Figure 1.
The expression and distribution of CLEC4F.
(A) Tissue distribution of CLEC4F transcripts were analyzed by qRT-PCR. F4/80 is the pan macrophage marker. (B) The protein expression of CLEC4F was examined by Western blot. GAPDH was used as internal control. (C) CLEC4F is a glycoprotein. The pFLAG-CMV-2/CLEC4F-transfected 293T cells were treated with tunicamycin at 1.5 µg/ml for indicated time periods to inhibit N-linked glycosylation, and the molecular weight of CLEC4F was analyzed by Western blot. DMSO is the vehicle control. Mock indicates 293T cells transfect with pFLAG-CMV-2. mLN, mesenteric lymph node; iLN, inguinal lymph node; KC, Kupffer cells; PBL, peripheral blood leukocytes; BM, bone marrow cells; BMDM, bone marrow derived macrophages.
Figure 2.
CLEC4F is co-expressed with F4/80 on liver Kupffer cells.
(A) CLEC4F and F4/80 immunohistochemistry of parafilm-embedded liver sections from wild-type and Clec4f−/− mice. (B) Double immunofluorescence of CLEC4F and F4/80 in wild-type livers was performed. Nuclei were counterstained with Hoechst 33342. Signals were determined by confocal microscope (magnification 10×63). (C) Coexpression of CLEC4F and F4/80 on Kupffer cells, but not peritoneal macrophages. Cells were double stained with Alexa Fluor 647-conjugated anti-F4/80 and PE-conjugated anti-CLEC4F mAb. Alexa Fluor 647-conjugated rat IgG2b and PE-conjugated mIgG1 were used as isotype controls.
Figure 3.
Expression of CLEC4F during embryogenesis.
Yolk sac, embryo and fetal liver in various embryonic stages were collected for CLEC4F detection by (A) qRT-PCR and (B) Western blot. Actin was used as internal control. (C) F4/80 and CLEC4F immunohistochemistry of fetal liver from E14.5 and E17.5, respectively.
Figure 4.
CLEC4F+ cells were appeared in the liver environment under Kupffer cell depletion and inflammatory stage.
(A) Kupffer cells were depleted by Cl2MBP-encapsulated liposome by intravenous injection (100 µl/mouse) at day 0 and livers were harvest at day 1, 7, 14 and 28. F4/80 and CLEC4F immunohistochemistry of liver sections were performed. (B) The numbers of F4/80+ or CLEC4F+ cells in livers were shown. For generating inflammatory stage, wild-type and Clec4f−/− littermates were infected with L. monocytogenes (1×105 CFU/mouse) intravenously. (C) The numbers of F4/80+ or CLEC4F+ cells in livers during L. monocytogenes infection. (D) Immunohistochemistry of L. monocytogenes infected livers of wild-type and Clec4f−/− mice at day 5 after infection. (E) Kaplan-Meier survival curves were shown for Clec4f−/− or wild-type littermates with L. monocytogenes infection. The p value was determined by Log-rank test.
Figure 5.
Glycan binding profile of Fc.CLEC4F.
Fc.CLEC4F (5 µg/ml) in binding buffer was applied onto glycan array slides. The “Average Binding Intensity” and “Standard Deviation (SD)” among eight glycan replicates were analyzed by ArrayVision software (GE Healthcare).
Figure 6.
CLEC4F is involved in the presentation of α-GalCer to NKT cells.
(A) The schematic structure of α-GalCer. (B) The binding curves were obtained from the function of Fc.CLEC4F concentration and fluorescence intensity determined from array images. (C) Competition experiment between solution and surface for Fc.CLEC4F binding to α-GalCer and three derivatives. At different concentration of the competitors, binding curves were obtained from the bound Fc.CLEC4F concentration and fluorescence. (D) Secretion of IFN-γ and IL-4 of NKT cells after incubation of α-GalCer presented by Kupffer cells isolated from wild-type and Clec4f−/− mice. Kupffer cells (1×105 cells) isolated form wild-type and Clec4f−/− littermates were treated with serial concentration of α-GalCer (10, 30, 100, 300 and 1000 ng/ml) then co-cultured with NKT cells (1×105 cells). The supernatant were collected at 72 h post-stimulation and detected the IL-4 and IFN-γ production by ELISA. (E) Effect of CD1d-blocking antibody in the α-GalCer presentation by wild-type and Clec4f−/− Kupffer cells. Kupffer cells were pretreated with CD1d blocking antibody. Three hours later, α-GalCer (300 ng/ml) was added and co-cultured with NKT cells. The supernatant were collected at 72 h post-stimulation and detected the IL-4 and IFN-γ production by ELISA.
Figure 7.
Clec4f−/− mice showed a similar response of α-GalCer induced activation to wild-type mice in vivo.
Both wild-type and Clec4f−/− littermates were treated with α-GalCer (1 µg/mouse) intravenously. Serum levels of (A) ALT and (B) cytokines, including IL-4 and IFN-γ were analyzed for samples taken at indicated time points after α-GalCer challenge. Data are representative of three independent experiments with similar results. (3 mice in each group).