Immunology and Inflammation

Downregulation of Let-7 miRNA promotes Tc17 differentiation and emphysema via de-repression of RORγt

Phillip A. Erice
Xinyan Huang
Matthew J. Seasock
Matthew J. Robertson
Hui-Ying Tung
Melissa A. Perez-Negron
Shivani L. Lotlikar
David B Corry
Farrah Kheradmand
Antony Rodriguez author has email address

Immunology Graduate Program, Baylor College of Medicine, Houston, TX, 77030
Department of Medicine, Immunology & Allergy Rheumatology, Baylor College of Medicine Houston TX, 77030
Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Sun Yat-sen University. Guangzhou, Guangdong Province, P.R. China
Dan Duncan Comprehensive Cancer Center, Baylor College of Medicine Houston, TX, 77030
Department of Pathology and Immunology, Baylor College of Medicine Houston, TX, 77030
Center for Translational Research on Inflammatory Diseases, Michael E. Debakey, Baylor College of Medicine, Houston, TX, 77030
Department of Medicine, Section of Pulmonary and Critical Care, Baylor College of Medicine. Houston, TX, 77030

https://doi.org/10.7554/eLife.92879.2

Open access
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Figures and data

Repression of Let-7 miRNA gene clusters in lung T cells from COPD patients and murine models of emphysema.
(A) Schematic representation of the polycistronic transcripts for the Let-7a1/Let-7f1/Let-7d- and Let-7b/Let-7a3-clusters in humans and let-7a1/let-7f1/let-7/d- and let-7b/let-7c2-clusters in mice. (B) In silico analysis of Mirlet7a1hg and Mirlet7bhg from the publicly available lung transcriptome dataset from RNA-seq of COPD and control patients (GEO: GSE57148). (C) Quantitative RT-PCR (qPCR) of mature Hsa-Let-7a from resected lung tissue of COPD (n=15) and control subjects (n=11). (D) QPCR and regression analysis of Hsa-Let-7a, Hsa-Let-7b, Hsa-Let-7d, and Hsa-Let-7f expression to emphysema severity score based on CT: 0=no, 1=upper lobes only, 2=upper/middle lobes, 3=extensive pan lobular emphysema (n=19). (E) Schematic diagram of experimental emphysema in mice induced by either intranasal (i.n.) instillation of nCB or exposure to CS by whole-body inhalation (w.b.i.). (F-H) QPCR analysis for pri-let-7a1/f1/d and pri-let-7b/c2 from lung tissue or lung-derived CD8⁺ and CD4⁺ T cells of mice with emphysema elicited by (F) nCB- or (G-H) CS (n=3-6 per group). Data are representative of three independent experiments displayed as mean±SEM. Mann-Whitney (B,C) or Student’s t-test (F,G,H). *p < 0.05, **p < 0.01, ***p <0.001, ****p<0.0001.

Deletion of the let-7bc2 cluster in T cells enhances nCB- or CS-triggered emphysema.
(A) Schematic representation of CD4-Cre (let-7bc2^LOF) or let-7bc2^f/f (Control) mice. (B) QPCR analysis of pri-let-7b/c2 from flow-sorted live,TCRβ⁺, CD4⁺CD8⁺ double-positive (DP) thymocytes of control and let-7bc2^LOF mice (n=3-5 per group). (C-G) Control and let-7bc2^LOF mice were exposed to vehicle (PBS) or nCB for 4 weeks, or alternatively air or cigarette smoke by whole body inhalation of cigarette smoke (CS) for 16 weeks. (C) Representative H&E stained lung sections from PBS-, nCB-, or CS-exposed mice as indicated on each panel (x20 magnification; scale bars, 50µm). (D-E) Mean linear intercept (MLI) measurement of lung morphometry. (F) Total and differential cell counts from bronchoalveolar lavage (BAL) fluid from controls and nCB-emphysemic mice (n=4-7 per group). (G) Mmp9 and Mmp12 mRNA expression from BAL cells of air- and smoke-exposed control and let-7bc2^LOF mice (n=4-6 per group). Data are representative of at least three independent experiments displayed as mean±SEM using Student’s t-test (B) or two-way ANOVA with post-hoc Tukey correction (D,E,F,G). *p < 0.05, **p < 0.01, ***p <0.001, ****p<0.0001.

In vivo T cell ablation of the let-7bc2-cluster enhances Tc17 inflammatory response to nCB-emphysema.
Representative flow plots with percentage and counts of live TCRβ⁺ (A) CD8⁺IL-17a⁺ and CD8⁺IFNγ⁺, (B) CD8⁺IFNγ⁺GzmA⁺, (C) CD4⁺IL-17a⁺ and CD4⁺IFNγ⁺, and (D) CD4⁺ Foxp3⁺CD25⁺ cells from the lungs of control (Ctrl) PBS vehicle- (n=5-6), control nCB- (n=6), and let-7bc2^LOF nCB-exposed mice. Data are representative of three independent experiments displayed as mean±SEM using ANOVA with post-hoc Sidak correction. *p < 0.05, **p < 0.01, ***p <0.001, ****p<0.0001.

