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. 2024 Nov 28;64(5):2301947.
doi: 10.1183/13993003.01947-2023. Print 2024 Nov.

Single-cell sequencing reveals cellular landscape alterations in the airway mucosa of patients with pulmonary long COVID

Firoozeh V Gerayeli  1   2 , Hye Yun Park  1   3   2 , Stephen Milne  1   4 , Xuan Li  1 , Chen Xi Yang  1 , Josie Tuong  1   5 , Rachel L Eddy  1   6 , Seyed Milad Vahedi  1   5 , Elizabeth Guinto  1 , Chung Y Cheung  1 , Julia S W Yang  1 , Cassie Gilchrist  1 , Dina Yehia, Tara Stach  7 , Hong Dang  8 , Clarus Leung  1   6 , Tawimas Shaipanich  6 , Jonathon Leipsic  9 , Graeme J Koelwyn  1   5 , Janice M Leung  1   6 , Don D Sin  10   6
Affiliations

Single-cell sequencing reveals cellular landscape alterations in the airway mucosa of patients with pulmonary long COVID

Firoozeh V Gerayeli et al. Eur Respir J. .

Abstract

Aim: To elucidate the important cellular and molecular drivers of pulmonary long COVID, we generated a single-cell transcriptomic map of the airway mucosa using bronchial brushings from patients with long COVID who reported persistent pulmonary symptoms.

Method: Adults with and without long COVID were recruited from the general community in Greater Vancouver, Canada. The cohort was divided into those with pulmonary long COVID, which was defined as persons with new or worsening respiratory symptoms following ≥12 weeks from their initial acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (n=9); and control subjects defined as SARS-CoV-2 infected persons whose acute respiratory symptoms had fully resolved or individuals who had no history of acute coronavirus disease 2019 (COVID-19) (n=9). These participants underwent bronchoscopy from which a single cell suspension was created from bronchial brush samples and then sequenced.

Results: A total of 56 906 cells were recovered for the downstream analysis, with 34 840 cells belonging to the pulmonary long COVID group, which strikingly showed a unique cluster of neutrophils in the pulmonary long COVID group (p<0.05). Ingenuity Pathway Analysis revealed that the neutrophil degranulation pathway was enriched across epithelial cell clusters. Differential gene expression analysis between the pulmonary long COVID and control groups demonstrated upregulation of inflammatory chemokines and epithelial barrier dysfunction across epithelial cell clusters, as well as over-expression of mucin genes across secretory cell clusters.

Conclusion: A single-cell transcriptomic landscape of the small airways suggest that neutrophils may play a significant role in mediating the chronic small airway inflammation driving pulmonary symptoms of long COVID.

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Conflict of interest statement

Conflict of interest: F.V. Geraveli reports grants from MiTACS Accelerate. S. Milne reports payment or honoraria for lectures, presentations, manuscript writing or educational events from Chiesi Australia, The Limbic Australia and Research Review Australia, support for attending meetings from Sanofi Australia, Chiesi Australia and AstraZeneca, and a leadership role with Thoracic Society of Australia and New Zealand (NSW/ACT). R.L. Eddy reports grants from Michael Smith Health Research BC, Canadian Respiratory Research Network and Natural Sciences and Engineering Research Council Canada, consultancy fees from VIDA Diagnostics Inc., payment or honoraria for lectures, presentations, manuscript writing or educational events from Thorasys Thoracic Medical Systems Inc., and support for attending meetings from Canadian Institutes of Health Research – Institute of Circulatory and Respiratory Health. C. Gilchrist reports grants from Canadian institutes of Health Research. J. Leipsic reports consultancy fees from Heartflow, and stock (or stock options) with Heartflow. J.M. Leung reports support for the present study from Canadian Institutes of Health Research, grants from Canadian Institutes of Health Research and BC Lung Foundation, payment or honoraria for lectures, presentations, manuscript writing or educational events from BC Lung Foundation and University of British Columbia, participation on a data safety monitoring board with Enhance Quality Safety and Patient experience in Chronic Obstructive Pulmonary Disorder (EQuiP COPD), and leadership roles with Canadian Respiratory Research Network and the CanCOLD Study. D.D. Sin reports payment or honoraria for lectures, presentations, manuscript writing or educational events from GSK, AstraZeneca and Boehringer Ingelheim, and participation on a data safety monitoring board or advisory board with NHLBI. The remaining authors have no potential conflicts of interest to disclose.

