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Syndecan-1 Promotes Staphylococcus aureus Corneal Infection by Counteracting Neutrophil-mediated Host Defense*

  • Atsuko Hayashida
    Affiliations
    From the Department of Medicine, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115 and
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  • Shiro Amano
    Affiliations
    the Department of Ophthalmology, University of Tokyo School of Medicine, 113-8655 Tokyo, Japan
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  • Pyong Woo Park
    Correspondence
    To whom correspondence should be addressed: Children's Hospital, Harvard Medical School, 320 Longwood Ave., Enders-461, Boston, MA 02115. Tel.: 617-919-4584; Fax: 617-730-0240;
    Affiliations
    From the Department of Medicine, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115 and
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant R01 HL94613.
    ♦ This article was selected as a Paper of the Week.
Open AccessPublished:December 02, 2010DOI:https://doi.org/10.1074/jbc.M110.185165
      Many microbial pathogens subvert cell surface heparan sulfate proteoglycans (HSPGs) to infect host cells in vitro. The significance of HSPG-pathogen interactions in vivo, however, remains to be determined. In this study, we examined the role of syndecan-1, a major cell surface HSPG of epithelial cells, in Staphylococcus aureus corneal infection. We found that syndecan-1 null (Sdc1−/−) mice significantly resist S. aureus corneal infection compared with wild type (WT) mice that express abundant syndecan-1 in their corneal epithelium. However, syndecan-1 did not bind to S. aureus, and syndecan-1 was not required for the colonization of cultured corneal epithelial cells by S. aureus, suggesting that syndecan-1 does not mediate S. aureus attachment to corneal tissues in vivo. Instead, S. aureus induced the shedding of syndecan-1 ectodomains from the surface of corneal epithelial cells. Topical administration of purified syndecan-1 ectodomains or heparan sulfate (HS) significantly increased, whereas inhibition of syndecan-1 shedding significantly decreased the bacterial burden in corneal tissues. Furthermore, depletion of neutrophils in the resistant Sdc1−/− mice increased the corneal bacterial burden to that of the susceptible WT mice, suggesting that syndecan-1 moderates neutrophils to promote infection. We found that syndecan-1 does not affect the infiltration of neutrophils into the infected cornea but that purified syndecan-1 ectodomain and HS significantly inhibit neutrophil-mediated killing of S. aureus. These data suggest a previously unknown bacterial subversion mechanism where S. aureus exploits the capacity of syndecan-1 ectodomains to inhibit neutrophil-mediated bacterial killing mechanisms in an HS-dependent manner to promote its pathogenesis in the cornea.

      Introduction

      Microbial pathogens express a multitude of factors that interact with host components. Pathogens use these host-pathogen interactions to their advantage to survive in the host environment. Studies during the last several decades have proposed that many viral, bacterial, and parasitic pathogens bind to cell surface HSPGs
      The abbreviations used are: HSPG, heparan sulfate proteoglycan; HBP, heparin-binding protein (azurocidin/CAP37); HS, heparan sulfate; KC, keratinocyte-derived chemokine (CXCL1); NET, neutrophil extracellular trap; NMuMG, normal murine mammary gland; RANTES, regulated upon activation normal T cell expressed and secreted; TSB, tryptic soy broth; cfu, colony-forming unit; HBSS, Hanks' balanced salt solution.
      to facilitate their initial attachment and subsequent invasion of host cells (
      • Bernfield M.
      • Götte M.
      • Park P.W.
      • Reizes O.
      • Fitzgerald M.L.
      • Lincecum J.
      • Zako M.
      ,
      • Rostand K.S.
      • Esko J.D.
      ,
      • Bartlett A.H.
      • Park P.W.
      ). Evidence that the HSPG interaction is biologically important is provided by the finding that HS-binding pathogens show markedly attenuated attachment or invasion of host cells whose HS expression has been reduced by enzymatic treatment or mutagenesis. Furthermore, exogenous HS or heparin (pharmaceutical functional mimic of HS) inhibits pathogen attachment and entry. Furthermore, where examined, mutant strains lacking the HSPG adhesin are viable and show normal growth rates, suggesting that the ability to bind to HSPGs is strictly a virulence activity. However, the significance of HSPG-pathogen interactions in infectious diseases has yet to be clearly established in vivo.
