Fig 2: LILRB4 deficiency in NK cells contributes to impaired viral clearance and ZIKV-induced death.(A) Purified, CFSE-labeled NK cells (3 × 105 cells) derived from naive WT or LILRB4-KO mice were injected i.p. into ZIKV-infected LILRB4-KO mice at 12 dpi. Three days later (15 dpi), mice were sacrificed to measure viral RNA in brain or received a second NK cell transfer and were followed to assess survival. (B) At 15 dpi, ZIKV RNA copies were quantified using real-time PCR from the brain, and the percentage of CD45hi cells and transferred NK cells (CFSE+CD45+NK1.1+) in the brain was determined by flow cytometry analysis. Data are representative of 2 independent experiments. Data were analyzed using 2-tailed unpaired Student’s t test. *P < 0.05. (C) Survival of ZIKV-infected LILRB4-KO mice (n = 10 for WT NK cell transfer and n = 11 for LILRB4-KO NK cell transfer) that received purified NK cells (3 × 105 cells i.p.) derived from naive WT or LILRB4-KO mice at 12 and 15 dpi. Statistical significance was determined by 2-tailed unpaired Student’s t test and the log-rank test.

Fig 3: TGFß1 inhibits LPS-induced upregulation of Lilrb4 in BV2 cells and primary microglia. Expression of Lilrb4 after treatment of BV2 cells with TGFß1 (5 ng/mL), LPS (1 µg/mL) or the combination of both factors (LPS + TGFß1) for 6 h (A), 12 h (B), and 24 h (C). Expression of Lilrb4 after treatment of primary microglia with TGFß1 (5 ng/mL), LPS (1 µg/mL) or the combination of both factors (LPS + TGFß1) for 6 h (D) and 24 h (E). Data are given as means ± SEM for at least three independent experiments. p-values derived from one-way ANOVA followed by Tukey’s multiple comparison tests are * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Fig 4: LILRB4 is highly expressed in peritoneal macrophages. Schematic of the experimental design to analyze LILRB4 expression in microglia, bone marrow-derived monocytes (BMMCs), and peritoneal macrophages (PMs). Created with BioRender.com (A). Percentages of LILRB4+ microglia (out of F4/80+ cells) after 3, 5, and 7 days in vitro as well as 7 days and 30 days ex vivo (B). Quantifications and statistical analyses of mean fluorescence intensities (MFI) of LILRB4 at indicated time points (C). Representative flow cytometry histograms (D). Comparison of percentages of LILRB4+ microglia, PMs, and BMMCs (E). Quantifications and statistical analyses of MFIs of LILRB4+ microglia, PMs, and BMMCs (F). Representative flow cytometry histograms (G). All data are given as means ± SEM for at least three independent experiments. p-values derived from one-way ANOVA followed by Tukey’s multiple comparison tests (E,F) or nonparametric Kruskal–Wallis tests followed by Dunn´s multiple comparisons tests (B,C) are # p < 0.05, * p < 0.05, ** p < 0.01, *** p < 0.0001, and **** p < 0.00001.

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.