Synthetic di-sulfated iduronic acid attenuatesasthmatic response by blocking T-cell recruitmentto inflammatory sitesMotohiro Nonakaa,1, Xingfeng Baoa,1, Fumiko Matsumuraa,b, Sebastian Götzeb,c, Jeyakumar Kandasamyb,c,Andrew Kononovb, David H. Broided, Jun Nakayamae, Peter H. Seebergerb,c, and Minoru Fukudaa,2

aGlycobiology Unit, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037; bDepartment of Biomolecular Systems, Max Planck Institute for Colloidsand Interfaces, 14476 Potsdam, Germany; cInstitute of Chemistry and Biochemistry, Freie Universität Berlin, 14195 Berlin, Germany; dDepartment of Medicine,University of California, San Diego, La Jolla, CA 92093; and eDepartment of Molecular Pathology, Shinshu University Graduate School of Medicine, Matsumoto390-8621, Japan

Edited by Carolyn R. Bertozzi, University of California, Berkeley, CA, and approved April 4, 2014 (received for review October 22, 2013)

Identification of carbohydrate sequences that determine affinity tospecific chemokines is a critical step for strategies to interfere withchemokine-mediated leukocyte trafficking. Here, we first charac-terized the development of allergic asthma in Tie2-dependent andinducible Ext1-knockout (Tie2-Ext1iKO) mice. We showed that hep-aran sulfate is essential for leukocyte recruitment in the peribron-chial region and bronchoalveolar lavage fluid (BALF), and is crucialfor induction of airway hyperresponsiveness. Our glycan microar-ray showed a unique affinity profile of chemokine CCL20 to sub-structures of heparin and heparin-like oligo/di/monosaccharides.Among them, we identified a synthetic and not naturally occurringmonosaccharide, 2,4-O-di-sulfated iduronic acid (Di-S-IdoA), asa potential inhibitor for CCL20–heparan sulfate interaction. Miceinjected with Di-S-IdoA via tail vain or nasal inhalation showed at-tenuated leukocyte recruitment into inflammatory sites and BALF.These results demonstrate a critical role of chemokine–heparansulfate interaction in the asthma development and Di-S-IdoA as apotential drug for asthma treatment.

sulfated monosaccharide | lymphocyte recruitment

Asthma is a common allergic disease characterized by chronicairway inflammation, mucus hypersecretion, and airway

hyperreactivity to inhaled allergens (1). Despite the importanceof T lymphocytes in adaptive immunity and host defense, theiraccumulation in airway in allergic asthma causes Th2-mediatedpulmonary inflammation. The asthmatic inflammatory responseis orchestrated by T-cell trafficking network among lung, bloodcirculation, secondary lymphoid organ, and peripheral tissue (2).Of note, the significant increase of T cells in the airway in asthmais mostly due to T-cell recruitment from regional lymph nodesrather than their proliferation at the inflamed site (3). Therefore,a therapeutic approach that shuts off the trafficking pathway ofpathogenic T cells should significantly inhibit the Th2-mediatedinflammation in allergic asthma.It is well known that the destination of T-cell trafficking

pathway is tightly restricted by the profile of chemokines, lipidchemoattractants, and T-cell chemokine receptors. As a part ofimmune surveillance, naïve T cells and central memory T cellsconstantly access secondary lymphoid organs from blood circu-lation via specialized high endothelial venules (HEVs). The in-teraction between T cells and HEV cells includes in a stepwisemanner (4, 5), L-selectin–dependent tethering and rolling, acti-vation, firm arrest, and transendothelial migration. Besides6-sulfo sialyl Lewis X as a L-selectin ligand, HEVs constitutivelyexpress chemokine CCL21 and CCL19 and attract T cells thatexpress its cognate receptor CCR7 (5). In contrast to this ho-meostatic homing, circulating T cells interact with inflamedblood vessels in lung after asthmatic exposure to an inhaled al-lergen. Among numerous combinations of chemokines and theirreceptors, there is considerable evidence that CCL20 and its

cognate receptor CCR6 may contribute to the pathogenesis ofasthma (6). CCL20-CCR6 plays a key role in the recruitment ofTh17 (7) cells and Th2 cells (8). Indeed, CCL20 is highly enrichedon inflammatory epithelium (9) and CCR6 is expressed on memoryT cells infiltrated in the lung during allergic inflammation (7). Inaddition, CCR6-deficient mice have decreased airway respon-siveness, and reduced recruitment of eosinophils into lung (10, 11).These findings suggest that CCL20-CCR6 axis is a putative tar-get for the treatment of asthma.Cumulative evidence in vivo and in vitro indicates that che-

mokines cannot be functionally active in HEVs and inflamedsites without their interaction with heparan sulfate (12). Heparansulfate protects chemokines from proteolysis, immobilizes themon the endothelium surface and produces chemokine gradientsin the vasculature. Heparan sulfate is composed of repeatingdisaccharide units of uronic acid [glucuronic acid (GlcA) oriduronic acid (IdoA)] and N-acetylglucosamine (GlcNAc) car-bohydrates. Some of GlcA (or IdoA) carbohydrates are sub-sequently O-sulfated, and GlcNAc carbohydrates are partiallymodified with N-deacetylation and N-sulfation (13). Previousreports have indicated that the sulfation patterns in heparansulfate are more restricted than expected (14), and the sulfation

Significance

Asthmatic inflammation is orchestrated by T-lymphocyte celltrafficking network within lungs, blood circulation, secondarylymphoid organ, and peripheral tissue. Here, we demonstratedthat T cell and following eosinophil recruitment was sub-stantially reduced in our recently generated mouse model,where heparan sulfate synthase exostoses-1 (Ext1) is knockoutin an inducible manner. Moreover, we discovered that evena monosaccharide, 2,4-disulfated iduronic acid (Di-S-IdoA),bound to chemokine CCL20 and significantly inhibited CCL20binding to heparan sulfate and to endothelial cell surface. Wefound that Di-S-IdoA attenuated asthmatic reaction, measuredby T cell, eosinophil, and CCL20 recruitment in asthmatic mice.These findings show for the first time (to our knowledge) thatsulfate monosaccharide can be developed into a potent ther-apeutic agent for treating asthma.