      Syndecans comprise a major family of cell surface HSPGs (
      • Bernfield M.
      • Götte M.
      • Park P.W.
      • Reizes O.
      • Fitzgerald M.L.
      • Lincecum J.
      • Zako M.
      ,
      • Park P.W.
      • Reizes O.
      • Bernfield M.
      ). Syndecans are type I transmembrane HSPGs composed of four members in mammals. At the cell surface, syndecans function primarily as a co-receptor for various HS-binding ligands and regulate cellular processes, such as adhesion, proliferation, migration, and differentiation. Although all syndecans harbor the ligand-binding HS chains in their extracellular domains, dramatic pathological phenotypes emerge when the single syndecan null mice are challenged with infectious or inflammatory stimuli (
      • Echtermeyer F.
      • Streit M.
      • Wilcox-Adelman S.
      • Saoncella S.
      • Denhez F.
      • Detmar M.
      • Goetinck P.
      ,
      • Haynes 3rd., A.
      • Ruda F.
      • Oliver J.
      • Hamood A.N.
      • Griswold J.A.
      • Park P.W.
      • Rumbaugh K.P.
      ,
      • Li Q.
      • Park P.W.
      • Wilson C.L.
      • Parks W.C.
      ,
      • Park P.W.
      • Pier G.B.
      • Hinkes M.T.
      • Bernfield M.
      ,
      • Hayashida A.
      • Bartlett A.H.
      • Foster T.J.
      • Park P.W.
      ,
      • Hayashida K.
      • Chen Y.
      • Bartlett A.H.
      • Park P.W.
      ,
      • Hayashida K.
      • Parks W.C.
      • Park P.W.
      ,
      • Stepp M.A.
      • Gibson H.E.
      • Gala P.H.
      • Iglesia D.D.
      • Pajoohesh-Ganji A.
      • Pal-Ghosh S.
      • Brown M.
      • Aquino C.
      • Schwartz A.M.
      • Goldberger O.
      • Hinkes M.T.
      • Bernfield M.
      ), indicating that certain post-developmental functions of each syndecan are specific and cannot be compensated by another syndecan or other HSPGs. How this is accomplished is incompletely understood, but syndecans likely perform specific functions in vivo because they are expressed on different cell types and locations at different levels and times (
      • Bernfield M.
      • Götte M.
      • Park P.W.
      • Reizes O.
      • Fitzgerald M.L.
      • Lincecum J.
      • Zako M.
      ,
      • Kim C.W.
      • Goldberger O.A.
      • Gallo R.L.
      • Bernfield M.
      ). For example, in adult tissues, syndecan-1 is abundantly expressed by both simple and stratified epithelial cells and expressed to a lesser degree by other cell types (e.g. fibroblasts) (
      • Bernfield M.
      • Götte M.
      • Park P.W.
      • Reizes O.
      • Fitzgerald M.L.
      • Lincecum J.
      • Zako M.
      ,
      • Kim C.W.
      • Goldberger O.A.
      • Gallo R.L.
      • Bernfield M.
      ,
      • Hayashi K.
      • Hayashi M.
      • Jalkanen M.
      • Firestone J.H.
      • Trelstad R.L.
      • Bernfield M.
      ).
      The role of syndecans in microbial infections is currently an active area of research. For instance, Neisseria gonorrhoeae binds to the HS moiety of syndecan-1 and -4 through the Opa protein, and this interaction mediates both bacterial attachment and invasion (
      • Freissler E.
      • Meyer auf der Heyde A.
      • David G.
      • Meyer T.F.
      • Dehio C.
      ). The intact syndecan cytoplasmic domain is essential in gonococcal invasion as N. gonorrhoeae attaches to but does not invade epithelial cells expressing syndecan mutant constructs lacking the cytoplasmic domain or those lacking specific signaling motifs in the cytoplasmic domain (
      • Freissler E.
      • Meyer auf der Heyde A.
      • David G.
      • Meyer T.F.
      • Dehio C.
