n e ws a n d v i e ws
Time to cast a larger net Matthew L Wheeler & David M Underhill
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© 2014 Nature America, Inc. All rights reserved.
Neutrophils sense the size of microbial targets and respond via ‘NETosis’ when targets are too big to internalize and contain via phagocytosis.
M
yeloid phagocytes, such as neutrophils, macrophages and dendritic cells, must detect and respond appropriately to a diverse pantheon of microbes ranging from small viruses to large multicellular parasites. Upon recognizing a microbe, these cells ‘eat’ (phagocytose), kill and degrade it, and also secrete inflammatory cytokines and chemokines that may orchestrate an ongoing inflammatory and adaptive immune response1. The process of phagocytosis is triggered when phagocytic receptors recognize a target and stimulate the cell to engulf it. However, this process requires that the target be sufficiently small that it can be engulfed. How does the immune system deal differently with pathogens that are too large to ‘eat’? In this issue of Nature Immunology, Branzk et al. show that neutrophils are able to sense microbe size, and although they phagocytose microbes that are sufficiently small, they are stimulated to release neutrophil extracellular traps (NETs) specifically in response to microbes that are too large to ‘eat’2. The idea that phagocytes are able to detect the size of a target and respond to this information has been around for a long time, but little has been done to apply this thinking to antimicrobial host defense. In the early 1970s, it was noted that neutrophils bound to antibodyopsonized surfaces engaged in a process called ‘frustrated phagocytosis’, in which the cells spread over surfaces as if attempting to ‘eat’ them3. In this situation, the neutrophils degranulated into the extracellular space3, a process that might contribute to local tissue damage in the context of antibody deposition Matthew L. Wheeler and David M. Underhill are in the Division of Biomedical Sciences and the F. Widjaja Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA. e-mail: [email protected]
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in tissue. In contrast, during the phagocytosis of ingestible particles, neutrophil granules fused with phagosomes3. Subsequent studies have reported that ‘frustrated phagocytosis’, or preventing phagocytosis by blocking actin polymerization, results in elevated production of proinflammatory cytokines, which occurs in part because downstream signaling events that are normally quenched during internalization become prolonged4,5. In addition, there is evidence that ingestible particles of different sizes may be able to elicit different responses. For example, human peripheral blood mononuclear cells stimulated with single-stranded RNA–coated protamine particles of different sizes selectively generate interferon-α in response to smaller particles (500 nm in diameter) tend to induce the production of tumor-necrosis factor6. This may be reflective of the types of cytokines that are needed for the immune system to combat smaller viruses compared with those needed to combat larger bacteria or fungi. Similarly, phagocytosis of larger particles leads to the presentation of antigens on major histocompatibility complex class II more efficiently than does phagocytosis of smaller particles, even under conditions in which the same total load of antigen is delivered7. This is due in part to differences in the trafficking of antigen-processing machinery to the phagosomes. Studies have also shown that in addition to size, other physical properties of particles, such as shape and rigidity, can influence the responses of phagocytes. Macrophages internalize immunoglobulin G–opsonized polyacrylamide particles engineered to have a more rigid surface more efficiently than they internalize ‘softer’ particles, owing to the inability of the phagocyte to induce actin polymerization upon contact with the less rigid target8. Also, polymeric microparticles engineered to
have a ‘budding’ surface morphology are more efficient at inducing inflammasome activation and neutrophil recruitment in tissues than are particles of a similar size with smooth surfaces9. Therefore, in addition to the combination of receptors that a phagocyte might use to recognize a particle, its physical size, shape and rigidity can influence the subsequent cellular responses. However, how these types of findings relate to antimicrobial host defense has been less clear. Neutrophils are one of the first cells of the immune system recruited to a site of infection, and they have potent antimicrobial properties owing to their ability to rapidly ingest and kill pathogens by exposing them to an arsenal of antimicrobial proteins stored in intracellular granules and by producing highly toxic reactive oxygen intermediates. Key to this study is that neutrophils have also been shown to use the curious antimicrobial strategy of ‘NETosis’, whereby the cell releases web-like NETs composed of decondensed chromatin in complex with many antimicrobial proteins that function to trap and neutralize pathogens to prevent their dissemination. The importance of NETs in host defense is illustrated by the observation that DNAses secreted by certain pathogens can degrade NETs and that this can be an important mechanism by which to evade the immune system10. In this issue, Branzk et al. investigate the role of NETosis in defense against the dimorphic fungal pathogen Candida albicans, which exists in a small yeast form necessary for dissemination, as well as a larger branching hyphal form required for penetration into tissues2. Through the use of a fluorescence-imaging approach to monitor the release of NETs in vitro, the authors note that neutrophils ‘preferentially’ trigger NETosis when exposed to the hyphal form of C. albicans, which, unlike the yeast form, is too large to be phagocytosed. To explore whether
volume 15 number 11 november 2014 nature immunology
news and v i ews
Hyphae
npg
© 2014 Nature America, Inc. All rights reserved.
