Global microRNA expression is essential for murine mast cell development in vivo
2014; Elsevier BV; Volume: 42; Issue: 10 Linguagem: Inglês
10.1016/j.exphem.2014.07.266
ISSN1873-2399
AutoresSun Young Oh, Stephanie Brandal, Reuben Kapur, Zhou Zhu, Clifford M. Takemoto,
Tópico(s)Reproductive System and Pregnancy
ResumoMicroRNAs (miRNAs) are small, noncoding RNAs that have been shown to play a critical role in normal physiology and disease, such as hematopoietic development and cancer. However, their role in mast-cell function and development is poorly understood. The major objective of this study was to determine how global miRNA expression affects mast-cell physiology. The RNase III endonuclease, Dicer, is required for the processing of pre-miRNAs into mature miRNAs. To investigate the effect of global miRNA depletion on mast cells in vivo, we generated a mast-cell-specific knock out of Dicer in mice. Transgenic mice (Mcpt5-Cre) that express Cre selectively in connective tissue mast cells were crossed with mice carrying the floxed conditional Dicer allele (Dicer fl/fl). Mcpt5-Cre × Dicer fl/fl mice with homozygous Dicer gene deletion in mast cells were found to have a profound mast-cell deficiency with near complete loss of peritoneal, gastrointestinal, and skin mast cells. We examined the in vivo functional consequence of mast-cell-specific Dicer deletion using an immunoglobulin-E-dependent passive systemic anaphylaxis murine model. Immunoglobulin-E-sensitized wild type Mcpt5-Cre × Dicer +/+ and heterozygous Mcpt5-Cre × Dicer fl/+ mice show marked hypothermia with antigen; however, homozygous Mcpt5-Cre × Dicer fl/fl mice were completely unresponsive to antigen challenge. These studies suggest a critical role for Dicer and miRNA expression for establishment of tissue compartments of functional mast cells in vivo. MicroRNAs (miRNAs) are small, noncoding RNAs that have been shown to play a critical role in normal physiology and disease, such as hematopoietic development and cancer. However, their role in mast-cell function and development is poorly understood. The major objective of this study was to determine how global miRNA expression affects mast-cell physiology. The RNase III endonuclease, Dicer, is required for the processing of pre-miRNAs into mature miRNAs. To investigate the effect of global miRNA depletion on mast cells in vivo, we generated a mast-cell-specific knock out of Dicer in mice. Transgenic mice (Mcpt5-Cre) that express Cre selectively in connective tissue mast cells were crossed with mice carrying the floxed conditional Dicer allele (Dicer fl/fl). Mcpt5-Cre × Dicer fl/fl mice with homozygous Dicer gene deletion in mast cells were found to have a profound mast-cell deficiency with near complete loss of peritoneal, gastrointestinal, and skin mast cells. We examined the in vivo functional consequence of mast-cell-specific Dicer deletion using an immunoglobulin-E-dependent passive systemic anaphylaxis murine model. Immunoglobulin-E-sensitized wild type Mcpt5-Cre × Dicer +/+ and heterozygous Mcpt5-Cre × Dicer fl/+ mice show marked hypothermia with antigen; however, homozygous Mcpt5-Cre × Dicer fl/fl mice were completely unresponsive to antigen challenge. These studies suggest a critical role for Dicer and miRNA expression for establishment of tissue compartments of functional mast cells in vivo. Mast cells are critical effectors of allergic and inflammatory responses [1Galli S.J. Tsai M. IgE and mast cells in allergic disease.Nat Med. 2012; 18: 693-704Crossref PubMed Scopus (1104) Google Scholar]. They also participate in normal innate immune responses to bacteria and parasites [2Galli S.J. Nakae S. Tsai M. Mast cells in the development of adaptive immune responses.Nat Immunol. 2005; 6: 135-142Crossref PubMed Scopus (1019) Google Scholar]. Dysregulated proliferation of mast cells manifests in diseases that range from benign cutaneous mastocytosis to mast cell leukemia [3Akin C. Mast cell activation disorders.J Allergy Clin Immunol Pract. 