IPS-1 Is Essential for the Control of West Nile Virus Infection and Immunity

Suthar, Mehul S.; Ma, Daphne Y.; Thomas, Sunil; Lund, Jennifer M.; Zhang, Nu; Daffis, Stephane; Rudensky, Alexander Y.; Bevan, Michael J.; Clark, Edward A.; Kaja, Murali-Krishna; Diamond, Michael S.; Gale, Michael

Draining lymph nodes from mice were isolated and digested with collagenase (Roche) and type I DNase in serum-free RPMI media at 37uC for 40 minutes with mechanical disruption. Cells were then incubated with RPMI media containing 10% FBS with EDTA and HEPES for 10 minutes at room temperature, pelleted, and resuspended in PBS containing 2% FBS and 0.1% sodium azide (FACS Staining buffer). Splenocytes were isolated, washed, and re-suspended in RPMI 1640 containing 10% FBS before in vitro stimulation. Cells were washed twice before FACS staining. For isolation of CNS immune cells, mice were euthanized and perfused extensively with PBS to remove residual intravascular leukocytes. Brains and spinal cords from 5 mice per experimental group were isolated and pooled. Tissues were minced in RPMI media, triturated, and digested with Liberase (Roche) and type I DNase in serum-free RPMI media at 37uC for 45 min. Immune cells were isolated after gradient centrifugation from a 37/70% Percoll interface and washed twice with FACS staining buffer. Immune cells were stained with antibodies specific to CD11c, CD11b, B220, CD3, CD25, CD4, CD8, NK1.1, Gr-1, siglec H, and CD45 (all reagents from eBiosciences). Intracellular FoxP3 staining was performed as previously described [26] . Intracellular IFN-c staining was performed on splenocytes and CNS immune cells as previous described [35, 36] . Briefly, lymphocytes were stimulated with 1 mg/ml of the WNV NS4B peptide (SSVWNAT-TAI) for 4 h at 37uC. Cells were washed and stained for cell surface markers followed by permeabilization-fixation using the Cytofix-Cytoperm Kit (BD-PharMingen) and stained with a Pacific-Blue conjugated IFN-c antibody (eBiosciences) at 4uC for 30 min, washed and analyzed by flow cytometry. Flow cytometry was performed on a BD LSRII machine using BD FACSDiva software. Cell analysis was performed on FlowJo (v.8.7.2) software.