Definition of the Anti-inflammatory Oligosaccharides Derived From the Galactosaminogalactan (GAG) From Aspergillus fumigatus

Abstract

Galactosaminogalactan (GAG) is an insoluble aminosugar polymer produced by Aspergillus fumigatus and has anti-inflammatory properties. Here, the minimum glycosidic sequences required for the induction of IL-1Ra by peripheral blood mononuclear cells (PBMCs) was investigated. Using chemical degradation of native GAG to isolate soluble oligomers, we have found that the de-N-acetylation of galactosamine residues and the size of oligomer are critical for the in vitro immune response. A minimal oligomer size of 20 galactosamine residues is required for the anti-inflammatory response but the presence of galactose residues is not necessary. In a Dextran sulfate induced colitis mouse model, a fraction of de-N-acetylated oligomers of 13 < dp < 20 rescue inflammatory damage like the native GAG polymer in an IL-1Ra dependent pathway. Our results demonstrate the therapeutic suitability of water-soluble GAG oligosaccharides in IL-1 mediated hyper-inflammatory diseases and suggest that α-1,4-galactosamine oligomers chemically synthesized could represent new anti-inflammatory glycodrugs.

Keywords: Aspergillus fumigatus; IL-Ra; anti-inflammatory response; galactosaminogalactan; glycodrug.

Figure 1

De-N-acetylation of GAG. Degree of acetylation (DA) of GAGs (A). DA was determined by an acetate assay (GAG, native polymer; dGAG, de-N-acetylated) (B). In vitro IL-1Ra secretion in PBMC supernatants in presence of GAG. LPS (10 ng/ml) served as external standard and were set to 100% in each biological replicate. Statistical analysis by WMW test in comparison to GAG: ns, non significative; *p < 0.05; ***p < 0.001.

Figure 2

Chemical analysis and IL-1Ra inducing activity of oligosaccharides generated by acid hydrolysis of dGAG. (A) Summerized MALDI-TOF MS analysis of fractions I-VIII. (DP, degree of polymerization; for detailed MS spectra see Figure S2). (B) IL-1Ra production by dGAG oligosaccharides. dGAG oligosaccharides fractions I-VIII were tested at 25, 5, and 1 μg/ml. dGAG (5 and 1 μg/ml) served as positive control. LPS (10 ng/ml) served as external standard and were set to 100% in each biological replicate. Wilcoxon-Mann-Whitney Test: **p < 0.01; ***p < 0.001 (compared to control, M).

Figure 3

IL-1Ra stimulation by GAG oligomers is abolished by GAGnase. (A) Fraction F-I and F-II are degraded by GAGnase. Fractions I and II (500 μg/ml) were enzymatically hydrolyzed by GAGnase (2 μg/ml) for 10 or 120 min or were kept in acetate buffer for 120 min (untreated). The degradation was followed by PABA assay to quantify sugar reducing end. (B) Induction of IL1-Ra expression in PBMCs by GAGnase treated fractions. (C) IL-1Ra induction of PBMCs in presence of GAGnase (20 ng/μl). GAGnase neither activate nor inhibit induction. LPS (10 ng/ml) served as reference control and were set to 100% in each biological replicate. bars indicate the SD, WMW Test: ***p < 0.001; ns, not significant (compared to control samples).

Figure 4

N-acetylation of de-N-acetylated GAG oligosaccharides and its impact on the IL-1Ra production. (A) Determination of the degree of acetylation (DA) by acetate assay of de-N-acetylated and acetylated fractions I, II and III. GalN and GalNAc served as positive and negative controls. (B) In vitro IL-1Ra production by de-N-acetylated and acetylated GAG fractions I, II, and III (10 and 1 μg/ml). LPS (10 ng/ml) served as positive control. WMW Test: **p < 0.01, ***p < 0.001.

Figure 5

Reduced severity of DSS colitis in C57BL/6 mice treated with modified GAG molecules. DSS or water vehicle was administered ad libitum in drinking water for 7 days. Native GAG polymer and GAG oligomers at the dose of 1 mg/kg were given intraperitoneally for 7 consecutive days after DSS treatment. (A) Survival, weight (grams) and clinical disease activity index. (B) Macroscopic image of colons. (C) Histological assessment of colitis severity (20X magnification, bar = 200 μm). (D) Levels of colonic cytokines. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001 GAGs-treated vs. untreated (None) C57BL/6 mice (n = 10 mice/group from one experiment). ns, not significant.