Expansion of CD14+HLA-DRneg/low monocytes and CD10neg neutrophils driving proinflammatory responses in patients with acute myocardial infarction

Background Immature myeloid cells expand in cancer, autoimmune diseases and viral infection, but their appearance and function after acute myocardial infarction (AMI) remain underexplored. Methods and Result Using flow cytometry, cell sorting and a mouse model of ischemia/reperfusion we investigated the role of immature granulocytic/monocytic cells in immune responses post-AMI. Seventy-one patients were categorized into unstable angina (n=11), Non-ST-elevation MI (NSTEMI, n=16), and ST-elevation MI (STEMI, n=44). Circulating CD14+HLA-DRneg/low monocytes and normal-density CD16+CD66b+CD10neg neutrophils are expanded in patients with large AMI and strongly correlated with cardiac damage, function and serum levels of S100A9/S100A8, MMP-9 and IL-1ß. Receiver operating characteristic curve analysis highlighted ability of CD14+HLA-DRneg/low and CD16+CD66b+CD10neg cells in discriminating acute coronary syndromes. CD14+HLA-DRneg/low monocytes did not suppress T-cell proliferation but expressed high amounts of IL1R1 and S100A9. Mechanistically, macrophages differentiated from CD14+HLA-DRneg/low monocytes produced more proinflammatory cytokines upon IFNγ stimulation as CD14+HLA-DRhigh monocyte-derived macrophages. CD10neg neutrophils expressed MMP9 and S100A9 at higher levels compared to CD10pos neutrophils. IFNγ production by CD4+ T-cells was increased in presence of CD10neg neutrophils. Remarkably, elevated circulating IFNγ levels were detected in cytomegalovirus-seropositive patients with expanded CD10neg neutrophils and increased frequency of CD4+CD28null T-cells. Lastly, we showed that murine homologs of human CD10neg neutrophils are Ly6GposCXCR2posCD11bdimCD101neg cells. CD101neg neutrophils are rapidly released into the bloodstream after AMI and migrate to ischemic sites, displaying increased expression of MMP-9 at 3 hours and of IL-1ß at 24 hours after reperfusion. Conclusion CD14+HLA-DRneg/low monocytes and normal-density CD10neg neutrophils inducing proinflammatory immune responses expand in patients with AMI.


INTRODUCTION
Despite advances in interventional therapies patients with large acute myocardial infarction (AMI) are at higher risk of heart failure morbidity and mortality. 1 Immunity and inflammation play a key role in the pathogenesis of ischemic heart failure and the complex role of immune cells during the wound healing process after injury is currently the focus of intensive research efforts. Understanding the immune mechanisms operating during AMI could pave the way to develop more effective strategies to prevent progressive dilative cardiac remodeling, functional deterioration and heart failure and to reduce cardiovascular adverse events.
HLA-DR neg/low monocytes and CD10 neg neutrophils expand in pathological conditions such as cancer, infection and inflammation, 2 and have recently been implicated in the pathogenesis of severe COVID-19, [3][4] but their role in immunoregulatory mechanisms operating during AMI remains largely unknown.
By integrating flow cytometry, cell sorting, and in vitro experiments this study investigated the appearance and function of HLA-DR neg/low monocytes and normal-density CD10 neg neutrophils in patients with AMI. We explored whether increased frequencies of HLA-DR neg/low monocytes and CD10 neg neutrophils are linked to circulating levels of G-CSF, S100A9/S100A8, MMP-9, NGAL, MPO, IL-6, TNFα and IL-1ß, immune regulators and acute inflammation markers. Moreover, we performed flow cytometric immunophenotyping of lymphocyte subsets and investigated circulating levels of IFNγ.
We found that CD14 + HLA-DR neg/low monocytes are not immunosuppressive but secrete high levels of TNFα, IL-6, and IL-1ß after differentiation to macrophages and IFNγ stimulation.
Further, our results point to a potential link among increased frequency of normal-density CD10 neg neutrophils, circulating CD4 + CD28 null T-cells and elevated IFNγ levels, especially in cytomegalovirus-seropositive patients. Lastly, using a mouse model of ischemia/reperfusion we showed that cardiac injury was associated with expansion of immature Ly6G pos CXCR2 pos CD11b dim CD101 neg cells in circulating blood and that immature neutrophils are a major source of MMP-9 and IL-1ß in the ischemic reperfused myocardium.

Patients and Study Design
The study protocol is in accordance with the ethical guidelines of the 1975 declaration of Helsinki and has been approved by the local ethics committee of Hannover Medical School.
Patient referred to your clinic for acute coronary syndrome (ACS) were included after providing written informed consent. Patients suffering from active malignant diseases or receiving immunosuppressive therapy were not included. Seventy-one patients (Table 1) were categorized into unstable angina (n=11), Non-ST-elevation MI (NSTEMI, n=16), and STelevation MI (STEMI, n=44). Left ventricular ejection fraction (LVEF) was measured in 2D echocardiographic studies using bi-plane Simpson's method. Seventeen healthy volunteers were recruited as control subjects.