Kim Caesar/Nature Publishing Group
Yeast o• • o• o• o
Failure of phagocytosis
Phagocytosis Fusion of granules with phagosomes
Intracellular killing, minimal extracellular damage
Delivery of granule enzymes to nucleus
o•
o• o• o• o•
NETosis, extracellular killing with collateral tissue damage
Figure 1 Neutrophils respond differently to fungal structures of different sizes. Neutrophils bind and internalize yeast cells via phagocytosis, which results in the sequestration of yeast in phagosomes that fuse with azurophilic granules. The production of ROS and the release of enzymes such as neutrophil elastase into the phagosome contribute to killing of the organism. When neutrophils encounter hyphae, they are not able to internalize them, and azurophilic granules are free to deliver their contents instead into the nucleus, which triggers chromatin decondensation and the release of NETs. NETs contribute to the immobilization and killing of extracellular organisms, but at the cost of some tissue damage.
selective release of NETs is a result of the larger size of the hyphal form of the fungus and is not due to differences between yeast and hyphae in their cell-wall composition, the authors expose neutrophils to hyphae fractionated into particles small enough to be internalized and find that NETosis is no longer induced. Additionally, when neutrophils are presented with the yeast form of C. albicans through the use of a Transwell system that allows the cells to engage yeast but not internalize them (because of physical separation by the Transwell), then NETosis occurs. NETs also form in response to large Aspergillus fumigatus filaments and to aggregates of A. fumigatus conidia but not to dispersed conidia. The phenomenon is not restricted to fungi, since large aggregates of Mycobacterium bovis induce NETosis, but small single bacteria do not. Whether this size-sensing mechanism also applies to other large pathogens such as multicellular parasites remains to be determined. Published work has demonstrated that NETosis requires the production of reactive oxygen species (ROS) by the NADPH phagocyte oxidase, as well as the ROS-related enzyme myeloperoxidase and the granule-stored protease neutrophil elastase6. Neutrophils from patients with chronic granulomatous disease, who have mutations in the gene encoding phagocyte oxidase, are unable to produce ROS and fail to release NETs. However, when the investigators examine ROS production, they find that differences between neutrophils
exposed to yeast and those exposed to hyphae could not explain the difference in NETosis. The authors propose a model in which NETosis is ‘decided’ by the trafficking of neutrophil elastase (Fig. 1). NETosis requires ROS- and myeloperoxidase-facilitated release of elastase from granules into the nucleus, where it cleaves histones to decondense chromatin and initiate the NETosis program. When phagocytosis is successful, granules fuse with phagosomes and elastase is delivered into the phagosome and is not available to be delivered into the nucleus. In support of this model, the investigators find that inhibiting phagocytosis or granule-phagosome fusion leads to the release of NETs in response to a yeast-locked strain of C. albicans, which suggests that blocking the trafficking of proteins such as elastase to phagosomes is sufficient to turn a yeast into a hyphae-like inducer of NETosis. The authors further test the hypothesis that phagocytosis is needed to sequester neutrophil elastase away from the nucleus to prevent the release of NETs by assessing the effect of blocking the phagocytic receptor dectin-1 on the release of NETs by human neutrophils. Blocking dectin-1 results in a substantial reduction in phagocytosis, which correlates with a significant increase in the release of NETs in response to the yeast-locked mutant of C. albicans in vitro and in vivo. Notably, the in vivo release of NETs in mice deficient in dectin-1 induces significant tissue damage that is ‘rescued’ by inhibition of neutrophil
nature immunology volume 15 number 11 november 2014
