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

Immature neutrophils and HLA-DRneg/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+CD10neg neutrophils and CD14+HLA-DRneg/low monocytes in AMI patients, correlating with cardiac damage, function and levels of immune-inflammation markers. Immature CD10neg neutrophils expressed high amounts of MMP-9 and S100A9, and displayed resistance to apoptosis. Moreover, we found that increased frequency of CD10neg neutrophils and elevated circulating IFN-γ levels were linked, mainly in patients with expanded CD4+CD28null T-cells. Notably, the expansion of circulating CD4+CD28null T-cells was associated with cytomegalovirus (CMV) seropositivity. Using bioinformatic tools, we identified a tight relationship among the peripheral expansion of immature CD10neg neutrophils, CMV IgG titers, and circulating levels of IFN-γ and IL-12 in patients with AMI. At a mechanistic level, CD10neg neutrophils enhanced IFN-γ production by CD4+ T-cells through a contact-independent mechanism involving IL-12. In vitro experiments also highlighted that HLA-DRneg/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 (CD11bposLy6GposCD101neg cells) are recruited to the injured myocardium and migrate to mediastinal lymph nodes shortly after reperfusion. In conclusion, immunoregulatory functions of CD10neg neutrophils play a dynamic role in mechanisms linking myeloid cell compartment dysregulation, Th1-type immune responses and inflammation after AMI.


Introduction
Despite advances in interventional therapies patients with large acute myocardial infarction (AMI) are at higher risk of heart failure morbidity and mortality (Heusch and Gersh, 2017). 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 adverse cardiovascular events.
HLA-DR neg/low monocytes and immature neutrophils expand in pathological conditions such as cancer, infection, and inflammation, (Gabrilovich and Nagaraj, 2009) and have recently been implicated in the pathogenesis of severe COVID-19 (Silvin et al., 2020;Deutsche COVID-19 OMICS Initiative (DeCOI) et al., 2020). A rapid depression of monocytic HLA-DR expression was observed in patients with AMI (Haeusler et al., 2012). Maréchal et al., 2020 studying neutrophil phenotypes in acute coronary syndrome found increased percentage of immature low density neutrophils in patients with ST-elevation MI. However, the role of immature neutrophils as well as HLA-DR neg/low monocytes in immune mechanisms operating during AMI remains largely unexplored.
By integrating flow cytometric immunophenotyping of monocyte, neutrophil, and lymphocyte subsets, ex vivo experiments with sorted cells as well as bioinformatic tools this study investigated the appearance and the functional immune properties of immature neutrophils and HLA-DR neg/low monocytes in patients with AMI. Moreover, we explored whether increased frequency of immature neutrophils and HLA-DR neg/low monocytes are linked to circulating levels of immune regulators and acute inflammation markers such as G-CSF, S100A9/S100A8, MMP-9, NGAL, MPO, IL-6, TNF-a, IL-1ß and IFN-g.
Since a crucial role for immature neutrophils in the orchestration of adaptive immunity has recently emerged, we mostly focused on neutrophil-mediated regulation of T-cell response. Environmental factors especially cytomegalovirus (CMV) infection are likely to provide a significant contribution to functional diversity of T-cells. Therefore, we also investigated the impact of CMV on CD4 + T-cell homeostasis and on the immunoregulatory properties of immature neutrophils derived from patients with AMI.
In addition, using a mouse model of reperfused AMI we addressed whether immature neutrophils migrate into the injured myocardium and populate heart-draining lymph nodes in response to acute ischemic injury.

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. Patients referred to our department 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 (UA, n=11), Non-ST-elevation MI (NSTEMI, n=16), and ST-elevation MI (STEMI,n=44). Left ventricular (LV) ejection fraction 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 hr of collection. White blood cell count was measured by an automated hematology analyzer (XT 2000i, Sysmex). Serum was separated within 45 min and stored at À80˚C. For multiparameter flow cytometry whole blood (100 mL) was incubated with fluorochrome-conjugated antibodies for 30 min at room temperature in the dark, followed by lysis of red blood cells with Versalyse Lysing Solution (Beckman Coulter) (Haghikia et al., 2018). Finally, the cells were washed twice with Hanks 0 Balanced Salt solution (4 mL). For cell sorting or flow cytometric analysis of monocyte-derived macrophages, cells were resuspended in ice-cold FACS-staining buffer (PBS, supplemented with 0.5% bovine serum albumin and 2 mM EDTA) and immunostaining was performed on ice. The following antibodies were used: anti-CD14 (

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). CD14 + HLA-DR neg/low /CD14 + HLA-DR high monocytes were FACS-sorted from PBMC. Granulocytes/neutrophils were isolated from the erythrocyte fraction by dextran sedimentation or from whole blood by immunomagnetic selection (

T-cell proliferation assays in presence of monocytes
Isolation of CD3 + T-cells was performed using Dynabeads Untouched Human T-cells Kit (11344D, Invitrogen). CD3 + T-cells were stained with CellTrace Violet Cell Proliferation Kit (C34571; Invitrogen) and resuspended at 1x10 6 /mL in T-Cell Activation Medium (OpTmizer CTS T-Cell Expansion culture medium supplemented with L-glutamine/PenStrep). CD3 + T-cells were co-cultured in 96-well plates with CD14 + HLA-DR neg/low and CD14 + HLA-DR high monocytes at a ratio of 1 to 1 (T-cells: monocytes). T-cells were stimulated with Dynabeads Human T-Activator CD3/CD28 (11131D; Gibco) and T-cell proliferation was assessed 4 days later by CellTrace Violet dilution by flow cytometry.

