Predestined neutrophil heterogeneity in homeostasis varies in transcriptional and phenotypic response to Candida
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eLife Assessment
In their study, Scherer and colleagues aim to use analyses of single-cell clones of murine granulocyte monocyte progenitors that are conditionally immortalized, and analyses of neutrophils derived from those clones to characterize an experimental system for studying neutrophil heterogeneity. The multi-omic and functional analyses reported are valuable but the strength of the evidence presented in support of them is incomplete because the study lacks a rigorous demonstration that the neutrophil-like cells that they derive are fully mature neutrophils.
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Abstract
Once perceived to be homogenous effector cells, neutrophils have since been shown to exhibit population heterogeneity. Here, we established an experimental model of clonal neutrophil heterogeneity using conditionally immortalized clonal granulocyte monocyte progenitors (GMPs) and their mature neutrophil progeny. Transcriptional and epigenetic profiling showed conserved genome-wide signatures of transcription and chromatin accessibility that were specific to individual GMP clones and their paired neutrophil progeny, suggesting that clone specificity is established as early as the GMP stage. Clone-specific genes in vital regulatory pathways were pre-programmed and exhibited delayed expression in the mature neutrophil stage. The clone-specific gene expression in the mature neutrophils paired to enhancer activation in their parental GMPs. To determine whether transcriptional heterogeneity predicted the response to fungal pathogens, neutrophil clones were functionally profiled. Clones demonstrated heterogeneous responses to fungal pathogens in vitro and revealed neutrophil subsets with evidence for tailored functional responses to Candida spp . as well as specific transcriptional and epigenetic patterns that may explain these differences. Together, this work establishes that heterogenous GMP and neutrophil compartments exist under homeostatic conditions and that these represent predefined clusters that are uniquely adapted to control invasive fungal pathogens.
Short Summary
Clonal neutrophil progenitors demonstrate heterogeneity in transcription and chromatin accessibility which may inform response to later fungal challenges.
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eLife Assessment
In their study, Scherer and colleagues aim to use analyses of single-cell clones of murine granulocyte monocyte progenitors that are conditionally immortalized, and analyses of neutrophils derived from those clones to characterize an experimental system for studying neutrophil heterogeneity. The multi-omic and functional analyses reported are valuable but the strength of the evidence presented in support of them is incomplete because the study lacks a rigorous demonstration that the neutrophil-like cells that they derive are fully mature neutrophils.
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Reviewer #1 (Public Review):
The heterogeneity within the neutrophil population is becoming clear. However, it was not clear if neutrophil progenitors are also heterogenous. Because neutrophils are short-lived, it is technically challenging to tackle the question. This study used a system to isolate and expand clonal neutrophil progenitors (granulocyte-monocyte progenitors; GMPs) to achieve molecular and functional profiling. In the study, transcriptional profiling was performed by RNAseq and ATACseq. Functional assays were performed ex vivo to examine phagocytosis, ROS production, NET formation, and neutrophil swarming using Candida albicans, as well as C. glabrata and C. auris. The strengths of this study include the use of the neutrophil clone system to track GMPs developing into neutrophils. The clone-based approach made it possible …
Reviewer #1 (Public Review):
The heterogeneity within the neutrophil population is becoming clear. However, it was not clear if neutrophil progenitors are also heterogenous. Because neutrophils are short-lived, it is technically challenging to tackle the question. This study used a system to isolate and expand clonal neutrophil progenitors (granulocyte-monocyte progenitors; GMPs) to achieve molecular and functional profiling. In the study, transcriptional profiling was performed by RNAseq and ATACseq. Functional assays were performed ex vivo to examine phagocytosis, ROS production, NET formation, and neutrophil swarming using Candida albicans, as well as C. glabrata and C. auris. The strengths of this study include the use of the neutrophil clone system to track GMPs developing into neutrophils. The clone-based approach made it possible to evaluate the functions of multiple neutrophil subpopulations. Limitations of this study include the dependency on ex vivo approaches and the modest degree of heterogeneity within presented neutrophils. Nevertheless, the finding - the heterogeneity of neutrophils can be traced back to the GMP stage - is significant.
