Real Time Observation of Neutrophil White Blood Cell Recruitment to Bacterial Infection In Vivo

A Fluorescence Microscopy Study Using Zebrafish Larvae as the Model Organism

July 27, 2017

The zebrafish (Danio rerio) is an emerging vertebrate model organism to study infection. The transparent larva comprises a fully functional innate immune system and enables live imaging of fluorescent immune cells in transgenic animals. Zebrafish infection models have been developed for both the human bacterial pathogen Shigella flexneri and the natural fish bacterial pathogen Mycobacterium marinum. Importantly, whilst S. flexneri causes acute infection and is typically used as an inflammatory paradigm, M. marinum causes a chronic disease similar to tuberculosis in humans. Here, we use real time fluorescence microscopy to image transgenic zebrafish larvae with neutrophils (granulocyte white blood cells) expressing the green fluorescent protein eGFP. This method allows the comparison of neutrophil recruitment for S. flexneri versus M. marinum. Using a low dose infection in the hindbrain ventricle (HBV) of 3 day old zebrafish larvae, we show robust and significant recruitment of neutrophils to S. flexneri. In contrast, we did not see significant neutrophil recruitment to a similar dose of M. marinum. These data agree with previous studies showing that S. flexneri induces tissue inflammation and chemotactic cues for neutrophil recruitment, whereas M. marinum largely avoids neutrophil recruitment. This comparative study highlights the value of zebrafish larvae to study host-pathogen interactions in vivo.


S. flexneri are human-adapted Escherichia coli bacteria that have gained the ability to invade the colonic mucosa (large intestine membrane lining), causing inflammation and bacillary dysentery disease. The intracellular lifestyle of this pathogen has been well-studied in vitro and Shigella has recently gained recognition as an inflammatory paradigm to study host innate immunity. However, in the absence of a natural mouse model for shigellosis, in vivo studies of Shigella pathogenesis (mechanism causing disease) have been limited [1].

M. marinum is a natural fish pathogen that results in granulomatous necrotic lesions (tissue damage from inflammation) in host tissues. These lesions are histologically similar to those generated by Mycobacterium tuberculosis, the causative agent of tuberculosis (TB) in humans. Again, the lack of a mouse model that fully recapitulates this disease, together with the risks to human health associated with studying M. tuberculosis, make it difficult to efficiently study TB in vivo. To this end, the M. marinum-zebrafish infection model has been an extremely valuable surrogate to understand the core mechanisms underlying mycobacterial infection [2, 3].

Non-mammalian models have been important in the understanding of diverse diseases (infection, cancer, neurodegeneration) and for the development of therapeutics to improve human health. In particular, the zebrafish is a vertebrate model which displays significant genetic, developmental, and physiological similarities with humans [4]. Zebrafish are highly fecund, externally fertilized, and have a short life cycle. From a practical point of view, these features mean that large quantities of embryos are readily and consistently available for study. Moreover, the larval stages of zebrafish are naturally translucent, providing a unique opportunity to visualize and dissect in vivo the behavior of fluorescent cells in transgenic larvae. In particular, infection models can benefit from zebrafish lines with fluorescently-tagged immune cells that enable the in vivo imaging of leukocytes (white blood cells: macrophages, neutrophils) and their role in inflammatory and infectious disease.

Here, we use S. flexneri and M. marinum zebrafish infection models to study neutrophil recruitment to infection in vivo. Live imaging of fluorescently-tagged bacteria during infection of transgenic larvae revealed highly pathogen-specific neutrophil recruitment. Strikingly, whilst we observed rapid and robust neutrophil recruitment for S. flexneri infection, similar doses of M. marinum did not stimulate significant neutrophil migration to the infected site. These data highlight the value of live imaging to enable the understanding of host response to bacterial infection.


