ORMDL2 Deficiency Potentiates the ORMDL3-Dependent Changes in Mast Cell Signaling

Abstract

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.

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

“…These data suggest the unique role of ORMDL3 in mast cells, presumably by engaging other signaling pathways. To corroborate the data and extend these results, we examined the properties of PDMCs isolated from WT and ORMDL3 KO mice prepared with the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 technology ( 30 ). PDMCs isolated from both WT and ORMDL3 KO mice exhibited properties of mast cells, including alcian blue/safranin or toluidine blue staining ( supplemental Fig.…”

“…Mast cells respond immediately to external stimuli by releasing preformed proinflammatory mediators stored in secretory lysosomes ( 44 ). We have previously reported that BMMCs with ORMDL3 knockdown or concurrently deficient in ORMDL2 and ORMDL3 exhibit increased responsiveness to antigen ( 29 , 30 ). In contrast, ORMDL3 is a positive regulator in eosinophils ( 28 ).…”

“…The regulatory role of ORMDL proteins in sphingolipid synthesis has been described in several cell types ( 10 , 11 , 12 , 13 ) including BMMCs ( 30 ). Mass spectrometry analysis indicated increased levels of total sphingosines, sphinganine C18:0, and sphingosine C18:1 in PDMCs from ORMDL3 KO ( Fig.…”

“…All mice were maintained and used in accordance with the Institute of Molecular Genetics guidelines (permit number 12135/2010-17210) and national guidelines (2048/2004-1020). We described the generation of mice deficient in ORMDL3, screening of founder animals, and the strategy of genotyping elsewhere ( 30 ).…”

“…Antigen-activated bone marrow derived mast cells (BMMCs) with ORMDL3 knockdown promoted phosphorylation of NF-κB inhibitor-α (IκB-α) followed by translocation of the p65 subunit of NF-κB into the nuclei and subsequent expression of proinflammatory cytokines, including tumor necrosis factor (TNF)-α, IL-6, and IL-13, CC motif chemokine ligand (CCL)3 and CCL4, and enhanced cyclooxygenase-2-dependent synthesis of prostaglandin (PG)D 2 (29). Recently, we found that BMMCs isolated from mice deficient in both ORMDL2 and ORMDL3 exhibited increased sphingolipid levels and enhanced release of IL-4, IL-6, and TNF-α cytokines when compared to wild-type (WT) cells (30). Similarly, increased expression of IL-2 was observed in activated primary memory CD4 + T cells with silenced ORMDL3 (31).These and other findings of frequently opposite roles of ORMDL3 in various cell types in vitro (19,28,29,31) and in vivo (24)(25)(26)(27) suggested that ORMDL3 could be involved in other signal transduction pathways than previously thought.…”