ORMDL3 regulates NLRP3 inflammasome activation by maintaining ER-mitochondria contacts in human macrophages and dictates ulcerative colitis patient outcome.

Genome-wide association studies in inflammatory bowel disease have identified risk loci in the orosomucoid-like protein 3/ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3) gene to confer susceptibility to ulcerative colitis (UC), but the underlying functional relevance remains unexplored. Here, we found that a subpopulation of the UC patients who had higher disease activity shows enhanced expression of ORMDL3 compared to the patients with lower disease activity and the non-UC controls. We also found that the patients showing high ORMDL3 mRNA expression have elevated interleukin-1ß cytokine levels indicating positive correlation. Further, knockdown of ORMDL3 in the human monocyte-derived macrophages resulted in significantly reduced interleukin-1ß release. Mechanistically, we report for the first time that ORMDL3 contributes to a mounting inflammatory response via modulating mitochondrial morphology and activation of the NLRP3 inflammasome. Specifically, we observed an increased fragmentation of mitochondria and enhanced contacts with the endoplasmic reticulum (ER) during ORMDL3 over-expression, enabling efficient NLRP3 inflammasome activation. We show that ORMDL3 that was previously known to be localized in the ER also becomes localized to mitochondria-associated membranes and mitochondria during inflammatory conditions. Additionally, ORMDL3 interacts with mitochondrial dynamic regulating protein Fis-1 present in the mitochondria-associated membrane. Accordingly, knockdown of ORMDL3 in a dextran sodium sulfate -induced colitis mouse model showed reduced colitis severity. Taken together, we have uncovered a functional role for ORMDL3 in mounting inflammation during UC pathogenesis by modulating ER-mitochondrial contact and dynamics.

Neutrophilia in severe asthma is reduced in Ormdl3 overexpressing mice.

Genome-wide association studies have linked the ORM (yeast)-like protein isoform 3 (ORMDL3) to asthma severity. Although ORMDL3 is a member of a family that negatively regulates serine palmitoyltransferase (SPT) and thus biosynthesis of sphingolipids, it is still unclear whether ORMDL3 and altered sphingolipid synthesis are causally related to non-Th2 severe asthma associated with a predominant neutrophil inflammation and high interleukin-17 (IL-17) levels. Here, we examined the effects of ORMDL3 overexpression in a preclinical mouse model of allergic lung inflammation that is predominantly neutrophilic and recapitulates many of the clinical features of severe human asthma. ORMDL3 overexpression reduced lung and circulating levels of dihydrosphingosine, the product of SPT. However, the most prominent effect on sphingolipid levels was reduction of circulating S1P. The LPS/OVA challenge increased markers of Th17 inflammation with a predominant infiltration of neutrophils into the lung. A significant decrease of neutrophil infiltration was observed in the Ormdl3 transgenic mice challenged with LPS/OVA compared to the wild type and concomitant decrease in IL-17, that plays a key role in the pathogenesis of neutrophilic asthma. LPS decreased survival of murine neutrophils, which was prevented by co-treatment with S1P. Moreover, S1P potentiated LPS-induced chemotaxis of neutrophil, suggesting that S1P can regulate neutrophil survival and recruitment following LPS airway inflammation. Our findings reveal a novel connection between ORMDL3 overexpression, circulating levels of S1P, IL-17 and neutrophil recruitment into the lung, and questions the potential involvement of ORMDL3 in the pathology, leading to development of severe neutrophilic asthma.

Histone acetylation regulates ORMDL3 expression-mediated NLRP3 inflammasome overexpression during RSV-allergic exacerbation mice.