Chemotaxis assay
Chemotaxis of CD14 + HLA-DR neg/low /CD14 + HLA-DR high monocytes was analyzed with the ChemoTx Disposable Chemotaxis System (NeuroProbe, NRP-106-8; Hö lzel Diagnostika) (Chen et al., 2015). Human S100A9 (A42590, Invitrogen) was diluted in RPMI 1640 medium and placed in the lower chamber. Cells were washed with RPMI 1640 medium and preincubated for 30 min at 37˚C before addition to the upper chamber. Monocytes were allowed to migrate for 3 hr and migration was analyzed by flow cytometry. The migration index was defined as the number of cells migrating in response to S100A9 divided by the number of cells migrating in response to medium alone (Chen et al., 2015). T-cell activation assays in presence of CD10 neg /CD10 pos neutrophils CD4 + T-cells were isolated from PBMC using the MojoSort Human CD4 T Cell Isolation Kit (480009; BioLegend) or by flow-cytometric sorting. The CD28 MicroBead Kit (130-093-247; Miltenyi Biotec) was used for isolation of CD4 + CD28 null T-cells from PBMC. CD4 + T-cells and CD4 + CD28 null T-cells were resuspended at 1x10 6 /mL in T-Cell Activation Medium and stimulated with Dynabeads Human T-Activator CD3/CD28. For transwell experiments CD4 + T-cells and CD10 neg /CD10 pos neutrophils were co-cultured in 24 well plates at a ratio of 1 to 2 (T-cells: neutrophils) for 24 hr. CD10 neg / CD10 pos neutrophils were cultured in 0.4 mm transwell inserts (140620, Thermo Scientific) and CD4 + T-cells in the well beneath the insert. In some experiments, CD10 neg /CD10 pos neutrophils were cultured overnight in T-Cell Activation Medium. The cell-free supernatants derived from CD10 neg / CD10 pos neutrophils were added to CD4 + T-cells cultured in 96-well plates (8Â10 4 cells/well) in the presence of neutralizing anti-IL-12 antibody (4 mg/mL; MAB219, R and D Systems; RRID:AB_ 2123616) or isotype control (4 mg/mL; MAB002, R and D Systems; RRID:AB_357344). CD4 + CD28 null T-cells were cultured with cell-free supernatants derived from CD10 neg neutrophils. Culture supernatants were collected after 24 hr incubation.

RT-quantitative PCR
RNA was isolated from cells sorted in RTL Lysis Buffer using the RNeasy Plus Mini Kit (QIAGEN) according to the manufactures' protocol. RNA quantification and quality testing were assessed by NanoDrop 2000 (Thermo Fisher Scientific) and Bioanalyzer 2100 (Agilent). cDNA synthesis was performed using 3 ng (human-neutrophils), 1 ng (mouse-neutrophils), and 10 ng (monocytes) of total RNA and iScript Reverse Transcription Supermix (Bio-Rad). Relative quantitation of mRNA expression levels was determined with CFX96 Touch Real Time PCR using SsoAdvanced Universal SYBR Green Supermix and PrimePCR Primers (Bio-Rad). ß-actin (ACTB) was chosen as an endogenous control. PCR amplification was performed at initially 95˚C for 30 s followed by 40 cycles at 95˚C for 5 s and terminated by 60˚C for 30 s. The delta-delta Ct method was employed for data analysis.

Mouse model of reperfused AMI
Myocardial ischemia was induced by transient left coronary artery ligation in age-and gendermatched mice. Briefly, mice were anesthetized with 2% isoflurane in a 100% oxygen mix, intubated, and ventilated using a ventilator (MINIVENT mouse ventilator model 845) with the tidal volume adjusted based on body weight (10 mL/g BW). Buprenorphine (0.1 mg/kg BW) was intraperitoneally administered for postoperative pain relief. The left coronary artery was ligated with a 6-0 silk suture just below the left auricular level (Fraccarollo et al., 2019;Galuppo et al., 2017;Fraccarollo et al., 2017;Fraccarollo et al., 2011). The suture was passed through a segment of PE-10 tubing. One hour after ischemia the tube was removed to allow for reperfusion. In sham-operated control mice, the ligature around the left anterior descending coronary artery was not tied.

RNA-Seq
Total RNA was isolated using PrepEase RNA Spin Kit according to the manufacturer's instructions (Fraccarollo et al., 2019;Galuppo et al., 2017). Sorted cells were directly collected in lysis buffer and immediately processed. RNA quantification and quality testing were assessed by NanoDrop 2000 (Thermo Fisher Scientific) and Bioanalyzer 2100 (Agilent). Libraries for RNA sequencing were prepared from 30 ng total RNA; from each sample, polyA RNA was purified, converted to cDNA and linked to Illumina adapters using the Illumina TruSeq stranded mRNA Kit according to the manufacturer's instructions. Samples were multiplexed and sequenced on an Illumina NextSeq 500 in a 75 nt single end setting using a high-output run mode. Raw BCL files were demultiplexed and converted to sample-specific FASTQ files using bcl2fastq v1.8.4 (Illumina). Residual adapter sequences present in the sequencing reads were removed with Cutadapt version 1.12. Reads (~40 million per sample) were aligned to the mouse reference sequence GENCODE vM8 using STAR version 2.5.2b. RNA sequencing data analysis was undertaken with the statistical programming language, R. The R package DeSeq2 (v1.14.1) was used to evaluate differential gene expression (Fraccarollo et al., 2019;Galuppo et al., 2017).

