Aberrant ORM (yeast)-like protein isoform 3 (ORMDL3) expression dysregulates ceramide homeostasis in cells and ceramide exacerbates allergic asthma in mice

Abstract

Background: Asthma, a chronic inflammatory condition defined by episodic shortness of breath with expiratory wheezing and cough, is a serious health concern affecting more than 250 million persons. Genome-wide association studies have identified ORM (yeast)-like protein isoform 3 (ORMDL3) as a gene associated with susceptibility to asthma. Although its yeast ortholog is a negative regulator of de novo ceramide biosynthesis, how ORMDL3 contributes to asthma pathogenesis is not known.

Objectives: We sought to decipher the molecular mechanism for the pathologic functions of ORMDL3 in asthma and the relationship to its evolutionarily conserved role in regulation of ceramide homeostasis.

Methods: We determined the relationship between expression of ORMDL3 and ceramide in epithelial and inflammatory cells and in asthma pathogenesis in mice.

Results: Although small increases in ORMDL3 expression decrease ceramide levels, remarkably, higher expression in lung epithelial cells and macrophages in vitro and in vivo increased ceramide production, which promoted chronic inflammation, airway hyperresponsiveness, and mucus production during house dust mite-induced allergic asthma. Moreover, nasal administration of the immunosuppressant drug FTY720/fingolimod reduced ORMDL3 expression and ceramide levels and mitigated airway inflammation and hyperreactivity and mucus hypersecretion in house dust mite-challenged mice.

Conclusions: Our findings demonstrate that overexpression of ORMDL3 regulates ceramide homeostasis in cells in a complex manner and suggest that local FTY720 administration might be a useful therapeutic intervention for the control of allergic asthma.

Keywords: Asthma; FTY720; ORMDL3; ceramide; house dust mite; sphingolipid.

FIG 1

Ceramide levels in lung epithelial cells are increased by depletion of ORMDL3 and reduced by its re-expression. A, A549 cells were transfected with scrambled siRNA (siControl) or specific siRNAs for each ORMDL isoform, as indicated. B, Cells were transfected with 1 µg of vector or ORMDL3 expression vector. C, After downregulation of ORMDL isoforms, cells were transfected with 1 µg of vector or ORMDL3. Ceramide species were measured by using LC-ESI-MS/MS. Experiments were performed in biological triplicates. Data are shown as means ± SDs and representative of 3 independent experiments. Fig 1, A, *P < .01 compared with siControl and **P < .001 compared with siORMDL3. Fig 1, B, *P < .01 compared with vector. Fig 1, C, *P < .05 compared with vector and **P < .005 compared with siORMDL1/2/3 transfected with vector. Fig 1, A and C, insets, Expression of ORMDL isoforms determined by means of quantitative PCR. Fig 1, B, inset, Cell lysates analyzed by means of immunoblotting with ORMDL3 antibody.

FIG 2

High overexpression of ORMDL3 enhances rather than decreases ceramide levels. A549 (A and B) or RAW264.7 (C and D) cells were transfected with vector or ORMDL3, as indicated. Ceramide species (Fig 2, A and C) and total ceramide (Fig 2, B) levels were determined by using LC-ESI-MS/MS. Fig 2, B and D, Cell lysates were analyzed by means of immunoblotting with ORMDL3 antibody and quantified by means of densitometry, and ORMDL3 expression was determined by using quantitative PCR. *P < .05 compared with vector.

FIG 3

Effect of ORMDL3 expression on incorporation of [¹³C]palmitate into ceramides. A, Scheme of ceramide formation by de novo biosynthesis and salvage/recycling pathways. B-E, A549 cells were transfected with vector or ORMDL3, as indicated, and then incubated for 4 hours with 0.1 mmol/L [¹³C] palmitic acid. Base-labeled (Fig 3, B), dual-labeled (Fig 3, C), fatty acid–labeled (Fig 3, D), and unlabeled (Fig 3, E) ceramides were determined by using LC-ESI-MS/MS. Data are means ± SDs. *P < .05 compared with vector.

FIG 4

High overexpression of ORMDL3 enhances LPS-induced cytokine and chemokine expression. RAW264.7 (A) and A549 lung epithelial (B) cells transfected with vector or 1 or 2 µg of ORMDL3 were treated with 10 ng/mL LPS for 4 hours (Fig 4, A) or 1 ng/mL LPS for 24 hours (Fig 4, B). Cytokine and chemokine expression was determined by using quantitative PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh). Experiments were performed in biological triplicates. Data are means ± SDs and representative of 3 independent experiments. *P < .05 compared with vector and **P < .05 compared with 1 µg of ORMDL3 plus LPS.

