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Illuminating Phagocyte Biology: The View from Zebrafish
Developmental Cell
Commentary Illuminating Phagocyte Biology: The View from Zebrafish Cong Huang1,2 and Philipp Niethammer1,* 1Cell
Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA Program in Biochemistry, Cell and Molecular Biology Allied Program, Weill Cornell Medical College, New York, NY 10065, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2016.07.003 2Graduate
Many phagocyte behaviors, including vascular rolling and adhesion, migration, and oxidative bursting, are better measured in seconds or minutes than hours or days. Zebrafish is ideally suited for imaging such rapid biology within the intact animal. We discuss how this model has revealed unique insights into various aspects of phagocyte physiology. Zebrafish are freshwater fish with an immune system that includes the innate and adaptive immune cell types found in higher vertebrates (Trede et al., 2004). Their larvae are thin and transparent and are excellently suited for real-time in vivo imaging of phagocyte development and behavior. Zebrafish were introduced in the 1960s as a model for nervous system development, in part because their larvae can be raised in large numbers in petri dishes at near-room-temperature (28.5 C). Their advantageous live-imaging properties have since led to their adoption by many other fields, including cell biology, cancer research, and immunology (Figure 1). But the promise of zebrafish as a model for human disease has also attracted skepticism: How can fish teach us anything new about mammalian physiology? And, conversely, how can we tell whether ‘‘new’’ findings in fish reflect general concepts rather than fish-specific biology? Below, we illustrate with a few examples how the zebrafish system has advanced phagocyte research in the past, and we propose how it may further it in the future. One area in which zebrafish provided clear insights involves the fate of neutrophils found at sites of tissue damage or infection. For a long time, it was thought that neutrophils follow one common path at infection sites: they migrate to the site of infection, ingest pathogens, and then die, their remains cleared by the macrophages that follow their footsteps. But in 1997, Savill and colleagues published experiments (Hughes et al., 1997) in which they tracked the cell fate of 111indium-labeled neutrophils in a rat inflammation model. Clearance from in-
flamed glomeruli in rats could not be explained by neutrophil death alone. Their data raised the intriguing possibility that neutrophil recruitment to sites of tissue damage or infection is not a one-way street, but rather is reversible. But how the neutrophils left the inflamed glomeruli was not obvious from the radiolabeling experiments. About a decade later, the Huttenlocher group used live microscopy to monitor how neutrophils, marked with a green fluorescent protein, trafficked between the vasculature and tiny wound sites in the zebrafish fin epithelium (Mathias et al., 2006). They noted that some leukocytes, after having arrived at the wound, returned to the vessels. This observation provided direct, in vivo proof for the existence of an anti-inflammatory mechanism that was termed ‘‘reverse leukocyte migration,’’ and later experiments revealed some of its molecular details (Elks et al., 2011; Tauzin et al., 2014). Furthermore, systematic compound screening in zebrafish identified pharmacological modulators of this process (Robertson et al., 2014). The discovery of regulatory H2O2 gradients was another proof of concept in the zebrafish system. For a long time, reactive oxygen species, such as H2O2, were viewed as toxic by-products of aerobic respiration. The possibility that H2O2 acts as biochemical signal for leukocyte chemotaxis was first raised by experiments with mouse peritoneal neutrophils (Klyubin et al., 1996). But direct in vivo evidence for the physiological relevance of this phenomenon was missing. In 2009, a zebrafish study that combined quantitative ratio-imaging of a transgenically expressed fluorescent H2O2 biosensor with
