Fig 1: HCMV ligands of Mincle are in the lipid envelope. (A) Density cushion‐purified HCMV viral particles were solubilized and separated into organic and aqueous phases. (B) The organic and aqueous phases were analyzed by HPTLC developed with chloroform/methanol/water (60:35:8, v/v/v). The HPTLC plate was stained by orcinol (left) and primulin (right) to visualize lipids and hexose, respectively. (C) hMincle‐Fc binding was directed at the organic phase only, whereas hDC‐SIGN‐Fc bound to both phases (representative of n = 3, duplicates). (D) hMincle‐Fc binding was noted only to the organic phase of the HCMV viral particle extract, with no binding observed to the aqueous phase of the viral particles or either phase prepared from a mock control (n = 3, duplicates). (E) Activation of the HEK hMincle reporter cell line was observed with the organic phase of the HCMV viral particle extract only, along with TDB as positive control (n = 3, duplicates). (F) The lipid extract (organic phase) was fractionated on a C18‐silica column using a range of eluents (chloroform to chloroform–methanol–water 25:25:8). Lipid fractions were analyzed by HPTLC developed with chloroform/methanol/water (65:25:4, v/v/v). The HPTLC plate was stained by primulin to visualize the migration of the lipids/glycolipids. (G) Binding of hMincle‐Fc was observed mostly to the first two fractions (representative of n = 2, duplicates). Data are presented as means ± SDs. C, chloroform; HCMV, human cytomegalovirus; M, methanol; OD, optical density; TDB, trehalose dibehenate; W, water.
Fig 2: Mincle binds HCMV and activates downstream signaling. (A) Viral particles were isolated by centrifugation through density cushions and used for Mincle binding and activation experiments or further purified on a density gradient and used for Mincle binding analysis only (data in Panel C only). The density cushion‐purified HCMV viral particles were characterized by nFC and TEM (Figure S1A,B, respectively), whereas density gradient‐purified HCMV was characterized by nFC only (Figure S1C). (B) Density cushion‐purified viral particles (Steps 1 and 2 shown in Panel A) of varying titers (8.8 × 109–1.6 × 1011 viral particles per mL), as determined by nFC, were diluted 10‐fold in DPBS and coated onto high‐binding plates. hMincle‐Fc binding was observed with the HCMV‐coated wells, but not with the mock controls prepared identically (n = 5 biological replicates; measured in duplicate). (C) Fractions from the density gradient purification of HCMV viral particles (Steps 3 and 4 in Panel A) were analyzed for hMincle‐Fc binding as described for Panel B. hMincle‐Fc binding was observed to fractions that contained the highest concentrations of HCMV viral particles, as determined by nFC (dotted cyan line; Figure S1C), whereas no binding was observed with the mock control fractions (n = 1, duplicates). (D) Inhibition of hMincle‐Fc binding to purified HCMV viral particles (titer: 1.6 × 1011) with 100 mM mannose and 20 mM EDTA (n = 3, duplicates). (E and F) Activation of the HEK hMincle cell line (E) and the matching HEK Null1 control cell line (F) by viral particles (n = 3, duplicates). (G) Infection of the HEK Null1 and hMincle reporter cell lines with eGFP‐expressing HCMV, assessed by flow cytometry (n = 4). Dot plots with gates are shown in Figure S2. Data are presented as means ± SEMs. Two‐tailed ratio t‐test (B); one‐way ANOVA (D and E); or two‐way ANOVA (G). eGFP, enhanced green fluorescent protein; HCMV, human cytomegalovirus; OD, optical density; TDM, trehalose dimycolate. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Fig 3: HCMV viral particles contain lipid/glycolipid ligands of Mincle, of which cholesterol activates Mincle signaling. (A) Mass spectrum of the CHCl3 fraction in negative‐ion detection mode. (B) Mass spectrum of the CHCl3 fraction in positive‐ion detection mode. (C) The product‐ion spectrum of the ion at m/z 465.3 (from Panel A) in negative ion mode. (D) Mass spectrum of the CH3OH fraction in negative‐ion mode. (E) Binding of hMincle‐Fc to pure lipids previously identified in HCMV, as well as TDB as positive control, coated at 1 µg per well (n = 3, triplicates). (F) Activation of the HEK hMincle cell line by pure lipids, coated at 1 µg per well (representative of n = 3). (G) EC50 values determined from the activation curves of the HEK hMincle reporter cells in panel F (n = 3, triplicates). (H) Lipid extracts generated from viral particles treated with 5 or 50 mM methyl‐β‐cyclodextrin were analyzed by HPTLC developed with chloroform/methanol/water (65:25:4, v/v/v) and stained with primulin to visualize lipids. (I) hMincle‐Fc binding to viral particles treated with methyl‐β‐cyclodextrin (coated at 1 µg of total protein per well), expressed as fold changes from untreated (n = 4, duplicates). (J) Activation of the HEK hMincle cell line by viral particles treated with methyl‐β‐cyclodextrin, coated at 1 µg of total protein per well (n = 3, triplicates). Additional MS data are found in Figure S3. Data are presented as means ± SDs (E) or SEMs (I and J). One‐way ANOVA (E, I, and J). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. HCMV, human cytomegalovirus; MβCD, methyl‐β‐cyclodextrin; OD, optical density; TDB, trehalose dibehenate; TLC, thin layer chromatography.
Fig 4: The recognition of HCMV by Mincle elicits a pro‐inflammatory immune response in primary human macrophages. (A and B) Macrophages were stimulated with 1 µg of plate‐coated lipids (TDB, cholesterol) or HCMV viral particles for 48 h, and TNF‐α (A) and IL‐6 (B) concentrations in the culture supernatant were determined by ELISA. Mincle dependence was investigated by preincubating cells with 5 µg/mL of a neutralizing anti‐hMincle antibody or a matching isotype control antibody for 30 min at 37°C before stimulation (n = 3, triplicates). Data are presented as mean ± SD (A and B). Two‐way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. HCMV, human cytomegalovirus; TDB, trehalose dibehenate.
Supplier Page from R&D Systems, a Bio-Techne Brand for CLEC4E Fc Chimera Protein
Available conjugates: Sizes Available: 50 ug