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. 2014 Jun 3;111(22):8173-8.
doi: 10.1073/pnas.1319870111. Epub 2014 May 16.

Synthetic di-sulfated iduronic acid attenuates asthmatic response by blocking T-cell recruitment to inflammatory sites

Motohiro Nonaka  1 , Xingfeng Bao  1 , Fumiko Matsumura  2 , Sebastian Götze  3 , Jeyakumar Kandasamy  3 , Andrew Kononov  4 , David H Broide  5 , Jun Nakayama  6 , Peter H Seeberger  3 , Minoru Fukuda  7
Affiliations

Synthetic di-sulfated iduronic acid attenuates asthmatic response by blocking T-cell recruitment to inflammatory sites

Motohiro Nonaka et al. Proc Natl Acad Sci U S A. .

Abstract

Identification of carbohydrate sequences that determine affinity to specific chemokines is a critical step for strategies to interfere with chemokine-mediated leukocyte trafficking. Here, we first characterized the development of allergic asthma in Tie2-dependent and inducible Ext1-knockout (Tie2-Ext1(iKO)) mice. We showed that heparan sulfate is essential for leukocyte recruitment in the peribronchial region and bronchoalveolar lavage fluid (BALF), and is crucial for induction of airway hyperresponsiveness. Our glycan microarray showed a unique affinity profile of chemokine CCL20 to substructures of heparin and heparin-like oligo/di/monosaccharides. Among them, we identified a synthetic and not naturally occurring monosaccharide, 2,4-O-di-sulfated iduronic acid (Di-S-IdoA), as a potential inhibitor for CCL20-heparan sulfate interaction. Mice injected with Di-S-IdoA via tail vain or nasal inhalation showed attenuated leukocyte recruitment into inflammatory sites and BALF. These results demonstrate a critical role of chemokine-heparan sulfate interaction in the asthma development and Di-S-IdoA as a potential drug for asthma treatment.

Keywords: lymphocyte recruitment; sulfated monosaccharide.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Reduced OVA-induced airway inflammation and airway responsiveness in endothelial bheparan sulfate-deficient mice. (A) Immunofluorescent staining of heparan sulfate in the lung endothelial cells from WT and endothelial heparan sulfate-deficient mice (Tie2-Ext1iKO). Quantification of fluorescent intensity is shown at Right. (B) Histological and immunohistochemical staining of the mouse lung sections from WT and Tie2-Ext1iKO mice challenged with OVA. Representative images of the infiltrating eosinophils and T cells near airway lumens are shown. (C) Quantification of leukocyte infiltration in the bronchoalveolar lavage fluid (BALF) from the OVA-challenged mice with the indicated genotypes. (D) Cytokine measurement in the lung lavage from OVA-challenged mice. (E) Reduced airway responsiveness in Tie2-Ext1iKO mice in OVA-challenged mice (n = 8 mice per group). The degree of airway responsiveness to PBS or methacholine (MCh) at 0, 3, 24, and 48 mg/mL was determined. Error bars indicate SD. Unpaired two-tailed Student t test was used for statistical analysis. P value less than 0.05 was considered significant (*). *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 2.
Fig. 2.
Reduced OVA-induced airway inflammation in L-selectin ligand-deficient mice. (A) Immunohistochemical staining of control or GlcNAc6ST-1/2 double-knockout (G6ST DKO) mice challenged with OVA. (B) Number of infiltrating cells in BALF recovered from control or G6ST DKO mice. Quantification of total BALF cells (Left) and each cell type (Right) are shown. Error bars indicate SD. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 3.
Fig. 3.
Binding of CCL20 to heparin-like oligosaccharides on a glycan microarray. (A) Compounds tested for glycan microarray. (B) Binding of recombinant human CCL20 to heparin oligosaccharide-like glycans on a microarray. Numbers 1–14 donate different glycan structures. Each sugar was printed on the slide at four different concentrations ranging from 1,000, 250, 63, and 16 μM in 10 replicas. (C) Quantification of the binding of CCL20 to the heparin oligosaccharide-like glycans shown in B (n = 10; concentration of printed compounds, 250 µM).
Fig. 4.
Fig. 4.
An affinity of unnatural 2,4-di-sulfated Iduronic acid (Di-S-IdoA) to CCL20 and its inhibitory effect on CCL20-heparan sulfate interaction. (A) Binding kinetics of CCL20 to immobilized Di-S-IdoA (monosaccharide 11) by SPR. (B) Inhibition assay with heparin oligosaccharide-immobilized plate. Binding of CCL20 to heparin-coated plate was assayed in the presence of soluble heparin, nonsulfated IdoA (Non-S-IdoA), and various concentration of Di-S-IdoA (n = 30). (C) Inhibitory activity of Di-S-IdoA in the binding of CCL20 to F2 endothelial cells. Heparin was included as a control. Heparinase treatment preceded the addition to the cells. The data are representative of two experiments with similar results. (D) Inhibitory activity of Di-S-IdoA in the binding of various proteins to F2 endothelial cells. Di-S-IdoA (800 µM) or heparin (20 µM) was used for the inhibition. Error bars indicate SD. *P < 0.05 and ***P < 0.001.
Fig. 5.
Fig. 5.
Di-S-IdoA administration attenuated OVA-induced airway inflammation. (A) Histology and immunohistochemical staining of lungs from mice receiving either PBS or Di-S-IdoA. (B) Quantification of infiltrating T cells in the airway of mice receiving PBS, Di-S-IdoA, Non-S-IdoA, or neutralizing anti-CCL20 antibody (n = 5–8 mice per group). Result are shown as mean ± SEM. *P < 0.05 and ***P < 0.001. (C) Immunofluorescent staining of CCL20, heparan sulfate, and endothelial cells in the lung from pulmonary Di-S-IdoA–inhaled mice. Images are representatives from mice treated with PBS or Di-S-IdoA (100 µg), respectively. Note that the primary antibody against CCL20 was injected through the tail vein before the collection of lung tissue. (D) Pulmonary administration of Di-S-IdoA decreases OVA-induced airway inflammation. Leukocyte infiltration into BALF following inhalation pretreatment with either saline or different concentration of Di-S-IdoA (50 and 100 µg) (n = 5–7 mice per group). *P < 0.05.
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