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In a recent study posted to the bioRxiv* pre-print server, researchers evaluated the underlying cellular mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced olfactory epithelium (OE) damage in golden Syrian hamsters.

Study: Neutrophils initiate the destruction of the olfactory epithelium during SARS-CoV-2 infection in hamsters. Image Credit: Nata Bene/Shutterstock

Anosmia is one of the most prevalent symptoms of coronavirus disease 2019 (COVID-19). Although it is well-known that anosmia occurs due to SARS-CoV-2-induced desquamation of the sustentacular cells (SCs) of the OE, the molecular mechanisms of this OE damage are unclear.

About the study

In the present study, researchers investigated the initial events in OE damage induced by SARS-CoV-2 infections.

The study was performed on 56 eight weeks-old male hamsters in a biosafety level 3 lab. The hamsters were infected with SARS-CoV-2 by nasal instillation under isoflurane. These animals were injected with cyclophosphamide and IcatCXPZ-01 intraperitoneally before SARS-CoV-2 infection to deplete the neutrophils and inhibit the activity of neutrophil elastase-like proteinases, respectively.

Additionally, famotidine pregnancy risk the hamsters’ nasal cavity was cryo-sectioned for immunohistochemistry (IHC) analysis to examine OE, the vomeronasal organ (VNO), Steno’s gland, and the olfactory bulb.  The IHC analysis also enabled the characterization of the ionized calcium-binding adapter molecule 1 (Iba1+), cluster of differentiation 68 (CD68+), and myeloperoxidase (MPO+) cells present in the lamina propria and the OE. The sections were incubated with antibodies against the viral nucleocapsid (N) protein to determine the percentage of infected desquamated cells in the nasal cavity lumen.

The extent of apoptosis was determined based on the cleaved caspases 3 (C3C) activity. The infiltration of immune cells such as microglia, neutrophils, and macrophages was assessed by the Iba1, MPO, and CD68 expression, respectively.

In addition, ribonucleic acid (RNA) was extracted from the nasal turbinates and subjected to quantitative polymerase chain reaction (qPCR) assays to determine the expression of Iba1, CD68, neutrophil cytosol factor 2 (ncf2), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) and the viral presence. The assay also assessed the impact of cyclophosphamide treatment on the expression of genes associated with innate immunity. Luminescence-based cell viability assays were performed to evaluate the loss of cell viability on the third day post-infection (dpi) which reflected the efficiency of the viral infection.

The tissues were divided into four zones for interpretation- infected, uninfected, intact, and damaged. The results were analyzed within two dpi to determine the initial OE response to SARS-CoV-2 infections.

Results

In infected OE, Iba1+ cells were majorly present whereas the CD68+ and MPO+ cells were absent. In contrast, the MPO+ and CD68+ cells were abundant in the exfoliated cells. This indicates that while the microglial cells were present in copious amounts in the infected OE, the macrophages and neutrophils were predominant in the exfoliated cells.

Additionally, increased C3C signal indicative of robust apoptotic activity was observed in the desquamated cells in the nasal cavity lumen that further increased by 14-fold on the second dpi, indicative of their exfoliation. On the other hand, in infected and damaged OE, the apoptotic activity was limited.

In infected OE, widespread infiltration by innate immune cells was observed. The ncf2 expression was substantially enhanced on the first dpi and further increased on the second dpi. This indicated that neutrophils were present on the first dpi and that their counts continued to increase on the second dpi along with the advent of abundant Iba1+ microglia and a few CD68+ macrophages. Surprisingly, on depleting neutrophils (by cyclophosphamide treatment) and inhibiting their proteinase activity, decreased Ncf2, MPO, TNF-α, and IL-6 expression were observed.

While reduced ncf2 and MPO expression indicated decreased neutrophil counts, reduced TNF-α and IL-6 indicated decreased viral inflammation. The expression of all genes related to innate immunity was also decreased on the second dpi. Surprisingly, neutrophil impairment corresponded with low N protein expression. This finding was confirmed by a significantly decreased nasal luminal area occupied by desquamated cells on inhibition of neutrophil action. This indicates that reduced neutrophil activity correlated with reduced SARS-CoV-2-induced olfactory damage.

To summarize, SARS-CoV-2 infections do not directly induce OE damage, but indirectly result in anosmia due to their impact on the neutrophils. These neutrophils have a major causative role in OE destruction following SARS-CoV-2 infection as they release elastase-like proteases, and their inhibition would limit viral replication. While the virus-infected OE had abundant microglia and showed limited apoptosis, the macrophages and neutrophils were predominant among the highly apoptotic exfoliated cells predominantly present in the nasal cavity lumen.

*Important notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Bourgon Clara, et al. (2022). Neutrophils initiate the destruction of the olfactory epithelium during SARS-CoV-2 infection in 2 hamsters.  bioRxivdoi: https://doi.org/10.1101/2022.03.15.484439 https://www.biorxiv.org/content/10.1101/2022.03.15.484439v1

Posted in: Medical Science News | Medical Research News | Disease/Infection News

Tags: Anosmia, Antibodies, Apoptosis, Assay, Calcium, Cell, Coronavirus, Coronavirus Disease COVID-19, covid-19, Cyclophosphamide, Genes, IHC, immunity, Immunohistochemistry, Inflammation, Interleukin, Interleukin-6, Microglia, Molecule, Myeloperoxidase, Necrosis, Neutrophils, Polymerase, Polymerase Chain Reaction, Protein, Protein Expression, Respiratory, Ribonucleic Acid, RNA, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Syndrome, Tumor, Tumor Necrosis Factor, Virus

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Written by

Pooja Toshniwal Paharia

Dr. based clinical-radiological diagnosis and management of oral lesions and conditions and associated maxillofacial disorders.

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