Skip to main content
Download PDF

ACE2 immunohistochemistry in salivary and tracheal glands related to age

BMC Research Notes volume 15, Article number: 144 (2022) Cite this article

Abstract

Objective

SARS-CoV-2 is the cause of COVID-19, the rapidly spreading pandemic. When SARS-CoV-2 enters the target cells in the respiratory system, the spike glycoprotein binds to a cellular receptor angiotensin converting enzyme 2 (ACE2). The susceptibility to infection in individuals under 20 years of age is approximately half that of adults aged over 20 years. In this study, we investigated the immunohistochemical protein expressions of ACE2 in mandibular salivary glands and tracheal glands from forensic autopsy specimens covering adults and children.

Results

The ACE2 immunohistochemistry of autopsy specimens was performed, and the percentages of the immuno-positive areas in the cell layers of the glands were calculated. Our results demonstrate that the ACE2 positivity in mandibular salivary gland and tracheal glands showed the statistically significant decrease with the increase of age, which indicates that the susceptibility of aged individuals to SARS-CoV-2 may be due to various factors including but not limited to ACE2 protein expressions.

Introduction

The novel coronavirus SARS-coronavirus 2 (SARS-CoV-2) is the cause of the coronavirus disease 2019 (COVID-19), the rapidly spreading pandemic. When SARS-CoV-2 enters the target cell, the spike (S) glycoprotein binds to a cellular receptor angiotensin converting enzyme 2 (ACE2) [1]. For a full understanding of the susceptibility for SARS-CoV-2 infection and the role of ACE2 in clinical manifestations of the disease, it is necessary to study the cell type-specific expression of ACE2 in human tissues, both on the mRNA and protein levels [2]. The respiratory system is of special interest because it is the major site of the disease [3]. The expression of ACE2 gene in nasal epithelium is lower in young children compared with adults [4]. In this study, we investigated the immunohistochemical protein expressions of ACE2 in salivary and tracheal glands from forensic autopsy specimens covering adults and children.

Main text

Materials and methods

The study was approved by Teikyo University Ethical Review Board for Medical and Health Research Involving Human Subjects (Approve No. 20-084). The profiles of human autopsy cases in this study are as follows: totally, 47 cases (male:female is 2:1) were studied for mandibular and bronchial glands, in which 37 cases were studied for both glands, whereas 5 cases were studied for either one of the glands. The ages range from 0 to 95 years old. The postmortem intervals range from 0.5 to 7.5 days. The causes of death are 3 cases of cardiac diseases, 5 cases of respiratory disease, 5 cases of trauma, 4 cases of asphyxia, 6 cases of drowning, 4 cases of burning, 3 cases of intoxication, 13 cases of other causes, 4 cases of unknown causes. None of the cases had the history of COVID-19 infection, and all cases were negative for COVID-19 PCR at the time of autopy.

Human autopsy samples were fixed in 16% formalin, embedded in paraffin, and sectioned. The immunohistochemistry was performed by the BOND RX automated immunostainer (Leica, Germany) using BOND polymer refine detection system [5]. The antibody used was 500 times diluted anti-ACE2 (rabbit polyclonal, ab15348, Abcam, UK). Diaminobenzidine (DAB) was used for the staining. The immunohistochemical positivity was measured using cellSence software (Olympus, Tokyo, Japan). The range of interest was set on the mandibular salivary glands and tracheal glands, and the brown color of DAB was selected. The percentages of the positive areas in the glands were calculated using the cellSense software (Olympus, Japan).

Statistical analysis

Linear regression test was used for statistical analyses, and graphs were made by Prism 7 (GraphPad Software, San Diego, USA). The p values less than 0.05 were considered statistically significant.

Results and discussion

Our study clearly proves the presence of ACE2 immunoreactivity in salivary and tracheal glands (Fig. 1A and B for 0 year old, and Fig. 2A and B for 69 years old). Our results demonstrate that ACE2 is immunohistochemically expressed in salivary glands and tracheal gland cells through all ages studied (Fig. 2A, B). COVID-19 has a characteristic in age distribution. That is, the susceptibility to infection in individuals under 20 years of age is approximately half that of adults aged over 20 years [6]. The ACE2 prevalence in children compared with adults would offer the important information as to the reason why the susceptibility is different in terms of age. Previously, the expression of ACE2 gene in nasal epithelium has been reported lower in young children compared with adults [4]. Since saliva is an important source of SARS-CoV-2 infection [7], we investigated the immunohistochemical expression of ACE2 in submandibular salivary glands as well as tracheal glands as the possible infection sites. Contrary to the above nasal result, our results reveal the novel finding that the ACE2 protein immunoreactivity in mandibular salivary glands (Fig. 2A) and tracheal glands (Fig. 2B) shows the statistically significant decrease with the increase of age. Thus, our results indicate that immunohistochemical ACE2 expressions may inversely correlate with the age. This indicates that the susceptibility of aged individuals to SARS-CoV-2 may be due to various factors including but not limited to ACE2 protein expressions.