      ). These data suggest that binding of N. gonorrhoeae to syndecan-1 and -4 induces signaling through the syndecan cytoplasmic domain, leading to internalization of the bacteria. Alternatively, syndecan-2 and -3 expressed on the surface of dendritic cells have been shown to bind to HIV and facilitate viral transmission to CD4-positive T cells (
      • Bobardt M.D.
      • Saphire A.C.
      • Hung H.C.
      • Yu X.
      • Van der Schueren B.
      • Zhang Z.
      • David G.
      • Gallay P.A.
      ,
      • de Witte L.
      • Bobardt M.
      • Chatterji U.
      • Degeest G.
      • David G.
      • Geijtenbeek T.B.
      • Gallay P.
      ). Here, syndecans are thought to prolong the infectivity of HIV, increase infectivity of dendritic cells in cis, and promote transmission to T cells.
      Syndecan-1 has also been proposed to modulate bacterial infections as a shed HSPG ectodomain. Syndecan-1 shedding is a highly regulated process that is stimulated in vitro by several inflammatory factors, and in vivo under certain pathological conditions (
      • Bernfield M.
      • Götte M.
      • Park P.W.
      • Reizes O.
      • Fitzgerald M.L.
      • Lincecum J.
      • Zako M.
      ,
      • Bartlett A.H.
      • Park P.W.
      ,
      • Bartlett A.H.
      • Hayashida K.
      • Park P.W.
      ,
      • Sanderson R.D.
      ). Bacterial pathogens, such as Staphylococcus aureus (
      • Park P.W.
      • Foster T.J.
      • Nishi E.
      • Duncan S.J.
      • Klagsbrun M.
      • Chen Y.
      ), Pseudomonas aeruginosa (
      • Park P.W.
      • Pier G.B.
      • Preston M.J.
      • Goldberger O.
      • Fitzgerald M.L.
      • Bernfield M.
      ), Streptococcus pneumoniae (
      • Chen Y.
      • Hayashida A.
      • Bennett A.E.
      • Hollingshead S.K.
      • Park P.W.
      ), and Bacillus anthracis (
      • Popova T.G.
      • Millis B.
      • Bradburne C.
      • Nazarenko S.
      • Bailey C.
      • Chandhoke V.
      • Popov S.G.
      ), secrete virulence factors that stimulate the host cell's metalloproteinase-mediated shedding mechanism at the cell surface. However, several strains of other Gram-positive and Gram-negative bacteria, including Streptococcus agalactiae (group B Streptococcus), Staphylococcus xylosus, Salmonella enteritidis, and Salmonella typhimurium, do not enhance shedding (
      • Park P.W.
      • Pier G.B.
      • Preston M.J.
      • Goldberger O.
      • Fitzgerald M.L.
      • Bernfield M.
      ), suggesting that certain opportunistic bacterial pathogens selectively induce syndecan-1 shedding. The physiological function of syndecan-1 shedding in bacterial infections is not fully understood, but syndecan-1 shedding is induced by established virulence factors, suggesting that this is a pathogenic mechanism. Moreover, syndecan-1 shedding is induced in mouse models of P. aeruginosa lung (
      • Park P.W.
      • Pier G.B.
      • Hinkes M.T.
      • Bernfield M.
      ) and burned skin infection (
      • Haynes 3rd., A.
      • Ruda F.
      • Oliver J.
      • Hamood A.N.
      • Griswold J.A.
      • Park P.W.
      • Rumbaugh K.P.
      ), and administration of purified syndecan-1 ectodomains enhances P. aeruginosa virulence in the lung (
      • Park P.W.
      • Pier G.B.
      • Hinkes M.T.
      • Bernfield M.
      ). Syndecan-1 ectodomains are thought to promote P. aeruginosa pathogenesis by interfering with innate host defense mechanisms, but the defense mechanisms inhibited by syndecan-1 ectodomains are not known. In addition, it is not known whether activation of syndecan-1 shedding is a general virulence mechanism used by various bacterial pathogens.
      All these properties of syndecan-1 suggest that it may have a prominent role in infection, likely with a critical function as a cell surface attachment site or a shed anti-host defense factor. In either case, syndecan-1 could be a suitable drug target for treatment of infectious diseases. To address these issues, we examined the role of syndecan-1 in the pathogenesis of S. aureus corneal infection. Bacterial keratitis is a serious public health concern with significant ocular morbidity that can lead to reduced acuity and irreversible scarring (
      • Bourcier T.
