Identification and characterization of a unique role for EDB fibronectin in phagocytosis

J Mol Med (2016) 94:567–581 DOI 10.1007/s00109-015-1373-0 ORIGINAL ARTICLE Identification and characterization of a unique role for EDB fibronectin in phagocytosis Sabrina Kraft 1,2 & Verena Klemis 1,2 & Carla Sens 1,2 & Thorsten Lenhard 3 & Christian Jacobi 3,4 & Yvonne Samstag 2 & Guido Wabnitz 2 & Michael Kirschfink 2 & Reinhard Wallich 2 & G. Maria Hänsch 2 & Inaam A. Nakchbandi 1,2 Received: 28 September 2015 / Revised: 12 November 2015 / Accepted: 19 November 2015 / Published online: 5 December 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Plasma fibronectin is a circulating protein that facilitates phagocytosis by connecting bacteria to immune cells. A fibronectin isoform, which includes a sequence of 90 AA called extra-domain B (EDB), is synthesized de novo at the messenger RNA (mRNA) level in immune cells, but the reason for its expression remains elusive. We detected an 80-fold increase in EDB-containing fibronectin in the cerebrospinal fluid of patients with bacterial meningitis that was most pronounced in staphylococcal infections. A role for this isoform in phagocytosis was further suggested by enhanced EDB fibronectin release after internalization of Staphylococcus aureus in vitro. Using transgenic mouse models, we established that immune cell production of fibronectin contributes to phagocytosis, more so than circulating plasma fibronectin, and that accentuated release of EDB-containing fibronectin by immune cells improved phagocytosis. In line with this, administration of EDB fibronectin enhanced in vitro phagocytosis to a larger extent than plasma fibronectin. This enhancement was medi- ated by αvβ3 integrin as shown using inhibitors or cells from β3 integrin knockout mice. Thus, we identified both a novel function for EDB fibronectin in augmenting phagocytosis over circulating plasma fibronectin, as well as the mediating receptor. Our data also establish for the first time, a direct role for β3 integrin in bacterial phagocytosis in mammals. Electronic supplementary material The online version of this article (doi:10.1007/s00109-015-1373-0) contains supplementary material, which is available to authorized users. Introduction * Inaam A. Nakchbandi 1 Max-Planck Institute of Biochemistry, 82152 Martinsried, Germany 2 Institute of Immunology, University of Heidelberg, 69120 Heidelberg, Germany 3 Department of Neurology, University of Heidelberg, 69120 Heidelberg, Germany 4 Department of Neurology, Krankenhaus Nordwest, 60488 Frankfurt, Germany Key messages • Fibronectin containing an extra domain called EDB is released in bacterial meningitis. • EDB-containing fibronectin enhances phagocytosis more than plasma fibronectin. • The enhancement is mediated by activation of αvβ3 integrin in the presence of EDB. Keywords Innate immunity . Bacterial infection . Phagocytosis . Fibronectin . EDB . EIIIB . αvβ3 integrin . Circulation . Plasma fibronectin Fibronectin is an extracellular matrix protein that is produced by almost all mammalian cells [1]. It affects proliferation, migration, differentiation, and survival [2, 3]. These different functions are made possible by both the presence of several isoforms and the binding to a variety of integrins. Two of the isoforms of fibronectin are defined by the presence of extra domains called extra domain A (EDA) and extra domain B (EDB). Even though most of the studies were performed with the circulating isoform of fibronectin, which lacks both EDA and EDB and is called plasma fibronectin (pFN), studies using isoform-specific knockouts lacking either EDA, EDB, or both domains found that these two isoforms contribute to 568 vasculogenesis in embryos and angiogenesis in cancer [4–7]. In addition, EDA-containing fibronectin plays a role in a variety of pathologic entities such as liver fibrosis and diabetes mellitus, but no further functions of EDB-containing fibronectin have been characterized [8, 9]. Furthermore, while a specific binding site to integrins was characterized for EDA, no receptor for EDB has been identified in vivo yet [1, 10]. Studies on the circulating isoform of fibronectin (plasma fibronectin) have shown that fibronectin facilitates adherence of bacteria to other cells and thus acts as an adhesion molecule on mammalian cells [11]. Strains of Staphylococcus aureus for example express several molecules such as fibronectinbinding proteins (Fnbp) that enable bacteria to attach to and invade tissues [12, 13]. The most widely known function of fibronectin in phagocytosis is as a bridge between the bacteria and integrin α5β1, the classical fibronectin receptor [14, 15]. Even though fibronectin was originally shown to act as an opsonin by marking the bacteria and enhancing phagocytosis [16, 17], experimental data also show that fibronectin enhances phagocytosis irrespective of whether it binds to bacteria or not [17]. Neither EDA- nor EDB-containing fibronectin was studied in the context of phagocytosis. Phagocytosis is evolutionally critical and beneficial. Therefore, much overlap in the stimulators and enhancers of phagocytosis exists, and several integrins are involved in phagocytosis. The only β2 subunit-containing integrin involved in phagocytosis is αMβ2 integrin (also called complement receptor 3 or CD11b/CD18) which affects complement-activated phagocytosis of several pathogens including lipopolysaccharide-expressing bacteria [18–20]. Therefore, upregulation of β2 enhances phagocytosis [18]. Another mechanism of phagocytosis involves the Fcγ receptor, which mediates phagocytosis of IgG-opsonized (i.e., IgG - coated) bacteria [21]. No evidence exists however that fibronectin directly binds to either β2 integrin or the Fcγ receptor. A report suggested that β3 integrin is able to induce phagocytosis in insect cells [22]. Apoptotic cells marked with the soluble glycoprotein called milk fat globule-EGF factor 8 (MFG-E8) were phagocytosed by macrophages through αvβ3 [23]. Furthermore, an interaction between β1 and β3, both of which bind to fibronectin, has been documented, whereby αvβ3 is required to allow α5β1-mediated phagocytosis [24]. Thus, while fibronectin supports phagocytosis and can bind to integrins involved in phagocytosis, it is not known whether its isoforms containing EDA and EDB play any role in phagocytosis and if they do, which receptors are involved. In this paper, we show that EDB-containing fibronectin is elevated in the cerebrospinal fluid of patients with bacterial meningitis. Indeed, phagocytosis is associated with increased production and release of EDB fibronectin, whereby this isoform by itself is able to enhance phagocytosis by up to 40 % compared to untreated cells. This effect is mediated through β3 integrin in cooperation with β2-mediated phagocytosis. J Mol Med (2016) 94:567–581 Furthermore, deletion of β1 does not diminish phagocytosis as suggested by the literature [24]. Instead, it increases β2 and β3 (...truncated)