Whether respiratory syncytial virus (RSV) infection in early life may induce orosomucoid 1-like protein 3 (ORMDL3) and lead to NOD-like receptor protein 3 (NLRP3) inflammasome overexpression in asthma, which could be alleviated by the inhibition of HAT p300. First, we explored the relationship between RSV, ORMDL3, and recurrent wheezing in the future through clinical data of infants with RSV-induced bronchiolitis. Then, we used bronchial epithelium transformed with Ad12-SV40 2B (BEAS-2B) and an asthmatic mouse model of repeated RSV infection and OVA sensitization and challenge (rRSV + OVA) in early life to assess the effects of ORMDL3 on NLRP3 inflammasome and that of histone acetylation on ORMDL3 regulation. ORMDL3 overexpression is the independent risk factor of recurrent wheezing in RSV-bronchiolitis follow-up. In BEAS-2B, ORMDL3-induced NLRP3 inflammasome expression. BEAS-2B infected by RSV resulted in overexpression of ORMDL3 and NLRP3 inflammasome and histone hyperacetylation, while ORMDL3-small interfering RNA and C646 interfered could decrease NLRP3 inflammasome. ORMDL3 overexpression in mouse lung increased NLRP3 inflammasome. The expression of ORMDL3 and NLRP3 inflammasome significantly increased, with histone hyperacetylation in the lung in rRSV + OVA mice. p300 and acetylH3 bound to ORMDL3 promoter. In C646 + rRSV + OVA mice, C646 alleviated lung inflammation and overexpression of ORMDL3 and NLRP3 inflammasome. RSV activated ORMDL3 overexpression through histone hyperacetylation and induced NLRP3 inflammasome expression.

Increased expression of ORMDL3 in allergic asthma: a case control and in vitro study.

BACKGROUND: Asthma is the most frequent chronic disease in children. One of the most replicated genetic findings in childhood asthma is the ORMDL3 gene confirmed in several GWA studies in several pediatric populations. OBJECTIVES: The purpose of this study was to analyze ORMDL3 variants and expression in childhood asthma in the Polish population. METHODS: In the study we included 416 subject, 223 asthmatic children and 193 healthy control subjects. The analysis of two SNPs (rs3744246 and rs8076131) was performed using genotyping with TaqMan probes. The methylation of the ORMDL3 promoter was examined with Methylation Sensitive HRM (MS-HRM), covering 9 CpG sites. The expression of ORMDL3 was analyzed in PBMCs from pediatric patients diagnosed with allergic asthma and primary human bronchial epithelial cells derived from healthy subjects treated with IL-13, IL-4, or co-treatment with both cytokines to model allergic airway inflammation. RESULTS: We found that ORMDL3 expression was increased in allergic asthma both in PBMCs from asthmatic patients as well as in human bronchial epithelial cells stimulated with the current cytokines. We did not observe significant differences between cases and controls either in the genotype distribution of analyzed SNPs (rs3744246 and rs8076131) nor in the level of promoter methylation. CONCLUSIONS: Increased ORMDL3 expression is associated with pediatric allergic asthma and upregulated in the airways upon Th2-cytokines stimulation, but further functional studies are required to fully understand its role in this disease.

ORMDL3 regulates cigarette smoke-induced endoplasmic reticulum stress in airway smooth muscle cells.

BACKGROUND: Orosomucoid 1-like protein 3 (ORMDL3), a transmembrane protein localized in the endoplasmic reticulum (ER), has been genetically associated with chronic obstructive pulmonary disease (COPD), in addition to childhood-onset asthma. However, the functional role of ORMDL3 in the pathogenesis of COPD is still unknown. OBJECTIVE: Because cigarette smoke is the major risk factor for COPD, we aimed to investigate the role of ORMDL3 in cigarette smoke-induced human airway smooth muscle cell (HASMC) injury. METHODS: The mRNA and protein expression of ORMDL3 was examined in HASMCs from nonsmokers and smokers without or with COPD. Knockdown of ORMDL3 in primary healthy HASMCs was performed using small interfering RNA before exposure to cigarette smoke medium (CSM) for 24 hours. Inflammatory, proliferative/apoptotic, ER stress, and mitochondrial markers were evaluated. RESULTS: Elevation of ORMDL3 mRNA and protein expression was observed in HASMCs of smokers without or with COPD. CSM caused significant upregulation of ORMDL3 expression in healthy nonsmokers. ORMDL3 knockdown regulated CSM-induced inflammation, cell proliferation, and apoptosis. Silencing ORMDL3 led to reduction of CSM-induced ER stress via inhibition of unfolded protein response pathways such as activating transcription factor 6 and protein kinase RNA-like ER kinase. ORMDL3 was also involved in CSM-induced mitochondrial dysfunction via the mitochondrial fission process. CONCLUSIONS: We report the induction of ORMDL3 in HASMCs after cigarette smoke exposure. ORMDL3 may mediate cigarette smoke-induced activation of unfolded protein response pathways during airway smooth muscle cell injury.