      • Thomas F.
      • Borderie V.
      • Chaumeil C.
      • Laroche L.
      ,
      • Limberg M.B.
      ,
      • Jett B.D.
      • Gilmore M.S.
      ). It is one of the major causes of blindness worldwide. S. aureus is a leading cause of bacterial keratitis, accounting for 10–25% of confirmed cases (
      • Green M.
      • Apel A.
      • Stapleton F.
      ,
      • Schaefer F.
      • Bruttin O.
      • Zografos L.
      • Guex-Crosier Y.
      ,
      • Ly C.N.
      • Pham J.N.
      • Badenoch P.R.
      • Bell S.M.
      • Hawkins G.
      • Rafferty D.L.
      • McClellan K.A.
      ). S. aureus binds to HS (
      • Liang O.D.
      • Ascencio F.
      • Fransson L.A.
      • Wadström T.
      ), and syndecan-1 has been shown to enhance the attachment of S. aureus to several types of host cells (
      • Henry-Stanley M.J.
      • Hess D.J.
      • Erickson E.A.
      • Garni R.M.
      • Wells C.L.
      ). Furthermore, S. aureus induces syndecan-1 shedding in vitro through α- and β-toxins (
      • Park P.W.
      • Foster T.J.
      • Nishi E.
      • Duncan S.J.
      • Klagsbrun M.
      • Chen Y.
      ), which are established exotoxin virulence factors in animal models of S. aureus keratitis (
      • Girgis D.O.
      • Sloop G.D.
      • Reed J.M.
      • O'Callaghan R.J.
      ,
      • O'Callaghan R.J.
      • Callegan M.C.
      • Moreau J.M.
      • Green L.C.
      • Foster T.J.
      • Hartford O.M.
      • Engel L.S.
      • Hill J.M.
      ). Our data surprisingly revealed that syndecan-1 does not bind to S. aureus and does not mediate S. aureus attachment to corneal epithelial cells. Instead, our results showed that S. aureus infection induces syndecan-1 shedding from the surface of corneal epithelial cells, and syndecan-1 ectodomains promote S. aureus corneal infection by interfering with the capacity of neutrophils to kill S. aureus.

      DISCUSSION

      In this study, we addressed the physiological relevance of syndecan-1 in S. aureus keratitis using a mouse model of scarified corneal infection. We found that ablation of syndecan-1 has a pronounced positive effect on S. aureus clearance in the cornea. The bacterial burden was similar in WT and Sdc1−/− corneas at earlier times post-infection, whereas it was significantly decreased in Sdc1−/− corneas compared with WT corneas at later times post-infection. Furthermore, syndecan-1 did not bind to S. aureus, and gene knockdown of syndecan-1 did not decrease the colonization of cultured epithelial cells by S. aureus. Collectively, these data suggest that syndecan-1 affects mechanisms of the host and not of the bacteria to promote S. aureus pathogenesis in the cornea. Indeed, we found that depletion of neutrophils enhances S. aureus virulence in the resistant Sdc1−/− corneas. However, the expression of KC and MIP-2 and extravasation of neutrophils into infected corneal tissues were similar between WT and Sdc1−/− mice, indicating that a more potent neutrophil-mediated host defense occurred in Sdc1−/− corneas despite a similar number of infiltrated neutrophils. Consistent with these data, we found that S. aureus infection induces syndecan-1 shedding from the surface of corneal epithelial cells and syndecan-1 ectodomains inhibit the capacity of neutrophils to kill S. aureus in an HS-dependent manner. These data reveal a new function of syndecan-1 in infectious diseases where it promotes pathogenesis by inhibiting the neutrophil arm of host defense and increasing bacterial survival in the host.
      One of the surprising observations from our studies was that syndecan-1 is not essential for the initial attachment of S. aureus to the injured corneal epithelium, suggesting that other HSPGs or other matrix components mediate S. aureus attachment to corneal tissues. Indeed, the collagen-binding adhesin has been shown to be a virulence factor in a rabbit model of soft contact lens-associated S. aureus keratitis (
      • Rhem M.N.
