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The Neurobeachin-Like 2 Protein Regulates Mast Cell Homeostasis

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The Neurobeachin-Like 2 Protein Regulates Mast Cell Homeostasis Published September 8, 2017, doi:10.4049/jimmunol.1700556 The Journal of Immunology The Neurobeachin-like 2 Protein Regulates Mast Cell Homeostasis Sebastian Drube,* Randy Grimlowski,* Carsten Deppermann,†,1 Julia Fro¨bel,‡ Florian Kraft,*,2 Nico Andreas,* David Stegner,† Jan Dudeck,‡ Franziska Weber,* Mandy Ro¨diger,* Christiane Go¨pfert,* Julia Drube,x Daniela Reich,x Bernhard Nieswandt,† Anne Dudeck,‡ and Thomas Kamradt* The neurobeachin-like 2 protein (Nbeal2) belongs to the family of beige and Chediak–Higashi (BEACH) domain proteins. Loss-of- function mutations in the human NBEAL2 gene or Nbeal2 deficiency in mice cause gray platelet syndrome, a bleeding disorder characterized by macrothrombocytopenia, splenomegaly, and paucity of a-granules in megakaryocytes and platelets. We found that in mast cells, Nbeal2 regulates the activation of the Shp1-STAT5 signaling axis and the composition of the c-Kit/STAT signalosome. Furthermore, Nbeal2 mediates granule formation and restricts the expression of the transcription factors, IRF8, GATA2, and MITF as well as of the cell-cycle inhibitor p27, which are essential for mast cell differentiation, proliferation, and cytokine production. These data demonstrate the relevance of Nbeal2 in mast cells above and beyond granule biosynthesis. The Journal of Immunology, 2017, 199: 000–000. he beige and Chediak–Higashi (BEACH) family members, platelets (7), and splenomegaly (8). Nbeal2 is highly expressed in such as neurobeachin-like 2 protein (Nbeal2), contain a cells of the hematopoietic system (4). However, alterations in T BEACH domain, a concavalin A-like lectin (ConA) do- immune reactions have not been studied in gray platelet syndrome main, WD40 domains, and pleckstrin homology domains (1). patients. Mast cells contain granules and are typically activated by BEACH domains are crucial for vesicle transport (1) and fusion of FcεRI cross-linking (9). FcεRI stimulation leads to NFAT activa- vesicles with the cell membrane. ConA domains bind glycosylated tion, degranulation, the release of cytokines, chemokines, hista- proteins and are therefore important for protein sorting and secretion mines, and proteases (10). Mast cells are effector cells of type I (1, 2), WD40 domains mediate protein–protein interaction (1) whereas hypersensitivity, and are central to the pathogenesis of allergic pleckstrin homology domains interact with 4,5-bisphosphoinositol diseases (11). Differentiation of mast cells depends on c-Kit and (3). In humans, loss-of-function mutations of NBEAL2 causes the IL-3R (12). Both receptors stimulate similar signaling path- gray platelet syndrome (4–6), which is characterized by macro- ways (e.g., STAT5 and PI3Ks), which are involved in mast cell thrombocytopenia, a paucity of a-granules in megakaryocytes and proliferation and/or differentiation (13–15). Mast cells also ex- press members of the TLR/IL-1 receptor family including IL-33R (16, 17), which mediates NF-kB–dependent (18) cytokine pro- *Institute of Immunology, Jena University Hospital, 07743 Jena, Germany; duction but not degranulation (19). Given that mast cell effector † Institute of Experimental Biomedicine, University Hospital and Rudolf Virchow functions depend on granules, and Nbeal2 is