Research Article: Neutrophils use superoxide to control bacterial infection at a distance

Date Published: July 17, 2018

Publisher: Public Library of Science

Author(s): Quang Tien Phan, Tamara Sipka, Catherine Gonzalez, Jean-Pierre Levraud, Georges Lutfalla, Mai Nguyen-Chi, Andreas J. Baumler.


Understanding the roles of neutrophils and macrophages in fighting bacterial infections is a critical issue in human pathologies. Although phagocytic killing has been extensively studied, little is known about how bacteria are eliminated extracellularly in live vertebrates. We have recently developed an infection model in the zebrafish embryo in which leukocytes cannot reach the injected bacteria. When Escherichia coli bacteria are injected within the notochord, both neutrophils and macrophages are massively recruited during several days, but do not infiltrate the infected tissue presumably because of its tough collagen sheath. Nevertheless, the bacteria are killed during the first 24 hours, and we report here that neutrophils, but not macrophages are involved in the control of the infection. Using genetic and chemical approaches, we show that even in absence of phagocytosis, the bactericidal action relies on NADPH oxidase-dependent production of superoxide in neutrophils. We thus reveal a host effector mechanism mediated by neutrophils that eliminates bacteria that cannot be reached by phagocytes and that is independent of macrophages, NO synthase or myeloperoxidase.

Partial Text

The innate immune system is the first line of defence of the host. It includes large phagocytes (such as macrophages and granulocytes) equipped with a battery of weapons to destroy the invader within minutes or hours. Since the seminal work of Elie Metchnikoff [1], the defence mechanisms relying on leukocytes remain a challenging subject. When microbes penetrate the epithelial barrier, macrophages and neutrophils are rapidly recruited and upon contact, engulf the bacteria into a vacuole called a phagosome that fuses with intracellular granules or lysosomes to form a lytic vacuole in which bacteria may be killed by a wide variety of mechanisms involving chemicals and enzymes [2,3]. Non-oxidative effectors include antimicrobial proteins, while the oxygen-dependent mechanism, also known as the respiratory burst, involves the generation of reactive oxygen species (ROS) [4,5,6]. ROS production inside the phagocytic vacuole involves NADPH oxidase and the major ROS, superoxide (O2-) and hydrogen peroxide (H2O2), can directly or indirectly promote the death of the microbe, according to the nature of the pathogens [7,8]. Nitric oxide (NO), produced by NO synthase, can contribute to microbicidal activity and is essential for the defence against intracellular organisms such as Salmonella enterica and mycobacteria [9,10].

Many studies have used the zebrafish embryo model to address the respective roles of neutrophils and macrophages in eliminating invading bacteria, but in all instances, at least one of these two cellular populations had direct access to the bacteria. In our model neither neutrophils nor macrophages could reach the bacteria. We first observed an active recruitment of both macrophages and neutrophils around the infected notochord that is correlated with the elimination of the bacteria in the notochord within 24 hours. Specifically depleting individual myeloid populations, we have investigated their contribution in the clearance of E. coli at a distance and describe molecular pathways involved in bacterial elimination by neutrophils.