Dicer is indispensable for the development of murine mast cells
2014; Elsevier BV; Volume: 135; Issue: 4 Linguagem: Inglês
10.1016/j.jaci.2014.10.005
ISSN1097-6825
AutoresAnja Förster, Birgit Blissenbach, Alzbeta Machova, Silke Leja, Anja Rabenhorst, Sarah Lea Wilmschen, Klaus Heger, Marc Schmidt‐Supprian, Axel Roers, Karin Hartmann, Nikoletta Papadopoulou,
Tópico(s)Asthma and respiratory diseases
ResumoMast cells (MCs) are major effector cells of type I allergy.1Voehringer D. Protective and pathological roles of mast cells and basophils.Nat Rev Immunol. 2013; 13: 362-375Crossref PubMed Scopus (270) Google Scholar In addition, there is growing evidence for the involvement of MCs in infection, autoimmunity, and tumor development. Murine MCs are categorized into connective tissue–type mast cells (CTMCs) and mucosal mast cells (MMCs). CTMCs reside predominantly in the dermis, peritoneal cavity, and submucosa of the gastrointestinal tract and mainly express mouse mast cell protease (mMCP) -4, -5, and -6, whereas MMCs are exclusively located in the mucosal epithelia of the airways and the gastrointestinal tract and primarily express mMCP-1 and mMCP-2. MicroRNAs (miRNAs) are small noncoding RNAs with critical functions in regulating the mammalian immune system by binding protein-coding mRNA targets, resulting in translational repression and mRNA decay.2Xiao C. Rajewsky K. MicroRNA control in the immune system: basic principles.Cell. 2009; 136: 26-36Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar Specific miRNAs, including miR-221/miR-222, miR-146, and miR-21, have been reported to regulate in vitro MC proliferation, cell cycling, cytokine production, and formation of the actin cytoskeleton.3Montagner S. Orlandi E.M. Merante S. Monticelli S. The role of miRNAs in mast cells and other innate immune cells.Immunol Rev. 2013; 253: 12-24Crossref PubMed Scopus (50) Google Scholar During miRNA biogenesis, the RNase III endoribonuclease Dicer is responsible for processing of mature miRNA duplexes. Ubiquitous deletion of Dicer in mice leads to early embryonic lethality, demonstrating a vital role for miRNAs in development.2Xiao C. Rajewsky K. MicroRNA control in the immune system: basic principles.Cell. 2009; 136: 26-36Abstract Full Text Full Text PDF PubMed Scopus (853) Google ScholarTo explore the role of Dicer in MCs in vivo, we generated mice with conditional deletion of Dicer in CTMCs. In particular, we analyzed MC counts and MC-specific responses. Furthermore, the development of MCs from Dicer-deficient bone marrow was assessed in vitro.To generate MC-specific Dicer knockout mice, we crossed the Mcpt5Cre strain,4Scholten J. Hartmann K. Gerbaulet A. Krieg T. Müller W. Testa G. et al.Mast cell-specific Cre/loxP-mediated recombination in vivo.Transgenic Res. 2008; 17: 307-315Crossref PubMed Scopus (0) Google Scholar which expresses Cre recombinase under control of the Mcpt5 promoter, to Dicerfl/fl mice.5Yi R. O'Carroll D. Pasolli H.A. Zhang Z. Dietrich F.S. Tarakhovsky A. et al.Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs.Nat Genet. 2006; 38: 356-362Crossref PubMed Scopus (417) Google Scholar Enumeration of MCs in different tissues was performed by means of microscopic counting and flow cytometry. MMC counts were additionally quantified by measurement of MMC-specific mMCP-1 levels in sera and corresponding Mcpt1 and Mcpt2 transcript levels in the small intestines of IL-3–treated mice. Passive systemic anaphylaxis after intraperitoneal injection with dinitrophenol (DNP)–specific IgE and subsequent challenge with dinitrophenylated human serum albumin (DNP-HSA) was analyzed by measurement of rectal body temperature. Blood cell counts were determined with the hematology system Hemavet 950FS (Drew Scientific, Waterbury, Conn), and numbers of myeloid and lymphoid cells in the spleen and peritoneal cavity were assessed by means of flow cytometry. Development of bone marrow–derived mast cells (BMMCs) was investigated in Mx1Cre/Dicerfl/fl mice. In these mice Cre expression under control of the Mx1 promoter was induced by administration of polyinosine-polycytidylic acid (polyI:C), leading to Dicer deletion in all interferon-responsive hematopoietic cells.6Kühn R. Schwenk F. Aguet M. Rajewsky K. Inducible gene targeting in mice.Science. 