Research ArticleIMMUNOLOGY

Epithelial cell–specific loss of function of Miz1 causes a spontaneous COPD-like phenotype and up-regulates Ace2 expression in mice

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Science Advances  14 Aug 2020:
Vol. 6, no. 33, eabb7238
DOI: 10.1126/sciadv.abb7238
  • Fig. 1 MIZ1 protein levels are reduced in human COPD lungs and cigarette smoke–exposed mouse lungs or cigarette smoke extract–treated lung epithelial cells.

    (A and B) Miz1 immunohistochemistry (IHC) staining of normal human lungs or lungs from patients with COPD at the time of lung transplantation (three subjects from each group are shown, which were representative of seven subjects from each group). Scale bars, 20 μm. (C) mRNA levels of Miz1 and Mule from normal human lungs or lungs from patients with COPD analyzed by RNA sequencing (RNA-seq) (normal, n = 13; COPD, n = 7). **P < 0.01; ns, not significant. (D) Mule IHC from human lungs mentioned in (A). (E to H) Western blot (E) or IF staining (F and G) of Miz1 or Mule IHC (H) in mouse lungs exposed to RA or CS for 7 days, 3 months, and 6 months. Representatives of n ≥ 4. (I and J) Western blot of Mule and Miz1 in CS extract–treated MLE-12 cells (I) or cells expressing tetracycline-inducible shRNA for Mule in the absence or presence of tetracycline (Tet) (J). Data are representative of at least three independent experiments.

  • Fig. 2 Lung epithelial cell–specific deletion of the Miz1 POZ domain causes spontaneous emphysema in mice.

    (A and E) Representative whole lung histology from ≤4-month-old control SPC-Cre+/Miz1(POZ)wt/wt or homozygous SPC-Cre+/Miz1(POZ)fl/fl mice (A) or ≥1-year-old control Miz1(POZ)fl/fl, heterozygous SPC-Cre+/Miz1(POZ)wt/fl, or homozygous SPC-Cre+/Miz1(POZ)fl/fl mice (E). (B, D, and F) Representative histological sections (10× or 20× objective) of lungs from ≤4-month-old control SPC-Cre+/Miz1(POZ)wt/wt or homozygous SPC-Cre+/Miz1(POZ)fl/fl mice (B), control Miz1(POZ)fl/fl or heterozygous SPC-Cre+/Miz1(POZ)wt/fl mice of different ages (D), or ≥1-year-old control SPC-Cre+/Miz1(POZ)wt/wt, Miz1(POZ)fl/fl, heterozygous SPC-Cre+/Miz1(POZ)wt/fl, or homozygous SPC-Cre+/Miz1(POZ)fl/fl mice (F). Scale bars, 20 μm. n = 4 to 8 for (A, B, and D to F). (C and G) MLI (micrometers ± SEM) or the ratio of surface area to alveolar air space volume (surface-to-volume ratio; means ± SEM) of alveolar septae measured in the lungs of ≤4-month-old control Miz1(POZ)fl/fl (n = 5) or homozygous SPC-Cre+/Miz1(POZ)fl/fl mice (n = 4) (C) or ≥ 1-year-old control Miz1(POZ)fl/fl (n = 6), heterozygous SPC-Cre+/Miz1(POZ)wt/fl (n = 5), or homozygous SPC-Cre+/Miz1(POZ)fl/fl mice (n = 4) (G). (H and I) Representative micro–computed x-ray tomography (micro-CT) images of the lung cross sections (H) or the three-dimensional lungs (I) of ≥1-year-old control Miz1(POZ)fl/fl or homozygous SPC-Cre+/Miz1(POZ)fl/fl mouse. In (I), the low attenuation area (below −700 HU) is colored in pink, and the whole lung field is colored in blue. (J) Representative HU of the lung parenchyma from ≥1-year-old control Miz1(POZ)fl/fl or homozygous SPC-Cre+/Miz1(POZ)fl/fl mouse. (K) Static lung compliance (Cst) of ≥1-year-old control Miz1(POZ)fl/fl (n = 8) or homozygous SPC-Cre+/Miz1(POZ)fl/fl mice (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001. Data are representative of at least three independent experiments.

  • Fig. 3 Mice with lung epithelial cell–specific loss of function of Miz1 also exhibit features of chronic bronchitis.

    (A and B) Representative histological sections (20×, 40×, or 100× objective) of pulmonary airways from ≤4-month-old control SPC-Cre+/Miz1(POZ)wt/wt or homozygous SPC-Cre+/Miz1(POZ)fl/fl mice (A) or ≥ 1-year-old control SPC-Cre+/Miz1(POZ)wt/wt, Miz1(POZ)fl/fl, heterozygous SPC-Cre+/Miz1(POZ)wt/fl, or homozygous SPC-Cre+/Miz1(POZ)fl/fl mice (B). Arrowheads indicate goblet cells. (C and D) MUC5AC IF staining from mice in (A) and (B). (E to G) Representative IHC staining of EMBP (eosinophils) (20× objective), F4/80 (macrophages) (20× objective) of lungs from young control SPC-Cre+/Miz1(POZ)wt/wt or homozygous SPC-Cre+/Miz1(POZ)fl/fl mice (E), Ly6G (PMNs) (20× objective) (F), or TUNEL (20× objective) (G) staining of ≥1-year-old control Miz1(POZ)fl/fl, SPC-Cre+/Miz1(POZ)wt/wt, or homozygous SPC-Cre+/Miz1(POZ)fl/fl mice. n ≥ 5. Data are representative of at least three independent experiments.