Single-base editing of rs12603332 on chromosome 17q21 with a cytosine base editor regulates ORMDL3 and ATF6α expression.

BACKGROUND: Genetic association studies have demonstrated that the SNP rs12603332 located on chromosome 17q21 is highly associated with the risk of the development of asthma. METHODS: To determine whether SNP rs1260332 is functional in regulating levels of ORMDL3 expression, we used a Cytosine Base Editor (CBE) plasmid DNA or a CBE mRNA to edit the rs12603332 C risk allele to the T non-risk allele in a human lymphocyte cell line (i.e., Jurkat cells) and in primary human CD4 T cells that carry the C risk alleles. RESULTS: Jurkat cells with the rs12603332 C risk allele expressed significantly higher levels of ORMDL3 mRNA, as well as the ORMDL3 regulated gene ATF6α as assessed by qPCR compared to Jurkat clones with the T non-risk allele. In primary human CD4 T cells, we edited 90 ± 3% of the rs12603332-C risk allele to the T non-risk allele and observed a reduction in ORMDL3 and ATF6α expression. Bioinformatic analysis predicted that the non-risk allele rs12603332-T could be the central element of the E-box binding motif (CANNTG) recognized by the E47 transcription factor. An EMSA assay confirmed the bioinformatics prediction demonstrating that a rs12603332-T containing probe bound to the transcription factor E47 in vitro. CONCLUSIONS: SNP rs12603332 is functional in regulating the expression of ORMDL3 as well as ORMDL3 regulated gene ATF6α expression. In addition, we demonstrate the use of CBE technology in functionally interrogating asthma-associated SNPs using studies of primary human CD4 cells.

Mapping the 17q12-21.1 Locus for Variants Associated with Early-Onset Asthma in African Americans.

Rationale: The 17q12-21.1 locus is one of the most highly replicated genetic associations with asthma. Individuals of African descent have lower linkage disequilibrium in this region, which could facilitate identifying causal variants.Objectives: To identify functional variants at 17q12-21.1 associated with early-onset asthma among African American individuals.Methods: We evaluated African American participants from SAPPHIRE (Study of Asthma Phenotypes and Pharmacogenomic Interactions by Race-Ethnicity) (n = 1,940), SAGE II (Study of African Americans, Asthma, Genes and Environment) (n = 885), and GCPD-A (Study of the Genetic Causes of Complex Pediatric Disorders-Asthma) (n = 2,805). Associations with asthma onset at ages under 5 years were meta-analyzed across cohorts. The lead signal was reevaluated considering haplotypes informed by genetic ancestry (i.e., African vs. European). Both an expression-quantitative trait locus analysis and a phenome-wide association study were performed on the lead variant.Measurements and Main Results: The meta-analyzed results from SAPPHIRE, SAGE II, and the GCPD-A identified rs11078928 as the top association for early-onset asthma. A haplotype analysis suggested that the asthma association partitioned most closely with the rs11078928 genotype. Genetic ancestry did not appear to influence the effect of this variant. In the expression-quantitative trait locus analysis, rs11078928 was related to alternative splicing of GSDMB (gasdermin-B) transcripts. The phenome-wide association study of rs11078928 suggested that this variant was predominantly associated with asthma and asthma-associated symptoms.Conclusions: A splice-acceptor polymorphism appears to be a causal variant for asthma at the 17q12-21.1 locus. This variant appears to have the same magnitude of effect in individuals of African and European descent.

Sphingolipids and Asthma.

