In mammals, the ORMDL family of evolutionarily conserved sphingolipid regulators consists of three highly homologous members, ORMDL1, ORMDL2 and ORMDL3. ORMDL3 gene has been associated with childhood-onset asthma and other inflammatory diseases in which mast cells play an important role. We previously described increased IgE-mediated activation of mast cells with simultaneous deletions of ORMDL2 and ORMDL3 proteins. In this study, we prepared mice with Ormdl1 knockout and thereafter, produced primary mast cells with reduced expression of one, two or all three ORMDL proteins. The lone deletion of ORMDL1, or in combination with ORMDL2, had no effect on sphingolipid metabolism nor IgE-antigen dependent responses in mast cells. Double ORMDL1 and ORMDL3 knockout mast cells displayed enhanced IgE-mediated calcium responses and cytokine production. Silencing of ORMDL3 in mast cells after maturation increased their sensitivity to antigen. Mast cells with reduced levels of all three ORMDL proteins demonstrated pro-inflammatory responses even in the absence of antigen activation. Overall, our results show that reduced levels of ORMDL proteins shift mast cells towards a pro-inflammatory phenotype, which is predominantly dependent on the levels of ORMDL3 expression.

CZ OPENSCREEN Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

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

Laboratory of Signal Transduction Institute of Molecular Genetics of the Czech Academy of Sciences Videnska 1083 14220 Prague 4 Czech Republic

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

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Simultaneous deletion of ORMDL1 and ORMDL3 proteins disrupts immune cell homeostasis