Effects of β(2) Agonists, Corticosteroids, and Novel Therapies on Rhinovirus-Induced Cytokine Release and Rhinovirus Replication in Primary Airway Fibroblasts

Abstract

Rhinovirus-(RV-) induced asthma exacerbations account for high asthma-related health costs and morbidity in Australia. The cellular mechanism underlying this pathology is likely the result of RV-induced nuclear-factor-kappa-B-(NF-κB-) dependent inflammation. NF-κB may also be important in RV replication as inhibition of NF-κB inhibits replication of other viruses such as human immunodeficiency virus and cytomegalovirus. To establish the role of NF-κB inhibitors in RV-induced IL- 6 and IL-8 and RV replication, we used pharmacological inhibitors of NF-κB, and steroids and/or β(2) agonists were used for comparison. Primary human lung fibroblasts were infected with RV-16 in the presence of NF-κB inhibitors: BAY-117085 and dimethyl fumarate; β(2) agonist: salmeterol; and/or corticosteroids: dexamethasone; fluticasone. RV-induced IL-6 and IL-8 and RV replication were assessed using ELISAs and virus titration assays. RV replicated and increased IL-6 and IL-8 release. Salmeterol increased, while dexamethasone and fluticasone decreased RV-induced IL-6 and IL-8 (P<0.05). The NF-κB inhibitor BAY-117085 inhibited only RV-induced IL-6 (P<0.05) and dimethyl fumarate did not alter RV-induced IL-6 and IL-8. Dimethylfumarate increased RV replication whilst other drugs did not alter RV replication. These data suggest that inhibition of NF-κB alone is unlikely to be an effective treatment compared to current asthma therapeutics.

Figure 1

(a) Time course of RV replication. Concentration is of RV from infected fibroblasts (MOI = 0.1) at 0, 3, 6, 24, 48 and 72 hours post infection were measured by RV titration. RV concentration was compared with each time point post infection using a 1-way ANOVA (n = 5). (b,c) Time course of RV-induced IL-6 and IL-8. Concentration of (b) IL-6 and (c) IL-8 release from noninfected fibroblast (constitutive release) or UVi-RV-(UVi-) or RV-16-(RV-) infected fibroblasts (MOI = 0.1) at 0, 3, 6, 24, 48 and 72 hours post infection were measured by ELISA. RV-induced IL-6 and IL-8 at 48, and 72 hours post infection compared to control and UVi (2-way ANOVA, n = 5). All data are presented as mean ± SEM. Significance of comparisons is represented as *P < 0.05, **P < 0.01, and ***P < 0.0001.

Figure 2

(a–f) Effect of dexamethasone (Dex), fluticasone (Flut) and salmeterol (Sal) on RV-induced IL-6 and IL-8. Concentration of IL-6 and IL-8 release from noninfected fibroblasts (constitutive release), UVi-RV-(UVi-) or RV-16-infected fibroblasts (RV) (MOI = 0.1), highest concentration of vehicle (Dex & Sal: 0.1% DMSO; Flut: 0.001% DMSO) and RV infected fibroblasts in the presence of Dex: 10⁻¹²–10⁻⁷ M (n = 7), Flut: 10⁻¹⁰–10⁻⁸ M (n = 7) and Sal: 10⁻⁸–10⁻⁶ M (n = 9) were measured 48 hrs post infection by ELISA. All IL-6 and IL-8 concentrations were compared to their respective RV-induced values (in the absence of drug and vehicle), using a 1-way ANOVA. All data are presented as mean ± SEM. Significance is represented as *P < 0.05, **P < 0.01, and ***P < 0.0001.

Figure 3

(a–d) Effect of BAY and DMF on RV-induced IL-6 and IL-8. Concentration of IL-6 and IL-8 release from noninfected fibroblast (constitutive release), UVi-RV-(UVi-) or RV-16-infected fibroblasts (RV) (MOI = 0.1), highest concentration of vehicle (0.1% DMSO) and RV infected fibroblasts in the presence of 10⁻⁸–10⁻⁶ M BAY (n = 10) and DMF (n = 9) measured 48 hrs post infection by ELISA. All IL-6 and IL-8 concentrations were compared to their respective RV-induced values (in the absence of drug and vehicle), using a 1-way ANOVA. All data are presented as mean ± SEM. Significance is represented as **P < 0.01 and ***P < 0.0001.

Figure 4

Effect of DMF on RV replication. Concentration of virus from RV-infected fibroblasts ± vehicle (0.1% DMSO); or 10⁻⁸–10⁻⁶ M DMF (n = 14) was measured 24 hrs post infection by RV titration. All RV concentrations were compared to RV concentration in the absence of drug and vehicle by 1-way ANOVA. All data are presented as mean ±SEM. Significance is represented as *P < 0.05 and **P < 0.01.

Figure 5

Salmeterol + fluticasone (Sal + Flut) and Sal + Flut + BAY inhibit RV-induced IL-6: the amount of IL-6 induced from fibroblasts infected with RV-16 (MOI = 0.1) was expressed as 100%. Inhibition caused by drug combinations, Sal + Flut (10⁻⁸ + 10⁻¹⁰ M) and Sal + Flut + BAY (10⁻⁸ + 10⁻¹⁰ + 10⁻⁶ M) (for all n = 4) was measured 48 hrs post infection by ELISA and expressed as a percentage of RV-induced IL-6. Percentage inhibition caused by drugs was compared using 1-way ANOVA with RV-induced IL-6. All data are presented as mean ± SEM. Significance is represented as **P < 0.01.