Budesonide and formoterol reduce early innate anti-viral immune responses in vitro

Abstract

Asthma is a chronic inflammatory airways disease in which respiratory viral infections frequently trigger exacerbations. Current treatment of asthma with combinations of inhaled corticosteroids and long acting beta2 agonists improves asthma control and reduces exacerbations but what impact this might have on innate anti-viral immunity is unclear. We investigated the in vitro effects of asthma drugs on innate anti-viral immunity. Peripheral blood mononuclear cells (PBMC) from healthy and asthmatic donors were cultured for 24 hours with the Toll-like receptor 7 agonist, imiquimod, or rhinovirus 16 (RV16) in the presence of budesonide and/or formoterol. Production of proinflammatory cytokines and expression of anti-viral intracellular signalling molecules were measured by ELISA and RT-PCR respectively. In PBMC from healthy donors, budesonide alone inhibited IP-10 and IL-6 production induced by imiquimod in a concentration-dependent manner and the degree of inhibition was amplified when budesonide and formoterol were used in combination. Formoterol alone had little effect on these parameters, except at high concentrations (10⁻⁶ M) when IL-6 production increased. In RV16 stimulated PBMC, the combination of budesonide and formoterol inhibited IFNα and IP-10 production in asthmatic as well as healthy donors. Combination of budesonide and formoterol also inhibited RV16-stimulated expression of the type I IFN induced genes myxovirus protein A and 2', 5' oligoadenylate synthetise. Notably, RV16 stimulated lower levels of type Myxovirus A and oligoadenylate synthase in PBMC of asthmatics than control donors. These in vitro studies demonstrate that combinations of drugs commonly used in asthma therapy inhibit both early pro-inflammatory cytokines and key aspects of the type I IFN pathway. These findings suggest that budesonide and formoterol curtail excessive inflammation induced by rhinovirus infections in patients with asthma, but whether this inhibits viral clearance in vivo remains to be determined.

Figure 1. Dose dependent effects of budesonide and formoterol on markers of innate immunity induced by imiquimod (IQ).

IP-10 (A, C) and IL-6 (B, D) production by PBMC from 20 healthy female donors cultured with IQ in the presence of increasing concentrations (10⁻¹⁰–10⁻⁶ M as indicated) of budesonide (Bud) and formoterol (Form). The background cytokine levels produced in unstimulated control cultures (median 69 pg/ml IP-10 and 181 pg/ml IL-6) have been subtracted. Box and whisker plots show the median and interquartile range with 10^th–90^th percentile. Differences between IQ-stimulated cultures and IQ-stimulated cultures with drugs were tested by Friedman ANOVA and Dunn's multiple comparison test (**, p<0.005; ***, p<0.001).

Figure 2. Effect of combination of budesonide and formoterol on imiquimod (IQ)-induced markers of innate immunity.

IP-10 (A) and IL-6 (B) production by PBMC from eight healthy female donors were cultured with IQ with budesonide (Bud, 10⁻⁸ M) and/or formoterol (Form,10⁻⁸ M). The background cytokine levels produced in unstimulated control cultures (median 211 pg/ml IP-10 and 238 pg/ml IL-6) have been subtracted. Box and whisker plots show the median and interquartile range with 10^th–90^th percentile. Differences between IQ-stimulated cultures and IQ-stimulated cultures with drugs were tested by Friedman ANOVA and Dunn's multiple comparison test (**, p<0.005; ***, p<0.001).

Figure 3. Effects of combination of budesonide and formoterol on IFNα-induced by rhinovirus.

Cytokines produced by PBMC from healthy (A) and asthmatic (B) donors cultured for 24 h with RV16 in the presence of budesonide (Bud, 10⁻⁸ M) and/or formoterol (Form,10⁻⁸ M) as indicated. Box and whisker plots show median, interquartile range, and 10^th and 90^th percentiles for data from 12 donors in each subject group. Significant differences by Friedman ANOVA and Dunn's multiple comparison test for unstimulated versus RV16-stimulated cultures (+++, p<0.005) and between RV16-stimulated and RV16-stimulated cultures treated with budesonide and/or formoterol (***, p<0.001).

Figure 4. Effect of budesonide and formoterol on rhinovirus-induced expression of type one IFN inducible genes.

IP-10 produced by PBMC from healthy (A) and asthmatic (B) donors cultured for 24 h with RV16 in the presence of budesonide (Bud, 10⁻⁸ M) and/or formoterol (Form, 10⁻⁸ M) as indicated. Box and whisker plots show median, interquartile range, 10^th and 90^th percentiles for data. Quantitative RT-PCR analysis of anti-viral genes MxA and OAS expression in healthy donors (C and E, n = 8) and asthmatic donors (D and F, n = 12). Significant differences by Friedman ANOVA and Dunn's multiple comparison test for unstimulated versus RV16-stimulated cultures (+++, p<0.005) and between RV16 versus RV16 with drugs were assessed by Friedman ANOVA and Dunn's multiple comparison test (*, p<0.05; **, p<0.005, ***, p<0.001). To show the effects of the drugs on gene expression the data for asthmatics is shown on a different scale to that from healthy donors.