elastase. These data suggest that selective release of NETs in response to large pathogens that cannot be cleared by phagocytosis may represent an important strategy for limiting NET-associated immunopathology only to cases in which this response is necessary for host defense. Of course, NETs are not released exclusively in response to large microbes; the original report about NETs demonstrated their release in response to soluble stimuli such as interleukin 8 (ref. 11). Thus, much remains to be learned about the various factors involved in ‘deciding’ when NETs are released. Abnormal release and/or clearance of NETs has been associated with several autoimmune and inflammatory disorders, and the prospect that this process could be targeted therapeutically is enticing10. This study2 provides an additional new twist in considering the mechanisms used by cells of the immune system to tailor their responses to different pathogens. Future studies should determine if other types of cells of the immune system, such as dendritic cells, exhibit similar size-sensing properties to dictate the different T cell responses needed for defense against viruses, bacteria, fungi and parasites. One major question that remains to be addressed, however, is why published studies have observed neutrophils undergoing robust NETosis in response to bacteria such as Staphylococcus aureus and Escherichia coli that are small enough to be phagocytosed10. Whether there are technical differences in the approaches that contribute to this discrepancy is not clear. Despite such issues, the study from Branzk et al. provides an enhanced view of how cells of the immune system sense microbes and offers important insight into the mechanisms involved in regulating the release of NETs from neutrophils2. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Underhill, D.M. & Goodridge, H.S. Nat. Rev. Immunol. 12, 492–502 (2012). 2. Branzk, N. et al. Nat. Immunol. 15, 1017–1025 (2014). 3. Henson, P.M. J. Exp. Med. 134, 114–135 (1971). 4. Rosas, M. et al. J. Immunol. 181, 3549–3557 (2008). 5. Hernanz-Falcón, P., Joffre, O., Williams, D.L. & Reis e Sousa, C. Eur. J. Immunol. 39, 507–513 (2009). 6. Rettig, L. et al. Blood 115, 4533–4541 (2010). 7. Brewer, J.M., Pollock, K.G., Tetley, L. & Russell, D.G. J. Immunol. 173, 6143–6150 (2004). 8. Beningo, K.A. & Wang, Y.L. J. Cell Sci. 115, 849–856 (2002). 9. Vaine, C.A. et al. J. Immunol. 190, 3525–3532 (2013). 10. Brinkmann, V. & Zychlinsky, A. J. Cell Biol. 198, 773–783 (2012). 11. Brinkmann, V. et al. Science 303, 1532–1535 (2004).
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Time to cast a larger net Matthew L Wheeler & David M Underhill
npg
© 2014 Nature America, Inc. All rights reserved.
Neutrophils sense the size of microbial targets and respond via ‘NETosis’ when targets are too big to internalize and contain via phagocytosis.
M
yeloid phagocytes, such as neutrophils, macrophages and dendritic cells, must detect and respond appropriately to a diverse pantheon of microbes ranging from small viruses to large multicellular parasites. Upon recognizing a microbe, these cells ‘eat’ (phagocytose), kill and degrade it, and also secrete inflammatory cytokines and chemokines that may orchestrate an ongoing inflammatory and adaptive immune response1. The process of phagocytosis is triggered when phagocytic receptors recognize a target and stimulate the cell to engulf it. However, this process requires that the target be sufficiently small that it can be engulfed. How does the immune system deal differently with pathogens that are too large to ‘eat’? In this issue of Nature Immunology, Branzk et al. show that neutrophils are able to sense microbe size, and although they phagocytose microbes that are sufficiently small, they are stimulated to release neutrophil extracellular traps (NETs) specifically in response to microbes that are too large to ‘eat’2. The idea that phagocytes are able to detect the size of a target and respond to this information has been around for a long time, but little has been done to apply this thinking to antimicrobial host defense. In the early 1970s, it was noted that neutrophils bound to antibodyopsonized surfaces engaged in a process called ‘frustrated phagocytosis’, in which the cells spread over surfaces as if attempting to ‘eat’ them3. In this situation, the neutrophils degranulated into the extracellular space3, a process that might contribute to local tissue damage in the context of antibody deposition Matthew L. Wheeler and David M. Underhill are in the Division of Biomedical Sciences and the F. Widjaja Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA. e-mail: [email protected]
1000