2014; 2: 252-257Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar]. They are derived from hematopoietic stem cells in the bone marrow, but they migrate and reside in the connective tissue of skin, lung, and gastrointestinal tract mucosa. Normal mast-cell development is dependent on a network of transcription factors that coordinate the expression of critical gene targets [4Takemoto C.M. Lee Y.N. Jegga A.G. et al.Mast cell transcriptional networks.Blood Cells Mol Dis. 2008; 41: 82-90Crossref PubMed Scopus (28) Google Scholar]. The role of epigenetic regulators of gene expression, such as microRNAs (miRNAs), in mast cells has not been extensively studied.MicroRNAs are small, noncoding RNA nucleotides, about 18 to 24 base pairs in size, and are expressed in a tissue-specific and developmentally regulated fashion. They function primarily as negative regulators of protein expression. The RNase III endonuclease, Dicer, is necessary for mature, double-stranded miRNAs. Inhibition of Dicer by RNA interference or gene targeting results in global depletion of miRNA expression [5Harfe B.D. McManus M.T. Mansfield J.H. Hornstein E. Tabin C.J. The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb.Proc Natl Acad Sci U S A. 2005; 102: 10898-10903Crossref PubMed Scopus (567) Google Scholar, 6Kumar M.S. Lu J. Mercer K.L. Golub T.R. Jacks T. Impaired microRNA processing enhances cellular transformation and tumorigenesis.Nat Genet. 2007; 39: 673-677Crossref PubMed Scopus (1237) Google Scholar]. MicroRNA profiling experiments have identified the expression pattern of miRNAs in bone-marrow-derived mast cells (BMMCs) during development [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, 8Monticelli S. Ansel K.M. Lee D.U. Rao A. Regulation of gene expression in mast cells: micro-rNA expression and chromatin structural analysis of cytokine genes.Novartis Found Symp. 2005; 271: 179-187Crossref PubMed Google Scholar]. Our group previously identified the miR-381 and miR-539 cluster that regulates microphthalmia-associated transcription factor (Mitf) expression in response to c-Kit signaling [9Lee Y.N. Brandal S. Noel P. et al.KIT signaling regulates MITF expression through miRNAs in normal and malignant mast cell proliferation.Blood. 2011; 117: 3629-3640Crossref PubMed Scopus (55) Google Scholar]. Other investigators have shown roles for miRNAs in cell cycle regulation and proliferation, as well as apoptosis and degranulation [10Mayoral R.J. Pipkin M.E. Pachkov M. van Nimwegen E. Rao A. Monticelli S. MicroRNA-221-222 regulate the cell cycle in mast cells.J Immunol. 2009; 182: 433-445Crossref PubMed Scopus (79) Google Scholar, 11Yamada Y. Kosaka K. Miyazawa T. Kurata-Miura K. Yoshida T. miR-142-3p enhances FcepsilonRI-mediated degranulation in mast cells.Biochem Biophys Res Commun. 2014; 443: 980-986Crossref PubMed Scopus (38) Google Scholar, 12Molnar V. Ersek B. Wiener Z. et al.MicroRNA-132 targets HB-EGF upon IgE-mediated activation in murine and human mast cells.Cell Mol Life Sci. 2012; 69: 793-808Crossref PubMed Scopus (30) Google Scholar, 13Rusca N. Deho L. Montagner S. et al.MiR-146a and NF-kappaB1 regulate mast cell survival and T lymphocyte differentiation.Mol Cell Biol. 2012; 32: 4432-4444Crossref PubMed Scopus (56) Google Scholar]. Global depletion of miRNAs through deletion of Dicer function has been demonstrated to have critical roles in normal differentiation and function of myeloid cells such as neutrophils, macrophages, and dendritic cells [14Alemdehy M.F. van Boxtel N.G. de Looper H.W. 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, 15Kuipers H. Schnorfeil F.M. Fehling H.J. Bartels H. Brocker T. Dicer-dependent microRNAs control maturation, function, and maintenance of Langerhans cells in vivo.J Immunol. 2010; 185: 400-409Crossref PubMed Scopus (64) Google Scholar, 16Sissons J.R. Peschon J.J. Schmitz F. Suen R. Gilchrist M. Aderem A. Cutting edge: microRNA regulation of macrophage fusion into multinucleated giant cells.J Immunol. 