In June 2020 an article entitled “Geranium and Lemon Essential Oils and Their Active Compounds Downregulate Angiotensin-Converting Enzyme 2 (ACE2), a SARS-CoV-2 Spike Receptor-Binding Domain, in Epithelial Cells” was published in the journal Plants (Basel).
I know, you’ve got questions. WE’VE got questions. What is ACE2, and how does it relate to SARS-CoV-2? What’s its normal function in the body? What is a spike receptor binding domain? Why does downregulation of ACE2 expression in epithelial cells matter for COVID19? What does it all meeeean?
No worries, we’ve got you. In this review we’ll:
- Take a look at the journal Plants, published by MDPI, to ensure that it’s legitimate.
- Define ACE2, its physiological function, and its relationship with SARS-CoV-2.
- Unpack the methodology and findings of this study, and discuss the value of in vitro vs. in vivo studies vs. clinical trials.
- Interpret these findings in the context of the scientific literature on ACE2 and SARS-CoV-2 infectivity and COVID19 pathogenesis.
Consider Your Source
First things first. Any time you come across an intriguing research article, take a moment to validate the journal. Just because something is a “scientific publication” doesn’t make it legitimate.
Over the last decade we’ve witnessed the rise of predatory journals, ubiquitous scourge of the scientific community.
A rigorous 2019 colloquium of international scholars and publishers defined predatory journals and publishers as “Entities that prioritize self-interest at the expense of scholarship and are characterized by false or misleading information, deviation from best editorial and publication practices, a lack of transparency, and/or the use of aggressive and indiscriminate solicitation practices.”
Predatory journals have been described as “a global threat.” They accept articles for publication for a significant fee, without executing promised quality checks for problems like plagiarism and ethical approval. Naive readers and seasoned researchers alike have fallen prey to these journals. Analyses indicate that predatory publishers amass millions of dollars in publication fees only to squander resources and produce shoddy scholarship.
The hydra-like multiplication of these low-quality journals rose to meet the institutional demand for academics to publish papers. Universities frequently use the number of papers a scholar has published as a benchmark for graduation or career advancement. In a “publish-or-perish” culture, many researchers turn to predatory journals to bolster their CVs (Grudniewicz et al., 2019).
Indeed, a new form of literacy is required to assess journal quality.
Keep an eye out for red flags like spelling errors, unprofessional-looking or extravagant graphic design, and the word “international” in the journal’s name. Reference an index of predatory journals to confirm that your source is not one of them. But keep in mind that these lists have limited value, as there are many such indexes and inconsistencies abound.
Let’s take a look at our present journal, Plants (Basel). Based on cursory googling, it does not appear to grace any such lists of predatory journals. That being said, its publisher MDPI (the Multidisciplinary Digital Publishing Institute) does have a somewhat shady history. The publisher, which produces 213 open-access journals, is highly incentivized by publication fees to prioritize content volume over quality (de Vrieze, 2018).
No need to assume that the present paper is mediocre, we just can’t be certain the journal engaged in conscientious quality control. We’ll have to do our own due diligence.
Let’s get into the science now, shall we?
ACE2 and the SARS-CoV-2 Spike Protein
Angiotensin-converting enzyme-2 (ACE2) is a predominantly membrane-bound host cell receptor involved in the regulation of blood pressure. ACE2 is expressed in various human tissues, at high levels in the small intestine, testis, kidneys, heart, thyroid, and adipose tissue, and moderate levels in the lungs, colon, liver, bladder, and adrenal gland (Li et al., 2020).
Image from: https://www.mdpi.com/1422-0067/21/8/3003/htm COVID-19 and RAS: Unravelling an Unclear Relationship Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
ACE2 can act as a vasodilator, antioxidant, and anti-inflammatory. Conversely, “ACE2 deficiency” is associated with hypertension, diabetes, and cardiovascular disease (Bosso et al., 2020). ACE2 has many physiological functions, but the majority involve protection against lung injury (Samavati and Uhal, 2020).