in tissue. In contrast, during the phagocytosis of ingestible particles, neutrophil granules fused with phagosomes3. Subsequent studies have reported that ‘frustrated phagocytosis’, or preventing phagocytosis by blocking actin polymerization, results in elevated production of proinflammatory cytokines, which occurs in part because downstream signaling events that are normally quenched during internalization become prolonged4,5. In addition, there is evidence that ingestible particles of different sizes may be able to elicit different responses. For example, human peripheral blood mononuclear cells stimulated with single-stranded RNA–coated protamine particles of different sizes selectively generate interferon-α in response to smaller particles (500 nm in diameter) tend to induce the production of tumor-necrosis factor6. This may be reflective of the types of cytokines that are needed for the immune system to combat smaller viruses compared with those needed to combat larger bacteria or fungi. Similarly, phagocytosis of larger particles leads to the presentation of antigens on major histocompatibility complex class II more efficiently than does phagocytosis of smaller particles, even under conditions in which the same total load of antigen is delivered7. This is due in part to differences in the trafficking of antigen-processing machinery to the phagosomes. Studies have also shown that in addition to size, other physical properties of particles, such as shape and rigidity, can influence the responses of phagocytes. Macrophages internalize immunoglobulin G–opsonized polyacrylamide particles engineered to have a more rigid surface more efficiently than they internalize ‘softer’ particles, owing to the inability of the phagocyte to induce actin polymerization upon contact with the less rigid target8. Also, polymeric microparticles engineered to
have a ‘budding’ surface morphology are more efficient at inducing inflammasome activation and neutrophil recruitment in tissues than are particles of a similar size with smooth surfaces9. Therefore, in addition to the combination of receptors that a phagocyte might use to recognize a particle, its physical size, shape and rigidity can influence the subsequent cellular responses. However, how these types of findings relate to antimicrobial host defense has been less clear. Neutrophils are one of the first cells of the immune system recruited to a site of infection, and they have potent antimicrobial properties owing to their ability to rapidly ingest and kill pathogens by exposing them to an arsenal of antimicrobial proteins stored in intracellular granules and by producing highly toxic reactive oxygen intermediates. Key to this study is that neutrophils have also been shown to use the curious antimicrobial strategy of ‘NETosis’, whereby the cell releases web-like NETs composed of decondensed chromatin in complex with many antimicrobial proteins that function to trap and neutralize pathogens to prevent their dissemination. The importance of NETs in host defense is illustrated by the observation that DNAses secreted by certain pathogens can degrade NETs and that this can be an important mechanism by which to evade the immune system10. In this issue, Branzk et al. investigate the role of NETosis in defense against the dimorphic fungal pathogen Candida albicans, which exists in a small yeast form necessary for dissemination, as well as a larger branching hyphal form required for penetration into tissues2. Through the use of a fluorescence-imaging approach to monitor the release of NETs in vitro, the authors note that neutrophils ‘preferentially’ trigger NETosis when exposed to the hyphal form of C. albicans, which, unlike the yeast form, is too large to be phagocytosed. To explore whether
volume 15 number 11 november 2014 nature immunology
news and v i ews
Hyphae
npg
© 2014 Nature America, Inc. All rights reserved.
Kim Caesar/Nature Publishing Group
Yeast o• • o• o• o
Failure of phagocytosis
Phagocytosis Fusion of granules with phagosomes
Intracellular killing, minimal extracellular damage
Delivery of granule enzymes to nucleus
o•
o• o• o• o•
NETosis, extracellular killing with collateral tissue damage
Figure 1 Neutrophils respond differently to fungal structures of different sizes. Neutrophils bind and internalize yeast cells via phagocytosis, which results in the sequestration of yeast in phagosomes that fuse with azurophilic granules. The production of ROS and the release of enzymes such as neutrophil elastase into the phagosome contribute to killing of the organism. When neutrophils encounter hyphae, they are not able to internalize them, and azurophilic granules are free to deliver their contents instead into the nucleus, which triggers chromatin decondensation and the release of NETs. NETs contribute to the immobilization and killing of extracellular organisms, but at the cost of some tissue damage.
