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 express 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). Cells were analyzed with the LSR II flow Legend). For stimulation experiments, BMMCs were left untreated, or cytometer (BD) and FlowJo (Tree Star). were treated with PMA/ionomycin (Sigma-Aldrich), or primed with anti-DNP IgE (clone: SPE-7) (Sigma-Aldrich) overnight, and stimulated Proliferation assays with HSA-DNP (Sigma-Aldrich). Subsequently, BMMCs were blocked BMMCs (106 cells per ml) were IL-3 starved (1 h), stimulated with IL-3 or with anti-CD16/CD32 (clone 2.4G2) and rat-IgG (Jackson Immuno- SCF (both PeproTech), and cultured for further 54 h. Then [3H]-thymidine Research) and stained with PacBlue-conjugated anti-murine CD107a, (1 mCi) in 25 ml complete IMDM (PAA) (without IL-3) was added to each PE-conjugated anti-murine c-Kit, or FITC-conjugated anti-murine well for an additional 18 h. Incorporated radioactivity was measured by ε Fc RI (all BioLegend). For intracellular cytokine detection, BMMCs using a b-scintillation counter (PerkinElmer). Shown is the stimulation 3 6 (4 10 cells per ml) were left untreated or were treated with brefeldin index (SI), where the cpm values of wt BMMCs were set as one. The cpm A and stimulated with PMA/ionomycin. Subsequently, cells were fixed values of stimulated BMMCs were the fold induction (SI) compared with in 2% formaldehyde in PBS at room temperature for 20 min. For cy- unstimulated wt BMMCs. tokine detection, fixed BMMCs were stained intracellularly with anti- CD117, and V450-conjugated anti-murine TNF-a (eBioscience) in 0.5% Transmission electron microscopy saponin, 0.5% BSA, and 2 mM EDTA. In all experiments cells were analyzed using an LSR II flow cytometer (BD) and FlowJo 9 (Tree Star, BMMCs (5 3 104 cells per ml) were fixed with 2.5% glutaraldehyde in Ashland, OR). 0.1 M cacodylate buffer (pH 7.2) and embedded in Epon. Sections were cut using an ultramicrotome (Leica Ultracut UCT), stained with 2% uranyl Cell stimulation and lysis acetate (in 100% ethanol), lead in citrate, and examined with an EM900 transmission electron microscope (Carl Zeiss). BMMCs (106 cells per ml) were IL-3 starved (1 h), stimulated with stem cell factor (SCF) or IL-3 (all from PeproTech), or were primed overnight Histology with anti-DNP IgE (clone: SPE-7) and stimulated with HSA-DNP (both Sigma-Aldrich). In all experiments cells were lysed in buffer containing Ear pinna were fixed in 4% (v/v) buffered formalin and embedded in paraffin 20 mM HEPES (pH 7.5), 10 mM EGTA, 40 mM b-glycerophosphate, ensuring a cross-sectional orientation. For detection of mast cells, 5 mm 2.5 mM MgCl2, 2 mM orthovanadate, 1 mM DTT, 20 mg/ml aprotinin, 20 sections were stained (Giemsa). Mast cells were counted per ear skin mg/ml leupeptin, and 1% Triton. For separation of the cytoplasmatic and section over a length of 1 cm. Shown is the mean of all sections per ge- nuclear fraction we used the ProteoJet-Kit (Fermentas) according to the notype 6 SD. experimental procedures. Protein concentration was determined by using the BCA-kit (Pierce). Protein samples were then boiled in 6 3 Laemmli Western blot quantification buffer. Western blots were quantified with ImageJ (National Institutes of Health). Immunoprecipitation To quantify the expression of proteins, the intensities of protein bands were determined and normalized to the respective internal controls. To quantify Cell lysates were incubated with Abs against STAT3a,STAT5a,STAT5b, protein phosphorylations, the intensities of the phosphorylation bands were or c-Kit (all from Santa Cruz) (5 mg Ab/500 mg protein). We used mouse-, determined and normalized to the respective total protein bands. The control rabbit- or goat-IgG as nonspecific control Igs (Gentaur). Protein-G Sepharose (unstimulated cells) of wt cells was set as 1. We calculated the fold increase (Invitrogen) was added and incubated for 4–6 h. Precipitates were washed of phosphorylations compared with the unstimulated wt control. and subsequently boiled in 6 3 Laemmli buffer. Statistical analysis Western blotting Western blotting, flow cytometry, and PCR experiments were performed Samples were separated on SDS-Laemmli gels, using 8% (for detection of three times (one representative