Dicer is indispensable for the development of murine mast cells

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Dicer is indispensable for the development of murine mast cells Anja Förster, MSc, Birgit Blissenbach, MSc, Alzbeta Machova, BSc, Silke Leja, MTA, Anja Rabenhorst, PhD, Sarah Wilmschen, MSc, Klaus Heger, MSc, Marc Schmidt-Supprian, PhD, Axel Roers, MD, Karin Hartmann, MD, Nikoletta Papadopoulou, PhD Journal of Allergy and Clinical Immunology Volume 135, Issue 4, Pages 1077-1080.e4 (April 2015) DOI: 10.1016/j.jaci.2014.10.005 Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Mcpt5Cre-mediated deletion of Dicer leads to the absence of CTMC and MC-specific responses. A-G, MC counts (n = 4-8). H, Serum levels of mMCP-1 (n = 4-7) upon injection of IL-3. I and J, Expression ratio of Mcpt1 and Mcpt2 mRNA relative to porphobilinogen deaminase (PBGD) mRNA in the small intestine (n = 4-7) upon injection of IL-3. K, IgE-mediated passive systemic anaphylaxis (n = 6-7, mean ± SD). L, BMMCs were cultured from Dicerfl/fl and Mx1Cre/Dicerfl/fl mice, and cell counts were assessed twice a week (n = 3, mean ± SD). M and N, Viability and numbers of MCs (Kit+FcεRIα+) in BMMC cultures were determined after 4 weeks of cultivation (100% refers to all cells in the culture; n = 3, mean ± SD). *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2015 135, 1077-1080.e4DOI: (10.1016/j.jaci.2014.10.005) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Numbers of other hematopoietic cell populations are unaffected in Mcpt5Cre/Dicerfl/fl mice. A, Hematocrit (HTC) and numbers of red blood cells (RBC), platelets (PLT), and the indicated white blood cell (WBC) subclasses in venous blood (n = 8-9). B and C, Numbers of myeloid and lymphoid cells in the spleen (Fig 2, B) and peritoneal cavity (Fig 2, C) were determined in Dicerfl/fl and Mcpt5Cre/Dicerfl/fl mice (n = 6) (mean ± SD). ***P < .001. Journal of Allergy and Clinical Immunology 2015 135, 1077-1080.e4DOI: (10.1016/j.jaci.2014.10.005) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Histologic analysis of different tissues reveals ablation of CTMCs in Mcpt5Cre/Dicerfl/fl mice. Representative Giemsa staining of dermis of back skin (×430 magnification; insert = ×1100 magnification; A and B), ears (×800 magnification; insert = ×1300 magnification; C and D), stomachs (×300 magnification; insert = ×800 magnification; E and F), and tongues (×200 magnification; insert = 800×; G and H) of Dicerfl/fl and Mcpt5Cre/Dicerfl/fl mice are depicted. Journal of Allergy and Clinical Immunology 2015 135, 1077-1080.e4DOI: (10.1016/j.jaci.2014.10.005) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Deletion of Dicer in mature MCs has no effect on MC counts. Mx1Cre/Dicerfl/fl (n = 6) and Dicerfl/fl mice (n = 6) were injected intraperitoneally with polyI:C. Numbers of MCs in the peritoneal cavity (A), dermis of back skin (B), and ears (C) were analyzed by using flow cytometry and histology. Journal of Allergy and Clinical Immunology 2015 135, 1077-1080.e4DOI: (10.1016/j.jaci.2014.10.005) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Dendritic cells develop from the bone marrow of Mx1Cre/Dicerfl/fl mice. Basophils and dendritic cells were cultured from bone marrow of Mx1Cre/Dicerfl/fl and Dicerfl/fl mice on induction of Cre expression. A, Representative fluorescence-activated cell sorting plots show gating of basophils (CD117 [Kit]− FcεRIα+) and CD11c+ and CD11chigh dendritic cells. Percentages of basophils (day 10) and dendritic cells (day 8 and 10) are depicted in bar charts. B and C, Cell counts (Fig E3, B) and apoptosis (Fig E3, C) were analyzed in cultures of basophils and dendritic cells by means of microscopic counting and flow cytometry (n = 3-4, mean ± SD). ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. Journal of Allergy and Clinical Immunology 2015 135, 1077-1080.e4DOI: (10.1016/j.jaci.2014.10.005) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions