Rationale: Polymorphisms on chromosome 17q21 confer the major genetic susceptibility to childhood-onset asthma. Risk alleles positively correlate with ORMDL3 (orosomucoid-like 3) expression. The locus influences disease severity and the frequency of human rhinovirus (HRV)–initiated exacerbations. ORMDL3 is known to regulate sphingolipid synthesis by binding serine palmitoyltransferase, but its role in inflammation is incompletely understood.
Objectives: To investigate the role of ORMDL3 in cellular inflammation.
Methods: We modeled a time series of IL1B-induced inflammation in A549 cells, using cytokine production as outputs and testing effects of ORMDL3 siRNA knockdown, ORMDL3 overexpression, and the serine palmitoyltransferase inhibitor myriocin. We replicated selected findings in normal human bronchial epithelial cells. Cytokine and metabolite levels were analyzed by analysis of variance. Transcript abundances were analyzed by group means parameterization, controlling the false discovery rate below 0.05.
Measurements and Main Results: Silencing ORMDL3 led to steroid-independent reduction of IL6 and IL8 release and reduced endoplasmic reticulum stress after IL1B stimulation. Overexpression and myriocin conversely augmented cytokine release. Knockdown reduced expression of genes regulating host–pathogen interactions, stress responses, and ubiquitination: in particular, ORMDL3 knockdown strongly reduced expression of the HRV receptor ICAM1. Silencing led to changes in levels of transcripts and metabolites integral to glycolysis. Increased levels of ceramides and the immune mediator sphingosine-1-phosphate were also observed.
Conclusions: The results show ORMDL3 has pleiotropic effects during cellular inflammation, consistent with its substantial genetic influence on childhood asthma. Actions on ICAM1 provide a mechanism for the locus to confer susceptibility to HRV-induced asthma.
Polymorphisms within a locus on chromosome 17q21 confer the major genetic susceptibility to childhood-onset asthma. They positively correlate with the transcript abundance of ORMDL3. The locus influences disease severity and the frequency of human rhinovirus (HRV)–initiated exacerbations, which are the major causes of morbidity in childhood asthma. ORMDL3 is known to regulate de novo sphingolipid synthesis and stress responses, but systematic knowledge about its actions in inflammation is limited.
We investigated the role of ORMDL3 in cellular inflammation, using transcriptomics and metabolomics. Silencing ORMDL3 led to steroid-independent reduction in inflammatory cytokine release and reduced endoplasmic reticulum stress after proinflammatory stimuli. Transcript abundances of genes regulating host–pathogen interactions, stress responses, and ubiquitination were altered. In particular, ORMDL3 silencing strongly reduced expression of the HRV receptor ICAM1, providing a mechanism for the 17q21 locus to modify the risk of HRV-induced asthma exacerbations. Mediators of glucose metabolism and metabolites integral to glycolysis were strongly affected by knockdown, providing a potential link between metabolic disease and asthma. Knockdown increased levels of ceramides and the immune mediator sphingosine-1-phosphate during inflammation. The results show that ORMDL3 has a wide range of effects during cellular inflammation, suggesting a range of possible therapeutic benefits from accessing ORMDL3-regulated pathways.
Childhood asthma is driven by environmental and genetic influences. Asthma-associated single-nucleotide polymorphisms within the ORMDL3 (orosomucoid-like 3) asthma locus (1) are highly positively correlated with the transcript abundance of ORMDL3 (1, 2). The locus carries a population-attributable fraction for childhood-onset asthma greater than 40% (3). Alleles associated with high levels of ORMDL3 transcription confer susceptibility to human rhinovirus (HRV)–induced wheeze, which is the major cause of morbidity in children with asthma (4). Conversely, low-transcription alleles predict which children will be protected against wheeze by a rich microbial environment (5). The ORMDL3 locus also affects susceptibility to type 1 diabetes (6) and may be associated with increased body mass index in individuals with asthma (7).
Information is limited about the mechanisms through which ORMDL3 exerts these important effects, beyond that ORM family proteins have known functions as rheostats on de novo sphingolipid synthesis (8), inhibiting the de novo synthesis of sphingolipids by the human SPT (serine palmitoyltransferase) complex. ORMDL3 has also been shown to facilitate the unfolded protein response to cellular stress by influencing SERCA (sarcoplasmic/endoplasmic reticulum calcium ATPase) and endoplasmic reticulum (ER)-mediated Ca2+ flux (9).
The pulmonary epithelia are highly active immunologically (10), and genes identified by asthma genome-wide association studies often communicate epithelial damage to the adaptive immune system (3). Studies of genetically modified mice showed Ormdl3 to be induced by allergens and helper T-cell type 2 cytokines and suggested that in the lung Ormdl3 was expressed predominantly in airway epithelial cells (11, 12). In humans ORMDL3 is expressed in diverse cell types that include lymphocytes (1, 2) as well as airway epithelial cells (13).
Consequently, we systematically studied the effects of ORMDL3 on inflammation. We used an established cellular model of lung innate immunity with human A549 cells (14, 15) stimulated by the major proinflammatory cytokine IL1B (15) (Figure 1 shows our study design). In light of previous findings, we concentrated on cytokine (12), transcriptomic (1), and metabolomic (8) consequences of siRNA silencing (knockdown) of ORMDL3. Positive findings were explored in normal human bronchial epithelial (NHBE) cells. We contrasted the effects of knockdown by ORMDL3 upregulation with plasmid transfection. We also investigated the effects of the SPT inhibitor myriocin (16), to explore whether small molecules (drugs) may mimic some of the effects of high ORMDL3 levels. Enhanced expression of ORMDL3 is accompanied by poor response to inhaled corticosteroid therapy (17), and so we benchmarked the degree of cytokine reductions against dexamethasone.
Full details of methods are given in the online supplement.
A549 human lung epithelial cells (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium. NHBE cells were obtained from Lonza and were grown in bronchial epithelial medium (BEGM; Lonza).
ORMDL3 siRNAs were obtained from Dharmacon Research Inc. Nontargeting pool–negative siRNAs were used as controls. siRNA transfections of A549 and NHBE cells were performed with DharmaFECT reagent 1. siRNAs and transfection reagents were mixed according to supplier protocols, and then added to single wells for 48 hours of incubation.
The human ORMDL3 gene was amplified with template control cDNAs from Clontech. Primer sequences were as follows: F.5′-CACCATGAATGTGGGCACAGCGCACAGCGAG-3′; R.5′-TCAGTACTTATTGATTCCAAAAATC-3′. The PCR product was cloned into the pcDNA3.1 directional expression vector from Invitrogen. Plasmids were introduced with 1 μl of Lipofectamine 2000 (Invitrogen) per well, and cells were cultured for 48 hours before stimulation.
Forty-eight hours after induction of ORMDL3 and other target gene silencing, cells were starved in serum-free medium for 24 hours before stimulation with IL1B (1 ng/ml; R&D Systems) or TNF-α (tumor necrosis factor-α) (10 ng/ml; R&D Systems). Cells were collected at 0, 2, 4, 6, 8, 10, and 24 hours with supernatants harvested for cytokine measurements as previously established (15), using three replicates for cytokines and four replicates for myriocin studies and other assays as exemplified (14, 18). For metabolite screening, experiments were performed in quadruplicate and cells and supernatants were collected at 0, 2, 4, 8, and 10 hours after stimulation. For dexamethasone pretreatment, dexamethasone was given 1 hour before IL1B stimulation, and cells and supernatants from triplicate samples were collected at 10 hours. The effects of SPT inhibition were tested by the addition of 40 μM myriocin to quadruplicate samples 2 hours before IL1B stimulation. This is a commonly used protocol to inhibit de novo sphingolipid biosynthesis.
Whole-cell protein extracts were prepared with an Active Motif nuclear extract kit (Active Motif Europe). Forty micrograms of protein was separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gels (Invitrogen) and transferred to nitrocellulose membranes. After blocking for 1 hour with 5% milk, the blot was immersed with the first antibody (rabbit anti-human ORMDL3, diluted 1:500 [Abgent Cat. No. AP10739c] or mouse anti-human ICAM1, diluted 1:500 [Santa Cruz Biotechnology Cat. No. sc-8439]) and then detected with secondary antibodies conjugated to detection solutions.
Cell-free supernatants were harvested after stimulation and used for cytokine measurements. Human IL-6 and IL-8 were measured with ELISA kits (R&D Systems). Arithmetic means were compared by one-way analysis of variance (ANOVA) (IBM SPSS Statistics 24).
Global biochemical profiling from cells was performed by gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry (MS/MS) platforms (Metabolon Inc.). Active and scrambled siRNA effects on the two treatment groups were measured in quadruplicate and compared across time points of 0, 2, 4, 8, and 10 hours after stimulation with IL1B. Data were analyzed by two-way ANOVA in R (https://cran.r-project.org/). To control for multiple comparisons, results with a q value less than 0.01 are reported. Sphingolipid levels were measured by liquid chromatography–electrospray ionization–MS/MS as previously described (19) and compared by one-way ANOVA (IBM SPSS Statistics 24).
RNA samples were hybridized to Affymetrix human gene 1.1 ST arrays on the Affymetrix GeneTitan. Differential expression across 14,488 transcripts and 38 samples was assessed with limma (version 3.22.7). A baseline shift in transcription between ORMDL3 knockdown and control cells at time point 0 was assessed via group-means parameterization. P values were adjusted for multiple testing, controlling the expected false discovery rate below 0.05. The median SD of gene expression for all samples was 0.19380 (first quartile, 0.14950; third quartile, 0.26570), giving 80% power to detect a fold change of 1.59 with a single false positive given four samples per group.
A549 cells were seeded into 24-well plates and cultured as described above. Thapsigargin (1 μM; Sigma), which inhibits endoplasmic reticulum calcium ATPase, was added to the cells for 8 hours, and results for triplicate samples were analyzed by ANOVA.
This study did not involve human subjects and did not require ethics board approval.
The data sets generated and analyzed during this study are deposited in the GEO database with accession number GSE92484 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE92484).
ORMDL3 mRNA was reduced by more than 95% by treating A549 cells with 25 nM siRNA for 48 hours, without affecting viability and with concomitant reduction of the ORMDL3 protein confirmed by Western blot (Figure 2).
Compared with controls transfected with scrambled siRNA, we observed during the time course after IL1B stimulation a small initial increase in IL8 supernatant after 2 hours, followed later by a greater than 50% reduction in IL8, which was most marked at 10 hours (Figure 2). Similar small initial increases followed by substantial reductions were seen with the production of IL6 (Figure 2) and after TNF-α stimulation (data not shown). In stimulated NHBE cells, ORMDL3 silencing led to late reduction in IL8 after 16 hours (Figure 2), whereas IL6 levels showed a more marked initial increase before falling significantly below control levels at 16 hours after stimulation (Figure 2). We therefore chose 10 hours after IL1B stimulation in A549 cells as a reference point for downstream experiments.
Next we overexpressed ORMDL3 in A549 cells for 48 hours, using a pcDNA3.1 plasmid construct containing the full-length gene. This resulted in significant increases in IL8 and IL6 production after IL1B stimulation (Figure 2). Application of the SPT inhibitor myriocin likewise led to higher IL8 and IL6 release after IL1B stimulation (Figure 2), thus confirming that SPT mediated effects on cytokine production.
Glucocorticoids are a mainstay of therapy for asthma, and enhanced expression of ORMDL3 is accompanied by a poor response to inhaled corticosteroid therapy (17). We therefore tested whether ORMDL3 was active in glucocorticoid-regulated pathways by administering increasing concentrations of dexamethasone to A549 cells after IL1B stimulation, with and without ORMDL3 silencing. Marked effects of silencing on cytokine production persisted in the presence of effective doses of dexamethasone, indicating a steroid-independent effect (Figure 2).
To investigate systematically the wider effects of ORMDL3 silencing, we measured global gene expression with Affymetrix human gene 1.1 ST arrays during the same time course of IL1B-induced inflammation in A549 cells. We found that the transcript abundances of 92 genes were altered at baseline by ORMDL3 knockdown (Padjusted < 0.05). These involved innate and adaptive immunity (BIRC3, EBI3, and TNFRSF9), stress responses (HMOX1, TRPA1, GFPT2, and ANKRD1), and ubiquitination (USP46 and OSTM1) (Table 1 lists the top hit transcripts altered with Padjusted < 0.01; Table E1 in the online supplement includes all results for Padjusted < 0.05).
|Gene||Functions||FC||Average Expression||Adjusted P Value|
|ORMDL3||Asthma susceptibility gene||−3.46||6.41||1.15 × 10−3|
|DIPK2A||Regulates cellular secretory traffic||−1.90||8.21||2.95 × 10−3|
|HMOX1||Host defense against physiological insults||−1.84||11.10||6.72 × 10−3|
|USP46||Deubiquitinating cysteine protease||−1.72||8.08||2.95 × 10−3|
|OSTM1||Ubiquitin-dependent degradation of G proteins||−1.65||7.97||6.72 × 10−3|
|ZP3||Structural component of zona pellucida||−1.62||8.82||6.72 × 10−3|
|ITGA2||Mediates cellular adhesion||1.64||10.91||6.72 × 10−3|
|GALNT5||Catalyzes O-glycosylation of Golgi proteins||1.88||7.53||6.86 × 10−3|
|EBI3*||IL27: induction of inflammation||1.89||6.69||2.95 × 10−3|
|TRPA1||Receptor for airway oxidative stress||1.94||7.66||7.87 × 10−3|
|SNORD38A||Small nucleolar RNA||1.96||4.91||5.46 × 10−3|
|GFPT2*||Hyperglycemia and oxidative stress||2.01||9.95||8.54 × 10−3|
|BIRC3*||Inhibits apoptosis by binding to TRAF1 and TRAF2||2.09||8.09||2.95 × 10−3|
|KLHL4||Kelch-like family member 4||2.09||6.68||1.60 × 10−3|
|TMOD1||NF-κB activation and MMP13 induction||2.11||8.49||6.72 × 10−3|
|ANKRD1||Inhibitor of ER stress–induced apoptosis||2.25||7.77||2.95 × 10−3|
|TNFRSF9*||Enhances development of T cells and monocytes||2.71||6.62||4.25 × 10−3|
|ICAM1†||Immune synapses, adhesion: HRV receptor||−2.42||10.97||1.01 × 10−4|
|NFKB2†||Subunit of NF-κB||−1.77||11.27||8.85 × 10−3|
Transcripts altered in abundance at baseline in general remained at the same levels relative to controls during the time course after IL1B stimulation (Figure E1). Using limma, we modeled dynamic treatment effects of ORMDL3 knockdown appearing during the time course. We observed changes in transcript abundance of 24 genes (Padjusted < 0.05; 9 of the 24 genes were also altered at baseline), with functions associated with apoptosis, inflammation and NF-κB; mitogenesis and cell division; inflammation-related metabolites (NO, Zn2+, Ca2+, dopamine, and ammonia); and lipid and glucose metabolism (Table 1).
The major HRV receptor ICAM1 (intercellular adhesion molecule 1) (20) exhibited a blunted transcriptional trajectory in ORMDL3-depleted A549 cells after IL1B stimulation that was not apparent at baseline (Figure 3). We showed by Western blot that the ICAM1 protein was also downregulated at baseline and at 10 hours after IL1B stimulation (Figure 3). Conversely, administration of myriocin led to increases in ICAM1 transcription at similar times after IL1B administration (2.0-fold increase at 6 h, P = 3.5 × 10−6; 2.2-fold increase at 8 h, P = 3.2 × 10−7).
ANKRD1, upregulated 2.2-fold by ORMDL3 knockdown (Table 1), also has a recognized role in antiviral immunity and viral entry (21); TNFRSF9 (CD137, 2.7-fold increased) moderates antiviral immunity (22); and EBI3 (a subunit of IL-27 and IL-35, 1.9-fold upregulated) is implicated in immune response to various pathogens (23, 24).
Two sphingolipid pathway transcripts (the phospholipases PLA2G15 and SGPP1) were significantly downregulated by ORMDL3 knockdown, and the abundance of sphingomyelin synthase SGMS2 was moderately increased (fold change, 1.36; Padjusted < 0.05; Table E1). Examination of other sphingolipid pathway genes (Table E2) showed ORMDL3 silencing not to induce compensatory increases in transcript abundances of ORMDL1 and ORMDL2 and other major sphingolipid modulators.
Global metabolomic profiling revealed that glucose and glycolytic intermediates including glucose 6-phosphate and lactate were significantly elevated in the ORMDL3 siRNA–treated samples at baseline and remained higher after IL1B stimulation (Figure 4 and Table 2). IL1B elevated these metabolites in both groups. Sorbitol and pentose phosphate pathway metabolites including ribose were similarly at higher levels in the ORMDL3 knockdown compared with controls, consistent with an observed increase in NADPH generation (Figure 4; Table 2 for changes with Padjusted < 0.01). The concomitant elevation in transcript abundance of GFPT2 (glutamine–fructose-6-phosphate transaminase 2) (Table 1) suggests that this gene may contribute to this effect. In addition, we saw levels of lysophospholipids become elevated compared with controls during the time course after IL1B stimulation (Figure 4 and Table 2), as were the amino acids kynurenine and creatine (Table 2).
|Pathway||Biochemical Name||P Value||q Value||Ratio 8 h|
|Glucose 6-phosphate (G6P)||<0.001||0.0007||2.07|
|Fructose 1,6-diphosphate, glucose 1,6-diphosphate||<0.001||0.0003||1.79|
|Nucleotide||Adenosine 2′-monophosphate (2′-AMP)||<0.001||0.0001||1.46|
|Cofactor||Nicotinamide adenine dinucleotide reduced (NADH)||0.0018||0.0067||1.65*|
Our metabolomic analyses of sphingolipids revealed that ORMDL3 silencing increased high molecular weight (C24:1, C24:0, and C26:1) ceramide levels in A549 and NHBE cells (Figure 5) at baseline (t = 0). IL1B stimulation reduced ceramide levels in both cell types, although the effects were more marked in NHBE cells. The ceramide reduction was substantially opposed by ORMDL3 knockdown at 10 hours in A549 cells and at 24 hours in NHBE cells (Figure 5).
We did not detect changes in sphinganine, dihydroceramides, sphingosine, or ceramide 1-phosphate. Statistically significant increases were observed in sphingosine-1-phosphate (S1P) levels (Figure 5), although the concentrations were at the lower limit of sensitivity of the assay.
Silencing of SPHK1 and SPHK2, which synthesize S1P, marginally increased IL6 production, whereas knockdown of SGPP2 (but not SGPP1), which degrade S1P, reduced cytokine production (Figure 6). These findings are consistent with the recognized antiinflammatory signal mediation by S1P (25).
The previously described facilitation of the unfolded protein response to cellular stress by ORMDL3 (9) was consistent with our global gene expression results (Table 1). We therefore induced ER stress by adding the SERCA inhibitor thapsigargin to ORMDL3-silenced cells. ORMDL3 knockdown corrected a thapsigargin-induced reduction of ceramide levels (Figure 5). These results confirm that ORMDL3 is a regulator of ER stress and add weight to the suggestion that ceramide or its metabolites may mediate this effect.
The results implicate manifold actions of ORMDL3 on pathways for epithelial–microbial interactions, inflammation, and glucose metabolism that are in addition to its recognized inhibition of de novo ceramide synthesis. ORM proteins are highly conserved in eukaryotes, which taken with our findings might suggest an ancient role for them in coordinating metabolism under conditions of stress.
Early HRV infection is a marker of subsequent asthma, and significant increases in the number of childhood HRV wheezing illnesses are observed in children with enhanced transcription genotypes at the 17q21 ORMDL3 locus (4). Our finding that ORMDL3 knockdown decreased the transcript and protein expression of ICAM1, the major HRV receptor (20), suggests a mechanism for the association between ORMDL3, HRV infections, and asthma. In addition, ICAM1 is a receptor for the bacterial airway pathogen Haemophilus influenzae (26), which is increased in abundance in asthmatic airways between exacerbations (27), potentially providing positive feedback between chronic bacterial colonization and acute viral infections.