Statistical analysis
Data are presented as mean ± SEM or as median [interquartile range] as indicated. Normality of data was assessed by Shapiro-Wilk test. Normal data were analyzed by one-way ANOVA with Tukey post hoc test, otherwise by Kruskal-Wallis test for comparisons of median values, with Mann-Whitney U test for multiple comparisons. Unpaired t-test or Mann-Whitney U test was used to compare two independent groups. Linear regression analysis or Spearman's rank correlation test was used to determine relationship between variables. Values of p 0.05 were considered statistically significant.

Results
Increased circulating levels of CD14 + HLA-DR neg/low monocytes in patients with acute MI Flow cytometric immunophenotyping was performed in whole blood from patients with unstable angina (UA) or acute MI within 24-72 hr of symptom onset (median 43.6 hr). A time course analysis of monocyte subset-frequencies and circulating levels up to day five after MI is shown in Figure 1figure supplement 1.
NSTEMI/STEMI patients displayed significantly higher absolute neutrophil and monocyte counts versus UA patients ( Table 2). Based on HLA-DR/CD14/CD16 expression, monocytes can be divided into different subsets. We detected increased circulating levels of intermediate (HLA-DR ++ CD14 + + CD16 + CX3CR1 + ) in ACS patients versus control, and of non-classical (HLA-DR + CD14 + CD16 + + CX3CR1 ++ ) in STEMI versus UA patients and controls (  There were no significant correlations between intermediate/non-classical monocytes and LV ejection fraction/CK max . We found increased percentages and absolute numbers of circulating CD14 + HLA-DR neg/low monocytes in STEMI/NSTEMI patients as compared to UA patients ( Figure 1A and B and Figure 1figure supplement 2B). Linear regression analysis revealed a positive correlation between circulating levels of CD14 + HLA-DR neg/low monocytes and CK max ( Figure 1C) and a negative correlation with LV ejection fraction (R=0.44, p<0.001).
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 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 (Bajpai et al., 2018).
CD14 + HLA-DR neg/low monocytes did not suppress T-cell proliferation ( Figure 2C), indicating that the expanded population of monocytic cells in infarct patients are not immunosuppressive. Remarkably, macrophages differentiated from CD14 + HLA-DR neg/low monocytes by 4-day culture with M-CSF produced more TNF-a, IL-6, and IL-1ß upon stimulation with IFN-g, as compared to macrophages generated from monocytes CD14 + HLA-DR high ( Figure 2D-F).
These results may indicate an important role for CD14 + HLA-DR neg/low monocytes in the inflammatory response during AMI.
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. Recently, the work of Maréchal et al., 2020 found higher frequency of  immature low-density neutrophils in STEMI patients. However, cancer patients were not excluded which might have influenced the proportion of low-density CD10 neg cells.
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 (Hardisty et al., 2021). Of note, linear regression analysis revealed a strong positive correlation between the percentages of CD16 + CD66b + CD10 neg cells and circulating levels of G-CSF ( Figure 4B). AMI patients with higher systemic concentrations of G-CSF have increased CD10 neg neutrophils levels, suggesting G-CSF-driven immature neutrophil release/expansion. Immature CD10 neg neutrophils from patients with AMI express high amounts of MMP-9 and S100A9 and display resistance to apoptosis In an attempt to identify-specific surface antigens that characterize normal-density CD10 neg neutrophils derived from AMI patients, we next examined the expression of markers associated with neutrophil maturation/activation (Hardisty et al., 2021;Silvestre-Roig et al., 2019). Flow cytometry showed that CD11b, CD101, CD114, and CD177 were expressed at equivalent levels on CD10 neg neutrophils versus CD10 pos neutrophils ( Figure 4C).
Next, we investigated the expression of inflammatory mediators playing a pathophysiological role in wound healing after AMI. CD10 neg neutrophils sorted from blood of AMI patients express higher amounts of MMP-9 and S100A9 than CD10 pos neutrophils ( Figure 4D Mouse neutrophils, unlike human granulocytes, lack CD10 expression (Kalled et al., 1995) Using next-generation RNA sequencing we identified CD101 among the genes down-regulated by ischemia in circulating neutrophils (Figure 4-figure supplement 1B).
Next, we found that CD101 can be used as a surface marker to identify the mature neutrophil subset among the heterogeneous Ly6G pos CXCR2 pos neutrophil populations, released into the bloodstream after ischemia/reperfusion (Figure 4-figure supplement 2A and B). As revealed by morphological analysis circulating CD11b bright CD101 pos neutrophils have a mature morphology, whereas CD11b dim CD101 neg cells are immature neutrophils with ring-shaped nuclei (Figure 4-figure supplement 2B). Recent study in mice showed that CD101 segregates immature neutrophils from mature neutrophils during G-CSF stimulation and in the tumor setting (Evrard et al., 2018). Notably, the levels of CD101 neg neutrophils in the bone marrow of mice subjected to ischemia followed by 6 hr of reperfusion were markedly reduced (Figure 4-figure supplement 2C), indicating early release of immature neutrophils from bone marrow into the circulation after AMI. Moreover, we found that CD11b pos Ly6G pos CD101 neg cells sorted from peripheral blood after AMI express higher amounts of Irg1 (Acod1), Il1r1, Mmp8, Nos2, and Stat3 than CD101 pos neutrophils and showed markedly reduced CD101 expression (Figure 4-figure supplement 1D and E). Taken together, our results emphasize interspecies differences in immune responses after ischemic injury, suggesting caution in applying findings in murine models of myocardial ischemia/reperfusion to patients with AMI.
To further define the functional properties of the immature neutrophil populations derived from AMI patients we investigated spontaneous apoptosis. The apoptotic rate of CD10 neg neutrophils was substantially reduced compared with CD10 pos neutrophils after 24 hr in vitro culture as determined by flow cytometric analysis using Vybrant DyeCycle Violet/SYTOX AADvanced stain ( Figure 4E) as well as Apotracker Green (Figure 4-figure supplement 3A). The decreased apoptosis rate of CD10 neg neutrophils was reflected by an increase in the percentage of live cells. Morphological assessment of cytospins (Figure 4-figure supplement 3B) confirmed the results obtained by flow cytometry.
Neutrophils may play a crucial role in cardiac healing after AMI by influencing macrophage polarization (Horckmans et al., 2017). Horckmans et al., 2017 found an induction of MerTK expression in macrophages (differentiated from peripheral blood monocytes of healthy donors) exposed to IL-4 in the presence of neutrophil supernatants from healthy individuals. Therefore, we next assessed MerTK expression on macrophages differentiated from CD14 + HLA-DR neg/low and CD14 + HLA-DR high monocytes and polarized in the presence of supernatants derived from CD10 neg neutrophils as well as CD10 pos neutrophils (Figure 4-figure supplement 3C and D). CD14 + HLA-DR neg/low /CD14 + HLA-DR high cells and CD10 neg /CD10 pos neutrophils were isolated from patients with AMI. We found a significantly higher expression of MerTK on CD14 pos CD163 pos macrophages differentiated from CD14 + HLA-DR neg/low monocytes and stimulated with IFN-g and IL-4 in presence of supernatants from CD10 pos neutrophils (Figure 4-figure supplement 3C). Efficient efferocytosis by human macrophages requires MerTK induction (Zizzo et al., 2012). Thus, we speculated that factors secreted by CD10 pos neutrophils from patients with AMI may enhance the capacity of HLA-DR neg/low monocyte-derived macrophages to phagocyte apoptotic cells by regulating MerTK expression.
The alarmin S100A9 have been shown to have chemotactic activity for monocytes (Chen et al., 2015). Previous experiments have focused on monocytic THP-1 cells and monocytes isolated from healthy human subjects (Chen et al., 2015). Here, we analyzed S100A9-induced chemotaxis of CD14 + HLA-DR neg/low /CD14 + HLA-DR high monocytes from patients with AMI. As shown in Figure 4figure supplement 3E, S100A9 significantly induced the migration of CD14 + HLA-DR high as well as CD14 + HLA-DR neg/low monocytes. Therefore, it is also possible that S100A9 regulates CD14 + HLA-DR neg/low /CD14 + HLA-DR high monocytes recruitment during AMI by functioning as a chemoattractant.
CD10 neg neutrophils and HLA-DR neg/low monocytes are linked to levels of immune-inflammation markers In a subgroup of patients we measured serum levels of immune inflammation markers ( Table 3). MMP-9, S100A9/S100A8, NGAL, IL-6, and IL-1ß levels were higher in STEMI patients versus UA patients.