Asthma is the most prevalent chronic respiratory disease worldwide and the leading serious chronic illness in children. Clinical characteristics are wheezing, reversible airway obstruction, airway inflammation, and airway hyperreactivity. Asthma susceptibility is influenced by genes and environment. 17q12-21 is the most significant genetic asthma susceptibility locus and single nucleotide polymorphisms (SNPs) within that high-risk locus are linked to increased expression of the Ormdl sphingolipid biosynthesis regulator (ORMDL) 3. ORMDL3 is an endoplasmic reticulum protein that stabilizes the serine palmitoyl transferase (SPT) complex that regulates sphingolipid de novo synthesis. Sphingolipids essential for formation and integrity of cellular membranes and bioactive molecules that regulate key cellular processes can be synthesized de novo and through recycling pathways. Their metabolism is tightly regulated through feedback regulation. ORMDL3 inhibits de novo synthesis when it engages subunit 1 of the SPT complex. This chapter focuses on the effect of decreased sphingolipid synthesis on asthma features and summarizes studies in mouse models and in children with and without asthma.

Single cell sequencing analysis identifies genetics-modulated ORMDL3+ cholangiocytes having higher metabolic effects on primary biliary cholangitis.

BACKGROUND: Primary biliary cholangitis (PBC) is a classical autoimmune disease, which is highly influenced by genetic determinants. Many genome-wide association studies (GWAS) have reported that numerous genetic loci were significantly associated with PBC susceptibility. However, the effects of genetic determinants on liver cells and its immune microenvironment for PBC remain unclear. RESULTS: We constructed a powerful computational framework to integrate GWAS summary statistics with scRNA-seq data to uncover genetics-modulated liver cell subpopulations for PBC. Based on our multi-omics integrative analysis, 29 risk genes including ORMDL3, GSNK2B, and DDAH2 were significantly associated with PBC susceptibility. By combining GWAS summary statistics with scRNA-seq data, we found that cholangiocytes exhibited a notable enrichment by PBC-related genetic association signals (Permuted P < 0.05). The risk gene of ORMDL3 showed the highest expression proportion in cholangiocytes than other liver cells (22.38%). The ORMDL3+ cholangiocytes have prominently higher metabolism activity score than ORMDL3- cholangiocytes (P = 1.38 × 10-15). Compared with ORMDL3- cholangiocytes, there were 77 significantly differentially expressed genes among ORMDL3+ cholangiocytes (FDR < 0.05), and these significant genes were associated with autoimmune diseases-related functional terms or pathways. The ORMDL3+ cholangiocytes exhibited relatively high communications with macrophage and monocyte. Compared with ORMDL3- cholangiocytes, the VEGF signaling pathway is specific for ORMDL3+ cholangiocytes to interact with other cell populations. CONCLUSIONS: To the best of our knowledge, this is the first study to integrate genetic information with single cell sequencing data for parsing genetics-influenced liver cells for PBC risk. We identified that ORMDL3+ cholangiocytes with higher metabolism activity play important immune-modulatory roles in the etiology of PBC.

ORMDL3 regulates poly I:C induced inflammatory responses in airway epithelial cells.

BACKGROUND: Oroscomucoid 3 (ORMDL3) has been linked to susceptibility of childhood asthma and respiratory viral infection. Polyinosinic-polycytidylic acid (poly I:C) is a synthetic analog of viral double-stranded RNA, a toll-like receptor 3 (TLR3) ligand and mimic of viral infection. METHODS: To investigate the functional role of ORMDL3 in the poly I:C-induced inflammatory response in airway epithelial cells, ORMDL3 knockdown and over-expression models were established in human A549 epithelial cells and primary normal human bronchial epithelial (NHBE) cells. The cells were stimulated with poly I:C or the Th17 cytokine IL-17A. IL-6 and IL-8 levels in supernatants,  mRNA levels of genes in the TLR3 pathway and inflammatory response from cell pellets were measured. ORMDL3 knockdown models in A549 and BEAS-2B epithelial cells were then infected with live human rhinovirus (HRV16) followed by IL-6 and IL-8 measurement. RESULTS: ORMDL3 knockdown and over-expression had little influence on the transcript levels of TLR3 in airway epithelial cells. Time course studies showed that ORMDL3-deficient A549 and NHBE cells had an attenuated IL-6 and IL-8 response to poly I:C stimulation. A549 and NHBE cells over-expressing ORMDL3 released relatively more IL-6 and IL-8 following poly I:C stimulation. IL-17A exhibited a similar inflammatory response in ORMDL3 knockdown and over-expressing cells, but co-stimulation of poly I:C and IL-17A did not significantly enhance the IL-6 and IL-8 response. Transcript abundance of IFNB following poly I:C stimulation was not significantly altered by ORMDL3 knockdown or over-expression. Dampening of the IL-6 response by ORMDL3 knockdown was confirmed in HRV16 infected BEAS-2B and A549 cells. CONCLUSIONS: ORMDL3 regulates the viral inflammatory response in airway epithelial cells via mechanisms independent of the TLR3 pathway.

