Fig 2: Analysis of differential gene expression upon NUP-MDSC differentiation and upon treatment with retinoic acid or 3-deazaneplanocin A. NUP cells were differentiated in vitro with GM-CSF/IL-6 for 4 days in the presence or absence of retinoic acid (RA) or 3-deazaneplanocin A (DZNep) or kept undifferentiated (4 replicates per condition). Gene transcription was analyzed by Next Generation RNA sequencing. The heatmap shows relative expression in all samples of 2.607 genes up-regulated in NUP-MDSC vs NUP cells (fold change = 2, adj P = 0.05).

Fig 3: Induction of ZBP1 by IAV infection and immune stimuli in primary mouse alveolar epithelial cells and immune cells. (A,B) Primary mAECs were treated with control PBS (- or control), infected with active H1N1 PR/8/34 strain at MOI of 5, 10, or 20, or treated with mouse TNFα (20 ng/ml), IFNα2 (50 ng/ml), IFNγ (50 ng/ml), TNFα plus IFNα2 (T+α2, 20 + 50 ng/ml), TNFα plus IFNγ (T+γ, 20 + 50 ng/ml), GM-CSF (30 ng/ml), GM-CSF plus IFNα2 (GM+α2, 30 + 50 ng/ml), GM-CSF plus IFNγ (GM+γ, 30 + 50 ng/ml) for 24 h. (C–F) BMDMs, BMDCs and RAW264.7 murine macrophages were treated with control PBS (mock or control), infected with H1N1 PR/8/34, H3N2 A/Hong Kong/8/68 (H3N2-HK), or H3N2 (x:31) A/Aichi/68 strains at MOI of 5, or treated with mouse IFNα2 (50 ng/ml), IFNγ (50 ng/ml), TNFα (20 ng/ml), LPS (100 ng/ml), poly(I:C) (PIC, 1 μg/ml), TNFα plus IFNα2 (T+α2, 20 + 50 ng/ml), or TNFα plus IFNγ (T+γ, 20 + 50 ng/ml) for 24 h. Equal amounts of cell lysates from (A–F) were subjected to Western blotting with indicated antibodies. Results represent the findings of three independent experiments. The production of IFNβ by 24 h treatment of LPS (100 ng/ml) and poly(I:C) (PIC, 1 μg/ml) was shown in the bar graph of (D) (n = 3).

Fig 4: VHH16 and VHH21 recognize mouse Ly-6C and Ly-6G. (A–D) Mouse BMDC were obtained after differentiation for 6 days with GM-CSF and IL-4. (A) MHCII vs CD11c expression on live, differentiated cells. (B) Binding of GFP-specific Enhancer, VHH16 and VHH21 on live cells. (C) MHCII vs CD11c expression on live cells negative or positive for VHH binding. (D) VHH binding vs Ly-6C expression on live cells. (E) Ly-6C, VHH16 or VHH21 vs Ly-6G expression gated on CD11b positive fresh bone cells. (F) Binding of Enhancer, VHH16 or VHH21 to control HEK 293 cells or cells transfected with Ly6c1, Ly6c2 or Ly6g constructs. Light grey histograms: unstained control. (G and H) Scattered plots show VHHs mean fluorescent intensity (MFI) binding on flow cytometry-sorted monocytic (CD11b+Ly-6C+Ly-6G−) (G) or granulocytes (CD11b+Ly-6ClowLy-6G+) (H) subsets from fresh bone marrow for indicated VHH concentrations. Each panel representative of 2 independent experiments or more.

Fig 5: Resolvin D1 enhances macrophage phagocytosis.(A) Bone marrow–derived macrophages (BMMs) from C57BL/6 mice were obtained by culturing myeloid precursors for 7 days in the presence of 20 ng/mL GM-CSF. BMMs were then pretreated for 15 minutes with RvD1 (100 nM) before incubation with pHrodo Green E. Coli Bio Particles for 60 minutes at 37°C in the continued presence of RvD1. (B) Quantification of phagocytosis by mouse GM-CSF BMMs treated with RvD1 (10–100 nM) as determined using a 96-well fluorescent plate reader (excitation/emission of 509/533 nm). (C) The effect of increasing doses of RvD1 (1–100 nM) on phagocytosis by human PBMC-derived M1-polarized macrophages. Data are presented as the percentage change in fluorescence intensity relative to matching vehicle-treated macrophage cultures from the same host. Bars show the mean ± SEM of cells from 3–4 donors with dots with representing data from each host. P values were determined by 1-way ANOVA followed by Holm-Šidák post hoc tests with vehicle-treated cells serving as a control group.