Immature CD101 neg neutrophils are recruited to sites of cardiac injury and exhibit resistance to apoptosis
We then investigated whether immature CD101 neg neutrophils are recruited to the injured myocardium using the mouse model of reperfused AMI. Preliminary experiments showed that current protocols for tissue dissociation and the recovery of neutrophils from ischemic myocardium involving long enzymatic digestion times resulted in cell activation/damage and phenotypic changes that can be easily over-interpreted. Therefore, using a modified Langendorff perfusion system, the infarcted hearts were perfused for 6 min to remove blood cells and subsequently digested for only 8 min to preserve cell surface antigens along with expression profiles. Flow cytometry analysis of immune cells isolated from the ischemic region 3 hr after reperfusion revealed marked infiltration of CD101 neg neutrophils, displaying increased expression of the matrix-degrading protease MMP-9 ( Figure 6A). Moreover, as shown in Figure 6B, we found that 24 hr after reperfusion CD101 neg neutrophils expressed IL-1ß at higher levels compared to CD101 pos cells. Morphological analysis of cytospin preparations of sorted neutrophils showed that CD101 neg neutrophils isolated from ischemic myocardium 24 hr after reperfusion still have an immature morphology ( Figure 6C). These findings suggest migration and homing of immature CD101 neg neutrophils to ischemic myocardium shortly after reperfusion. Effective resolution of inflammation as well as a favorable wound healing response requires timely phagocytic clearance of apoptotic neutrophils by macrophages. As CD10 neg neutrophils derived from AMI patients showed reduced spontaneous apoptosis, we studied the apoptosis rate of CD101 neg and CD101 pos neutrophils isolated from the ischemic region 24 hr after reperfusion. We found that CD101 neg neutrophils were more resistant to apoptosis than their CD101 pos counterparts. After 24 hr in vitro culture the majority of CD101 neg neutrophils remained alive, as assessed by flow cytometry ( Figure 6D).

