Expression analysis of altered neutrophils from septic mice

Neutrophils are critical for effective anti-fungal immune responses. However, in fungal sepsis neutrophils undergo a phenotypical (Ly6G downregulation) and functional alteration (defective ROS production), impairing their anti-fungal capacity. Therefore we wish to characterize the neutrophils in septic mice transcriptionally to describe their role in driving sepsis. We included several control groups. Mice that lack T cells (TCRaKO mice) have increased resistance to the infection and their neutrophils appear unaltered. We identified G-CSF and DNA/histones to be responsible for affecting granulopoiesis, as rG-CSF and Clodronate liposomes combined expand Ly6Glow neutrohils in naive mice. Overall design: The spleens were gently meshed and filtered to obtain a single cell suspension. ACK treatment was performed to lyse erythrocytes. A neutrophil enrichment was performed using a commercially available kit (StemCell), to obtain a semi-pure neutrophil suspension. These cells were stained with fluorescently labeled antibodies and DAPI, and live neutrophils were sorted as CD3/CD19-, CD11b+ and either high or low expression of Ly6G. 20.000-100.000 cells were sorted per condition. Sorted neutrophils were washed and resuspended in 300ul RLT. RNA extraction was performed using the Qiagen RNeasy mini-kit, and the Nugen cDNA synthesis kit was used for library prep. GCSFCLODRONATE treatment: Mice were injected intravenously with 1mg of clodronate liposomes (Liposoma) and the subsequent day intraperitoneally with 2.5ug recombinant G-CSF (rG-CSF; BioLegend). Splenic neutrophils were collected for RNA extraction two days after rG-CSF injection.