Author contributions: M.N., X.B., and M.F. designed research; M.N., X.B., F.M., S.G., A.K.,and J.N. performed research; F.M., S.G., J.K., A.K., D.H.B., and P.H.S. contributed newreagents/analytic tools; D.H.B., J.N., P.H.S., and M.F. analyzed data; and M.N., X.B., andM.F. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1M.N. and X.B. contributed equally to this work.2To whom correspondence should be addressed. E-mail: minoru@sanfordburnham.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1319870111/-/DCSupplemental.

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is associated with respiratory distress (15) and asthma (16). It isbelieved that there are specific interactions between heparansulfate and chemokines (17). Nevertheless, the nontemplatenature of long carbohydrate chains (<25,000 disaccharide units)and conformational plasticity still make it difficult to identify thecommon sequences of heparan sulfate that display affinity tospecific chemokines. In this regard, our previously establishedglycan microarray system (18, 19) is a powerful tool to define theselectivity in heparan sulfate–chemokine interactions.Recently, we established the exostoses-1 (Ext1) gene condi-

tional knockout Tie2-Ext1iKO mouse model, in which GlcA/IdoA-GlcNAc repeat of heparan sulfate can be abrogated inendothelial cells in a tetracycline-inducible manner (20). In thisstudy, Tie2-Ext1iKO mouse showed significant reduction of bothleukocyte recruitment to lung tissues and of airway hyper-responsiveness in ovalbumin (OVA) asthma model. Moreover,glycan microarray analysis surprisingly identified that an un-natural and synthetic monosaccharide, 2,4-O-di-sulfated iduronicacid (Di-S-IdoA) has a high affinity to recombinant CCL20.Intravenous and inhalation challenges of Di-S-IdoA significantlyinhibited the leukocyte infiltration in bronchoalveolar lavage fluid(BALF). Our finding that even a monosaccharide can attenuateairway inflammation suggests its potential use as an antiasthmatherapy that can be administered by inhalation.

ResultsTie2-Ext1iKO Mice Show Diminished T-Cell Accumulation andInflammatory Response in the Lung in a Mouse Model of OVA-Induced Asthma. Recently, we have developed Tie2-Ext1iKO

mouse line in which embryonic lethality of homozygous Ext1−/−

mouse is avoidable. Indeed, there was little expression of en-dothelial heparan sulfate in the lungs of Tie2-Ext1iKO mice (Fig.1A). To determine the role of endothelial heparan sulfate inasthma, we sensitized and challenged these mice with OVA al-lergen. In OVA-challenged control mice, periodic acid–Schiff(PAS) staining showed significant mucus accumulation in airwayepithelium. In addition, a large number of CD3+ T cells andeosinophils were detected in the peribronchial regions (Fig. 1B).In contrast, there was little infiltration of CD3+ T cells and eosin-ophil cells, as well as mucus secretion in Tie2-Ext1iKO mice. Therewas also a significant reduction of total cell number includinglymphocytes, macrophages, and eosinophils in BALF in Tie2-Ext1iKO mice (Fig. 1C). Moreover, whereas BALF in controlmice contained high levels of Th2 cytokines IL-5 and IL-13 butnot the Th1 cytokine IFN-γ, only low levels of IL-5 and IL-13could be detected in that of Tie2-Ext1iKO (Fig. 1D), suggesting

that OVA-triggered Th2 activation was abolished in Tie2-Ext1iKO mice.To determine the effect of deficiency in heparan sulfate on

airway responsiveness, methacholine (MCh) responsivenesswas assessed in OVA-challenged Tie2-Ext1iKO mice. Comparedwith OVA-challenged control mice, OVA-challenged Tie2-Ext1iKO

mice showed significantly less response to MCh at 48 mg/mL(P = 0.0033) (Fig. 1E). Taken together, these results indicatethat endothelial heparan sulfate is essential for lymphocyte ac-cumulation at sites of allergic inflammation in the lung, andthat the reduced accumulation is associated with less airwayresponsiveness.

Interruption of Lymphocyte Trafficking Pathway Attenuates AsthmaticResponse. We have previously reported that endothelial heparansulfate is required for lymphocyte recruitment to secondarylymph nodes. Here, we hypothesized that the attenuation of theOVA induced asthmatic response in Tie2-Ext1iKO mice is due toblocking of lymphocyte recruitment from regional lymph nodes.Previous studies have demonstrated that the homeostatic lym-phocyte trafficking pathway was impaired in L-selectin ligand-deficient [GlcNAc6ST-1/2 double knockout (G6ST DKO)] mice(21, 22). To evaluate the contribution of homeostatic lymphocytetrafficking to the lung, G6ST DKO mice were challenged withOVA. Similar to Tie2-Ext1iKO mice, G6ST DKO mice had sig-nificantly fewer CD3+ T cells and eosinophil cells around theairway walls compared with control mice (Fig. 2A). Moreover,G6ST DKO mice BALF contained significantly fewer eosino-phils and lymphocytes (Fig. 2B). These data support that accu-mulation of lymphocytes in lung tissue is mainly contributed bytheir recruitment from regional lymph nodes.

Glycan Microarray Showed Chemokine CCL20 Binds Short Heparin-Like Sulfated Oligosaccharide. Our previous studies have in-dicated that heparan sulfate is important to maintain morpho-logic localization of the homeostatic chemokine CCL21 (20).Here, the results from OVA-challenged Tie2-Ext1iKO asthmaticmice led us suggest that heparan sulfate also interacts with in-flammation-related chemokines such as CCL20. Thus, we analyzedthe binding profile of CCL20 to a heparin-like carbohydrate li-brary, which contains carbohydrates with various sulfation patternsranging from monosaccharides to hexasaccharides (Fig. 3A) byour glycan microarray system (18, 19). The screening resultsshowed that CCL20 strongly bound to hexasaccharides 1, 2,and moderately bound to hexasaccharide 5 and tetrasaccharide 7.This indicates that the affinity of CCL20 toward heparan sulfatebecomes higher with increasing sulfations levels (Fig. 3 B and C).