ORMDL3/GSDMB genotype as a risk factor for early-onset adult asthma is linked to total serum IgE levels but not to allergic sensitization.

BACKGROUND: An orosomucoid-like 3 (ORMDL3)/gasdermin B (GSDMB) gene locus on chromosome 17q is consistently associated with childhood-onset asthma, which is highly atopic. As some evidence suggests the relationship between asthma and allergic sensitization reflects asthma patient susceptibility to augmented IgE responses driven by common environmental allergens rather than an increased asthma risk after allergen exposure, we aimed to determine any relationships between this locus region and childhood-onset adult asthma with regard to serum total IgE levels or allergic sensitization. METHODS: We conducted a case-control association study using three independent Japanese populations (3869 total adults) and analyzed the ORs for association of rs7216389, an expression quantitative trait locus for ORMDL3/GSDMB, with adult asthma according to onset age. Additionally, associations between the rs7216389 genotype and total serum IgE levels or allergic sensitization was examined. RESULTS: Rs7216389 was associated with both childhood-onset adult asthma (OR for asthmatic patients afflicted at the age of 10 years or younger = 1.61, p = 0.00021) and asthmatic patients with higher levels of total serum IgE (OR for asthmatic patients with IgE ≥1000IU/mL = 1.55, p = 0.0033). In both healthy controls and in the combined healthy and asthmatic individuals, rs7216389 was correlated with increased total serum IgE levels (p < 0.0005), but not allergic sensitization (p > 0.1). CONCLUSIONS: ORMDL3/GSDMB is an important susceptibility gene for childhood-onset adult asthma in Japanese populations and this association is linked to elevated total serum IgE levels but not to allergic sensitization.

Orosomucoid-like 3 Supports Rhinovirus Replication in Human Epithelial Cells.

Polymorphism at the 17q21 gene locus and wheezing responses to rhinovirus (RV) early in childhood conspire to increase the risk of developing asthma. However, the mechanisms mediating this gene-environment interaction remain unclear. In this study, we investigated the impact of one of the 17q21-encoded genes, ORMDL3 (orosomucoid-like 3), on RV replication in human epithelial cells. ORMDL3 knockdown inhibited RV-A16 replication in HeLa, BEAS-2B, A549, and NCI-H358 epithelial cell lines and primary nasal and bronchial epithelial cells. Inhibition varied by RV species, as both minor and major group RV-A subtypes RV-B52 and RV-C2 were inhibited but not RV-C15 or RV-C41. ORMDL3 siRNA did not affect expression of the major group RV-A receptor ICAM-1 or initial internalization of RV-A16. The two major outcomes of ORMDL3 activity, SPT (serine palmitoyl-CoA transferase) inhibition and endoplasmic reticulum (ER) stress induction, were further examined: silencing ORMDL3 decreased RV-induced ER stress and IFN-ß mRNA expression. However, pharmacologic induction of ER stress and concomitant increased IFN-ß inhibited RV-A16 replication. Conversely, blockade of ER stress with tauroursodeoxycholic acid augmented replication, pointing to an alternative mechanism for the effect of ORMDL3 knockdown on RV replication. In comparison, the SPT inhibitor myriocin increased RV-A16 but not RV-C15 replication and negated the inhibitory effect of ORMDL3 knockdown. Furthermore, lipidomics analysis revealed opposing regulation of specific sphingolipid species (downstream of SPT) by myriocin and ORMDL3 siRNA, correlating with the effect of these treatments on RV replication. Together, these data revealed a requirement for ORMDL3 in supporting RV replication in epithelial cells via SPT inhibition.

