Human Type II Taste Cells Express Angiotensin-Converting Enzyme 2 and Are Infected by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

Abstract

Chemosensory changes are well-reported symptoms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The virus targets cells for entry by binding of its spike protein to cell-surface angiotensin-converting enzyme 2 (ACE2). It is not known whether ACE2 is expressed on taste receptor cells (TRCs), or whether TRCs are infected directly. in situ hybridization probe and an antibody specific to ACE2 indicated presence of ACE2 on a subpopulation of TRCs (namely, type II cells in taste buds in taste papillae). Fungiform papillae of a SARS-CoV-2⁺ patient exhibiting symptoms of coronavirus disease 2019 (COVID-19), including taste changes, were biopsied. Presence of replicating SARS-CoV-2 in type II cells was verified by in situ hybridization. Therefore, taste type II cells provide a potential portal for viral entry that predicts vulnerabilities to SARS-CoV-2 in the oral cavity. The continuity and cell turnover of a patient's fungiform papillae taste stem cell layer were disrupted during infection and had not completely recovered 6 weeks after symptom onset. Another patient experiencing post-COVID-19 taste disturbances also had disrupted stem cells. These results demonstrate the possibility that novel and sudden taste changes, frequently reported in COVID-19, may be the result of direct infection of taste papillae by SARS-CoV-2. This may result in impaired taste receptor stem cell activity and suggest that further work is needed to understand the acute and postacute dynamics of viral kinetics in the human taste bud.

Keywords: ACE2; COVID-19; Taste; Type II Receptor Cell.

Figure 1

The receptor for SARS-CoV-2 angiotensin-converting enzyme 2 (ACE2) is on type II taste bud cells in taste papillae of the tongue. A: The distribution of taste buds and chemosensory cells in the oropharyngeal cavity and how virus may infect the tongue and oropharyngeal areas. Branches of three cranial nerves (VII, IX, and X) are involved in relaying taste information to the central nervous system. Taste is first discriminated in taste receptor cells (TRCs) within taste buds located in circumvallate papillae (CVP), foliate papillae (FLP), and fungiform papillae (FP) in the tongue. Three defined TRCs relay five prototypic tastes. Stem cells immediately surrounding the taste bud receive signals from taste cells, prompting differentiation into a replacement TRC. Circles on tongue, uvula, epiglottis, and oropharyngeal areas represent taste buds and chemosensory cells. Arrows indicate the probable routes of viral entry on air eddies and flowing through and around (double-headed arrows) the oral cavity. B: Hematoxylin and eosin (H&E) staining and immunofluorescence staining (IFS) of CVP toprow (post-mortem) and FP bottomrow with taste buds embedded in the epithelial layer. Keratin 8 (KRT8) is a cytoskeletal marker of all TRCs, whereas phospholipase C β₂ (PLCβ₂) is an obligatory signal molecule in all type II cells. ACE2 and PLCβ₂ were colocalized (merged signals) in IFS images. Nuclei are shown in blue, stained with DAPI. Likewise, H&E staining of a fresh FP with two taste buds (inset) and IFS for KRT8, PLCβ₂, and ACE2 show colocalization of the latter two proteins. Dashed lines in H&E of CVP and FP indicate the location of the line of stem cells. C:In situ hybridization (ISH) images of FP. Top row: Probes for PLCB2 and ACE2 confirm their colocalization in a fresh FP taste bud; nuclei are shown in blue. The yellow arrows indicate two areas outside the taste bud where ACE2 signal is found in the absence of PLCB2.Middle row: Colocalization of ACE2 and PLCB2 in the same cell is observed, and there is no overlap of the type III cell marker neural cell adhesion molecule 1 (NCAM1; light blue arrows) with either of these two markers. Bottom row: No overlap of ACE2 (taste cell positivity indicated by two white arrows) with the probe for the transcript of the type I cell marker ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2; orange arrow) and the type III marker NCAM1 (taste cell positivity indicated by two pink arrows). Scale bars = 50 μm (B and C).

Figure 2

Evidence of SARS-CoV-2 in human fungiform papillae (FP). A: Tongue images of Patient 114 moments before biopsy of her FP during the course of COVID-19 and 3 months later. B: Hematoxylin and eosin staining of a section through the FP from Patient 114 that contained two taste buds; the consecutive section was used for in situ hybridization (ISH), outlining the presence of viral particle (SARS-CoV-2 S) probe for the spike mRNA and the SARS-CoV-2 (ORF1ab) probe for the replicating virus, shown in Figure 2C. C: An antisense probe specific to the genomic positive strand RNA of the spike protein (S) sequence of SARS-CoV-2 and a sense probe to the SARS-CoV-2 ORF1ab negative strand RNA indicate the presence of replicating virus in PLCB2-positive cells (Participant 114). Taste bud is outlined by white dashed lines. Note the arrow pointing to another viral positive cell in the neighboring taste bud. D: The proliferation of the stem cell layer of the FP by immunostaining for the marker of all active phases of cell cycle Ki-67 and the late G₂ and M phase marker phosphorylated histone H3 (PHH3). Left columns: An FP from an age- and sex-matched control participant for Participant 114 compared with during and after SARS-CoV-2 infection. White arrows indicate the breaks in the otherwise continuous layer of stem cells. Right columns: A continuous stem cell layer in Participant 089 before, but multiple breaks especially at 6 weeks, and less at 10 weeks, after SARS-CoV-2. E: The percentages of total cells (as determined by DAPI-stained nuclei) positive for Ki-67 and the percentage of Ki-67–positive cells that are also positive for PHH3. F: The proliferating taste stem cells do not express cleaved caspase 3. Data are given as means ± SEM (E). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗∗P < 0.0001. Scale bars = 50 μm (B–D and F). IFS, immunofluorescence staining.