Polysialic acid blocks mononuclear phagocyte reactivity, inhibits complement activation and protects from vascular damage in the retina (2016)
Figure 3

Figure 3. PolySia avDP20 inhibits TNFSF2, VEGF, and superoxide production in human macrophages and prevents activation of the alternative complement pathway.

A. Analysis of relative TNFSF2 gene transcription in human control and SIGLEC11/16 knockout THP1-macrophages. The levels of gene transcripts were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and concentrations of 0.15 µM and 1.5 µM of polySia avDP20 in the human wild type macrophages. No response to polySia avDP20 was detectable in the knockout line. Data show mean +/- SEM. ** p < 0.01, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=7 and p< 0.0001, PolySia avDP20 1.5 µM n=4 and p=0.0002, LPS n=7, LPS/PolySia avDP20 0.15µM n=3 and p=0.009, LPS/PolySia avDP20 1.5µM n=5 and p=0.002. Siglec11/16 KO: no treatment n=5 and p=0.01, PolySia avDP20 n=4 and p=0.122, LPS n=7, LPS/PolySia avDP20 0.15µM n=3 and p=1.0, LPS/PolySia avDP20 1.5µM n=4 and p=1.0.

B. Analysis of relative TNFSF2 protein release in human control and SIGLEC11/16 knockout THP1-macrophages. The released protein levels were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and concentrations of 0.15 µM and 1.5 µM of polySia avDP20 in the human wild type macrophages. No response to polySia avDP20 was detectable in the knockout line. Data show mean +/- SEM. ** p < 0.01, *** p < 0.001, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=8 and p<0.0001, PolySia avDP20 1.5 µM n=5 and p<0.001, LPS n=7, LPS/PolySia avDP20 0.15µM n=5 and p=0.002, LPS/PolySia avDP20 1.5µM n=4 and p=0.0003. Siglec11/16 KO: no treatment n=6 and p<0.001, PolySia avDP20 1.5 µM n=5 and p<0.001, LPS n=7, LPS/PolySia avDP20 0.15µM n=5 and p=1.0, LPS/PolySia avDP20 1.5µM n=5 and p=1.0.

C. Analysis of relative VEGFA gene transcription in human control and SIGLEC11/16 knockout THP1-macrophages. Gene transcripts were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and polySia avDP20 (0.15 µM and 1.5 µM) in the human wild type macrophages. No response to polySia avDP20 was detectable in the knockout macrophages. Data show mean +/- SEM. *** p < 0.001, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=6 and p<0.0001, PolySia avDP20 1.5 µM n=5 and p<0.0001, LPS n=5, LPS/PolySia avDP20 0.15µM n=5 and p=0.0002, LPS/PolySia avDP20 1.5µM n=5 and p<0.0001. Siglec11/16 KO: no treatment n=5 and p=0.022, PolySia avDP20 n=3 and p=0.063, LPS n=4, LPS/PolySia avDP20 0.15µM n=3 and p=1.0, LPS/PolySia avDP20 1.5µM n=4 and p=1.0.

D. Analysis of relative VEGFA protein release in human control and SIGLEC11/16 knockout THP1-macrophages. Released protein levels were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and polySia avDP20 (0.15 µM and 1.5 µM) in the human macrophages. Data show mean +/- SEM. * p < 0.05, *** p < 0.001, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=9 and p<0.0001, PolySia avDP20 1.5 µM n=6 and p=0.009, LPS n=9, LPS/PolySia avDP20 0.15µM n=6 and p=0.043, LPS/PolySia avDP20 1.5µM n=7 and p=0.0001. Siglec11/16 KO: no treatment n=9 and p=0.349, PolySia avDP20 1.5 µM n= 6 and p=0.249, LPS n=7, LPS/PolySia avDP20 0.15µM n=6 and p=1.0, LPS/PolySia avDP20 1.5µM n=7 and p=1.0.

E. Quantification of human control and SIGLEC11/16 knockout THP1-macrophages having ingested cellular debris. PolySia avDP20 (1.5 µM) reduced the percentage of phagocytic cells having ingested drusen-like debris. No response to polySia avDP20 was detectable in the knockout macrophages. Data are presented as mean +/- SEM. n=6. *p ≤ 0.05, *** p ≤ 0.001, ANOVA followed by Bonferroni. Debris treated WT macrophages vs debris plus polySia avDP20 treated WT macrophages p = 0.028, debris plus polySia avDP20 treated WT macrophages vs KO macrophages p = 0.00012.

F. Prevention of superoxide release in activated human control and SIGLEC11/16 knockout THP1-macrophages by polySia avDP20. Cultured human THP1-macrophages were stimulated with RPE cell debris or co-stimulated with debris and polySia avDP20. Addition of debris stimulated the production of superoxide. 1.5 µM polySia avDP20 completely prevented the release of superoxide induced by debris challenge. No response to polySia avDP20 was detectable in the knockout macrophages. Data are presented as mean +/- SEM. n=6. p* ≤ 0.05, *** p ≤ 0.001, ANOVA followed by Bonferroni. Untreated WT macrophages vs debris treated WT macrophages p = 0.037, debris treated WT macrophage vs debris plus polySia avDP20 1.5 µM treated WT macrophages p < 0.001, debris plus polySia avDP20 WT macrophages vs KO macrophages p = 0.001,

G. PolySia avDP20 (0.15 to 50 µM) was added to normal human serum (NHS) to evaluate any interference with activation of the classical complement pathway by IgM. Complement activation was determined by C3b deposition. PolySia avDP20 had no effect on C3b deposition induced by activation of the classical complement pathway. Data show mean +/- SEM. n = 3, n.s.= not significant.

H. PolySia avDP20 inhibits the alternative complement pathway. PolySia avDP20 (0.15 to 50 µM) was added to NHS and activation of the alternative pathway was induced by LPS. C3b deposition was monitored by ELISA. Data show mean +/- SEM (n = 4. * p < 0.05, ** p < 0.01, p***<0.001, ANOVA followed by Bonferroni).