The SARS-CoV-2 virus accesses human host cells by means of a spike protein embedded in the viral envelope. These spike proteins give the virus its characteristic crown-like “corona” appearance. The spike protein, with the help of local cofactors, binds to ACE2 receptors in human (primarily nasal) epithelium. This allows the virus to enter the cell, where it can hijack the cellular machinery, replicate itself, and establish an infection.
SARS-CoV-2 binding cofactors are more widely expressed throughout the body than ACE2, suggesting that ACE2 receptor availability may be a limiting factor for viral entry at the initial infection stage (Sungnak et al., 2020).
In sum, ACE2 is a receptor with two faces: it performs a crucial protective function in the cardiovascular and pulmonary systems, while simultaneously serving as the entry point of SARS-CoV-2 into its human host.
Geranium and Lemon Essential Oils Downregulate ACE2 Receptors
This study investigated the effect of various essential oils on the expression of ACE2 receptors in intestinal epithelium.
The researchers used the HT-29 colon adenocarcinoma cell line for their experiments, a cell type which was chosen because they are thought to overexpress ACE2. In other words, cells with a surfeit of ACE2 receptors were used to clarify the impact of essential oils on ACE2 expression in vitro.
HT-29 cells are a common choice in preclinical in vitro studies, often those involving intestinal cell differentiation and permeability (Martínez-Maqueda et al., 2015). Cancer cell lines are widely used for in vitro experiments, particularly in cancer research and drug development, as they generate an indefinite source of biological material to work with (Mirabelli, et al., 2019).
First, the researchers tested for cytotoxic activity of essential oils on these cells, and used this data to determine ideal experimental concentrations, or “optimum non-cytotoxic concentrations.” The HT-29 cells were incubated with these optimum concentrations of essential oils for 48 hours.
The essential oils tested were:
- Petitgrain
- Tea tree
- Eucalyptus
- Bergamot
- Juniper berry
- Tangerine
- Cypress
- Neroli
- Lemon
- Geranium
All oils significantly reduced ACE2 expression in HT-29 cells relative controls. Lemon and geranium oils exerted the greatest effect, and were found to inhibit ACE2 expression in a dose-dependent manner.
Lemon and geranium oils were further tested, and found to inhibit the expression of TMPRSS2, an important cofactor that primes SARS-CoV-2 to bind to ACE2.
Next, the major constituents of lemon and geranium essential oil were determined.
Geranium essential oil:
- Citronellol (27.1%)
- Geraniol (21.4%)
- Neryl acetate (10.5 %)
Lemon essential oil:
- Limonene (73.0%)
- γ-terpinene (9.2%)
- β-pinene (8.6%)
These constituents were likewise assessed for their cytotoxic concentrations, and optimal experimental concentrations determined. Treatment with citronellol, geraniol, limonene, and neryl acetate was found to significantly inhibit ACE2 protein expression and reduce TMPRSS2 mRNA levels in HT-29 cells.
The authors claim that this study is the first of its kind. They note that the majority of in vitro research investigating the antiviral properties of essential oils have identified the primary mechanisms to be:
- Inhibition of viral replication by blocking viral biosynthesis.
- Perturbing the structure or glycoproteins of the virus, thereby reducing or eliminating infectivity of the virus particles.
This study is unique in that the authors propose the novel antiviral mechanism of reducing viral infectivity by modulating the host terrain:
- By downregulating the expression of ACE2 receptors, the primary target molecule for viral invasion.
- Inhibiting expression of TMPRSS2, a crucial cofactor for viral spike protein binding to ACE2.
The authors conclude that these essential oils and their major constituents may be used to alter the epithelial environment in order to block invasion by SARS-CoV-2 (Senthil Kumar et al., 2020).