leukocyte recruitment assays provided this evidence (Niethammer et al., 2009). Quantitative biosensor imaging in living tissues is a nontrivial task. Inhomogeneous illumination, spatial variations of tissue thickness, autofluorescence, and out-of-focus blur can confound the biological signal of interest. Because larval zebrafish tail fins are flat (50 mm thick), transparent, and simple-structured (i.e., two double-layered epithelial layers folded onto each other), they exhibit low and regularly distributed background fluorescence, which interferes only minimally with fluorescent biosensor signals. These favorable imaging properties helped the discovery of physiological H2O2 gradients, which was followed by research into the mechanisms by which phagocytes detect H2O2 (Yoo et al., 2011). Proof-of-concept studies aside, the zebrafish system has also served as a useful disease model, e.g., for illuminating macrophage-pathogen interactions during mycobacterial infections (Meijer, 2016). Granulomas, infection foci consisting of mycobacteria surrounded by macrophages (i.e., a special type of innate immune cells), are key pathological structures of tuberculosis. Some important features of human granulomas, such as their central necrotic core and flanking hypoxic regions, have been difficult to reconstitute in early mouse models of tuberculosis (Rhoades et al., 1997) but are readily observable in zebrafish models of mycobacterial infection (using M. marinum). Given that macrophages are sufficient for granuloma formation in zebrafish (Davis et al., 2002), it is possible to study granuloma initiation mechanisms
Developmental Cell 38, July 25, 2016 ª 2016 Elsevier Inc. 133
Developmental Cell
Commentary processes, whether involving phagocyte responses to infections, wounds, cancer cells, or other types of homeostatic perturbations. It is a safe bet that the implementation of new imaging assays and genome-editing technologies will produce a bloom of disease models, in which the contribution of phagocytes can be observed in physiological detail. Now is a better time than ever for phagocyte research to discover its inner fish. ACKNOWLEDGMENTS
Figure 1. Zebrafish Allows Convenient Imaging of Phagocyte Behavior in Complex Pathophysiological Situations Depicted is a still image from a confocal time-lapse movie showing neutrophils (red) migrating toward a tail fin wound (right edge of the image), as well as interacting with metastasized melanoma cells (green). The purpose of this particular experiment was to test whether tumor-neutrophil interactions are altered by tissue-damage-induced inflammation. White box, individual neutrophil attracted by the wound. Scale bar, 50 mm.
and dynamics by intravitral imaging in transparent larvae, which initially only contain innate—not adaptive—immune cells. More than 10 years of experimentation in zebrafish models of tuberculosis have contributed to a conceptual shift away from the idea that granulomas are mere host defense structures that wall off mycobacteria and toward the notion that they also constitute a bacterial strategy to promote infection (Davis and Ramakrishnan, 2009). A number of potential drug targets have emerged from research using the zebrafish tuberculosis model (Meijer, 2016). The above examples show that the zebrafish model, through direct in vivo imaging, is well suited for following up on preliminary observations in mammalian systems. They also illustrate that at times it is smart to trade in the relative evolutionary proximity to humans (e.g., mice or rats) for the unique imaging capabilities of fish and the ability to study many animals in a short time frame. Given their crucial importance for defense against pathogens, it seems probable that many, if not most, phagocyte functions and regulatory principles are conserved. Hence, a thorough understanding of phagocyte regulation in fish may also produce unexpected perspectives on apparently well-understood aspects of mammalian physiology and cell biology. One example of the latter involves the mechanism by which neutrophils detect epithelial breaches. In zebrafish larvae, which live in a low-osmolarity solu134 Developmental Cell 38, July 25, 2016
tion (i.e., fresh water), neutrophils rapidly detect epithelial breaches through the drop of interstitial osmolarity after injury. The osmolarity decrease causes cell swelling, which leads to the production of chemotactic lipid mediators of the eicosanoid class (Enyedi et al., 2013). Eicosanoid synthesis is mechanically triggered by nuclear swelling via nuclear envelope stretch in response to cell swelling (Enyedi et al., 2016). At first glance, this finding appears to be a specific adaption of an epithelial immune defense mechanism to a freshwater environment, which may not be relevant to land-dwelling animals not immersed in hypotonic liquid. Surprisingly, a closer look at human physiology shows that the upper digestive tract of humans is, in fact, a fresh water-like environment. The linings of mouth and