The project

Coronavirus disease 2019 (COVID-19) represents a serious global public health issue. Although effective vaccines have successfully reduced both viral transmission and disease burden, an urgent need remains to elucidate the mechanisms that lead to severe COVID-19, predict its severity and develop new treatments. Although multiple single-cell RNA-sequencing (scRNA-seq) studies of peripheral blood samples have highlighted dysregulated immune responses in patients with COVID-19 (Ref. 1), the mechanisms that underly the dysfunctional immune response in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are not yet fully understood. In addition, genome-wide association studies (GWASs) of patients with COVID-19 have demonstrated that the host’s genetic background influences their susceptibility to and/or the severity of COVID-19 (Refs. 2,3). However, the functional roles of the genetic variants that have been identified by these GWASs remain elusive, especially in non-European populations. Our goal was to elucidate the immune responses in COVID-19 from both a pathophysiological and host genetic perspective in the Japanese population.

The discovery

We used a multi-omics approach including scRNA-seq, T cell receptor and immunoglobulin heavy chain (VDJ) sequencing to analyze 895,460 peripheral blood mononuclear cells (PBMCs) from 73 patients with COVID-19 and 75 healthy controls, all of Japanese ancestry, to investigate the systemic immune response in COVID-19 (Fig. 1a). Next, we integrated our scRNA-seq data with polygenic signals from the largest COVID-19 GWAS2 using a single-cell disease-relevance score (scDRS)4 to evaluate the contribution of genome-wide host genetics to risk of COVID-19 and identify subpopulations of disease-associated PBMCs. Finally, to understand how transcriptional dynamics are regulated by COVID-19-associated variants, we examined single-cell expression quantitative trait loci (eQTL) effects of the COVID-19 risk variants that were replicated in a GWAS of a Japanese population3,5.

Fig. 1: Associations of cell types with host genetic risk of COVID-19.
figure 1

a, Uniform manifold approximation and projection (UMAP) of 895,460 PBMCs, colored by 13 cell types. b, COVID-19 PBMCs colored by scDRS4 calculated from GWAS summary statistics of three phenotypes2. c, Heatmaps depicting each cell type–disease association for three disease phenotypes. Colors denote uncorrected P values for cell type–disease associations evaluated using a scDRS4. False discovery rates (FDRs) were calculated via the Benjamini–Hochberg method across all pairwise comparisons. cDC, conventional dendritic cells; DC, dendritic cells; int, intermediate; MAIT, mucosal-associated invariant T cells; NK, natural killer cells; PB, plasmablasts; pDC, plasmacytoid dendritic cells; pro, proliferative; sig, significant; Treg, regulatory T cells. © 2023, Edahiro, R. et al. CCBY 4.0.

We found that the decreased fraction of non-classical monocytes (ncMono), a known COVID-19-specific feature1, is partially due to downregulation of the cellular transition from classical monocytes (cMono) to ncMono. Differential expression analysis revealed reduced CXCL10 expression in ncMono in individuals with severe COVID-19. Cell–cell communication analysis inferred that cellular interactions involving ncMono are decreased in severe disease, which suggests that their dysfunction might be closely involved in the immunopathology of COVID-19 severity. We also observed enriched expression of the putative disease genes that were identified by the GWAS of patients who were hospitalized and those with very severe COVID-19 in the monocytes and dendritic cells, whereas no cell type showed enriched expression of these genes in the GWAS of people with self-reported infection (Fig. 1b,c). The single-cell eQTL analysis revealed that COVID-19-associated variants (such as the IFNAR2 variant rs13050728) had context (COVID-19)-specific and cell-type (monocyte)-specific eQTL effects. These multimodal and integrative data analyses consistently indicated an enrichment of host genetic factors that confer an increased risk of severe COVID-19 in innate immune cells.

The implications

This study highlights the essential role of innate immune cells in determining COVID-19 severity. We revealed that ncMono are involved in the pathogenesis of COVID-19 severity, which suggests a potential new drug target. We also clearly demonstrated cell-type-specific and context-specific eQTL effects of COVID-19-associated gene variants, which implies that such analysis could aid in the understanding of its pathogenesis and the potential for personalized therapy.

Comprehensive evaluation of the latest GWAS-identified COVID-19 risk variants is desirable. We adopted a pseudo-bulk approach for our eQTL analysis that was restricted to coarse cell states that imperfectly partition a continuous transcriptional landscape. Therefore, conducting a genome-wide and dynamic eQTL analysis at single-cell resolution would provide a more comprehensive understanding of dynamic gene regulation in the context of COVID-19.

To this end, we aim to further expand our single-cell dataset via an international collaboration network to cover the global diversity of host genetic data and perform genome-wide single-cell eQTL analysis. In parallel, we want to explore the immunological mechanisms that underpin the dysfunction of ncMono using other single-cell modalities such as assay for transposase-accessible chromatin (ATAC)-seq and in vivo experiments.

Ryuya Edahiro & Yukinori Okada

Osaka University Graduate School of Medicine, Suita, Japan.

Expert opinion

“The researchers conducted a scRNA-seq study of peripheral blood cells from 73 patients with COVID-19 and a genetic association analysis in which these patients were compared with 75 healthy control individuals. The data included in this study form a rich resource for deep mining the immunological changes caused by COVID-19 and the associated genetic background.” Xianwen Ren, Changping Laboratory, Beijing, China.

Behind the paper

In early 2020, the Japan COVID-19 Task Force was established as a nationwide multi-center consortium and conducted a COVID-19 GWAS and eQTL analysis in the Japanese population3,5. In parallel, we conducted PBMC scRNA-seq analysis of Japanese patients with COVID-19 and healthy controls, supported by the Team Osaka University Research Project and the Nippon Foundation. The COVID-19 GWAS by the Japan COVID-19 Task Force reported the COVID-19-specific eQTL effect of identified risk alleles in a cell-type-specific manner3. Motivated by these findings, we chose to focus on ncMono in our study. The 2022 report from the COMBAT Consortium1, a comprehensive multi-omics study that compared patients with COVID-19 and individuals who had influenza or sepsis with healthy controls, convinced us that ncMono have an important role in the pathogenesis of COVID-19. R.E & Y.O.

From the editor

“This study provides an invaluable resource for those interested in studying the role of the innate immune system in COVID-19 at single-cell resolution. By integrating these data with host genetic data, the authors provide much-needed insight into the factors that govern disease severity.” Safia Danovi, Senior Editor, Nature Genetics.