experiment is shown). Proliferation assays Nbeal2) or 10% poly acrylamide gels (for detection of phosphorylated/ and ELISAs were performed three times (thereby, one experiment includes nonphosphorylated proteins), and were transferred onto nitrocellulose the BMMCs generated from n = 3 mice per genotype and two samples per The Journal of Immunology 3 mouse). Shown is the mean 6 SD. For statistical analysis we used IBM sequence does not contain nuclear localization sequences, and we SPSS Statistics (version 20.0; IBM, Ehningen, Germany). Statistical only detected Nbeal2 in the cytoplasm but not in the nucleus of significance was assessed by unpaired Student t test. Statistical significance BMMCs (Supplemental Fig. 2A, 2B), excluding nuclear locali- was accepted for p , 0.05. zation. Next, we investigated the cell-cycle progression, and the SCF- and IL-3–induced proliferation of mature wt and Nbeal22/2 Results BMMCs. Compared to wt BMMCs, in Nbeal22/2 BMMCs we Nbeal2 mediates the expansion of BMMC in vitro found a cell-cycle arrest (Fig. 2A), and a reduced proliferation rate To characterize the role of Nbeal2 in mast cells, we used in vitro– (Fig. 2B, 2C). Thus these data indicate that Nbeal2 controls cell- generated BMMCs from wt and Nbeal22/2 mice. To determine if cycle progression and BMMC expansion in vitro. BMMCs express Nbeal2 we performed Nbeal2-specific PCRs. Nbeal2 regulates the activation of STAT3 and STAT5 Additionally, we used a polyclonal Ab, which recognizes the C terminus of Nbeal2 (Supplemental Fig. 1A). Both the Ab and the Next, we investigated the effects of Nbeal2 deficiency on the SCF- PCRsdetectedNbeal2inwtbutnotinNbeal22/2 BMMCs and IL-3–induced STAT signaling, which is critically involved in (Supplemental Fig. 1B–D). Next, we investigated the influence mast cell differentiation and proliferation (13, 14, 27). Nbeal2 of Nbeal2 in in vitro BMMC differentiation in the presence of deficiency did not influence the SCF-induced c-Kit activation but IL-3. Compared to wt bone marrow cultures, in Nbeal22/2 bone resulted in a reduced SCF- (Fig. 2D, 2E; for statistical analysis of marrow cultures the fraction of BMMCs (c-Kit+/FcεRI+)was the Western blots, see Supplemental Fig. 3A–C) and IL-3– (data increased in the first but decreased in the second week. After 3 wk, not shown) induced STAT3 and STAT5 activation. STATs are the fraction of BMMCs was similar in bone marrow cultures negatively regulated by Shp1 in mast cells (27). Therefore, we 2/2 investigated the SCF-induced activation of Shp1 in wt and from wt and Nbeal2 mice (Fig. 1A, 1B). In contrast, the total 2/2 2/2 cell numbers and absolute BMMC numbers (Fig. 1C, 1D) were Nbeal2 BMMCs. Compared to wt BMMCs, in Nbeal2 decreased in Nbeal22/2 cultures throughout the whole culture BMMCs we found a strongly increased SCF-induced Shp1 acti- period. We also tested the in vitro differentiation in the presence vation (Fig. 2F; for statistical analysis of the Western blots see of IL-3 in combination with SCF, where we found similar results Supplemental Fig. 3D), indicating that Nbeal2 negatively reg- as shown in the presence of IL-3 alone (Supplemental Fig. 1E– ulates Shp1 and thus suppresses the activation of STAT5. H). These data indicate that Nbeal2 is not critical for mast cell During these Western blot analyses we also found that Nbeal2 is differentiation but mediates the expansion of BMMC in vitro. expressed as the 300 kDa and additionally in a 200 kDa variant in Mast cell expansion is regulated by the MAPK-kinase MKK7 BMMCs (Fig. 2F). Thereby, the 200 kDa variant is destabilized in and cell-cycle inhibitors such as p27 (21). Therefore, we tested response to SCF (Fig. 2F). Together, our data show that Nbeal2 the expression of both proteins in lysates of wt and Nbeal22/2 regulates the activation of Shp1, STAT3, and STAT5, and is bone marrow cultures. As shown in Fig. 1E, Nbeal2 deficiency expressed as a 300 kDa and a yet-uncharacterized 200 kDa variant resulted in increased levels of MKK7 and p27 (for statistical in mast cells. analysis of the Western blots, see Supplemental Fig. 1I). This Nbeal2 interacts with STATs and controls the 2/2 indicates that the reduced BMMC numbers in the Nbeal2 c-Kit/STAT interaction bone marrow cultures result from the increased