selective release of NETs is a result of the larger size of the hyphal form of the fungus and is not due to differences between yeast and hyphae in their cell-wall composition, the authors expose neutrophils to hyphae fractionated into particles small enough to be internalized and find that NETosis is no longer induced. Additionally, when neutrophils are presented with the yeast form of C. albicans through the use of a Transwell system that allows the cells to engage yeast but not internalize them (because of physical separation by the Transwell), then NETosis occurs. NETs also form in response to large Aspergillus fumigatus filaments and to aggregates of A. fumigatus conidia but not to dispersed conidia. The phenomenon is not restricted to fungi, since large aggregates of Mycobacterium bovis induce NETosis, but small single bacteria do not. Whether this size-sensing mechanism also applies to other large pathogens such as multicellular parasites remains to be determined. Published work has demonstrated that NETosis requires the production of reactive oxygen species (ROS) by the NADPH phagocyte oxidase, as well as the ROS-related enzyme myeloperoxidase and the granule-stored protease neutrophil elastase6. Neutrophils from patients with chronic granulomatous disease, who have mutations in the gene encoding phagocyte oxidase, are unable to produce ROS and fail to release NETs. However, when the investigators examine ROS production, they find that differences between neutrophils
exposed to yeast and those exposed to hyphae could not explain the difference in NETosis. The authors propose a model in which NETosis is ‘decided’ by the trafficking of neutrophil elastase (Fig. 1). NETosis requires ROS- and myeloperoxidase-facilitated release of elastase from granules into the nucleus, where it cleaves histones to decondense chromatin and initiate the NETosis program. When phagocytosis is successful, granules fuse with phagosomes and elastase is delivered into the phagosome and is not available to be delivered into the nucleus. In support of this model, the investigators find that inhibiting phagocytosis or granule-phagosome fusion leads to the release of NETs in response to a yeast-locked strain of C. albicans, which suggests that blocking the trafficking of proteins such as elastase to phagosomes is sufficient to turn a yeast into a hyphae-like inducer of NETosis. The authors further test the hypothesis that phagocytosis is needed to sequester neutrophil elastase away from the nucleus to prevent the release of NETs by assessing the effect of blocking the phagocytic receptor dectin-1 on the release of NETs by human neutrophils. Blocking dectin-1 results in a substantial reduction in phagocytosis, which correlates with a significant increase in the release of NETs in response to the yeast-locked mutant of C. albicans in vitro and in vivo. Notably, the in vivo release of NETs in mice deficient in dectin-1 induces significant tissue damage that is ‘rescued’ by inhibition of neutrophil
nature immunology volume 15 number 11 november 2014
elastase. These data suggest that selective release of NETs in response to large pathogens that cannot be cleared by phagocytosis may represent an important strategy for limiting NET-associated immunopathology only to cases in which this response is necessary for host defense. Of course, NETs are not released exclusively in response to large microbes; the original report about NETs demonstrated their release in response to soluble stimuli such as interleukin 8 (ref. 11). Thus, much remains to be learned about the various factors involved in ‘deciding’ when NETs are released. Abnormal release and/or clearance of NETs has been associated with several autoimmune and inflammatory disorders, and the prospect that this process could be targeted therapeutically is enticing10. This study2 provides an additional new twist in considering the mechanisms used by cells of the immune system to tailor their responses to different pathogens. Future studies should determine if other types of cells of the immune system, such as dendritic cells, exhibit similar size-sensing properties to dictate the different T cell responses needed for defense against viruses, bacteria, fungi and parasites. One major question that remains to be addressed, however, is why published studies have observed neutrophils undergoing robust NETosis in response to bacteria such as Staphylococcus aureus and Escherichia coli that are small enough to be phagocytosed10. Whether there are technical differences in the approaches that contribute to this discrepancy is not clear. Despite such issues, the study from Branzk et al. provides an enhanced view of how cells of the immune system sense microbes and offers important insight into the mechanisms involved in regulating the release of NETs from neutrophils2. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Underhill, D.M. & Goodridge, H.S. Nat. Rev. Immunol. 12, 492–502 (2012). 2. Branzk, N. et al. Nat. Immunol. 15, 1017–1025 (2014). 3. Henson, P.M. J. Exp. Med. 134, 114–135 (1971). 4. Rosas, M. et al. J. Immunol. 181, 3549–3557 (2008). 5. Hernanz-Falcón, P., Joffre, O., Williams, D.L. & Reis e Sousa, C. Eur. J. Immunol. 39, 507–513 (2009). 6. Rettig, L. et al. Blood 115, 4533–4541 (2010). 7. Brewer, J.M., Pollock, K.G., Tetley, L. & Russell, D.G. J. Immunol. 173, 6143–6150 (2004). 8. Beningo, K.A. & Wang, Y.L. J. Cell Sci. 115, 849–856 (2002). 9. Vaine, C.A. et al. J. Immunol. 190, 3525–3532 (2013). 10. Brinkmann, V. & Zychlinsky, A. J. Cell Biol. 198, 773–783 (2012). 11. Brinkmann, V. et al. Science 303, 1532–1535 (2004).
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