The Flavoring Diacetyl Found In Many Foods Increases Susceptibility To SARS-CoV-2 As It Alters Airway Cell Morphology, Inflammatory And Antiviral Response!

Think before you reach for processed foods like microwave popcorn, potato chips, crackers, corn chips, chocolate, cookies, cocoa-flavor products, gelatin, candy, flour mixes, syrup with flavoring, frostings, chewing gum, ice cream, soft drinks and sauces at your supermarket aisles as most of these products contain a food flavoring called Diacetyl (DA)  which according to a new study by researchers from Duke Clinical Research Institute and Department of Medicine at Duke University Medical Center-USA increases the susceptibility to SARS-CoV-2 as it alters airway cell morphology, inflammatory and antiviral response! Diacetyl is also used in e-cigarettes flavorings which are just as dangerous in terms of increasing COVID-19 susceptibility.
 
Diacetyl is most prevalent in processed foods that contain butter flavoring. It is used as a flavoring agent in butter, butter sprays, margarine, shortening, oil, oil sprays and other butter-flavored substances. If a product is advertised as having “buttery flavor,” then that product likely contains diacetyl.


 
Diacetyl is an α-diketone that is used to flavor many processed foods including microwave popcorn, coffee, and e-cigarettes. Occupational exposure to high levels of causes impaired lung function and obstructive airway disease.
 
Furthermore, lower levels of diacetyl exposure dampen host defenses in vitro.
 
Comprehending diacetyl’s impact on lung epithelium is important for delineating exposure risk on lung health.
 
The study team assessed the impact of diacetyl on normal human bronchial epithelial cell (NHBEC) morphology, transcriptional profiles, and susceptibility to SARS-CoV-2 infection.
 
Transcriptomic analysis demonstrated cilia dysregulation, an increase in hypoxia and sterile inflammation associated pathways, and decreased expression of interferon-stimulated genes after diacetyl exposure.
 
Importantly, diacetyl exposure resulted in cilia loss and increased hyaluronan production. After SARS-CoV-2 infection, both genomic and subgenomic SARS-CoV-2 RNA were increased in diacetyl-exposed compared to vehicle-exposed NHBECs.
 
The Diacetyl-SARS-CoV-2-Susceptibility study finding suggests that transcriptomic and physiologic changes induced by diacetyl exposure damage cilia and increase host susceptibility to SARS-CoV-2.
 
The study findings were published the peer reviewed journal by Nature: Cell Death Discovery. https://www.nature.com/articles/s41420-022-00855-3
 
Diacetyl exposure promotes oxidative stress, upregulates inflammatory processes including IL-8 secretion, cilia loss, and dedifferentiation of the epithelial layer in vitro. This is relevant considering clinical and experimental evidence links the development of bronchiolitis obliterans (“popcorn lung”) and irreversible lung disease to occupational diacetyl exposure.
https://pubmed.ncbi.nlm.nih.gov/29375399/
 
https://pubmed.ncbi.nlm.nih.gov/30851105/
 
< a href="https://pubmed.ncbi.nlm.nih.gov/24816162/">https://pubmed.ncbi.nlm.nih.gov/24816162/
 
https://pubmed.ncbi.nlm.nih.gov/28248570/
 
https://pubmed.ncbi.nlm.nih.gov/27966365/
 
https://pubmed.ncbi.nlm.nih.gov/27326900/
 
The airway epithelium contains motile cilia which are an integral part of the mucociliary escalator, removing fluids, mucins, and particulates from the airway.
 
Past studies have shown that expression of multiple genes involved in cilia biogenesis are significantly downregulated in NHBECs (Normal human bronchial epithelial cells) when diacetyl liquid was added to cell culture media. https://pubmed.ncbi.nlm.nih.gov/30710127/
 
The study team observed loss of cilia on the surface of NHBECs and broad suppression of transcripts related to cilia structure and function after diacetyl vapor exposure. For example, FOXJ1, a master regulator of motile ciliogenesis, and RFX2 and RFX3, transcriptional coactivators of FoxJ1, were significantly downregulated in the RNA-seq analysis. Considering cilia assembly is under strict transcriptional control, diacetyl-induced suppression of these transcripts could result in an overall decrease in the cell’s cilia production program.
 
