Correlation of ACE2 and TMPRSS2 expression levels in nasopharyngeal swab samples of COVID-19 patients with disease severity

Abstract

Background: Angiotensin converting enzyme-2 (ACE2) and Transmembrane serine protease 2 (TMPRSS2) are key proteins that serve as receptors and co-receptor in the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infective process, which could affect host susceptibility or severity in response to infection. We evaluated the expression levels of above genes to consider the probable relationship with age, sex, and respiratory distress.

Method and Result: Here, in a case-control study, we compared the expression levels of ACE2 and TMPRSS2 genes in nasopharyngeal swab samples of one hundred Coronavirus disease of 2019 (COVID-19) patients against 50 negative samples. In the positive group, 50 patients selected with mild symptoms and fifty patients were included in the severe / critically ill subgroup We determine the expression levels of ACE2 and TMPRSS2 by Quantitative Real-Time Reverse Transcription PCR (qRT-PCR) and statistical analyzes were implemented to consider the probable relationship between the expression levels of the above genes with sdisease severity, age, and  sex. Our results showed ACE2 was down regulated in laboratory-confirmed COVID-19 patients comparing normal control group. In addition, considerable downregulation of ACE2 levels was detected in the severe subgroup compared to mild patients. ACE2 mRNA levels were negatively correlated to age, while there was no significant association between the expression levels of the ACE2 and TMPRSS2 genes and sex. No association was found between the expression levels of TMPRSS2 and the clinical findings of the patients.

Conclusion: This data indicate there is a probable prognostic value of ACE2 expression in the follow-up of the COVID-19 patients.

Introduction

Coronavirus disease 2019 (COVID-19) originated in December 2019 in Wuhan, China, is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The disease was disseminated nearly all over the world as a pandemic (The disease was officially announced as a pandemic by the World Health Organization on March 11, 2020). More than 0.5 billion morbidities and more than six million deaths have been documented until May 2022 (https://www.worldometers.info/coronavirus/).

The SARS-CoV2 virus is an enveloped single-stranded RNA virus that comprises a 29.9 kilobase (kb) genome. The virus has four structural proteins: Envelope (E), Membrane (M), Nucleocapsid (N), and Spike (S) proteins. In addition, the RNA genome of the virus contains at least 15 nonstructural proteins that are necessary for virus replication, evasion of the immune system, inhibition of host gene expression, etc. [1]. Like the SARS-CoV virus, the SARS-CoV2 uses angiotensin-converting enzyme 2 (ACE2) as a receptor to enter the cells [2]. In fact, spike (S) glycoprotein of the SARS-CoV2 binds to the human ACE2 receptor in the cell membrane through the S1 subunit [3]. The transmembrane serine protease-2 (TMPRSS-2) of the host cell is required for priming of S protein and fusion of the virus to the cell membrane via the S2 subunit. Then the virus is internalized into the cell by endocytosis, and after the endosome escape, the viral RNA replicates and translates in the ribosome of the host cell. Finally, the assembly of virion and exocytosis of new viral particles happens and infects other cells [4].

The median incubation period of the COVID-19 disease is around five days. The disease's clinical spectrum ranges from asymptomatic to more severe forms of the disease, including respiratory distress, septic shock, and multi-organ failure [5]. Many risk factors have been considered for the prediction of disease severity, hospitalization, and death. Among th`em, age (higher age), sex (male), obesity, A blood group and underlying disorders like hypertension, diabetes, and pulmonary disorders have been identified [6]. In addition, host genomic factors have also been investigated in some populations and variants associated with susceptibility to COVID-19 and disease severity in candidate genes have been determined [7]. The ACE2 has been described as the receptor for binding of SARS-CoV-2 and the TMPRSS2 is an important priming enzyme required during this process [2]. The ACE2 gene is located on the short arm of chromosome X (Xp22) chromosome, contains 18 exons and encodes a protein consisting of 805 amino acids. In the renin-angiotensin-aldosterone system (RAAS), ACE2 catalyzes the cleavage of Ang I to Ang (1–9) and Ang II into Ang (1–7).[8] The activation of ACE2- Ang (1–7)-Mas axis results in vasodilation and consequently activation of anti-fibrosis, anti-apoptosis and anti-inflammation pathways [4].

The ACE2 expression has been detected in different tissues, including the small intestine, lung, kidney, heart, testis, adipose tissue, and mammary tissue of the breast (https://gtexportal.org/home/gene/ACE2). The TMPRSS2 gene is located at the long arm of chromosome 21 (21q22.3) and prominently expressed in normal and cancerous prostate tissue [9]. TMPRSS2 is the major host protease that contributes to the cell entry of SARS-CoV-2 and coronaviruses by cleavage of S protein into subunit1 (S1) and subunit2 (S2) to allow the fusion of the virus to the cell membrane. [2], [10] In addition to the prostate, TMPRSS2 is expressed in some other tissues, including lung and bronchial transient secretory cells [11]. Collectively, the expression level of ACE2 and TMPRSS2 genes is a critical parameter in the infectivity of SARS-CoV2. However, the association between the expression level of ACE2 and TMPRSS2 transcripts and the severity of the COVID-19 disease is not well determined.

