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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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.).
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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).
(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).
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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