Other transcripts overrepresented in the top 10 downregulated pathways were associated with the radial spoke and spokehead, dynein proteins, IFT proteins KIF member genes and CCNO, HYDIN and TUBA1A transcripts.
 
The study team further validated the transcriptomic data and observed loss of cilia by cell histology and decreased protein expression of acetylated tubulin.
 
Acetylation of α-tubulin marks stable, long-lived subpopulations of cilia and flagella, and decreased acetylated tubulin indicates primary cilia loss. The suppression of cilia-related genes in addition to cilia loss is likely to result in decreased mucociliary clearance and increased cellular susceptibility to pathogens. Indeed, genetic defects in DNAH5, HYDIN, and TUBA1A and others result in primary cilia dyskinesia (PCD); a variety of clinical manifestations including ineffective mucociliary clearance and recurrent lower respiratory tract infections. https://pubmed.ncbi.nlm.nih.gov/25386990/
 
https://pubmed.ncbi.nlm.nih.gov/23022101/
 
https://pubmed.ncbi.nlm.nih.gov/12142464/
 
https://pubmed.ncbi.nlm.nih.gov/25789548/
 
Various endogenous danger-associated molecular patterns (DAMPs) are produced after non-pathogenic cellular injury that activate PRRs and induce sterile inflammation. Hyaluronan or HA is an extracellular matrix glycosaminoglycan released after sterile injury or pathogenic infection and exerts size-dependent effects. For example, low molecular weight (LMW) HA acts as a proinflammatory DAMP via toll-like receptor (TLR) signaling.
 
The study findings demonstrated that diacetyl exposure increased HAS2 transcript expression and HA production in NHBECs.
 
Though the study team did not measure LMW HA specifically, they observed an increase in transcripts associated with PRR signaling. For example, CD14 and LY96 gene expression were significantly upregulated after diacetyl treatment.

Membrane-bound CD14 is a PRR that recognizes diverse microbial products and LY96 (MD-2) physically associates with TLR4 to mediate LPS signaling via CD14.
 
Interestingly, HA is demonstrated to signal through a unique complex of TLR4, MD-2 and CD44. While HA has not been demonstrated to signal through CD14 directly, the interaction of HA binding proteoglycans like versican and CD14 can stimulate downstream cellular responses. Additionally, HA exudates have been observed in the lungs of deceased COVID-19 patients, suggesting a role for this molecule in acute respiratory distress syndrome caused by SARS-CoV-2.
 
The team also observed mobilization of NHBECs after diacetyl vapor exposure. After mechanical damage to airway epithelium, epithelial cell migration is integral to tissue repair.
 
Previous studies demonstrate that rats that inhale diacetyl exhibit ultrastructural changes in the trachea consistent with epithelial spreading and migration.
https://pubmed.ncbi.nlm.nih.gov/18474946/
 
Interestingly, increased basal cell mobilization is also observed during SARS-CoV-2 infection in NHBECs in vitro. https://pubmed.ncbi.nlm.nih.gov/34272374/
 
These findings suggest that damage to the airway epithelium, whether by toxin exposure or viral infection, results in epithelial cell mobilization that will likely impact the processes involved in epithelium repair.
 
The study findings also identified downregulated Hallmark pathways associated with DNA replication and DNA damage repair after diacetyl exposure.
 
This is consistent with previous literature demonstrating diacetyl covalently binds to guanyl nucleotides leading to DNA unwinding and cellular apoptosis. https://pubmed.ncbi.nlm.nih.gov/22385266/
 
Damage is likely further exacerbated by an increase in diacetyl-induced hypoxia and sterile inflammation as indicated in our Hallmark pathway analysis.
 
Recent studies have demonstrated a link between hypoxia and sterile inflammation via IL-1β priming of the NLRP3 inflammasome.
 
The upregulation of the Hypoxia and Inflammatory response Hallmark pathways and significant increase in the IL1B gene observed in our RNA-seq analysis suggests a similar mechanism after diacetyl exposure in NHBECs. Hypoxia induces p53 which mediates cellular apoptosis, both of which are upregulated in the Hallmark gene set analysis (i.e. P53 pathway and Apoptosis).
 
