Identification of TLR4 as the Receptor That Recognizes Shiga Toxins in Human Neutrophils This information is current as Maurizio Brigotti, Domenica Carnicelli, Valentina Arfilli, of September 29, 2021. Nicola Tamassia, Francesca Borsetti, Elena Fabbri, Pier Luigi Tazzari, Francesca Ricci, Pasqualepaolo Pagliaro, Enzo Spisni and Marco A. Cassatella J Immunol 2013; 191:4748-4758; Prepublished online 25 September 2013; Downloaded from doi: 10.4049/jimmunol.1300122 http://www.jimmunol.org/content/191/9/4748 Supplementary http://www.jimmunol.org/content/suppl/2013/09/25/jimmunol.130012 http://www.jimmunol.org/ Material 2.DC1 References This article cites 81 articles, 32 of which you can access for free at: http://www.jimmunol.org/content/191/9/4748.full#ref-list-1 Why The JI? Submit online. by guest on September 29, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Identification of TLR4 as the Receptor That Recognizes Shiga Toxins in Human Neutrophils Maurizio Brigotti,* Domenica Carnicelli,*,1 Valentina Arfilli,*,1 Nicola Tamassia,† Francesca Borsetti,‡ Elena Fabbri,‡ Pier Luigi Tazzari,x Francesca Ricci,x Pasqualepaolo Pagliaro,x Enzo Spisni,‡ and Marco A. Cassatella† Hemolytic uremic syndrome (HUS) caused by intestinal Shiga toxin–producing Escherichia coli infections is a worldwide health problem, as dramatically exemplified by the German outbreak occurred in summer 2011 and by a constant burden of cases in children. Shiga toxins (Stx) play a pivotal role in HUS by triggering endothelial damage in kidney and brain through globo- triaosylceramide (Gb3Cer) receptor targeting. Moreover, Stx interact with human neutrophils, as experimentally demonstrated in vitro and as observed in patients with HUS. A neutrophil-protective role on endothelial damage (sequestration of circulating toxins) and a causative role in toxin delivery from the gut to the kidney (piggyback transport) have been suggested in different Downloaded from studies. However, the receptor that recognizes Stx in human neutrophils, which do not express Gb3Cer, has not been identified. In this study, by competition and functional experiments with appropriate agonists and antagonists (LPS, anti-TLR4 Abs, respec- tively), we have identified TLR4 as the receptor that specifically recognizes Stx1 and Stx2 in human neutrophils. Accordingly, these treatments displaced both toxin variants from neutrophils and, upon challenge with Stx1 or Stx2, neutrophils displayed the same pattern of cytokine expression as in response to LPS (assessed by quantitative RT-PCR, ELISA, or multiplexed Luminex- based immunoassays). Moreover, data were supported by adequate controls excluding any potential interference of contaminating http://www.jimmunol.org/ LPS in Stx-binding and activation of neutrophils. The identification of the Stx-receptor on neutrophils provides additional elements to foster the understanding of the pathophysiology of HUS and could have an important effect on the development of therapeutic strategies. The Journal of Immunology, 2013, 191: 4748–4758. higa toxin (Stx)–producing Escherichia coli (STEC) in- observed in adult patients during the dramatic STEC outbreak, fections are a worldwide health problem because of the which occurred in summer 2011, causing ∼4000 cases of bloody severity of the deriving illnesses, which occur in sporadic diarrhea and more than 800 HUS cases in Europe (4–7). Thus, the S by guest on September 29, 2021 form or as community-wide outbreaks (1–3). In humans, the main risks of STEC infections are 2-fold: the sudden appearance of clinical manifestations of STEC infections are bloody diarrhea and food-borne outbreaks and the burden of a constant incidence rate its life-threatening sequela, hemolytic uremic syndrome (HUS). The of childhood HUS. latter represents the main cause of acute renal failure in early Infectious diseases always develop as a consequence of a balance childhood, and it is characterized by thrombocytopenia and between the virulence factors expressed by a pathogen and the host microangiopathic hemolytic anemia (1–3). Such a triad was also response to those challenges. In this view, Stx as STEC virulence factors, along with neutrophils as effectors of innate immunity, play an important role in the pathogenesis of HUS. STEC produce two *Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Universita` di