      • Lech E.M.
      • Patti J.M.
      • McDevitt D.
      • Höök M.
      • Jones D.B.
      • Wilhelmus K.R.
      ), and deletion of S. aureus fibronectin-binding proteins A and B has been shown to reduce S. aureus attachment and invasion of human corneal epithelial cells by 99% (
      • Jett B.D.
      • Gilmore M.S.
      ). These observations suggest that collagen and/or fibronectin, and not HSPGs, are the key host determinants that mediate the initial attachment of S. aureus to injured corneal tissues.
      We also conclude that S. aureus takes advantage of the capacity of syndecan-1 ectodomains to inhibit neutrophil-mediated bacterial killing mechanisms by inducing syndecan-1 shedding from the surface of corneal epithelial cells. How S. aureus induces syndecan-1 shedding in corneal epithelial cells is not understood. However, we previously reported that S. aureus α- and β-toxin induce syndecan-1 shedding in lung epithelial and mammary gland epithelial cells (
      • Park P.W.
      • Foster T.J.
      • Nishi E.
      • Duncan S.J.
      • Klagsbrun M.
      • Chen Y.
      ). Furthermore, studies of S. aureus keratitis in rabbit and mouse models showed that α-toxin mediates the majority of the virulence activities (
      • O'Callaghan R.J.
      • Callegan M.C.
      • Moreau J.M.
      • Green L.C.
      • Foster T.J.
      • Hartford O.M.
      • Engel L.S.
      • Hill J.M.
      ,
      • McCormick C.C.
      • Caballero A.R.
      • Balzli C.L.
      • Tang A.
      • O'Callaghan R.J.
      ,
      • Hume E.B.
      • Dajcs J.J.
      • Moreau J.M.
      • O'Callaghan R.J.
      ), whereas β-toxin is also important but contributes less to virulence in the cornea (
      • O'Callaghan R.J.
      • Callegan M.C.
      • Moreau J.M.
      • Green L.C.
      • Foster T.J.
      • Hartford O.M.
      • Engel L.S.
      • Hill J.M.
      ). Together, these observations suggest that S. aureus induces syndecan-1 shedding in the cornea through α- and β-toxin, and the capacity to enhance syndecan-1 shedding and generate soluble ectodomains is an important virulence activity of these exotoxin virulence factors.
      The pro-pathogenic effects of HS on S. aureus corneal infection suggest that other HSPGs may function similarly to syndecan-1. However, because other syndecans and HSPGs are intact in Sdc1−/− mice, the significant difference in the capacity of WT and Sdc1−/− corneas to clear S. aureus suggests that syndecan-1 ectodomains function specifically to increase bacterial survival. Precisely how syndecan-1 ectodomains act in this manner is not known, but previous studies have also shown that loss of syndecan-1 alone results in dramatic pathological phenotypes in animal models of various infectious and inflammatory diseases (
      • Haynes 3rd., A.
      • Ruda F.
      • Oliver J.
      • Hamood A.N.
      • Griswold J.A.
      • Park P.W.
      • Rumbaugh K.P.
      ,
      • Li Q.
      • Park P.W.
      • Wilson C.L.
      • Parks W.C.
      ,
      • Park P.W.
      • Pier G.B.
      • Hinkes M.T.
      • Bernfield M.
      ,
      • Hayashida A.
      • Bartlett A.H.
      • Foster T.J.
      • Park P.W.
      ,
      • Hayashida K.
      • Chen Y.
      • Bartlett A.H.
      • Park P.W.
      ,
      • Hayashida K.
      • Parks W.C.
      • Park P.W.
      ), suggesting that other HSPGs cannot compensate for the loss of syndecan-1 in vivo. In the corneal epithelium, both syndecan-1 and -4 are expressed, but syndecan-4 is expressed at a lower level than syndecan-1. Furthermore, although syndecan-4 shedding was also induced during S. aureus corneal infection, syndecan-4 ectodomain levels did not increase as rapidly or significantly as those of syndecan-1. These observations suggest that the specificity of syndecan-1 functions in S. aureus corneal infection may be a reflection of its abundant expression and prominent shedding in corneal epithelial cells. Alternatively, because syndecan-1 acts in an HS-dependent manner, the potential unique structural features of syndecan-1 HS chains may mediate its specific functions in S. aureus corneal infection. However, the structure and function of syndecan-1 and -4 isolated from mammary gland epithelial cells are indistinguishable, and syndecan HS chains are considered to be cell type-specific and not core protein-specific (
      • Zako M.