In Vitro, In Vivo, Clinical: Not All Studies are Created Equal
The study reviewed in this article was in vitro, meaning, executed in a petri dish. That’s a good place to start, but it goes without saying that the human body is a touch more complex than a clump of cells in a plastic tray. As such, in vitro findings should not be extrapolated to clinical practice without in vivo confirmation: animal studies, and, ultimately clinical trials.
Consider metabolism. Just as essential oil acts on the body, the body acts on the oil. Human metabolism breaks down essential oils, converting them to various metabolites, each with their own unique bioactivity. It is impossible to know what action a substance will have on the body based on in vitro studies alone. Furthermore, methods of delivery (aromatherapy, transdermal absorption, orally ingested capsules, etc.) will affect absorption and metabolism of the oils and their constituents. This will significantly impact their bioavailability and activity.
Another major limitation of this study’s methodology is the use of HT-29 cells, which, as with other cancer-derived cell line models, are known to exhibit significant differences in gene expression relative to normal cells (Martínez-Maqueda et al., 2015). Even within the confines of in vitro research, we can’t be sure that lemon and geranium essential oils and their major constituents would exert the same effects on normal intestinal cells as they did on the HT-29 adenocarcinoma cells.
Finally, ACE2 is differentially expressed in tissues throughout the body. In regards to SARS-CoV-2, the most significant location for viral transmissibility is the nasal epithelium. It’s unclear from this study how essential oils would influence ACE2 expression in these types of cells.
In Context of the Scientific Literature
The entire premise of this study is based on the notion that reduced expression of ACE2 receptors will lower susceptibility to infection by SARS-CoV-2. Theoretically it makes sense: if SARS-CoV-2 primarily binds to ACE2 receptors in order to invade host cells, then reducing the number of receptors will reduce the risk of infection.
The idea is plausible, sure, but has it been proven? Let’s take a look at the research and see if we can find some answers.
Is there any evidence that reducing ACE2 receptor expression is protective against COVID19?
The authors of a paper published June 5th, 2020 lament in their concluding remarks, “Surprisingly, little is known about the effect of SARS-CoV-2 virus binding to ACE2.” Their “critical question” yet to be answered is much the same as ours: “Do known inhibitors or activators of ACE2 have any effect(s) on the binding of SARS-CoV-2 to the ACE2 receptor and/or infection of lung epithelial cells?” (Samavati and Uhal, 2020).
One way to approach this question is to consider the effects of medications known to increase ACE2 receptor expression. These are primarily antihypertensive drugs, including Angiotensin II receptor blockers (ARBs) and Angiotensin-converting enzyme inhibitors (ACEIs). As of April 2020, no clinical data existed concerning the effects of ARBs and ACEIs on human tissue ACE2 expression or activity. Neither are there animal studies investigating these effects. There are only in vitro findings.
In other words, we have no idea if ARBs and ACEIs either augment susceptibility to SARS-CoV-2 or aggravate the severity and outcomes of COVID-19.
We know that in mice, knocking out the ACE2 gene greatly reduces viral infection and replication after experimental SARS-CoV infection. It remains unclear, however, whether downregulating ACE2 as opposed to rendering the ACE2 gene utterly inoperative, will have comparable effects. Furthermore, we still don’t know if ACE2 is the only receptor for SARS-CoV-2 infection.
Researchers investigating influenza viruses, and other coronaviruses (but not SARS-CoV-2) found that there’s a possibility that viral transmissibility is contingent on the spatial distribution of ACE2 receptors and the cofactors required for SARS-CoV-2 binding along the respiratory tract (Sungnak et al., 2020). This suggests that receptor availability may play a role in viral infectivity, but does not account for the complexity of the cofactors required for SARS-CoV-2 binding, and their tissue distribution.
The authors of a recent review entitled “Interactions of coronaviruses with ACE2, angiotensin II, and RAS inhibitors—lessons from available evidence and insights into COVID-19” published in Hypertension Research make it crystal clear: “We don’t know whether changes in ACE2 levels facilitate greater SARS-CoV-2 viral entry into cells, even in animal models, let alone humans. In other words, it is pure speculation that upregulation of ACE2 would lead to higher rates of infection, or that inhibited ACE2 infection would be preventative” (Kai and Kai, 2020).