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Fig. 1. Reduced OVA-induced airway inflam-mation and airway responsiveness in endothelialbheparan sulfate-deficient mice. (A) Immunofluo-rescent staining of heparan sulfate in the lung en-dothelial cells from WT and endothelial heparansulfate-deficient mice (Tie2-Ext1iKO). Quantificationof fluorescent intensity is shown at Right. (B) His-tological and immunohistochemical staining of themouse lung sections from WT and Tie2-Ext1iKO

mice challenged with OVA. Representative imagesof the infiltrating eosinophils and T cells near airwaylumens are shown. (C) Quantification of leukocyteinfiltration in the bronchoalveolar lavage fluid(BALF) from the OVA-challenged mice with the in-dicated genotypes. (D) Cytokine measurement inthe lung lavage from OVA-challenged mice. (E) Re-duced airway responsiveness in Tie2-Ext1iKO mice inOVA-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.

8174 | www.pnas.org/cgi/doi/10.1073/pnas.1319870111 Nonaka et al.

Notably, CCL20 exhibited high affinity to disaccharide 10 and evenmonosaccharide 11. Observed fluorescent intensities of thosecarbohydrates were comparable to 5-kDa natural heparin.

A Unique and Unnatural Monosaccharide Di-S-IdoA Inhibits HeparanSulfate–CCL20 Interaction.Monosaccharide 11 is Di-S-IdoA, whichcontains two axial sulfate groups. Because Di-S-IdoA is an un-

natural synthetic monosaccharide that has never been isolatedfrom a natural source, we were prompted to further characterizeits interaction with CCL20 in more detail. The data from surfaceplasmon resonance (SPR) showed that CCL20 binds to immo-bilized Di-S-IdoA in the micromolar range (KD = 2.9 × 10−6 M)(Fig. 4A). We next analyzed the inhibitory effect of Di-S-IdoA.Interaction between immobilized hexasaccharides 1 and CCL20was blocked by Di-S-IdoA in a concentration-dependent mannerand 500 μM Di-S-IdoA inhibited at a comparable level of 5,000μM heparin (Fig. 4B), whereas nonsulfated IdoA showed no suchinhibitory activity at 5 mM. We next synthesized Di-S-IdoA withaminopentyl linker (Figs. S1 and S2). Interestingly, in the cul-tured F2 cell, which is known to express endogenous heparansulfate, Di-S-IdoA also interfered with the CCL20–heparansulfate interactions in a dose-dependent manner (Fig. 4C). Theseresults suggest that Di-S-IdoA is an effective as a functional in-hibitor of CCL20 chemokine activity. To next study the spec-ificity of Di-S-IdoA, the inhibitory effect of Di-S-IdoA on thebindings between the various proteins and endothelial cellswas assayed. It is known that CCL21 (23, 24), IL-8 (23, 25),L-selectin (26, 27), and complement component 5a (C5a) (28,29) are involved both in the binding to heparin/heparan sulfatein vitro and in the asthma pathogenesis. The result showed thatDi-S-IdoA did not block the attachment of CCL21 to mouseendothelial F2 cells, whereas heparin efficiently blocked (Fig.4D). Di-S-IdoA significantly blocked the binding of L-electin

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Fig. 2. Reduced OVA-induced airway inflammation in L-selectin ligand-deficient mice. (A) Immunohistochemical staining of control or GlcNAc6ST-1/2double-knockout (G6ST DKO) mice challenged with OVA. (B) Number ofinfiltrating cells in BALF recovered from control or G6ST DKO mice. Quan-tification of total BALF cells (Left) and each cell type (Right) are shown. Errorbars indicate SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 3. Binding of CCL20 to heparin-like oligosaccharides on a glycan microarray. (A) Compounds tested for glycan microarray. (B) Binding of recombinanthuman CCL20 to heparin oligosaccharide-like glycans on a microarray. Numbers 1–14 donate different glycan structures. Each sugar was printed on the slideat 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 oligo-saccharide-like glycans shown in B (n = 10; concentration of printed compounds, 250 μM).

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to F2 cells. However, Di-S-IdoA showed even higher inhibitoryeffect than heparin in IL-8 binding. In this experimentalmodel, C5a did not show any bindings to F2 cells. Those resultsindicate Di-S-IdoA has unique binding preferences distinctfrom heparin.

Inhibitory Effect of Di-S-IdoA in OVA-Challenged WT Mice. To test thepossibility of Di-S-IdoA as an inhibitor of CCL20 activity invivo, we injected either PBS or Di-S-IdoA into tail vein rightbefore the reexposure of OVA in WT mice. Pretreatment ofDi-S-IdoA decreased the mucus secretion and recruitment ofCD3+ T cells in the lung tissues (Fig. 5A). We also observeda reduced total leukocyte number in Di-S-IdoA–treated andneutralizing anti-CCL20 antibody-treated mice but not non-sulfated IdoA (Non-S-IdoA)-treated mice (Fig. 5B). Theseresults suggest that i.v. administered Di-S-IdoA can access at theinflamed postcapillary venule, where it inhibits the activity ofchemoattractants.Because CCL20 has been demonstrated to be mainly synthe-

sized from airway epithelial cells, we hypothesized that admin-istration of Di-S-IdoA through intranasal inhalation wouldinterfere the localization and presentation on the vascular en-dothelial cell surface in inflamed lung. To test the accessibility ofDi-S-IdoA through the lung alveoli, we administered Di-S-IdoAby inhalation before OVA challenge in WT mice. Indeed, wefound robust expression of CCL20 on the vascular endothelialcell wall in the OVA-challenged WT mice. In contrast, there waslittle expression of CCL20 in the Di-S-IdoA–pretreated mice,although heparan sulfate is present on vascular surface (Fig. 5C).These data suggest that CCL20 presentation on the endothelialcells at the lung inflammatory site was blocked by Di-S-IdoAadministration. Indeed, the number of total leukocytes in BALFwas dramatically decreased in 100 μg of Di-S-IdoA treatment(P < 0.05) compared with saline-treated mice (Fig. 5D). Bloodtests from the WT mice treated with Di-S-IdoA showed no sideeffect in liver function, kidney function, and blood cell count(Table S1). Taken together, our results demonstrated that in-halation administration of Di-S-IdoA was effective in reducingairway inflammation in allergen-challenged WT mice.

