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

Figures
Tables
Additional files

11 figures, 5 tables and 1 additional file

Figures

Figure 1 with 2 supplements

Download asset Open asset

Increased circulating levels of CD14⁺HLA-DR^neg/low monocytes in patients with AMI.

(A) Gating strategy to identify CD14⁺HLA-DR^neg/low monocytes. (B) Circulating levels of CD14⁺HLA-DR^neg/low monocytes in patients with unstable angina (UA; n=11), non-ST-elevation MI (NSTEMI, n=16), and ST-elevation MI (STEMI, n=44). (C) Linear regression analysis between circulating levels of CD14⁺HLA-DR^neg/low monocytes and maximum CK (CK_max) in patients with acute coronary syndrome. (D) Receiver operator characteristic (ROC) curve of CD14⁺HLA-DR^neg/low monocytes discriminating UA/STEMI and UA/NSTEMI patients and the combination of CD14⁺HLA-DR^neg/low monocytes (n/µL) with CK_max. *p<0.05, vs. UA; †p<0.05, vs. NSTEMI.

Figure 1—figure supplement 1

Download asset Open asset

Time course analysis of subset-frequencies and circulating levels of monocytes in patients with unstable angina (UA; n=11), non-ST-elevation MI (NSTEMI, n=16), and ST-elevation MI (STEMI, n=44).

Phenotypic characterization was performed within the initial 24 hr and up to day 5 after onset of symptoms in patients with ACS. *p<0.01 vs. ≤ 24 hr. Error bars represent SEM.

Figure 1—figure supplement 2

Download asset Open asset

Monocyte subset-frequencies.

(A) Gating strategy to identify classical (HLA-DR⁺⁺CD14⁺⁺CD16^-CX3CR1⁺), intermediate (HLA-DR⁺⁺CD14⁺⁺CD16⁺CX3CR1⁺) and non-classical (HLA-DR⁺CD14⁺CD16⁺⁺CX3CR1⁺⁺) monocytes. (B) Percentages of CD14⁺HLA-DR^neg/low, intermediate and non-classical monocytes in control subjects (CTR, n=17) and in patients with unstable angina (UA; n=11), non-ST-elevation MI (NSTEMI, n=16), and ST-elevation MI (STEMI, n=44). (C) Mean fluorescence intensity (MFI) of CX3CR1 and CCR2 on monocyte subsets. *p<0.05 vs. CTR; †p<0.05 vs. UA; ‡p<0.05 vs. NSTEMI.

Figure 2 with 1 supplement

Download asset Open asset

CD14⁺HLA-DR^neg/low monocytes from patients with AMI are not immunosuppressive but exhibit an inflammatory phenotype.

(A) May-Grünwald Giemsa stained cytospin preparations of CD14⁺HLA-DR^neg/low and CD14⁺HLA-DR^high monocytes. (B) Relative RNA expression of S100A9 and IL1R1 in CD14⁺HLA-DR^neg/low versus CD14⁺HLA-DR^high monocytes. (C) T-cell proliferation in the presence of CD14⁺HLA-DR^neg/low or CD14⁺HLA-DR^high monocytes assessed by CellTrace Violet dilution after 96 hr of co-culture. (D) Macrophages differentiated from CD14⁺HLA-DR^neg/low monocytes and (E) CD14⁺HLA-DR^high cells by 4‐day culture with M-CSF. (F) TNF-α, IL-6, and IL-1ß in supernatants of macrophage cultures upon stimulation with IFN-γ. M^b=baseline. CD14⁺HLA-DR^neg/low/CD14⁺HLA-DR^high cells were isolated by flow-cytometric sorting from patients with AMI (n=5–6). Data are presented as mean ± SEM. *p<0.05.

Figure 2—figure supplement 1

Download asset Open asset

Expression of genes dysregulated in circulating monocytes and infarct macrophages in a mouse model of reperfused AMI.

(A) RT-qPCR showing the expression of CAT, CCR1, IL1R2, LCN2, MMP8, NOS2, STAT3, SAAP3 in CD14⁺HLA-DR^neg/low (lo) versus CD14⁺HLA-DR^high (hi) monocytes FACS-sorted from blood of patients with AMI. Data are presented as mean ± SEM, (n=3–5); n.d., not detectable. (B) MA plots showing genes regulated in circulating monocytes in a mouse model of reperfused AMI. RNA sequencing was performed on monocytes FACS-sorted from blood of sham-operated mice (Basal) and mice subjected to 1 hr of coronary occlusion followed by 6 hr of reperfusion. Genes upregulated/downregulated by AMI in monocytes were similarly regulated in (C) infarct macrophages FACS–sorted from the ischemic region 24 hr after AMI.

Figure 3 with 1 supplement

Download asset Open asset

Circulating normal-density CD10^neg neutrophils increase in patients with AMI.

(A) Gating strategy to identify CD10^neg neutrophils. (B) Circulating levels of CD16⁺CD66b⁺CD10^neg neutrophils in patients with unstable angina (UA; n=11), non-ST-elevation MI (NSTEMI, n=16), and ST-elevation MI (STEMI, n=44). (C) Linear regression analysis between circulating levels of CD10^neg neutrophils and maximum CK (CK_max). (D) Receiver operator characteristic (ROC) curve of CD10^neg neutrophils (n/µL) discriminating UA/STEMI and UA/NSTEMI patients and the combination of CD10^neg neutrophils with CK_max in patients with acute coronary syndrome. *p<0.05 vs. UA.

