Expansion of CD10neg neutrophils and CD14+HLA-DRneg/low monocytes driving proinflammatory responses in patients with acute myocardial infarction
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Evaluation Summary:
This paper will be of broad interest to cardiologist and scientists studying acute myocardial infarction (AMI), especially to those focussing on the immune responses during AMI. Using combination of in vivo and in vitro model, as well as tissue from patients, the authors reveal new insights regarding the immune mechanisms during AMI, highlighting the importance of neutrophils and monocytes during the early days of its process. The findings in this paper add to the understanding of how immune mechanisms may contribute to subsequent adverse events after AMI.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors)
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Abstract
Immature neutrophils and HLA-DR neg/low monocytes expand in cancer, autoimmune diseases and viral infections, but their appearance and immunoregulatory effects on T-cells after acute myocardial infarction (AMI) remain underexplored. We found an expansion of circulating immature CD16 + CD66b + CD10 neg neutrophils and CD14 + HLA-DR neg/low monocytes in AMI patients, correlating with cardiac damage, function and levels of immune-inflammation markers. Immature CD10 neg neutrophils expressed high amounts of MMP-9 and S100A9, and displayed resistance to apoptosis. Moreover, we found that increased frequency of CD10 neg neutrophils and elevated circulating IFN-γ levels were linked, mainly in patients with expanded CD4 + CD28 null T-cells. Notably, the expansion of circulating CD4 + CD28 null T-cells was associated with cytomegalovirus (CMV) seropositivity. Using bioinformatic tools, we identified a tight relationship among the peripheral expansion of immature CD10 neg neutrophils, CMV IgG titers, and circulating levels of IFN-γ and IL-12 in patients with AMI. At a mechanistic level, CD10 neg neutrophils enhanced IFN-γ production by CD4 + T-cells through a contact-independent mechanism involving IL-12. In vitro experiments also highlighted that HLA-DR neg/low monocytes do not suppress T-cell proliferation but secrete high levels of pro-inflammatory cytokines after differentiation to macrophages and IFN-γ stimulation. Lastly, using a mouse model of AMI, we showed that immature neutrophils (CD11b pos Ly6G pos CD101 neg cells) are recruited to the injured myocardium and migrate to mediastinal lymph nodes shortly after reperfusion. In conclusion, immunoregulatory functions of CD10 neg neutrophils play a dynamic role in mechanisms linking myeloid cell compartment dysregulation, Th1-type immune responses and inflammation after AMI.
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Reviewer #3 (Public Review):
This is a well-executed study with interesting and novel findings. The main strength is the combined use of well-executed flow cytometry studies in human patients with MI and in vitro experiments to suggest a role for immature neutrophils in infarction. The main weakness is the descriptive/associative nature of the data. What is lacking is in vivo experimentation documenting the proposed pro-inflammatory role of immature neutrophils. This limits the conclusions. The following specific concerns are raised:
Major:
1.In some cases, conclusions are not supported by robust data. For example, the authors conclude that CD14+HLA-DRneg/lo monocytes play a crucial role in post-infarction inflammation based exclusively on in vitro experiments. Moreover, conclusions regarding the pro-inflammatory role of immature …
Reviewer #3 (Public Review):
This is a well-executed study with interesting and novel findings. The main strength is the combined use of well-executed flow cytometry studies in human patients with MI and in vitro experiments to suggest a role for immature neutrophils in infarction. The main weakness is the descriptive/associative nature of the data. What is lacking is in vivo experimentation documenting the proposed pro-inflammatory role of immature neutrophils. This limits the conclusions. The following specific concerns are raised:
Major:
1.In some cases, conclusions are not supported by robust data. For example, the authors conclude that CD14+HLA-DRneg/lo monocytes play a crucial role in post-infarction inflammation based exclusively on in vitro experiments. Moreover, conclusions regarding the pro-inflammatory role of immature neutrophils are based on in vitro data and associative studies.
2.Immature neutrophils have a short lifespan. Information on the fate of immature neutrophils in the infarct is lacking. The in vivo mouse model may be ideal to address whether immature neutrophils undergo apoptosis or mature within the infarct environment
3.The rationale for selective assessment of specific genes and for the specific neutrophil-lymphocyte co-culture system is unclear. In neutrophils, the basis for selective assessment of some specific genes (MMP9, IL1R1, IL1R2, STAT3 etc), vs. other inflammatory genes known to be expressed at high levels by neutrophils is not explained. Similarly, the rationale for the experiment examining interactions of CD10neg neutrophils with T cells is not clear. Considering the effects of neutrophils on macrophage phenotype and on cardiomyocytes, study of interactions with other cell types may have made more sense.
4.The concept of CMV seropositivity is suddenly introduced without a clear rationale. The data show infiltration of the infarcted heart with immature neutrophils and CD14+HLA-DRneg monocytes. One would have anticipated more experiments investigating the (proposed) role of these cells in the post-infarction inflammatory response, rather than comparison of CMV+ vs negative patients.