expression of MKK7 and p27. Because Nbeal2 mediates effective STAT activation, we hypoth- GATA2 and MITF regulate the expression of p27 (22–24). esized that Nbeal2 interacts with STATs. In STAT3 (Fig. 3A) and Furthermore, the expression of GATA2 and MITF is mediated by STAT5 (Fig. 3B) precipitates we predominantly detected the 200 IRF8 (13, 25, 26). Therefore we hypothesized that the increased kDa Nbeal2 variant indicating complex formation between these protein level of p27 results from increased levels of IRF8, GATA2, proteins. Given that Nbeal2 interacts with STATs and regulates and MITF. Indeed, the protein levels of IRF8, GATA2, and MITF their SCF-induced activation, we hypothesized that Nbeal2 defi- are increased in Nbeal22/2 compared with wt BMMCs (Fig. 1E; ciency might influence the interaction between c-Kit and STATs. for statistical analysis of the Western blots, see Supplemental Fig. Unexpectedly, we could not detect Nbeal2 in c-Kit precipitates but 2/2 confirmed that c-Kit interacts with STAT3/5 in wt BMMCs (Fig. 4). 1I). In Nbeal2 cultures the increased MITF protein level per- 2/2 sisted for 5 wk, whereas the protein levels of IRF8 and GATA2 In Nbeal2 BMMCs, the interactions between c-Kit and STAT3/5 were upregulated in the third but downregulated in the fourth and were strongly increased (Fig. 4). Together, these data show that fifth week (Fig. 1E; for statistical analysis of the Western blots, see Nbeal2 does not directly interact with c-Kit but controls the as- Supplemental Fig. 1I). These data indicate that NBeal2 negatively sembly of the c-Kit/STAT signalosome. regulates the IRF8-MITF/GATA2-p27 pathway. The role of Nbeal2 in Fc«RI-mediated signaling and Nbeal2 controls the number of mast cells in vivo cytokine response Next, we compared the mast cell numbers in wt and Nbeal22/2 Next, we investigated FcεRI-mediated signaling, the major re- mice in vivo. To do this, we investigated the peritoneal fluid and ceptor system pivotal to mediate mast cell effector functions. the ear skin. Compared to wt mice, the mast cell numbers in the FcεRI signaling leads to a Syk-dependent Lat1 phosphorylation peritoneal fluid (Fig. 1F, 1G) and in ear skin (Fig. 1H, 1I) were (28) and therefore to membrane localization and to activation of reduced in Nbeal22/2 mice. Taken together, these data indicate the PLCg1 (28–30). Furthermore, Lat1 phosphorylation is critical that Nbeal2 controls the number of mast cells in vitro and in vivo. for MAP-kinase activation (31). Compared to wt BMMCs the activation of Syk was strongly, and of the PLCg1 slightly in- Nbeal2 controls cell-cycle progression and proliferation in creased in Nbeal22/2 BMMCs (Fig. 5A; for statistical analysis of mature BMMCs the Western blots see Supplemental Fig. 3E, 3F). However, the We found that Nbeal2 deficiency results in overexpression of phosphorylation of the Syk substrate Lat1 (Fig. 5A; for statistical transcription factors and of the cell-cycle inhibitor p27 in BMMC analysis of the Western blots see Supplemental Fig. 3G, 3H), and cultures. Therefore, we speculated that Nbeal2 is a nuclear protein the activation of IKK2, p65, JNK1/2, p38, and of Erk1/2 (Fig. 5B; that controls the cell cycle and proliferation. However, the Nbeal2 for statistical analysis of the Western blots see Supplemental Fig. 4 Nbeal2 REGULATES MAST CELL HOMEOSTASIS

4A–E) was similar in wt and Nbeal22/2 BMMCs. This demon- strates that Nbeal2 controls the FcεRI-mediated Syk and PLCg1 activation in BMMCs. In platelets, Nbeal2 controls the release of cytokines (32) by regulating the biogenesis of a-granules (5, 20). Mast cell functions also depend on granules. Therefore, we investigated whether Nbeal2 mediates granule formation and thus mast cell effector functions. Flow cytometry and transmission electron microscopy showed that Nbeal22/2 BMMCs are smaller and less granular than wt BMMCs (Fig. 5C–E), but express the same amount of the FcεRI and of the IL-33R (Supplemental Fig. 4F). Next, we determined whether Nbeal2 regulates the fast mast cell response induced by the FcεRI by investigating degranulation, and the release of preformed cytokines. To trigger the FcεRI, BMMCs were primed with the DNP-speciﬁc IgE Ab, SPE-7, and were stimulated with HSA-DNP for 1 h. Afterwards, we