Heparan Sulfate Modulates Neutrophil and Endothelial Function in Antibacterial Innate Immunity Ding Xu,a,c,f Joshua Olson,b Jason N. Cole,a,b,g Xander M. van Wijk,a,c Volker Brinkmann,h Arturo Zychlinsky,h Victor Nizet,a,b,d Jeffrey D. Esko,a,c Yung-Chi Changa,b,e Glycobiology Research and Training Center,a Department of Pediatrics,b Department of Cellular and Molecular Medicine,c and Skaggs School of Pharmacy and d Pharmaceutical Sciences, University of California, San Diego, La Jolla, California, USA; Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Downloaded from Taipei, Taiwane; Department of Oral Biology, University at Buffalo, School of Dental Medicine, The State University of New York, Buffalo, New York, USAf; School of Chemistry and Molecular Biosciences and Australian Infectious Diseases Research Centre, The University of Queensland, St. Lucia, Queensland, Australiag; Department of Cellular Microbiology, Max Planck Institute for Infection Biology, Berlin, Germanyh Recently, we showed that endothelial heparan sulfate facilitates entry of a bacterial pathogen into the central nervous system. Here, we show that normal bactericidal activity of neutrophils is influenced by the sulfation pattern of heparan sulfate. Inactiva- tion of heparan sulfate uronyl 2-O-sulfotransferase (Hs2st) in neutrophils substantially reduced their bactericidal activity, and Hs2st deficiency rendered mice more susceptible to systemic infection with the pathogenic bacterium group B Streptococcus. http://iai.asm.org/ Specifically, altered sulfation of heparan sulfate in mutant neutrophils affected formation of neutrophil extracellular traps while not influencing phagocytosis, production of reactive oxygen species, or secretion of granular proteases. Heparan sulfate pro- teoglycan(s) is present in neutrophil extracellular traps, modulates histone affinity, and modulates their microbial activity. Hs2st-deficient brain endothelial cells show enhanced binding to group B Streptococcus and are more susceptible to apoptosis, likely contributing to the observed increase in dissemination of group B Streptococcus into the brain of Hs2st-deficient mice fol- lowing intravenous challenge. Taken together, our data provide strong evidence that heparan sulfate from both neutrophils and the endothelium plays important roles in modulating innate immunity. on August 12, 2015 by UNIV OF CALIF SAN DIEGO eparan sulfate (HS) proteoglycans (HSPGs) are ubiquitously cation can influence intrinsic neutrophil functions relevant to an- Hexpressed by animal cells and play essential roles in cell sig- tibacterial defense and microbicidal activity. naling and cell-cell interactions (1, 2). To a large extent, these Several potential neutrophil bactericidal effectors, including properties depend on the interaction of HS with various signaling proteases (elastase and cathepsin G) and ␣-defensins, have been receptors and their ligands (3). Many pathogens have evolved to shown to interact with HSPGs and affect their intracellular local- exploit cell surface HS molecules as counterreceptors for adhesion ization and retention within granules (14–16). During NET for- and invasion (4–7). Cell surface HSPGs also modulate the innate mation at tissue foci of infection, antimicrobial effectors, includ- immune response by regulating chemokine presentation and ing elastase, cathepsin G, and strongly cationic histones, are growth factor responses (8). Thus, HSPGs lie at a nexus between displayed across the chromatin-based network to act against bac- pathogen invasion and host defense. teria ensnared in the extracellular structures (17, 18). The polyan- Neutrophils, the most abundant circulating leukocyte, repre- ionic charge of HS should favor interaction with positively sent a critical first line of mammalian host defense against a wide charged histones with the potential to modulate their cytotoxic range of infectious pathogens. Neutrophils are recruited to sites of effects (19–22). Thus, we sought to understand whether modifi- infection, recognize and phagocytose microbes, and kill patho- cation of HS sulfation in neutrophils could regulate their antimi- gens through a combination of microbicidal mechanisms, includ- crobial activity, especially in the context of NETs during in vivo ing production of reactive oxygen species, release of antimicrobial infection. peptides, and expulsion of their nuclear contents to form neutro- phil extracellular traps (NETs) (9, 10). HS modulates the rapid recruitment of neutrophils to sites of infection or injury at differ- Received 27 April 2015 Returned for modification 20 May 2015 ent levels, in particular through its interactions with cytokines, Accepted 26 June 2015 chemokines, and selectins (8). We reported previously that re- Accepted