Zebrafish care and maintenance

All experiments were performed on zebrafish larvae less than 5 days post fertilization (dpf), prior to the free-feeding stage. Therefore, none of the experiments reported here fall under the animal experimentation regulation of the UK and the European Union. Adult breeding fish were maintained according to European Union guidelines for handling of laboratory animals and the Imperial College London Animals (Scientific Procedures) Act of 1986. All experiments and procedures were approved by the Home Office (Project license: PPL 70/7446). Eggs were obtained by natural spawning, cleaned and maintained in Petri dishes containing 0.5X E2 embryo medium supplemented with 0.3 mg/ml methylene blue as previously described. For microscopy, the embryo medium was supplemented with 0.003% 1- phenyl-2-thiourea at 24 hours post fertilization (hpf) to prevent melanin synthesis. Figure 1 shows the zebrafish holding facility at Imperial College London.

Figure 1: S. Mostowy (left) and A. Willis (right) in the Imperial College facility where adult zebrafish are maintained. Larvae are spawned in this facility in similar tanks containing breeding boats.

Bacterial preparation

Bacterial strains used in this study were:

1)  Wild-type (wt) S. flexneri (M90T strain) expressing mCherry (a red fluorescent protein);

2)  Wt M. marinum (M strain) expressing mCherry [5].

For microinjection of zebrafish larvae, bacteria were harvested by centrifugation, washed, and resuspended at the desired concentration (~500 CFU/nl) in phosphate buffered saline (PBS).

Bacterial infection of zebrafish larvae

For injection procedures, larvae were anesthetized with 200 mg/ml tricaine. At 3 dpf, larvae positioned on agarose injection plates were microinjected into the hindbrain ventricle (HBV) with 1 nl (~500 CFU) of either S. flexneri or M. marinum bacterial suspension or a clean PBS solution to act as a control. Injections were performed using an IM 300 microinjector (Narishige) and a M80 stereo microscope (Leica Microsystems) as described previously [6]. Inocula were confirmed a posteriori by injection into a drop of PBS and bacterial plating. Figure 2a-b show the lab facility for bacteria and larva preparation and microinjection.

Figure 2a: CMBI, ICL laboratory showing equipment for preparation of zebrafish larvae for microinjection of bacteria.

Figure 2b: A. Willis explains preparation of the S. flexneri bacteria and zebrafish larvae for microinjection.

Live imaging of zebrafish larvae

The zebrafish HBV is highly amenable to imaging and enables injected pathogenic bacteria and leukocytes to be followed over time. To perform in vivo imaging of neutrophil recruitment to the site of injection, anesthetized zebrafish larvae were oriented and immobilized in 1% low melting point agarose and covered with 0.5X E2 medium containing tricaine. Brightfield and fluorescence widefield imaging was performed using a M205 FA fluorescence stereo microscope (Leica Microsystems). Multiple field z-stacks (multi-focus imaging) were acquired at 15 min intervals. The automated stage permitted several animals to be followed simultaneously (3 PBS injected control larvae, 4 M. marinum infected larvae, and 5 S. flexneri infected larvae). Figures 3a-c show 2 of the co-authors imaging the larvae.

Figure 3a: V. Torraca confirming infection in live, anesthetized zebrafish larvae after microinjection of mCherry-fluorescent M. marinum in the HBV. Imaging is performed using a M205 FA stereo microscope.

Figure 3b: A. Willis imaging a live, anesthetized zebrafish larvae after microinjection of mCherry-fluorescent S. flexneri in the HBV. Imaging is done with a M205 FA stereo microscope.

Figure 3c. Live imaging of infected, anesthetized zebrafish larva using a M205 FA stereo microscope in both brightfield and fluorescence modes.

Neutrophil quantification

Quantifications were performed using the previously published Tg(mpx:eGFP) transgenic zebrafish line with neutrophils expressing the enhanced green fluorescent protein eGFP [7]. Images taken by fluorescence stereo microscopy were processed into movies and analyzed using ImageJ software [8]. Recruited eGFP-expressing neutrophils were quantified in the hindbrain at 1 and 6 hours post infection (hpi). Statistics were calculated using the Student’s t test via Graph pad prism.