esophagus are exposed to saliva, and for little-understood reasons, our bodies invest much energy to maintain saliva roughly as hypotonic as fresh water. Furthermore, cell and nuclear swelling are general hallmarks of any severe tissue damage. Thus, it seems possible that a similar mechanism for detecting epithelial breaches exists in mammals, a hypothesis that should be examined in future studies. Another promise of the zebrafish system is to inspire novel working hypotheses that may be hard to infer from studying mammalian models alone. We believe zebrafish researchers will continue to capitalize on its unique technical strengths for investigating the initiation and rapid dynamics of disease
We apologize to the colleagues whose excellent work we could not mention due the space limitations. This work was supported by NIH grant GM099970 and an American Asthma Foundation Scholar grant to P.N. REFERENCES Davis, J.M., and Ramakrishnan, L. (2009). Cell 136, 37–49. Davis, J.M., Clay, H., Lewis, J.L., Ghori, N., Herbomel, P., and Ramakrishnan, L. (2002). Immunity 17, 693–702. Elks, P.M., van Eeden, F.J., Dixon, G., Wang, X., Reyes-Aldasoro, C.C., Ingham, P.W., Whyte, M.K., Walmsley, S.R., and Renshaw, S.A. (2011). Blood 118, 712–722. Enyedi, B., Kala, S., Nikolich-Zugich, T., and Niethammer, P. (2013). Nat. Cell Biol. 15, 1123–1130. Enyedi, B., Jelcic, M., and Niethammer, P. (2016). Cell 165, 1160–1170. Hughes, J., Johnson, R.J., Mooney, A., Hugo, C., Gordon, K., and Savill, J. (1997). Am. J. Pathol. 150, 223–234. Klyubin, I.V., Kirpichnikova, K.M., and Gamaley, I.A. (1996). Eur. J. Cell Biol. 70, 347–351. Mathias, J.R., Perrin, B.J., Liu, T.X., Kanki, J., Look, A.T., and Huttenlocher, A. (2006). J. Leukoc. Biol. 80, 1281–1288. Meijer, A.H. (2016). Semin. Immunopathol. 38, 261–273. Niethammer, P., Grabher, C., Look, A.T., and Mitchison, T.J. (2009). Nature 459, 996–999. Rhoades, E.R., Frank, A.A., and Orme, I.M. (1997). Tuber. Lung Dis. 78, 57–66. Robertson, A.L., Holmes, G.R., Bojarczuk, A.N., Burgon, J., Loynes, C.A., Chimen, M., Sawtell, A.K., Hamza, B., Willson, J., Walmsley, S.R., et al. (2014). Sci. Transl. Med. 6, 225ra29. Tauzin, S., Starnes, T.W., Becker, F.B., Lam, P.Y., and Huttenlocher, A. (2014). J. Cell Biol. 207, 589–598. Trede, N.S., Langenau, D.M., Traver, D., Look, A.T., and Zon, L.I. (2004). Immunity 20, 367–379. Yoo, S.K., Starnes, T.W., Deng, Q., and Huttenlocher, A. (2011). Nature 480, 109–112.
Commentary Illuminating Phagocyte Biology: The View from Zebrafish Cong Huang1,2 and Philipp Niethammer1,* 1Cell
Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA Program in Biochemistry, Cell and Molecular Biology Allied Program, Weill Cornell Medical College, New York, NY 10065, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2016.07.003 2Graduate
Many phagocyte behaviors, including vascular rolling and adhesion, migration, and oxidative bursting, are better measured in seconds or minutes than hours or days. Zebrafish is ideally suited for imaging such rapid biology within the intact animal. We discuss how this model has revealed unique insights into various aspects of phagocyte physiology. Zebrafish are freshwater fish with an immune system that includes the innate and adaptive immune cell types found in higher vertebrates (Trede et al., 2004). Their larvae are thin and transparent and are excellently suited for real-time in vivo imaging of phagocyte development and behavior. Zebrafish were introduced in the 1960s as a model for nervous system development, in part because their larvae can be raised in large numbers in petri dishes at near-room-temperature (28.5 C). Their advantageous live-imaging properties have since led to their adoption by many other fields, including cell biology, cancer research, and immunology (Figure 1). But the promise of zebrafish as a model for human disease has also attracted skepticism: How can fish teach us anything new about mammalian physiology? And, conversely, how can we tell whether ‘‘new’’ findings in fish reflect general concepts rather than fish-specific biology? Below, we illustrate with a few examples how the zebrafish system has advanced phagocyte research in the past, and we propose how it may further it in the future. One area in which zebrafish provided clear insights involves the fate of neutrophils found at sites of tissue damage or infection. For a long time, it was thought that neutrophils follow one common path at infection sites: they migrate to the site of infection, ingest pathogens, and then die, their remains cleared by the macrophages that follow their footsteps. But in 1997, Savill and colleagues published experiments (Hughes et al., 1997) in which they tracked the cell fate of 111indium-labeled neutrophils in a rat inflammation model. Clearance from in-
