Figure 2. PolySia acts anti-inflammatory on human macrophages via SIGLEC-11 receptors.

(a) Flow cytometry analysis of HEK-T-cells transfected with either control plasmid, SIGLEC11 plasmid or SIGLEC16 plasmid. GFP-positive cells were gated and stained with a SIGLEC-11 (clone 3 EH) antibody. SIGLEC-11 expression was detected on the SIGLEC11 transfected cells. Control antibody: irrelevant isotype antibody. Representative histograms of n = 3 independent experiments. (b) Gene transcription of SIGLEC7, SIGLEC10 and SIGLEC11 in human macrophages. SIGLEC11 shows the highest transcript levels. GAPDH: internal control. H₂O: control without cDNA. Representative image of at least n = 3 independent experiments. (c) TNFSF2 transcription of macrophages treated with LPS (1 μg/ml) alone or with LPS plus polySia avDP20 (1.5 μM), oligoSia with degree of polymerization 6 (SiaDP6; 1.5 μM) or sialic acid (Sia; 1.5 μM). Only polySia avDP20 reduced the LPS induced gene transcription of TNFSF2. Mean +/−  SEM of n = 3 independent experiments. ***p < 0.001; ANOVA followed by Bonferroni. (d) Gene transcripts for SIGLEC11 were analyzed by qRT-PCR. Knock-down of SIGLEC11 by short hairpin lentiviral vectors (ShSiglec-11) in macrophages reduced the gene transcription of SIGLEC11. ShControl: scrambled short hairpin vector. Mean +/− SEM of n = 4 independent experiments. ***p < 0.001; t-test. (e) SIGLEC-11 expression on macrophages was analyzed by flow cytometry. Knock-down of SIGLEC11 by short hairpin vectors (ShSiglec-11) reduced the expression of SIGLEC-11. ShControl: scrambled short hairpin vector. (f) Effects of SIGLEC11 knock-down on TNFSF2 gene transcription. Macrophages were treated with LPS (1 μg/ml) alone or with LPS plus polySia avDP20 (1.5 μM). PolySia avDP20 reduced the TNFSF2 gene transcription that were transduced with the control vector (ShControl), but failed to reduce the TNFSF2 gene transcription levels after knock-down of SIGLEC11 (ShSiglec-11). Mean +/− SEM of at least n = 3 independent experiments. **p < 0.01, ***p < 0.001; ANOVA followed by Bonferroni. (g) Effects of SIGLEC11 knock-down on TNFSF2 protein secretion. Macrophages were treated 24 hours with LPS (1 μg/ml) alone or with LPS plus polySia avDP20 (1.5 μM). PolySia avDP20 reduced the TNFSF2 protein secretion that were transduced with the control vector (ShControl), but failed to reduce the TNFSF2 protein secretion levels after knock-down of SIGLEC11 (ShSiglec-11). Mean +/− SEM of at least n = 3 independent experiments. **p < 0.01; ANOVA followed by Bonferroni.