ORMDL3 silencing affected gluconeogenesis during inflammation. We observed concomitant upregulation in the transcription of GFPT2 (Table 1), which is associated in diabetics with insulin resistance, hyperglycemia, and oxidative stress (28). Obesity and asthma form a complex multifactorial syndrome with poor responses to therapy (29, 30). Our results may suggest a mechanism for a reported genetic component to obese asthma from the ORMDL3 locus (7).
Taken in the context of the reduced airway responsiveness observed in Ormdl3 knockout mice (12) and the increased responsiveness of Spt+/− knockout (31) and Ormdl3 transgenic mice (12, 32), our study identified candidates that may regulate the nonspecific airway hyperresponsiveness that accompanies human asthma (33). TRPA1 is a receptor that recognizes oxidative stress and modulates airway hyperresponsiveness (34), and ATP2B4 varies the production of the bronchodilator nitric oxide (35). The elevated metabolite kynurenine is a vasodilator and immune signaling molecule (36) that by inference may also alter bronchial smooth muscle tone.
ORMDL3 is known to inhibit de novo synthesis of sphingolipids by SPT (8). We found that ORMDL3 knockdown increased intracellular levels of ceramide, some lysophospholipids, and S1P. We were not able to distinguish completely between direct effects of these sphingolipids on the various changes we have observed and as yet unknown consequences of ORMDL3 activation, but our administration of myriocin and knockdowns of other pathway enzymes support central roles for sphingolipids in the observed effects of ORMDL3 on inflammation and metabolism.
The results of our study should be considered in light of several limitations. Ceramide synthesis is known to be multifaceted, and ceramide effects vary in different cellular compartments (25) that we have not tested independently in our experiments. We have studied mostly A549 cells, because the scope of our experiments required cell lines that grow robustly and at a reasonable cost, and because there is a substantial literature underpinning A549 cell use in inflammation research (14, 18). A549 cells are, however, derived from an alveolar malignancy, so in selected experiments we investigated NHBE cells to represent bronchial epithelium. Primary NHBE cells grow much more slowly than malignant A549 cells, complicating direct comparisons. The results from both cell types nevertheless show that ORMDL3 knockdown reduced late cytokine responses to IL1B, although an early increase in supernatant cytokine release was more marked after knockdown in NHBE cells. NHBE cells also showed larger increases than A549 cells in C24 ceramides after knockdown, but concomitant downstream increases in S1P were qualitatively similar. Fully differentiated NHBE cells, which best reflect the airway epithelium, may show further differences. Further caution should be applied to extrapolating the results to the complex mixed cell interactions that are present in asthmatic airway inflammation.
Polymorphisms within the ORMDL3 locus carry the highest known risk for childhood asthma (37). Genetic associations between disease and a drug target more than double the chance of success in phase 2 trials (38, 39). Our results suggest a range of possible therapeutic benefits from accessing ORMDL3-regulated pathways on airway viral infection, inflammation, and bronchial responsiveness. S1P is a recognized target for therapy of inflammatory disorders (40, 41) and manipulation of other steps in sphingolipid assembly may be of therapeutic benefit (42). Investigation of upstream regulatory phosphorylation of ORMDL3 (8) and its interactions with SPT may also provide novel opportunities.
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*Joint senior authors.
Supported by the Wellcome Trust under grants WT097117 and WT096964, and through an unrestricted grant from Merck and Co. Y.Z. is an Asmarley Lecturer. M.F.M. and W.O.C.M.C. are Joint Wellcome Trust Senior Investigators. C.M.L. is a Wellcome Senior Fellow. S.S. was supported by NIH grant 5R01AI125433.
Author Contributions: Y. Z., M.F.M., and W.O.C.M.C. planned the overall study with input from C.M.L. Y.Z. designed individual experimental components with advice from M.F.M. and performed all the experimental work except metabolomic measurements. S.S. provided assays of complex sphingolipids and advice on sphingolipid metabolism and the interpretation of results. Y.Z. and S.A.G.W.-O. performed statistical analyses of the data with input from W.O.C.M.C. Y.Z. wrote the first draft of the paper.
This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.
Originally Published in Press as DOI: 10.1164/rccm.201803-0438OC on October 19, 2018