2012; 189: 23-27Crossref PubMed Scopus (19) Google Scholar]. The role of global depletion of miRNA in mast cells is, to our knowledge, yet unreported. To address this question, we generated mice with a mast-cell-selective deletion of Dicer by crossing the Mcpt5-Cre strain with Dicer fl/fl strain and found a profound loss of tissue mast cells. These studies demonstrate a critical role for miRNA expression in mast-cell development.Materials and methodsAnimalsMcpt5-Cre mice were provided by Dr. Axel Roers from Dresden, Germany [17Scholten J. Hartmann K. Gerbaulet A. et al.Mast cell-specific Cre/loxP-mediated recombination in vivo.Transgenic Res. 2008; 17: 307-315Crossref PubMed Scopus (148) Google Scholar]. These mice harbor a transgene in which the coding region of the first exon of the Mcpt5 gene is replaced with a Cre-encoding cDNA; Cre is expressed in the connective tissue mast cells of skin and peritoneum [17Scholten J. Hartmann K. Gerbaulet A. et al.Mast cell-specific Cre/loxP-mediated recombination in vivo.Transgenic Res. 2008; 17: 307-315Crossref PubMed Scopus (148) Google Scholar, 18Dudeck A. Dudeck J. Scholten J. et al.Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens.Immunity. 2011; 34: 973-984Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar]. Dicer fl/fl mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and previously described [5Harfe B.D. McManus M.T. Mansfield J.H. Hornstein E. Tabin C.J. The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb.Proc Natl Acad Sci U S A. 2005; 102: 10898-10903Crossref PubMed Scopus (567) Google Scholar]. These two strains are on the C57BL/6 background. Six-to twelve-week-old Mcpt5-Cre × Dicer fl/fl mice were used to obtain splenocytes and bone marrow and were also used for the anaphylaxis experiments. Mice were maintained in the Johns Hopkins University Animal Facilities (Baltimore, MD) in strict accordance with institutional guidelines. All experiments were approved by the Johns Hopkins University Animal Care and Use Committee.Passive systemic anaphylaxis and active systemic anaphylaxisThe passive systemic anaphylaxis (PSA) and active systemic anaphylaxis (ASA) experiments were previously described [19Zhou L. Oh S.Y. Zhou Y. et al.SHP-1 regulation of mast cell function in allergic inflammation and anaphylaxis.PLoS One. 2013; 8: e55763Crossref PubMed Scopus (14) Google Scholar]. For PSA, mice were given 10 μg of anti-dinitrophenyl immunoglobulin E (IgE) and were challenged 24 hours later with 1 mg of dinitrophenylated-human serum albumin (Sigma-Aldrich, St. Louis, MO) intravenously. For ASA, mice were immunized by intraperitoneal injection of 50 μg ovalbumin (OVA) mixed with 1 mg Alum and challenged 2 weeks later by 1 mg OVA intravenously. For both PSA and ASA models, body temperature and clinical scores, including survival, were recorded every 10 min up to 90 min after challenge. The t test was used for analysis of body temperature change and clinical scores. The log-rank (Mantel-Cox) test (X2) was used for survival analysis. At the completion of the PSA and ASA experiments, mice were euthanized with a combination of ketamine/xylazine (400 mg/40 mg/kg) given intraperitoneally.Quantification of tissue mast cellsIndicated tissues from mice of different genotypes were harvested, fixed in 10% buffered formalin, sectioned, and stained with 0.5% toluidine blue (Sigma-Aldrich). For each sample of mast cells, as identified by the presence of metachromatic granules stained by toluidine blue, 5 to 10 fields were counted under 50X magnification. Average numbers of mast cells in a given field are represented. For identification of mast cells in the peritoneal cavity, peritoneal fluid was obtained by lavage, cytospined, and stained with Wright Giemsa. Total cells and mast-cell percentage were determined by counting cells from 5 to 10 fields under 50X magnification.Results and discussionMast-cell-specific deletion of Dicer results in loss of tissue mast cellsMice with homozygous mast-cell deletion of Dicer (Mcpt5-Cre/Dicer fl/fl, herein referred to as MC-Dicer −/−) and heterozygous deletion of Dicer (MC-Dicer +/−) were viable, healthy, and indistinguishable from wild type (Mcpt5-Cre/Dicer +/+) mice. However, there was a striking reduction of peritoneal mast cells in MC-Dicer −/− mice compared with wild type mice. In addition, mast cells in the skin and submucosa of the stomach