Flow cytometry
Venous blood was collected in EDTA tubes, stored at room temperature and processed within 1 hour of collection. White blood cell count was measured by an automated hematology analyzer (XT 2000i, Sysmex). Serum was separated within 45 minutes and stored at −80°C.
For multiparameter flow cytometry whole blood (100μL) was incubated with fluorochromeconjugated antibodies for 30 minutes at room temperature in the dark, followed by lysis of red blood cells with Versalyse Lysing Solution® (Beckman Coulter). 5

Isolation of blood mononuclear cells and neutrophils
Peripheral blood was collected in EDTA tubes and mononuclear cells (PBMC) were isolated by density gradient centrifugation using Ficoll®-Paque Premium (GE Healthcare Biosciences

RNA was isolated from cells sorted in RTL Lysis
An expanded Methods section is available in the online-only Data Supplement.

Statistical Analysis
Data are presented as mean ± SEM, SD or as median [interquartile range] as indicated.
Normality of data was assessed by Shapiro-Wilk test. Non-parametric data from two independent groups were compared using Mann-Whitney U test and for more than two groups using Kruskal-Wallis test. Normal data were analyzed by one-way ANOVA with Tukey post hoc test. Pearson's r was used to test for correlation between two variables. Values of P≤0.05 were considered statistically significant.

Increased circulating levels of CD14 + HLA-DR neg/low monocytes in patients with acute MI.
Phenotypic characterization of monocytes and neutrophils was performed in whole blood from patients with unstable angina (UA) or acute MI within 24 to 72 hours of symptom onset (median 43.6 hours). A time-course analysis within the initial 24 hours and up to day 5 after MI is shown in Figure I in the online-only Data Supplement.
Next, we analyzed the immunoregulatory features of CD14 + HLA-DR neg/low monocytes. Using FACS-sorting, CD14 + HLA-DR neg/low /CD14 + HLA-DR high cells were isolated from blood of patients with AMI ( Figure 2A). Quantitative RT-PCR showed that CD14 + HLA-DR neg/low monocytes express high amounts of S100A9 and IL1R1 ( Figure 2B). Of interest, studies in heart failure patients have provided evidence for the presence of HLA-DR neg/low cells within myocardial tissue expressing high levels of S100A9. 7  Noticeable, CD10 neg neutrophils, which expand proportional to the degree of myocardial injury/dysfunction, also significantly correlated with circulating levels of MMP-9, S100A9/S100A8, NGAL, MPO, IL-6, and IL-1ß ( Figure IIIA  CD16 + CD66b + CD10 neg neutrophils co-purified with the erythrocyte fraction following density gradient centrifugation. Low-density neutrophils were not present in mononuclear cell fraction obtained from AMI patients. Cytospin slides were made after FACS-sorting to examine nuclear morphology ( Figure 4A). We found that the majority of the CD16 + CD66b + CD10 neg cells has an immature morphology with a lobular nucleus, while CD16 + CD66b + CD10 pos cells are mature neutrophils with segmented nuclei ( Figure 4A). These findings were obtained when neutrophils were isolated by dextran sedimentation as well as by negative selection using magnetic beads, indicating that the differences between the neutrophil subpopulations cannot be considered an artifact due to the isolation technique used. 8 CD10 neg neutrophils sorted from blood of AMI patients express higher amounts of MMP-9 and S100A9 than CD10 pos neutrophils ( Figure 4B). The significant positive correlation between the frequency of CD10 neg neutrophils and MMP-9/S100A9 serum levels A crucial role for neutrophils in the orchestration of adaptive immunity is emerging. [9][10] In an effort to define the immunoregulatory properties of CD10 neg neutrophils we found that IFNγ production by CD4 + T-cells was increased when co-cultured with CD10 neg neutrophils using a transwell system ( Figure 4C). Thus, normal-density CD10 neg neutrophils from MI patients may modulate CD4 + T-cell immune response in a cell contact-independent manner through soluble factors. Further studies aiming at deciphering the underlying mechanisms are mandatory.

