In a recent study posted to the medRxiv* preprint server, researchers assessed the cellular and molecular level immunopathology of post-coronavirus disease 2019 (COVID-19) lung disease (PCLD), characterized radiographically by inflammation and fibrosis.
Study: Single-cell analysis of bronchoalveolar cells in inflammatory and fibrotic post-COVID lung disease. Image Credit: createjobs51/Shutterstock.com
*Important notice: medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
Background
Studies have reported that pulmonary manifestations persist among COVID-19 patients in the post-acute period; however, the underlying molecular mechanisms of post-COVID-19 or long COVID immunopathogenesis are not well-characterized and require further investigation.
In the lungs of post-COVID-19 patients, radiological abnormalities comprise pneumonia, denoting sub-acute inflammation, and reticulation, denoting fibrosis.
Data on whether the PCLD radiological patterns represent different pathologies requiring different therapeutic strategies are limited. The investigation of which could aid in developing therapies tailored to the stage of the disease to improve the overall standard of care of long COVID patients.
About the study
In the present study, researchers evaluated T cell receptor (TCR) repertoires and single-cell level transcriptomes of immunological cells collected from the inflammation and/or fibrosis site in COVID-19 convalescents' lungs.
The convalescents had predominant computed tomography (CT) features of inflammation and fibrosis and were severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected during the first or second COVID-19 waves.
Additionally, they underwent bronchoscopy for persistent pulmonary symptomatology and radiographic features of inflammation (five individuals) or fibrosis (five individuals) after three to 12 months of acute COVID-19.
Bronchoalveolar lavage (BAL) was performed to obtain cells from the pathology site in COVID-19 convalescents, subjected to single-cell ribonucleic acid sequencing (scRNAseq) analysis.
Thoracic CT scan features were determined by interstitial lung disease (ILD) inter-disciplinary team members, including radiologists with expertise in ILD.
Further, single-cell TCR sequencing (scTCRseq) was performed to compare the TCR repertoires of the different radiological phenotypes. Differentially expressed genes (DEGs) were identified, and the abundance of different cell types was assessed.
T-cell receptor clustering, upstream regulator, and pathway enrichment analyses were performed. The VDJdb database was referred to for obtaining TCRs of Epstein-Barr virus (EBV), SARS-CoV-2, and cytomegalovirus (CMV).
Results
Single-cell level transcriptomes of the inflammatory and fibrosis PCLD phenotypes were similar for all types of cells.
However, the central memory-type T (TCM) and effector memory-type T (TEM) subsets of the helper T/ cluster of differentiation 4 (CD4) T lymphocytes and cytotoxic/CD8 lymphocytes, respectively, showed significantly greater abundance in those with PCLD-associated inflammation, with few regulatory T lymphocytes (Treg).
A greater percentage of CD4 central memory T lymphocytes also showed clonal expansion among tissues with PCLD-associated inflammation.
Strikingly, comparable TCRs from various donors with either PCLD phenotypes were highly clustered, indicative of immunological responses specific to antigens, localized at the tissue level.
Enrichment of known anti-SARS-CoV-2 TCRs was not observed. Macrophages were dominant among all individuals, with small proportions of T lymphocytes, natural killer (NK) T lymphocytes, epithelial cells, B lymphocytes, and dendritic cells, and the cells showed elevated expression of known marker genes, including interferon (IFN)-stimulated genes.
Overlapping pathways and processes were enriched despite differentially expressed gene (DEG) expression, indicating that the findings of the single-cell analysis and the pseudobulk analysis corresponded.
Interleukins (IL)-2 and -15 were the most significant upstream regulators of CD4 TCM and CD8 TEM DEGs in those with inflammation but not among those with fibrosis, indicating cytokine-mediated proliferation resulting in an elevated abundance of T lymphocytes in the inflammatory phenotype.
Inflammatory cytokines, T lymphocyte activation factors, tumor growth factor-beta (TGF-b), and osteopontin were more enriched upstream regulators of DEGs in the inflammatory post-COVID-19 lung disease monocytes.
However, considerable overlapping between molecules estimated to drive gene expression differences in the PCLD phenotypes was observed, indicating that the DEG expression was not representative of differential biological pathways between the two PCLD patterns.
The sharing of identical complementarity-determining region 3 (CDR3) sequences was minimal between individuals with different PCLD patterns.
Anti-SARS-CoV TCRs were fewer than anti-EBV or anti-CMV TCRs, indicating no enrichment of anti-SARS-CoV-2 T lymphocytes at the site of pathology. Significant enrichment of anti-CMV TCRs was observed in fibrotic PCLD.
Myeloid cell phenotype, consistent with monocytes and macrophages in healthy airspaces, was observed in both groups without statistically significant evidence of exaggerated pro-fibrogenic activities.
Conclusions
Overall, the study findings showed that inflammatory post-COVID-19 lung disease was characterized by airway T lymphocyte infiltration and long-term antigen-targeted T lymphocyte responses.
Single-cell bronchoalveolar transcriptomes of PCLD-associated inflammation and fibrosis closely resembled each other across cell types, but CD4 TCM and CD8 TEM showed greater abundance in inflammatory PLCD. Marked TCR clustering, indicative of antigen-specific responses, was evident in both phenotypes.
The findings highlight opportunities for early therapeutic intervention with T lymphocyte-targeted therapies to decrease long-term PLCD-associated morbidity and the health burden of PCLD.
*Important notice: medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
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