Persistent loss of smell after COVID-19 associated with altered olfactory epithelial gene expression

In a recent study posted to the bioRxiv* preprint server, researchers performed histology of the olfactory epithelium (OE) for smell loss measurements in patients with post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (PASC).

Study: Persistent post-COVID-19 smell loss is associated with inflammatory infiltration and altered olfactory epithelial gene expression. Image Credit: megaflopp/Shutterstock
Study: Persistent post-COVID-19 smell loss is associated with inflammatory infiltration and altered olfactory epithelial gene expression. Image Credit: megaflopp/Shutterstock

Background

Studies have established the OE, which also houses sustentacular cells, as the site of SARS-CoV-2 infection in the nasal cavity. Studies in animals suggest that loss, dysfunction, or inflammation in these cells drive transient gene expression changes in olfactory sensory neurons (OSNs) or alterations in the mucus layer surrounding neuronal cilia. Notably, the olfactory receptors (ORs) nested inside the neuronal cilia detect volatile odors.

In most coronavirus disease 2019 (COVID-19) patients, transient olfactory loss, termed anosmia, is restored as the virus gets cleared. Subsequently, neurogenic basal cells reconstitute the sustentacular cell population, restoring the impaired olfactory function. However, in some patients, loss of smell may be persistent following recovery. Thus, it would be intriguing to determine what prevents recovery from anosmia in some PASC patients.

To date, studies utilizing single-cell ribonucleic acid (RNA)-sequencing (sc-RNA-seq) for directly examining the olfactory tissue obtained from PASC patients are sparse.

About the study

In the present study, researchers examined OE biopsy samples from PASC patients with olfactory dysfunction lasting more than four months using sc-RNA-seq and immunohistochemistry (IHC) to identify transcriptional alterations related to PASC-olfactory dysfunction.

They assessed the olfactory function with a smell identification test (SIT) to confirm hyposmia. The samples for scRNA-seq included six PASC hyposmics, of which five were female, and one was male in the age group of 22-58 years.

For cytokine/chemokine assays, a separate group comprised of 15 PASC hyposmics and 13 normosmic controls donated the olfactory cleft mucus. Interestingly, the respiratory epithelium within the olfactory cleft region is constituted by several cell populations, including secretory cells, ciliated cells, basal cells, submucosal Bowman’s glands, stromal cells, and immune cells.

The team dissociated biopsies and processed live-cell suspensions for scRNA-seq to examine all these cell states and transcriptional profiles. They confirmed the distribution of olfactory, respiratory, and immune cells using uniform manifold approximation projection (UMAP) plots. Likewise, pseudotime analysis confirmed the gene expression in the OE and OE lineage relationships.

Study findings

The authors observed changes in several gene transcripts related to olfactory function in neuron clusters from PASC hyposmics. Additionally, they observed diminished functionality of the adenylyl cyclase (ADCY3), a protein that couples ORs to action potentials.

Although the team normalized olfactory sensory neuron (OSN) counts to sustentacular cell counts in the PASC hyposmic samples, PASC samples were not different in the frequency of cells expressing ORs, the expression levels of the OR genes, or their distribution across OSNs compared to controls. The selective reduction in OSN numbers raises the possibility that PASC-related alterations in OSN cell numbers might be responsible for persistent changes in smell.

The researchers identified 780 high-quality sustentacular cells from PASC hyposmic or control samples. Notably, these cells strongly expressed UDP Glucuronosyltransferase Family 2 Member A1 Complex Locus (UGT2A1), a gene shown to elevate the risk of olfactory loss in COVID-19 via a genome-wide association study.

Similarly, differential gene expression analysis validated transcriptional alterations between PASC and control sustentacular cells. Gene set enrichment analysis assessed greater than 0.6 log2 fold change in the transcripts upregulated in PASC hyposmics, which indicated the occurrence of several biological processes, including interferon signaling and antigen presentation.

Together, these results suggested that sustentacular cells respond to pro-inflammatory cytokines in their microenvironment rather than directly to the SARS-CoV-2 infection.

IHC analyses further validated these findings. The authors used anti-neuron beta-III tubulin (TUJ1) antibody to stain immature OSN somata and neurites. Two regions in the post-COVID-19 hyposmic samples showed abnormal labeling patterns; while one showed intact sustentacular cells but few neurons, the other showed disorganized neurons and patchy sustentacular labels.

Conclusions   

The current study is the first to examine the olfactory biopsies from PASC hyposmic patients with olfactory dysfunction lasting at least four months following COVID-19 and perform an objective measurement of COVID-19-induced damage within the human OE. The results demonstrated altered immune cell – OE interactions which likely altered functions of sustentacular cells and OSNs, thereby explaining sensory dysfunctions, including anosmia, hyposmia, and parosmia. Further, these OSN transcriptomic changes suggested a persistent underlying non-cell-autonomous signal.

The location of the OE seems ideal for topical drug delivery. Therefore, mechanistic insights provided by the study could help develop potential novel therapeutic strategies, e.g., selectively blocking local pro-inflammatory immune cells and initiating timely medical intervention.

*Important notice

bioRxiv 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:
Neha Mathur

Written by

Neha Mathur

Neha is a digital marketing professional based in Gurugram, India. She has a Master’s degree from the University of Rajasthan with a specialization in Biotechnology in 2008. She has experience in pre-clinical research as part of her research project in The Department of Toxicology at the prestigious Central Drug Research Institute (CDRI), Lucknow, India. She also holds a certification in C++ programming.

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