Impaired local intrinsic immunity to SARS-CoV-2 infection in severe COVID-19

Abstract

Infection with SARS-CoV-2, the virus that causes COVID-19, can lead to severe lower respiratory illness including pneumonia and acute respiratory distress syndrome, which can result in profound morbidity and mortality. However, many infected individuals are either asymptomatic or have isolated upper respiratory symptoms, which suggests that the upper airways represent the initial site of viral infection, and that some individuals are able to largely constrain viral pathology to the nasal and oropharyngeal tissues. Which cell types in the human nasopharynx are the primary targets of SARS-CoV-2 infection, and how infection influences the cellular organization of the respiratory epithelium remains incompletely understood. Here, we present nasopharyngeal samples from a cohort of 35 individuals with COVID-19, representing a wide spectrum of disease states from ambulatory to critically ill, as well as 23 healthy and intubated patients without COVID-19. Using standard nasopharyngeal swabs, we collected viable cells and performed single-cell RNA-sequencing (scRNA-seq), simultaneously profiling both host and viral RNA. We find that following infection with SARS-CoV-2, the upper respiratory epithelium undergoes massive reorganization: secretory cells diversify and expand, and mature epithelial cells are preferentially lost. Further, we observe evidence for deuterosomal cell and immature ciliated cell expansion, potentially representing active repopulation of lost ciliated cells through coupled secretory cell differentiation. Epithelial cells from participants with mild/moderate COVID-19 show extensive induction of genes associated with anti-viral and type I interferon responses. In contrast, cells from participants with severe lower respiratory symptoms appear globally muted in their anti-viral capacity, despite substantially higher local inflammatory myeloid populations and equivalent nasal viral loads. This suggests an essential role for intrinsic, local epithelial immunity in curbing and constraining viral-induced pathology. Using a custom computational pipeline, we characterized cell-associated SARS-CoV-2 RNA and identified rare cells with RNA intermediates strongly suggestive of active replication. Both within and across individuals, we find remarkable diversity and heterogeneity among SARS-CoV-2 RNA+ host cells, including developing/immature and interferon-responsive ciliated cells, KRT13+ "hillock"-like cells, and unique subsets of secretory, goblet, and squamous cells. Finally, SARS-CoV-2 RNA+ cells, as compared to uninfected bystanders, are enriched for genes involved in susceptibility (e.g., CTSL, TMPRSS2) or response (e.g., MX1, IFITM3, EIF2AK2) to infection. Together, this work defines both protective and detrimental host responses to SARS-CoV-2, determines the direct viral targets of infection, and suggests that failed anti-viral epithelial immunity in the nasal mucosa may underlie the progression to severe COVID-19.

Dynamic innate immune response determines susceptibility to SARS-CoV-2 infection and early replication kinetics

“…Recent work on the host response to SARS-CoV-2 in the airway epithelium or model cell lines has shown that SARS-CoV-2 can trigger IFN and ISG expression via the viral RNA sensor MDA5, albeit with delayed kinetics, but that this response in some cases was ineffective at curtailing viral replication; furthermore, while pretreatment with exogenous IFN blocked SARS-CoV-2 infection, IFN was much less effective if added after the infection was established ( Yin et al, 2021 ; Rebendenne et al, 2021 ; Hsin et al, 2021 Preprint ). Single-cell analysis of epithelial cells from the nasopharynx of COVID-19 patients showed ISG induction in epithelial cells primarily from patients with mild-to-moderate disease, but not severe disease, consistent with the observation that patients with severe disease are more likely to have genetic or acquired deficiencies in IFN signaling pathways ( Ziegler et al, 2021 Preprint ). Together, these findings indicate that epithelial cells are capable of mounting an IFN response to SARS-CoV-2 but that the timing and magnitude of the IFN response relative to viral replication may be critical for determining the course of infection.…”

The local and systemic response to SARS-CoV-2 infection in children and adults

“…Furthermore, we detect squamous cells with the characteristic expression of SPRR3 and KRT78 . In addition to the unsupervised clustering, we visualised markers ( KRT14 , KRT6A , KRT13 , KRT24 ) that have been associated with “Hillock” cells 44, 49, 50 which seem to align along a distinct differentiation trajectory at the edge of the secretory cluster from basal towards squamous cells ( Extended Data Figure 4a ). Within the ciliated cluster, the differentiation trajectory points from ciliated 1 ( PIFO , OMG ) to ciliated 2 ( CFAP54 , DZIP1L ) cells.…”

“…In contrast, basal 1 cells are notably reduced. Ciliated and goblet cells contain the highest number of viral reads ( Figure 1f ), most likely leading to cell death 50, 51 . We hypothesize that increased ciliated 1 and goblet 2 cell numbers reflect an ongoing compensatory replacement of these dying cells by their precursors to maintain homeostasis as seen in lower airways upon infection 52, 53 .…”

“…In adults viral infection is strongly associated with an interferon response 54 and for SARS-CoV-2 a higher response to interferon has been reported for mild and moderate disease than for severe disease 50, 55, 56 . We therefore carried out differential gene expression analysis (using a multivariate random effect model; see methods) between healthy controls and COVID-19 patients, followed by a GO term enrichment analysis.…”

Pharmacokinetic-based failure of a detergent virucidal for SARS-COV-2 nasal infections

“…From nasal surfaces, SARS-CoV-2 can spread locally to the olfactory region and more widely to the oral cavity and lung 1,3 . Accordingly, nasal protection represents an important strategy to limit SARS-CoV-2 infection and transmission 4 . In addition to physical means, e.g., masks, topical administration of a spectrum of virucidal agents to nasal surfaces has been advocated for this purpose, including iodine, metal, and detergent-based molecular entities [5][6][7] .…”