Bologna, 40126 Bologna, Italy; †Dipartimento di Patologia e Diagnostica, Sezione main AB5 toxin variants—Shiga toxin 1 (Stx1) and Shiga toxin 2 di Patologia Generale, Universita` di Verona, 37134 Verona, Italy; ‡Dipartimento di (Stx2)—consisting of five B chains that are linked noncovalently Scienze Biologiche, Geologiche e Ambientali, Universita` di Bologna, 40126 Bologna, x to a single A chain (8, 9). The B pentamer directs the toxins Italy; and Servizio di Immunoematologia e Trasfusionale, Ospedale Sant’Orsola- Malpighi, 40138 Bologna, Italy toward cells harboring glycosphingolipids of the globoseries 1D.C. and V.A. contributed equally to this work. called globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) (10). Few human cells possess these receptors (11–14) Received for publication January 14, 2013. Accepted for publication August 28, 2013. whose expression characterizes the endothelial lining of the intestine, This work was supported by grants from the University of Bologna and Progetto brain, and kidney, which are indeed the body sites mainly targeted Organizzazione non Lucrativa di Utilita` Sociale Alice–Associazione per la Lotta alla by Stx. In particular, glomerular endothelial injury induced by Stx Sindrome Emolitico Uremica (to M.B.) and Ministero dell’Istruzione, dell’Universita` is considered central to the pathogenesis of HUS (9). The A chain e della Ricerca Grant 2009MFXE7L_001 and Associazione Italiana per la Ricerca sul Cancro Grant AIRC, IG-11782 (both to M.A.C.). N.T. is the recipient of a fellowship is endowed with the enzymatic activity (15–17) and is responsible from Fondazione Italiana per la Ricerca sul Cancro. for an array of damage within intoxicated cells, as well as multiple Address correspondence and reprint requests to Prof. Maurizio Brigotti, Dipartimento cellular and host responses, culminating in the typical prothrombotic di Medicina Specialistica, Diagnostica e Sperimentale, Universita` di Bologna, Via state and renal involvement seen in HUS. Among the toxin actions on San Giacomo 14, 40126 Bologna, Italy. E-mail address: [email protected] endothelial cells involved in HUS pathogenesis, damage to DNA, The online version of this article contains supplemental material. ribotoxic stress, endoplasmic reticulum stress, activation of the apo- Abbreviations used in this article: Gb3Cer, globotriaosylceramide; Gb34Cer, globo- tetraosylceramide; HUS, hemolytic uremic syndrome; MCV, mean channel value of ptotic program, stimulation of the production of proinflammatory fluorescence; MNE, mean normalized expression; PMN, polymorphonuclear leuko- cytokines, and upregulation of adhesion molecules have been de- cyte; QRT-PCR, quantitative RT-PCR; STEC, Shiga toxin–producing Escherichia scribed (17–23). Moreover, activation of complement, formation of coli; Stx, Shiga toxin; Stx1, Shiga toxin 1; Stx2, Shiga toxin 2. platelet-leukocyte complexes, and production of tissue factor–bearing Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 microparticles in blood by Stx have been demonstrated in vitro and www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300122 The Journal of Immunology 4749 in the blood from patients with HUS (24–26). In this context, several Materials and Methods lines of evidence indicate that Stx bind in vitro to platelets through Toxins Gb3Cer lipoforms similar to those on target endothelial cells (27– The Stx1 producer E. coli C600 (H19J) and the Stx2 producer C600 29). A role of platelets as Stx carriers in blood has been proposed (933W) were supplied by Dr. Alison O’Brien (Department of Microbiol- by different authors; however, platelets internalize Stx within 2 h, ogy and Immunology, Uniformed Services University of the Health Sci- becoming activated and aggregated (28). Because platelet aggre- ences, Bethesda, MD). Stx1 and Stx2 were purified by receptor analog gates are cleared by the reticuloendothelial system, direct binding affinity chromatography, the former on globotriose-Fractogel (IsoSep, a b of the toxins to these cells seems to be involved in thrombocy- Lund, Sweden) (45) and the latter on (Gal 1-4Gal -O-spacer)-BSA- Sepharose 4B (Glycorex, Lund, Sweden) (19), followed in both cases by topenia, rather than in the passive transfer of Stx to the kidney and a passage through ActiClean Etox columns (Sterogene Bioseparations,