investigated the surface expression of CD107a, which correlates with degranulation (33), and examined the release of preformed cytokines such as MCP-1 and TNF-a (34, 35), as well as the production of IL-6 and IL-13. In Nbeal22/2 BMMCs, the CD107a surface expression (Fig. 5F) and the release of MCP-1, TNF-a, and IL-6 were increased (Fig. 5G–I). In contrast, FcεRI stimulation did not induce an IL-13 release from wt or Nbeal22/2 BMMCs (Supplemental Fig. 4G), indicating that IL-13 is not stored in granules as a preformed cytokine. The increased surface expression of CD107a and the increased release of cytokines indicated enhanced degranulation, despite fewer granules in Nbeal22/2 BMMCs. To test this hypothesis, we compared the total amount of CD107a and the histamine release in wt and Nbeal22/2 BMMCs. Compared to wt BMMCs, in Nbeal22/2 BMMCs, the total amount of CD107a was strongly increased (Fig. 5J). In contrast, the histamine release was decreased in Nbeal22/2 BMMCs (Fig. 5K). Furthermore, we investigated the late mast cell response, which depends on the de novo cytokine synthesis. Therefore, we stimulated the FcεRI for 4–8 h. Nbeal2 deﬁciency resulted in an increased release of MCP-1, TNF-a, and IL-6, whereas the release of IL-13 was similar in wt an Nbeal22/2 BMMCs (Fig. 6A–D). Next, we wanted to determine whether Nbeal2 regulates the synthesis of cytokines. Thus we investigated the intracellular content of cytokines in response to stimulation with PMA/ ionomycin. Similar to the FcεRI stimulation, PMA/ionomycin treatment also induced an increased CD107a surface expression, and increased the release of TNF-a in Nbeal22/2 BMMCs (Fig. 6E, 6F). Furthermore, the intracellular content of cytokines was also increased in Nbeal22/2 BMMCs (Fig. 6G). Together, these data indicate that Nbeal2 mediates granule formation, and negatively controls the cytokine production. Nbeal2 is dispensable for anaphylactic reactions To investigate the role of Nbeal2 in IgE-mediated anaphylaxis, we sensitized mice with SPE-7 and treated them with HSA-DNP. We

FIGURE 1. Nbeal2 mediates mast cell expansion in vitro and in vivo. marrow cultures (from three mice per genotype). Shown is the mean 6 SD. (A) Bone marrow from wt and Nbeal22/2 was cultured in presence of IL-3 (E) Lysates from wt and Nbeal22/2 bone marrow cultures were analyzed (20 ng/ml). Cultures from wt and Nbeal22/2 were analyzed for CD117+/ by Western blotting at different time points of the in vitro differentiation. FcεRI+ cells by flow cytometry every week (shown is one representa- (F) Shown is the percentage of mature mast cells (CD117+/IL-33R+ tive experiment out of three). (B) Statistical analysis of in vitro BMMC /FcεRI+) in the peritoneal fluid of wt and Nbeal22/2 mice (shown is one generation. The percentage of CD117+/FcεRI+ cells in three wt and three representative experiment out of three). (G) The statistic shows mast cell Nbeal22/2 bone marrow cultures (from three mice per genotype) was de- numbers (CD117+/IL-33R+) per peritoneum. Shown is the mean 6 SD of termined by flow cytometry every week. Shown is the mean 6 SD. (C)Cells n = 3 mice per genotype. (H) The statistic shows mast cell numbers per ear from three wt and three Nbeal22/2 bone marrow cultures (from three mice section over a length of 1 cm. Shown is the mean 6 SD of all sections per per genotype) were treated with toluidine blue and viable cells were coun- genotype (n = 4 mice per genotype and four sections per mouse). (I) ted. Shown is the mean 6 SD. (D) Shown are the calculated mast cell Representative ear histologies from wt and Nbeal22/2 mice stained with numbers (CD117+/FcεRI+ cells) in three wt and three Nbeal22/2 bone Giemsa are shown (scale bar, 50 mm). *p , 0.05, **p , 0.01 The Journal of Immunology 5

did not detect differences in the IgE-mediated anaphylaxis between wt and Nbeal22/2 mice (Fig. 6H). This result was conﬁrmed by the similar content of histamine, IL-6, and TNF-a in the serum of wt and Nbeal22/2 mice in response to FcεRI-mediated anaphylaxis (Fig. 6I). These results support the hypothesis that the decreased mast cell number caused by Nbeal2 deﬁciency is compensated by the hyper-reactive phenotype, which is characterized by an increased production of cytokines in Nbeal22/2 mast cells (Fig. 7).