Immature CD101 neg neutrophils are present in mediastinal lymph nodes after acute MI
A crucial role for neutrophils in the orchestration of adaptive immunity is emerging (Costa et al., 2019;Silvestre-Roig et al., 2020). During homeostasis and inflammation, neutrophils can migrate to lymph nodes, which are critical sites for T-cell priming (Voisin and Nourshargh, 2019). Therefore, we next addressed whether immature CD101 neg neutrophils migrate into heart-draining lymph nodes early after AMI. We found a small population of neutrophils in heart-draining lymph nodes at steady state, consistent with recent studies (Lok et al., 2019). Notably, in mice subjected to coronary occlusion followed by 3 hr of reperfusion the percentage of neutrophils in mediastinal lymph nodes increased significantly ( Figure 7A). About 50% of infiltrating neutrophils were CD101 neg cells. CD101 neg neutrophils were nearly absent in mediastinal lymph nodes at steady state ( Figure 7A). Analysis of cytospins of mediastinal lymph node cell suspensions revealed the presence of neutrophils with ring-shaped nuclei as well as mature neutrophils with segmented nuclei after AMI Figure 5. Multiple intercorrelations among CD10 neg neutrophils, HLA-DR neg/low monocytes and immuneinflammation markers. Spearman-correlation matrix of CD16 + CD66b + CD10 neg neutrophils (%), CD14 + HLA-DR neg/ low monocytes (%) and circulating levels of MMP-9, S100A9/S100A8, IL-6, IL-1ß, TNF-a, MPO, and NGAL (levels). UA (n=11), NSTEMI (n=10), and STEMI (n=26). Each circle illustrates a significant correlation between different couples of parameters (p<0.05). The correlation coefficient is colored and sized up according to the value; square leaved blank indicates not significant correlation.
( Figure 7B). These findings provide evidence that immature CD101 neg neutrophils are able to traffic into lymph nodes after AMI, where they could contribute to regulation of lymphocyte effector functions.
Elevated circulating levels of IFN-g in patients with expanded CD10 neg neutrophils and increased frequency of CD4 + CD28 null T-cells In order to investigate the potential immunoregulatory effects on T-cells of immature CD10 neg neutrophils we first performed flow cytometric analysis of CD4 + T-cells and analyzed circulating levels of IFN-g in a subgroup of patients. Contrary to some reports, circulating levels of naive (CCR7 + -CD45RA + ), central memory (CCR7 + CD45RA -), effector memory (CCR7 -CD45RA -), terminally differentiated effector cells (EMRA, CCR7 -CD45RA + ) and CD4 + CD28 null T-cells were not significantly different among patients with ACS (Table 4). Figure 6. Immature CD101 neg neutrophils are rapidly recruited to ischemic sites, are a major source of MMP-9 and IL-1ß in the reperfused myocardium and exhibit resistance to apoptosis. (A) Flow cytometric gating strategy to identify neutrophils in the ischemic region, mean fluorescent intensity (MFI) of MMP-9 on CD101 neg and CD101 pos neutrophils, number of CD101 neg and CD101 pos neutrophils 3 hr after reperfusion. (B) Flow cytometry identifying infarct neutrophils, mean fluorescent intensity of IL-1ß on CD101 neg and CD101 pos neutrophils, number of CD101 neg and CD101 pos neutrophils 24 hr after reperfusion. (C) Wright-Giemsa stained cytospin preparations of CD101 neg and CD101 pos neutrophils isolated by cell sorting from ischemic myocardium 24 hr after reperfusion. (D) Rate of apoptosis of infarct CD101 neg /CD101 pos neutrophils determined by flow cytometry using Vybrant DyeCycle Violet stain after 24 hr in vitro culture. CD11b pos Ly6G pos CD101 neg (CD101 neg ) and CD11b pos Ly6G pos CD101 pos (CD101 pos ) cells were isolated by cell sorting from ischemic myocardium 24 hr after reperfusion. Data are presented as mean ± SEM (n=3-4). *p<0.05.
Notably, we found that elevated circulating IFN-g levels and increased frequency of CD10 neg neutrophils were linked, mainly in patients with expanded CD4 + CD28 null T-cells ( Figure 8A and B). To better highlight the relationship among IFN-g, CD10 neg neutrophils and CD4 + CD28 null T-cells we  also performed principal component analysis (PCA) that showed clustering according to elevated circulating levels of IFN-g, high levels of CD10 neg neutrophils and peripheral expansion of CD4 + -CD28 null T-cells ( Figure 8C). The highest IFN-g levels were found in STEMI patients with expanded CD10 neg neutrophils (>10%) and increased frequency of CD4 + CD28 null T-cells ( Figure 8C).