Elevated circulating levels of IFNγ in cytomegalovirus-seropositive patients with expanded CD10 neg neutrophils and increased frequency of CD4 + CD28 null T-cells.
In a subgroup of patients we also performed flow cytometric immunophenotyping of lymphocyte subsets and investigated circulating levels of IFNγ ( Figure 5 and Table in the online-only Data Supplement). Patients with ACS were stratified in 4 subgroups based on frequency of CD10 neg neutrophils (≤10% or >10%) and frequency of CD4 + CD28 null T-cells (≤ 0.5% or >0.5%). Principal component analysis showed clustering according to elevated circulating levels of IFNγ, high levels of CD10 neg neutrophils and peripheral expansion of CD4 + CD28 null T-cells. The highest levels IFNγ were found in STEMI patients with expanded CD10 neg neutrophils (> 10%) and increased frequency of CD4 + CD28 null T-cells ( Figure 5A and   5B). Across all patients with ACS an increased frequency of CD4 + CD28 null T-cells was observed in CMV-seropositive individuals ( Figure 5D), indicating that the expansion of CD4 + CD28 null T-cells may not be a direct result of coronary events. Increased frequencies of CD28 null T-cells have been linked to cytomegalovirus (CMV) seropositivity. 11 Strikingly, when stratified according to CMV serostatus, the highest circulating levels of IFNγ were detected in CMV-seropositive STEMI patients displaying increased levels of CD10 neg neutrophils ( Figure 5D). Altogether, these findings suggest that CD10 neg neutrophils may play an important role in determining the type of T helper cell response, especially in cytomegalovirus-seropositive patients with expanded peripheral CD4 + CD28 null T cells.
Immature CD101 neg neutrophils are rapidly recruited to sites of cardiac injury during ischemia/reperfusion.  Figure 6B). A recent study in mice showed that banded neutrophils can be distinguished from mature neutrophils through CD101 expression and are associated with tumor progression. 12 We then asked whether CD101 neg neutrophils are recruited to site of ischemic injury after reperfusion. Flow cytometry analysis of immune cells isolated from the ischemic region 3 hours after reperfusion revealed infiltration of CD11b pos Ly6G pos CD101 neg neutrophils, displaying increased expression of the matrix-degrading protease MMP-9 ( Figure 6C). Moreover, we found that 24 hours after reperfusion CD11b pos Ly6G pos CD101 neg neutrophils expressed IL-1ß at higher levels compared to CD11b pos Ly6G pos CD101 pos cells ( Figure 6D). These findings suggest that immature neutrophils may play an important role in the ischemic reperfused myocardium.