1995; 269: 1427-1429Crossref PubMed Google Scholar The methods used are further detailed in the Methods section in this article's Online Repository at www.jacionline.org.Enumeration of MCs in different tissues revealed comparable MC counts in Mcpt5Cre/Dicerfl/wt mice and Dicerfl/wt control animals (Fig 1, A-E). Interestingly, homozygous deletion of Dicer significantly reduced the number of CTMCs in the peritoneal cavity (85.8%) and resulted in nearly complete ablation (93.4% to 100%) of CTMCs in the dermis of back skin, ears, mesentery, muscularis of the glandular stomach, tongue, and heart compared with levels seen in Dicerfl/fl control animals (Fig 1, A-G, and see Fig E1 in this article's Online Repository at www.jacionline.org). In contrast to these alterations in CTMCs, MMC numbers were comparable between Mcpt5Cre/Dicerfl/fl mice and Dicerfl/fl control animals, as indicated by unchanged serum levels of mMCP-1 (Fig 1, H) and comparable expression of Mcpt1 and Mcpt2 mRNA in the small intestine in both groups (Fig 1, I and J).Because MCs are major effector cells of anaphylaxis, we next sought to investigate the response of Mcpt5Cre/Dicerfl/fl mice in IgE-mediated passive systemic anaphylaxis (Fig 1, K). We observed that Mcpt5Cre/Dicerfl/fl mice were completely protected from the anaphylaxis-associated decrease in body temperature, whereas Dicerfl/fl control mice showed the expected temperature decrease. These findings demonstrate that ablation of CTMCs is highly efficient in Mcpt5Cre/Dicerfl/fl mice and results in loss of systemic MC functions.We observed no alterations in MC counts upon deletion of Dicer in mature MCs in vivo using polyI:C-treated Mx1Cre/Dicerfl/fl mice (see Fig E2 in this article's Online Repository at www.jacionline.org), suggesting that Dicer might be dispensable for survival of MCs. To further investigate the role of Dicer for MC development, we also assessed MC differentiation from bone marrow of polyI:C-treated Mx1Cre/Dicerfl/fl mice (Fig 1, L-N). In Mx1Cre/Dicerfl/fl cultures we observed very low cell numbers and massive cell death (Fig 1, L and M). Furthermore, analysis of Kit+ and FcεRIα+ cells after 4 weeks in culture revealed that only 3.09% ± 0.05% MCs developed in Mx1Cre/Dicerfl/fl cultures (Fig 1, N). In contrast, Dicerfl/fl cultures showed expression of Kit and FcεRIα in 91.29% ± 1.18% of cells. Similarly, basophils did not develop, whereas low numbers of dendritic cells arose from the bone marrow of Mx1Cre/Dicerfl/fl mice (see Fig E3 in this article's Online Repository at www.jacionline.org). These findings indicate that Dicer is indispensable for development of MCs, as well as basophils, but not for survival of hematopoietic stem cells. Different miRNAs, such as miR-342-3p, miR-125b-5p, and miR-132, have recently been reported to be crucially involved in the development of MCs.7Xiang Y. Eyers F. Young I.G. Rosenberg H.F. Foster P.S. Yang M. Identification of microRNAs regulating the developmental pathways of bone marrow derived mast cells.PLoS One. 2014; 9: e98139Crossref PubMed Scopus (17) Google Scholar Hence the absence of these miRNAs upon deletion of Dicer might explain the deficiency of CTMCs. In line with our findings, Dicer ablation in vivo has been shown to also affect maturation of granulocyte/macrophage progenitors toward neutrophils, macrophages, and dendritic cells, as well as T-cell differentiation, B-cell development, and germinal center formation.2Xiao C. Rajewsky K. MicroRNA control in the immune system: basic principles.Cell. 2009; 136: 26-36Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar, 8Alemdehy M.F. van Boxtel N.G. de Looper H.W. van den Berge I.J. Sanders M.A. Cupedo T. et al.Dicer1 deletion in myeloid-committed progenitors causes neutrophil dysplasia and blocks macrophage/dendritic cell development in mice.Blood. 2012; 119: 4723-4730Crossref PubMed Scopus (43) Google ScholarExpression of Mcpt5Cre has previously been demonstrated to be highly specific for CTMCs,4Scholten J. Hartmann K. Gerbaulet A. Krieg T. Müller W. Testa G. et al.Mast cell-specific Cre/loxP-mediated recombination in vivo.Transgenic Res. 2008; 17: 307-315Crossref PubMed Scopus (0) Google Scholar with the exception of TH2 cell–mediated expansion of MMCs.9Burton O.T. Noval Rivas M. Zhou J.S. Logsdon S.L. Darling A.R. Koleoglou K.J. et al.Immunoglobulin E signal inhibition during allergen ingestion leads to reversal of established food allergy and induction of regulatory T cells.Immunity. 2014; 41: 141-151Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar To further assess the specificity of Mcpt5Cre-mediated deletion of Dicer, we next analyzed the number of different myeloid and lymphoid cells in peripheral blood, the spleen, and the peritoneal cavity (Fig 2). We found that all these cell populations showed comparable numbers in Mcpt5Cre/Dicerfl/fl mice and Dicerfl/fl control animals, confirming that Mcpt5Cre is specifically expressed in MCs.Fig 2Numbers 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)For previous investigations of MC functions, the Kit mutant mouse strains WBB6F1-KitW/W-v and C57BL/6-KitW-sh/W-sh have been widely used. However, these strains also exhibit other major alterations in the hematopoietic system and in fertility. To overcome these limitations, we and others recently generated mouse models of more specific deficiency of MCs, which depend on the Cre/loxP recombination system.1Voehringer D. Protective and pathological roles of mast cells and basophils.Nat Rev Immunol. 