New Study Finds Connection Between Immune Response & Persistent Smell Loss Post-COVID-19

Most SARS-CoV-2 patients experience anosmia, or a loss of smell, which can linger for months after recovery. According to olfactory epithelium samples taken from 24 biopsies, T cell-mediated inflammation continues in the olfactory epithelium even after SARS-CoV-2 has been removed from the tissue, indicating a mechanism for long-term post-COVID-19 smell loss.

The severe acute respiratory syndrome coronavirus 2 is thought to harm the olfactory epithelium, a specialized tissue involved in smell sense that runs along the top of the nasal cavity (SARS-CoV-2). The olfactory epithelium comprises three key components: odor-detecting olfactory receptor cells, a sustentacular cell layer for support, and basal stem cells that replenish the cells continually.

In animal models of SARS-CoV-2 infection, transient gene expression changes in olfactory sensory neurons, changes in the nature of the mucus layer surrounding their cilia, and inflammation are hypothesized to induce acute anosmia. The virus infects sustentacular cells rather than neurons, according to research in both animal models and human postmortem tissues.

Hypotheses for inhibition of recovery

Although the typical reparative processes restore function following viral clearance in the majority of patients with COVID-19-associated anosmia, the reasons underlying what inhibits recovery in patients with post-acute sequelae SARS-CoV-2 infection (PASC) remain unknown. There are many different hypotheses, such as severe tissue damage that eliminates basal stem cells, neuroinflammation or autoimmune phenomena that disrupt normal olfactory functions via gene alterations, or central processes that cause damage to the olfactory cortex of the brain.

Despite the fact that the examination of human autopsy tissue derived from patients who died of PASC revealed persistent infection of sustentacular cells and diverse molecular changes in olfactory sensory neurons that could lead to changes in smell detection, no damage was detected in olfactory sensory neurons, and the epithelial anatomy was found to be intact.

Analyses performed using the biopsies and results

Prior to this study, no direct analysis of olfactory tissue from patients with chronic olfactory impairment had been conducted. The researchers utilized olfactory epithelium biopsies obtained from nine PASC patients with chronic anosmia, which was confirmed by a smell identification test before the biopsies. The researchers examined changes associated with PASC-related olfactory dysfunctions via the use of immunohistochemistry as well as single-cell RNA sequencing.

An initial immunohistochemistry analysis was performed on olfactory epithelium biopsies collected from the nine PASC patients. Samples from non-COVID and post-COVID persons who had a normal sense of smell were used as controls in this study. 

T cells, which were identified by CD3 expression, appeared to be more widespread in PASC hyposmic samples, and many of these were localized within the upper layers of the epithelium itself rather than being confined to the deeper stroma, as was observed in control tissue. This was in contrast to what was seen in control tissue, where T cells were observed to be confined to the deeper stroma. Post–COVID-19 normosmic samples and non–COVID-19 normosmic samples did not include cells with the same markers.

Given the presence of T cell infiltration into the PASC olfactory epithelium, additional nasal biopsy samples were obtained from individuals who reported that their olfactory dysfunction had persisted for at least four months since the onset of COVID-19. These samples were then analyzed using single-cell sequencing. There were no transcripts from SARS-Cov-2 retrieved from any of our biopsies, which is consistent with the fact that there was neither an active infection nor a chronic level of viral RNAemia. 

A broad range of T cell subtypes was identified in both the control and PASC hyposmic olfactory epithelium based on the expression of cell type-specific markers (canonical markers). There was an abundance of resident CD8⁺ T cells, which were recognized as γδ T cells based on immunological marker gene expression. The γδ T cells identified here express the inflammatory cytokine interferon-γ, which facilitates immunity regulation and adaptive response. 

Myeloid lineage received more attention as a result of the predominance of T cells in PASC olfactory epithelium. Myeloid lineage has the ability to coordinate changes in lymphocyte populations. The anti-CD68 labeling revealed the presence of myeloid cells in the olfactory epithelium of both the control and the PASC cells. Further analysis using scRNA-seq revealed alterations in the population of myeloid cells, which led to an increase in CD207⁺ dendritic cells and a reduction in anti-inflammatory M2 macrophages.

Despite the lack of SARS-CoV-2 proteins or ribonucleic acid, further gene expression analyses indicated responses in the sustentacular cells to continued inflammatory signaling (RNA). As the apical barrier cell lining the olfactory epithelium, sustentacular cells serve multiple purposes. These include the detoxification of harmful chemicals and the feedback regulation of olfactory epithelium stem cells. Antigen presentation genes were enriched in sustentacular cells derived from PASC hyposmic samples, which is consistent with sustentacular cells mounting a response to inflammation. However, typical markers of an active viral infection, such as PTX3 or CD46, showed minimal changes.

It was shown that the number of olfactory sensory neurons, particularly mature neurons that presented olfactory marker proteins, was much lower when compared to the amount of sustentacular cells found in the olfactory epithelium. Based on the data obtained from scRNA-seq, olfactory sensory neuron counts were normalized to sustentacular cell counts in order to provide a quantitative estimate of the number of olfactory sensory neurons. This was subsequently validated by immunohistochemical labeling on additional samples, including mature olfactory sensory neurons stained with anti–olfactory marker protein antibody and sustentacular cells stained with an apical layer anti-SOX2 antibody. In PASC hyposmic biopsies, the ratio of mature olfactory sensory neurons to sustentacular cells was considerably lower compared to normosmic groups.

A comparison of immune cell phenotypes in humans with anosmia during PASC and those in hamsters with acute SARS-CoV-2 infections revealed that infiltrates in infected hamsters’ olfactory epithelia resolved within two weeks, whereas T lymphocytes penetrated anosmic PASC patients’ olfactory epithelia for months after COVID-19. These disparities revealed that immunological responses during PASC-related hyposmia or anosmia differed significantly from those seen during acute SARS-CoV-2 infections.

Concluding remarks

The researchers used immunohistochemistry and sc-RNA sequencing to investigate olfactory epithelium biopsy samples from anosmic PASC patients. T-cells infiltrated the olfactory epithelia of anosmic PASC patients, and gene expression showed continuing inflammatory signaling despite the lack of SARS-CoV-2 proteins or RNA in olfactory tissue. 

The pandemic has revealed the urgent need for novel, effective therapies for olfactory loss. The findings suggest the possibility of creating treatment alternatives that may be directly given to the olfactory epithelium, perhaps avoiding additional systemic effects of the medications. 

Article Source: Reference Paper

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