Fig. 1
figure 1

Representative ACE2 immunohistochemistry of mandibular salivary glands (A) and tracheal glands (B) from 0 year old, whereas mandibular salivary glands (C) and tracheal glands (D) from 69 years old in contrast. Brown diaminobenzidine-positivity shows ACE2 immunoreactivity. The scales at bottom right show 50 µm

Full size image
Fig. 2
figure 2

A Correlation between ACE2 immunohistochemistry and age for mandibular salivary glands. The statistically significant linear regression line is drawn (n = 42, R2 = 0.1265, p < 0.05). Some dots overlap due to close values. B Correlation between ACE2 immunohistochemistry and age for tracheal glands. The statistically significant linear regression line is drawn (n = 42, R2 = 0.119, p < 0.05). Some dots overlap due to close values

Full size image

Limitations

Since this is an immunohistochemical study, the functional aspects of ACE2 in the glands are not covered.

Availability of data and materials

The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

ACE2:

Angiotensin converting enzyme 2

SARS-CoV-2:

SARS coronavirus-2

COVID-19:

Coronavirus infection 2019

References

  1. He J, Tao H, Yan Y, Huang SY, Xiao Y. Molecular mechanism of evolution and human infection with SARS-CoV-2. Viruses. 2020;12(4):428.

    Article  CAS  PubMed Central  Google Scholar 

  2. Hikmet F, Mear L, Edvinsson A, Micke P, Uhlen M, Lindskog C. The protein expression profile of ACE2 in human tissues. Mol Syst Biol. 2020;16(7):e9610.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579(7798):270–3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Bunyavanich S, Do A, Vicencio A. Nasal gene expression of angiotensin-converting enzyme 2 in children and adults. J Am Med Assoc. 2020;323(23):2427–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Sato A, Nakamura N, Kojima M, Ohmachi K, Carreras J, Kikuti YY, et al. Clinical outcome of Epstein–Barr virus-positive diffuse large B-cell lymphoma of the elderly in the rituximab era. Cancer Sci. 2014;105(9):1170–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Davies NG, Klepac P, Liu Y, Prem K, Jit M, Group CC-w, et al. Age-dependent effects in the transmission and control of COVID-19 epidemics. Nat Med. 2020;26(8):1205–11.

    Article  CAS  PubMed  Google Scholar 

  7. Xu R, Cui B, Duan X, Zhang P, Zhou X, Yuan Q. Saliva: potential diagnostic value and transmission of 2019-nCoV. Int J Oral Sci. 2020;12(1):11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors express gratitude to Rie Yonemoto, Shouichi Tomisawa, and Etsuko Higuchi for their excellent technical assistance.

Funding

This study was supported in part by JSPS KAKENHI [20K10564 (M.N.) and 19K 12783 (T.N.)].

Author information

Authors and Affiliations

  1. Department of Legal Medicine, Teikyo University School of Medicine, 2-11-1, Kaga, Itabashi-ku, Tokyo, 173-8605, Japan

    Makoto Nogami, Tomoaki Hoshi, Yoko Toukairin, Tomomi Arai & Tadashi Nishio

Authors
  1. Makoto Nogami
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tomoaki Hoshi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yoko Toukairin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tomomi Arai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tadashi Nishio
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

MN: CONCEPTUALIZATION, investigation, methodology, writing-original draft, writing-review and editing. HT: investigation, YT: investigation, TA: investigation, TN: conceptualization, investigation, methodology, writing-review and editing. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Makoto Nogami.

Ethics declarations

Ethics approval and consent to participate

The study was approved by Teikyo University Ethical Review Board for Medical and Health Research Involving Human Subjects (Approve No. 20-084). Pursuant to the chapter 4 of the ethical guidelines for medical health and health research involving human subjects by the Ministry of Health, Labor and Welfare, individual informed consent was not required.

Consent for publication

Not applied.

Competing interests

There are no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nogami, M., Hoshi, T., Toukairin, Y. et al. ACE2 immunohistochemistry in salivary and tracheal glands related to age. BMC Res Notes 15, 144 (2022). https://doi.org/10.1186/s13104-022-06031-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13104-022-06031-1

Keywords

  • ACE2
  • SARS-CoV-2
  • COVID-19
  • Immunohistochemistry
  • Salivary gland
  • Tracheal gland

BMC Research Notes

ISSN: 1756-0500

Contact us