were profoundly reduced compared with tissues of wild type mice (Fig. 1). Other tissues, including lung, small intestine, submucosa of stomach, and colon, were also examined. From both wild type and MC-Dicer −/− mice, these tissues had almost no mast cells detectable by toluidine blue staining. The expression of Cre in the Mcpt5-Cre mice is restricted to mast cells; other hematopoietic cell types, including basophils, are not affected (Supplementary Figure E1, online only, available at www.exphem.org).MC-Dicer −/− mice are resistant to anaphylaxisWe examined the in vivo functional consequence of mast-cell-specific Dicer deletion using an OVA-allergen-induced ASA murine model [19Zhou L. Oh S.Y. Zhou Y. et al.SHP-1 regulation of mast cell function in allergic inflammation and anaphylaxis.PLoS One. 2013; 8: e55763Crossref PubMed Scopus (14) Google Scholar]. As shown in Figures 2A and 2B, wild type mice showed clear anaphylactic responses, including significant drop in body temperature, severe clinical symptoms, and death. However, MC-Dicer −/− mice showed only mild changes in body temperature, mild clinical symptoms, and no death. Of 10 control wild type mice, 5 did not survive to completion of the experiment, compared with 0 of 7 MC-Dicer −/− mice (p = 0.034). There was also a statistically significance difference in clinical symptoms (score 3.7 in WT versus 1.0 in MC-Dicer −/−, p = 0.0003).Figure 2Mast cell deletion of Dicer protects against anaphylaxis. (A) MC-Dicer −/− mice are protected against active systemic anaphylaxis. MC-Dicer −/− mice show blunted drop in body temperature after challenge with antigen compared with wild type mice. Wild type (n = 10); MC-Dicer −/− (n = 7). (B) Increased survival of MC-Dicer −/− mice in active systemic anaphylaxis model. Half of wild type mice do not survive after antigen challenge, whereas all MC-Dicer −/− mice survive. (C) MC-Dicer −/− mice are resistant to passive systemic anaphylaxis. MC-Dicer −/− mice show no drop in body temperature with antigen challenge, whereas wild type mice show significant decrease in body temperature. Wild type (n = 4); MC-Dicer −/− (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)We also examined-mast cell-specific Dicer deletion in the PSA murine model, which is dependent on mast cells [19Zhou L. Oh S.Y. Zhou Y. et al.SHP-1 regulation of mast cell function in allergic inflammation and anaphylaxis.PLoS One. 2013; 8: e55763Crossref PubMed Scopus (14) Google Scholar, 20Oka T. Rios E.J. Tsai M. Kalesnikoff J. Galli S.J. Rapid desensitization induces internalization of antigen-specific IgE on mouse mast cells.J Allergy Clin Immunol. 2013; 132: 922-932Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar]. As shown in Figure 2C, IgE-sensitized wild type mice showed symptoms of anaphylaxis, as evidenced by marked hypothermia with antigen; however, MC-Dicer −/− mice were completely protected against systemic anaphylaxis. These results indicate that, in the absence of Dicer function, mast cells do not mature normally; mast-cell-dependent allergic responses require connective tissue mast cells rather than mucosal mast cells. The mild responses of MC-Dicer −/− mice in the ASA model may be mediated by basophils and macrophages, which would not be expected to be affected in the Mcpt5-Cre model [21Reber L.L. Marichal T. Mukai K. et al.Selective ablation of mast cells or basophils reduces peanut-induced anaphylaxis in mice.J Allergy Clin Immunol. 2013; 132: 881-888Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar].Taken together, these studies suggest a critical role for Dicer and miRNA expression for establishment of tissue compartments of connective tissue mast cells in vivo. In our model, Dicer is deleted in committed mast cell precursors, with expression of the protease, Mcpt-5. The absence of connective tissue mast cells may be due to the requirement for Dicer in differentiation, migration, survival, or proliferation. However, this central question of how Dicer and microRNAs regulate the population of tissue mast cell compartments is yet to be addressed.A challenge to further examining the mechanism