involved in granule Center for Experimental Biomedicine, University Wurzburg,€ 97080 Wurzburg,€ Ger- many; ‡Medical Faculty, Institute for Molecular and Clinical Immunology, 39120 biogenesis, we determined the functional relevance of Nbeal2 in Magdeburg, Germany; and xCenter for Molecular Biomedicine, University Hospital mast cells. Jena, 07745 Jena, Germany 1Current address: The Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada. Materials and Methods Mice 2Current address: Medical Faculty, Institute of Human Genetics, Rheinisch-Westfa¨lische Technische Hochschule Aachen University, Aachen, Germany. We used sex- and age-matched (8–10 wk old) Nbeal22/2 mice (20) and ORCIDs: 0000-0003-3740-8635 (C.D.); 0000-0002-4958-6343 (J.F.); 0000-0002- wild type (wt) littermates. 5324-9155 (F.K.); 0000-0002-4591-7037 (N.A.); 0000-0003-1059-9865 (D.S.); 0000-0002-1311-9620 (A.D.). Genotyping PCR and quantitative PCR Received for publication April 19, 2017. Accepted for publication August 21, 2017. For genotyping PCR we used the forward (fw)-primer 59-GTCCT- S.D. developed the concept, designed the research, performed experiments, analyzed GCTTGACCTACCGTC-39, and the reverse (rw)-primers 59-CAGGGAGGA- data, made the figures, and drafted and wrote the paper; R.G., C.D., J.F., D.S., N.A., TAACGAGATAGTCTT-39 (rw-primer 1), 59-CCTAGGAATGCTCGTCAAGA- F.W., C.G., and F.K. performed experiments and analyzed data; J. Dudeck., M.R., 39 (IRES-GT-Primer). Nbeal22/2 mice were generated by targeting the J. Drube, and D.R. performed experiments; B.N. provided samples and reagents, exons 4 to 11 of the Nbeal2 gene. The exons 4–11 were replaced by an and edited the manuscript; A.D. designed the research, performed experiments, and IRES element containing a selection cassette. The wt PCR with the fw- analyzed data; and T.K. wrote and edited the manuscript. primer and the rw-primer 1 generates a 223 bp product (wt product). In Address correspondence and reprint requests to Dr. Sebastian Drube, Institute of contrast, the mutant PCR with the fw-primer and the IRES-GT-Primer Immunology, Jena University Hospital, Leutragraben 3, 07743 Jena, Germany. generates a 401 bp product (mutant product). E-mail address: [email protected] For RT-PCR experiments, total RNAwas isolated by using the peqGOLD The online version of this article contains supplemental material. TriFast kit (PEQLAB). The RNA was transcribed into cDNA by using the Abbreviations used in this article: BMMC, bone marrow–derived mast cell; ConA, first-strand cDNA synthesis kit (Thermo Fisher Scientific) and random concavalin A-like lectin; fw, forward; Nbeal2, neurobeachin-like 2; rw, reverse; SCF, hexamer primers. Subsequently, cDNA was subjected to quantitative PCR stem cell factor; SI, stimulation index; wt, wild type. by using the Maxima SYBR Green/ROX qPCR Master mix (Thermo Fisher Scientific). We used the fw-Nbeal2 primer 59-TGTGAAGGGCTCTTT- Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 GACCC-39 and the rw-Nbeal2 primer 59-GGCCGGAGGGAACTTG- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700556 2 Nbeal2 REGULATES MAST CELL HOMEOSTASIS TATT-39 (both Sigma-Aldrich). For housekeeping genes we used the b2 membranes (Biostep) by Western blotting. Membranes were blocked with microglobulin (fw-primer 59-CTGACCGGCCTGTATGCTATC-39 and rw- dry milk and incubated with Abs detecting phosphorylated or non- primer: 59-TGCAGTCCCGCATAGTTGAA-39, both Sigma-Aldrich). The phosphorylated proteins. We used