Kawasaki disease (KD) is a childhood vasculitis characterized by coronary artery lesions. The COVID-19 pandemic highlighted similarities between KD and the Multisystem Inflammatory Syndrome in Children (MIS-C), a severe complication of COVID-19. Both diseases involve severe vasculitis, yet the underlying mechanisms remain unclear. This study used single-cell RNA sequencing (scRNA-seq) to analyze peripheral blood mononuclear cells (PBMCs) from KD and COVID-19 patients, aiming to identify shared and distinct molecular mechanisms of vascular injury. The research is crucial because understanding these mechanisms could lead to better prevention and treatment strategies for both KD and COVID-19. The study leveraged the unexpected opportunity presented by the COVID-19 pandemic to compare the immunological responses in two seemingly distinct vasculitides with overlapping clinical features. The researchers hypothesized that shared molecular pathways in immune cell responses underlie the similar vascular complications observed in both KD and COVID-19.

Previous research has explored the immunological features of KD and COVID-19 separately. Studies on KD have focused on the role of inflammation and immune dysregulation in coronary artery damage, but the precise cellular and molecular mechanisms have remained elusive. Bulk transcriptomic analyses have offered insights but lacked the single-cell resolution needed to fully understand the complexity of immune responses. Similarly, studies on COVID-19 have revealed its impact on the vasculature, but a direct comparison with KD has been lacking. The emergence of MIS-C further emphasized the need to understand the shared immunological pathways between KD and COVID-19-related vasculitis. The current study builds on previous work by employing single-cell resolution, allowing a more precise comparison of immune responses in these two conditions.

The study used scRNA-seq to analyze PBMCs from six children with complete KD, three age-matched healthy controls (KHC), six COVID-19 patients (COV), three influenza patients (FLU), and four healthy controls (CHC). Peripheral blood samples were collected, and single-cell suspensions were prepared using the 10x Genomics platform. The data were processed using Cell Ranger and Seurat software, with quality control steps to filter out low-quality cells and doublets. Cell types were annotated using Azimuth, and differential gene expression analysis was performed using DESeq2. Pseudo-bulk analysis was conducted to analyze gene expression at the cell-type level. GO biological process and pathway enrichment analyses were performed using clusterProfiler. In vitro experiments, including flow cytometry, immunofluorescence, monocyte adhesion assays, and qPCR, were performed to validate the scRNA-seq findings. Specifically, the researchers used human umbilical vein endothelial cells (HUVECs) and THP-1 cells (a human monocytic cell line) in co-culture experiments to assess monocyte adhesion and endothelial cell damage. The effects of silencing genes identified in the scRNA-seq analysis (SELL, CCR1, DYSF, LMNB1, and XAF1) were evaluated to determine their roles in monocyte-endothelial cell interactions and subsequent endothelial cell damage.

scRNA-seq analysis revealed differences in immune cell composition between KD and COVID-19 patients. Both KD and COVID-19 patients showed an increased proportion of CD14+ classic monocytes. However, there were differences in the activation of other immune cell types, such as B cells and T cells. The analysis identified a set of genes upregulated in CD14+ monocytes in both KD and COVID-19 patients, but not in influenza patients. These genes were enriched in pathways related to type I interferon response and response to viruses. Further analysis focused on five genes (SELL, CCR1, DYSF, LMNB1, and XAF1) highly expressed in CD14+ monocytes of both KD and COVID-19 patients. In vitro experiments showed that the SELL+/CCR1+/XAF1+ CD14+ monocyte subset enhanced adhesion to and damage of endothelial cells. Silencing of SELL, CCR1, and XAF1 reduced monocyte adhesion and endothelial cell damage. Analysis of B cells revealed different subtypes participating in KD and COVID-19. KD involved naïve and intermediate B cells, while COVID-19 primarily involved plasmablasts. Analysis of T cells showed activation of NK cells and CD8 TEM cells in KD, while COVID-19 showed differences in other T cell subsets. This highlights a critical role for innate immunity in both diseases but indicates differences in adaptive immune responses. The study validated scRNA-seq findings through in vitro experiments, confirming the role of SELL, CCR1, and XAF1 in monocyte-mediated endothelial damage. Immunostaining of coronary artery sections from KD patients showed increased endothelial cell damage compared to healthy controls.

The findings highlight the importance of innate immunity, particularly CD14+ monocytes, in the pathogenesis of both KD and COVID-19-associated vasculitis. The study identified a specific monocyte subset (SELL+/CCR1+/XAF1+) that contributes to endothelial damage in both diseases. This suggests potential therapeutic targets for interventions in both conditions. The differences in adaptive immunity activation between KD and COVID-19 could explain variations in clinical presentation. The study also highlights the usefulness of scRNA-seq in dissecting the complex immune responses in these conditions. The validation experiments provide strong support for the scRNA-seq findings, underscoring the functional relevance of the identified genes in monocyte-endothelial cell interactions. The findings suggest that targeting the identified genes (SELL, CCR1, XAF1) could represent a novel therapeutic strategy for both KD and COVID-19-related vasculitis. Further research is needed to investigate these targets and to explore potential therapeutic approaches.

This study demonstrates the pivotal role of innate immune responses in endothelial dysfunction in both KD and COVID-19, with differences in adaptive immunity activation. The study identifies SELL, CCR1, and XAF1 as potential therapeutic targets. Future studies should explore the specific mechanisms of action of these genes and develop targeted therapies.

The study had a relatively small sample size, especially for the COVID-19 cohort. The absence of coronary artery samples from COVID-19 patients limits the direct comparison of vascular damage. Further studies with larger cohorts and direct comparison of vascular tissues are needed to validate the findings more comprehensively.