Scientists find main reason for continued loss of sense of smell in long COVID

In a recent study published in the journal Science Translational Medicineresearchers have reported that long-term anosmia after coronavirus disease 2019 (COVID-19) may be the result of persistent T cell-mediated inflammation occurring in the olfactory epithelium even after respiratory syndrome has been ruled out Severe acute coronavirus 2 (SARS-CoV-2) from olfactory tissue.

To study: Persistent post-COVID-19 odor loss is associated with immune cell infiltration and altered gene expression in the olfactory epithelium. Image Credit: Nicole Rerk/Shutterstock

Background

A commonly reported symptom of COVID-19 and post-acute sequelae of COVID-19 (PASC) is anosmia or loss of olfactory senses. Anosmia is thought to be caused by the effect of SARS-CoV-2 on the olfactory epithelium, which consists of the primary olfactory sensory neurons detecting odors, a layer of barrier-forming sustentacular cells, and stem cells basal or progenitor cells that restore the olfactory epithelium. Autopsy studies in humans and animal models have suggested various mechanisms explaining anosmia in COVID-19 patients, including changes in gene expression in olfactory neurons, changes in the mucus layer around the ciliated neuronal cells, altered gene expression in sustentacular cells and inflammation.

However, the mechanisms underlying the persistent anomie in PASC patients are still unclear. Possible explanations include severe epithelial damage, neuroinflammation, and damage to the olfactory bulbs or olfactory cortex of the brain. While autopsy studies on PASC patients found persistent infections in sustentacular cells and molecular changes in sensory neurons, epithelial anatomy was found intact without infection in olfactory neurons.

About the study

In the current study, the researchers used olfactory epithelium biopsy samples from nine PASC patients with persistent anosmia, which was verified using an odor identification test before biopsy. Olfactory biopsies and mucus samples from normosmic individuals with and without a history of COVID-19 were used as a control.

Biopsy specimens were processed for single-cell ribonucleic acid sequencing (sc-RNA seq) to determine cellular or transcriptional alterations associated with the persistent olfactory dysfunction observed in PASC patients. Additionally, immunohistochemical analysis was performed on the tissues using primary antibodies against a wide range of antigens including tubulin, cluster of differentiation (CD) 45, CD3, CD68, CD207, receptor gamma /delta cells (TCR γ/δ), sustentacular cell specific marker Ermin (ERMN), olfactory marker protein (OMP), SARS-CoV-2 nucleocapsid protein and sex determining region Y box 2 (SOX2).

In addition, mucosa samples were subjected to a multiplex fluorescent bead assay and flow cytometry to test for 13 chemokines and cytokines.

Results

The results reported infiltration of the olfactory epithelium with T cells secreting interferon-γ (IFN-γ) and changes in myeloid cell population resulting in increased CD207⁺ dendritic cells and a decrease in anti-inflammatory macrophages. Moreover, gene expression analysis revealed responses to continued inflammatory signaling in sustentacular cells despite the absence of SARS-CoV-2 proteins or ribonucleic acid (RNA). Compared to the number of sustentacular cells in the olfactory epithelium, the number of olfactory sensory neurons, especially mature neurons exhibiting OMP, was observed to be lower.

T cell infiltrates in nasal olfactory epithelial biopsies from hyposmic PASC patients. (A) Representative immunohistochemical images of nasal biopsy tissue from non-COVID-19 normosmic hyposmic, post-COVID-19 normosmic, or PASC individuals. Tissue sections were immunostained for the neuronal marker TUJ1, the pan-immune cell marker CD45, the T cell marker CD3 and the myeloid cell marker CD68. PASC hyposmic tissue showed dense CD45⁺ immune cell infiltration, including prominent CD3⁺ lymphocyte infiltration, which was absent in normosmic groups; scattered CD68⁺ cells were present in all conditions. (B) Magnified area (yellow box) of (A) shows CD3⁺ lymphocytes with prominent infiltration in the olfactory epithelium (white arrows); the dotted white line marks the basal lamina. Scale bar, 50 μm. (VS) Additional nasal biopsies were processed for scRNA-seq to allow for quantitative analyses. Visualization by uniform multiple approximation projection (UMAP) of combined hyposmic and normosmic control scRNA-seq PASC datasets integrating 16 human nasal biopsies enabled robust cell cluster analysis and annotation. red blood cells, red blood cells; pDC, plasmacytoid DC.

Unlike acute COVID-19 cases, there was an absence of transcriptomic changes in olfactory sensory neurons, nor was severe inflammation observed in PASC-related anosmic patients. Instead, interferon response signatures and lymphocyte populations expressing γ/δ T cells and IFN-γ were observed in olfactory epithelium samples from PASC patients with anosmia.

Comparison of immune cell phenotypes of humans with anosmia during PASC and those of hamsters with acute SARS-CoV-2 infections revealed that infiltrates in the olfactory epithelium of infected hamsters contained monocytes , neutrophils, and macrophages during the first week after infection, which resolved by the second week. In contrast, the olfactory epithelia of anosmic PASC patients were infiltrated with T cells for months after COVID-19. These differences indicated that immunological responses during PASC-related hyposmia or anosmia differ significantly from immunological responses during acute SARS-CoV-2 infections.

While the mechanisms underlying sensory dysfunction affecting olfactory sensory neurons, sustentacular cells, horizontal basal cells, and immune cells remain unclear, analysis of macrophages in patients with severe COVID-19 indicated that pro-inflammatory reprogramming during acute SARS-CoV-2 infections induces long-term changes in other immune cell functions.

The results offer potential for developing therapeutic options that can be delivered topically to the olfactory epithelium or olfactory cleft, which would also help avoid further systemic reactions to therapies.

conclusion

Briefly, the study examined olfactory epithelial biopsy specimens from anosmic PASC patients using immunohistochemical analyzes and sc-RNA sequencing. The results revealed significant differences between immunological responses during acute SARS-CoV-2 infections and PASC-related anosmia. Moreover, olfactory epithelia in anosmic PASC patients were infiltrated by IFN-γ-secreting T cells, and gene expression indicated continued inflammatory signaling despite the absence of SARS-CoV- proteins or RNAs. 2 in the olfactory tissue.