Long noncoding RNA SOX21-AS1 regulates the progression of triple-negative breast cancer through regulation of miR-520a-5p/ORMDL3 axis.

Recently, long noncoding RNAs (lncRNAs) have been reported as a new kind of controllers about cancer processes in biology. In spite of the dysregulation of lncRNAs in various kinds of cancers, only a little of the information was effective on the expression configuration and inner effects of lncRNAs in triple-negative breast cancer (TNBC). This study valued the expression of lncRNA SOX21-AS1 and the biological role it played in TNBC. In our research, SOX21-AS1 had a high expression in TNBC cell lines. The functional experiments showed that knockdown of SOX21-AS1 obviously restrained cell proliferation, migration, invasion, and epithelial-mesenchymal transition process and promoted cell apoptosis. Mechanistically, SOX21-AS1 was found to bind with miR-520a-5p. Besides, ORMDL3 was identified as a downstream target of miR-520a-5p, and the suppressed ORMDL3 expression induced by silenced SOX21-AS1 could be restored by miR-520a-5p inhibition. Further, data from rescue assays revealed that SOX21-AS1 could regulate the malignancy of TNBC via miR-520a-5p/ORMDL3 axis. All in all, we identified that SOX21-AS1 regulated the cellular process of TNBC cells via antagonizing miR-520a-5p availability to upregulate ORMDL3 expression.

ORMDL3 modulates airway epithelial cell repair in children with asthma under glucocorticoid treatment via regulating IL-33.

BACKGROUND: Study found that glucocorticoids, as first-line treatments for asthma, fails to prevent asthma recurrence. Orosomucoid-like (ORMDL) 3 is associated to childhood asthma onset and involved in the inflammation and repair of airway epithelium. We explored the functional role of ORMDL3 in glucocorticoid treatment for childhood asthma. METHODS: Mice were sensitized with Ovalbumin (OVA) and treated with Dexamethasone (Dex), followed by OVA challenge to establish a mouse model of asthma. Histopathological changes in lung tissues were observed by hematoxylin-eosin and masson staining. Human bronchial epithelial (16HBE-14°) cells were transfected with ORMDL3 overexpression plasmid and siRNA-interleukin (IL)-33 alone or in combination, followed by Dex. Cell viability was measured by MTT assay. Cell migration was evaluated by wound healing assay. The expressions of E-cadherin and Vimentin and the activation of NF-κB and MAPK/ERK in 16HBE-14° cells were assessed by Western blot. The expressions of ORMDL3 and IL-33 in lung tissues and 16HBE-14° cells were analyzed by qRT-PCR or Western blot. RESULTS: Dex treatment alleviated the histopathological abnormality and reversed the overexpressions of ORMDL3 and IL-33 in the lung tissues of asthmatic mice. Overexpressed ORMDL3 enhanced migration and viability, decreased E-cadherin level, increased the levels of IL-33 and Vimentin, and promoted the phosphorylation of NF-κB and MAPK/ERK in Dex-treated 16HBE-14° cells, thus reversing the effect of Dex treatment. However, siRNA-IL-33 inhibited viability and migration, increased E-cadherin level, decreased Vimentin level, and suppressed the phosphorylation of NF-κB and MAPK/ERK, thus reversing the effect of overexpressed ORMDL3 in Dex-treated 16HBE-14° cells. CONCLUSION: ORMDL3 overexpression helped airway epithelial cellrepairin asthma via regulating IL-33 expression.

Pulmonary delivery of ORMDL3 short hairpin RNA - a potential tool to regulate allergen-induced airway inflammation.