flamed glomeruli in rats could not be explained by neutrophil death alone. Their data raised the intriguing possibility that neutrophil recruitment to sites of tissue damage or infection is not a one-way street, but rather is reversible. But how the neutrophils left the inflamed glomeruli was not obvious from the radiolabeling experiments. About a decade later, the Huttenlocher group used live microscopy to monitor how neutrophils, marked with a green fluorescent protein, trafficked between the vasculature and tiny wound sites in the zebrafish fin epithelium (Mathias et al., 2006). They noted that some leukocytes, after having arrived at the wound, returned to the vessels. This observation provided direct, in vivo proof for the existence of an anti-inflammatory mechanism that was termed ‘‘reverse leukocyte migration,’’ and later experiments revealed some of its molecular details (Elks et al., 2011; Tauzin et al., 2014). Furthermore, systematic compound screening in zebrafish identified pharmacological modulators of this process (Robertson et al., 2014). The discovery of regulatory H2O2 gradients was another proof of concept in the zebrafish system. For a long time, reactive oxygen species, such as H2O2, were viewed as toxic by-products of aerobic respiration. The possibility that H2O2 acts as biochemical signal for leukocyte chemotaxis was first raised by experiments with mouse peritoneal neutrophils (Klyubin et al., 1996). But direct in vivo evidence for the physiological relevance of this phenomenon was missing. In 2009, a zebrafish study that combined quantitative ratio-imaging of a transgenically expressed fluorescent H2O2 biosensor with
leukocyte recruitment assays provided this evidence (Niethammer et al., 2009). Quantitative biosensor imaging in living tissues is a nontrivial task. Inhomogeneous illumination, spatial variations of tissue thickness, autofluorescence, and out-of-focus blur can confound the biological signal of interest. Because larval zebrafish tail fins are flat (50 mm thick), transparent, and simple-structured (i.e., two double-layered epithelial layers folded onto each other), they exhibit low and regularly distributed background fluorescence, which interferes only minimally with fluorescent biosensor signals. These favorable imaging properties helped the discovery of physiological H2O2 gradients, which was followed by research into the mechanisms by which phagocytes detect H2O2 (Yoo et al., 2011). Proof-of-concept studies aside, the zebrafish system has also served as a useful disease model, e.g., for illuminating macrophage-pathogen interactions during mycobacterial infections (Meijer, 2016). Granulomas, infection foci consisting of mycobacteria surrounded by macrophages (i.e., a special type of innate immune cells), are key pathological structures of tuberculosis. Some important features of human granulomas, such as their central necrotic core and flanking hypoxic regions, have been difficult to reconstitute in early mouse models of tuberculosis (Rhoades et al., 1997) but are readily observable in zebrafish models of mycobacterial infection (using M. marinum). Given that macrophages are sufficient for granuloma formation in zebrafish (Davis et al., 2002), it is possible to study granuloma initiation mechanisms
Developmental Cell 38, July 25, 2016 ª 2016 Elsevier Inc. 133
Developmental Cell
Commentary processes, whether involving phagocyte responses to infections, wounds, cancer cells, or other types of homeostatic perturbations. It is a safe bet that the implementation of new imaging assays and genome-editing technologies will produce a bloom of disease models, in which the contribution of phagocytes can be observed in physiological detail. Now is a better time than ever for phagocyte research to discover its inner fish. ACKNOWLEDGMENTS
Figure 1. Zebrafish Allows Convenient Imaging of Phagocyte Behavior in Complex Pathophysiological Situations Depicted is a still image from a confocal time-lapse movie showing neutrophils (red) migrating toward a tail fin wound (right edge of the image), as well as interacting with metastasized melanoma cells (green). The purpose of this particular experiment was to test whether tumor-neutrophil interactions are altered by tissue-damage-induced inflammation. White box, individual neutrophil attracted by the wound. Scale bar, 50 mm.