Therefore, in this study, we aim to identify the relationship between ACE2 and TMPRSS2 expression levels in Covid-19 patients with disease severity.

Materials And Methods

Results

In this study, we investigated the clinical characteristics of COVID-19 patients by sex and age.Then we examined the expression profile of ACE2 and TMPRSS2 genes with disease severity.

Fifty mild and fifty severe/critically ill COVID-19 patients were included in the study. The mean ± SD of age in mild and severe cases were 46 ± 12.5 and 57 ± 13.9, respectively. The evaluation of demographic and clinical characteristics of patients showed a direct correlation between increasing age and severity of COVID-19. Our study found that COVID-19 patients aged ≥ 59 had a higher rate of respiratory failure and needed more frequent intensive care than those aged < 59 (Fig. 1 and Table 2). However, no significant association between sex and respiratory distress was observed in this study.

Then, the relationship between the expression profile of the ACE2 and TMPRSS2 genes and the age of COVID-19 patients was investigated. Data showed that the expression level of the ACE2 gene in nasopharyngeal swab samples is decreased in patients with older ages (age ≥ 59 years), while the expression of TMPRSS2 is not significantly changed (Fig. 1)

To determine whether the expression levels of ACE2 and TMPRSS2 in nasopharyngeal swab samples are related to disease severity in COVID-19 patients, we compared the mRNA levels of the above genes in mild and severe forms of COVID-19 patients.

The expression levels of ACE2 is downregulated in swab samples of all COVID-19 patients compared to SARS-CoV2 negative controls (Fig. 2a), while no significant diffrernec was detected in the expression level of TMPRSS2 in mild and severe/critical illness groups. Our findings showed significant differences in transcription levels of ACE2 among COVID-19 patients with mild and severe forms. In fact, ACE2 expression in the severe/critically ill subgroup was significantly lower than in the mild subgroup (Fig. 2c), whereas no significant difference was detected in TMPRSS2 expression in mild and severe/critical ilness subgroups. (Fig. 2d) Furthermore,

Discussion

A wide range of clinical symptoms of COVID-19 disease has been reported from the first weeks after the identification of the disease in Wuhan, China[13]. The range of clinical manifestations has been grouped as follows: Asymptomatic or pre-symptomatic infection, mild illness (repetitive), moderate illness, severe illness, and critical illness with multi-organ failure.[12] It is estimated that 80 percent of patients are asymptomatic or have mild to moderate symptoms, 15 percent have severe symptoms and 5 percent are in critical status [14]. In addition to virus genotype, the wide range of the disease manifestations are attributed to host factors like age, sex, smoking, underlying disorders such as hypertension, diabetes mellitus, blood groups and genomic, transcriptomic and epigenomics factors [15]. Among genetic factors, the association of variants of the ACE2 gene and also the expression level of this gene in different tissues have been investigated [16][17]. Furthermore, the expression of TMPRSS2 and associated variants have also been investigated as a contributing factor mainly in sex difference in clinical outcomes in covid-19 patients [19–21].

Given the vast range of clinical morbidity in the patients affected with covid-19, predicting the clinical severity of the disease is of great importance. In this context, we hypothesized the potential relationship between the expression level of ACE2 and TMPRSS2 genes in nasopharyngeal swabs of COVID-19 patients with disease severity.

We found that the process of aging is positively correlated to the severity of the disease. This finding has been reported in previous studies, in which the association of higher age with susceptibility to severe forms of COVID-19, hospitalization and mortality has been determined (Fig. 2) [19] [20].

While a substantial number of studies so far have focused on the higher risk of mortality, mainly in the male sex, despite the influence of sex differences on COVID-19 fatality, no significant association between sex and respiratory distress was observed in this study. This could be attributed to the type of sampling in this study and also relative to the low number of cases examined in this project [24] [25].

However, previous studies have identified the necessity of co-expression of ACE2 and TMPRSS2 in different cell lines for infectivity of SRAS-CoV2, and controversial findings have been obtained regarding the relationship between the expression profile of these two genes and the severity of the disease [22].

According to the obtained results during this survey, comparing the expression levels of the ACE2 gene among SARS-CoV2 positive cases and negative groups showed decreasing in ACE2 expression in positive samples. Indeed, we suggest that the reduction of ACE2 expression is compatible with the molecular mechanism of viral infection, as the virus uses the cell's translational machinery to replicate and assemble virus particles [23]. Moreover, our evidence supports that decreasing ACE2 expression will result in higher morbidities, hospitalization and death in COVID-19 patients. This finding is consistent with previous findings, as many investigations revealed a positive correlation between the downregulation of ACE2 and the respiratory distress in COVID-19 patients [24]. Accordingly, some studies showed that higher expression of ACE2 was significantly associated with protection against respiratory distress by various mechanisms [25].