Discussion Nbeal2 mediates granule formation in megakaryocytes and platelets (5, 20, 36). We found that Nbeal2 is also critical for biogenesis of granules and therefore for storage and release of cytokines in mast cells. Deppermann et al. (20) showed that Nbeal2 is not involved in megakaryocyte differentiation, but controls the proliferation of megakaryocytes. We investigated the role of Nbeal2 in mast cell differentiation and proliferation. We found that Nbeal2 deficiency leads to reduced mast cell numbers in vivo. It is possible that the number of mast cells might be influenced by the surrounding environment, resulting in fewer mast cells in vivo. However, in in vitro cultures the exogenous influence of surrounding cells is minimized, and in Nbeal22/2 cultures the number of BMMC is also strongly reduced, instead indicating intrinsic defects of Nbeal22/2 mast cells. Intrinsic defects could be caused by an altered expression of transcription factors or of cell-cycle inhibitors in Nbeal22/2 BMMCs. Mast- cell differentiation is driven by the IRF8-dependent expression of GATA2 (25, 26) and the PI3K-MITF signaling axis (13). Fur- thermore, the expansion of mature mast cells is regulated by cell- cycle inhibitors such as p27 (21). Interestingly, in Nbeal22/2 bone marrow cultures, the protein levels of IRF8, MITF, GATA2, and p27 are strongly increased. Given that the expression of p27 depends on GATA2 (23) and MITF (24), we conclude that the increased protein level of the IRF8-GATA2/MITF transcription factor cascade results in an increased expression of p27 in mature mast cells. Consequently, the increased expression of p27 induces cell-cycle arrest and therefore a blockade of the growth factor– induced proliferation of mature mast cells. Together, our data indicate that Nbeal2 is not important for mast cell differentiation but controls their proliferation. Therefore, Nbeal2 maintains appropriate numbers of mature mast cells in the periphery by regulating the amount of IRF8, GATA2, MITF, and finally of p27. How Nbeal2 regulates the amount of these proteins is unknown. Nbeal2 is not located in the nucleus, excluding a direct influence of Nbeal2 on the transcriptional machinery. We hypothesize an essential role of Shp1 and STAT5 in the Nbeal2-mediated regulation of IRF8, GATA2, MITF, and p27 as well as in the resulting mast cell proliferation. The SCF-induced STAT5 activation and proliferation is reduced in Nbeal22/2 BMMCs. We suppose that strongly activated Shp1 is causative for the reduced STAT5 activity, and the decreased proliferation rate of Nbeal22/2 BMMCs. This is underpinned by the fact that 1) overexpression of wt Shp1 leads to inhibition of STAT5 (27); and that 2) either over-

FIGURE 2. Nbeal2 is crucial for the SCF-induced mitogenic STAT signaling. (A) Wt and NBeal22/2 BMMCs were stimulated with IL-3 (50 ulated with SCF (50 ng/ml) as indicated. Lysates were analyzed by Western ng/ml), ﬁxed, and treated with PI. The cell-cycle progression was deter- blotting. (E)WtandNbeal22/2 BMMCs were stimulated with SCF (50 ng/ml) mined by ﬂow cytometry (left). Shown is the mean 6 SD (BMMCs from as indicated. Lysates were subjected to immunoprecipitation for STAT5a three mice per genotype, one sample per mouse) (right). (B and C) Wt and or STAT5b. Precipitates and total cell lysates (input) were analyzed by Nbeal22/2 BMMCs (from three mice per genotype, four samples per Western blotting. (F)WtandNbeal22/2 BMMCs were stimulated with mouse) were stimulated with IL-3 (B) or SCF (C) (both 50 ng/ml). Shown SCF (50 ng/ml) as indicated. Lysates were analyzed by Western blotting. is the mean 6 SD of the SI. (D) Wt and Nbeal22/2 BMMCs were stim- ***p , 0.001 6 Nbeal2 REGULATES MAST CELL HOMEOSTASIS