DISCUSSION
This study uncovered that CD10 can be used as a surface marker to identify the banded neutrophil subset that expands and drives proinflammatory effects in patients with AMI. We believe CD10 neg neutrophils to derive from MI-induced altered myelopoiesis. Both mature and immature neutrophils are released presumably to meet the high demand for more neutrophils, especially in patients with large MI. Increased systemic levels of G-CSF, the primary regulator of emergency granulopoiesis, [9][10] are likely to drive the production/release of proinflammatory neutrophils from the bone marrow into the circulation. Accordingly, G-CSFmediated immature CD10 neg neutrophil expansion may be an adverse consequence of treating AMI patients with G-CSF. However, neutrophils may be released from the bone marrow in response to increased damage-associated molecular patterns such as S100A8/S100A9, secreted from necrotic/necroptotic cells and neutrophils as mediators of sterile inflammation. 13 We found that circulating CD10 neg neutrophils express high amounts of S100A9, indicating banded neutrophils as a major source for this alarmin during AMI.
Under inflammatory conditions neutrophils traffic to inflamed tissues as well as to draining lymph nodes 9, 14 modulating T cell-mediated immune responses. Recently, CD10 has been proposed as a marker that distinguishes mature from immature neutrophils in healthy volunteers receiving G-CSF for stem cell mobilization. 15 Immunostimulatory CD10 neg neutrophils and immunosuppressive CD10 pos appear in the circulation of G-CSF-treated donors. However, human neutrophils can mimic myeloid-derived suppressor cells and suppress T-cell proliferation through artefactual mechanisms. 16 Of note, by performing transwell experiments we found that IFNγ production by CD4 + T-cells was increased in the presence of CD10 neg neutrophils. In systemic lupus erythematosus immature granulocytes have been shown to display a proinflammatory phenotype and to induce the production of IFNγ from CD4 + T-cells by a contact-independent mechanism. 17 Several cellular and molecular mechanisms may lead to increased circulating levels of IFNγ after AMI. Our principal component analysis showed clustering according to elevated circulating levels of IFNγ, high levels of CD10 neg neutrophils and peripheral expansion of CD4 + CD28 null T-cells. The function/role of CD4 + CD28 null T-cells in coronary artery disease and atherogenesis is far from clear. CD4 + CD28 null T-cells are long-lived cytotoxic T helper cells, secreting high levels of IFNγ. 18 A recent study revealed complex associations between of CD4 + CD28 null T-cells and cardiovascular disease. 19 CD4 + CD28 null T cells are associated with a lower risk for first-time coronary events in a population-based cohort. In contrast, in patients with advanced atherosclerotic disease an increased frequency of CD4 + CD28 null T-cells was associated with more frequent major adverse cardiovascular events. 19 Noteworthy, in our study the expansion of CD4 + CD28 null T-cells was closely linked to CMV infection across all patients with ACS. CMV has been associated with atherosclerosis and increased risk for cardiovascular diseases. Recent clinical data showed that myocardial ischemia in CMVseropositive patients leads to significant changes in the composition of the CD8 + T-cell repertoire, accelerating immunosenescence. 20 Overall, our findings point to a potential link among increased frequency of normal-density CD10 neg neutrophils, circulating CD4 + CD28 null T-cells and elevated IFNγ levels, especially in cytomegalovirus-seropositive patients. Thus, determination of circulating CD10 neg neutrophils levels, particularly in the context of CMV infection, might help to identify patients at risk for excessive inflammatory immune response.
In spite of numerous studies on polymorphonuclear myeloid cells the role of immature neutrophils is underexplored in the setting of AMI in mice. We found that CD101 can be used as a surface marker to identify the banded neutrophil subset among the heterogeneous CD11b pos Ly6G pos neutrophil populations in blood and infarct tissues during ischemia/reperfusion. Previous studies in a human model of experimental endotoxemia showed that banded neutrophils exhibit efficient migration to sites of infection. 21 Moreover, developmental analysis of bone marrow neutrophils revealed that immature neutrophils are recruited to the periphery of tumor-bearing mice. 12 Of note, we found that CD101 neg neutrophils are released into the bloodstream within minutes after reperfusion and are capable of efficient migration to ischemic tissues, displaying increased expression of MMP-9 and IL-1ß at 3 and 24 hours after reperfusion, respectively. There are significant differences between mouse and human immunology 22 and the transit time of leukocytes may be quite different. 23 During homeostasis, trafficking of neutrophils/myeloid cells from bone marrow into the circulation takes between 1-2 days in mice and 5-8 days in humans. 23 Such differences should be considered when comparing animal and human studies on immune mechanisms underlying wound healing.
The recruitment of immune cells to sites of tissue repair is a complex process involving cytokines, chemokines, and interactions between infiltrating immune cells. CD14 + HLA-DR neg/ low monocytes from patients with AMI are not immunosuppressive but express high amounts of IL1R1. Thus, immature neutrophils, as an important source of IL1ß in the reperfused heart, may be actively involved in the recruitment of CD14 + HLA-DR neg/low monocytes. Saxena et al. 24 showed that IL1R1 signaling mediates early recruitment of Ly6C hi monocytes to the infarcted myocardium. Reperfused myocardial infarction had intense infiltration with Ly6C hi monocytes expressing IL1R1 that peaked after 24 hours of reperfusion. 24 Noteworthy, recent studies demonstrated that the failing human heart also contains HLA-DR neg/low monocytes. 7 Several immune mechanisms operate during cardiac wound healing and IFNγ plays different roles depending on the cellular and microenvironmental context intrinsically linked to the stages of ischemic injury. By integrating cell sorting and in vitro experiments we found that macrophages differentiated from CD14 + HLA-DR neg/low monocytes produced more TNFα, IL-6, and IL-1ß upon IFNγ stimulation as CD14 + HLA-DR high monocyte-derived macrophages.
These findings may support a role for CD14 + HLA-DR neg/low cells in pathogenic mechanisms operating during AMI and may, at least in part, explain why increased immature monocytic cells frequency correlate with circulating levels of TNFα, IL-6, and IL-1ß.
The interleukin-1 pathway has been shown to play a key pathogenetic role in post-MI inflammation and the progression to heart failure. 25 Our in vitro mechanistic experiments with neutrophils/monocytes from patients with AMI as well as mouse studies provide a potential linkage between the induction of immature myeloid cells and increased interleukin-1 activity during AMI. Emerging evidences highlight that targeting interleukin-1 may hold promise for patients after MI. 26 In STEMI patients the interleukin-1 receptor antagonist anakinra significantly reduced the systemic inflammatory response. Moreover, in the CANTOS trial, administration of canakinumab (a monoclonal antibody targeting IL1β) prevented the recurrence of ischemic events, reduced heart failure-related hospitalizations and mortality in patients with prior AMI. 26 In summary, we demonstrate that CD14 + HLA-DR lo/neg monocytes and normal-density      Relative RNA expression of MMP9 and S100A9 in CD10 neg versus CD10 pos neutrophils. (C) IFNγ production by CD4 + T cells stimulated with anti-CD3/CD28 beads and co-cultured for 48 hours in presence/absence of CD10 neg or CD10 pos neutrophils using a transwell system. CD10 neg /CD10 pos neutrophils were isolated by flow-cytometric sorting from patients with AMI (n=5). *P<0.05.