Mechanisms of allergy and clinical immunology
Let-7 microRNA–mediated regulation of IL-13 and allergic airway inflammation

https://doi.org/10.1016/j.jaci.2011.04.034Get rights and content

Background

IL-13, a cytokine secreted by TH2 lymphocytes and other cells, critically modulates allergic inflammation and tissue remodeling in allergic asthma. Although much is known about transcriptional regulation of IL-13, posttranscriptional regulation is poorly understood.

Objective

Because many inflammatory pathways are known to be regulated by microRNAs, permitting a rapid and fine-tuned response, the role of microRNA-mediated regulation of IL-13 was investigated using both in vitro and in vivo studies.

Methods

A combination of in silico approaches and in vitro transfections in A549 cells and primary cultured T cells was used to demonstrate the involvement of let-7 in IL-13 regulation. Furthermore, intranasal delivery of let-7 microRNA mimic in mice was performed to study its effects in allergic airway inflammatory conditions.

Results

Using a combination of bioinformatics and molecular approaches, we demonstrate that the let-7 family of microRNAs regulates IL-13 expression. Induced levels of IL-13 in cultured T cells were inversely related to let-7 levels. In an IL-13–dependent murine model of allergic airway inflammation, we observed that inflammation was associated with a reduction in most of the members of the let-7 family. Exogenous administration of let-7 mimic to lungs of mice with allergic inflammation resulted in a decrease in IL-13 levels, resolution of airway inflammation, reduction in airway hyperresponsiveness, and attenuation of mucus metaplasia and subepithelial fibrosis.

Conclusion

Let-7 microRNAs inhibit IL-13 expression and represent a major regulatory mechanism for modulating IL-13 secretion in IL-13–producing cell types and thereby TH2 inflammation.

Section snippets

In silico identification of microRNA binding sites

A database search for all human microRNAs (miRBase v9) was made against the human IL13 3′ untranslated region (UTR) using miRanda, RNAhybrid, and TargetScan.16, 17, 18 A consensus approach was used, as described earlier19 and listed in Table I, to minimize false-positive hits.

Plasmids

The human IL13 3′UTR was amplified from genomic DNA, cloned in pMIR-REPORT vector (Ambion, Austin, Tex), and designated as pMIR-REPORT-IL13 3′UTR. PCR-based site-directed mutagenesis was performed as described previously

Let-7 targets IL13 3′ UTR

Consensus prediction using 3 independent software programs indicated potential binding of 11 microRNAs to the IL13 3′UTR. Five of these belonged to the let-7 family (namely miR-98, let-7d, let-7f, let-7g, and let-7i), showing a high degree of conservation in the let-7 family26 and its predicted targeting of IL13 (Table I). The entire IL13 3′UTR was cloned into a luciferase reporter system and cotransfected with individual microRNA mimics (10 nmol/L) in A549 cells to experimentally validate

Discussion

IL-13 is a key effector TH2 cytokine. Because of its well-established role in modulating TH2 immune responses, including asthma, efforts are being made to therapeutically modulate IL-13 function. For instance, the anti–IL-13 antibodies, such as QAX576, were under clinical trials for the treatment of IL-13–related disorders32; the human anti–IL-13 IgG4 mAb CAT-354 has shown a significant reduction in airway eosinophilia and airway reactivity in a murine model of airway and esophageal

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    Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest.

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