Massive transient damage of the olfactory epithelium associated with infection of sustentacular cells by SARS-CoV-2 in golden Syrian hamsters

Abstract

Anosmia is one of the most prevalent symptoms of SARS-CoV-2 infection during the COVID-19 pandemic. However, the cellular mechanism behind the sudden loss of smell has not yet been investigated. The initial step of odour detection takes place in the pseudostratified olfactory epithelium (OE) mainly composed of olfactory sensory neurons surrounded by supporting cells known as sustentacular cells. The olfactory neurons project their axons to the olfactory bulb in the central nervous system offering a potential pathway for pathogens to enter the central nervous system by bypassing the blood brain barrier. In the present study, we explored the impact of SARS-CoV-2 infection on the olfactory system in golden Syrian hamsters. We observed massive damage of the OE as early as 2 days post nasal instillation of SARS-CoV-2, resulting in a major loss of cilia necessary for odour detection. These damages were associated with infection of a large proportion of sustentacular cells but not of olfactory neurons, and we did not detect any presence of the virus in the olfactory bulbs. We observed massive infiltration of immune cells in the OE and lamina propria of infected animals, which may contribute to the desquamation of the OE. The OE was partially restored 14 days post infection. Anosmia observed in COVID-19 patient is therefore likely to be linked to a massive and fast desquamation of the OE following sustentacular cells infection with SARS-CoV-2 and subsequent recruitment of immune cells in the OE and lamina propria.

Keywords: Central nervous system; Nasal cavity; Olfaction; Respiratory virus; SARS.

Fig. 1

SARS-CoV-2 nasal instillation induces massive damage of the olfactory epithelium 2 DPI which has partially healed 14 DPI. A Global view of the olfactory nasal turbinates surrounding the septum (2 DPI, Virus UCN19). Some parts of the olfactory epithelium are mildly damaged (black asterisk) while other parts are totally destroyed and released into the lumen of the nasal cavity (red asterisk), leaving the lamina propria in direct contact with the environment along with axon bundles (arrows). B Evolution of the dorsal septal region of the nasal cavity from mock infected hamsters to 14 DPI in UCN19 virus-infected hamsters. C Thickness of the olfactory epithelium in the same region. D Damage score of the olfactory epithelium for both viruses. Each histogram represents a different animal except for mock (n = 2). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Localization of SARS-CoV-2 in the nasal cavity. A Representative image of double staining of the olfactory mucosa against SARS-CoV-2 and a marker of olfactory sensory neurons (OMP, 4 DPI, UCN19). The OE is mostly disorganized but axon bundles expressing OMP are well preserved (white asterisk). The nasal cavity lumen contains many cellular aggregates revealed by the nuclear Hoechst staining along with the presence of SARS-CoV-2 antigens and OMP (white arrows). The proximity to the damaged OE suggests that these cellular aggregates could originate from the disassociation of the OE from the lamina propria. B Same double staining in a relatively preserved area of the OE from the same animal showing an absence of overlap between OMP and SARS-CoV-2 staining. C Representative image of double staining of the olfactory mucosa against SARS-CoV-2 and a marker of sustentacular cells (CK18, 4 DPI, UCN19) which colocalize.

Fig. 3

OSNs cilia are severely impaired following SARS-CoV-2 infection in the nasal cavity. A Representative images of immunostaining against G_olf (left) and SARS-CoV-2 (right) on two successive slides of the olfactory mucosa (4DPI, UCN19). Some parts of the OE still hold some of G_olf staining (white arrow), but most of it was eliminated in the cellular aggregates present in the nasal cavity lumen (orange arrow). Other OE apical regions were devoid of G_olf signal (red arrow) while retaining the signal in the axon bundles (white asterisk) B Representative images of G_olf staining from mock treated animals and 2 to 14 DPI in UCN19 virus-infected hamsters. Olfactory epithelium (OE) and lamina propria (LP) are indicated on the left of the first image. The cilia layer stained with G_olf signal is indicated by a white arrow. C Percentage of apical border of the septal OE stained with G_olf. D Scores of G_olf presence in the apical part of the OE for both viruses. Each histogram represents a different animal except for mock (n = 2). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Infiltration of immune cells in the olfactory mucosa following SARS-CoV-2 infection. A Representative images from successive slides of immunostaining against Iba1 and SARS-CoV-2 (2 DPI, UCN19). Iba1+ cells are massively present in both the olfactory epithelium and the underlying lamina propria. Both Iba1+ cells and SARS-CoV-2 are also present in the cellular aggregates in the lumen of the nasal cavity near the disorganized olfactory epithelium (upper panels, white arrows). We also observed a similar localization of infected cells and immune cells in relatively preserved part of the olfactory epithelium (lower panel). B Representative images of Iba1 staining from mock treated animals and 2 to 14 DPI in infected animals (UCN19). Olfactory epithelium (OE) and lamina propria (LP) are indicated on the left of the first image. The typical phenotypes of a resting Iba1+ cell presenting ramified shape (mock) and of an active phenotype of amoeboid shape are presented in the upper panel of the image. C Percentage of Iba1+ cells area in the olfactory epithelium. D Scores of Iba1+ cells presence and shape in the olfactory epithelium (OE) and lamina propria (LP). Each histogram represents a different animal except for mock (n = 2).

Supplementary Fig. 1

(A) Representative images of the presence of SARS-CoV-2 in the olfactory epithelium from Mock to 14 DPI (white arrows indicate the presence of the virus in the lumen of the nasal cavity) with (B ) associated score of presence in the olfactory epithelium. Olfactory epithelium (OE) and lamina propria (LP) are indicated on the left of the first image. (C) Presence of the virus in the lungs.

Supplementary Fig. 2

Presence of SARS-CoV-2 and immune cells outside of the olfactory epithelium in the nasal cavity. (A) In the vomeronasal organ and (B) in the Steno's gland.

Supplementary Fig. 3

Presence of SARS-CoV-2 and immune cells (Iba1+) in the central nervous system (J4, ICN19). (A) In the olfactory bulb (white arrow indicates a glomerulus) and (B) in different areas of the central nervous system (Upper image is the Nissl stain of the corresponding area on successive slide): Brainstem (focus on the ventral respiratory column and the nucleus of the solitary tract); Olfactory cortex (piriform cortex and olfactory tubercle), hypothalamus (paraventricular nucleus).