2013; 13: 362-375Crossref PubMed Scopus (270) Google Scholar, 4Scholten J. Hartmann K. Gerbaulet A. Krieg T. Müller W. Testa G. et al.Mast cell-specific Cre/loxP-mediated recombination in vivo.Transgenic Res. 2008; 17: 307-315Crossref PubMed Scopus (0) Google Scholar The reported Cpa3Cre-dependent mouse models exhibited deficiency of MCs and a reduction in basophil counts. In contrast, crossing Mcpt5Cre to R-DTA mice, which express diphtheria toxin A in Cre-expressing cells, induced depletion of CTMCs. The present study reporting on Mcpt5Cre-mediated deletion of Dicer thus introduces a new mouse model of highly specific and efficient CTMC deficiency.In conclusion, our study demonstrates that posttranscriptional regulation by Dicer-dependent mechanisms is essential for development of the MC lineage. Furthermore, the Mcpt5Cre/Dicerfl/fl cross represents a novel mouse model with specific CTMC deficiency. Mast cells (MCs) are major effector cells of type I allergy.1Voehringer D. Protective and pathological roles of mast cells and basophils.Nat Rev Immunol. 2013; 13: 362-375Crossref PubMed Scopus (270) Google Scholar In addition, there is growing evidence for the involvement of MCs in infection, autoimmunity, and tumor development. Murine MCs are categorized into connective tissue–type mast cells (CTMCs) and mucosal mast cells (MMCs). CTMCs reside predominantly in the dermis, peritoneal cavity, and submucosa of the gastrointestinal tract and mainly express mouse mast cell protease (mMCP) -4, -5, and -6, whereas MMCs are exclusively located in the mucosal epithelia of the airways and the gastrointestinal tract and primarily express mMCP-1 and mMCP-2. MicroRNAs (miRNAs) are small noncoding RNAs with critical functions in regulating the mammalian immune system by binding protein-coding mRNA targets, resulting in translational repression and mRNA decay.2Xiao C. Rajewsky K. MicroRNA control in the immune system: basic principles.Cell. 2009; 136: 26-36Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar Specific miRNAs, including miR-221/miR-222, miR-146, and miR-21, have been reported to regulate in vitro MC proliferation, cell cycling, cytokine production, and formation of the actin cytoskeleton.3Montagner S. Orlandi E.M. Merante S. Monticelli S. The role of miRNAs in mast cells and other innate immune cells.Immunol Rev. 2013; 253: 12-24Crossref PubMed Scopus (50) Google Scholar During miRNA biogenesis, the RNase III endoribonuclease Dicer is responsible for processing of mature miRNA duplexes. Ubiquitous deletion of Dicer in mice leads to early embryonic lethality, demonstrating a vital role for miRNAs in development.2Xiao C. Rajewsky K. MicroRNA control in the immune system: basic principles.Cell. 2009; 136: 26-36Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar To explore the role of Dicer in MCs in vivo, we generated mice with conditional deletion of Dicer in CTMCs. In particular, we analyzed MC counts and MC-specific responses. Furthermore, the development of MCs from Dicer-deficient bone marrow was assessed in vitro. To generate MC-specific Dicer knockout mice, we crossed the Mcpt5Cre strain,4Scholten J. Hartmann K. Gerbaulet A. Krieg T. Müller W. Testa G. et al.Mast cell-specific Cre/loxP-mediated recombination in vivo.Transgenic Res. 2008; 17: 307-315Crossref PubMed Scopus (0) Google Scholar which expresses Cre recombinase under control of the Mcpt5 promoter, to Dicerfl/fl mice.5Yi R. O'Carroll D. Pasolli H.A. Zhang Z. Dietrich F.S. Tarakhovsky A. et al.Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs.Nat Genet. 2006; 38: 356-362Crossref PubMed Scopus (417) Google Scholar Enumeration of MCs in different tissues was performed by means of microscopic counting and flow cytometry. MMC counts were additionally quantified by measurement of MMC-specific mMCP-1 levels in sera and corresponding Mcpt1 and Mcpt2 transcript levels in the small intestines of IL-3–treated mice. Passive systemic anaphylaxis after intraperitoneal injection with dinitrophenol (DNP)–specific IgE and subsequent challenge with dinitrophenylated human serum albumin (DNP-HSA) was analyzed by measurement of rectal body temperature. Blood cell counts were determined with the hematology system Hemavet 950FS (Drew Scientific, Waterbury, Conn), and numbers of myeloid and lymphoid cells in the spleen and peritoneal cavity were assessed by means of flow cytometry. Development of bone marrow–derived mast cells (BMMCs) was investigated in Mx1Cre/Dicerfl/fl mice. In these mice Cre expression under control of the Mx1 promoter was induced by administration of polyinosine-polycytidylic acid (polyI:C), leading to Dicer deletion in all interferon-responsive hematopoietic cells.6Kühn R. Schwenk F. Aguet M. Rajewsky K. Inducible gene targeting in mice.Science. 