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Reviewer #2 (Public Review):
The stated goal of the authors is to establish and characterize an experimental system to study neutrophil heterogeneity in a manner that allows for functional outcomes to be probed. To do so, they start with murine GMPs that are conditionally immortalized by ER-HoxB8 expression and make single-cell clonal populations to ask whether those GMPs or neutrophils derived by differentiating such clonal GMPs harbor heterogeneity. At a conceptual level, this is an innovative approach that could shed light on mechanisms of neutrophil heterogeneity that have been described in both health and disease. They perform bulk multi-omics and functional analyses of both the clonal GMPs and neutrophil-like cells, including transcriptional and epigenetic profiling. However, the major weakness of the study is that the authors do …
Reviewer #2 (Public Review):
The stated goal of the authors is to establish and characterize an experimental system to study neutrophil heterogeneity in a manner that allows for functional outcomes to be probed. To do so, they start with murine GMPs that are conditionally immortalized by ER-HoxB8 expression and make single-cell clonal populations to ask whether those GMPs or neutrophils derived by differentiating such clonal GMPs harbor heterogeneity. At a conceptual level, this is an innovative approach that could shed light on mechanisms of neutrophil heterogeneity that have been described in both health and disease. They perform bulk multi-omics and functional analyses of both the clonal GMPs and neutrophil-like cells, including transcriptional and epigenetic profiling. However, the major weakness of the study is that the authors do not provide rigorous or convincing data that the cells they derive are truly mature neutrophils. To the contrary, the neutrophil-like cells lack Ly6G expression and so the authors fall back on using CD11b as the primary marker for delineating neutrophils; however, CD11b is expressed by both myeloid progenitors and some premature and mature myeloid lineages that are not neutrophils. They acknowledge this shortcoming, but they make an assumption that Ly6G expression is the only way in which the cells they derive are different from primary neutrophils without presenting any evidence indicating such. The authors use only SCF during the maturation of ER-HoxB8 GMPs into leukocytes, rather than including other cytokines such as G-CSF (or use in vivo maturation) that could have better-induced differentiation and maturation into granulocytes/neutrophils. The authors did not use their transcriptional analyses to further establish that the cells they derive from ER-HoxB8 GMPs are similar/different from primary murine neutrophils. Unfortunately, this shortcoming means that all of the analyses of neutrophil-like cells derived from clonal GMPs may or may not represent the transcriptional, epigenetic, etc. profile of a true mature neutrophil. It is also not rigorously addressed whether what they call PMNs derived from clonal GMPs are a transcriptionally uniform population or if they harbor heterogeneity within the bulk population. Overall, while conceptually intriguing and in pursuit of an experimental system that would be impactful for the field, the study as performed has critical flaws.
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Author response:
Reviewer #1 (Public Review):
The heterogeneity within the neutrophil population is becoming clear. However, it was not clear if neutrophil progenitors are also heterogenous. Because neutrophils are short-lived, it is technically challenging to tackle the question. This study used a system to isolate and expand clonal neutrophil progenitors (granulocyte-monocyte progenitors; GMPs) to achieve molecular and functional profiling. In the study, transcriptional profiling was performed by RNAseq and ATACseq. Functional assays were performed ex vivo to examine phagocytosis, ROS production, NET formation, and neutrophil swarming using Candida albicans, as well as C. glabrata and C. auris. The strengths of this study include the use of the neutrophil clone system to track GMPs developing into neutrophils. The clone-based …
Author response:
Reviewer #1 (Public Review):
The heterogeneity within the neutrophil population is becoming clear. However, it was not clear if neutrophil progenitors are also heterogenous. Because neutrophils are short-lived, it is technically challenging to tackle the question. This study used a system to isolate and expand clonal neutrophil progenitors (granulocyte-monocyte progenitors; GMPs) to achieve molecular and functional profiling. In the study, transcriptional profiling was performed by RNAseq and ATACseq. Functional assays were performed ex vivo to examine phagocytosis, ROS production, NET formation, and neutrophil swarming using Candida albicans, as well as C. glabrata and C. auris. The strengths of this study include the use of the neutrophil clone system to track GMPs developing into neutrophils. The clone-based approach made it possible to evaluate the functions of multiple neutrophil subpopulations. Limitations of this study include the dependency on ex vivo approaches and the modest degree of heterogeneity within presented neutrophils. Nevertheless, the finding - the heterogeneity of neutrophils can be traced back to the GMP stage - is significant.
