The systemic anaphylactic reaction is a life-threatening allergic response initiated by activated mast cells. Sphingolipids are an essential player in the development and attenuation of this response. De novo synthesis of sphingolipids in mammalian cells is inhibited by the family of three ORMDL proteins (ORMDL1, 2, and 3). However, the cell and tissue-specific functions of ORMDL proteins in mast cell signaling are poorly understood. This study aimed to determine cross-talk of ORMDL2 and ORMDL3 proteins in IgE-mediated responses. To this end, we prepared mice with whole-body knockout (KO) of Ormdl2 and/or Ormdl3 genes and studied their role in mast cell-dependent activation events in vitro and in vivo. We found that the absence of ORMDL3 in bone marrow-derived mast cells (BMMCs) increased the levels of cellular sphingolipids. Such an increase was further raised by simultaneous ORMDL2 deficiency, which alone had no effect on sphingolipid levels. Cells with double ORMDL2 and ORMDL3 KO exhibited increased intracellular levels of sphingosine-1-phosphate (S1P). Furthermore, we found that concurrent ORMDL2 and ORMDL3 deficiency increased IκB-α phosphorylation, degranulation, and production of IL-4, IL-6, and TNF-α cytokines in antigen-activated mast cells. Interestingly, the chemotaxis towards antigen was increased in all mutant cell types analyzed. Experiments in vivo showed that passive cutaneous anaphylaxis (PCA), which is initiated by mast cell activation, was increased only in ORMDL2,3 double KO mice, supporting our in vitro observations with mast cells. On the other hand, ORMDL3 KO and ORMDL2,3 double KO mice showed faster recovery from passive systemic anaphylaxis, which could be mediated by increased levels of blood S1P presented in such mice. Our findings demonstrate that Ormdl2 deficiency potentiates the ORMDL3-dependent changes in mast cell signaling.

Czech Centre for Phenogenomics Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czechia

Department of Signal Transduction Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czechia

Research Unit for Rare Diseases Department of Pediatrics and Inherited Metabolic Disorders 1st Faculty of Medicine Charles University and General University Hospital Prague Prague Czechia

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Olivera A, Rivera J. Sphingolipids and the balancing of immune cell function: lessons from the mast cell. J Immunol (2005) 174:1153–8.  10.4049/jimmunol.174.3.1153 PubMed DOI

Hanada K. Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism. Biochim Biophys Acta (2003) 1632:16–30.  10.1016/s1388-1981(03)00059-3 PubMed DOI

Hannun YA, Obeid LM. Principles of bioactive lipid signalling: lessons from sphingolipids. Nat Rev Mol Cell Biol (2008) 9:139–50.  10.1038/nrm2329 PubMed DOI

Schauberger E, Peinhaupt M, Cazares T, Lindsley AW. Lipid mediators of allergic disease: pathways, treatments, and emerging therapeutic targets. Curr Allergy Asthma Rep (2016) 16:48.  10.1007/s11882-016-0628-3 PubMed DOI PMC

Wennekes T, van den Berg RJ, Boot RG, van der Marel GA, Overkleeft HS, Aerts. Glycosphingolipids-nature JM. function, and pharmacological modulation. Angew Chem Int Ed Engl (2009) 48:8848–69.  10.1002/anie.200902620 PubMed DOI

Breslow DK, Collins SR, Bodenmiller B, Aebersold R, Simons K, Shevchenko A, et al. Orm family proteins mediate sphingolipid homeostasis. Nature (2010) 463:1048–53.  10.1038/nature08787 PubMed DOI PMC

Hjelmqvist L, Tuson M, Marfany G, Herrero E, Balcells S, Gonzalez-Duarte R. ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins. Genome Biol (2002) 3(6):RESEARCH0027.  10.1186/gb-2002-3-6-research0027 PubMed DOI PMC

Davis DL, Gable K, Suemitsu J, Dunn TM, Wattenberg BW. The ORMDL/Orm-serine palmitoyltransferase (SPT) complex is directly regulated by ceramide: Reconstitution of SPT regulation in isolated membranes. J Biol Chem (2019) 294:5146–56.  10.1074/jbc.RA118.007291 PubMed DOI PMC

Clarke BA, Majumder S, Zhu H, Lee YT, Kono M, Li C, et al. The Ormdl genes regulate the sphingolipid synthesis pathway to ensure proper myelination and neurologic function in mice. Elife (2019) 8:e51067.  10.7554/eLife.51067 PubMed DOI PMC

Hojjati MR, Li Z, Jiang XC. Serine palmitoyl-CoA transferase (SPT) deficiency and sphingolipid levels in mice. Biochim Biophys Acta (2005) 1737:44–51.  10.1016/j.bbalip.2005.08.006 PubMed DOI