1995; 269: 1427-1429Crossref PubMed Google Scholar The methods used are further detailed in the Methods section in this article's Online Repository at www.jacionline.org. Enumeration of MCs in different tissues revealed comparable MC counts in Mcpt5Cre/Dicerfl/wt mice and Dicerfl/wt control animals (Fig 1, A-E). Interestingly, homozygous deletion of Dicer significantly reduced the number of CTMCs in the peritoneal cavity (85.8%) and resulted in nearly complete ablation (93.4% to 100%) of CTMCs in the dermis of back skin, ears, mesentery, muscularis of the glandular stomach, tongue, and heart compared with levels seen in Dicerfl/fl control animals (Fig 1, A-G, and see Fig E1 in this article's Online Repository at www.jacionline.org). In contrast to these alterations in CTMCs, MMC numbers were comparable between Mcpt5Cre/Dicerfl/fl mice and Dicerfl/fl control animals, as indicated by unchanged serum levels of mMCP-1 (Fig 1, H) and comparable expression of Mcpt1 and Mcpt2 mRNA in the small intestine in both groups (Fig 1, I and J). Because MCs are major effector cells of anaphylaxis, we next sought to investigate the response of Mcpt5Cre/Dicerfl/fl mice in IgE-mediated passive systemic anaphylaxis (Fig 1, K). We observed that Mcpt5Cre/Dicerfl/fl mice were completely protected from the anaphylaxis-associated decrease in body temperature, whereas Dicerfl/fl control mice showed the expected temperature decrease. These findings demonstrate that ablation of CTMCs is highly efficient in Mcpt5Cre/Dicerfl/fl mice and results in loss of systemic MC functions. We observed no alterations in MC counts upon deletion of Dicer in mature MCs in vivo using polyI:C-treated Mx1Cre/Dicerfl/fl mice (see Fig E2 in this article's Online Repository at www.jacionline.org), suggesting that Dicer might be dispensable for survival of MCs. To further investigate the role of Dicer for MC development, we also assessed MC differentiation from bone marrow of polyI:C-treated Mx1Cre/Dicerfl/fl mice (Fig 1, L-N). In Mx1Cre/Dicerfl/fl cultures we observed very low cell numbers and massive cell death (Fig 1, L and M). Furthermore, analysis of Kit+ and FcεRIα+ cells after 4 weeks in culture revealed that only 3.09% ± 0.05% MCs developed in Mx1Cre/Dicerfl/fl cultures (Fig 1, N). In contrast, Dicerfl/fl cultures showed expression of Kit and FcεRIα in 91.29% ± 1.18% of cells. Similarly, basophils did not develop, whereas low numbers of dendritic cells arose from the bone marrow of Mx1Cre/Dicerfl/fl mice (see Fig E3 in this article's Online Repository at www.jacionline.org). These findings indicate that Dicer is indispensable for development of MCs, as well as basophils, but not for survival of hematopoietic stem cells. Different miRNAs, such as miR-342-3p, miR-125b-5p, and miR-132, have recently been reported to be crucially involved in the development of MCs.7Xiang Y. Eyers F. Young I.G. Rosenberg H.F. Foster P.S. Yang M. Identification of microRNAs regulating the developmental pathways of bone marrow derived mast cells.PLoS One. 2014; 9: e98139Crossref PubMed Scopus (17) Google Scholar Hence the absence of these miRNAs upon deletion of Dicer might explain the deficiency of CTMCs. In line with our findings, Dicer ablation in vivo has been shown to also affect maturation of granulocyte/macrophage progenitors toward neutrophils, macrophages, and dendritic cells, as well as T-cell differentiation, B-cell development, and germinal center formation.2Xiao C. Rajewsky K. MicroRNA control in the immune system: basic principles.Cell. 2009; 136: 26-36Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar, 8Alemdehy M.F. van Boxtel N.G. de Looper H.W. van den Berge I.J. Sanders M.A. Cupedo T. et al.Dicer1 deletion in myeloid-committed progenitors causes neutrophil dysplasia and blocks macrophage/dendritic cell development in mice.Blood. 2012; 119: 4723-4730Crossref PubMed Scopus (43) Google Scholar Expression of Mcpt5Cre has previously been demonstrated to be highly specific for CTMCs,4Scholten J. Hartmann K. Gerbaulet A. Krieg T. Müller W. Testa G. et al.Mast cell-specific Cre/loxP-mediated recombination in vivo.Transgenic Res. 2008; 17: 307-315Crossref PubMed Scopus (0) Google Scholar with the exception of TH2 cell–mediated expansion of MMCs.9Burton O.T. Noval Rivas M. Zhou J.S. Logsdon S.L. Darling A.R. Koleoglou K.J. et al.Immunoglobulin E signal inhibition during allergen ingestion leads to reversal of established food allergy and induction of regulatory T cells.Immunity. 2014; 41: 141-151Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar To further assess the specificity of Mcpt5Cre-mediated deletion of Dicer, we next analyzed the number of different myeloid and lymphoid cells in peripheral blood, the spleen, and the peritoneal cavity (Fig 2). We found that all these cell populations showed comparable numbers in Mcpt5Cre/Dicerfl/fl mice and Dicerfl/fl control animals, confirming that Mcpt5Cre is specifically expressed in MCs. For previous investigations of MC functions, the Kit mutant mouse strains WBB6F1-KitW/W-v and C57BL/6-KitW-sh/W-sh have been widely used. However, these strains also exhibit other major alterations in the hematopoietic system and in fertility. To overcome these limitations, we and others recently generated mouse models of more specific deficiency of MCs, which depend on the Cre/loxP recombination system.1Voehringer D. Protective and pathological roles of mast cells and basophils.Nat Rev Immunol. 