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Reviewer #2 (Public Review):
In this study, Fraccarollo and colleagues describe the existence and higher prevalence of subpopulations of immature monocytes and neutrophils with pro-inflammatory responses in patients with acute myocardial infarction. CD14+HLA-DRneg/low monocytes and CD16+CD66b+CD10neg neutrophils correlate with markers of systemic inflammation and parameters of cardiac damage. In particular in patients positive for cytomegalovirus and elevated levels of CD4+CD28null T cells, the expansion of immature neutrophils associates with increased levels of circulating IFNg. Mechanistically, immature neutrophils regulate T-cell responses by inducing IFN release through IL-12 production in a contact-independent manner. Besides, CD14+HLA-DRneg/low monocytes differentiate into macrophages with a potent pro-inflammatory phenotype …
Reviewer #2 (Public Review):
In this study, Fraccarollo and colleagues describe the existence and higher prevalence of subpopulations of immature monocytes and neutrophils with pro-inflammatory responses in patients with acute myocardial infarction. CD14+HLA-DRneg/low monocytes and CD16+CD66b+CD10neg neutrophils correlate with markers of systemic inflammation and parameters of cardiac damage. In particular in patients positive for cytomegalovirus and elevated levels of CD4+CD28null T cells, the expansion of immature neutrophils associates with increased levels of circulating IFNg. Mechanistically, immature neutrophils regulate T-cell responses by inducing IFN release through IL-12 production in a contact-independent manner. Besides, CD14+HLA-DRneg/low monocytes differentiate into macrophages with a potent pro-inflammatory phenotype characterized by the release of pro-inflammatory cytokines upon IFNg stimulation.
This very interesting study provides new insights into the diversity and complexity of myeloid populations and responses in the context of cardiac ischemia. It is technically well performed and the results sufficiently support the conclusions of the study.
Strengths
The authors provide a detailed analysis of the phenotype and function of two subpopulations of CD14+HLA-DRneg/low monocytes and CD16+CD66b+CD10neg neutrophils in the context of acute myocardial infarction (AMI). Extensive phenotyping of these immune populations at different time-points after the onset of the disease provides strong correlations with multiple parameters of inflammation and severity of the disease. Hence, these subpopulations emerge as biomarkers of heart ischemic diseases with predictive potential. Using in vitro approaches, the authors support these correlations with mechanistic analyses of the inflammatory and immunomodulatory function of these populations. Finally, the authors use mouse models of ischemia-reperfusion injury to mimic the conditions observed in the AMI patients and supporting the pro-inflammatory role of immature neutrophils in this disease.
Weaknesses
The associations between immature neutrophils, IFNg, and CD4+CD28null T cells found in AMI patients positive for cytomegalovirus are not well supported by the mechanistic findings observed in vitro. Here, the induction of IFNg production by immature neutrophils is restricted to CD4+CD28+ T cells but not CD4+CD28null T cells.
The experimental data obtained from mouse models of AMI to support their findings in humans would require a more extensive study. Causality between the expansion of these immature populations and the course of the disease is missing. Also, although expected, substantial differences are found between equivalent subpopulations in mice and humans thus limiting the relevance of the mouse data.
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Reviewer #1 (Public Review):
In this paper, the authors tried to investigate complex roles of immune cells during acute myocardial infarction (AMI) by examining immune cells in blood samples from acute coronary syndrome (ACS) patients. They found an increase in the circulating levels of CD14+HLA-DRneg/low monocytes and CD16+CD66b+CD10neg neutrophils in the blood of ACS patients compared to healthy people, all of which were correlated with elevated levels of inflammatory markers in serum. Those findings were then further explored at a mechanistic level by using in vitro and in vivo experiments. Interestingly, the researchers also found that high cytomegalovirus (CMV) antibody titers could affect the immunoregulatory mechanisms in AMI patients. Taken together, the findings of the researchers could potentially contribute to the development …
Reviewer #1 (Public Review):
In this paper, the authors tried to investigate complex roles of immune cells during acute myocardial infarction (AMI) by examining immune cells in blood samples from acute coronary syndrome (ACS) patients. They found an increase in the circulating levels of CD14+HLA-DRneg/low monocytes and CD16+CD66b+CD10neg neutrophils in the blood of ACS patients compared to healthy people, all of which were correlated with elevated levels of inflammatory markers in serum. Those findings were then further explored at a mechanistic level by using in vitro and in vivo experiments. Interestingly, the researchers also found that high cytomegalovirus (CMV) antibody titers could affect the immunoregulatory mechanisms in AMI patients. Taken together, the findings of the researchers could potentially contribute to the development of a more effective strategy to prevent cardiac deterioration and cardiovascular adverse events after AMI.
Strengths:
This paper contains novel insight regarding role of neutrophil and monocyte subset in pathophysiology of AMI. Although the increased level of CD10neg subsets of neutrophils in AMI patients has recently been reported (Marechal, P., et al. 2020. Neutrophil phenotypes in coronary artery disease. Journal of Clinical Medicine), the current paper aptly complemented the previous findings obtained by using its in vitro and in vivo mice model. This study also has robust methods to support their conclusion.
Weakness:
To further improve the strength of their conclusion, the experiments investigating the effects of immunoregulatory function of immature neutrophils and HLA-DRneg/low monocytes subsets would be advised.
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Evaluation Summary:
This paper will be of broad interest to cardiologist and scientists studying acute myocardial infarction (AMI), especially to those focussing on the immune responses during AMI. Using combination of in vivo and in vitro model, as well as tissue from patients, the authors reveal new insights regarding the immune mechanisms during AMI, highlighting the importance of neutrophils and monocytes during the early days of its process. The findings in this paper add to the understanding of how immune mechanisms may contribute to subsequent adverse events after AMI.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors)
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