(A) C57Bl/6N (WT; black) or Ifi35^-/- (red) mice were intranasally infected with 10⁴ TCID₅₀ of highly pathogenic H5N1 (A/Hong Kong/213/2003), or (B) 100 EID₅₀ of H5N1-Vietnam/PR8 (H5N1-VN/PR8; described in Materials and Methods), or (C) 10⁴ TCID₅₀ of pandemic H1N1 A/California/04/2009 and monitored for weight loss. (D) Littermate Ifi35^+/+ (black), Ifi35^+/- (grey), or Ifi35^-/- (red) mice were infected with 100 EID₅₀ of H5N1-VN/PR8 and monitored for weight loss. Starting weight is indicated with a dashed line at 100%. Values are the means of at least three independent experiments ± SEM. n = 18–29 per group (E) Viral titer (log₁₀ TCID₅₀/mL) in bronchoalveolar lavage fluid from WT (black circle) or Ifi35^-/- (red circle) mice at the indicated time-points post infection. n = 8–14 mice for each time-point from at least three independent experiments. Each symbol represents an individual mouse. Bars represent the mean from at least three different experiments (± SEM). Assay limit of detection (LOD) indicated at 1x10² TCID₅₀/mL; dotted line. *P < 0.05, **P < 0.001, ***P < 0.0001 using multiple student t-test for (A-C) and (E), One-way ANOVA multiple-comparison for (D).

(A) Gating strategy for analysis of myeloid and lymphoid cells from bronchoalveolar lavage (BAL) of naïve and H5N1-VN/PR8 infected mice. Cells were gated based on CD11b, CD11c, Ly6G, Ly6C, F4/80, and MHC-II expression for myeloid populations or B220, CD3, CD4, CD8, γδ-TCR, and CD44 expression for lymphoid populations. The parental gate is shown above each plot. Representative plots are shown from a WT mouse at 3 dpi for myeloid gating or 5 dpi for lymphoid gating. (B) Total number of cells isolated from bronchoalveolar lavage from naïve, infected WT (black), and Ifi35^-/- (red) mice at indicated times post infection. (C) Total number of CD45⁺ alveolar macrophage (CD11c⁺, CD11b^-, MHC-II^-, F4/80⁺), neutrophil (PMNs; CD11b⁺, CD11c^-, Ly6G⁺), monocyte (CD11b⁺, Ly6G^-, Ly6C^hi), and dendritic cell (CD11c⁺, CD11b^-, MHC-II⁺) numbers in BAL. (D) Percentage of myeloid cell populations in BAL at 3 dpi following pre-gating on CD45⁺ cells. (E) Total number of CD3⁺, CD44⁺ lymphoid CD4⁺, CD8⁺, or γδ-TCR⁺ T cells in BAL. n = 8–11 for each group/time point post-infection. n = 3–4 for naïve samples. Each symbol represents an individual mouse. Bars represent the mean from at least three different experiments (± SEM). PMN, polymorphonuclear cells; n.s., not significant; *P < 0.01, **P < 0.001, ***P < 0.0001 using multiple student t-test.

(A) CXCL1, (B) G-CSF, and (C) IL-12p40 concentrations (pg/mL) in bronchoalveolar lavage fluid from WT (black circle) and Ifi35^-/- mice (red circle) at indicated times post infection with H5N1-VN/PR8 as measured by Bio-Plex Pro Mouse Cytokine 23-Plex assay. Dotted line (red) indicates limit of detection (LOD) for individual cytokines and chemokines based on internal standard curve values. (D) Serum IL-12p40 concentration (pg/mL) in WT (black circle) and Ifi35^-/- (red square) mice at 3 hours post stimulation with 100μg HMW poly I:C via intraperitoneal injection. Serum cytokine levels measured using an IL-12p40 specific ELISA; dotted line indicates LOD based on standard curve values. Each symbol represents an individual mouse. Bars represent the mean from at least three different experiments (± SEM). n = 6–19 per group per time-point. *P < 0.01, **P < 0.001, ***P < 0.0001 using multiple student t-test.