2013; 13: 362-375Crossref PubMed Scopus (270) Google Scholar, 4Scholten J. Hartmann K. Gerbaulet A. Krieg T. Müller W. Testa G. et al.Mast cell-specific Cre/loxP-mediated recombination in vivo.Transgenic Res. 2008; 17: 307-315Crossref PubMed Scopus (0) Google Scholar The reported Cpa3Cre-dependent mouse models exhibited deficiency of MCs and a reduction in basophil counts. In contrast, crossing Mcpt5Cre to R-DTA mice, which express diphtheria toxin A in Cre-expressing cells, induced depletion of CTMCs. The present study reporting on Mcpt5Cre-mediated deletion of Dicer thus introduces a new mouse model of highly specific and efficient CTMC deficiency. In conclusion, our study demonstrates that posttranscriptional regulation by Dicer-dependent mechanisms is essential for development of the MC lineage. Furthermore, the Mcpt5Cre/Dicerfl/fl cross represents a novel mouse model with specific CTMC deficiency. Dicerfl/fl mice were kindly provided by Alexander Tarakhovsky (Rockefeller University, NY). MethodsMiceAll mice were kept in the animal facilities of the Institute for Medical Microbiology, Immunology and Hygiene and the Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany. Dicerfl/fl miceE1Yi R. O'Carroll D. Pasolli H.A. Zhang Z. Dietrich F.S. Tarakhovsky A. et al.Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs.Nat Genet. 2006; 38: 356-362Crossref PubMed Scopus (445) Google Scholar were crossed to heterozygous Mcpt5CreE2Scholten J. Hartmann K. Gerbaulet A. Krieg T. Müller W. Testa G. et al.Mast cell-specific Cre/loxP-mediated recombination in vivo.Transgenic Res. 2008; 17: 307-315Crossref PubMed Scopus (148) Google Scholar or Mx1CreE3Kühn R. Schwenk F. Aguet M. Rajewsky K. Inducible gene targeting in mice.Science. 1995; 269: 1427-1429Crossref PubMed Scopus (1523) Google Scholar mice. Littermates negative for the respective Cre served as control mice. All mice were on a C57BL/6 background and used for experiments at the age of 8 to 12 weeks. The experiments were carried out in accordance with institutional and state guidelines (approvals AZ 84-02.04.2011.A302 and 84-02.04.2013.A064). Mcpt5Cre/Dicerfl/fl, Mcpt5Cre/Dicerfl/wt, and Mx1Cre/Dicerfl/fl mice were viable and fertile. Weekly monitoring of body weight, fur and skin condition over 10 weeks revealed no apparent defects in Mcpt5Cre/Dicerfl/fl or Mcpt5Cre/Dicerfl/wt mice compared with Dicerfl/fl control animals. Mx1Cre/Dicerfl/fl mice and Dicerfl/fl littermate control mice were injected intraperitoneally twice within 2 days with polyI:C (GE Healthcare, Fairfield, Conn) to induce Cre expression in interferon-responsive hematopoietic cells.Enumeration of MCs in different tissuesFor enumeration of MCs in different tissues, paraffin-embedded sections were stained with Giemsa, and MCs were counted in 15 medium-power fields (×200 magnification, dermis of back skin, tongue, and stomach) and 15 high-power fields (×400 magnification, ear) of Giemsa-stained, paraffin-embedded sections by using a Leica DM4000B microscope (Leica Microsystems, Wetzlar, Germany) with Diskus software (Hilgers, Königswinter, Germany). MC counts in the peritoneal cavity (Kit+ and FcεRIα+) were determined by means of flow cytometry.Analysis of MC proteases expressed by MMCsMice were injected intraperitoneally for 5 consecutive days with 0.5 mL of IL-3–containing supernatant of X63/0 cells, as previously described,E4Heger K. Seidler B. Vahl J.C. Schwartz C. Kober M. Klein S. et al.CreER(T2) expression from within the c-Kit gene locus allows efficient inducible gene targeting in and ablation of mast cells.Eur J Immunol. 2014; 44: 296-306Crossref PubMed Scopus (24) Google Scholar to induce expansion of intestinal MCs. Three days later, mice were killed to harvest serum, as well as the small intestine, for measurement of MC proteases, which are specific for MMCs. mMCP-1 serum levels were determined by using the mMCP-1 ELISA (eBioscience, Frankfurt, Germany), according to the manufacturer's instructions. To quantify expression of Mcpt1 and Mcpt2 mRNA as a marker for MMCs in the small intestine, quantitative real-time PCR was used, as previously described.E4Heger K. Seidler B. Vahl J.C. Schwartz C. Kober M. Klein S. et al.CreER(T2) expression from within the c-Kit gene locus allows efficient inducible gene targeting in and ablation of mast cells.Eur J Immunol. 2014; 44: 296-306Crossref PubMed Scopus (24) Google Scholar Porphobilinogen deaminase (PBGD) was used as a housekeeping gene.IgE-mediated passive systemic anaphylaxisMice were injected intraperitoneally with 150 μL of DNP-specific IgE (83 μg/mL; clone SPE7, Sigma-Aldrich, Steinheim, Germany) and challenged 24 hours later with 200 μL of DNP-HSA (1 mg/mL). The extent of anaphylaxis was measured based on rectal body temperature every 10 minutes for 80 minutes.Cell cultureFive days after the first polyI:C injection, femoral and tibial bone marrow was isolated from Mx1Cre/Dicerfl/fl mice and Dicerfl/fl littermate control animals. To gain BMMCs, cells were cultured in Dulbecco modified Eagle medium (high glucose; Gibco, Invitrogen, Carlsbad, Calif) supplemented with 