Fig 2: LILRB4 expression is upregulated by microglia and infiltrated myeloid cells into brains during ZIKV infection.(A) Diagram depicting the disease progression of ZIKV infection. (B) Heatmap for the fold changes in RNA expression of immune checkpoint receptors in the brains of ZIKV-infected mice at the indicated time points (n = 3–8, each time point). RNA expression was assessed by NanoString analysis using the nCounter mouse immunology panel. (C) Relative mRNA levels of Lilrb4 and Lilrb3 in the mouse brain during ZIKV infection compared with uninfected mouse brain. (D–F) Flow cytometry analysis of LILRB4 expression on cells isolated from the brains of ZIKV-infected mice at the indicated time points. The graph shows the percentage of LILRB4-expressing cells in total live cells (D). Live cells were separated based on CD45 and LILRB4 expression. The pie chart shows the percentage of macrophages (CD45hiCD11b+F4/80+), neutrophils (CD45hiCD11b+Ly6G+F4/80–), DCs (CD45hiCD11c+F4/80–Ly6G–), T cells (CD45hiCD3+NK1.1–), and NK cells (CD45hi NK1.1+CD3–) within CD45hiLILRB4+ cell lation (indicated by the dashed circle) at 15 dpi (E). The histogram shows LILRB4 expression on microglia (CD45loCD11b+), macrophages (CD45hiCD11b+F4/80+), neutrophils (CD45hiCD11b+Ly6G+F4/80–), and DCs (CD45hiCD11c+F4/80–Ly6G–). Gray histograms indicate staining with IgG isotype control antibodies (F). Data are representative of 2 independent experiments (n = 3–5, each time point) (E and F). Data were analyzed using 1-way ANOVA with Dunnett’s multiple comparisons, ***P < 0.001.

Fig 3: LILRB4 deficiency worsens clinical outcomes in ZIKV infection.(A and B) P1 WT (n = 30) and P1 LILRB4-KO mice (n = 18) were challenged with 1000 TCID50/mL of ZIKV and monitored for weight changes (A) and survival (B). Statistical significance was determined by a 2-way ANOVA (A) and the log-rank test (B), respectively. (C) Quantification of ZIKV RNA copies using real-time PCR was performed in ZIKV-infected WT and LILRB4-KO mice at 9 and 15 dpi in the brain, blood, kidney, eye, liver, and spleen (n = 3–8 per group). (D) Virus and infiltrating cell distribution in the brains of ZIKV-infected WT and LILRB4-KO mice at 15 dpi. The images show representative immunofluorescence staining for CD45 (green), ZIKV (pink), and DAPI (blue) in brain sections from WT and LILRB4-KO mice. (E and F) Flow cytometry analysis was performed on cells isolated from the brains of uninfected (P16) or ZIKV-infected WT and LILRB4-KO mice at 15 dpi (n = 3–4, each group). Live cells were gated and separated based on CD45 expression. The graph shows the percentage of infiltrating cells (CD45hi) in total live cells (E). CD45hi cells were gated and the population of the following cells was determined: macrophages (CD45hiCD11b+F4/80+), DCs (CD45hiCD11c+F4/80–Ly6G–), neutrophils (CD45hiCD11b+Ly6G+F4/80–), T cells (CD45hiCD3+NK1.1–), and NK cells (CD45hiNK1.1+CD3–). Both graph and pie charts show the percentage of indicated cells within CD45hi cell population in the brain (F). Data were analyzed using 2-tailed unpaired Student’s t test (C, E, and F). (G) Histopathology of the brains from ZIKV-infected WT and LILRB4-KO mice at 22 dpi. The upper images show representative H&E staining of cerebellum or hippocampus (original magnification, 20×); scale bars: 500 µm. The bottom images show representative brain lesions observed in cerebellum or hippocampus (original magnification, 400×); scale bars: 20 µm. White arrows indicate multifocal malacia or focal gliosis. *P < 0.05, ***P < 0.001. N.D., not detected.

Fig 4: The anti-tumoral effect of chemerin is unrelated to leukocyte recruitment.(A) Flow cytometric gating used to quantify leukocyte subsets in a representative sample. (B and C) B16 cells were grafted to control and CMKLR1-/- mice (B) or to control and K5-chemerin mice (C). Six days after the graft, the tumors were collected and digested, the cell suspension stained with combinations of antibodies, and analyzed by flow cytometry. The percentage of CD45+ cells and the proportion of various leukocyte subsets (% of CD45+ cells) recruited to the tumors are represented, including T cells (CD3+ NK1.1-), macrophages (CD11b+ CD11c- Gr1-), DCs (CD11c+), NK cells (CD3- NK1.1+) and MDSCs (CD11b+ CD11c- Gr1+). The data combine three independent experiments with 5 mice per condition in each experiment. (D) B16 cells expressing or not chemerin were grafted to NOD/SCID/IL2R?-/- mice and the tumor size monitored daily. The results are from a representative experiment out of two performed (mean ± SEM, n = 6 mice per group, *** P < 0.001, Mann-Withney test).

Fig 5: LILRB4 expression on B and T cells is not a determining factor in protecting mice from ZIKV-induced death.B and T cells’ responses were evaluated in WT and LILRB4-KO mice at 15 dpi. (A) IgG levels in sera were measured by ELISA (n = 3–5, each group). (B) Neutralization assay using Vero E6 cells infected with ZIKV-GFP in the presence of sera (1:10 dilution). The percentage of infectivity was determined using ImageJ (NIH). Upper panel: Bright-field. Lower panel: GFP expression. Scale bar: 100 µm. (C and D) The percentages of LILRB4-expressing B cells (CD45hiCD19+CD3–) (C) and T cells (CD45hiCD3+NK1.1–) (D) were determined by flow cytometry in the brains of ZIKV-infected WT and LILRB4-KO mice. Data shown as mean ± SD of 6 mice per group. (E) LILRB4-expressing T cells characterized by CD4 and CD8 expression. (F) CD69 and PD-1 staining of T cells infiltrating the brain. (G and H) IFN-? expression in T cells isolated from the brains of ZIKV-infected WT and LILRB4-KO mice (G) and in naive splenic T cells stimulated with anti-CD3/CD28 for 24 hours (H). Data are representative of 3 independent experiments. Left: Representative flow data of 3 independent experiments. Right: Percentage of IFN-?+ in T cells. Data were analyzed using 2-tailed unpaired Student’s t test. *P < 0.05.