manuscript posted online 6 July 2015 duced expression and altered sulfation of HS on endothelial cells, Citation Xu D, Olson J, Cole JN, van Wijk XM, Brinkmann V, Zychlinsky A, Nizet V, achieved by inactivating glucosamine N-deacetylase-N-sulfo- Esko JD, Chang Y-C. 2015. Heparan sulfate modulates neutrophil and endothelial function in antibacterial innate immunity. Infect Immun 83:3648–3656. transferase-1 (Ndst1) or uronyl 2-O-sulfotransferase (Hs2st), al- doi:10.1128/IAI.00545-15. tered neutrophil extravasation to sites of sterile inflammation by Editor: B. A. McCormick modifying interactions between endothelial HS and L-selectins on Address correspondence to Jeffrey D. Esko, [email protected], or Yung-Chi Chang, neutrophils and chemokines released during the inflammatory [email protected]. response (11, 12). In contrast, the same modifications of HS on the Supplemental material for this article may be found at http://dx.doi.org/10.1128 neutrophil cell surface did not affect trafficking during sterile in- /IAI.00545-15. flammation (11, 12) and had only mild effects on the acquired Copyright © 2015, American Society for Microbiology. All Rights Reserved. immune responses (13). Here we extended this line of investiga- doi:10.1128/IAI.00545-15 tion to live-bacterial-infection models to probe how HS modifi- 3648 iai.asm.org Infection and Immunity September 2015 Volume 83 Number 9 Hs2st Modulates Neutrophil Bactericidal Activity Here we took advantage of conditional knockout mice in BMDM bactericidal activity and cytokine release. For bacterial kill- which Hs2st is deleted in myeloid cells (including neutrophils) ing, 105 CFU of the GBS strain NCTC 10/84 were added to 5 ϫ 105 and endothelial cells by excision of a loxP-flanked allele under the BMDMs (multiplicity of infection [MOI] ϭ 0.2) for 1 h; then 50 ␮l of control of Cre recombinase driven by the Tie2 promoter (Hs2stf/f 0.6% Triton X-100 was added to lyse cells. The number of CFU of GBS ϩ recovered was determined. To detect cytokine secretion after GBS infec- Tie2 Cre mice, referred to here as Hs2st-deficient or mutant 5 mice). Analysis of these animals revealed that neutrophil-derived tion, 10 macrophages were stimulated for 30 min with GBS (MOI ϭ 1), followed by addition of penicillin (5 ␮g/ml) and gentamicin (10 ␮g/ml) to HS plays a significant role in controlling bactericidal activity. prevent bacterial overgrowth. Culture supernatants were collected 24 h Whereas inactivation of Hs2st on endothelial cells facilitates neu- after infection, and secretion of tumor necrosis factor alpha (TNF-␣) and trophil infiltration to sterile inflammatory stimuli (11), the im- interleukin 6 (IL-6) was analyzed by enzyme-linked immunosorbent as- paired intrinsic bactericidal activity of Hs2st-deficient neutrophils say (ELISA) (BD Biosciences). For quantitative RT-PCR analysis, RNA Downloaded from results in increased susceptibility of Hs2st-deficient mice to inva- was isolated from BMDMs using TRIzol (Life Technologies) 30 min after sive bacterial infection. The bactericidal defect in Hs2st-deficient GBS stimulation (MOI ϭ 10) and used as a template for cDNA prepara- mice correlates with reduced NET formation and altered histone tion by iScript (Bio-Rad). Quantitative PCR was performed using iQ affinity to HS within the NETs. SYBR green supermix (Bio-Rad) per standard protocols. Primer combi- nations were as follows: for IL-1␤,5-ACTACAGGCTCCGAGATGAA = MATERIALS AND METHODS C-3 plus 5 -CCCAAGGCCACAGGTATTTT-3 ; for TNF-␣,5-AGCCC ACGTCGTAGCAAACCACCAA-3= = plus 5 -ACACCCATTCCCTTCAC= = Bacteria and mouse strains. Human serotype V group B Streptococcus AGAGCAAT-3 ; for GAPDH, 5 -AACTTTGGCATTGTGGAAGGGC-3= = = = = (GBS) clinical isolate NCTC 10/84 (ATCC 49447) was propagated in plus 5 -GGTAGGAACACGGAAGGCCATG-3 . Primers for IL-10 were http://iai.asm.org/ Todd-Hewitt broth (THB; Difco, BD Diagnostics) at 37°C without shak- obtained= from the QuantiTect primer assay (Qiagen).= ing. For infection studies, bacteria were grown at to mid-exponential Neutrophil bactericidal activity and extracellular-trap (NET) for- ϳ ϫ phase, resuspended to an optical density at 600 nm (OD600) of 0.4 ( 2 mation. For bactericidal assays, murine bone marrow neutrophils were 108 CFU per ml), and serially diluted to the desired inoculum. Hs2stf/f Tie2 pretreated with 160 ␮M phorbol myristate acetate (PMA) for 90 min, Creϩ mice were generated as described previously (11) and back- whereas LPS-elicited neutrophils were used directly upon isolation. GBS crossed Ͼ10 generations on a C57BL/6 background. Littermate Hs2stf/f (2 ϫ 105 CFU) were added to 106 neutrophils (MOI ϭ 0.2) and incubated Tie2 CreϪ mice were used as wild-type controls in all experiments. Mice at 37°C with rotation in a siliconized microtube;