infected with RSV (MOI = 1). B: quantification of results from A; n = 3 independent experiments. #P < 0.05, ##P < 0.01 non-asthmatic compared with asthmatic. C: [Ca²⁺]_i in primary HBE cells from children without (1, 2, 5, 11) and with asthma (12–15) after treatment with rhNGF (50 ng/mL) for 1 h before activation of TRPV₁ with capsaicin (150 µM); n = 3 independent experiments. D: primary HBE cells from children without asthma (1, 2) or with asthma (13, 15) were transiently transfected with nontargeting siRNA (NTsiRNA) or 50 nM of NGF-specific siRNA (NGFsiRNA) for 48 h, then infected with sterile medium or RSV (MOI = 1) for 12 h, and tested by qPCR for NGF mRNA expression normalized to GAPDH as the housekeeping gene. Data are expressed as means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001. E: inhibition of [Ca²⁺]_i in primary HBE from children with asthma (12–15) transiently transfected with NGF-specific siRNA (NGFsiRNA, 50 nM) for 48 h, then incubated with RSV (MOI = 1) or sterile medium for 12 h before activation of TRPV₁ with capsaicin. Controls were transfected with nontargeting siRNA (NTsiRNA). All experiments were repeated ≥2 times in quadruplicate. Data are expressed as means ± SE and were analyzed using the Kruskal–Wallis test with Dunn’s multiple comparisons. *P < 0.05, ***P < 0.001 compared with untreated controls; ###P < 0.001 compared with controls treated with NTsiRNA. [Ca²⁺]_i, intracellular calcium; HBE, human bronchial epithelium; MOI, multiplicity of infection; NGF, nerve growth factor; qPCR, quantitative polymerase chain reaction; rhNGF, recombinant human nerve growth factor; RSV, respiratory syncytial virus; TRPV1, transient receptor potential vanilloid 1.