suppression in Nbeal22/2 BMMCs leads to upregulation of IRF8, MITF/GATA2, and p27, and finally to cell-cycle arrest and the blockade of proliferation. This indicates that dysregulated Nbeal2 expression or gain-of-function mutations of Nbeal2 result in altered activation of STATs and thus in altered expression patterns of several transcription factors. This model fits the concept that Nbeal2 variants interact with STATs, and mediate their effective activation. Unexpectedly, STATs predominantly interact with a novel and yet uncharacterized short 200 kDa Nbeal2 variant in BMMCs. We screened the Uniprot database but could not find a murine Nbeal2 variant with a molecular mass of 200 kDa. All listed short Nbeal2 splice variants (e.g., the Nbeal2 variant with the UniProt Accession number F6VTL9; http://www.uniprot.org/uniprot/F6VTL9) lack the ConA domain which contains the N-terminal part. Therefore, we pre- dict a 200 kDa Nbeal2 variant, which also lacks the N-terminal part but is identical to the C-terminal part of the 300 kDa Nbeal2 variant (UniProt Accession number E9Q9L6; http://www.uniprot. org/uniprot/E9Q9L6) (Fig. 7A). However, the structural orga- nization of the 200 kDa Nbeal2 variant and the specific roles of the 300 kDa and the 200 kDa variant are unknown, but are under investigation in our laboratory. Nevertheless, we conclude that Nbeal2 interacts with STATs and controls the assembly of the c-Kit/STAT signalosome and its regulation by Shp1 (Fig. 7B, 7C).

FIGURE 3. Nbeal2 interacts with STATs. (A and B) Wt BMMCs were stimulated with SCF (50 ng/ml). Lysates were subjected to STAT3- (A), STAT5a-, or b-(B) speciﬁc immunoprecipitations. Precipitates and total cell lysates (input) were analyzed by Western blotting. expression of wt Shp1 or a dominant negative STAT5 mutant results in strongly reduced BMMC proliferation (27). FIGURE 4. Nbeal2 controls the assembly of the c-Kit/STAT signal- Thus, we hypothesize that the reduced STAT5 activation results osome. Wt and Nbeal22/2 BMMCs were stimulated with SCF (50 ng/ml) 2/2 in increased expression of IRF8 in Nbeal2 BMMCs. Interest- as indicated. Lysates were subjected to c-Kit–speciﬁc immunoprecipita- ingly, Esashi et al. (37) demonstrated that STAT5 negatively reg- tions. Precipitates and total cell lysates (input) were analyzed by Western ulates IRF8 expression. Consequently, the Shp1-mediated STAT5 blotting. The Journal of Immunology 7

Importantly, these NBeal2 functions are not limited to the growth factor–induced signaling. The FcεRI-triggered signaling is also partly regulated by Nbeal2. Thereby, Nbeal2 negatively regulates the activation of Syk and PLCg1. The FcεRI-triggered Lat1 phosphorylation and the resulting downstream signaling (e.g., the activation of JNK1/2, Erk1/2, and p38) is described as a Syk- dependent event (28, 31). However, we found increased Syk activation, but neither increased phosphorylation of Lat1 nor increased activation of JNK1/2, Erk1/2, or p38. This indicates uncoupled and Syk-independent signaling in Nbeal22/2 BMMCs. Therefore, we hypothesize that Nbeal2 controls the localization and therefore the activation of Syk in mast cells. In addition to the regulation of signaling pathways, Nbeal2 also controls compartmentalization and effector functions of mast cells. In Nbeal22/2 BMMCs, the numbers of granules and therefore the release of histamine were reduced. In contrast, the expression of CD107a is increased. This indicates that Nbeal22/2 BMMCs have fewer granules, which contain more CD107a compared with wt BMMCs. Furthermore, in Nbeal22/2 BMMCs we also found an increased production and release of cytokines, as well as an increased amount of GATA2. GATA2 is essential for cytokine production in mast cells (38). Thus we conclude that the increased cytokine production is mediated by the increased amount of GATA2. Therefore, the reduced number of granules and thus the decreased storage capacity is compensated for by enhanced production of preformed or de novo–synthesized cytokines. How Nbeal2 regulates granule formation and the release of preformed or de novo–synthesized mediators in mast cells remains unknown. Together, these results demonstrate that Nbeal2 mediates granule biogenesis and controls the