and dynamics by intravitral imaging in transparent larvae, which initially only contain innate—not adaptive—immune cells. More than 10 years of experimentation in zebrafish models of tuberculosis have contributed to a conceptual shift away from the idea that granulomas are mere host defense structures that wall off mycobacteria and toward the notion that they also constitute a bacterial strategy to promote infection (Davis and Ramakrishnan, 2009). A number of potential drug targets have emerged from research using the zebrafish tuberculosis model (Meijer, 2016). The above examples show that the zebrafish model, through direct in vivo imaging, is well suited for following up on preliminary observations in mammalian systems. They also illustrate that at times it is smart to trade in the relative evolutionary proximity to humans (e.g., mice or rats) for the unique imaging capabilities of fish and the ability to study many animals in a short time frame. Given their crucial importance for defense against pathogens, it seems probable that many, if not most, phagocyte functions and regulatory principles are conserved. Hence, a thorough understanding of phagocyte regulation in fish may also produce unexpected perspectives on apparently well-understood aspects of mammalian physiology and cell biology. One example of the latter involves the mechanism by which neutrophils detect epithelial breaches. In zebrafish larvae, which live in a low-osmolarity solu134 Developmental Cell 38, July 25, 2016
tion (i.e., fresh water), neutrophils rapidly detect epithelial breaches through the drop of interstitial osmolarity after injury. The osmolarity decrease causes cell swelling, which leads to the production of chemotactic lipid mediators of the eicosanoid class (Enyedi et al., 2013). Eicosanoid synthesis is mechanically triggered by nuclear swelling via nuclear envelope stretch in response to cell swelling (Enyedi et al., 2016). At first glance, this finding appears to be a specific adaption of an epithelial immune defense mechanism to a freshwater environment, which may not be relevant to land-dwelling animals not immersed in hypotonic liquid. Surprisingly, a closer look at human physiology shows that the upper digestive tract of humans is, in fact, a fresh water-like environment. The linings of mouth and esophagus are exposed to saliva, and for little-understood reasons, our bodies invest much energy to maintain saliva roughly as hypotonic as fresh water. Furthermore, cell and nuclear swelling are general hallmarks of any severe tissue damage. Thus, it seems possible that a similar mechanism for detecting epithelial breaches exists in mammals, a hypothesis that should be examined in future studies. Another promise of the zebrafish system is to inspire novel working hypotheses that may be hard to infer from studying mammalian models alone. We believe zebrafish researchers will continue to capitalize on its unique technical strengths for investigating the initiation and rapid dynamics of disease
We apologize to the colleagues whose excellent work we could not mention due the space limitations. This work was supported by NIH grant GM099970 and an American Asthma Foundation Scholar grant to P.N. REFERENCES Davis, J.M., and Ramakrishnan, L. (2009). Cell 136, 37–49. Davis, J.M., Clay, H., Lewis, J.L., Ghori, N., Herbomel, P., and Ramakrishnan, L. (2002). Immunity 17, 693–702. Elks, P.M., van Eeden, F.J., Dixon, G., Wang, X., Reyes-Aldasoro, C.C., Ingham, P.W., Whyte, M.K., Walmsley, S.R., and Renshaw, S.A. (2011). Blood 118, 712–722. Enyedi, B., Kala, S., Nikolich-Zugich, T., and Niethammer, P. (2013). Nat. Cell Biol. 15, 1123–1130. Enyedi, B., Jelcic, M., and Niethammer, P. (2016). Cell 165, 1160–1170. Hughes, J., Johnson, R.J., Mooney, A., Hugo, C., Gordon, K., and Savill, J. (1997). Am. J. Pathol. 150, 223–234. Klyubin, I.V., Kirpichnikova, K.M., and Gamaley, I.A. (1996). Eur. J. Cell Biol. 70, 347–351. Mathias, J.R., Perrin, B.J., Liu, T.X., Kanki, J., Look, A.T., and Huttenlocher, A. (2006). J. Leukoc. Biol. 80, 1281–1288. Meijer, A.H. (2016). Semin. Immunopathol. 38, 261–273. Niethammer, P., Grabher, C., Look, A.T., and Mitchison, T.J. (2009). Nature 459, 996–999. Rhoades, E.R., Frank, A.A., and Orme, I.M. (1997). Tuber. Lung Dis. 78, 57–66. Robertson, A.L., Holmes, G.R., Bojarczuk, A.N., Burgon, J., Loynes, C.A., Chimen, M., Sawtell, A.K., Hamza, B., Willson, J., Walmsley, S.R., et al. (2014). Sci. Transl. Med. 6, 225ra29. Tauzin, S., Starnes, T.W., Becker, F.B., Lam, P.Y., and Huttenlocher, A. (2014). J. Cell Biol. 207, 589–598. Trede, N.S., Langenau, D.M., Traver, D., Look, A.T., and Zon, L.I. (2004). Immunity 20, 367–379. Yoo, S.K., Starnes, T.W., Deng, Q., and Huttenlocher, A. (2011). Nature 480, 109–112.