Aim/Purpose: Exposure to various allergens has been shown to increase expression of ORMDL3 in the lung in models of allergic asthma. Studies using genetically modified (transgenic or knock out) mice have revealed some of the functions of ORMDL3 in asthma pathogenesis, although amid debate. The goal of this study was to use targeted post-transcriptional downregulation of ORMDL3 in allergen-challenged wild-type (WT) mice by RNA interference to further elucidate the functional role of ORMDL3 in asthma pathogenesis and evaluate a potential therapeutic option.Methods: Allergen (ovalbumin [OVA])-challenged WT mice were administered intranasally (i.n) with a single dose of five short hairpin RNA (shRNA) constructs with different target sequence for murine ORMDL3 cloned in a lentiviral vector or with the empty vector (control). Mice were evaluated for allergen-induced airway hyperresponsiveness (AHR) and various features of airway inflammation after 72 hours.Results: I.n administration of a single dose of ORMDL3 shRNAs to OVA-challenged mice resulted in reduction of ORMDL3 gene expression in the lungs associated with a significant reduction in AHR to inhaled methacholine and in the number of inflammatory cells recruited in the airways, specifically eosinophils, as well as in airway mucus secretion compared to OVA-challenged mice that received the empty vector. Administration of ORMDL3 shRNAs also significantly inhibited levels of IL-13, eotaxin-2 and sphingosine in the lungs. Additionally, ORMDL3 shRNAs significantly inhibited the allergen-mediated increase in monohexyl ceramides C22:0 and C24:0.Conclusions: Post-transcriptional down regulation of ORMDL3 in allergic lungs using i.n-delivered ORMDL3 shRNA (akin to inhaled therapy) attenuates development of key features of airway allergic disease, confirming the involvement of ORMDL3 in allergic asthma pathogenesis and serving as a model for a potential therapeutic strategy.

MiR-200a and miR-200b restrain inflammation by targeting ORMDL3 to regulate the ERK/MMP-9 pathway in asthma.

BACKGROUND: Asthma is one of the most frequent and serious diseases worldwide. Inflammation has been reported to correlate with airway remodeling, which is critical for the progression of asthma. Better understanding of novel molecules modulating asthma and the underlying mechanism will benefit explorations of new treatments. Method: To explore the role of miR-200a and miR-200b in asthma, miR-200a mimics/inhibitor and miR-200b mimics/inhibitor were employed in A549 cells, respectively. Expression levels of inflammatory cytokines, including TNF-α, IL-4, IL-5, IL-13 and IL-1ß, were measured by quantitative real time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). A dual luciferase reporter assay was performed to identify whether miR-200a/200b directly bound to Orosomucoid 1-like 3 (ORMDL3). ERK, p-ERK and MMP-9, involved in downstream pathways of ORMDL3, were detected using qRT-PCR and western blotting. Results: MiR-200a/200b silencing significantly increased the expression of inflammatory cytokines, including TNF-α, IL-4, IL-5, IL-13 and IL-1ß, in A549 cells. ORMDL3 was the target gene of miR-200a/200b, with high expression levels in miR-200a inhibitor and miR-200b inhibitor groups. MiR-200a and miR-200b played synergistic roles in the regulation of the inflammatory effect in A549 cells. Expression levels of p-ERK and MMP-9 were significantly increased in miR-200a inhibitor and miR-200b inhibitor groups and were rescued by ERK inhibitor and MMP-9 inhibitor, respectively. Conclusion: These findings suggest that miR-200a and miR-200b are required to regulate asthma inflammation. Reduction in miR-200a/200b promotes the development of asthma inflammation by targeting ORMDL3 to activate the ERK/MMP-9 pathway. Therefore, elevating miR-200a and miR-200b and decreasing ORMDL3 might be potential strategies for inhibition of the asthma process.

The ORMDL3 Asthma Gene Regulates ICAM1 and Has Multiple Effects on Cellular Inflammation.

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.

The ORMDL3 asthma susceptibility gene regulates systemic ceramide levels without altering key asthma features in mice.