(A) CD45.1⁺ B6-Ly5.1/Cr and CD45.2⁺ Ifi35^-/- mice were Busulfan treated and reconstituted with CD45.1⁺ B6-Ly5.1/Cr, CD45.2⁺ WT, or CD45.2⁺ Ifi35^-/- bone marrow cells (10⁷ cells). Bone marrow donor and recipient pairs for the experiment are shown. (B) Twelve-week-old chimeric mice were infected with 100 EID₅₀ of H5N1-VN/PR8 and BAL fluid was collected at 3 dpi and assessed for total BAL cell number or (C) assessed for IL-12p40 levels via ELISA. Each symbol represents an individual mouse. Bars represent the mean from at least three different experiments (± SEM). n = 8–17 per group. (D) IL-12p40-IRES-eYFP reporter (IL12b-yet40) mice were infected with 100 EID₅₀ of H5N1-VN/PR8 and on 0, 3, and 5 dpi whole lungs were taken for collagenase digestion and flow cytometry analysis of eYFP expression (FITC⁺) and CD45 surface staining. FITC⁺ cells were separated based on CD45 staining and enumerated per 1x10⁶ single cells collected. Bars represent the mean from at least three different experiments (± SEM). n = 4 for naïve and 11–12 for infected groups. n.s., not significant; *P < 0.01, **P < 0.001, ***P < 0.0001, using one-way ANOVA with multiple comparisons correction (Tukey’s comparison correction).

(A) Supernatant IL-12p40 levels at different time points post treatment from bone marrow derived macrophages (BMDMs) stimulated with 2.5μg/mL HMW poly I.C. (B) Supernatant IL-12p40 levels following either R848 (25ng/mL) or LPS (5μg/mL) treatment for 12 hrs. n = 3–6 per group from at least three independent experiments. (C) BMDM IL-12p40 mRNA levels measured by qRT-PCR following poly I:C or LPS treatment for 6 hrs. Values are fold change relative to C57Bl/6N untreated samples. n = 3 per group from three independent experiments. (D) Non-reducing western blot of 10X concentrated BMDM supernatant following HMW poly I:C stimulation at indicated time-points. (E) Non-reducing western blot of cell lysate from Brefeldin-A treated BMDMs stimulated with HMW poly I:C. Blot probed for accumulated cellular IL-12p40 and subsequently, β-actin for loading control. (F) Non-reducing western blot of 10X concentrated BMDM supernatant 12hrs following either LPS or R848 treatment. Supernatant IL-12p40 levels were measured using an IL-12p40 specific ELISA. Western blots were probed with anti-IL-12p40 antibody and run with rIL-12p40 as a standard. All graphed values are the means ± SEM from at least three independent experiments. Original, uncropped blot images are shown in . *P < 0.01, **P < 0.001, ***P < 0.0001, using multiple student t-test.

(A) Representative image of IL-12p40 species (anti-IL12p40 mAb) in WT and Ifi35^-/- bronchoalveolar lavage fluid (BALF) at 5 dpi visualized by western blotting under non-reducing conditions and detected by fluorescent secondary antibody (Alexa Fluor 790 goat anti-rat). Bands that correspond to the IL-12p40 homodimer (IL-12p80) and IL-12p40 monomer (IL-12p40) are indicated by solid and open arrowheads, respectively. IL-17 was used as loading control given similar levels of production in both WT and Ifi35^-/- mice in two independent assays ( and ). (B) Mean band intensity of IL-12p80 and IL-12p40 bands quantified relative to IL-17 production using LiCor Image Studio software for WT (n = 8) and Ifi35^-/- mice (n = 8). (C) Weight loss in H5N1-VN/PR8 infected WT mice that received 100μg of either anti-IL-12p80 mAb a3-1d (blue line) or Armenian Hamster IgG (black line) on day -1 and day +3 (indicated by open triangle). n = 9–16 per group. (D) Total number of cells in BAL on day 3 post infection from H5N1-VN/PR8 infected WT female mice that received either 100μg anti-IL-12p80 mAb a3-1d (blue squares) or 100μg Armenian Hamster IgG (black circles) at day -1. n = 6–7 per group. (E) Total number of cells in BAL on day 3 post infection from H5N1-VN/PR8 infected WT male mice that received either 50μL PBS/0.01% BSA (black circles) or 50ng recombinant IL-12p80 in 50μL (blue squares) intranasally at 1 dpi. n = 7–8 per group. Each symbol represents an individual mouse in D-E. All graphed values are the means ± SEM from at least three (B, C) or two (D, E) independent experiments. Original, uncropped blot images are shown in . *P < 0.05, **P < 0.001, ***P < 0.0001 using multiple student t-test.