Transcriptomic Profiling of Neutrophils and Low-Density Granulocytes in COVID-19 Patients

The severity of COVID-19 is linked to excessive inflammation. Neutrophils represent a critical arm of the innate immune response and are major mediators of inflammation, but their role in COVID-19 pathophysiology remains poorly understood. We conducted transcriptomic profiling of neutrophils obtained from patients with mild and severe COVID-19, as well as from non-infected healthy controls. Additionally, low-density granulocytes (LDGs) from patients with severe COVID-19 were included to understand their unique role. Transcriptomic analysis of polymorphonuclear cells (PMNs), consisting mainly of mature neutrophils, revealed a striking type I interferon (IFN-I) gene signature in severe COVID-19 patients, contrasting with mild COVID-19 and healthy controls. LDGs from severe COVID-19 patients exhibited an immature neutrophil phenotype and lacked this IFN-I signature. These findings underscore the crucial role of neutrophil inflammasomes in driving inflammation during severe COVID-19. The study provides insights into the pathological mechanisms of severe COVID-19 and highlights potential targets for therapeutic intervention. Overall design: Neutrophils were isolated from peripheral blood of three different cohorts: patients with severe COVID-19, patients with mild COVID-19, and non-infected healthy controls. Additionally, low-density granulocytes (LDGs) were isolated from patients with severe COVID-19. cDNA synthesis from total RNA was performed using the Takara SMARTseq v4 Ultra-low input RNA kit for Sequencing. This was followed by library preparation using the Illumina Nextera XT Library preparation kit. Unique Dual Index (UDI) setup was used for the Nextera XT libraries. Library quality was assessed using the LabChip GX Touch HT High Sensitivity assay, and libraries were pooled based on the concentration measurements. The pooled libraries were quantified for sequencing using the KAPA Library Quantification Kit and sequenced on the Illumina NovaSeq6000 system using an S1 flow cell. The sequencing read length was 2x101 bp, and the paired-end run was conducted for 200 cycles. The study aims to elucidate the transcriptomic changes in neutrophils and LDGs during SARS-CoV-2 infection and their role in the inflammation associated with severe COVID-19.