Occurrence of foamy macrophages during the innate response of zebrafish to trypanosome infections
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Summary: This study investigates the role of the innate immune response in controlling bloodstream trypanosome infection in the zebrafish infection model recently developed by the authors. The study found that an innate immune response, characterized by controlled inflammatory response was sufficient to control infection in some individuals, while failure to control infection was associated with a strong inflammatory response characterized by expansion of foamy macrophages. The findings highlight the importance of a balanced immune response in controlling bloodstream trypanosome infections that are likely relevant to mammalian infections.
Reviewer #1 and Reviewer #2 opted to reveal their name to the authors in the decision letter after review.
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Abstract
A tightly regulated innate immune response to trypanosome infections is critical to strike a balance between parasite control and inflammation-associated pathology. In this study, we make use of the recently established Trypanosoma carassii infection model in larval zebrafish to study the early response of macrophages and neutrophils to trypanosome infections in vivo. We consistently identified high- and low-infected individuals and were able to simultaneously characterise their differential innate response. Not only did macrophage and neutrophil number and distribution differ between the two groups, but also macrophage morphology and activation state. Exclusive to high-infected zebrafish, was the occurrence of foamy macrophages characterised by a strong pro-inflammatory profile and potentially associated with an exacerbated immune response as well as susceptibility to the infection. To our knowledge, this is the first report of the occurrence of foamy macrophages during an extracellular trypanosome infection.
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Reviewer #3:
Overall the manuscript is a valuable contribution and represents an important advance using the model that the authors have recently established in Doro et al. 2019.
I have however a few suggestions for improvement, that I present below.
Suggestions to strengthen the manuscript:
Fig. 1 diagram is very useful. However, it would be very informative if the diagram could be followed by a representative quantification. For example, when injecting 200 T. carassii, what % of larvae is classified in the two infection categories? Could the authors also further discuss the % of T. low larvae where no parasites were observed during the clinical scoring? Have these larvae (or some of them) cleared the infection completely? Shouldn't they be classified/followed on their own?
Fig. 2: Is the clinical scoring predictive of early death onset …
Reviewer #3:
Overall the manuscript is a valuable contribution and represents an important advance using the model that the authors have recently established in Doro et al. 2019.
I have however a few suggestions for improvement, that I present below.
Suggestions to strengthen the manuscript:
Fig. 1 diagram is very useful. However, it would be very informative if the diagram could be followed by a representative quantification. For example, when injecting 200 T. carassii, what % of larvae is classified in the two infection categories? Could the authors also further discuss the % of T. low larvae where no parasites were observed during the clinical scoring? Have these larvae (or some of them) cleared the infection completely? Shouldn't they be classified/followed on their own?
Fig. 2: Is the clinical scoring predictive of early death onset (or likelihood of death)? To show this, the authors could, for example, divide the T. car 200 survival curve into 2 separate curves, based on the clinical scoring at day 4-5.
In Fig. 5 and Fig. 6 and related text, the authors describe their results as "macrophage proliferation" and "neutrophil proliferation". I would encourage them to avoid these terms and rephrase these sections. Normally "macrophage proliferation" is used to refer to resident tissue macrophages that occasionally are seen to divide/proliferate. To my knowledge, neutrophil proliferation in a similar manner has not been described. Most likely what the authors describe is myelopoiesis (in agreement, the authors also indicate that Edu staining most commonly is seen in hematopoietic tissues) and the EdU staining in mature macrophages/neutrophils is the result of a (recent) cell division of a hematopoietic progenitor cell. The authors do not have evidence that the terminally-differentiated cells (macrophages and neutrophils) are actually "proliferating". In lack of a more specific mechanistic insight, I would encourage the use of much broader terms, such as "increased production/number of macrophages/neutrophils" rather than "macrophage/neutrophil proliferation", throughout.