20% FCS, 2 mmol/L l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin (Biochrom AG, Berlin, Germany), and 30% WEHI-3B–conditioned medium (as a source of IL-3) for at least 4 weeks. After 3 weeks in culture, 0.5% CHO-conditioned medium (as a source of stem cell factor) was added. Dendritic cells were cultured in RPMI 1640 medium (Biochrom AG) supplemented with 10% heat-inactivated FCS, 2 mmol/L l-glutamine, 50 μmol/L 2-mercaptoethanol (Gibco, Invitrogen), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco, Invitrogen), and 20 ng/mL rmGM-CSF (rhesus macaque GM-CSF; PeproTech, Hamburg, Germany). Basophils were cultured with 30 ng/mL IL-3 (PeproTech) instead of rmGM-CSF.Analysis of different cell populations in peripheral blood, the spleen, and the peritoneal cavityWhole blood samples were taken from the superficial temporal vein. Counting of blood cells was performed with the hematology system Hemavet 950FS (Drew Scientific), according to the manufacturer's protocol. Numbers of T cells (CD3e+), B cells (CD19+), dendritic cells (CD11c+), macrophages (F4/80+), neutrophils (Gr-1+), eosinophils (Siglec-F+), and basophils (CD49b+ and FcεRIα+) in the spleen and numbers of B cells (CD19+), macrophages (F4/80+), and MCs (Kit+ and FcεRIα+) in the peritoneal cavity were determined by using flow cytometry.Flow cytometryThe following antibodies were used for flow cytometry: anti-mouse CD16/32 (clone 93; eBioscience, San Diego, Calif), phycoerythrin (PE)–conjugated anti-mouse FcεRIα (clone MAR-1; eBioscience), allophycocyanin (APC)–conjugated anti-mouse CD117 (Kit; clone 2B8, eBioscience), PE-conjugated anti-mouse CD3e (clone 145-2C11, eBioscience), fluorescein isothiocyanate–conjugated anti-mouse CD19 (clone 1D3, eBioscience), PE-conjugated anti-mouse CD11c (clone N418, eBioscience), fluorescein isothiocyanate–conjugated anti-mouse F4/80 (clone BM8, eBioscience), PE-conjugated anti-mouse Gr-1 (clone RB6-8C5; Miltenyi Biotec, Bergisch-Gladbach, Germany), PE-conjugated anti-mouse Siglec-F (clone E50-2440; BD Biosciences, Heidelberg, Germany), and APC-conjugated anti-mouse CD49b (integrin alpha 2; clone DX5, eBioscience). Propidium iodide (Sigma-Aldrich) and APC-conjugated Annexin V (eBioscience) were used to determine viability and apoptosis, respectively.Statistical analysisFig E2Deletion 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Dendritic 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) MiceAll mice were kept in the animal facilities of the Institute for Medical Microbiology, Immunology and Hygiene and the Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany. Dicerfl/fl miceE1Yi R. O'Carroll D. Pasolli H.A. Zhang Z. Dietrich F.S. Tarakhovsky A. et al.Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs.Nat Genet. 2006; 38: 356-362Crossref PubMed Scopus (445) Google Scholar were crossed to heterozygous Mcpt5CreE2Scholten J. Hartmann K. Gerbaulet A. Krieg T. Müller W. Testa G. et al.Mast cell-specific Cre/loxP-mediated recombination in vivo.Transgenic Res. 2008; 17: 307-315Crossref PubMed Scopus (148) Google Scholar or Mx1CreE3Kühn R. Schwenk F. Aguet M. Rajewsky K. Inducible gene targeting in mice.Science. 1995; 269: 1427-1429Crossref PubMed Scopus (1523) Google Scholar mice. Littermates negative for the respective Cre served as control mice. All mice were on a C57BL/6 background and used for experiments at the age of 8 to 12 weeks. The experiments were carried out in accordance with institutional and state guidelines (approvals AZ 84-02.04.2011.A302 and 84-02.04.2013.A064). Mcpt5Cre/Dicerfl/fl, Mcpt5Cre/Dicerfl/wt, and Mx1Cre/Dicerfl/fl mice were viable and fertile. Weekly monitoring of body weight, fur and skin condition over 10 weeks revealed no apparent defects in Mcpt5Cre/Dicerfl/fl or Mcpt5Cre/Dicerfl/wt mice compared with Dicerfl/fl control animals. Mx1Cre/Dicerfl/fl mice and Dicerfl/fl littermate control mice were injected intraperitoneally twice within 2 days with polyI:C (GE Healthcare, Fairfield, Conn) to induce Cre expression in interferon-responsive hematopoietic cells. All mice were kept in the animal facilities of the Institute for Medical Microbiology, Immunology and Hygiene and the Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany. Dicerfl/fl miceE1Yi R. O'Carroll D. Pasolli H.A. Zhang Z. Dietrich F.S. Tarakhovsky A. et al.Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs.Nat Genet. 2006; 38: 356-362Crossref PubMed Scopus (445) Google Scholar were crossed to heterozygous Mcpt5CreE2Scholten J. Hartmann K. Gerbaulet A. Krieg T. Müller W. Testa G. et al.Mast cell-specific Cre/loxP-mediated recombination in vivo.Transgenic Res. 2008; 17: 307-315Crossref PubMed Scopus (148) Google Scholar or Mx1CreE3Kühn R. Schwenk F. Aguet M. Rajewsky K. Inducible gene targeting in mice.Science. 