      • Dong J.
      • Goldberger O.
      • Bernfield M.
      • Gallagher J.T.
      • Deakin J.A.
      ), suggesting that the structure and function of syndecan-1 and -4 are also similar in the corneal epithelium. Nonetheless, these observations imply that despite similar cellular distribution and HS structures, corneal syndecan-1 and -4 have nonredundant roles in vivo. Future studies determining the response of Sdc4−/− mice in S. aureus corneal infection and analyzing the fine structure of syndecan-1 and -4 HS chains should address these issues.
      Both passive and active defense mechanisms effectively protect the cornea from infections. The mechanical action of the eyelid physically removes pathogen; the outermost lipid layer of the tear film serves as a physical barrier, and the washing effects of tears also remove pathogens (
      • Sack R.A.
      • Nunes I.
      • Beaton A.
      • Morris C.
      ). Furthermore, the aqueous tear fluid contains a wide array of antimicrobial factors (
      • Sack R.A.
      • Nunes I.
      • Beaton A.
      • Morris C.
      ). However, all of these defense mechanisms are likely not affected by syndecan-1 because S. aureus was cleared similarly in uninjured corneas of both WT and Sdc1−/− mice. Once the passive host defense mechanisms have failed or the corneal epithelium is breached, neutrophils are actively recruited to the site of infection by various neutrophil-chemotactic factors, such as KC, MIP-2, and lipopolysaccharide-induced CXC chemokine (CXCL5). Syndecan-1 shedding has been shown to mediate the generation of a KC gradient across the lung alveolar epithelium (
      • Li Q.
      • Park P.W.
      • Wilson C.L.
      • Parks W.C.
      ) and to facilitate the removal of KC and MIP-2 tethered to endothelial cells in the lung, liver, and kidney (
      • Hayashida K.
      • Parks W.C.
      • Park P.W.
      ). Hence, we were most surprised to find that syndecan-1 moderates neutrophil activity and not its infiltration into corneal tissues. Although increased mononuclear leukocyte adhesion onto retinal blood vessels was observed in TNFα-stimulated Sdc1−/− mice (
      • Götte M.
      • Joussen A.M.
      • Klein C.
      • Andre P.
      • Wagner D.D.
      • Hinkes M.T.
      • Kirchhof B.
      • Adamis A.P.
      • Bernfield M.
      ), we found no differences in the expression of KC and MIP-2 and infiltration of neutrophils into infected WT and Sdc1−/− corneas, suggesting that mechanisms governing neutrophil influx in response to a single cytokine and an active bacterial infection may be different. Furthermore, because lumican and keratocan core proteins have been shown to bind to CXC chemokines and form a chemokine gradient that directs neutrophil migration during a corneal inflammatory response (
      • Lin M.
      • Carlson E.
      • Diaconu E.
      • Pearlman E.
      ,
      • Carlson E.C.
      • Lin M.
      • Liu C.Y.
      • Kao W.W.
      • Perez V.L.
      • Pearlman E.
      ), mechanisms governing the generation of a CXC chemokine gradient in corneal tissues may be different from those of other tissues. In addition, because TLR2 and MyD88 are required for the induction of KC and MIP-2 in injured corneas and for the recruitment of neutrophils in response to S. aureus corneal infection (
      • Sun Y.
      • Hise A.G.
      • Kalsow C.M.
      • Pearlman E.
      ), our results suggest that signaling events through TLR2 and MyD88 are also not affected by syndecan-1 in S. aureus corneal infection.