So the answer is, we don’t know. And we won’t know until there are clinical trials.
Does inhibiting ACE2 expression have negative physiological consequences?
As discussed earlier in this review, ACE2 is an essential regulator of blood pressure homeostasis; deficiencies of this receptor have been associated with diabetes, cardiovascular disease, and lung injury.
ACE2 deficiency has been noted in people with hypertension, diabetes, and cardiovascular disease and the elderly — precisely the populations that are more likely to become infected, and, once infected, suffer more severe complications of COVID19.
Interestingly, SARS-CoV-2 binding to ACE2 receptors can lead to subsequent ACE2 deficiency. Furthermore, reduced ACE2 expression is linked with COVID19 severity and mortality. Once SARS-CoV-2 binds to ACE2 receptors and enters the host cell, ACE2 receptors are subsequently downregulated, which contributes to ACE2 deficiency. This inhibition of ACE2 leads to marked dysregulation of blood pressure homeostasis, and enhances the progression of inflammatory and thrombotic processes (Verdecchia et al., 2020).
Downregulation of ACE2 receptor expression leads to elevations in a substance called angiotensin II. An imbalance of angiotensin II signalling caused by ACE2 deficiency was seen to exaggerate acute lung injury in animal models (Milne et al., 2020).
Angiotensin II harms the lungs by decreasing the stability of pulmonary endothelium, which can aggravate respiratory distress. It also leads to increased secretion of aldosterone, a steroid hormone involved in sodium conservation, which increases sodium reabsorption, loss of potassium via urine, and inflammation (Silhol et al., 2020).
Angiotensin II has been shown to exert adverse reactions, including:
- Myocardial hypertrophy and dysfunction
- Interstitial fibrosis
- Endothelial dysfunction
- Enhanced inflammation
- Obesity-associated hypertension
- Oxidative stress
- Increased coagulation
Angiotensin II also interferes with adaptive immunity by activating macrophages and other immune cells, which triggers increased production of pro-inflammatory IL-6, TNFα, and other inflammatory cytokines (Verdecchia et al., 2020).
People with comorbid hypertension suffer higher mortality rates due to COVID19. One might ask if this due to the pathogenesis of hypertension, or due to the commonly associated therapies like ARBs and ACEIs. Many antihypertensive medications are known to upregulate ACE2 receptors, and researchers have investigated whether this increases patient susceptibility to SARS-CoV-2 infection. The general consensus appears to be no. Researchers seem to agree that the cardiovascular and lung protective benefits of increasing ACE2 expression outweigh any theoretical risk of increased susceptibility to infection (Bosso et al., 2020).
In sum, there’s no conclusive evidence that downregulating ACE2 receptors blocks infection with SARS-CoV-2, but an abundance of evidence that ACE2 is essential for cardiovascular and pulmonary health.
As usual, this study raises more questions than it answers. What effect would essential oils have on ACE2 receptors in nasal epithelium? Do essential oils actually protect against SARS-CoV-2 infection in humans via a mechanism of ACE2 downregulation? How do modes of essential oil administration influence infectivity outcomes? Are there negative pulmonary and cardiovascular consequences of downregulating ACE2 expression?
It’s sort of a letdown… there’s nothing in this study that you can run with and apply in your clinical practice or home or self-care routine. Scientific research is a process, often unwieldy, and there are rarely clear answers or satisfying conclusions.
Sometimes it’s the simple stuff that works: Listen to your body, wash your hands, and social distance like your life depends on it.
References
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de Vrieze, J. (2018, September 4). Open-access journal editors resign after alleged pressure to publish mediocre papers. American Association for the Advancement of Science; Science. https://www.sciencemag.org/news/2018/09/open-access-editors-resign-after-alleged-pressure-publish-mediocre-papers
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