Frequency distribution of CD4 + CD28 null T-cells (log 10 transformed to improve visualization) appeared bimodal and analyzing separately in CMV + and CMV-seronegative (CMV -) patients, the median was significantly higher by a factor 14.1 in CMV + patients. (Figure 8-figure supplement  1A). Moreover, CD4 + CD28 null frequency positively correlated with CMV-IgG antibody levels (R=0.6, p<10 À5 ). Therefore, we believe that the expansion of CD4 + CD28 null T-cells may not be a direct result of coronary events but appears to be related to the CMV-induced immune changes secondary to repeated antigen exposure.
We defined expansion of CD4 + CD28 null T-cells frequency a non-parametric, upper outlier limit (upper quartile+1.5Âinterquartile range) as previously reported (Pera et al., 2018). The subgroup of CMVpatients with unstable angina was considered as reference group. (Figure 8-figure supplement 1B). Similarly, we derived a cut-off for expansion of CD10 neg neutrophils but taking as reference group the whole cohort of patients with UA since frequency expansion appeared prevalently due to the grade of coronary disease, not to CMV-seropositivity and both contributions to cell expansion could not be dissected (Figure 8-figure supplement 1C and D). Then, in order to highlight relationship among IFN-g levels, CD10 neg neutrophils, CD4 + T-cell subsets and CMV-seropositivity we performed hierarchical clustering stratifying patients with ACS according to the expansion cut-offs above described. This individuated four subgroups of patients with frequency of CD10 neg neutrophils ( 10% or >10%) and frequency of CD4 + CD28 null T-cells ( 0.5% or >0.5%) as depicted in Figure 8C by principal component analysis (PCA). In the derived heatmap IFN-g, CD10 neg neutrophils, CD4 + CD28 null , EMRA, and EM CD4 + T-cells were grouped together showing similar patterns (Figure 8-figure supplement 1E), indicating that persistent CMV infection is associated with expansion of the effector memory CD4 + T-cell compartment and higher IFN-g levels in patients with increased frequency of CD10 neg neutrophils. Not surprisingly, when stratified according to CMV serostatus, maximum levels of circulating IFN-g among ACS patients were detected in CMV-seropositive STEMI patients displaying increased levels of CD10 neg neutrophils ( Figure 8E), indicating a relation among expansion of immature CD10 neg neutrophils, CMV seropositivity and strongly enhanced levels of IFN-g in patients with large AMI.Of note, PCA performed replacing the percentage of CD4 + CD28 null cells with CMV-IgG titer (stratified by a cut-off equal to 0.55) showed a better cluster grouping (Figure 8-figure supplement 2A), with similar represented total variance (PC1+PC2: 79.5% vs. 83% respectively). The heatmap derived stratifying patients by frequency of CD10 neg neutrophils and CMV-IgG cut-offs (Figure 8-figure supplement 2B) shows hierarchical subgrouping of CMV-IgG titers and CD4 + CD28 null cells. Moreover, Spearman's correlation matrix revealed a strong positive correlation between CMV-IgG titers and the percentage of CD4 + CD28 null cells (Figure 8figure supplement 2C). Taken together, these results highlight the complex relationship in vivo among CMV seropositivity, CD4 + CD28 null cells, CD10 neg neutrophils and IFN-g levels.
CD10 neg neutrophils via induction of interleukin-12 enhance priming for IFN-g production by CD4 + T-cells Environmental factors such as CMV infection can induce changes in CD4 + T-cell phenotype and function. Consequently, to provide a mechanistic understanding of the cellular basis for raised IFN-g in CMV-seropositive patients with expanded CD10 neg neutrophils, we investigated IFN-g secretion by CD4 + T-cells isolated from CMV -/CMV + patients and its potential link to interleukin 12 (IL-12), potent inducer of IFN-g (Trinchieri, 2003). In cell-to-cell contact-dependent conditions human neutrophils can mimic myeloid-derived suppressor cells and suppress T-cell activation through artefactual mechanisms (Negorev et al., 2018). Therefore, CD10 neg /CD10 pos neutrophils were evaluated for their ability to enhance IFN-g production in cell contact-independent manner. We found that CD10 neg neutrophils strongly enhanced IFN-g and IL-12 production by CD4 + T-cells from CMV + patients ( Figure 9A and B), when co-cultured using a transwell system where CD4 + T-cells in the lower chamber were separated from neutrophils in the upper chamber. Of note, CD4 + T-cells equally responded to cell-free supernatants derived from CD10 neg neutrophils. IFN-g and IL-12 production were significantly higher in CD4 + T-cells from CMV + than CMVpatients. The addition of neutralizing anti-IL-12 antibody abrogated the IFN-g production by CD4 + T-cells from CMV + patients in presence of supernatants derived from CD10 neg neutrophils ( Figure 9A). Our data indicate that CD10 neg neutrophils release soluble factors that efficiently induce a strong Th1 type response. Further studies aiming at characterizing the neutrophil-secreted immunomodulatory factors are ongoing.