      The underlying mechanisms of how syndecan-1 ectodomains inhibit the capacity of neutrophils to kill S. aureus in an HS-dependent manner remain unknown. Because syndecan-1 ectodomain does not bind to S. aureus and does not affect the viability of isolated neutrophils, ectodomains must be inhibiting key processes of neutrophils that kill bacteria. Furthermore, because our preliminary data suggest that sulfated domains in HS interfere with the killing of S. aureus by neutrophils, cationic defense factors and their mechanisms are presumably inhibited by syndecan-1 ectodomains. However, given that cell surface receptors for syndecan-1 or HS on neutrophils have not been reported and syndecan-1 ectodomains are not membrane-permeable, syndecan-1 ectodomains are likely acting on extracellular killing mechanisms of neutrophils. Activated neutrophils secrete a highly cationic heparin-binding protein (HBP, azurocidin/CAP37) that has antimicrobial activities against several bacterial pathogens, including S. aureus (
      • Pereira H.A.
      • Erdem I.
      • Pohl J.
      • Spitznagel J.K.
      ). HBP has also been shown to increase the phagocytosis of S. aureus by monocytes and macrophages (
      • Soehnlein O.
      • Kai-Larsen Y.
      • Frithiof R.
      • Sorensen O.E.
      • Kenne E.
      • Scharffetter-Kochanek K.
      • Eriksson E.E.
      • Herwald H.
      • Agerberth B.
      • Lindbom L.
      ). These observations suggest that syndecan-1 HS chains may bind to HBP and interfere with its bacterial killing and phagocytosis-potentiating activities, although it is not known if HBP affects bacterial phagocytosis by neutrophils.
      Alternatively, syndecan-1 ectodomains may inhibit a recently described extracellular killing mechanism of neutrophils called neutrophil extracellular traps (NETs) (
      • Brinkmann V.
      • Reichard U.
      • Goosmann C.
      • Fauler B.
      • Uhlemann Y.
      • Weiss D.S.
      • Weinrauch Y.
      • Zychlinsky A.
      ). NETs are made by activated neutrophils, but not naive neutrophils, and consist of negatively charged, decondensed chromatin fibers with cationic antimicrobial factors, such as HBP, cathelicidins, defensins, and elastase, embedded in them. NETs trap pathogens and facilitate killing by bringing the pathogens and neutrophil-derived antimicrobial factors to proximity. Importantly, impaired NET formation in vivo predisposes the host to bacterial infections (
      • Yost C.C.
      • Cody M.J.
      • Harris E.S.
      • Thornton N.L.
      • McInturff A.M.
      • Martinez M.L.
      • Chandler N.B.
      • Rodesch C.K.
      • Albertine K.H.
      • Petti C.A.
      • Weyrich A.S.
      • Zimmerman G.A.
      ,
      • Li P.
      • Li M.
      • Lindberg M.R.
      • Kennett M.J.
      • Xiong N.
      • Wang Y.
      ). Because functional NETs are formed by the ionic interactions between anionic chromatin fibers and cationic antimicrobial factors, the highly anionic HS chains of syndecan-1 ectodomains may bind to and displace the cationic antimicrobial factors from anionic NET fibers and inhibit NET-mediated killing of S. aureus. Altogether, these observations raise the possibility that syndecan-1 ectodomains may inhibit one of more bacterial killing mechanisms of neutrophils. Further studies will be required to elucidate the impact of syndecan-1 ectodomains on these host defense mechanisms of neutrophils in corneal tissues.
      In summary, our findings extend the biological functions of syndecan-1 from that of a cell surface proteoglycan that regulates cell adhesion, proliferation, and migration to that of a soluble HSPG ectodomain capable of modulating neutrophil-mediated host defense mechanisms crucial to the clearance of S. aureus in injured corneas. Interestingly, the majority of bacterial pathogens that can induce syndecan-1 shedding in vitro are major etiological agents of bacterial keratitis in vivo (e.g. S. aureus, P. aeruginosa, and S. pneumoniae), suggesting that subversion of the capacity of syndecan-1 ectodomains to counteract neutrophil-mediated bacterial killing is a general virulence mechanism of various ocular surface pathogens. Although the normal functions of syndecan-1 in the resting cornea remain to be defined, our findings suggest a possible beneficial role of inhibiting syndecan-1 shedding or neutralizing syndecan-1 ectodomains in treating bacterial keratitis.

      Acknowledgments

      We thank Dr. Joram Piatigorsky (NEI, National Institutes of Health, Rockville, MD) for the A6(1) corneal epithelial cell line.

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