The authors observe several very interesting phenotypes that they report in Fig. 7, 8, 9 & 10. The frequency of these phenotypes (association with infection and with each other) however is not quantified and tested statistically. In particular:
The authors report that macrophages, but not neutrophils, infiltrate in the cardinal vein, although both cell populations are accumulating on the outer side of the vasculature during infection. Can the authors quantify and test statistically these phenomena, i.e. by counting cells inside the vessel and associated (externally) with the vessel in the PVP, T. car-low and T. car-high groups? Also, do neutrophils ever interact with trypanosomes in other sections of the vasculature, if not in the cardinal vein? Do trypanosomes ever escape from the circulation and interact with neutrophils elsewhere?
The authors report that foamy macrophages occur inside the vasculature and are exclusive to high-infected larvae. Can the authors show some quantifications of these associations and perform statistical tests (i.e. count foamy/non-foamy mpeg+ cells inside/outside the vessels in the PVP, T. car-low and T. car-high groups)? Also, macrophages do not phagocytose T. carassii, but foamy macrophages are seen in the context of other (intracellular) Trypanosoma infection. Are macrophages here perhaps scavenging dead Trypanosoma from the circulation, and is this leading to the foamy macrophage phenotype? Trypanosomes are also leading to hemolysis and this could lead to increased phagocytosis of red blood cell debris by macrophages. Could this be linked to the foamy appearance? How specific is BODIPY, to distinguish cholesterol (typical of foamy macrophages), vs lipids derived by phagocytosis of cell debris (i.e. high in membrane phospholipids?)
The authors report that foamy macrophages occurring in T. car-infected larvae are characterised by a strong proinflammatory profile and are all il1beta and all tnfa positive. Significant differences are observed in the inflammatory response of macrophages in high- and low-infected individuals and in their susceptibility to infection. Can the authors quantify and test statistically these observations? For example, can the authors show that foamy macrophages are indeed more frequently il1b positive/tnfa positive than neighbouring non-foamy mpeg+ cells?
The authors report that a strong inflammatory profile is associated with the occurrence of foamy macrophages. However, it is not clear how widely spread the inflammation is and only images of macrophages and endothelial cells in the cardinal vein are shown. Moreover, only tnfa and il1b are assessed (using transgenic reporters). The authors also mention that they observe a mild inflammatory response in low-infected individuals and that this is strongly associated with control of parasitaemia and survival to the infection. Can they confirm strong vs mild inflammatory profiles and different association with survival in the 2 infection categories and PVP control with a panel of qRT-PCR for several inflammatory markers (i.e. il1beta, tnfa and other relevant cytokines and chemokines)?
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Reviewer #2:
Using this new Trypanosoma carassii infectious model in larval zebrafish, Jacobs et al. have developed a new clinical scoring system to reliably separate high-and low-infected larvae in order to investigate their individual innate immune responses, with a special emphasis on macrophages and neutrophils.
In summary the separation system used in this allows us i) to identify a strong macrophage and neutrophil proliferation response by high-and low-infected larvae, although happening a bit earlier, 5 dpi, for macrophages in low-infected larvae, and ii) to observe a differential distribution and morphology of macrophages, associated to the unique presence of more rounded foamy macrophages with a high pro-inflammatory profile into the vessels of high-infected zebrafish larvae. Together, this study constitutes the first report of …
Reviewer #2:
Using this new Trypanosoma carassii infectious model in larval zebrafish, Jacobs et al. have developed a new clinical scoring system to reliably separate high-and low-infected larvae in order to investigate their individual innate immune responses, with a special emphasis on macrophages and neutrophils.
In summary the separation system used in this allows us i) to identify a strong macrophage and neutrophil proliferation response by high-and low-infected larvae, although happening a bit earlier, 5 dpi, for macrophages in low-infected larvae, and ii) to observe a differential distribution and morphology of macrophages, associated to the unique presence of more rounded foamy macrophages with a high pro-inflammatory profile into the vessels of high-infected zebrafish larvae. Together, this study constitutes the first report of the occurrence of foamy macrophages during an extracellular trypanosome infection.