  • Fig. 4 AM- and dendritic cell–specific loss of function of Miz1 is not sufficient to cause a COPD-like phenotype in mice.

    (A, C, and D) Representative lung histological sections (A and D) or whole lung histology (C) of ≤4-month-old (A) or ≥ 1-year-old control Miz1(POZ)fl/fl or CD11c-Cre+/Miz1(POZ)fl/fl mice (C and D). (B) BALF cell differentials from young or ≥1-year-old control Miz1(POZ)fl/fl or CD11c-Cre+/Miz1(POZ)fl/fl mice (n = 4 to 11). Mȹ, macrophages; PMN, neutrophils; Lym, lymphocytes. **P < 0.01, ***P < 0.0001. (E) Representative histological sections (20×, 40×, or 100× objective) of pulmonary airways from ≥1-year-old control Miz1(POZ)fl/fl or CD11c-Cre+/Miz1(POZ)fl/fl mice. Data are representative of at least three independent experiments.

  • Fig. 5 Loss of the Miz1 POZ domain results in deregulated expression profiles enriched in innate immunity/inflammation.

    (A and D to G) GO analysis of differentially expressed genes (analyzed by RNA-seq) in AT2 (A and D), AMs (E and F), or whole lungs (G) from 6-month-old control Miz1(POZ)fl/fl, heterozygous SPC-Cre+/Miz1(POZ)wt/fl, or homozygous SPC-Cre+/Miz1(POZ)fl/fl mice. (B) Ace2 and Tmprss2 mRNA levels analyzed by RNA-seq from control Miz1(POZ)fl/fl or homozygous SPC-Cre+/Miz1(POZ)fl/fl mice of similar age [control, n = 3; knockout (KO), n = 4]. (C and H) Heatmaps of differentially expressed genes involved in NF-κB, TNF, or TLR signaling by KEGG PATHWAY mapping from (A) (C) or differentially expressed genes of those identified as COPD susceptibility genes by GWAS from (A) to (G) (H), respectively. n ≥ 4 for (A) to (H). Red underlines indicate pathways involved in innate immunity. Control, Miz1(POZ)fl/fl mice; Het, heterozygous SPC-Cre+/Miz1(POZ)wt/fl mice; Homo, homozygous SPC-Cre+/Miz1(POZ)fl/fl mice.

  • Fig. 6 RelA haploinsufficiency rescues the phenotype of Miz1-deficient mice, and lung epithelial cell–specific loss of function of Miz1 in the adulthood is also sufficient to induce emphysema.

    (A and C) Representative whole lung histology (A) or histological sections of pulmonary airways (C) in the lungs of ~1-year-old control SPC-Cre+/Miz1(POZ)wt/wt, heterozygous SPC-Cre+/Miz1(POZ)wt/fl, or SPC-Cre+/Miz1(POZ)wt/flRelAwt/fl mice. (B) MLI (micrometers ± SEM) or surface-to-volume ratio (means ± SEM) of alveolar septae measured in the lungs of ~1-year-old control Miz1(POZ)fl/fl (n = 6), heterozygous SPC-Cre+/Miz1(POZ)wt/fl (n = 5), or SPC-Cre+/Miz1(POZ)wt/flRelAwt/fl mice (n = 4). **P < 0.01 and ***P < 0.001. (D) Representative lung histological sections of control SPC-CreERT2−/Miz1(POZ)f/f or SPC-CreERT2+/Miz1(POZ)f/f mice after treatment with tamoxifen for 14 months. Three subjects from each group were shown, which were the representative of five to eight subjects from each group. Scale bars, 500 μm. (E) MLI (micrometers ± SEM) or the ratio of surface area to alveolar air space volume (surface-to-volume ratio; means ± SEM) of alveolar septae measured in the lungs of control SPC-CreERT2−/Miz1(POZ)f/f or SPC-CreERT2+/Miz1(POZ)f/f mice after treatment with tamoxifen for 14 months. n = 5 to 8. Data are representative of at least three independent experiments.

Supplementary Materials

  • Supplementary Materials

    Epithelial cell–specific loss of function of Miz1 causes a spontaneous COPD-like phenotype and up-regulates Ace2 expression in mice

    Hanh Chi Do-Umehara, Cong Chen, Qiao Zhang, Alexander V. Misharin, Hiam Abdala-Valencia, S. Marina Casalino-Matsuda, Paul A. Reyfman, Kishore R. Anekalla, Francisco J. Gonzalez-Gonzalez, Marc A. Sala, Chao Peng, Ping Wu, Catherine C. L. Wong, Ravi Kalhan, Ankit Bharat, Harris Perlman, Karen M. Ridge, Jacob I. Sznajder, Peter H. S. Sporn, Navdeep S. Chandel, Jindan Yu, Xiangdong Fu, Irina Petrache, Rubin Tuder, G. R. Scott Budinger, Jing Liu

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