DiscussionDespite extensive research efforts in the past decades, the prev-alence and severity of asthma are still increasing in the de-veloped countries. The pathology of allergic asthma is directly

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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) Bindingkinetics of CCL20 to immobilized Di-S-IdoA (monosaccharide 11) by SPR. (B) Inhibition assay with heparin oligosaccharide-immobilized plate. Binding of CCL20to 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 additionto 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 F2endothelial 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.

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Fig. 5. Di-S-IdoA administration attenuated OVA-induced airway inflam-mation. (A) Histology and immunohistochemical staining of lungs from micereceiving either PBS or Di-S-IdoA. (B) Quantification of infiltrating T cells inthe 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 orDi-S-IdoA (100 μg), respectively. Note that the primary antibody againstCCL20 was injected through the tail vein before the collection of lung tissue.(D) Pulmonary administration of Di-S-IdoA decreases OVA-induced airwayinflammation. Leukocyte infiltration into BALF following inhalation pre-treatment with either saline or different concentration of Di-S-IdoA (50 and100 μg) (n = 5–7 mice per group). *P < 0.05.

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linked to the consequence of long-term chronic Th2-mediatedinflammation in the lung at the site of continuous exposure toallergens (30). Previous report has showed that a partial lossof N-sulfate in heparan sulfate contributes to attenuation ofairway inflammation (16). However, the role of the main chainof heparan sulfate in the pathogenesis of asthma had not beenaddressed because of the embryonic lethality of homozygousExt1−/− mouse. In this study, we used our established induciblegene knockout system to circumvent this lethality (20). In con-trast to WT mice, Th2-mediated allergic inflammation in the lungin OVA-challenged Tie2-Ext1iKO mice was abolished as assessedby eosinophil recruitment to the lung, and eosinophil, mac-rophage, and lymphocyte infiltration into BALF. Moreover,Tie2-Ext1iKO mice showed lower airway hyperresponsiveness toMCh than control mice did, indicating that GlcA/IdoA-GlcNAcrepeat of endothelial heparan sulfate is not only essential forlymphocyte, eosinophil, and macrophage recruitment, but alsofor airway responsiveness a cardinal feature of asthma. Impor-tantly, we have shown that the proliferation and rolling ability oflymphocytes in Tie2-Ext1iKO mice do not change during homing(20). In another report, lymphocyte-specific deletion of heparansulfate has little effects on their differentiation (31). Our resultsalso showed that selectin ligand-deficient G6ST DKO mice haddecreased lymphocyte recruitment into the lung. These resultsnot only reemphasize the importance of T-cell trafficking in thepathogenesis of asthma, but also indicate the potential impor-tance of blocking of this T-cell trafficking pathway as an effectivetherapeutic strategy for asthma.Among the asthma-associated chemokines, the role of CCL20

in asthma is only beginning to be understood and a large part ofits physiological significance remains to be elucidated. Given thataffinity between chemokine and heparan sulfate also variesdepending on type of chemokines (18), we hypothesized thereare specific oligosaccharide patterns preferable for binding toCCL20. In this study, a glycan microarray identified unnaturaland synthetic monosaccharide Di-S-IdoA, which has two axialO-sulfations at C2 and C4 positions, as a potent inhibitor ofCCL20. This finding was surprising because previously seven otherchemokines including CCL21 did not show high affinities for Di-S-IdoA as equivalent to sulfated hexasaccharides (18). In addition,the data that nonsulfated IdoA did not show any binding toCCL20 suggests that two sulfate groups are important to forma pool of negative charge on one side and this ionic charge con-tributed to interaction with CCL20. Furthermore, inhalation aswell as i.v. administration of this compound significantly decreasedlymphocyte recruitment into the allergen-challenged lung in-flammatory site, suggesting that CCL20-mediated Th17 and Th2cell recruitments in early phase is critical for following diseasedevelopment including eosinophil, macrophage recruitments.Heparin, a soluble analog of heparan sulfate, and its deriva-

tives have an antiinflammatory activity as well as an anticoagulanteffect. A role for heparin in improving symptoms in inflammatorydisease was demonstrated in neonatally immunized rabbits (32),naturally sensitized sheep (33), and asthma patients in clinicaltrials (34, 35). The mechanism of its antiinflammatory effect islikely due to the interference with the chemokine retention onvascular heparan sulfate, blockage of interleukins, selectins, andcomplement components. In this regard, a therapeutic approachusing heparin-derived low–molecular-weight saccharides wouldbe beneficial (12). However, so far, X-ray crystallography ofnatural heparin revealed that a disaccharide unit of heparin isnot sufficient and at least tetrasaccharide is necessary to inhibitactivity of chemokine RANTES in vivo (36). In this context, ourresult that a monosaccharide with unusual sulfation pattern caninterfere with the chemokine–heparan sulfate interaction is anovel finding. Taken together, our findings strongly suggest thatDi-S-IdoA binds to CCL20, resulting in inhibiting CCL20 bindingto heparan sulfate and to the cellular receptor, CCR6.

As a whole, our results have demonstrated an essential role ofendothelial heparan sulfate in T-cell trafficking into lung andthus in the pathogenesis of allergic asthma. We identified a novelmonosaccharide Di-S-IdoA as a potent inhibitor of chemokineCCL20. With the establishment of this new tool for CCL20, wedetermined the effect of Di-S-IdoA in lymphocyte recruitmentinto the allergen-challenged lung inflammation site. Pulmonaryinhalation drug delivery of this compound may help to attenuateasthmatic symptoms by suppressing chemokine-mediated inflam-matory responses, mucus production, and airway responsiveness.