Worgall TS, Veerappan A, Sung B, Kim BI, Weiner E, Bholah R, et al. Impaired sphingolipid synthesis in the respiratory tract induces airway hyperreactivity. Sci Trans Med (2013) 5(186):186ra67. 10.1126/scitranslmed.3005765 PubMed DOI

Li Z, Kabir I, Tietelman G, Huan C, Fan J, Worgall T, et al. Sphingolipid de novo biosynthesis is essential for intestine cell survival and barrier function. Cell Death Dis (2018) 9:173.  10.1038/s41419-017-0214-1 PubMed DOI PMC

Parthibane V, Acharya D, Srideshikan SM, Lin J, Myerscough DG, Abimannan T, et al. Sptlc1 is essential for myeloid differentiation and hematopoietic homeostasis. Blood Adv (2019) 3:3635–49.  10.1182/bloodadvances.2019000729 PubMed DOI PMC

Cantero-Recasens G, Fandos C, Rubio-Moscardo F, Valverde MA, Vicente R. The asthma-associated ORMDL3 gene product regulates endoplasmic reticulum-mediated calcium signaling and cellular stress. Hum Mol Genet (2010) 19:111–21.  10.1093/hmg/ddp471 PubMed DOI

Carreras-Sureda A, Cantero-Recasens G, Rubio-Moscardo F, Kiefer K, Peinelt C, Niemeyer BA, et al. ORMDL3 modulates store-operated calcium entry and lymphocyte activation. Hum Mol Genet (2013) 22:519–30.  10.1093/hmg/dds450 PubMed DOI

Miller M, Tam AB, Cho JY, Doherty TA, Pham A, Khorram N, et al. ORMDL3 is an inducible lung epithelial gene regulating metalloproteases, chemokines, OAS, and ATF6. P Natl Acad Sci USA (2012) 109:16648–53.  10.1073/pnas.1204151109 PubMed DOI PMC

Moffatt MF, Kabesch M, Liang L, Dixon AL, Strachan D, Heath S, et al. Genetic variants regulating ORMDL3 expression contribute to the risk of childhood asthma. Nature (2007) 448:470–3.  10.1038/nature06014 PubMed DOI

Moffatt MF, Gut IG, Demenais F, Strachan DP, Bouzigon E, Heath S, et al. A large-scale, consortium-based genomewide association study of asthma. N Engl J Med (2010) 363:1211–21.  10.1056/NEJMoa0906312 PubMed DOI PMC

Ono JG, Kim BI, Zhao Y, Christos PJ, Tesfaigzi Y, Worgall TS, et al. Decreased sphingolipid synthesis in children with 17q21 asthma-risk genotypes. J Clin Invest (2020) 130:921–6.  10.1172/JCI130860 PubMed DOI PMC

Loser S, Gregory LG, Zhang Y, Schaefer K, Walker SA, Buckley J, et al. Pulmonary ORMDL3 is critical for induction of Alternaria-induced allergic airways disease. J Allergy Clin Immunol (2016) 139(5):1496–507.e3. 10.1016/j.jaci.2016.07.033 PubMed DOI PMC

Miller M, Tam AB, Mueller JL, Rosenthal P, Beppu A, Gordillo R, et al. Cutting edge: Targeting epithelial ORMDL3 increases, rather than reduces, airway responsiveness and is associated with increased sphingosine-1-phosphate. J Immunol (2017). 198(8):3017–22. 10.4049/jimmunol.1601848 PubMed DOI PMC

Debeuf N, Zhakupova A, Steiner R, Van GS, Deswarte K, Fayazpour F, et al. The ORMDL3 asthma susceptibility gene regulates systemic ceramide levels without altering key asthma features in mice. J Allergy Clin Immunol (2019) 144(6):1648–59.e9.  10.1016/j.jaci.2019.06.041 PubMed DOI PMC

Siow DL, Wattenberg BW. Mammalian ORMDL proteins mediate the feedback response in ceramide biosynthesis. J Biol Chem (2012) 287:40198–204. 10.1074/jbc.C112.404012 PubMed DOI PMC

Bugajev V, Halova I, Draberova L, Bambouskova M, Potuckova L, Draberova H, et al. Negative regulatory roles of ORMDL3 in the FcϵRI-triggered expression of proinflammatory mediators and chemotactic response in murine mast cells. Cell Mol Life Sci (2016) 73:1265–85.  10.1007/s00018-015-2047-3 PubMed DOI PMC

Galli SJ, Tsai M, Piliponsky AM. The development of allergic inflammation. Nature (2008) 454:445–54.  10.1038/nature07204 PubMed DOI PMC