Discussion
Innate immune mechanisms play a paramount role during AMI and the functional heterogeneity of monocytes and neutrophils have been the focus of intensive research in recent years. This study highlights for the first time that immature CD16 + CD66b + CD10 neg neutrophils and CD14 + HLA-DR neg/ low monocytes promoting proinflammatory immune responses expand in the peripheral blood from patients with large AMI. We also show that immature neutrophils are recruited to the injured myocardium and migrate to mediastinal lymph nodes shortly after reperfusion, using a mouse model of AMI. Furthermore, we found a potential link among increased frequency of immature CD10neg neutrophils and elevated IFN-g levels, especially in cytomegalovirus-seropositive patients with expanded CD4 + CD28 null T-cells. Finally, we could show that CD10 neg neutrophils enhance CD4 + T-cells IFN-g production by a contact-independent mechanism involving IL-12 ( Figure 10).
This study uncovered that CD10 can be used as a surface marker to identify the immature neutrophil population that expands and promotes proinflammatory effects in patients suffering from AMI. We believe that immature CD10 neg neutrophils derive from MI-induced emergency granulopoiesis. Both mature (segmented) and immature banded neutrophils are released from the bone marrow presumably to meet the high demand for more neutrophils, especially in patients with large AMI. Not surprisingly, in our study higher frequency of circulating CD10 neg neutrophils was associated with increased systemic concentrations of G-CSF, an essential regulator of neutrophil trafficking from the bone marrow to the blood and important neutrophil survival factor (Costa et al., 2019;Silvestre-Roig et al., 2020). Recently, CD10 has been proposed as a marker that distinguishes mature from immature neutrophils in healthy volunteers receiving G-CSF for stem cell mobilization (Marini et al., 2017).
Multiple clinical trials have evaluated the use of G-CSF in patients with AMI after successful revascularization. The majority of these studies found that effective stem cell mobilization with G-CSF therapy failed to improve left ventricular recovery (Traverse, 2019). Our findings suggest that the therapeutic benefits of G-CSF therapy after AMI might be compromised due to the release of immature proinflammatory CD10 neg neutrophils.
However, neutrophils may be released from the bone marrow in response to increased damageassociated molecular patterns such as S100A8/S100A9, secreted from neutrophils as mediators of sterile inflammation (Sreejit et al., 2020). Of interest, we found that circulating CD10 neg neutrophils express high amounts of S100A9, indicating that immature neutrophils could be an important source of this alarmin in patients with AMI.
Under inflammatory conditions neutrophils traffic to inflamed tissues as well as to draining lymph nodes (Costa et al., 2019;Leliefeld et al., 2015) modulating T cell-mediated immune responses. Our data from the mouse model of AMI are first to provide evidence that immature neutrophils can populate heart-draining lymph nodes in response to acute ischemic injury. It is intriguing to speculate that CD10 neg neutrophils migrate into lymph nodes where they could encounter T-cells and shape adaptive immune responses.
Emerging evidence indicates that immature neutrophils can be T-cell suppressive or do possess T-cell stimulatory capacities, displaying disease-specific functional plasticity (Costa et al., 2019;Rahman et al., 2019). Immunostimulatory immature CD10 neg neutrophils appear in the circulation of G-CSF-treated healthy volunteers and contact-dependent mechanisms account for their immunoregulatory functions (Marini et al., 2017). Here, we provide mechanistic evidence that immature CD10 neg neutrophils from patients with AMI, in a contact-independent way involving IL-12, enhance priming for IFN-g production in activated CD4 + T-cells. Thus, through diverse mechanisms immature CD10 neg neutrophils may exert immunostimulatory/proinflammatory functions actively participating in the regulation of adaptive immunity.
Genetic and environmental factors shape the immune system over time. Several studies have demonstrated that persistent CMV infection is associated with changes in T-cell phenotype and function (Nikolich-Zugich, 2008;Wertheimer et al., 2014;Davenport et al., 2020). Our results highlight that CD4 + CD28 + T-cells from CMV-seropositive AMI patients are skewed toward a Th1 phenotype, producing large amounts of IFN-g in presence of CD10 neg neutrophils. However, results obtained in vitro cannot be translated directly to the in vivo situation and several cellular and molecular mechanisms could have led to increased circulating levels of the pleiotropic cytokine IFN-g after AMI. Notably, using bioinformatic tools (PCA and hierarchical clustering) we were able to highlight the tight relationship among the peripheral expansion of immature CD10 neg neutrophils, CMValtered CD4 + T-cell homeostasis and high levels of IFN-g in patients with large AMI. Thus, determination of circulating CD10 neg neutrophils levels, particularly in the context of persistent CMV infection, might help to identify patients at risk for excessive inflammatory immune response.
Although a pathogenetic role of CD4 + CD28 null T-cells in coronary artery disease and atherogenesis have been recognized, important issues have remained unresolved (Dumitriu et al., 2009). A recent study revealed complex associations between of CD4 + CD28 null T-cells and cardiovascular disease (Tomas et al., 2020). 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 (Tomas et al., 2020). Our findings point to a potential link between CMV induced immune alterations following repeated antigen exposure and the peripheral expansion of CD4 + CD28 null T-cells in ACS patients. CMV has been associated with atherosclerosis and increased risk for cardiovascular diseases. Recent clinical data showed that myocardial ischemia in CMV-seropositive patients leads to significant changes in the composition of the CD8 + T-cell repertoire, accelerating immunosenescence (Hoffmann et al., 2015).
