Maturing Neutrophils of Lower Density Associate with Thrombocytopenia in Puumala Orthohantavirus-Caused Hemorrhagic Fever with Renal Syndrome - Human samples

Puumala orthohantavirus-caused hemorrhagic fever with renal syndrome (PUUV-HFRS) is characterized by significant neutrophil activation. Neutrophils, the most abundant immune cells in circulation, are equipped to respond rapidly to infections and exhibit a notable heterogeneity. This study aims to identify and characterize different neutrophil subsets in the circulation of PUUV-HFRS patients, focusing on low-density granulocytes (LDGs) and normal density polymorphonuclear cells (PMNs). The study finds that PMNs show activation of antiviral pathways, while circulating LDGs increase following acute PUUV-HFRS. Additionally, PUUV-associated LDGs, primarily immature, likely reflect increased bone marrow neutrophil production. Notably, the frequency of LDGs and a "left shift" in blood are associated with the extent of thrombocytopenia, a hallmark of severe HFRS, suggesting a role for maturing neutrophils in disease pathogenesis. Unlike COVID-19 associated LDGs, PUUV LDGs did not exhibit significant immunosuppressive ability, indicating inherent biological differences in LDG responses based on the causative virus or infection kinetics. RNA sequencing was performed on neutrophils isolated from four cohorts: healthy control PMNs, PUUV-infected PMNs, PUUV-infected CD16- LDGs, and PUUV-infected CD16+ LDGs. The RNA sequencing and subsequent bioinformatics analysis were conducted by the Biomedicum Functional Genomics Unit at the Helsinki Institute of Life Science and Biocenter Finland at the University of Helsinki. In this study, we investigated the gene expression profiles of neutrophil subsets in patients with Puumala orthohantavirus-caused hemorrhagic fever with renal syndrome (PUUV-HFRS). Neutrophils were isolated from four distinct cohorts: healthy control polymorphonuclear neutrophils (HC PMNs), PUUV-infected polymorphonuclear neutrophils (PUUV PMNs), PUUV-infected CD16- low-density granulocytes (LDGs), and PUUV-infected CD16+ LDGs. Each cohort comprised four samples, resulting in a total of sixteen samples. The neutrophils were isolated and lysed in Trizol, followed by RNA extraction using the RNeasy micro kit. RNA sequencing was performed using the NEBNext Ultra II directional RNA library preparation method, and sequencing was conducted on the Illumina NextSeq platform. The raw and processed RNA-seq data were analyzed to quantify gene expression, enabling us to identify and characterize the different neutrophil subsets and their activation pathways during PUUV-HFRS. The findings reveal insights into the role of neutrophil heterogeneity and density in the immune response and pathogenesis of PUUV-HFRS.