Although the paper is well-written and the findings are interesting as they bring new insights into the development of foamy macrophages in response to an extracellular pathogen, i.e. Trypanosoma carassii, using a zebrafish larvae model, I have a few concerns regarding the following:
The experimental infectious model in zebrafish: figure 2 summarizes that only 15% of the infected larvae, named low-infected larvae, are able to survive the infection. As an explanation the authors refer to the trypanosuceptible vs. trypanotolerant background of the host observed in non-zebrafish models. However, in this particular setting, all the larvae possess an identical genetic background. Therefore, why would the larvae behave differently in response to a similar pathogen? In addition, there is no clear differences in neither parasitic load at 2 dpi (figure 3F) nor myeloid cells accumulation at 3 dpi (figure 4AB), which could lead to a drastic difference in parasitic load based on mRNA expression at 4 dpi (figure 3F). The authors should discuss this shortly.
Figure 4: the representative pictures from Fig4B do not seem to clearly match the histograms depicted in Fig4C. For example, from the pictures in Fig4B, it seems that there is a decrease in red fluorescence in the representative pictures from 7 dpi to 9 dpi low-infected larvae, which is not reflected in the histogram. Also, a representative picture of 7 hi-infected larvae seems to show at least equal or even more red fluorescence compared to 9 dpi low-infected larvae.
Lines 494-496 states "No significant difference was observed between high-and low-infected fish, confirming that macrophages react to the presence and not to the number of trypanosomes.", reflecting that there is no differences in total macrophages nor in their proliferation between low- and high-infected zebrafish larvae (Figure 5B&C). Therefore it is not sufficiently clear on which basis the authors states a few lines later as a conclusion that "Altogether, these data confirm that T. carassii infection triggers macrophage proliferation and that proliferation is higher in low-infected compared to high-infected individuals, possibly due to a higher haematopoietic activity." Therefore the authors should revise this conclusion or bring stronger data to reinforce their results. Also, similar conclusions need to be adjusted in the discussion section and bring new elements to explain the higher number of macrophages observed in figure 4.
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Reviewer #1:
The authors devised clinical criteria for identifying Zebrafish larvae with high or low T. cassari infections in order to track. Using transgenic fish line marking macrophages and neutrophils, the authors showed that both groups of larvae increase macrophage (and to lesser extent neutrophil) levels in response to infection. However, the macrophages in high parasitaemia animals migrated into the capillaries and had elevated levels of inflammatory markers (TNF, IL-1) and lipids, indicative of a foamy phenotype. The authors conclude that a measured inflammatory response allows animals to control the initial infection, while an exaggerated inflammatory response leads to an environment in which the bloodstream trypanosomes can proliferate. The findings support and extend data from murine models of infection, by allowing direct …
Reviewer #1:
The authors devised clinical criteria for identifying Zebrafish larvae with high or low T. cassari infections in order to track. Using transgenic fish line marking macrophages and neutrophils, the authors showed that both groups of larvae increase macrophage (and to lesser extent neutrophil) levels in response to infection. However, the macrophages in high parasitaemia animals migrated into the capillaries and had elevated levels of inflammatory markers (TNF, IL-1) and lipids, indicative of a foamy phenotype. The authors conclude that a measured inflammatory response allows animals to control the initial infection, while an exaggerated inflammatory response leads to an environment in which the bloodstream trypanosomes can proliferate. The findings support and extend data from murine models of infection, by allowing direct visualization of host immune response.
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Summary: This study investigates the role of the innate immune response in controlling bloodstream trypanosome infection in the zebrafish infection model recently developed by the authors. The study found that an innate immune response, characterized by controlled inflammatory response was sufficient to control infection in some individuals, while failure to control infection was associated with a strong inflammatory response characterized by expansion of foamy macrophages. The findings highlight the importance of a balanced immune response in controlling bloodstream trypanosome infections that are likely relevant to mammalian infections.
Reviewer #1 and Reviewer #2 opted to reveal their name to the authors in the decision letter after review.
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