Reviewer #2 (Public Review):
The stated goal of the authors is to establish and characterize an experimental system to study neutrophil heterogeneity in a manner that allows for functional outcomes to be probed. To do so, they start with murine GMPs that are conditionally immortalized by ER-HoxB8 expression and make single-cell clonal populations to ask whether those GMPs or neutrophils derived by differentiating such clonal GMPs harbor heterogeneity. At a conceptual level, this is an innovative approach that could shed light on mechanisms of neutrophil heterogeneity that have been described in both health and disease. They perform bulk multi-omics and functional analyses of both the clonal GMPs and neutrophil-like cells, including transcriptional and epigenetic profiling. However, the major weakness of the study is that the authors do not provide rigorous or convincing data that the cells they derive are truly mature neutrophils. To the contrary, the neutrophil-like cells lack Ly6G expression and so the authors fall back on using CD11b as the primary marker for delineating neutrophils; however, CD11b is expressed by both myeloid progenitors and some premature and mature myeloid lineages that are not neutrophils. They acknowledge this shortcoming, but they make an assumption that Ly6G expression is the only way in which the cells they derive are different from primary neutrophils without presenting any evidence indicating such. The authors use only SCF during the maturation of ER-HoxB8 GMPs into leukocytes, rather than including other cytokines such as G-CSF (or use in vivo maturation) that could have better-induced differentiation and maturation into granulocytes/neutrophils.
Thank you. Of note, reviewer #1 also commented on the question of including other cytokines during the neutrophil differentiation process. We have included our response to reviewer #1 below, which includes the use of GM-CSF and IL-4.
“We have now demonstrated enhanced Ly6G expression with GM-CSF and IL-4 treatment in a new Supplementary Figure 1.
GMPs were washed out of estradiol-containing media and placed in fresh media containing 10 ng/ml GM-CSF and/or 1 ng/ml IL-4 for four days. Cells were collected and stained with CD117 (APC), F4/80 (AlexaFluor 488), Ly6G (PE), and CD11b (BV421). Neutrophil clones were run in biological triplicates, and undifferentiated GMPs were included as a negative control.
GMPs stain as CD117POS / F4/80NEG / Ly6GNEG / CD11bNEG, indicating they are immature. The clones removed from estradiol differentiate and lose their CD117 expression. The mature cells remain F4/80NEG, as expected for mature neutrophils.
The addition of GM-CSF to the media led to a significant increase in the expression of Ly6G. The addition of both GM-CSF + IL-4 did not further increase the proportion of Ly6G+ cells, and we have altered our statement slightly in the main text to reflect this finding (line 139).”
The authors did not use their transcriptional analyses to further establish that the cells they derive from ER-HoxB8 GMPs are similar/different from primary murine neutrophils. Unfortunately, this shortcoming means that all of the analyses of neutrophil-like cells derived from clonal GMPs may or may not represent the transcriptional, epigenetic, etc. profile of a true mature neutrophil.
Thank you. The ER-Hoxb8 system has been well-characterized by many authors at the function and at the transcriptional level, confirming that the cells highly reflect that same gene expression pattern as mature neutrophils. This was actually recently reviewed by Lail et al. (Traffic, 2022, PMID: 36117140). In terms of our analysis, we used transcriptional profiling to examine heterogeneity between different single-cell clones and not to re-validate the similarity with primary neutrophils.
It is also not rigorously addressed whether what they call PMNs derived from clonal GMPs are a transcriptionally uniform population or if they harbor heterogeneity within the bulk population.
Thank you. The reviewer poses an interesting, albeit challenging, question of whether even a single GMP clone can differentiate and result in mature neutrophil heterogeneity. To address this would require single cell sequencing of the resulting cells which we did not perform. We relied on single cell subcloning of the immature granulocyte monocyte progenitors to ensure a genetically identical clonal population. This was then additional confirmed by the retroviral insertional analysis. These analyses confirmed the clonal nature of our starting population, from which we posed the question of as whether the neutrophils derived from these clonal GMPs resulted in mature cells with consistent functional heterogeneity, which was indeed the case.
Overall, while conceptually intriguing and in pursuit of an experimental system that would be impactful for the field, the study as performed has critical flaws.
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