In spite of numerous studies on polymorphonuclear myeloid cells the presence and functional characteristics of immature neutrophils is underexplored in the setting of AMI in mice. The present study demonstrated for the first time that CD101 can be used as a marker to define the maturation status of neutrophils mobilized into the peripheral blood in response to ischemia and recruited to sites of ischemic injury after reperfusion. Previous studies in a human model of experimental endotoxemia showed that banded neutrophils exhibit efficient migration to sites of infection (van Grinsven et al., 2019). Moreover, developmental analysis of bone marrow neutrophils revealed that immature neutrophils possess functional migratory machinery and are recruited to the periphery of tumor-bearing mice (Evrard et al., 2018). Of note, we found that immature 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 hr after reperfusion, respectively. There are significant differences between mouse and human immunology. In mice, 10-25% of circulating leukocytes are neutrophils, while in humans neutrophils are the predominant leukocyte population, accounting for 50-70% of leukocytes. Moreover, the transit time of leukocytes may be quite different (Mestas and Hughes, 2004;Mackey et al., 2019;Riehle and Bauersachs, 2019). 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 (Mackey et al., 2019). Such differences should be considered when comparing animal and human studies on immune mechanisms underlying wound healing. However, future studies that employ intravital microscopy are required to track the fate of immature neutrophils mobilized from the bone marrow and recruited to the ischemic myocardium as well as lymph nodes after AMI.
The recruitment of immune cells to sites of tissue repair is a complex highly regulated process involving cytokines, chemokines, and interactions between infiltrating immune cells. 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 IL-1ß in the reperfused heart, may be actively involved in the recruitment of HLA-DR neg/low cells. Saxena et al., 2013 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 hr of reperfusion (Saxena et al., 2013). Noteworthy, recent studies demonstrated that the failing human heart also contains HLA-DR neg/low monocytes (Bajpai et al., 2018).
Several immune mechanisms operate during cardiac wound healing and IFN-g 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 HLA-DR neg/low monocytes produced more TNF-a, IL-6, and IL-1ß upon IFN-g stimulation as HLA-DR high monocyte-derived macrophages. These findings may support a role for HLA-DR neg/low monocytes in pathogenic mechanisms operating during AMI and may, at least in part, explain why an expansion of circulating HLA-DR neg/low monocytes correlates with circulating levels of TNF-a, IL-6, and IL-1ß.
The interleukin-1 pathway plays a key role in post-MI inflammation and the progression to heart failure (Abbate et al., 2020). Our in vitro mechanistic experiments with immune cells from AMI patients as well as mouse studies provide a potential linkage between the induction of immature CD10 neg neutrophils/HLA-DR neg/low monocytes and increased interleukin-1 activity during AMI. Emerging evidences highlight that targeting interleukin-1 may hold promise for patients after MI (Buckley and Abbate, 2018). 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 IL-1b) prevented the recurrence of ischemic events, reduced heart failure-related hospitalizations and mortality in patients with prior AMI (Buckley and Abbate, 2018).
Neutrophils may orchestrate post-MI healing through induction of MerTK expression and increased efferocytosis capacity of macrophages (Horckmans et al., 2017). Of interest, we found significantly higher expression of MerTK on macrophages differentiated from CD14 + HLA-DR neg/low monocytes and polarized in the presence of secretome derived from CD10 pos neutrophils. It is tempting to speculate that HLA-DR neg/low monocytes had the potential to differentiate into macrophages with enhanced capacity to phagocyte apoptotic cells when exposed to CD10 pos neutrophils after AMI. Nevertheless, in vitro studies have limited ability to explain the complexity of macrophage activation within the infarct environment.
The apoptosis rate of CD10 neg neutrophils was strongly reduced compared with CD10 pos neutrophils. This finding is consistent with recent studies showing that cellular immaturity influences apoptosis (Marini et al., 2017;Wang et al., 2020). We also observed that CD101 neg neutrophils sorted from reperfused ischemic hearts were resistant to apoptosis. Apoptosis is essential for neutrophil functional shutdown, phagocytic clearance by macrophages and timely resolution of inflammation (Koedel et al., 2009;Elliott et al., 2017). Therefore, apoptosis resistance of immature neutrophils may play a crucial role, hitherto overlooked, after MI. Further depletion/modulation studies in mice will shed light on the immunoregulatory functions of immature neutrophils in the inflammatory response during wound healing.
In conclusion, this study shows that immature CD10 neg neutrophils and CD14 + HLA-DR neg/low monocytes expand in patients with AMI and highlights their potential role as triggers of immune/ inflammatory dysregulation after ischemic injury. These findings could have major implications for understanding immunoregulatory mechanisms operating during AMI and for the development of future therapeutic strategies. Nevertheless, further studies deciphering the relationship between elevated CD14 + HLA-DR neg/low monocytes/CD10 neg neutrophils and ensuing mortality and morbidity after ischemic injury are necessary and ongoing. Verbraucherschutz und Lebensmittelsicherheit (Oldenburg, Germany;permit No. 33.12-42502-04-11/0644;33.9-42502-04-13/1124 and33.12-42502-04-17/2702

Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for