Deletion of either the let7bc2- or let7afd-cluster in T cells enhances RORγt expression in vivo.
(A) Left: Schematic repre-sentation of the murine Rorc 3’UTR with let-7 miRNA binding sites as identified by TargetScan. Right: Schematic of a conserved let-7 miRNA target sequence in the 3’UTR of Rorc. (B-C) Flow analysis of RORγt expression by MFI quantification in live TCRβ⁺CD8⁺ or CD4⁺ T cells from indicated tissues of (B) naïve control (Ctrl) and let-7bc2^LOF mice or (C) nCB-treated lungs by representative flow plot and MFI quantification (n=5 per group). (D) RORγt expression by MFI quantification in naïve mice let-7afd^LOF mice thymus, spleen, and lungs (n=3-4 per group), or (E) nCB-exposed lungs (n=5 per group). Data are representative of at least three independent experiments displayed as mean±SEM using student’s t-test. *p < 0.05, **p < 0.01, ***p <0.001, ****p<0.0001.

Let-7 restricts Tc17 in vitro differentiation in part via direct targeting of Rorc mRNA.
(A) Representative flow plots of live TCRβ⁺ CD8⁺, IL-17a⁺ and IFNγ⁺ populations from Tc1 and Tc17 polarized naïve splenic CD8⁺ cells from control and let-7bc2^LOF mice and (B) quantification of CD8⁺IL-17a⁺ cells (n=5 per group). (C) ELISA of IL-17a from the supernatant of Tc1 and Tc17 polarized control and let-7bc2^LOF cells (n=5-6 per group). (D) Flow quantification of CD8⁺IFNγ⁺ populations in Tc1 and Tc17 polarized control and let-7bc2^LOF cells (n=5 per group). (E) Representative flow plot and quantification of RORγt from Tc0 or Tc17 differentiated naïve splenic CD8⁺ T cells isolated from control and let-7bc2^LOF mice (n=5 per group). (F) Representative flow plots of CD8⁺IL-17a⁺ population frequency and quantification of Tc17 polarized naive splenic CD8⁺ cells of indicated mice polarized under Tc1 or Tc17 conditions. (G) ELISA of IL-17a from control, Tc1 (n=4), control Tc17 (n=4), and let-7afd^LOF Tc17 (n=3) polarized cells. (H) Quantification of RORγt from Tc0 or Tc17 in vitro polarized naive CD8⁺ T cells from control and let-7afd^LOF mice (n=3-4 per group). (I) Control (Rorc WT) or binding site mutant (Rorc Mut) 3’ UTRs of Rorc were cloned downstream of the renilla luciferase reporter. Plasmids were cotransfected with either a control-miR (black bars) or let-7b mimic (blue bars) duplex into cultured cells. Reporter activity was measured 24 hours after transfection and normalized to firefly activity. Data are representative of two (H), three independent experiments (A-G), or carried out in triplicate (I) and displayed as mean±SEM using student’s t-test. *p < 0.05, **p < 0.01, ***p <0.001, ****p<0.0001.

Enforced let-7 expression in T cells restrains induction of RORγt and Tc17/Th17 inflammation in lungs of nCB-exposed mice.
(A) Schematic outlining our T cell-inducible let-7g mouse model (let-7^GOF). (B) Flow analysis of RORγt expression in live, TCRβ⁺CD8⁺ or CD4⁺ T cells from (B) naïve control and let-7^GOF mice in thymus, spleen, and lungs (n=3-5 per group). (C) Control and let-7^GOF mice were treated with PBS vehicle or nCB then analyzed. Representative H&E-stained lung sections from PBS- and nCB-exposed mice as indicated on each panel (x20 magnification; scale bars, 50µm) (D) MLI measurements from indicated mice (n=5-6 per group). (E) Flow analysis of lungs gated on live TCRβ⁺ CD8⁺ or CD4⁺ cells for (E) IL-17a⁺ population frequency (n=3-4 per group) or (F) RORγt expression by representative flow plot and MFI quantification (n=4-5 per group). (G) Figure model for let-7/RORγt axis in emphysema pathogenesis. Data are representative of two (B) or three (C-F) independent experiments and displayed as mean±SEM using student’s t-test (B) or two-way ANOVA with Tukey’s multiple correction (D-F). *p < 0.05, **p < 0.01, ***p <0.001, ****p<0.0001.

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