1995; 269: 1427-1429Crossref PubMed Scopus (1523) Google Scholar mice. Littermates negative for the respective Cre served as control mice. All mice were on a C57BL/6 background and used for experiments at the age of 8 to 12 weeks. The experiments were carried out in accordance with institutional and state guidelines (approvals AZ 84-02.04.2011.A302 and 84-02.04.2013.A064). Mcpt5Cre/Dicerfl/fl, Mcpt5Cre/Dicerfl/wt, and Mx1Cre/Dicerfl/fl mice were viable and fertile. Weekly monitoring of body weight, fur and skin condition over 10 weeks revealed no apparent defects in Mcpt5Cre/Dicerfl/fl or Mcpt5Cre/Dicerfl/wt mice compared with Dicerfl/fl control animals. Mx1Cre/Dicerfl/fl mice and Dicerfl/fl littermate control mice were injected intraperitoneally twice within 2 days with polyI:C (GE Healthcare, Fairfield, Conn) to induce Cre expression in interferon-responsive hematopoietic cells. Enumeration of MCs in different tissuesFor enumeration of MCs in different tissues, paraffin-embedded sections were stained with Giemsa, and MCs were counted in 15 medium-power fields (×200 magnification, dermis of back skin, tongue, and stomach) and 15 high-power fields (×400 magnification, ear) of Giemsa-stained, paraffin-embedded sections by using a Leica DM4000B microscope (Leica Microsystems, Wetzlar, Germany) with Diskus software (Hilgers, Königswinter, Germany). MC counts in the peritoneal cavity (Kit+ and FcεRIα+) were determined by means of flow cytometry. For enumeration of MCs in different tissues, paraffin-embedded sections were stained with Giemsa, and MCs were counted in 15 medium-power fields (×200 magnification, dermis of back skin, tongue, and stomach) and 15 high-power fields (×400 magnification, ear) of Giemsa-stained, paraffin-embedded sections by using a Leica DM4000B microscope (Leica Microsystems, Wetzlar, Germany) with Diskus software (Hilgers, Königswinter, Germany). MC counts in the peritoneal cavity (Kit+ and FcεRIα+) were determined by means of flow cytometry. Analysis of MC proteases expressed by MMCsMice were injected intraperitoneally for 5 consecutive days with 0.5 mL of IL-3–containing supernatant of X63/0 cells, as previously described,E4Heger K. Seidler B. Vahl J.C. Schwartz C. Kober M. Klein S. et al.CreER(T2) expression from within the c-Kit gene locus allows efficient inducible gene targeting in and ablation of mast cells.Eur J Immunol. 2014; 44: 296-306Crossref PubMed Scopus (24) Google Scholar to induce expansion of intestinal MCs. Three days later, mice were killed to harvest serum, as well as the small intestine, for measurement of MC proteases, which are specific for MMCs. mMCP-1 serum levels were determined by using the mMCP-1 ELISA (eBioscience, Frankfurt, Germany), according to the manufacturer's instructions. To quantify expression of Mcpt1 and Mcpt2 mRNA as a marker for MMCs in the small intestine, quantitative real-time PCR was used, as previously described.E4Heger K. Seidler B. Vahl J.C. Schwartz C. Kober M. Klein S. et al.CreER(T2) expression from within the c-Kit gene locus allows efficient inducible gene targeting in and ablation of mast cells.Eur J Immunol. 2014; 44: 296-306Crossref PubMed Scopus (24) Google Scholar Porphobilinogen deaminase (PBGD) was used as a housekeeping gene. Mice were injected intraperitoneally for 5 consecutive days with 0.5 mL of IL-3–containing supernatant of X63/0 cells, as previously described,E4Heger K. Seidler B. Vahl J.C. Schwartz C. Kober M. Klein S. et al.CreER(T2) expression from within the c-Kit gene locus allows efficient inducible gene targeting in and ablation of mast cells.Eur J Immunol. 2014; 44: 296-306Crossref PubMed Scopus (24) Google Scholar to induce expansion of intestinal MCs. Three days later, mice were killed to harvest serum, as well as the small intestine, for measurement of MC proteases, which are specific for MMCs. mMCP-1 serum levels were determined by using the mMCP-1 ELISA (eBioscience, Frankfurt, Germany), according to the manufacturer's instructions. To quantify expression of Mcpt1 and Mcpt2 mRNA as a marker for MMCs in the small intestine, quantitative real-time PCR was used, as previously described.E4Heger K. Seidler B. Vahl J.C. Schwartz C. Kober M. Klein S. et al.CreER(T2) expression from within the c-Kit gene locus allows efficient inducible gene targeting in and ablation of mast cells.Eur J Immunol. 2014; 44: 296-306Crossref PubMed Scopus (24) Google Scholar Porphobilinogen deaminase (PBGD) was used as a housekeeping gene. IgE-mediated passive systemic anaphylaxisMice were injected intraperitoneally with 150 μL of DNP-specific IgE (83 μg/mL; clone SPE7, Sigma-Aldrich, Steinheim, Germany) and challenged 24 hours later with 200 μL of DNP-HSA (1 mg/mL). The extent of anaphylaxis was measured based on rectal body temperature every 10 minutes for 80 minutes. Mice were injected intraperitoneally with 150 μL of DNP-specific IgE (83 μg/mL; clone SPE7, Sigma-Aldrich, Steinheim, Germany) and challenged 24 hours later with 200 μL of DNP-HSA (1 mg/mL). The extent of anaphylaxis was measured based on rectal body temperature every 10 minutes for 80 minutes. Cell cultureFive days after the first polyI:C injection, femoral and tibial bone marrow was isolated from Mx1Cre/Dicerfl/fl mice and Dicerfl/fl littermate control