production of cytokines in mast cells. Unexpectedly, the fast FcεRI-mediated anaphylactic reaction, which strongly depends on histamine-producing mast cells (11, 39), was not altered in Nbeal22/2 mice. This result is conﬁrmed by similar histamine and cytokine levels in wt and Nbeal22/2 mice in response to the anaphylactic reaction. Together, these data indicated that the lower mast cell number is compensated for by an increased responsiveness of the remaining mast cells in Nbeal22/2 mice. Indeed, in vitro–generated Nbeal22/2 BMMCs produce more cytokines than wt BMMCs, indicating an increased responsiveness of Nbeal22/2 BMMCs. However, in contrast to the release of cytokines, in vitro–generated Nbeal22/2 BMMCs release less histamine compared with wt BMMCs. The discrepancy in the histamine release might be explained by mast cell extrinsic factors, which might compensate for the reduced histamine response of mast cells in NBeal22/2 mice. Thereby, endothelial cells can be excluded because they do not express Nbeal2, and show normal synthesis and storage of several factors in Nbeal22/2

were stimulated with HSA-DNP (10 ng/ml) for 1 h. Cells were analyzed for CD107a surface expression by flow cytometry. (G–I)WtandNbeal22/2 BMMCs (from three mice per genotype; two samples per mouse per condition) were primed with anti-DNP IgE [clone: SPE-7 (1 mg/ml)] overnight, FIGURE 5. Nbeal2 mediates granule formation, and cytokine release. and were stimulated with HSA-DNP (1 h) as indicated. Supernatants were (A and B) Wt and Nbeal22/2 BMMCs were primed with anti-DNP IgE analyzed for MCP-1, TNF-a, and IL-6 by ELISA. Shown is the mean 6 SD. [clone: SPE-7 (1 mg/ml)] overnight, and stimulated with HSA-DNP (10 (J) Lysates of wt and Nbeal22/2 BMMCs were analyzed by Western blot- ng/ml) as indicated. Lysates were analyzed by Western blotting. (C)Wt ting. Shown are the statistics of normalized units from four independent and Nbeal22/2 BMMCs were analyzed by flow cytometry. (D and E) experiments. The intensities of CD107a bands were normalized to the in- Shown is the ultrastructure of BMMCs. Representative transmission elec- tensities of the respective tubulin bands. Shown is the mean 6 SD. (K)Wt tron microscopy images of resting wt and Nbeal22/2 BMMCs. Black and Nbeal22/2 BMMCs (from three mice per genotype, two samples per arrows indicate granules and white arrows indicate mitochondria. Quan- mouse per condition) were primed with anti-DNP IgE [clone: SPE-7 (1 mg/ml)] tification was performed by counting granules in wt and Nbeal22/2 overnight, and were stimulated with HSA-DNP (10 ng/ml) as indicated. BMMCs (granules per phenotype 6 SD). (F) Wt and Nbeal22/2 BMMCs Supernatants were collected and analyzed for histamine by ELISA. Shown were primed with anti-DNP IgE [clone: SPE-7 (1 mg/ml)] overnight, and is the mean 6 SD. **p , 0.01, ***p , 0.001. 8 Nbeal2 REGULATES MAST CELL HOMEOSTASIS

FIGURE 6. (A–D)WtandNbeal22/2 BMMCs (from three mice per genotype) were primed with anti-DNP IgE [clone: SPE-7 (1 mg/ml)] overnight and stimulated with HSA-DNP for 4 h. Supernatants were analyzed for MCP-1, TNF-a, IL-6, and IL-13 by ELISA (six samples per condition; two samples per mouse). Shown is the mean 6 SD. (E)WtandNbeal22/2 BMMCs were treated with PMA/ionomycin (10 nM/2 mM) for 1 h. Cells were analyzed for CD107a surface expression by flow cytometry. (F)WtandNbeal22/2 BMMCs (from three mice per genotype) were stimulated with PMA/ionomycin (10 nM/2 mM) for 1 or 5 h. Supernatants were analyzed for TNF-a by ELISA (six samples per condition, two samples per mouse). (G)WtandNbeal22/2 BMMCs were left untreated or treated with brefeldin A (BrefA) (3 mg/ml), and stimulated with PMA/ionomycin (10 ng/ml/1 mg/ml) for 1 or 5 h. Fixed cells were stained intracellularly with anti-CD117 and anti–TNF-a. (H) Mice were sensitized with anti-DNP IgE (3 mg/ml) intravenously. After 20 h the basal body temperature was measured (control). HSA-DNP was injected to induce anaphylaxis. The body temperature was measured. The experiment was performed twice with five mice per genotype. Shown is the mean of the temperature of five mice per genotype 6 SD. (I) Mice were treated as mentioned in (H). Blood was collected and the serum was analyzed for histamine, IL-6, and TNF-a (n =6mice per genotype). Shown is the mean of the histamine, IL-6, and TNF-a.*p , 0.05, **p , 0.01, ***p , 0.001. ns, not significant. The Journal of Immunology 9