Furthermore, the upregulation of TNF-α signaling via NF-κB and IL-6 JAK STAT3 signaling Hallmark pathways highlight a potential role for TNF-α and IL-6 as triggers of sterile inflammation. The increase in Kras and TGF-β signaling pathways after diacetyl exposure suggests a role for TGF-β in mediating tissue remodeling after hypoxia-induced injury. KRAS is a member of the Ras protein family, which functions as a signal transducer between epidermal growth factor receptor (EGFR) signaling and the MAPK pathway (Ras/MAPK pathway). Ras/MAPK activity induces p53 phosphorylation, enabling the interaction of p53 and TGF-β. Overall, the Hallmark gene set analysis suggests that diacetyl exposure results in changes in NHBECs similar to those observed in hypoxia leading to inflammation and compensatory TGF-β-mediated repair signatures.
 
In the study, the team observed suppression of multiple ISG (interferon stimulated gene) transcripts important in antiviral protection of epithelial cells after diacetyl vapor exposure in normal human bronchial epithelial cells or NHBECs. ISG products exhibit many diverse functions but collectively are highly effective at resisting and controlling viral infections. https://pubmed.ncbi.nlm.nih.gov/24555472/
 
For example, RSAD2 (Viperin) was one of the most significantly decreased ISG after diacetyl exposure.
 
Viperin has been demonstrated to inhibit replication of multiple respiratory viruses including measles, respiratory syncytial virus, and influenza.
https://pubmed.ncbi.nlm.nih.gov/30684519/
 
https://pubmed.ncbi.nlm.nih.gov/22377585/
 
 
Past studies demonstrate that e-cig exposure significantly impaired antiviral host defenses in the lungs. https://pubmed.ncbi.nlm.nih.gov/31390877/
 
https://pubmed.ncbi.nlm.nih.gov/25651083/
 
This current study findings however are the first evidence that a flavoring chemical directly reduces expression of multiple ISG transcripts in NHBECs. These data demonstrate that diacetyl suppressed the transcriptomic expression of important antiviral and immune factors that could lead to increased susceptibility to SARS-CoV-2.
 
The evidence that diacetyl exposure induces a loss of cilia and acetylated tubulin combined with a decrease in transcriptomic expression of important ISGs led the study team to hypothesize that diacetyl-exposed NHBECs cells would be more susceptible to SARS-CoV-2 infection.
 
Furthermore, recent evidence demonstrates a loss of motile cilia and dedifferentiation of the epithelial cells after SARS-CoV-2 infection both in vitro and in vivo. https://pubmed.ncbi.nlm.nih.gov/34272374/
 
After confirming SARS-CoV-2 receptor ACE2 and cellular protease TMPRSS2 were still present after diacetyl exposure, the study team infected diacetyl vapor and PBS vehicle-exposed NHBECs with SARS-CoV-2. The increase in the mean of both genomic and subgenomic SARS-CoV-2 RNA levels in NHBECs demonstrate that diacetyl increases susceptibility of NHBECs to SARS-CoV-2 infection.
 
The study findings are in line with a recent report demonstrating that direct cigarette smoke exposure reduces innate immune responses and increases the number of SARS-CoV-2 infected cells. https://pubmed.ncbi.nlm.nih.gov/33259798/
 
Furthermore, this is consistent with other studies demonstrating negative impacts of e-cigs on the airway and host defenses. For example, mice exposed to e-cig vapor had altered lung lipid homeostasis and downregulated innate immune responses in alveolar macrophages and epithelial cells. When infected with influenza, e-cig vapor-exposed mice had increased lung inflammation and tissue damage. https://pubmed.ncbi.nlm.nih.gov/31483291/
 
The study findings demonstrate that Diacetyl induces cilia loss and specific pathological changes in the airway epithelium that increase susceptibility to viral infection, specifically against SARS-CoV-2, suggesting a potential mechanism for increased susceptibility in e-cigarette users.
 
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