of Dicer deletion on mast cell function and development is the lack of tissue mast cells to study in our in vivo model. Bone-marrow-derived mast cells are a useful tool to examine mast cell differentiation and function ex vivo. However, because of low expression of Cre, BMMCs from these MC-Dicer −/− mice are not a suitable model system to examine Dicer function [17Scholten J. Hartmann K. Gerbaulet A. et al.Mast cell-specific Cre/loxP-mediated recombination in vivo.Transgenic Res. 2008; 17: 307-315Crossref PubMed Scopus (148) Google Scholar, 22Gerbaulet A. Wickenhauser C. Scholten J. et al.Mast cell hyperplasia, B-cell malignancy, and intestinal inflammation in mice with conditional expression of a constitutively active kit.Blood. 2011; 117: 2012-2021Crossref PubMed Scopus (47) Google Scholar]. In BMMCs from MC-Dicer −/− mice, we found incomplete deletion of Dicer and no discernable phenotypic differences in granule expression by toluidine blue stain and c-KIT/FcεRIα expression compared with wild type BMMCs (Supplementary Figure E1, online only, available at www.exphem.org). We found that MC-Dicer −/+ mice have no significant differences in tissue mast-cell populations or susceptibility to active or systemic anaphylaxis, suggesting that subphysiologic expression of Dicer is sufficient for normal mast cell development. Further studies of the mechanisms of Dicer function in mast cells will be aided by other approaches, such as inducible systems to delete Dicer in vivo and ex vivo.In hematopoietic stem cells, Dicer has been shown to regulate the stem cell and progenitor cell pool; conditional deletion of Dicer results in increased apoptosis of this compartment [23Guo S. Lu J. Schlanger R. et al.MicroRNA miR-125a controls hematopoietic stem cell number.Proc Natl Acad Sci U S A. 2010; 107: 14229-14234Crossref PubMed Scopus (269) Google Scholar]. In committed hematopoietic cells, loss of Dicer affects different physiologic functions. Dicer deletion in early myeloid progenitors (using a Cre driven by the CCAAT-enhancer-binding protein alpha promoter) results in severe reduction of macrophages and dysplastic neutrophils [14Alemdehy M.F. van Boxtel N.G. de Looper H.W. 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]. Using a different model with a Lyz2-Cre strain, Dicer deletion resulted in an increase in giant cell formation of macrophages [16Sissons J.R. Peschon J.J. Schmitz F. Suen R. Gilchrist M. Aderem A. Cutting edge: microRNA regulation of macrophage fusion into multinucleated giant cells.J Immunol. 2012; 189: 23-27Crossref PubMed Scopus (19) Google Scholar]. In both T and B lymphocytes, Dicer deletion can result in aberrations in differentiation or activation, depending the cell type and stage of development in which Dicer function is inhibited [24Devasthanam A.S. Tomasi T.B. Dicer in immune cell development and function.Immunol Invest. 2014; 43: 182-195Crossref PubMed Scopus (20) Google Scholar]. In our study, loss of Dicer function in committed mast-cell progenitors resulted in profound loss of tissue mast cell compartments, suggesting a critical role for miRNAs in mast-cell differentiation, growth, or migration. Further studies are needed to determine the essential miRNAs that mediate these functions and their potential impact on allergic and inflammatory diseases. Mast cells are critical effectors of allergic and inflammatory responses [1Galli S.J. Tsai M. IgE and mast cells in allergic disease.Nat Med. 2012; 18: 693-704Crossref PubMed Scopus (1104) Google Scholar]. They also participate in normal innate immune responses to bacteria and parasites [2Galli S.J. Nakae S. Tsai M. Mast cells in the development of adaptive immune responses.Nat Immunol. 2005; 6: 135-142Crossref PubMed Scopus (1019) Google Scholar]. Dysregulated proliferation of mast cells manifests in diseases that range from benign cutaneous mastocytosis to mast cell leukemia [3Akin C. Mast cell activation disorders.J Allergy Clin Immunol Pract. 