BACKGROUND: Genome-wide association studies in asthma have repeatedly identified single nucleotide polymorphisms in the ORM (yeast)-like protein isoform 3 (ORMDL3) gene across different populations. Although the ORM homologues in yeast are well-known inhibitors of sphingolipid synthesis, it is still unclear whether and how mammalian ORMDL3 regulates sphingolipid metabolism and whether altered sphingolipid synthesis would be causally related to asthma risk. OBJECTIVE: We sought to examine the in vivo role of ORMDL3 in sphingolipid metabolism and allergic asthma. METHODS: Ormdl3-LacZ reporter mice, gene-deficient Ormdl3-/- mice, and overexpressing Ormdl3Tg/wt mice were exposed to physiologically relevant aeroallergens, such as house dust mite (HDM) or Alternaria alternata, to induce experimental asthma. Mass spectrometry-based sphingolipidomics were performed, and airway eosinophilia, TH2 cytokine production, immunoglobulin synthesis, airway remodeling, and bronchial hyperreactivity were measured. RESULTS: HDM challenge significantly increased levels of total sphingolipids in the lungs of HDM-sensitized mice compared with those in control mice. In Ormdl3Tg/wt mice the allergen-induced increase in lung ceramide levels was significantly reduced, whereas total sphingolipid levels were not affected. Conversely, in liver and serum, levels of total sphingolipids, including ceramides, were increased in Ormdl3-/- mice, whereas they were decreased in Ormdl3Tg/wt mice. This difference was independent of allergen exposure. Despite these changes, all features of asthma were identical between wild-type, Ormdl3Tg/wt, and Ormdl3-/- mice across several models of experimental asthma. CONCLUSION: ORMDL3 regulates systemic ceramide levels, but genetically interfering with Ormdl3 expression does not result in altered experimental asthma.

Methylation status of ORMDL3 regulates cytokine production and p-ERK/MMP9 pathway expression.

Orosomucoid like-3 (ORMDL3) has been identified to be associated with the development of asthma according to previous studies. However, the definite role of ORMDL3 in the pathogenesis of asthma remains unclear. In this study, we found ORMDL3 was highly expressed in PBMC specimens from childhood asthma patients. Cytokines production and p-ERK/MMP-9 pathway expression was also increased in childhood asthma patients compared with controls. In addition, ORMDL3 overexpression induced IL-6 and IL-8 release and activated p-ERK/MMP-9 pathway in vitro. Increased ORMDL3 expression was observed after treated with 5-Aza-CdR. 5-Aza-CdR decreased the percentage of the CpG island in the ORMDL3 promoter region and increased its promoter activity. In addition, 5-Aza-CdR significantly increased IL-6 and IL-8 levels in NHBE cells while there was no obvious alteration after knocking down ORMDL3. Knockdown of ORMDL3 also significantly decreased the expression of p-ERK/MMP-9 pathway in the presence or absence of 5-Aza-CdR. In conclusion, our study provided novel evidence for the association between ORMDL3 and asthma-associated cytokines. Moreover, DNA methylation plays an important role in ORMDL3-mediated increased IL-6 and IL-8 levels and p-ERK/MMP-9 pathway expression.

STING positively regulates human ORMDL3 expression through TBK1-IRF3-STAT6 complex mediation.

Orosomucoid 1-like protein 3 (ORMDL3) is an asthma candidate gene associated with virus-triggered recurrent wheeze. Stimulator of interferon gene (STING) controls TLR-independent cytosolic responses to viruses. However, the association of STING with ORMDL3 is unclear. Here, we have shown that ORMDL3 expression shows a linear correlation with STING in recurrent wheeze patients. In elucidating the molecular mechanisms of the ORMDL3-STING relationship, we found that STING promoted the transcriptional activity of ORMDL3, which was significantly associated with increased levels of interferon regulatory factor 3 (IRF3) and signal transducer and activator of transcription 6 (STAT6). Further study showed that via activation of TANK binding kinase 1 (TBK1), STING enhanced the phosphorylation and binding of IRF3 and STAT6, which upregulated ORMDL3 by binding to the promoter. Our results showed that STING positively regulated ORMDL3 through the TBK1-IRF3-STAT6 complex.