Materials and MethodsReagents and Animals. Tie2-Ext1iKO mice were generated and maintained asreported (20). To induce the deletion of endothelial heparan sulfate, mice of4-wk age were treated with doxycycline for 3 consecutive weeks beforeexperiments. All protocols for animal experiments were approved by theInstitutional Animal Care and Use Committee (IACUC) of the Sanford-Burnham Medical Research Institute, and were conducted in accordancewith National Institutes of Health guidelines. OVA was purchased fromSigma, heparan sulfate antibody 3G10 from US Biological, recombinanthuman CCL20 from R&D and Shenandoah Biotech, recombinant humanCCL21 from Sino Biological, recombinant human IL-8 from Cell Sciences,recombinant mouse L-selectin from Life Technologies, recombinant human C5afrom BioVision, eosinophil anti-MBP antibody (Dr. James Lee, Mayo Clinic,Scottsdale, AZ) (37), anti-CD3e antibody from Santa Cruz, anti-macrophage in-flammatory protein 3α (CCL20) antibody from Abcam, neutralizing anti-CCL20antibody and anti-CCL21 antibody from R&D, anti-His antibody from Clontech,and anti-von Willebrand factor antibody from Dako.

OVA-Induced Model of Asthma in Mice. For sensitization, mice were in-traperitoneally administrated a suspension containing 50 μg of OVA and0.5 mg of aluminum hydroxide (Sigma) in 200 μL of PBS on day 0 and day 12.On days 23, 25, and 27, the mice were briefly anesthetized with isofluraneand challenged intranasally with 20 μg of OVA in 50 μL of PBS throughthe nose. For studies with a therapeutic intervention, 100 μg of Di-S-IdoAor Non-S-IdoA, or 50 μg of neutralizing anti-CCL20 antibody was adminis-tered either i.v. or inhalationally 30 min before each OVA challenge. Twenty-four hours after the last OVA challenge, the mice were killed and the lungbronchoalveolar lavage was performed by infusion of 1 mL of 0.1% PBSthrough the trachea with Surflash IV catheters (Terumo). The lung lavage wassubjected to leukocyte counting by a cytometer, and each subpopulation wasanalyzed by cytospin followed by a stain with Hema-3 (Fisher).

Immunohistochemistry. Mouse lung lobes were fixed with 4% (wt/vol) PFAfollowed by paraffin embedding and histological examination by H&Estaining and PAS staining. For evaluation of immune infiltration into lunginflammatory sites, tissues were stained by anti-CD3e antibody and anti-MBPantibody described above.

Airway Responsiveness to MCh. Airway responsiveness to MCh was assayed24 h after the final OVA challenge in anesthetized, intubated, and ventilatedmice as previously described (38). Intubated and ventilated mice were anes-thetized intraperitoneally. The dynamic airway resistance was determined inmice exposed to nebulized PBS or MCh at 0, 3, 24, and 48 mg/mL.

Glycan Microarray. Microarrays were fabricated as previously reported (39).Microarray slide was incubated with 100 μL (5 μg) of rhCCL20 (R&D Systems)in HBS-N containing 0.01% Tween 20 for 1 h at room temperature undermild shaking, washed three times with HBS-N, and dried by centrifugation.The slide was then reacted with 100 μL (2 μg) of anti-hCCL20 goat IgG (R&DSystems) for 1 h, followed by incubation with anti-goat Alexa Fluor 594antibody (Invitrogen) for 1 h. Slides were scanned with a GenePix 4300Amicroarray scanner and analyzed by GenePix Pro software. Binding of CCL20to immobilized heparin-like oligosaccharide 1 in the presence of Non-S-IdoAand Di-S-IdoA was observed in the same manner. The amino group of eachmonosaccharide sample was acylated before use by treatment with asolution of acetic anhydride–triethylamine–methanol (2:3:5, vol/vol/vol)followed by condensation and lyophilization. The inhibition solution con-tained 5 mM 5-kDa heparin (Santa Cruz Biotechnology), 5 mM Non-S-IdoA,or 5, 50, and 500 μM Di-S-IdoA.

Immunohistochemistry. For heparan sulfate staining, tissue sections weretreated with heparatinase at 37 °C for 1 h before incubation with anti-

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heparan sulfate 3G10 antibody. Alexa Fluor 568 anti-mouse IgG2b was usedfor secondary reaction. Vascular endothelial cells were visualized by anti-vonWillebrand factor antibody (1:100 dilution). Lung endothelial CCL20 could notbe visualized by the general fluorescent staining protocol. Instead, localizationof blood vessel CCL20 was examined by i.v. injection of anti-CCL20 antibody(25 μg per mouse) through mouse tail vein. Thirty minutes after the injection,lungs were collected and frozen in OCT compound (Tissue Tek). Frozen tissuesections were then incubated with Alexa Fluor 568 anti-rabbit IgG (Invitrogen).

To avoid the cross-reaction in double staining with CCL20, sections wereblocked with anti-rabbit IgG antibody and protein A. Then the sections wereincubated with anti-von Willebrand factor antibody, followed by incubationwith FITC–anti-rabbit IgG-Fc antibody (Jackson ImmunoResearch).

Statistical Analyses. All data are showed as means ± SD. Unpaired two-tailedStudent t test was used for statistical analyses. We considered P values of lessthan 0.05 as statistically significant. Degrees of statistical significance arepresented as *P < 0.05, **P < 0.01, and ***P < 0.001.

ACKNOWLEDGMENTS. We thank Dr. S. Rosen (University of California, SanFrancisco) for helpful suggestions for lung CCL20 staining, Dr. P. Rosenthal(University of California, San Diego) for help with assay of methacholineresponsiveness, and Dr. M. N. Hecht for help with the microarray and SPRexperiments. This work was supported by National Institutes of HealthGrants P01 CA71932 (to M.F.) and AI 107779, AI 38425, AI 70535, and AI 72115(to D.H.B.).