FIGURE 7. Proposed model of the Nbeal2-mediated STAT activation. (A) Shown is the 300 kDa Nbeal2 variant (UniProt: E9Q9L6) in comparison with the predicted structural composition of the 200 kDa Nbeal2 variant. (B) In wt BMMCs Nbeal2 prevents an un- controlled interaction between c-Kit and STATs. Stimulation with SCF (S) leads to activation of STATs, which is regulated by Shp1. This balanced activation of STATs leads to suppression of the IRF8 expression and thus to controlled expression of GATA2 and p27. This is the precondition for mature mast cells to proliferate. Furthermore, Nbeal2 regulates the granule biogenesis and the FcεRI-mediated cytokine production. (C)In Nbeal22/2 BMMCs c-Kit strongly associates with STATs. Stimulation with SCF (S) leads to a strong activation of Shp1 and thus to a reduced activation of STAT5. The reduced activity of STAT5 results in an increased expression of IRF8, and thus to an uncon- trolled expression of GATA2 and p27. This leads to cell-cycle arrest and strongly reduced mast cell proliferation. Nbeal2 deﬁciency furthermore results in a reduced number of granules and leads to increased FcεRI-mediated cytokine production.

mice (20, 40). We rather speculate that Nbeal2 influences the dif- Disclosures ferentiation of other cell lineages (e.g., basophils), which are also The authors have no financial conflicts of interest. important for anaphylactic reactions and therefore might compensate the reduced histamine release from mast cells in Nbeal22/2 mice. Together, we conclude that the reduced mast cell number might References 1. Cullinane, A. R., A. A. Scha¨ffer, and M. Huizing. 2013. The BEACH is hot: a be compensated by 1) an increased responsiveness of the remaining LYST of emerging roles for BEACH-domain containing proteins in human mast cells; and/or by 2) an altered differentiation of other cell disease. Traffic 14: 749–766. lineages in Nbeal22/2 mice. In summary, we show that Nbeal2 is 2. Burgess, A., J. P. Mornon, G. de Saint-Basile, and I. Callebaut. 2009. A con- canavalin A-like lectin domain in the CHS1/LYST protein, shared by members a multifunctional protein regulating mitogenic signaling, cell- of the BEACH family. Bioinformatics 25: 1219–1222. cycle arrest, and mast cell effector functions. Together with a 3. Harlan, J. E., P. J. Hajduk, H. S. Yoon, and S. W. Fesik. 1994. Pleckstrin ho- recent report that shows that a homozygous nonsense NBEAL2 mology domains bind to phosphatidylinositol-4,5-bisphosphate. Nature 371: 168–170. mutation can mimic the clinical symptoms of the autoimmune 4. Gunay-Aygun, M., T. C. Falik-Zaccai, T. Vilboux, Y. Zivony-Elboum, lymphoproliferative syndrome (41), our data support the relevance F. Gumruk, M. Cetin, M. Khayat, C. F. Boerkoel, N. Kfir, Y. Huang, et al. 2011. of Nbeal2 in immunity above and beyond granule biosynthesis. NBEAL2 is mutated in gray platelet syndrome and is required for biogenesis of platelet a-granules. Nat. Genet. 43: 732–734. 5.Kahr,W.H.,J.Hinckley,L.Li,H.Schwertz,H.Christensen,J.W.Rowley, Acknowledgments F. G. Pluthero, D. Urban, S. Fabbro, B. Nixon, et al. 2011. Mutations in NBEAL2, encoding a BEACH protein, cause gray platelet syndrome. Nat. Genet. 43: 738–740. We thank Dr. F.-D. Bo¨hmer (Centre of Medical Biomedicine, Jena) for 6. Albers, C. A., A. Cvejic, R. Favier, E. E. Bouwmans, M. C. Alessi, P. Bertone, critical reading. G. Jordan, R. N. Kettleborough, G. Kiddle, M. Kostadima, et al. 2011. Exome 10 Nbeal2 REGULATES MAST CELL HOMEOSTASIS

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