FIG 5

HDM increases and FTY720 administration attenuates ORMDL3 expression and increase of lung ceramide and dihydroceramide levels. Mice (n = 5 per group) were challenged intranasally with saline or HDM extract and treated intranasally with vehicle (saline) or FTY720 (0.3 mg/kg) 30 minutes before challenge, and lungs were examined on day 15. A, Expression of ORMDL3 in the lungs was determined by means of Western blot analysis with anti-ORMDL3 antibody and quantified by means of densitometry. B, Lung sections were immunostained with anti-ORMDL3 antibody. Scale bar = 100 µm. C–F, On day 15, lung (Fig 5, C and D) and BALF (Fig 5, E and F) ceramide (Fig 5, C and E) and dihydroceramide (Fig 5, D and F) species were analyzed by using LC-ESI-MS/MS. Data are mean ± SEMs and representative of 2 independent experiments. *P < .05 compared with HDM and **P < .05 compared with vehicle.

FIG 6

Blocking ceramide level increase mitigates HDM-induced airway hyperreactivity and allergic airway inflammation. Mice (n = 5 per group) challenged intranasally with HDM or saline were injected intraperitoneally with vehicle, myriocin (Myr; 0.3 mg/kg), or FB1 (0.5 mg/kg) from days 10 to 12, as indicated. A, Expression of ORMDL3 in the lungs was determined by means of Western blot analysis with anti-ORMDL3 antibody and quantified by using densitometry. ORMDL3+, Extract from cells overexpressing ORMDL3 as a positive control. B and C, Lung ceramide and dihydroceramide species were analyzed by using LC-ESI-MS/MS. D, Lung resistance in response to inhaled methacholine was measured with the Flexi-Vent system. Note that the responses to methacholine in the unsensitized mice treated with vehicle, FB1, or myriocin were indistinguishable. E, BALF accumulation of eosinophils determined by using fluorescence-activated cell sorting. F, mRNA expression of cytokines and chemokines in the lung were determined by using quantitative PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh). Data are means ± SEMs. *P < .05 compared with HDM and **P < .05 compared with vehicle.

FIG 7

HDM-induced ORMDL3expression, airway resistance, mucusproduction, andceramidelevel increase are suppressed in IL-13 knockout (KO) mice. IL-13^−/− mice and wild-type (WT) littermates (n = 5 per group) were challenged intranasally with saline or HDM extract, and lungs were examined on day 15. A, Expression of ORMDL3 in the lungs was determined by means of Western blot analysis with anti-ORMDL3 antibody and quantified by using densitometry. B, Lung resistance in response to inhaled methacholine was measured with the FlexiVent apparatus on day 15. C, BALF accumulation of eosinophils determined by using fluorescence-activated cell sorting. D, mRNA expression of Muc5AC determined by using quantitative PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and mucin levels in BALF determined by means of ELISA. E and F, BALF ceramide (Fig 7, E) and dihydroceramide (Fig 7, F) levels were analyzed by using LC-ESI-MS/MS. Data are means ± SEMs. *P < .05, IL-13 knockout compared with WT mice challenged with HDM; **P < .05, WT mice challenged with HDM compared with vehicle.

FIG 8

FTY720 attenuates HDM-induced airway hyperreactivity and allergic airway inflammation. Mice (n = 5 per group) were challenged intranasally with saline or HDM extract and treated intranasally with vehicle (saline) or FTY720 (0.3 mg/kg) 30 minutes before challenge. A, Lung resistance in response to inhaled meth-acholine was measured with the FlexiVent apparatus on day 15. Note that the responses to methacholine in the unsensitized mice treated with vehicle or FTY720 were indistinguishable. B, Eosinophils, neutrophils, and lymphocytes in BALF. C, mRNA expression of cytokines and chemokines and Muc5AC in the lung was determined by using quantitative PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh). D, Mucin levels in BALF were determined by means of ELISA. E and F, Lung sections were stained with H&E or PAS. Scale bars = 100 µm. Fig 8, E, Number of peribronchial, PAS-positive, mucus-producing epithelial cells. Data are means ± SEMs and representative of 2 independent experiments. *P < .05 compared with HDM and **P < .05 compared with vehicle.