Reactive oxygen species suppress phagocyte surveillance by oxidizing cytoskeletal regulators
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Curated by eLife
eLife assessment
In this study, Ferling and colleagues provide convincing evidence demonstrating that myeloid cells exert distinct, cargo-dependent responses during and after phagocytosis. These important findings establish previously unrecognized insights into the function(s) of myeloid cells in immunosurveillance and are thus likely to be broadly impactful across the spectrum of biomedical disciplines including immunology and cell biology. Notwithstanding these clear strengths of the article, some minor issues were noted pertinent to the relative opaqueness of the mechanisms underpinning context-specific RhoA activation.
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Abstract
Despite their superficial similarities, the phagocytosis of pathogens differs from that of apoptotic cells in their recognition mechanisms and downstream signaling pathways. While the initial stages of these processes have been studied, the cytoskeletal reorganization that follows particle uptake is not well understood. By comparing the uptake of phosphatidylserine (PS)- coated targets versus IgG-opsonized targets of identical size, shape, and rigidity, we noted remarkable differences in the accompanying changes in cell morphology, adhesion and migration that persisted long after phagocytosis. While myeloid cells continued to survey their microenvironment after engulfing PS-coated targets, the uptake of IgG-opsonized targets caused phagocytes to round up, decreased their membrane ruffling, and led to the complete disassembly of podosomes. These changes were associated with increased activation of Rho and a concomitant decrease of Rac activity that collectively resulted in the thickening and compaction of the cortical F-actin cytoskeleton. Rho/formin-induced actin polymers were fastened to the membrane by their preferential interaction with Ezrin-Radixin-Moesin (ERM) proteins, which were necessary for cell compaction and podosome disassembly following ingestion of IgG-coated particles. The source of the distinct responses to PS- versus IgG-targets was the differential activation of the respiratory burst mediated by the NADPH oxidase: reactive oxygen species (ROS), emanating from phagosomes containing IgG-opsonized targets – but not those containing PS-coated ones – directly led to the activation of Rho. Similar findings were made with phagocytes that encountered pathogens or microbial-associated molecular patterns (MAMPS) that instigate the activation of the NADPH oxidase. These results implicate a connection between sensing of harmful particulates, the oxidation of cytoskeletal regulators, and the immune surveillance by myeloid cells that have potentially important consequences for the containment of pathogens.
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eLife assessment
In this study, Ferling and colleagues provide convincing evidence demonstrating that myeloid cells exert distinct, cargo-dependent responses during and after phagocytosis. These important findings establish previously unrecognized insights into the function(s) of myeloid cells in immunosurveillance and are thus likely to be broadly impactful across the spectrum of biomedical disciplines including immunology and cell biology. Notwithstanding these clear strengths of the article, some minor issues were noted pertinent to the relative opaqueness of the mechanisms underpinning context-specific RhoA activation.
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Reviewer #1 (Public review):
Summary:
This manuscript uses PS-coated and IgG-opsonized targets to model the engulfment of apoptotic cells and pathogens. It demonstrates that differential activation of the respiratory burst accounts for variations in cell morphology, adhesion, and migration following phagocytosis of different particles. Specifically, reactive oxygen species produced by phagosomes containing IgG-opsonized targets activate Rho GTPases. This activation triggers Formin- and ERM-dependent compaction of the cortical actin network, leading to rounded cell morphology, reduced membrane ruffling, disassembly of podosomes, and decreased migration. Some of these findings are validated in cells exposed to pathogens or soluble MAMPs.
Strengths:
The manuscript presents well-executed and controlled experiments. It proposes an intriguing …
Reviewer #1 (Public review):
Summary:
This manuscript uses PS-coated and IgG-opsonized targets to model the engulfment of apoptotic cells and pathogens. It demonstrates that differential activation of the respiratory burst accounts for variations in cell morphology, adhesion, and migration following phagocytosis of different particles. Specifically, reactive oxygen species produced by phagosomes containing IgG-opsonized targets activate Rho GTPases. This activation triggers Formin- and ERM-dependent compaction of the cortical actin network, leading to rounded cell morphology, reduced membrane ruffling, disassembly of podosomes, and decreased migration. Some of these findings are validated in cells exposed to pathogens or soluble MAMPs.
Strengths:
The manuscript presents well-executed and controlled experiments. It proposes an intriguing model to explain the distinct behaviors of myeloid cells when confronted with different phagocytic cargoes and offers fresh insights into immune surveillance.
Weaknesses:
Certain aspects of the proposed model require further experimental evidence. The significance of the cellular behavioral differences in response to various phagocytic cargoes warrants further exploration within physiological contexts.
Specific comments:
How do reactive oxygen species lead to an increase in Rho activation while simultaneously reducing Rac activity? The underlying molecular mechanisms remain unresolved, although potential regulatory pathways are discussed.
Given that the number of phagocytosed particles affects cell behavior (SF1), it is important to ensure that an equivalent number of particles are phagocytosed when comparing cells treated with PS-beads and IgG-beads (Figure 1a). How was this experimentally controlled, and how many particles are phagocytosed under each condition?
Why were experiments conducted in BMDM, Raw264.7, and PMN cells under different conditions? For Raw264.7 and PMN cells, cell behavior was only compared between those treated with IgG-RBC and untreated cells. What occurs to these cells when they are exposed to PS-beads as opposed to IgG-beads?
How long does it take for cells treated with IgG-beads to recover and regain their mobility and surveillance activity? Does this recovery occur following a reduction in reactive oxygen species production?
A contractile actin cortex usually requires the activity of both Formin and myosin II. It is a bit surprising that inhibitors of ROCK and myosin II, when added to Raw cells engulfing IgG-RBC, did not affect podosome disassembly. Is the cytoskeletal rearrangement observed in Figure 2 also independent of myosin II activity?
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Reviewer #2 (Public review):
Summary:
The manuscript by Ferling et al. describes how phagocytosis of IgG but not PS-opsonized targets induces the cells to round up and disassemble their podosomes. The mechanism downstream of the FcR is then dissected. The authors show that RhoA-mediated actin polymerization is involved, as well as actin nucleators of the Formin family, but not ROCK or Myosin II. ERM proteins and ROS production play a role in podosome loss and RhoA activation. Similar observations were made after cells were put in contact with Candida albicans or with soluble LPS.
Strengths:
The manuscript is of very good scientific standards, based on solid cell biology and biochemistry approaches, both in a murine macrophage cell line and in murine primary macrophages. It reaches the criteria for a significant advance in the field.
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