2014; 2: 252-257Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar]. They are derived from hematopoietic stem cells in the bone marrow, but they migrate and reside in the connective tissue of skin, lung, and gastrointestinal tract mucosa. Normal mast-cell development is dependent on a network of transcription factors that coordinate the expression of critical gene targets [4Takemoto C.M. Lee Y.N. Jegga A.G. et al.Mast cell transcriptional networks.Blood Cells Mol Dis. 2008; 41: 82-90Crossref PubMed Scopus (28) Google Scholar]. The role of epigenetic regulators of gene expression, such as microRNAs (miRNAs), in mast cells has not been extensively studied. MicroRNAs are small, noncoding RNA nucleotides, about 18 to 24 base pairs in size, and are expressed in a tissue-specific and developmentally regulated fashion. They function primarily as negative regulators of protein expression. The RNase III endonuclease, Dicer, is necessary for mature, double-stranded miRNAs. Inhibition of Dicer by RNA interference or gene targeting results in global depletion of miRNA expression [5Harfe B.D. McManus M.T. Mansfield J.H. Hornstein E. Tabin C.J. The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb.Proc Natl Acad Sci U S A. 2005; 102: 10898-10903Crossref PubMed Scopus (567) Google Scholar, 6Kumar M.S. Lu J. Mercer K.L. Golub T.R. Jacks T. Impaired microRNA processing enhances cellular transformation and tumorigenesis.Nat Genet. 2007; 39: 673-677Crossref PubMed Scopus (1237) Google Scholar]. MicroRNA profiling experiments have identified the expression pattern of miRNAs in bone-marrow-derived mast cells (BMMCs) during development [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, 8Monticelli S. Ansel K.M. Lee D.U. Rao A. Regulation of gene expression in mast cells: micro-rNA expression and chromatin structural analysis of cytokine genes.Novartis Found Symp. 2005; 271: 179-187Crossref PubMed Google Scholar]. Our group previously identified the miR-381 and miR-539 cluster that regulates microphthalmia-associated transcription factor (Mitf) expression in response to c-Kit signaling [9Lee Y.N. Brandal S. Noel P. et al.KIT signaling regulates MITF expression through miRNAs in normal and malignant mast cell proliferation.Blood. 2011; 117: 3629-3640Crossref PubMed Scopus (55) Google Scholar]. Other investigators have shown roles for miRNAs in cell cycle regulation and proliferation, as well as apoptosis and degranulation [10Mayoral R.J. Pipkin M.E. Pachkov M. van Nimwegen E. Rao A. Monticelli S. MicroRNA-221-222 regulate the cell cycle in mast cells.J Immunol. 2009; 182: 433-445Crossref PubMed Scopus (79) Google Scholar, 11Yamada Y. Kosaka K. Miyazawa T. Kurata-Miura K. Yoshida T. miR-142-3p enhances FcepsilonRI-mediated degranulation in mast cells.Biochem Biophys Res Commun. 2014; 443: 980-986Crossref PubMed Scopus (38) Google Scholar, 12Molnar V. Ersek B. Wiener Z. et al.MicroRNA-132 targets HB-EGF upon IgE-mediated activation in murine and human mast cells.Cell Mol Life Sci. 2012; 69: 793-808Crossref PubMed Scopus (30) Google Scholar, 13Rusca N. Deho L. Montagner S. et al.MiR-146a and NF-kappaB1 regulate mast cell survival and T lymphocyte differentiation.Mol Cell Biol. 2012; 32: 4432-4444Crossref PubMed Scopus (56) Google Scholar]. Global depletion of miRNAs through deletion of Dicer function has been demonstrated to have critical roles in normal differentiation and function of myeloid cells such as neutrophils, macrophages, and dendritic cells [14Alemdehy M.F. van Boxtel N.G. de Looper H.W. 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, 15Kuipers H. Schnorfeil F.M. Fehling H.J. Bartels H. Brocker T. Dicer-dependent microRNAs control maturation, function, and maintenance of Langerhans cells in vivo.J Immunol. 2010; 185: 400-409Crossref PubMed Scopus (64) Google Scholar, 16Sissons J.R. Peschon J.J. Schmitz F. Suen R. Gilchrist M. Aderem A. Cutting edge: microRNA regulation of macrophage fusion into multinucleated giant cells.J Immunol. 