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Supporting InformationNonaka et al. 10.1073/pnas.1319870111SI Materials and MethodsPreparation of 2,4-Disulfated Iduronic Acid with Aminopentyl Linker.2,4-Disulfated iduronic acid (Di-S-IdoA) was synthesized from S1(1). A brief reaction scheme is shown in Fig. S1.Methyl (ethyl 3-O-benzyl-1-thio-β-L-idopyranosyl) uronate (S2). A solu-tion of S1 (450 mg, 1.2 mmol) in THF (50 mL) was bubbled byAr for 15 min. To the solution was added Ir[COD(PCH3Ph2)2]PF6 (55 mg, cat) at room temperature (RT), and the light pinksolution was bubbled with Ar for 10 min, and then bubbled withH2 for 10 min. The reaction mixture was stirred at RT for 3 hunder H2 and 21 h under Ar. The mixture was concentrated anddiluted to MeOH (15 mL). To the solution was added TsOH·H2O(1.1 g). The reaction was stirred at RT for 21 h, quenched withpyridine (5 mL), concentrated, and azeotroped twice with toluene.Purification by flash column chromatography on silica gel (1:0→9:1,toluene/acetone) gave S2 (71 mg, 18%) as a colorless oil. S1 wasrecovered as a colorless oil. 1H NMR (CDCl3) δ 7.32–7.23 (m, 5H),4.94 (d, 1H, J = 2 Hz), 4.57 (dd, 2H, J = 10 Hz, 18 Hz), 4.43 (d, 1H,J = 4 Hz), 4.03 (br, 1H), 3.84 (t, 1H, J = 4 Hz), 3.78–3.61 (m, 6H),2.73 (br, 1H), 2.70 (q, 2H, J = 8 Hz), 1.21 (t, 3H, J = 8 Hz).13C NMR (CDCl3) δ 169.6, 137.3, 128.6, 128.2, 127.7, 82.9,76.2, 75.0, 72.4, 69.3, 67.5, 52.4, 25.1, 15.1.Methyl (ethyl 3-O-benzyl-2,4-di-O-pivaloyl-1-thio-β-L-idopyranosyl) uronate(S3). To a solution of S2 (71 mg, 0.21 mmol) in pyridine (1.0 mL)were added PivCl (0.50 mL) and 4-dimethylaminopyridine (5.0mg). The reaction mixture was stirred at RT for 15 h, and thendiluted with CHCl3 (20 mL). The mixture was washed with brine(20 mL) and saturated (sat.) NaHCO3 aqueous (aq.) (20 mL).The aqueous layer was extracted with CHCl3 (3 × 10 mL). Theorganic layers were combined, dried over Na2SO4, filtered, andconcentrated. Purification by silica gel column chromatography(toluene→AcOEt 0–7%) gave S3 (101 mg, 95%) as a colorlessfoam. 1H NMR (CDCl3) δ 7.38–7.28 (m, 5H), 5.18 (t, 1H, J = 2Hz), 5.02 (t, 1H, J = 2 Hz), 4.97 (d, 1H, J = 2 Hz), 4.81 (dd, 2H,J = 11 Hz, 13 Hz), 4.55 (d, 1H, J = 2 Hz), 3.83 (t, 1H, J = 2 Hz),3.74 (s, 3H), 2.79–2.68 (m, 2H), 1.28 (t, 3H, J = 7 Hz), 1.24 (s, 9H), 1.17 (s, 9H). 13C NMR (CDCl3) δ 178.2, 178.1, 167.9, 137.2,128.5, 128.1, 127.7, 82.5, 75.0, 74.5, 73.0, 69.0, 67.3, 52.2, 39.3,39.0, 27.3, 27.2, 25.9, 14.8. MS [electrospray ionization (ESI)]m/zcalculated (calcd.) [M-H]+ 509.2, found 509.9.Methyl {N-benzyl-(benzyloxycarbonyl)-5-aminopentyl 3-O-benzyl-2,4-di-O-pivaloyl-α-L-idopyranosyl} uronate (S4). To a solution S3 (90 mg, 0.18mmol) and HO(CH2)5NBnCbz (170 mg, 0.5 mmol) in CH2Cl2(5 mL), was added N-iodosuccinimide (79 mg, 352 mmol). Themixture was cooled to –40 °C and TfOH (5 μL) was added. Thereaction mixture was stirred at –40 °C for 5 min, and then at 0 °Cfor 10 min. Reaction mixture was quenched by the addition ofsat. NaHCO3 aq (5 mL) and Na2S2O3 (0.50 g). The mixture wasdiluted with CH2Cl2 (10 mL) and organic layer was washed withsat. NaHCO3 aq. (10 mL). The aqueous layer was extracted withCH2Cl2 (2 × 10 mL). The organic layers were combined, driedNa2SO4, filtered, and concentrated. The crude product was pu-rified by a silica gel column (toluene–AcOEt, 1:0–10:1) to giveS4 (89.7 mg, 66%). 1H NMR (CDCl3) δ 7.30–7.05 (m, 15H), 5.14(t, 1H, J = 3 Hz), 5.09 (d, 2H, J = 9 Hz), 4.88 (s, 1H), 4.84 (t, 1H,J = 3 Hz), 4.78 (s, 1H), 4.66 (dd, 2H, J = 12 Hz, 21 Hz), 3.68–3.44(m, 4H), 3.37 (br, 1H), 3.11 (br, 2H), 1.35–1.57 (m, 4H), 1.05–1.28 (m, 21H). MS (ESI) m/z calcd. [M+Na]+ 798.4, found 797.8.N-Benzyl-(benzyloxycarbonyl)-5-aminopentyl 3-O-benzyl-α-L-idopyranosyluro-nate (S5). To the solution of S4 (80.1 mg, 0.10 mmol) in anhydrousTHF (3.2 mL) were added H2O2 (30%, 1.1 mL) and 1 M aqueousLiOH (1.8 mL) at 0 °C. The reaction mixture was stirred at RT for

