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. 2013;8(3):e59397.
doi: 10.1371/journal.pone.0059397. Epub 2013 Mar 22.

Targeted ablation of miR-21 decreases murine eosinophil progenitor cell growth

Thomas X Lu  1 , Eun-Jin Lim, Svetlana Itskovich, John A Besse, Andrew J Plassard, Melissa K Mingler, Joelle A Rothenberg, Patricia C Fulkerson, Bruce J Aronow, Marc E Rothenberg
Affiliations

Targeted ablation of miR-21 decreases murine eosinophil progenitor cell growth

Thomas X Lu et al. PLoS One. 2013.

Abstract

MiR-21 is one of the most up-regulated miRNAs in multiple allergic diseases associated with eosinophilia and has been shown to positively correlate with eosinophil levels. Herein, we show that miR-21 is up-regulated during IL-5-driven eosinophil differentiation from progenitor cells in vitro. Targeted ablation of miR-21 leads to reduced eosinophil progenitor cell growth. Furthermore, miR-21(-/-) eosinophil progenitor cells have increased apoptosis as indicated by increased levels of annexin V positivity compared to miR-21(+/+) eosinophil progenitor cells. Indeed, miR-21(-/-) mice have reduced blood eosinophil levels in vivo and reduced eosinophil colony forming unit capacity in the bone marrow. Using gene expression microarray analysis, we identified dysregulation of genes involved in cell proliferation (e,g, Ms4a3, Grb7), cell cycle and immune response as the most significant pathways affected by miR-21 in eosinophil progenitors. These results demonstrate that miR-21 can regulate the development of eosinophils by influencing eosinophil progenitor cell growth. Our findings have identified one of the first miRNAs with a role in regulating eosinophil development.

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Conflict of interest statement

Competing Interests: M. E. Rothenberg has equity interest in reslizumab through Cephalon, is a consultant for Immune Pharmaceuticals, is on the American Partnership for Eosinophilic Disorders Medical Advisory Board, and is on the International Eosinophil Society's Executive Council. The rest of the authors declare that they have no relevant conflicts of interest. Concerning the potential conflict of interest reported, this does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. MiR-21 is induced during eosinophil differentiation.
(A) Purity of cultured eosinophils at day 14. Eosinophils are identified as CCR3+Siglec-F+ cells. (B) Levels of miR-21 during the eosinophil differentiation culture determined by qPCR normalized to U6 small nuclear RNA. N = 3 per group; data are represented as mean ± S.E.M. Data are representative of 3 independent experiments.
Figure 2
Figure 2. Growth of eosinophil progenitor cells from miR-21−/− mice and miR-21+/+ controls during the ex vivo eosinophil culture.
Total cell number of (A) eosinophil and (B) neutrophil cultures derived from miR-21+/+ and miR-21−/− mice is shown. N = 6 per group; data are represented as mean ± S.E.M. **: p<0.01. (C) Morphology of miR-21+/+ and miR-21−/− cultured eosinophils at day 12 determined by Diff-Quik staining. Data are representative of 3 independent experiments for panels A and C; data are representative of 2 independent experiments for panel B.
Figure 3
Figure 3. Levels of apoptosis in the eosinophil progenitor culture from the miR-21+/+ and miR-21−/− mice.
Levels of Annexin V and 7AAD staining during eosinophil differentiation culture as determined by FACS. The viable cells are AnnexinV− 7AAD−. The early apoptotic cells are AnnexinV+ 7AAD−. The late apoptotic cells are AnnexinV+ 7AAD+. Data are representative of 3 independent experiments.
Figure 4
Figure 4. Blood eosinophil percentage and bone marrow eosinophil colony forming unit capacity in the miR-21+/+ and miR-21−/− mice.
(A) Blood eosinophil percentage from miR-21+/+ and miR-21−/− mice determined by FACS staining for CCR3+Siglec-F+ cells; n = 9–10 mice per group. (B) Bone marrow eosinophil colony-forming unit (CFU-Eos) and (C) neutrophil colony-forming unit (CFU-G) capacity from miR-21+/+ and miR-21−/− mice; n = 4 per group. Data are represented as mean ± S.E.M. Data are representative of 3 independent experiments.
Figure 5
Figure 5. Differentially regulated genes between miR-21+/+ and miR-21−/− eosinophil progenitor cultures at day 8 and day 12.
(A) Heat map of differentially regulated genes at day 8 of the eosinophil differentiation culture. Red: up-regulated in miR-21−/− eosinophil progenitor culture compared to miR-21+/+ eosinophil progenitor culture. (B) Heat map of differentially regulated genes at day 12 of the eosinophil differentiation culture. Red: up-regulated in miR-21−/− eosinophil progenitor culture compared to miR-21+/+ eosinophil progenitor culture; blue: down-regulated in miR-21−/− eosinophil progenitor culture compared to miR-21+/+ eosinophil progenitor culture. (C) Quantitative RT-PCR verification of a selected set of differentially expressed genes between miR-21+/+ and miR-21−/− eosinophil progenitor cultures. (D) Western blot showing levels of PSRC1 in miR-21+/+ and miR-21−/− eosinophil progenitor cultures at day 8 and day 12. GAPDH is shown as a loading control. (E) Functional enrichment analysis of differentially regulated genes in the eosinophil progenitor cultures at day 12. The networks are shown as Cytoscape graph networks generated from ToppCluster network analysis.
Figure 6
Figure 6. Esophageal eosinophil level in an allergen-induced experimental eosinophil esophagitis model in miR-21+/+ and miR-21−/− mice.
Eosinophil levels in the esophagus were determined by morphometric analysis following anti-MBP staining. N = 5–6 mice per group. Data are represented as mean ± S.E.M. Data are representative of 2 independent experiments.
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