2012; 189: 23-27Crossref PubMed Scopus (19) Google Scholar]. The role of global depletion of miRNA in mast cells is, to our knowledge, yet unreported. To address this question, we generated mice with a mast-cell-selective deletion of Dicer by crossing the Mcpt5-Cre strain with Dicer fl/fl strain and found a profound loss of tissue mast cells. These studies demonstrate a critical role for miRNA expression in mast-cell development. Materials and methodsAnimalsMcpt5-Cre mice were provided by Dr. Axel Roers from Dresden, Germany [17Scholten J. Hartmann K. Gerbaulet A. et al.Mast cell-specific Cre/loxP-mediated recombination in vivo.Transgenic Res. 2008; 17: 307-315Crossref PubMed Scopus (148) Google Scholar]. These mice harbor a transgene in which the coding region of the first exon of the Mcpt5 gene is replaced with a Cre-encoding cDNA; Cre is expressed in the connective tissue mast cells of skin and peritoneum [17Scholten J. Hartmann K. Gerbaulet A. et al.Mast cell-specific Cre/loxP-mediated recombination in vivo.Transgenic Res. 2008; 17: 307-315Crossref PubMed Scopus (148) Google Scholar, 18Dudeck A. Dudeck J. Scholten J. et al.Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens.Immunity. 2011; 34: 973-984Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar]. Dicer fl/fl mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and previously described [5Harfe B.D. McManus M.T. Mansfield J.H. Hornstein E. Tabin C.J. The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb.Proc Natl Acad Sci U S A. 2005; 102: 10898-10903Crossref PubMed Scopus (567) Google Scholar]. These two strains are on the C57BL/6 background. Six-to twelve-week-old Mcpt5-Cre × Dicer fl/fl mice were used to obtain splenocytes and bone marrow and were also used for the anaphylaxis experiments. Mice were maintained in the Johns Hopkins University Animal Facilities (Baltimore, MD) in strict accordance with institutional guidelines. All experiments were approved by the Johns Hopkins University Animal Care and Use Committee.Passive systemic anaphylaxis and active systemic anaphylaxisThe passive systemic anaphylaxis (PSA) and active systemic anaphylaxis (ASA) experiments were previously described [19Zhou L. Oh S.Y. Zhou Y. et al.SHP-1 regulation of mast cell function in allergic inflammation and anaphylaxis.PLoS One. 2013; 8: e55763Crossref PubMed Scopus (14) Google Scholar]. For PSA, mice were given 10 μg of anti-dinitrophenyl immunoglobulin E (IgE) and were challenged 24 hours later with 1 mg of dinitrophenylated-human serum albumin (Sigma-Aldrich, St. Louis, MO) intravenously. For ASA, mice were immunized by intraperitoneal injection of 50 μg ovalbumin (OVA) mixed with 1 mg Alum and challenged 2 weeks later by 1 mg OVA intravenously. For both PSA and ASA models, body temperature and clinical scores, including survival, were recorded every 10 min up to 90 min after challenge. The t test was used for analysis of body temperature change and clinical scores. The log-rank (Mantel-Cox) test (X2) was used for survival analysis. At the completion of the PSA and ASA experiments, mice were euthanized with a combination of ketamine/xylazine (400 mg/40 mg/kg) given intraperitoneally.Quantification of tissue mast cellsIndicated tissues from mice of different genotypes were harvested, fixed in 10% buffered formalin, sectioned, and stained with 0.5% toluidine blue (Sigma-Aldrich). For each sample of mast cells, as identified by the presence of metachromatic granules stained by toluidine blue, 5 to 10 fields were counted under 50X magnification. Average numbers of mast cells in a given field are represented. For identification of mast cells in the peritoneal cavity, peritoneal fluid was obtained by lavage, cytospined, and stained with Wright Giemsa. Total cells and mast-cell percentage were determined by counting cells from 5 to 10 fields under 50X magnification. AnimalsMcpt5-Cre mice were provided by Dr. Axel Roers from Dresden, Germany [17Scholten J. Hartmann K. Gerbaulet A. et al.Mast cell-specific Cre/loxP-mediated recombination in vivo.Transgenic Res. 2008; 17: 307-315Crossref PubMed Scopus (148) Google Scholar]. These mice harbor a transgene in which the coding region of the first exon of the Mcpt5 gene is replaced with a Cre-encoding cDNA; Cre is expressed in the connective tissue mast cells of skin and peritoneum [17Scholten J. Hartmann K. Gerbau
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