24 h, and then MeOH (1.7 mL) and 3 M KOH aq. (3.2 mL) wereadded. The reaction mixture was stirred at RT for 18 h. Themixture was then neutralized with Amberite IR-120 (H+), filtered,and concentrated. The residue was purified by LH-20 (CHCl3–MeOH, 2:1, vol/vol) to give S5 (61.6 mg, 99%) as a colorlesspowder. 1H NMR (CDCl3) δ 7.35–7.03 (m, 15H), 5.07 (d, 2H, J =12 Hz), 4.85 (d, 1H, J = 6 Hz), 4.66 (s, 1H), 4.51 (dd, 2H, J = 10Hz, 45 Hz), 4.36 (d, 2 H, J = 12 Hz), 4.10 (s, 1H), 3.76 (s, 1H), 3.67(t, 3H), 3.62 (br, 1H), 3.33 (br, 1H), 3.08 (br, 2H), 1.08–1.54 (m,6H). MS (ESI) m/z calcd. [M+Na]+ 752.2, found 752.1.N-Benzyl-(benzyloxycarbonyl)-5-aminopentyl 3-O-benzyl-2,4-di-O-sulfo-α-L-idopyranosyluronate (S6). To a solution of S5 (60.0 mg) in anhy-drous pyridine (4.0 mL), was added SO3·pyridine (0.16 g). Thereaction mixture was stirred at RT for 24 h, quenched by theaddition of MeOH (0.5 mL) and triethylamine (0.1 mL), andconentrated. The residue was purified by a Sephadex LH-20chromatograpy (MeOH–CH2Cl2, 1:1, vol/vol). The obtainedproduct was converted into the Na salt by passing through a col-umn of Dowex 50WX8 (Na+) in MeOH–H2O (9:1, vol/vol) to giveS6 (64 mg, 85%). 1H NMR (CD3OD) δ 7.38–7.07 (m, 15H), 5.20–5.05 (m, 3H), 4.87–4.64 (m, 4H), 4.50–4.35 (m, 4H), 3.61 (br, 1H),3.44 (br, 1H), 3.13 (br, 4H), 1.08–1.66 (m, 6H). MS (ESI) m/zcalcd. [M3−+2H]− 616.3, found 616.3.5-Aminopentyl 2,4-di-O-sulfo-α-L-idopyranosyluronate (di-S-IdoA withaminopentyl linker). A solution of S6 (39.0 mg) in MeOH–H2O(2:1, vol/vol, 3 mL) was bubbled by Ar for 10 min. To the solu-tion, was added 10% Pd/C (16 mg). The mixture was bubbled byAr for 10 min then by H2 for 15 min, and stirred at RT for 24 h.The suspension was filtered and concentrated to give 1 (21.7 mg,96%) as a colorless foam. 1H NMR (D2O) δ 4.98 (br s, 1H), 4.45(br, 2H), 4.37 (br s, 1H), 4.07 (br s, 1H), 3.61 (br, 1H), 3.53 (br,1H), 2.87 (br, 2H), 1.55 (br, 4H), 1.33 (br, 2H). 13C NMR (D2O)δ 174.8, 98.3, 74.6, 73.5, 68.2, 66.4, 66.3, 39.4, 27.8, 26.3, 22.2.[M3+2H]− 438.0, found 438.0.

Surface Plasmon Resonance Immobilization.Di-S-IdoA was covalentlybound to the sensor surface using primary amine coupling and aBiacore T100 (GEHealthcare). HBS-N containing 0.005% (vol/vol)P20 was used as running buffer. The carboxymethylated dex-tran matrix (CM5 chip; GE Healthcare) was first activated at aflow rate of 10 μL/min using an 8-min injection pulse of anaqueous solution containing N-hydroxysuccinimide (NHS)(0.05 M; GE Healthcare) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) (0.2 M; GE Health-care). Next, a solution of the oligosaccharide (50 μg/mL) containing1 mM hexadecyltrimethylammonium chloride (Sigma-Aldrich) wasflowed over the activated surface for 8 min at 5 μL/min. Remainingreactive groups on the chip surface were quenched by injection ofa 1 M ethanolamine hydrochloride (pH 8.5; GE Healthcare) so-lution for 7.5 min at 10 μL/min. A parallel flow cell was coupledwith HS/heparin 9 as a reference cell. The following binding levelswere established [in response units (RU)]: HS/heparin 11, 299;HS/heparin 9, 407.

Kinetic Analysis by Surface Plasmon Resonance. Di-S-IdoA was co-valently bound to the sensor surface using primary amine couplingand a Biacore T100 (GE Healthcare) as described above. For KDdetermination between immobilized Di-S-IdoA and rhCCL20,the following protein concentrations were injected at a flow rateof 30 μL/min and 25 °C: 0.125, 0.25, 0.5, 1, 2, and 4 μM. Runningbuffer was then flowed over the sensor surface for 10 min toenable dissociation. The sensor surface was regenerated for the

Nonaka et al. www.pnas.org/cgi/content/short/1319870111 1 of 4

next sample using 0.1% SDS and 0.085% H3PO4 injected for1 min at a flow rate of 80 μL/min. The signal from the referenceflow cell containing oligosaccharide 9 was subtracted to correctfor the contribution of nonspecific interactions and systematicerrors. Kinetic analysis was performed using the BIAevaluationsoftware for T100. Association and dissociation phase data wereglobally fitted to a simple 1:1 interaction model (A + B = AB).

