Ifnα2

Neutrophils from the lung, spleen, and bone marrow as well as bone marrow-derived T and B cells were isolated from naïve wild-type, IFNAR^−/−, IFNLR1^−/−, IFNAR^−/−IFNLR1^−/−(DKO), MRP8^cre Ifnlr1^fl/fl, MRP8^cre Stat1^fl/fl, MRP8^cre, Ifnlr1^fl/fl, and Stat1^fl/fl mice. Neutrophils and B cells were stimulated with 100 ng of IFNα2 (Novoprotein) or IFNλ2 (PBL). B cells were sorted as Live CD45⁺B220⁺, T cells were sorted as Live CD45⁺Thy1.2⁺, and neutrophils were sorted as Live CD45⁺CD11b⁺Ly6C^intLy6G⁺. These populations were sorted to >99% purity. Cell lysates were collected in lysis buffer containing protease and phosphatase inhibitors. Equal amounts of total protein was separated on 7.5% SDS-PAGE gels, transferred to Nitrocellulose 0.45 µm membrane (BIO-RAD), and subsequently probed with antibody against phosphorylated STAT1 (BD #612133) and β-actin (Sigma #A5441). For PCR genotyping, DNA from sorted cells was isolated using Qiagen DNeasy kit, and run on a 4% agarose gel. Control DNA was extracted from tail snips.

For depletion of CCR2⁺ cells, CCR2-DTR mice and control CCR2-DTR negative littermates received 250 ng of diphtheria toxin i.p. one day prior to infection and every other day thereafter in order to maintain depletion. Mice were injected i.p. with 1.0 µg of either IFNα2 (Novoprotein), IFNλ3 (PBL), or both on D0 and D+1 for ROS analysis 48 hours post-infection. For survival, mice were injected i.p. with 1.0 µg of either IFNα2, IFNλ3, or 500 ng of both for a total of 1.0 µg on D0 and every other day. Mice were euthanized as they developed severe symptoms of IA, and survival was terminated at D+14.
For CCR2⁺monocyte transfer experiments, CD45.1⁺CCR2-GFP⁺ monocytes were sorted from mice that were infected with 4 × 10⁷Af conidia for 3 hours (peak of IFN-α transcription). CD45.1⁺CCR2-GFP⁺ monocytes (~850,000) were transferred via retro-orbital injection into CD45.2⁺CCR2-DTR mice that have been infected with 4 × 10⁷Af conidia for 3 hours. CD45.2⁺CCR2-DTR mice were treated with DT on D-1, D0, and D+1. Congenic markers were used to track transferred cells in recipients by flow cytometry. ROS and fungal burden was assessed 48 hours after infection. Monocytes were sorted as (Live CD45.1⁺CD11b⁺NK1.1⁻CCR2-GFP⁺Ly6C⁺)