Asthma is a complex disease affecting many individuals in the developed world. Genome-wide association studies have recently been used to identify genetic causes for such complex diseases. One particular gene, ORMDL3, is of interest because of its association with asthma, IBD, and Type I diabetes – all of which are caused by immune-mediated pathology [6, 10, 22, 38, 39]. The gene ORMDL3 is an ER-membrane protein and is potentially involved in Ca2+-signaling in the ER and sphingolipid synthesis [20, 21, 40]. It has also been correlated to activation of the UPR, though the mechanisms remain unclear . Activation of the UPR may be biologically relevant, as ER stress, the UPR, and inflammation have all been linked . However, the functional role ORMDL3 in the pathogenesis of asthma has yet to be elucidated.
Airway epithelial cells play an important role in innate immunity and in the development of asthma. Current findings in literature indicate that ORMDL3 is involved in immunity and that asthmatics have higher expression of the gene than non-asthmatics [18, 21, 22]. A recent study by Miller et al. also investigated the role of ORMDL3 in airway epithelial cells. They reported that in vitro overexpression of ORMDL3 activated the ATF6 pathway of the UPR and induced expression of several genes with potential importance in the pathogenesis of asthma . Our investigation, in contrast, focuses on the effect of variation of ORMDL3 expression levels, at baseline, on the innate immune responsiveness of airway epithelial cells. By manipulating ORMDL3 expression in vitro to mimic differences in gene expression established between asthmatics and healthy individuals, we aimed to understand the role of ORMDL3 on the innate immune response and UPR activation status in airway epithelial cells. This method ensured control and the confidence that any effect on the innate immune response was in fact correlated with a change in ORMDL3 expression levels. If the same experiments were performed on ex vivo airway cells of patients, genetic and other differences between individuals could have affected the results.
After knockdown of ORMDL3 in vitro, cells were stimulated with cytokines (TNF-α, IL-1β) or common microbial components (LPS, flagellin). We monitored production of interleukin-6 (IL-6) and interleukin-8 (IL-8) (alias CXCL8), two pro-inflammatory cytokines produced by airway cells that are relevant in asthma pathogenesis. Specifically, IL-6 is elevated in individuals with asthma  and is also regulated by ATF6 during activation of the UPR . Similarly, transfection of ORMDL3 into human airway epithelial cells triggers ATF6 activation and IL-8 secretion . However, in our experimental system, variation in ORMDL3 expression levels did not affect NF-κB-induced innate immune production of IL-6 and IL-8 in airway epithelial cells.
We next explored the effects of ORMDL3 expression on activation of the UPR. UPR signaling cascades are initiated in response to ER stress, and restoration of homeostasis is achieved by attenuating translation, restoring protein folding, or degrading misfolded proteins . Although often associated with abnormal physiological conditions, the UPR plays a central beneficial role in normal physiology; as illustrated by the role of the UPR in terminal B cell differentiation which requires a massive increase in the biosynthetic capacity to synthesize antibodies in response to infection . However, the ER stress response and UPR can also initiate inflammation through induction of cytokine production or activation of transcriptional regulators of inflammatory genes. Cytokines IL-6 and IL-8 are examples of genes that may be induced by UPR activation . ER stress and the UPR have been implicated in many immune-related diseases including IBD, diabetes, chronic obstructive pulmonary disease (COPD), arthritis, and neurodegenerative inflammatory diseases . It is poorly understood whether ER stress is an underlying cause of disease or if its induction is a result of chronic inflammation. Indeed, it is possible that environment factors such as infection or inhalation of smoke particles can activate the UPR, triggering the onset of lung disease in genetically predisposed individuals . However, it is also possible that ER stress is exacerbated by inflammation and contributes to the perpetuation of the disease.
Cantero-Recasens et al. previously reported that ORMDL3 overexpression activated the PERK pathway, but did not affect the IRE1 pathway of the UPR . In contrast, Miller et al. reported that ORMDL3 overexpression activated the ATF6 pathway, but not the PERK or IRE1 pathways . In our study, we chose four markers of UPR activation: XBP-1u, XBP-1s, CHOP, and p-eIF2α. With activation of the UPR, we expect downregulation of XBP-1u and upregulation of XBP-1s and CHOP. However, our results demonstrate that knockdown of ORMDL3 does not activate the UPR, in either unstimulated or stimulated cells. Immunoblot analysis also showed no change in p-eIF2α levels with ORMDL3 knockdown. Furthermore, downstream markers of UPR activation, IL-6 and IL-8 cytokines, were produced at similar levels in unstimulated cells with varying ORMDL3 levels. This further supports our results that ORMDL3 does not activate the UPR. Differences in our results compared to previous work might be due to the different types of cells, conditions, or markers used. It is possible that the effects of variation in ORMDL3 expression are a cell type-dependent phenomenon. While no effect on the inflammatory response was detected in airway cells, other cells types such as dendritic cells or T cells may be affected by altered ORMDL3 expression. Observations made by Lluis et al. suggest that the 17q21 locus may potentially play a role in T-cell development .
Taking a broader approach, PCR arrays looking at expression of 168 common immunity genes were performed. We reasoned that although ORMDL3 levels may not affect the production of IL-6 or IL-8 cytokines, perhaps they were impacting gene expression of other important immune genes, such as IL-33, IL-25 and TSLP, which have all been implicated in asthma pathogenesis . Verification of differential expression of these genes at a transcript level, however, did not show any significant changes between the ORMDL3 knockdown conditions. This suggests that altering ORMDL3 expression does not have a profound effect on the expression of innate immune genes upon stimulation in the airway epithelia. However, there may be other genes that are affected that were not investigated in this study. Pfeifer et al. recently showed that IL-17C cytokine is expressed by human bronchial epithelial cells and is induced by bacterial infection . It may be worthwhile in future experiments to investigate a broader range of immune-related genes. Interestingly, we did not observe changes to expression of the genes reported by Miller et al., MMP-9, CCL-20, CXCL-10, CXCL-11, or IL-8. This variance may be explained by differences in experimental conditions. Our study examined outcomes in gene expression after stimulation of cells co-transfected with ORMDL3 and ORMDL3- specific siRNA, while the other study used a different experimental approach.
Although this study focused exclusively on the potential role of ORMDL3 in asthma pathogenesis, it is possible that neighboring genes such as GSDML contribute to disease susceptibility at this locus. Many groups consider ORMDL3 as an ‘asthma gene’; however, it should be acknowledged that the identified SNPs associating this gene to asthma susceptibility are not located in the gene itself. Even so, these polymorphisms have been consistently correlated with increased odds of asthma risk, highlighting the importance of this locus in disease susceptibility [6–11, 13, 14].
Our data show that variation in ORMDL3 expression is not correlated with differential innate immune responses to stimuli or activation of the UPR in vitro in airway epithelial cells. Taken together, our results are biologically relevant because they suggest that normal human variation of ORMDL3 expression is not likely an important factor in increasing the innate immune response of airway cells we observe in asthmatics. Despite these results, this gene remains an important candidate for asthma susceptibility. More research is required to elucidate its role in asthma pathogenesis and its potential role as an initial trigger of inflammation. By increasing our understanding of the mechanisms responsible for allergic and atopic disease development, new treatments can then be developed. Thus, we can reduce inflammatory responses by targeting the potential triggers, rather than the symptoms, of the disease. In doing so, we will ultimately reduce the morbidity, mortality, and socio-economic burden of asthma and related allergic diseases.