Binding of Recombinant Proteins to Endothelial Cells. Endothelialcell line F2 was cultured in 96 wells to confluency. Wells weregently washed with PBS twice. For binding of CCL20, rhCCL20 at1 μg/mL was added to wells and incubated at 4 °C for 30 min.After washing with PBS for three times, biotinylated goat anti-IgG at 0.5 μg/mL was added to wells and incubated at 4 °C for30 min. HRP-streptavidin at 100 ng/mL was added after three

times of washing with PBS. Following a PBS washing, ortho-phenylenediamine was added to wells and incubated for 15 minfollowed by addition of 3 M sulfuric acid. Last, the wells wereread at 490 nm. In case of heparitinase treatment, the cellswere first incubated with 0.1 mI/U of each enzyme at 37 °C for1 h before addition of rhCCL20. For bindings of CCL21, IL-8,C5a, and L-selectin, 0.1 μg of proteins was added to wells.After washing, F2 cell-bound proteins were incubated withanti-CCL21 or anti-His antibody, followed by reaction with HRP-conjugated secondary antibodies. Color reaction was done by3,3′,5,5′-tetramethylbenzidine substrate solution (eBioscience).In case of competitive inhibition, recombinant proteins weremixed with the Di-S-IdoA or heparin at RT for 30 min beforeadding to the wells.

1. Bindschädler P, Adibekian A, Grünstein D, Seeberger PH (2010) De novo synthesis ofdifferentially protected L-iduronic acid glycosylating agents. Carbohydr Res 345(7):948–955.

OMeOOC

OR2 OR1

OBnSEt

S1: R1 = Allyl, R2 = HS2: R1 = R2 = HS3: R1 = R2 = Piv

OR5

OR4 OR3

OBnO N

Cbz

Bn

S4: R3 = R4 = Piv, R5 = COOMeS5: R3 = R4 = H, R5 = COOHS6: R3 = R4 = SO3 Na+, R5 = COO Na+

O-OOC

OSO3- OSO3

-

OHO NH2

a)

b)

d)

e)

c) f)

a) (i) Ir[COD(PCH3Ph2)2]PF6, H2, THF; (ii) TsOH·H2O, MeOH, 18%; b) PivCl, DMAP, pridine, 95%; c) HO(CH2)5NBnCbz, NIS, TfOH, CH2Cl2, 66%; d) (i) LiOH, H2O2, THF; (ii) KOH, H2O, MeOH, 99%; e) (i) SO3·pyridine, pyridine; (ii) Dowex50WX8 (Na+

form), 85%, f) Pd/C, MeOH, H 2O, 96%

Fig. S1. Preparation scheme for Di-S-IdoA with aminopentyl linker. Di-S-IdoA was synthesized from S1 (1). The spectrum of each intermediate was analyzed by1H NMR and 13C NMR as shown in Fig. S2.

1. Bindschädler P, Adibekian A, Grünstein D, Seeberger PH (2010) De novo synthesis of differentially protected L-iduronic acid glycosylating agents. Carbohydr Res 345(7):948–955.

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B

A 1H NMR

13C NMR

Di-S-IdoA

(D2O)

(CD3OD)

(CDCl3)

(CDCl3)

(CDCl3)

(CDCl3)

S2

S3

S4

S5

S6

Di-S-IdoA

(D2O)

(CD3OD)

(CDCl3)

(CDCl3)

(CDCl3)

(CDCl3)

S2

S3

S4

S5

S6

Fig. S2. 1H NMR and 13C NMR spectra of Di-S-IdoA with aminopentyl linker and its intermediates. The synthesis method of Di-S-IdoA is shown in Fig. S1.1H NMR (A) and 13C NMR (B) spectra showed that the structures of six compounds (S2, S3, S4, S5, S6, and Di-S-IdoA) are consistent with the expected structures.The spectra also showed no indication of other contaminating compounds.

Nonaka et al. www.pnas.org/cgi/content/short/1319870111 3 of 4

Table S1. Blood test of mice subjected to intranasal injection ofDi-S-IdoA

Blood profiles WT Di-S-IdoA treated Unit P*

WBC 5.70 ± 4.06 3.69 ± 3.24 ×109/L 0.47LYM 5.37 ± 3.76 3.28 ± 2.59 ×109/L 0.40MON 0.16 ± 0.10 0.33 ± 0.56 ×109/L 0.58GRA 0.16 ± 0.24 0.07 ± 0.10 ×109/L 0.51LY% 95.0 ± 1.9 93.1 ± 6.33 % 0.57MO% 2.90 ± 1.21 5.43 ± 5.64 % 0.44GR% 2.08 ± 1.89 1.53 ± 0.75 % 0.61RBC 5.59 ± 2.35 5.25 ± 1.18 ×1012/L 0.81HGB 10.7 ± 4.7 9.50 ± 2.60 g/dL 0.67HCT 22.6 ± 9.3 21.8 ± 5.3 % 0.89PLT 123 ± 85 197 ± 310 ×109/L 0.67PCT 0.125 ± 0.137 0.14 ± 0.23 % 0.90ALT 161 ± 152 72 ± 22 U/L 0.33AMY 916 ± 55 1,753 ± 1,431 U/L 0.42TBIL 4.75 ± 0.50 4.33 ± 1.53 μmol/L 0.62CA 2.90 ± 0.13 2.83 ± 0.08 mmol/L 0.46PHOS 3.19 ± 0.46 3.29 ± 0.31 mmol/L 0.75CRE 9.25 ± 12.70 11.33 ± 11.37 μmol/L 0.83GLU 15.7 ± 1.5 14.4 ± 0.4 mmol/L 0.20TP 50.5 ± 2.4 55.0 ± 6.0 g/L 0.22

Di-S-IdoA was administered intranasally every other day for three times.Twenty-four hours after the last OVA challenge, blood samples were takenfrom WT mice (n = 4) and Di-S-IdoA–treated mice (n = 3). ALT, alanineaminotransferase; AMY, amylase; CA, calcium; CRE, creatinine; GLU, glucose;GR%, granulocyte percentage; GRA, granulocyte; HCT, hematocrit; HGB,hemoglobin; LY%, lymphocyte percentage; LYM, lymphocyte; MO%, mono-cyte percentage; MON, monocyte; PCT, platelet hematocrit; PHOS, phos-phate; PLT, platelet; RBC, red blood cell count; TBIL, bilirubin; TP, totalprotein; WBC, total white blood cell count.*Analyzed by means of Student t test.

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