Abstract Introduction Post-COVID syndrome (PCS) affects up to 17% of individuals after acute SARS-CoV-2 infection. PCS is a multi-organ pathology presenting with fatigue, dyspnoea and cognitive dysfunction, greatly reducing patient quality of life. Despite the growing burden of PCS, little is known regarding diagnosis, biomarkers, mechanisms or potential intervention targets. We aimed to dissect the molecular and cellular landscape underlying the severity of post-COVID syndrome using single-cell RNA sequencing (scRNA-seq) of nasal biopsies. Methods PCS cases were classified when symptoms persisted 12 weeks post-acute SARS-CoV-2 infection, with PCS severity graded via symptom-based scoring (Bahmer et al. 2022, EClinMed). Nasal biopsies collected from moderate- (n = 6F/5M; avg age=37.56) and severe-PCS (n = 9F/5M; avg age=40.41) were analysed by scRNA-seq to investigate differential cell abundance, pathway signalling, and cell-cell interaction networks. Transcriptome analysis of the ALLIANCE cohort (healthy=22/asthma=189) provided comparative cell composition analysis with healthy and chronic diseased tissues. The TriNETx cohort (PCS=52,833/controls=51,310) was analysed for comorbidity risk in PCS. To validate in vivo findings, scRNA-seq was conducted on air-liquid interface (ALI) models incubated with candidate cytokines TNF-alpha and TGF-beta. All analyses were completed using R software (v4.2.1). Results scRNA-seq revealed 17 unique cell populations with reduced ciliated cells and increased basal, myeloid dendritic and T-cells cells in severe-PCS vs moderate-PCS (scProportion;FDR0.05), with no viral sequence detected. Reduced ciliated cells were observed compared to healthy tissue (25%vs35%) with a lesser change in asthma (27%). The TriNETx cohort revealed PCS is significantly associated with chronic disease such as asthma, respiratory failure and bronchitis. Cell-cell interaction analysis (CellChat) highlighted basal proliferating cells as central communicators, interacting with myeloid dendritic cells in worsening PCS. In particular, the MIF-CD64+CXCR4 axis was a critical signalling cascade in myeloid cells (p 0.05). TGF-beta and TNF-alpha were highly enriched for signalling (PROGENy) in proliferating basal cells and stimulation in myeloid/T-cells. Altered signalling, induced divergent pseudo-time trajectory maps with moderate-PCS reporting 8.1x10-3 centrality scores vs 3.1x10-3 in severe-PCS (monocle). ALI validation recapitulated the dysmorphic in vivo mucosa, where combined TGF-beta and TNF-alpha stimulation induced reduced ciliated cells (-6.6-fold difference, FDR0.05) and increased epithelial-mesenchymal transition (EMT) as observed in the in vivo analysis (GSEA). Conclusions Our study provides robust insights into PCS at a single-cell resolution. We highlight TGF-beta and TNF-alpha as critical and functional pathways associated with worse PCS. In vivo results could be recreated in vitro, underlining this as a pipeline for screening potential therapeutic interventions to improve PCS patient outcomes. This abstract is funded by: COVID-19 Research Initia- tive Schleswig-Holstein, the Follow-Up of Respiratory Infections in Schleswig-Holstein (FRISH), the German Center of Lung Research (DZL, Funding No. 82DZL001B6), intramural funding of the Christian- Albrechts-University Kiel, the University of Lübeck, and the Leibniz Lung Center, Research Center Borstel
Reddy et al. (Fri,) studied this question.