Figure 2.

Production of polySia fractions with defined DPs and effects on proinflammatory activation. (A) Analytical anion exchange chromatography of the polySia batch used as a starting material. DPs of individual peaks were determined by spiking with α2,8-linked tetrasialic acid (DP4, blue spectrum). N-acetyl groups of sialic acid were detected by absorption at 214 nm. (B) BV2 microglia were activated by the application of 10 ng/ml of LPS, 50 ng/ml of LPS-binding protein, and 10 μg/ml of zymosan (LLZ; joint activation of TLR4 and TLR2) in serum-free medium and treated without (–) or with polySia of the indicated DP, or with polySia with an average DP of ~50 (avDP50, 250 nM, each). NO production was assessed by the detection of the NO breakdown product nitrite using the colorimetric Griess assay, and % inhibition of LLZ-induced NO production is plotted. The inhibition of ~30% in the presence of FPLC-purified avDP50 corresponds to the inhibition obtained by polySia applied to LPS-induced primary or stem cell-derived murine microglia (Werneburg et al., 2015, 2016), or to LPS-induced BV2 cells (Thiesler et al., 2021). Per group, individual values and means ± SEM from n = 4 independently treated cultures are plotted. The one-way ANOVA revealed significant differences (P < 0.0001), and Tukey's post-hoc tests were applied. Significant group differences are indicated (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). (C) Spectra of polySia (with DP4 added) after partial hydrolysis on a preparative scale (upper trace), and of pooled fractions with DP24–30 (middle) and DP8–14 (lower) analyzed after lyophilization, determination of yield by weighing, and rehydration. Salt gradient for elution same as in a, but with a step at 432 mM to 1 M NaCl.