Single-Cell Multi-Omic Profiling of Human Influenza Challenge Uncovers Dynamic Peripheral Innate Responses Modulated by Disease Severity and Viral Shedding

Aaron Wilk, MD, PhD – Stanford University; Purvesh Khatri, PhD – Professor of Medicine, Stanford University; Catherine Blish, MD, PhD – Professor in Medicine, Stanford University

Abstract Text: Innate immune cells play a crucial role in early antiviral defenses, mediating initial interferon (IFN) responses for pathogen clearance, and may exhibit trained immunity through lasting epigenomic changes. We hypothesized that disease severity and shedding status may modulate the magnitude of acute and long-term innate responses. To test this, we performed paired single-cell RNA and ATAC sequencing on peripheral blood samples from a cohort of 12 healthy adults challenged with influenza (H1N1), and measured pre-infection, acute stage, and one-month post-infection timepoints.
We found that people who developed an infection had reduced proportions of NK cells at baseline compared to non-shedders. During acute infection, we found that IFN-stimulated genes (ISG) responses in CD14+ and CD16+ monocytes positively correlated with viral shedding. Notably, ISG responses were negatively correlated with disease severity at day 3 and shifted to a positive correlation by day 6. Additionally, we found that there was a day 6 emergence of intermediate monocyte-like cells across all subjects with symptomatic infections.
Further, we updated our single-cell analysis method, called single-cell MetaIntegrator, to analyze paired single-cell ATAC-seq data. Using this new method, we show that one-month post-infection, CD14+ monocytes of viral shedders showed persistent changes, including upregulated immune activation genes and significant differences in chromatin accessibility compared to baseline, while asymptomatic non-shedders showed minimal to no differences.
In summary, our findings emphasize the importance of coordinated early IFN responses in managing disease severity and suggest that symptomatic influenza may persistent epigenetic changes, offering new insights into disease.