Strategies of professional phagocytes in vivo: unlike macrophages, neutrophils engulf only surface- associated microbes Emma Colucci-Guyon 1,2, *, Jean-Yves Tinevez 3 , Stephen A. Renshaw 4 and Philippe Herbomel 1,2, * 1 Institut Pasteur, Unite ´ Macrophages et De ´ veloppement de l’Immunite ´, De ´ partement de Biologie du De ´ veloppement, F-75015 Paris, France 2 CNRS, URA2578, F-75015 Paris, France 3 Institut Pasteur, Imagopole, Plate-forme d’Imagerie Dynamique, F-75015 Paris, France 4 MRC Centre for Developmental and Biomedical Genetics and Department of Infection and Immunity, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom *Authors for correspondence (emma.colucci@pasteur.fr; philippe.herbomel@pasteur.fr) Accepted 17 2011 Journal of Cell Science 000, 1–7 ß 2011. Published by The Company of Biologists Ltd doi: 10.1242/jcs.082792 Summary The early control of potentially invading microbes by our immune system primarily depends on its main professional phagocytes – macrophages and neutrophils. Although the different functions of these two cell types have been extensively studied, little is known about their respective contributions to the initial control of invading microorganisms before the onset of adaptive immune responses. The naturally translucent zebrafish larva has recently emerged as a powerful model vertebrate in which to visualise the dynamic interactions between leukocytes and microbes in vivo. Using high-resolution live imaging, we found that whereas macrophages efficiently engulf bacteria from blood or fluid-filled body cavities, neutrophils barely do so. By contrast, neutrophils very efficiently sweep up surface-associated, but not fluid-borne, bacteria. Thus the physical presentation of unopsonised microbes is a crucial determinant of neutrophil phagocytic ability. Neutrophils engulf microbes only as they move over them, in a ‘vacuum-cleaner’ type of behaviour. This context-dependent nature of phagocytosis by neutrophils should be of particular relevance to human infectious diseases, especially for the early phase of encounter with microbes new to the host. Key words: Neutrophils, Macrophages, Professional phagocytes, Live imaging, Host–microbe interaction, Innate immunity, Zebrafish Introduction When potentially infectious microbes penetrate epithelial barriers and invade the host’s tissues, they first encounter innate antimicrobial mechanisms. These mechanisms mainly rely on the activities of the two dedicated so-called ‘professional phagocytes’, macrophages and neutrophils. The differential features of these two cell types and the molecular mechanisms underlying microbe phagocytosis and killing have been exten- sively studied. However, these studies have been mostly conducted in cell culture, using macrophage or neutrophil cell lines, human blood or mouse bone-marrow-derived phagocytes (Kantari et al., 2008; Nathan, 2006). Therefore, little is known about the relative contribution of macrophages and neutrophils in the initial phase of encounter with a potentially invasive microbe in vivo. Originally introduced as a new model vertebrate organism in developmental biology (Streisinger et al., 1981), the zebrafish (Danio rerio) has emerged in the last decade as a powerful non- mammalian model to study the development and function of the immune system (Lieschke and Trede, 2009). The small size and natural translucency of swimming zebrafish larvae make it possible to follow leukocyte deployment and behaviour in vivo throughout the organism, at high resolution. As the immune system develops gradually, its adaptive arm becomes operational – in terms of ability to mount an antibody response – only when the larva develops into a juvenile fish (Lam et al., 2004). Thus, the larva has a purely innate immune system, consisting of macro- phages and neutrophils (Bennett et al., 2001; Herbomel et al., 1999; Lieschke et al., 2001). It is therefore especially suitable for an in vivo investigation of innate immune responses to invading microorganisms in real time (Davis et al., 2002; Tobin et al., 2010). In a previous study of zebrafish neutrophil development, we began to study neutrophil behaviour towards microbes. Upon injecting non-pathogenic Escherichia coli into the bloodstream or otic cavity of zebrafish larvae, we found that both neutrophils and macrophages were able to sense and migrate towards the injected microbes, but surprisingly, neutrophils ineffectively engulfed these bacteria, whereas macrophages engulfed them in large numbers (Le Guyader et al., 2008). Here, we analysed microbe– neutrophil interactions after microbe inoculation of zebrafish by live-imaging confocal time-lapse microscopy. We found that zebrafish neutrophils very efficiently engulf bacteria on tissue surfaces but are virtually unable to phagocytose microbes in fluid environments. In stark contrast, macrophages are able to engulf microbes regardless of how they are presented. Results and Discussion Unlike macrophages, neutrophils ineffectively engulf microbes in fluid-filled body cavities To image neutrophil–microbe interactions, we performed con- focal time-lapse microscopy, using transgenic mpx:GFP zebrafish Journal of Cell Science JCS082792.3d 18/7/11 17:36:50 The Charlesworth Group, Wakefield +44(0)1924 369598 - Rev 7.51n/W (Jan 20 2003) Short Report 1