In contrast, some other studies suggested a correlation between ACE2 or TMPRSS2 mRNA levels and COVID-19 associated clinical manifestation[26]. Preliminary data suggested that the increased severity of COVID-19 in obese young patients may be linked to increased ACE2 expression in lung epithelial cells.[27]

In addition, higher ACE2 expression levels could be expected in individuals with hypertension [32] and also in the respiratory tract of smokers, supporting that increasing available binding site for SARS-CoV-2 could enhance viral entry into the cell and leads to higher viral load and disease morbidities [29].

On the other hand, some studies have compared the expression levels of ACE2 and TMPRSS2 in children and adults [30]. Berni Cannani et al. compared the expression level of ACE2 in nasal epithelium of 30 children with 30 adults. They found no significant difference between ACE2 expression between the two groups.[31] Bunyavanich and Vicencio investigated the expression level of ACE2 in 305 individuals aged 4–60. They found age-dependent ACE2 gene expression in the nasal epithelium, with the lowest expression in younger children (< 10 years) [32]. Felsenstein and Hedrich discussed increased ACE2 expression in young COVID-19 patients facilitate virus entry into the cell, but its anti-inflammatory role causes mild symptoms in children [33].

In addition, ACE2 plays a critical role in counterbalancing Ang2 in RAS, deficiencies of ACE2, which might be due to the aging or induced by a virus infection, can lead to increased Ang2 subsequently [34]. As a result, increasing Ang2, a product of ACE cleaving angiotensin1, can cause vasoconstriction, inflammation and fibrosis by signaling through angiotensin2 type1 receptors (ATR1) [28].

Therefore, the results so far clearly indicate that the equilibrium concentration of ACE2 can abbreviate the harmful effect of vasoconstriction, inflammation, and fibrosis and generate vasodilation by mediating the conversion of angiotensin2 to angiotensin 1–7, which seems to have a protective effect in lung injury. In fact, the complexity towards the role of ACE2 in COVID-19 disease is entirely due to the dual impact of its' actions on the severity of the disease [35].

On the other hand, in the present study, the functional impact of TMPRSS2 in S protein priming facilitates the process of SARS-CoV-2 fusion and entry after its' recognition by the ACE2 receptor, no significant difference in TMRRSS2 transcription levels was observed between mild and severe cases. However, due to the risk effect of TMPRSS2 on SARS-CoV-2 severity, TMPRSS2 was expected as an important player host for SARS-CoV-2 susceptibility in many studies.[36] [37].

Therefore, considering that TMPRSS2 function is depended on the presence of ACE2 during SARS-CoV-2 entry and since ACE2 is subjected to extensive transcriptional modulation by an epigenetic mechanism, we suggest further expression studies on SARS-CoV-2 pathogenesis in larger size of samples to evaluate the co-expression of both genes and clarify the plausible association of TMPRSS2 levels with SARS-CoV-2 severity.

Furthermore, recent studies have shown the expression level of the ACE2 gene is comparable in nasal swab bronchial samples and broncho-alveolar lavage (BAL) [25] [38]. Therefore, it is reasonable to use nasopharyngeal samples instead of bronchial samples.

One important strength of this study is the samples included in the study were obtained at the time of virus detection at the early viremia phase of the disease instead inflammatory phase of the disease. Therefore, the data could be used as a biomarker for the potential prediction of disease severity. Yet, our study has some limitations that should be considered to improve the next studies. One is the lack of samples from children and also the lack of samples from broncho-alveolar lavage (BAL) and bronchial samples to compare the expression profile of studied genes in different tissues.

Another limitation of this study was that our examination was done after COVID-19 infection, so our data does not consist of the expression levels of our target genes before and after the infection. Therefore, we suggest that prospective studies follow a case-control study to evaluate the probable changes in expression levels of these two genes before and during the infection course and after the recovery.

Conclusion

To conclude, our current study certainly showed a significant correlation between down regulation of ACE2 and respiratory distress. We have noticed that the evaluation of ACE2 expression could help explain whether the patients' condition could portend a higher risk of acute respiratory distress. Our findings revealed the critical role of ACE2 in predicting respiratory distress, particularly among cases that have already lacked the expression levels of ACE2, such as the elderly.

Declarations

Acknowledgments

The authors thank all the patients and participants included in this study. We also thank Covid-19 lab at Pasteur Institute of Iran, Gholhak pathobiology lab, Sajad and Valie-Asr hospitals for providing the samples for this study.

Funding

This work was supported by the Pasteur Institute of Iran and North Tehran Branch, Islamic Azad University, Tehran, Iran [grant number iau-tnb162279836]

Competing Interests

The authors declare no relevant financial or non-financial interests to disclose.

Authorship Contributions

Author1: Investigation, writing-orginal draft, Application of statistical. Author 2: Formal analysis, Supervision. Author3: Recources-provision of laboratory samples. Author4: Supervision. Author5: Resources -provision of laboratory samples. MK* designed and outlined the framework of this experiment and edited the final manuscript.

Ethics approval

This study was performed in line with the principles of the Declaration of Pasteur Institute of Iran. Approval was granted by the Ethics Committee of Pasteur Institute of Iran (No: IR.PII.REC.1399.012) with the written informed consent obtained from all enrolled participants.

Consent to participate

Informed consent was obtained from all individual participants included in the study.

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