scRNA-seq of SARS-CoV-2-infected lung organoids finds increased NFKB inhibitor alpha in infected cells

The ongoing coronavirus disease 2019 (COVID-19) pandemic is having a major impact on global healthcare systems and economies. Since the beginning of the pandemic caused by the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), researchers have continuously investigated the replication mechanisms of this virus.

study: NF-κB inhibitor alpha plays a cross-variant role during SARS-CoV-2 infection in ACE2-overexpressing human airway organoids. Image credit: marianstock / Shutterstock.com

Background

SARS-CoV-2 infects humans through its interactions. spike protein and host cell angiotensin-converting enzyme 2 (ACE2) receptors.

Previous studies have reported that SARS-CoV-2 primarily replicates in airway epithelial cells that express high levels of ACE2 receptors. Compared to the high levels of ACE2 expression observed in the airways and lungs, the alveolar space shows much lower levels of his ACE2 expression.

The main function of the airway epithelium is to remove and neutralize harmful substances and pathogens present in inhaled air. For example, club cells produce immunomodulatory club cell secreted proteins.

By comparison, goblet cells secrete mucin and form mucus on the airway lining that protects the underlying epithelium. Ciliated cells facilitate the movement of mucus through the airways.

Viral proteins have been detected in respiratory epithelium and lung tissue from COVID-19 patients. Although ciliated cells appear to be the natural target of SARS-CoV-2, viral proteins have also been detected in both basal and secretory cells. in vivo When ex vivo Infection.

A three-dimensional (3D) organoid model was developed to mimic the complex cellularity of human airway epithelia. These organoids consist of different types of cells grown in his 3D structures that mimic human organs.

These 3D organoid models are typically derived from pluripotent stem cells (iPSCs). embryonic stem cells (ESC), or progenitor cells. In the context of SARS-CoV-2, iPSC- or ESC-derived human airway organoids (HAOs) are often used to study their replication patterns.

Since the original strain of SARS-CoV-2 was detected in 2019, it has undergone genomic mutations that have led to the emergence of several SARS-CoV-2 variants. These variants are classified by the World Health Organization (WHO) as variants of concern (VOC) and variants of interest (VOI).

Survey results

In a recent study published on the preprint server bioRxiv*, Researchers have developed a genetically engineered airway organoid system that overcomes challenges associated with natural fluctuations in ACE2 levels. This adult stem cell-derived undifferentiated HAO model provides high infection rates without long differentiation while allowing researchers to analyze the effects of infection using genetically diverse SARS-CoV-2 variants. .

In the current study, scientists applied single-cell ribonucleic acid (RNA) sequencing to decipher cell-specific responses to SARS-CoV-2. To this end, they predicted that increased infection rates of SARS-CoV-2 variants could also be observed with other ACE2-utilizing viruses. coronavirus Such as hCOV-NL63 and SARS-CoV.

Transcriptional profiling of secretory goblet cells, clavate cells, and basal cells was also performed. These cells exhibited common transcriptional changes, indicated by clustering after infection with SARS-CoV-2 mutants.

The nuclear factor κB inhibitor alpha (NFKBIA) gene was proposed to be effective in controlling SARS-CoV-2 infection with various variants. When the NFKBIA gene is highly induced, messenger ribonucleic acid (mRNA) levels are positively correlated with viral RNA levels.

Cells infected with SARS-CoV-2 express high levels of IκBα protein. The presence of NF-κB in the nucleus of infected but not healthy cells despite the upregulation of IκBα indicates a continuous triggering of NF-κB signaling. This finding is consistent with previous studies observing a defective feedback loop in NF-κB regulation during respiratory syncytial virus (RSV) infection.

This defective feedback loop in NF-κB may represent an ongoing competition between SARS-CoV-2 and the host. Although a high proportion of her NFKBIA transcripts are present, the continued upregulation of antiviral NF-κB signaling within the host due to the persistent presence of virus affects a feedback loop. Furthermore, continuous synthesis of IκBα protein due to constant degradation of IκBα inhibitors affects viral replication.

Organoid models support the phenomenon that overexpression of mutant non-phosphorylated IκBα proteins is readily degraded. This study reported that IκBα is a proviral factor that supports viral replication in the host.

The present study supports previous observations that IκBα promotes viral infection by partially limiting the antiviral activity of NF-κB. In contrast to this observation, knockdown of p65 shows a positive effect of NF-κB signaling in SARS-CoV-2 infection.

Conclusion

The organoid model described in the current study could be a powerful tool for studying SARS-CoV-2 infection in primary airway cells. This model reduces donor-dependent variable infection rates and provides a fully differentiated primary cell model. Importantly, it also helps us understand the transcriptional reprogramming that occurs in airway progenitor cells in response to COVID-19 infection.

*Important Notices

Bio Rxiv We publish a non-peer-reviewed, preliminary scientific report and should not be taken as conclusive, to guide clinical practice/health-related actions, or to be treated as established information.