Figures

None
Overview of the study. IL: interleukin; TSLP: thymic stromal lymphopoietin; SLPI: secretory leukocyte protease inhibitor.
FIGURE 1
FIGURE 1
A flow diagram summarising the participants who were enrolled between January 2021 and August 2023 and underwent bronchoscopy. 13 subjects were identified as having persistent respiratory symptoms ≥3 months post-COVID-19 (coronavirus disease 2019) infection and scored >10 in the St George's Respiratory Questionnaire (pulmonary long COVID). Nine subjects were selected as controls due to either no history of COVID-19 infection or lack of persistent respiratory symptoms post-infection. The final number of subjects used for analysis was nine in the pulmonary long COVID group and nine in the control group. #: subjects with pre-existing chronic respiratory disorders and participants who were heavily smoking tobacco cigarettes or using cannabis or e-cigarettes at the time of recruitment were excluded.
FIGURE 2
FIGURE 2
Uniform manifold approximation (UMAP) of 56 906 of profiled cells from both pulmonary long COVID and control groups, which were annotated using legacy markers. Final annotation of the cluster revealed 16 clusters that were of epithelial origin and 15 clusters that were immune cells. a) A dimensionality reduction plot of pulmonary long COVID versus control reveals distribution of clusters across both categories. It prominently highlights a specific cluster derived from the pulmonary long COVID group. b) Various clusters of cells originating from both epithelial and immune cell lineages were observed. As anticipated, the diversity among these identified cell clusters serves as a representation of the cellular heterogeneity within the airway mucosa. Additionally, distinct neutrophil cluster was highlighted. MAIT: mucosal-associated invariant T-cell; TCM: central memory T-cell; NK: natural killer.
FIGURE 3
FIGURE 3
A four-part composition plot, each representing the distribution of epithelial and immune cells identified in the sample. TCM: central memory T-cell; MAIT: mucosal-associated invariant T-cell; NK: natural killer.
FIGURE 4
FIGURE 4
Bubble plots of top 15 canonical pathways from differentially expressed genes (false discovery rate <0.05 and log2 fold change >0.5) between pulmonary long COVID and control participants across a) basal, b) secretory and c) ciliated cell clusters. n: number of differentially expressed genes regulated per pathway, with colour denoting cell subclusters. HMOX: haem oxygenase; Ca2+: calcium; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; RHOGDI: Ras homology GDP-dissociation inhibitor; ER: endoplasmic reticulum; IGF: insulin-like growth factor; IGFBP: IGF binding protein; HSP: heat shock protein; IL: interleukin; TCR: T-cell receptor; IQGAP: Ras GTPase-activating-like protein; MSP-RON: macrophage stimulating protein receptor d'origine Nantais.
FIGURE 5
FIGURE 5
The expression pattern of the commonly recognised neutrophil chemoattractant and markers of epithelial barrier dysfunction was assessed among the differentially expressed genes within our dataset. a) The cell level differential gene expression of the chemoattractant and markers of epithelial dysfunction across all epithelial cells that have >2% representation in the sample. b) The cell level differential gene expression of the all the known human mucin genes that we were able to identify across all epithelial cells that have >2% representation in the sample.
FIGURE 6
FIGURE 6
Top 20 predicated biological and chemical drug targets from differentially expressed genes (false discovery rate <0.05 and log2 fold change >0.5) between the pulmonary long COVID group and control participants across epithelial cell clusters. Negative z-scores indicate that drug administration in pulmonary long COVID would reorient cell-cluster-specific differentially expressed genes to those of controls. n: number of differentially expressed genes regulated per target, with colour denoting cell subclusters. GABA: γ-aminobutyric acid; GnRH: gonadotropin hormone-releasing hormone.
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