animals. To gain BMMCs, cells were cultured in Dulbecco modified Eagle medium (high glucose; Gibco, Invitrogen, Carlsbad, Calif) supplemented with 20% FCS, 2 mmol/L l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin (Biochrom AG, Berlin, Germany), and 30% WEHI-3B–conditioned medium (as a source of IL-3) for at least 4 weeks. After 3 weeks in culture, 0.5% CHO-conditioned medium (as a source of stem cell factor) was added. Dendritic cells were cultured in RPMI 1640 medium (Biochrom AG) supplemented with 10% heat-inactivated FCS, 2 mmol/L l-glutamine, 50 μmol/L 2-mercaptoethanol (Gibco, Invitrogen), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco, Invitrogen), and 20 ng/mL rmGM-CSF (rhesus macaque GM-CSF; PeproTech, Hamburg, Germany). Basophils were cultured with 30 ng/mL IL-3 (PeproTech) instead of rmGM-CSF. Five days after the first polyI:C injection, femoral and tibial bone marrow was isolated from Mx1Cre/Dicerfl/fl mice and Dicerfl/fl littermate control animals. To gain BMMCs, cells were cultured in Dulbecco modified Eagle medium (high glucose; Gibco, Invitrogen, Carlsbad, Calif) supplemented with 20% FCS, 2 mmol/L l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin (Biochrom AG, Berlin, Germany), and 30% WEHI-3B–conditioned medium (as a source of IL-3) for at least 4 weeks. After 3 weeks in culture, 0.5% CHO-conditioned medium (as a source of stem cell factor) was added. Dendritic cells were cultured in RPMI 1640 medium (Biochrom AG) supplemented with 10% heat-inactivated FCS, 2 mmol/L l-glutamine, 50 μmol/L 2-mercaptoethanol (Gibco, Invitrogen), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco, Invitrogen), and 20 ng/mL rmGM-CSF (rhesus macaque GM-CSF; PeproTech, Hamburg, Germany). Basophils were cultured with 30 ng/mL IL-3 (PeproTech) instead of rmGM-CSF. Analysis of different cell populations in peripheral blood, the spleen, and the peritoneal cavityWhole blood samples were taken from the superficial temporal vein. Counting of blood cells was performed with the hematology system Hemavet 950FS (Drew Scientific), according to the manufacturer's protocol. Numbers of T cells (CD3e+), B cells (CD19+), dendritic cells (CD11c+), macrophages (F4/80+), neutrophils (Gr-1+), eosinophils (Siglec-F+), and basophils (CD49b+ and FcεRIα+) in the spleen and numbers of B cells (CD19+), macrophages (F4/80+), and MCs (Kit+ and FcεRIα+) in the peritoneal cavity were determined by using flow cytometry. Whole blood samples were taken from the superficial temporal vein. Counting of blood cells was performed with the hematology system Hemavet 950FS (Drew Scientific), according to the manufacturer's protocol. Numbers of T cells (CD3e+), B cells (CD19+), dendritic cells (CD11c+), macrophages (F4/80+), neutrophils (Gr-1+), eosinophils (Siglec-F+), and basophils (CD49b+ and FcεRIα+) in the spleen and numbers of B cells (CD19+), macrophages (F4/80+), and MCs (Kit+ and FcεRIα+) in the peritoneal cavity were determined by using flow cytometry. Flow cytometryThe following antibodies were used for flow cytometry: anti-mouse CD16/32 (clone 93; eBioscience, San Diego, Calif), phycoerythrin (PE)–conjugated anti-mouse FcεRIα (clone MAR-1; eBioscience), allophycocyanin (APC)–conjugated anti-mouse CD117 (Kit; clone 2B8, eBioscience), PE-conjugated anti-mouse CD3e (clone 145-2C11, eBioscience), fluorescein isothiocyanate–conjugated anti-mouse CD19 (clone 1D3, eBioscience), PE-conjugated anti-mouse CD11c (clone N418, eBioscience), fluorescein isothiocyanate–conjugated anti-mouse F4/80 (clone BM8, eBioscience), PE-conjugated anti-mouse Gr-1 (clone RB6-8C5; Miltenyi Biotec, Bergisch-Gladbach, Germany), PE-conjugated anti-mouse Siglec-F (clone E50-2440; BD Biosciences, Heidelberg, Germany), and APC-conjugated anti-mouse CD49b (integrin alpha 2; clone DX5, eBioscience). Propidium iodide (Sigma-Aldrich) and APC-conjugated Annexin V (eBioscience) were used to determine viability and apoptosis, respectively. The following antibodies were used for flow cytometry: anti-mouse CD16/32 (clone 93; eBioscience, San Diego, Calif), phycoerythrin (PE)–conjugated anti-mouse FcεRIα (clone MAR-1; eBioscience), allophycocyanin (APC)–conjugated anti-mouse CD117 (Kit; clone 2B8, eBioscience), PE-conjugated anti-mouse CD3e (clone 145-2C11, eBioscience), fluorescein isothiocyanate–conjugated anti-mouse CD19 (clone 1D3, eBioscience), PE-conjugated anti-mouse CD11c (clone N418, eBioscience), fluorescein isothiocyanate–conjugated anti-mouse F4/80 (clone BM8, eBioscience), PE-conjugated anti-mouse Gr-1 (clone RB6-8C5; Miltenyi Biotec, Bergisch-Gladbach, Germany), PE-conjugated anti-mouse Siglec-F (clone E50-2440; BD Biosciences, Heidelberg, Germany), and APC-conjugated anti-mouse CD49b (integrin alpha 2; clone DX5, eBioscience). Propidium iodide (Sigma-Aldrich) and APC-conjugated Annexin V (eBioscience) were used to determine viability and apoptosis, respectively. Statistical analysisFig E3Dendritic 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)
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