Jolly PS, Bektas M, Olivera A, Gonzalez-Espinosa C, Proia RL, Rivera J, et al. Transactivation of sphingosine-1-phosphate receptors by FcεRI triggering is required for normal mast cell degranulation and chemotaxis. J Exp Med (2004) 199:959–70.  10.1084/jem.20030680 PubMed DOI PMC

Izawa K, Yamanishi Y, Maehara A, Takahashi M, Isobe M, Ito S, et al. The receptor LMIR3 negatively regulates mast cell activation and allergic responses by binding to extracellular ceramide. Immunity (2012) 37:827–39.  10.1016/j.immuni.2012.08.018 PubMed DOI

Olivera A, Eisner C, Kitamura Y, Dillahunt S, Allende L, Tuymetova G, et al. Sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 are vital to recovery from anaphylactic shock in mice. J Clin Invest (2010) 120:1429–40.  10.1172/JCI40659 PubMed DOI PMC

Rudolph AK, Burrows PD, Wabl MR. Thirteen hybridomas secreting hapten-specific immunoglobulin E from mice with Iga or Igb heavy chain haplotype. Eur J Immunol (1981) 11:527–9.  10.1002/eji.1830110617 PubMed DOI

Tolar P, Dráberová L, Dráber P. Protein tyrosine kinase Syk is involved in Thy-1 signaling in rat basophilic leukemia cells. Eur J Immunol (1997) 27:3389–97.  10.1002/eji.1830271238 PubMed DOI

Dráberová L, Amoui M, Dráber P. Thy-1-mediated activation of rat mast cells: the role of Thy-1 membrane microdomains. Immunology (1996) 87:141–8. PubMed PMC

Lee H, Fenske RJ, Akcan T, Domask E, Davis DB, Kimple ME, et al. Differential Expression of Ormdl Genes in the Islets of Mice and Humans with Obesity. iScience (2020) 23:101324. 10.1016/j.isci.2020.101324 PubMed DOI PMC

Haeussler M, Schonig K, Eckert H, Eschstruth A, Mianne J, Renaud JB, et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol (2016) 17:148.  10.1186/s13059-016-1012-2 PubMed DOI PMC

Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science (2013) 339:819–23.  10.1126/science.1231143 PubMed DOI PMC

Ota S, Hisano Y, Muraki M, Hoshijima K, Dahlem TJ, Grunwald DJ, et al. Efficient identification of TALEN-mediated genome modifications using heteroduplex mobility assays. Genes Cells (2013) 18:450–8.  10.1111/gtc.12050 PubMed DOI PMC

Horakova H, Polakovicova I, Shaik GM, Eitler J, Bugajev V, Draberova L, et al. 1,2-propanediol-trehalose mixture as a potent quantitative real-time PCR enhancer. BMC Biotechnol (2011) 11:41.  10.1186/1472-6750-11-41 PubMed DOI PMC

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (1970) 227:680–5.  10.1038/227680a0 PubMed DOI

Haan C, Behrmann I. A cost effective non-commercial ECL-solution for Western blot detections yielding strong signals and low background. J Immunol Methods (2007) 318:11–9.  10.1016/j.jim.2006.07.027 PubMed DOI

Aerts JM, Groener JE, Kuiper S, Donker-Koopman WE, Strijland A, Ottenhoff R, et al. Elevated globotriaosylsphingosine is a hallmark of Fabry disease. Proc Natl Acad Sci USA (2008) 105:2812–7.  10.1073/pnas.0712309105 PubMed DOI PMC

Kuchar L, Sikora J, Gulinello ME, Poupetova H, Lugowska A, Malinova V, et al. Quantitation of plasmatic lysosphingomyelin and lysosphingomyelin-509 for differential screening of Niemann-Pick A/B and C diseases. Anal Biochem (2017) 525:73–7.  10.1016/j.ab.2017.02.019 PubMed DOI

Kuchar L, Faltyskova H, Krasny L, Dobrovolny R, Hulkova H, Ledvinova J, et al. Fabry disease: renal sphingolipid distribution in the α-Gal A knockout mouse model by mass spectrometric and immunohistochemical imaging. Anal Bioanal Chem (2015) 407:2283–91.  10.1007/s00216-014-8402-7 PubMed DOI

Surviladze Z, Draberova L, Kubinova L, Draber P. Functional heterogeneity of Thy-1 membrane microdomains in rat basophilic leukemia cells. Eur J Immunol (1998) 28:1847–58. 10.1002/(SICI)1521-4141(199806)28:06<1847::AID-IMMU1847>3.0.CO;2-O PubMed DOI

Volna P, Lebduska P, Draberova L, Simova S, Heneberg P, Boubelik M, et al. Negative regulation of mast cell signaling and function by the adaptor LAB/NTAL. J Exp Med (2004) 200:1001–13.  10.1084/jem.20041213 PubMed DOI PMC

