Neutrophils play a major role in the destruction of the olfactory epithelium during SARS-CoV-2 infection in hamsters

Abstract

The loss of smell (anosmia) related to SARS-CoV-2 infection is one of the most common symptoms of COVID-19. Olfaction starts in the olfactory epithelium mainly composed of olfactory sensory neurons surrounded by supporting cells called sustentacular cells. It is now clear that the loss of smell is related to the massive infection by SARS-CoV-2 of the sustentacular cells in the olfactory epithelium leading to its desquamation. However, the molecular mechanism behind the destabilization of the olfactory epithelium is less clear. Using golden Syrian hamsters infected with an early circulating SARS-CoV-2 strain harboring the D614G mutation in the spike protein; we show here that rather than being related to a first wave of apoptosis as proposed in previous studies, the innate immune cells play a major role in the destruction of the olfactory epithelium. We observed that while apoptosis remains at a low level in the damaged area of the infected epithelium, the latter is invaded by Iba1⁺ cells, neutrophils and macrophages. By depleting the neutrophil population or blocking the activity of neutrophil elastase-like proteinases, we could reduce the damage induced by the SARS-CoV-2 infection. Surprisingly, the impairment of neutrophil activity led to a decrease in SARS-CoV-2 infection levels in the olfactory epithelium. Our results indicate a counterproductive role of neutrophils leading to the release of infected cells in the lumen of the nasal cavity and thereby enhanced spreading of the virus in the early phase of the SARS-CoV-2 infection.

Keywords: Innate immunity; Pathophysiology; Post-viral olfactory disorder (PVOD).

Fig. 1

Apoptosis occurs in desquamated cells in the lumen of the nasal cavity following SARS-CoV-2 infection but not in the olfactory epithelium. Representative images of an infected intact (A), infected damaged (B) area of the olfactory epithelium at 2 days post-infection (dpi) and in a control animal (C). Apoptotic cells in the olfactory epithelium are indicated by a white arrow (OE; olfactory epithelium/LP lamina propria). The lumen of the nasal cavity is indicated by a white asterisk and is filled with cells, some of which colocalize in their nucleus cleaved caspase 3 signal (orange arrow). (D) Cleaved caspase 3 signal in the olfactory epithelium normalized to control (log 10, Mean ± SEM, n = 4, *p < 0.01 (Mann–Whitney test))

Fig. 2

Iba1⁺ (microglia/monocyte lineage), CD68⁺ (macrophages) and MPO⁺ (neutrophils) cells presence in the olfactory epithelium before and during SARS-CoV-2 infection. Immunostaining on successive slides of the olfactory epithelium from a non-infected (A) or 1 dpi hamster (B). Only Iba1⁺ cells are present in the uninfected olfactory epithelium (OE) and in the lamina propria (LP). In the infected epithelium, Iba1⁺ cells are massively present in the OE while CD68⁺ and MPO cells are mostly present in the desquamated cells (red asterisk) in the lumen of the nasal cavity (white asterisk)

Fig. 3

Iba1⁺ cell infiltration increases with the damage in the OE. Representative images of the olfactory epithelium from an uninfected animal (A), infected but undamaged (B) and infected and damaged (C) area of the olfactory epithelium (OE) at 2 days post-infection (dpi). The lumen of the nasal cavity is indicated by a white asterisk. (D) Iba1⁺ signal in the olfactory epithelium (OE, left) and lamina propria (LP, right) in control animals (CTL) or at 1 or 2 dpi (Mean normalized to control ± SEM, n = 4, *p < 0.01 (Mann–Whitney test)). (E) Correlation between score damage of the olfactory epithelium and the percentage of Iba1⁺ signal in the olfactory epithelium (left panel) and the lamina propria (right panel). Spearman test p value

Fig. 4

CD68⁺ macrophage and MPO⁺ neutrophil cells are associated with damage of the olfactory epithelium during SARS-CoV-2 infection. CD68⁺ (A₁) and MPO⁺ (B₂) signal in the olfactory epithelium (OE, left) and lamina propria (LP, right) in either control animals (CTL) or at 1 or 2 days post-infection (dpi) (Mean normalized to control ± SEM, n = 4, *p < 0.05 (Mann–Whitney test)). Correlation between score damage and percentage of CD68⁺ (A₁) and MPO⁺ (B₂) signal in the olfactory epithelium (left panel) and the lamina propria (right panel). Spearman test p value

Fig. 5

Immunosuppression induced by cyclophosphamide reduces damage of the olfactory epithelium as well as OE infection area. (A) Expression of innate immune genes in the nasal turbinates with or without cyclophosphamide treatment at 1 and 2 days post-infection (dpi). Iba1, CD68 and Ncf2 are related to the presence of microglia/macrophages, monocytes/macrophages and neutrophils, respectively; TNFα and IL6 are two cytokines expressed during inflammation; SARS-CoV-2 N expression is related to the SARS-CoV-2 infection. Results represent the Mean ± SEM relative to vehicle-treated hamsters (n = 4, *p < 0.05; Mann–Whitney test). Representative images of the infected olfactory epithelium immunostained for MPO (neutrophil marker) and SARS-CoV-2 N protein in (B) vehicle and (C) cyclophosphamide treated animal (olfactory epithelium (OE), lamina propria (LP)). In the vehicle condition, the lumen (white asterisk) is filled with desquamated cells (red asterisk) containing MPO signal. In the cyclophosphamide condition, MPO signal is absent and the lumen is mostly free of cellular debris. Quantification in the OE of (D₁) MPO⁺ neutrophil presence (D₂) damage score (D₃) SARS-CoV-2-infected area and in the lumen of the nasal cavity of (D₄) desquamated cells area and (D₅) percentage of SARS-CoV-2-infected area in the desquamated cells (Mean ± SEM, n = 8 areas of the nasal cavity from 4 different animals, *p < 0.05, **p < 0.01, ***p < 0.001 (Mann–Whitney))

Fig. 6

Inhibition of neutrophil proteinases reduces damage of the olfactory epithelium as well as infected area. Representative images of the infected olfactory epithelium immunostained for MPO (neutrophil marker) and SARS-CoV-2 N protein in (A) vehicle and (B) cathepsin C inhibitor (IcatC_XPZ-01) treated animal (olfactory epithelium (OE), lamina propria (LP)). In the vehicle condition, the lumen (white asterisk) is filled with desquamated cells (red asterisk) containing MPO signal. Under cathepsin C inhibition, MPO signal is less abundant and the lumen is mostly free of cellular debris. Quantification (C₁) in the OE of MPO⁺ neutrophil presence; damage score; SARS-CoV-2-infected area and (C₂) in the lumen of the nasal cavity of desquamated cell area and percentage of SARS-CoV-2-infected area in the desquamated cells (Mean ± SEM, n = 8 areas of the nasal cavity from 4 different animals, *p < 0.05, **p < 0.01, ***p < 0.001 (Mann–Whitney test))

Fig. 7

Model of innate immune cell signaling leading to olfactory epithelium desquamation. The olfactory epithelium is mainly composed of olfactory sensory neurons (OSN) surrounded by supporting cells (sustentacular cells) and basal cells able to regenerate all cell types of the epithelium. During the infection of sustentacular cells (turning red), Iba1⁺ cells become activated and infiltrate the olfactory epithelium followed by neutrophils and macrophages. Neutrophils release elastase-like proteinase leading to destabilization of the epithelium structures and the expulsion of cells including non-infected neurons into the lumen of the nasal cavity. The release of infected cells may contribute to an increased spreading of the virus in the OE