Proinflammatory role for let-7 microRNAS in experimental asthma

Abstract

MicroRNAs (miRNAs) are short, non-coding RNAs that target and silence protein coding genes through 3'-UTR elements. Evidence increasingly assigns an immunosuppressive role for miRNAs in immunity, but relatively few miRNAs have been studied, and an overall understanding of the importance of these regulatory transcripts in complex in vivo systems is lacking. Here we have applied multiple technologies to globally analyze miRNA expression and function in allergic lung disease, an experimental model of asthma. Deep sequencing and microarray analyses of the mouse lung short RNAome revealed numerous extant and novel miRNAs and other transcript classes. Similar to mRNAs, lung miRNA expression changed dynamically during the transition from the naive to the allergic state, suggesting numerous functional relationships. A possible role for miRNA editing in altering the lung mRNA target repertoire was also identified. Multiple members of the highly conserved let-7 miRNA family were the most abundant lung miRNAs, and we confirmed in vitro that interleukin 13 (IL-13), a cytokine essential for expression for allergic lung disease, is regulated by mmu-let-7a. However, inhibition of let-7 miRNAs in vivo using a locked nucleic acid profoundly inhibited production of allergic cytokines and the disease phenotype. Our findings thus reveal unexpected complexity in the miRNAome underlying allergic lung disease and demonstrate a proinflammatory role for let-7 miRNAs.

Figures

FIGURE 1.

Characterization and distribution of small RNAs in mouse lung. A, frequency of NGS-derived sequences as a function of nucleotide length. The 21–23-nt peak is typical for miRNAs. B, pie charts show absolute numbers of sequenced transcripts from distinct lung RNA classes comparing allergen-challenged with naive mice. C, distribution of nucleotide modifications along the length of mature lung miRNAs comparing allergen-challenged with naive mice. D, editing of mmu-let-7a-1 as detected by NGS comparing allergen challenged with naive lungs in which the ninth nucleotide of the seed sequence, U, has been modified to G. *, canonical mature sequence. Also see supplemental Fig. 1 and Tables S1–S5.

FIGURE 2.

Gene and miRNA expression profiling of allergen-challenged and naive mouse lungs. A, heat map of genes (mRNAs) induced or repressed (p < 0.01, >1.5-fold) in allergen-challenged versus naive lung. B, validation of gene microarray findings by quantitative RT-PCR for selected genes. C, heat map of miRNAs induced or repressed (p < 0.01, >1.5-fold) in allergen-challenged versus naive lung. D, validation of miRNA microarray findings by quantitative RT-PCR for selected miRNAs. Bar graph data are presented as means ± S.E. (error bars), n = 3; *, p < 0.05. Also see supplemental Tables S6 and S7.

FIGURE 3.

Inverse expression of il-13 and let-7a suggests a functional association. A, the let-7a target sequence in the il-13 3′-UTR is conserved across mammalia (Targetscan 5.1). B, mature let-7a sequence (green) aligned with the mouse il-13 3′-UTR target site (red) and predicted minimum free energy (mfe) value. The let-7a seed sequence is shown. C and D, quantitative RT-PCR analysis of il-13 and ifn-γ (C) and mmu-let-7a (D) transcripts from in vitro cultured Th1 and Th2 cells. Data are presented as means ± S.E. (error bars), n = 3; *, p < 0.05.

FIGURE 4.

IL-13 expression is suppressed by let-7a. A and B, let-7a suppresses mouse and human IL-13 in HEK293T cells. HEK293T cells were transfected with plasmids containing firefly luciferase under the control of the mouse (A) or human (B) il-13 3′-UTR or control 3′-UTR and simultaneously with plasmids expressing pre-mmu-miR-705, scrambled pre-miR, or pre-let-7a, as indicated. After 2 days, gene expression was quantitated as firefly relative light units after normalizing for transfection efficiency based on Renilla luciferase activity (Firefly/Renilla). C and D, anti-let-7a rescues mouse IL-13 expression. HEK293T cells were transfected simultaneously with mouse (C) or human (D) IL-13 3′-UTR and pre-mmu-let-7a plasmids as in A and additionally scrambled, irrelevant (anti-miR-705) or anti-let-7a locked nucleic acids. After 2 days, IL-13 expression was assessed as firefly/Renilla relative light units. E–G, let-7a suppresses IL-13 gene expression in primary T cells. E, mouse splenic CD4+ T cells were electroporated with FITC-labeled anti-let-7a LNAs (black curve) or sham (red curve), and the efficiency of transfection was assessed by flow cytometry. Additional T cells were transfected with control or anti-let-7a LNA, and relative expression of let-7a (F) and IL-13 (G) transcripts were determined by quantitative RT-PCR 24 h later. H, editing of let-7a to let-7e reduces efficiency of targeting of il-13. HEK293T cells were transfected with mouse IL-13 3′-UTR-containing luciferase plasmid, as in A, and either plasmids for expression of let-7a or edited let-7a(U→G) and either scrambled or anti-let-7a(U→G) LNA, as indicated, and the effect on il-13 gene expression was assessed as relative light units. I, pre-let-7a(U→G) is fully processed to let-7e. Quantitative RT-PCR quantitation of let-7e or let-7a in HEK293T cells transfected with either pre-let-7a or pre-let-7a(U→G) expression plasmids. Data are presented as means ± S.E. (error bars), n = 3 or 4 replicates/condition; *, p < 0.05 for the indicated comparisons.

FIGURE 5.

let-7 miRNAs are required for allergic lung disease. A, protocol timeline for ovalbumin (OVA) immunization intraperitoneally (IP) and challenge intranasally (IN) and anti-miRNA LNA administration intravenously (IV). B, anti-let-7 LNA suppresses T cell let-7 and il-13 in vivo. Quantitative RT-PCR analysis of let-7a, il-13, and ifn-γ transcripts in splenic CD4 T cells from mice treated under the indicated conditions. C, airway responsiveness as assessed by the change in respiratory system resistance (RRS) in response to graded intravenous acetylcholine (Ach) challenge. *, p < 0.05 relative to naive or ovalbumin or ovalbumin + control LNA groups. D, total bronchoalveolar lavage fluid (BALF) inflammatory cells (eosinophils, macrophages, neutrophils, lymphocytes, total cells). E, relative expression of Muc5AC transcripts from whole lung. *, p < 0.05 relative to naive. F, bronchoalveolar lavage fluid levels of the indicated cytokines. *, p < 0.05 for the indicated comparisons. Data are presented as means ± S.E. (error bars), n = 5 mice/group.