Draberova L, Paulenda T, Halova I, Potuckova L, Bugajev V, Bambouskova M, et al. Ethanol inhibits high-affinity immunoglobulin E receptor (FcϵRI) signaling in mast cells by suppressing the function of FcϵRI-cholesterol signalosome. PloS One (2015) 10:e0144596.  10.1371/journal.pone.0144596 PubMed DOI PMC

Dahlin JS, Ding Z, Hallgren J. Distinguishing mast cell progenitors from mature mast cells in mice. Stem Cells Dev (2015) 24:1703–11.  10.1089/scd.2014.0553 PubMed DOI PMC

Davis D, Suemitsu J, Wattenberg B. Transmembrane topology of mammalian ORMDL proteins in the endoplasmic reticulum as revealed by the substituted cysteine accessibility method (SCAM). Biochim Biophys Acta Proteins Proteom (2019) 1867:382–95.  10.1016/j.bbapap.2019.01.005 PubMed DOI PMC

Saluja R, Kumar A, Jain M, Goel SK, Jain A. Role of sphingosine-1-phosphate in mast cell functions and asthma and its regulation by non-coding RNA. Front Immunol (2017) 8:587.  10.3389/fimmu.2017.00587 PubMed DOI PMC

Dillahunt SE, Sargent JL, Suzuki R, Proia RL, Gilfillan A, Rivera J, et al. Usage of sphingosine kinase isoforms in mast cells is species and/or cell type determined. J Immunol (2013) 190:2058–67.  10.4049/jimmunol.1201503 PubMed DOI PMC

Olivera A, Mizugishi K, Tikhonova A, Ciaccia L, Odom S, Proia RL, et al. The sphingosine kinase-sphingosine-1-phosphate axis is a determinant of mast cell function and anaphylaxis. Immunity (2007) 26:287–97.  10.1016/j.immuni.2007.02.008 PubMed DOI

Schmiedel BJ, Seumois G, Samaniego-Castruita D, Cayford J, Schulten V, Chavez L, et al. 17q21 asthma-risk variants switch CTCF binding and regulate IL-2 production by T cells. Nat Commun (2016) 7:13426.  10.1038/ncomms13426 PubMed DOI PMC

Ha SG, Ge XN, Bahaie NS, Kang BN, Rao A, Rao SP, et al. ORMDL3 promotes eosinophil trafficking and activation via regulation of integrins and CD48. Nat Commun (2013) 4:2479.  10.1038/ncomms3479 PubMed DOI PMC

Bugajev V, Bambouskova M, Draberova L, Draber P. What precedes the initial tyrosine phosphorylation of the high affinity IgE receptor in antigen-activated mast cell? FEBS Lett (2010) 584:4949–55.  10.1016/j.febslet.2010.08.045 PubMed DOI

Toncheva AA, Potaczek DP, Schedel M, Gersting SW, Michel S, Krajnov N, et al. Childhood asthma is associated with mutations and gene expression differences of ORMDL genes that can interact. Allergy (2015) 70:1288–99.  10.1111/all.12652 PubMed DOI

Kanjarawi R, Dy M, Bardel E, Sparwasser T, Dubois B, Mecheri S, et al. Regulatory CD4+Foxp3+ T cells control the severity of anaphylaxis. PloS One (2013) 8:e69183.  10.1371/journal.pone.0069183 PubMed DOI PMC

Makabe-Kobayashi Y, Hori Y, Adachi T, Ishigaki-Suzuki S, Kikuchi Y, Kagaya Y, et al. The control effect of histamine on body temperature and respiratory function in IgE-dependent systemic anaphylaxis. J Allergy Clin Immunol (2002) 110:298–303.  10.1067/mai.2002.125977 PubMed DOI

Oskeritzian CA, Milstien S, Spiegel S. Sphingosine-1-phosphate in allergic responses, asthma and anaphylaxis. Pharmacol Ther (2007) 115:390–9.  10.1016/j.pharmthera.2007.05.011 PubMed DOI PMC

Deciphering pathophysiological mechanisms underlying cystathionine beta-synthase-deficient homocystinuria using targeted metabolomics, liver proteomics, sphingolipidomics and analysis of mitochondrial function

Simultaneous deletion of ORMDL1 and ORMDL3 proteins disrupts immune cell homeostasis

Enhanced Membrane Fluidization and Cholesterol Displacement by 1-Heptanol Inhibit Mast Cell Effector Functions

Simultaneous reduction of all ORMDL proteins decreases the threshold of mast cell activation

Crosstalk between ORMDL3, serine palmitoyltransferase, and 5-lipoxygenase in the sphingolipid and eicosanoid metabolic pathways