The microRNA miR-22 inhibits the histone deacetylase HDAC4 to promote TH17 cell–dependent emphysema

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Abstract

Smoking-related emphysema is a chronic inflammatory disease driven by the TH17 subset of helper T cells through molecular mechanisms that remain obscure. Here we explored the role of the microRNA miR-22 in emphysema. We found that miR-22 was upregulated in lung myeloid dendritic cells (mDCs) of smokers with emphysema and antigen-presenting cells (APCs) of mice exposed to smoke or nanoparticulate carbon black (nCB) through a mechanism that involved the transcription factor NF-κB. Mice deficient in miR-22, but not wild-type mice, showed attenuated TH17 responses and failed to develop emphysema after exposure to smoke or nCB. We further found that miR-22 controlled the activation of APCs and TH17 responses through the activation of AP-1 transcription factor complexes and the histone deacetylase HDAC4. Thus, miR-22 is a critical regulator of both emphysema and TH17 responses.

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Figure 1: Enhanced miR-22 expression in human emphysema and cigarette smoke–activated lung APCs.
Figure 2: miR-22 deficiency protects mice from nCB-induced emphysema.
Figure 3: miR-22 expression is transcriptionally regulated through NF-κB and AP-1 complexes.
Figure 4: miR-22 promotes the activation of lung APCs.
Figure 5: miR-22 promotes the transcriptional activity of AP-1 in APCs.
Figure 6: miR-22 targets HDAC4 in APCs to promote IL-6 production.
Figure 7: HDAC4 inhibits the activation of APCs and the progression of emphysema.
Figure 8: Neutralization of lung miR-22 reverses nCB-induced emphysema.

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Acknowledgements

We thank J. Levitt (Baylor College of Medicine) for CD11c-Cre mice; E. Olson (UT Southwestern Medical Center) for Hdac4fl mice; and W. Decker, J. Sederstrom, Y. Qian and L.-Z. Song for technical assistance. Supported by the US National Institutes of Health (R01HL117181 to F.K.; R01HL110883 to F.K. and D.B.C.; and AI036211, CA125123 and RR024574 to the Cytometry and Cell Sorting Core at Baylor College of Medicine) and the US Veterans Administration Office of Research and Development (1I01BX002221 to D.B.C.).

Author information

W.L. and D.B.C. conceptualized the project and all studies and wrote the manuscript with assistance from all co-authors; W.L. designed and performed all experiments, with R.Y., X.Y. and T.Y. contributing to selected mouse experiments and micro-CT analysis; E.L.G.S., D.C.M., W.K.A.S. and J.M.T. prepared nCB; A.R. provided miR-22 deficient mice; and F.K. and D.B.C. provided grant support.

Correspondence to Farrah Kheradmand or David B Corry.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 miR-22 is required for airway MMP activities but not for the development of γδ+ T cells in emphysema.

(a) Gelatin zymogram depicting matrix metalloproteinase (MMP) 2 and MMP9 activities in bronchoalveolar lavage fluid (BALF) of wild type (WT; lanes 3-5) and Mir22–/– mice (lanes 6-9) previously challenged with nCB. Lanes 1 and 2 are BALF from naïve wild type mice. (b) Total lung IL-17A+ γδ+ T cells were quantified by flow cytometry.

Supplementary Figure 2 Mir22–/– mice develop reduced TH17 responses upon sensitization with ovalbumin.

Both WT and Mir22–/– mice were intraperitoneally injected with 25µg ovalbumin precipitated in alum weekly for three consecutive weeks. Total splenocytes were harvested at the end of the fifth week. (a) IFN-γ, (b) IL-4 and (c) IL-17A positive cells with ovalbumin (OVA; 0.5 mg/ml overnight) and without (Media) were quantified by ELISpot. *: p<0.05, Kruskal Wallis test. n=4.

Supplementary Figure 3 nCB-induced production of pro-inflammatory cytokines and chemokines from whole lung is in part miR-22 dependent.

WT and Mir22–/– mice were challenged intranasally with nCB over one month. (a-d) Lungs were then collected and the concentrations of the indicated cytokines and chemokines from lung homogenate fluids were determined by Bio-plex. (e-g) Lung CD11c+ cells were isolated and cultured ex vivo overnight. Concentrations of the indicated supernatant chemokines were determined by Bio-plex. *: p<0.05; **: p<0.01, ***: p<0.001, Kruskal Wallis test. n=4-5.

Supplementary Figure 4 Smoke-exposed wild-type lung APCs are sufficient to induce emphysema in mice with and without miR-22.

Lung CD11c+ APC from four month cigarette SMK or air exposed WT mice were isolated and adaptively transferred to WT and Mir22–/– recipients. (a) Micro-CT quantification of recipient mouse lung volume after 3 months. (b) Total macrophages in BALF. (c) Relative abundance of lung IL-17A+ TH17 cells as assessed by flow cytometry. *: p<0.05; **: p<0.01, Kruskal Wallis test. n=3-4 as indicated in (A).

Supplementary Figure 5 PPAR-γ regulates miR-22 expression in lung APCs.

(a) MiR-22 expression in CD11c+ lung APCs from eight month old Ppargflox and PpargCD11c mice. (b) MiR-22 expression in CD11c+ lung APCs from air or cigarette smoke (SMK) exposed mice with or without intranasally challenge of ciglitazone (Cig). *: p<0.05; unpaired t-test in (a) one-way ANOVA test in (b). (n=3)

Supplementary Figure 6 miR-22 is required for pro-inflammatory gene expression in nCB-exposed lung APCs.

Lung CD11c+ cells from WT and Mir22–/– mice exposed to PBS or nCB were isolated. Expression of indicated genes was determined by quantitative PCR. *, p<0.05; **, p<0.01; ***, p<0.001, One-way ANOVA test. (n=3)

Supplementary Figure 7 miR-22 deficient APCs induce a TH17 cytokine in vitro with supplementation of IL-6.

MiR-22 sufficient (Mir22flox) and deficient BMDCs (Mir22CD11c) were primed with or without 1000ng nCB and co-cultured with WT naïve CD4+ T cells for 3 days and secreted IL-17A was measured by ELISA (n=4-5). 10ng/ml IL-6 supplement was added as indicated. *: p<0.05; Mann-Whitney test (n=4-5).

Supplementary Figure 8 Silencing miR-22 alters pro- and anti-inflammatory gene expression in APCs.

Mice were sacrificed at the 5 month point in Figure (8a). (a) MiR-22 expression level in lung CD11c+ cells. IL-6 (b) and IL-17A (c) concentration in lung homogenates as assessed by Bioplex. (d-f) Indicated gene expression in isolated lung CD11c+ cells determined by RT-qPCR. *, p<0.05; **, p<0.01; ***, p<0.001, Kruskal Wallis test (n=4-6). Data in are from one of two comparable experiments.

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Lu, W., You, R., Yuan, X. et al. The microRNA miR-22 inhibits the histone deacetylase HDAC4 to promote TH17 cell–dependent emphysema. Nat Immunol 16, 1185–1194 (2015) doi:10.1038/ni.3292

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