MicroRNA-125a and -b inhibit A20 and MAVS to promote inflammation and impair antiviral response in COPD
Alan C-Y. Hsu, Kamal Dua, Malcolm R. Starkey, Tatt-Jhong Haw, Prema M. Nair, Kristy Nichol, Nathan Zammit, Shane T. Grey, Katherine J. Baines, Paul S. Foster, Philip M. Hansbro, Peter A. Wark
Alan C-Y. Hsu, Kamal Dua, Malcolm R. Starkey, Tatt-Jhong Haw, Prema M. Nair, Kristy Nichol, Nathan Zammit, Shane T. Grey, Katherine J. Baines, Paul S. Foster, Philip M. Hansbro, Peter A. Wark
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Research Article Immunology Pulmonology

MicroRNA-125a and -b inhibit A20 and MAVS to promote inflammation and impair antiviral response in COPD

Abstract

Influenza A virus (IAV) infections lead to severe inflammation in the airways. Patients with chronic obstructive pulmonary disease (COPD) characteristically have exaggerated airway inflammation and are more susceptible to infections with severe symptoms and increased mortality. The mechanisms that control inflammation during IAV infection and the mechanisms of immune dysregulation in COPD are unclear. We found that IAV infections lead to increased inflammatory and antiviral responses in primary bronchial epithelial cells (pBECs) from healthy nonsmoking and smoking subjects. In pBECs from COPD patients, infections resulted in exaggerated inflammatory but deficient antiviral responses. A20 is an important negative regulator of NF-κB–mediated inflammatory but not antiviral responses, and A20 expression was reduced in COPD. IAV infection increased the expression of miR-125a or -b, which directly reduced the expression of A20 and mitochondrial antiviral signaling (MAVS), and caused exaggerated inflammation and impaired antiviral responses. These events were replicated in vivo in a mouse model of experimental COPD. Thus, miR-125a or -b and A20 may be targeted therapeutically to inhibit excessive inflammatory responses and enhance antiviral immunity in IAV infections and in COPD.

Authors

Alan C-Y. Hsu, Kamal Dua, Malcolm R. Starkey, Tatt-Jhong Haw, Prema M. Nair, Kristy Nichol, Nathan Zammit, Shane T. Grey, Katherine J. Baines, Paul S. Foster, Philip M. Hansbro, Peter A. Wark

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Figure 5

miR-125a and -b target a functional binding site of the 3′-UTR of the mRNA of MAVS to suppress its expression.

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miR-125a and -b target a functional binding site of the 3′-UTR of the mR...
(A) Representation of MAVS gene structure and location of miR-125a and -b binding site. (B) The binding site on 3′-UTR of MAVS is 100% conserved between human and mouse MAVS. (C) pBECs were infected with H3N2 or H1N1, and MAVS protein was detected at 6 hours (left) and 24 hours (right). Densitometry results (Supplemental Figure 5A, representative immunoblot) were calculated as MAVS/GAPDH ratios and expressed as fold change from untreated, uninfected controls. Data are mean ± SEM, n = 15 per group. *P ≤ 0.05 versus uninfected healthy or smoker controls, +P ≤ 0.05 versus infected or uninfected healthy controls. (D) BALB/c mice were exposed to cigarette smoke (Smk) or normal air (Air) for 8 weeks, inoculated with IAV H1N1 (A/PR/8/34, 8 pfu) or media (Sham) on the last day of smoke exposure, and sacrificed 7 days postinoculation (dpi). The levels of MAVS protein were measured in lung homogenates. Densitometry results (Supplemental Figure 5B, representative immunoblot) were calculated as MAVS/β-actin ratios in mouse and expressed as fold change from untreated, uninfected controls. Data are mean ± SEM, n = 6 per group. *P ≤ 0.05 versus Sham-treated controls, +P ≤ 0.05 versus infected Air controls. (E) The miR-125a and -b binding site on 3′-UTR was cloned into a pMIR luciferase reporter construct and transfected into HEK293 cells with miR-125a or -b mimetics. The luciferase reporter assay was performed to determine binding. Data are mean ± SEM, n = 3 per group.*P ≤ 0.05 versus miRNA scrambled controls. (F) Ago2 was immunoprecipitated from miR-125a or -b mimetic-transfected HEK293, and (G) A20 and MAVS mRNA was detected by qPCR in Ago2-immunoprecipitate. Data are mean ± SEM, n = 3 per group. *P ≤ 0.05 versus IgG control IP. Statistical differences were determined with one-way ANOVA followed by Bonferroni post-test.