Toll-like Receptor 7 Is Reduced in Severe Asthma and Linked to an Altered MicroRNA Profile

Abstract

Rationale: Asthma is one of the most common chronic diseases worldwide, and individuals with severe asthma experience recurrent exacerbations. Exacerbations are predominantly viral associated and have been linked to defective airway IFN responses. Ascertaining the molecular mechanisms underlying this deficiency is a major research goal to identify new therapeutic targets.

Objectives: We investigated the hypothesis that reduced Toll-like receptor 7 (TLR7)-derived signaling drove the impaired IFN responses to rhinovirus by asthmatic alveolar macrophages (AMs); the molecular mechanisms underlying this deficiency were explored.

Methods: AMs were recovered from bronchoalveolar lavage from healthy subjects and patients with severe asthma. Expression of pattern-recognition receptors and microRNAs was evaluated by quantitative polymerase chain reaction and Western blotting. A TLR7-luciferase reporter construct was created to evaluate binding of microRNAs to the 3' untranslated region of TLR7. IFN production was measured by quantitative polymerase chain reaction and ELISA.

Measurements and main results: The expression of TLR7 was significantly reduced in severe asthma AMs and was associated with reduced rhinovirus and imiquimod-induced IFN responses by these cells compared with healthy AMs. Severe asthma AMs also expressed increased levels of three microRNAs, which we showed were able to directly reduce TLR7 expression. Ex vivo knockdown of these microRNAs restored TLR7 expression with concomitant augmentation of virus-induced IFN production.

Conclusions: In severe asthma, TLR7 deficiency drives impaired innate immune responses to virus by AMs. Blocking a group of microRNAs that are up-regulated in these cells can restore antiviral innate responses, providing a novel approach for therapy in asthma.

Keywords: alveolar macrophage; interferon; rhinovirus.

Figure 1.

Toll-like receptor-7 (TLR7) expression and function is reduced in severe asthma alveolar macrophages (SA-AM). (A) Rhinovirus-dependent induction of IFN-β and IFN-α mRNA and protein occurs in both SA-AMs (n = 10) and healthy AMs (n = 10), but the magnitude of the response is significantly lower in SA-AMs compared with healthy AMs. mRNA is quantified by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and is normalized to glyceraldehyde phosphate dehydrogenase (GAPDH). Protein levels are quantified using a Meso Scale Discovery electrochemiluminescence platform. (B) TLR7 mRNA is reduced in SA-AMs (n = 23) compared with healthy AMs (n = 23), quantified using qRT-PCR, and normalized to GAPDH. TLR7 protein is reduced in SA-AMs compared with healthy AMs using Western blot, relative to β-actin (mean + SEM). The TLR7/β-actin ratio was arbitrarily adjusted to one in the healthy control subjects. The panel below is a representative Western blot image of TLR7 and β-actin in healthy (HC) and SA-AMs. (C) TLR8, TLR3, retinoic acid–inducible gene 1 (RIG-1) and melanoma differentiation–associated gene 5 (MDA5) mRNA expression is similar in SA-AMs (n = 23) and healthy AMs (n = 23), quantified using qRT-PCR, and normalized to GAPDH. (D) Imiquimod-induced production of IFNβ, IFNα, 2′5′ oligoadenylate synthetase (OAS), and myxovirus resistance protein (MxA) mRNA is reduced in SA-AMs (n = 11) compared with healthy AMs (n = 10), quantified using qRT-PCR, and normalized to GAPDH. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns = not significant; RV = rhinovirus; UV = ultraviolet.

Figure 2.

Expression of Toll-like receptor 7 (TLR7) correlates with clinical parameters of asthma control. Expression of TLR7 mRNA in severe asthma alveolar macrophages are inversely correlated with the (A) number of exacerbations experienced in the previous 12 months and with the (B) patients’ Asthma Control Questionnaire (ACQ) score.

Figure 3.

MicroRNAs (miRNAs) targeting Toll-like receptor 7 (TLR7) are increased in severe asthma alveolar macrophages (SA-AMs). (A) Heat map representing Taqman low-density array expression of miRNAs comparing AMs from four patients with mild steroid–naive asthma (A1–4) and four healthy (H1–4) subjects. The 3′ untranslated region (UTR) of TLR7 is also shown with predicted targeting by miRNAs that are increased in patients with asthma. (B) Expression of miR-150, miR-152, and miR-375 is elevated in SA-AMs (n = 15) compared with healthy AMs (n = 15) and quantified by quantitative reverse-transcriptase polymerase chain reaction and normalized to RNU44. *P < 0.05; **P < 0.01.

Figure 4.

Ex vivo steroids and prototypical T helper 1 (Th1) and Th2 cytokines do not increase expression of microRNA-150 (miR-150), miR-152, and miR-375. (A) Expression of miR-150, miR-152, and miR-375 in alveolar macrophages (AMs) from healthy subjects is not altered by dexamethasone treatment at concentrations of 10, 100, and 1,000 nM at 24 or 48 hours. (B) Expression of miR-150, miR-152, and miR-375 in healthy AMs is not altered by treatment with IL-4, IL-13, tumor necrosis factor-α (TNFα), or IFN-γ for 24 hours (all cytokines used at 10 ng/ml). Data are from seven subjects, quantified by quantitative reverse-transcriptase polymerase chain reaction, and normalized to RNU44.

Figure 5.

miR-150, miR-152, and miR-375 directly target Toll-like receptor 7 (TLR7) to reduce its expression. (A) Transfection of miR-150, miR-152, or miR-375 into HeLa cells reduces renilla-luciferase (rLUC) activity of the TLR7-reporter (WT-TLR7-3′ untranslated region [UTR]). This effect is abrogated when the predicted binding sequence for miR-150 (MUT-150_1-TLR7-3′UTR and MUT-150_2-TLR7-3′UTR), miR-152 (MUT-152-TLR7-3′UTR), or miR-375 (MUT-152-TLR7-3′UTR) are mutated. (B) Expression of all three microRNAs shows further reduced expression of rLUC in WT-TLR7-3′UTR compared with individual microRNAs (mean + SEM). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. miR = microRNA; ns = not significant; WT = wild type.

Figure 6.

Blocking miR-150, miR-152, and miR-375 in alveolar macrophages (AMs) augments Toll-like receptor 7 (TLR7) expression and increases IFN responses to rhinovirus (RV). (A) Knockdown of mir-150, miR-152, and miR-375 (anti-miR Mix) increases TLR7 mRNA expression as quantified by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and normalized to glyceraldehyde phosphate dehydrogenase (GAPDH). Data are from 12 independent experiments. (B) Knockdown of miR-150, miR-152, and mir375 (anti-miR Mix) increases RV-induced production of IFN-β and IFN-α mRNA and protein compared with cells transfected with scrambled anti-miR (Control). mRNA are quantified by qRT-PCR and normalized to GAPDH, and protein is quantified using a Meso Scale Discovery electrochemiluminescence platform. AMs are from three healthy subjects and four patients with severe asthma. (C) Knockdown of miR-150, miR-152, and miR-375 (anti-miR Mix) increases imiquimod induction of IFN-β, IFN-α, 2′5′ oligoadenylate synthetase (OAS), and myxovirus resistance protein (MxA) mRNA, quantified by qRT-PCR and normalized to GAPDH. AMs are from two healthy subjects and three patients with severe asthma. Anti-miR Mix = alveolar macrophages transfected with anti-miR-150, anti-miR-152, and anti-miR-375, Control = alveolar macrophages transfected with an anti-miR scrambled control. Nonparametric test. *P < 0.05; **P < 0.01. miR = microRNA; UV = ultraviolet.