anti–pY719-c-Kit, anti–pY694-STAT5, quantitative PCR was performed by using ROCHE LightCycler 480. anti-STAT5, anti–pS463-p65, anti-p65, anti-pY525/pY526-Syk, anti-Syk, anti–pY783-PLCg1, anti–pY191-Lat1, anti–pY171-Lat1, anti-Lat1, anti– Passive systemic anaphylaxis pS176/pS180-IKK2, anti–pY705-STAT3, anti-STAT3, anti-MKK7, anti- MITF, anti-GATA2, anti-IRF8, anti-lamin, and anti-CD107a (all from Mice were treated intravenously with 3 mg rat anti-DNP IgE (D8406; Cell Signaling). Furthermore, we used anti–c-Kit, anti-STAT3a,anti- Sigma-Aldrich) in a total volume of 100 ml PBS. After 20 h the body STAT5a, anti-STAT5b, anti-p27, anti–pY536-Shp1, anti-PLCg1, anti- temperature was measured rectally. HSA-DNP (A6661; Sigma-Aldrich) IKK2 (all Santa Cruz), anti-pNFAT, anti-tubulin (Sigma-Aldrich), and (250 mg/100 ml PBS) was injected to induce anaphylaxis and body tem- anti-Nbeal2 (Thermo Fisher Scientific) Abs. Membranes were washed in perature was measured rectally. All animal experiments were approved by 0.1% Tween/TBS and incubated with HRP-conjugated secondary Abs: the appropriate institutional and governmental committees for animal anti–rabbit-Ig, anti–goat-Ig (both Santa Cruz), and anti–mouse-Ig welfare. (Thermo Fisher Scientific). Detection was performed using ECL re- Cell culture agent (Pierce). For generation of bone marrow–derived mast cells (BMMCs), bone marrow ELISAs 2/2 was obtained from the femurs and tibias of wt and Nbeal2 mice. Bone 6 marrow cells were cultured in IMDM (PAA) supplemented with 10% FCS, BMMCs (10 cells per ml) were IL-3 starved, primed with SPE-7 (1 100 U/ml penicillin, 100 mg/ml streptomycin, 50 mM 2-ME, and 20 ng/ml mg/ml) overnight, and stimulated with HSA-DNP (both Sigma-Aldrich). IL-3 (conditioned media from WEHI-3 cells). In the first week of BMMC Furthermore, we stimulated with PMA and/or ionomycin (Sigma-Aldrich). generation, the medium was changed every second day and the adherent Supernatants were analyzed by cytokine ELISAs with matched-paired Abs cells were discarded. In weeks 2 until 4 of culture, the medium was (BioLegend) or by the histamine ELISA (Histamine FAST ELISA; DRG, changed twice a week. After weeks 4 and 5, cell culture consisted of 95% Springfield, NJ) according to the experimental procedures. For determi- BMMCs (identified by FcεRI and c-Kit). BMMCs were used for 4–6 wk nation of the serum TNF-a and IL-6, we used ProcartaPlex Simplex Kit, after differentiation. High Sensitivity in combination with the ProcartaPlex Mouse High Sen- sitivity Basic Kit (Invitrogen) according to the experimental procedures. Flow cytometry Serum histamine was determined by using the Histamine FAST ELISA (DRG) according to the experimental procedures. For analysis of the in vitro cultures, cells were blocked with anti-CD16/ CD32 (clone 2.4G2) and rat-IgG (Jackson ImmunoResearch) and stained Cell cycle analysis with PE-conjugated anti-murine c-Kit and FITC-conjugated anti-murine FcεRI (all BioLegend). For determination of mast cells in vivo, cells BMMC were stimulated with IL-3 (20 ng/ml) (24 h), and were fixed in 70% 2 were blocked with anti-CD16/CD32 (clone 2.4G2) and rat-IgG (Jackson ethanol (3 h; 20˚C). Subsequently, cells were stained with a solution ImmunoResearch) and stained with FITC-conjugated anti-murine containing 2.5 mg/ml propidium iodide, 0.1 mg/ml RNase A, and 0.05% IL-33R (MD Bioproducts) and PE-conjugated anti-murine c-Kit (Bio- Triton X-100 (30 min).
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