Results and Discussion

Neutrophils are highly motile innate immune cells and often respond rapidly to invading pathogens. Neutrophil recruitment, phagocytosis, and pathogen destruction are, therefore, very important for the initial containment of many infections. We show here that zebrafish larval neutrophils display a strong recruitment to S. flexneri. In contrast, M. marinum largely avoids detection by neutrophils (Fig. 4a-b). Quantifications reveal that S. flexneri injected fish show a 2.7-fold increase in neutrophil number in the hindbrain ventricle relative to the control fish (PBS only) already by 1 hpi and 3.0-fold increase at 6 hpi (Fig. 5). In contrast, neutrophil localization upon M. marinum infection of the hindbrain ventricle is not significantly different from control larvae at either of the time points (1 or 6 hpi) tested.

These observations are in line with previous studies, where it has been shown that S. flexneri infection of the HBV can trigger the activation of various inflammatory mediators and induce chemotactic cues that actively recruit neutrophils [9,10]. On the other hand, studies using M. marinum infection of the HBV have demonstrated that chemotactic cues are negligible [11]. Data has shown that neutrophils interact with mycobacteria only at a later, more advanced and inflammatory stage of infection, i.e. upon the formation of granulomas (inflammation from aggregated macrophages) in tissue [11]. In this case, neutrophils are attracted by the debris derived from dying infected macrophages unable to control M. marinum infection.

The different inflammatory and chemoattractant properties of S. flexneri and M. marinum demonstrate the ‘fine-tuning’ of host-pathogen interactions in vivo. Shigella is an enterobacterium and relies on inflammatory destruction of the intestinal epithelium lining to invade, survive, and replicate within its host. Inflammation is mediated by pathogen-secreted virulence factors. Invasion of the intestinal epithelium is followed by transcriptional reprogramming of the host to produce chemotactic factors, such as the potent neutrophil attractant Interleukin-8 (IL-8) [12]. While neutrophils are important for bacterial killing, they can also disrupt the intestinal epithelium and facilitate increased invasion of cells unable to control Shigella.

In contrast, M. marinum (similar to M. tuberculosis) preferentially establishes a chronic infection. The bacteria mask their pathogen associated molecular patterns (PAMPs) beneath a layer of surface lipids to evade early stages of innate immune recognition [13]. Mycobacteria are well-known to establish a niche within macrophages using sophisticated mechanisms (e.g. induction of Ccl2 and Cxcl11) to specifically chemoattract macrophages [13,14]. Within the macrophage niche, M. marinum can replicate and subsequently disseminate within the host. Disseminated bacteria form granulomatous lesions, hallmarks of infection by mycobacteria, which serve as a bacteria-protective barrier and reduces immune cell access [15].

Figure 4a: Neutrophil recruitment by S. flexneri infection: 3 day old Tg(mpx:eGFP) larvae with green neutrophils were injected in the HBV with ~500 CFU mCherry-S. flexneri. Anesthetized larvae were immobilized in agarose and imaged every 15 min for 9 h using a M205 FA fluorescence stereo microscope. Frames were processed using ImageJ to produce movies. The unit “mpi” refers to minutes post injection.
Figure 4b: Lack of neutrophil recruitment by M. marinum infection: 3 day old Tg(mpx:eGFP) larvae with green neutrophils were injected in the HBV with ~500 CFU mCherry-M. marinum. Anesthetized larvae were immobilized in agarose and imaged every 15 min for 9 h using a M205 FA fluorescence stereo microscope. Frames were processed using ImageJ to produce movies. The unit “mpi” refers to minutes post injection.


The zebrafish is an emerging model organism for the study of Shigella and mycobacterial infection, and is contributing significantly to our understanding of how bacteria cause disease in vivo. Using infection of the zebrafish larva, interactions between bacteria and host cells can be imaged at high resolution. Here, we exploit the optical accessibility of transparent zebrafish larvae to investigate the interactions of S. flexneri and M. marinum with neutrophils (granulocyte white blood cells). These observations can help us better understand host defenses against important bacterial pathogens and illuminate new ways to control bacterial infection in humans.




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