Figure 3—figure supplement 1

Download asset Open asset

Increased frequency of CD10^negneutrophils in patients with AMI.

(A) Percentages of circulating CD16⁺CD66b⁺CD10^neg neutrophils in patients with unstable angina (UA; n=11), non-ST-elevation MI (NSTEMI, n=16), and ST-elevation MI (STEMI, n=44). (B) Time course analysis of circulating levels and frequencies of CD16⁺CD66b⁺CD10^neg neutrophils. Phenotypic characterization was performed within the initial 24 hr and up to day 5 after onset of symptoms in patients with ACS. *p<0.05, vs. CTR; †p<0.05 vs. UA. Error bars represent SEM.

Figure 4 with 3 supplements

Download asset Open asset

Immature CD10^neg neutrophils from patients with AMI express high amounts of MMP-9 and S100A9 and display resistance to apoptosis.

(A) May-Grünwald Giemsa stained cytospin preparations of CD16⁺CD66b⁺CD10^neg (CD10^neg) and CD16⁺CD66b⁺CD10^pos (CD10^pos) neutrophils. (B) Linear regression analysis between the percentages of CD16⁺CD66b⁺CD10^neg neutrophils and circulating levels of G-CSF in patients with acute coronary syndrome (n=71). (C) Mean fluorescence intensity (MFI) of CD11b, CD101, CD114, and CD177 on CD10^neg versus CD10^pos neutrophils (n=25). (D) Relative RNA expression of MMP-9 and S100A9 in CD10^neg versus CD10^pos neutrophils. (E) Percentage of apoptotic neutrophils assessed by flow cytometry using Vybrant DyeCycle Violet stain and SYTOX AADvanced stain. CD10^neg/CD10^pos neutrophils were isolated by flow-cytometric sorting from patients with AMI (n=4–5). *p<0.05.

Figure 4—figure supplement 1

Download asset Open asset

Expression of genes regulated in circulating neutrophils and infarct neutrophils in a mouse model of reperfused AMI.

(A) RT-qPCR showing the expression of IRG1, IL1R1, IL1R2, MMP8, NOS2, OLFM4, STAT3 in CD10^neg (lo) versus CD10^pos (hi) neutrophils FACS-sorted from blood of patients with AMI, (n=3–5); n.d., not detectable. (B) MA plots showing genes regulated in circulating neutrophils in a mouse model of reperfused AMI. RNA sequencing was performed on neutrophils FACS-sorted from blood of sham-operated mice (Basal) and mice subjected to 1 hr of coronary occlusion followed by 6 hr of reperfusion. Genes upregulated/downregulated by AMI in neutrophils were similarly regulated in (C) infarct neutrophils FACS–sorted from the ischemic region 24 hr after AMI. (D) RT-qPCR showing Cd101 expression in bone marrow CD101^neg (BM) and peripheral blood CD101^neg (lo) versus CD101^pos (hi) neutrophils. (E) Expression of Irg1, Il1r1, Il1r2, Mmp8, Nos2, Olfm4, and Stat3 in circulating CD101^neg (lo) versus CD101^pos (hi) neutrophils. CD11b^posLy6G^posCD101^neg (lo) and CD11b^posLy6G^posCD101^pos (hi) cells were isolated by FACS sorting after AMI. (n=3–5). *p<0.05.

Figure 4—figure supplement 2

Download asset Open asset

Immature CD101^neg neutrophils are rapidly released into the bloodstream after reperfusion in a mouse model of AMI.

(A) Representative gating strategy to identify circulating immature neutrophils among CD11b^posLy6G^posCXCR2^pos cells and number of CD11b^dimCD101^neg and CD11b^brightCD101^pos neutrophils released into the bloodstream 90 min after reperfusion. (B) May-Grünwald Giemsa stained cytospin preparations of sorted CD11b^dimCD101^neg and CD11b^brightCD101^pos neutrophils. (C) Flow cytometric analysis and number of CD101^neg neutrophils in bone marrow from sham-operated mice (Sham) and mice subjected to coronary occlusion followed by 6 hr of reperfusion (AMI). Data are presented as mean ± SEM (n=3–4). *p<0.05.

Figure 4—figure supplement 3

Download asset Open asset

Interactions between CD10^negneutrophils and CD14⁺HLA-DR^neg/lowmonocytes.

(A) Percentage of live CD10^neg/CD10^pos neutrophils assessed by flow cytometry using Apotracker Green. Flow plots show a small number of apoptotic CD10^neg neutrophils after 24 hr in vitro culture. (B) Wright-Giemsa stained cytospin preparations of CD10^neg and CD10^pos neutrophils after 24 hr in vitro culture. (**C, D**) Mean fluorescence intensity (MFI) of MerTK on CD14^posCD163^pos monocyte-derived macrophages. CD14⁺HLA-DR^neg/low and CD14⁺HLA-DR^high monocytes were differentiated to macrophages by 4‐day culture with M-CSF and stimulated with IFN-γ M(IFN-γ), IL-4 M(IL-4) and dexamethasone M(Dexa) in presence or absence of neutrophil supernatants from CD10^neg neutrophils or CD10^pos neutrophils. (E) Chemotactic response to human S100A9 of CD14⁺HLA-DR^neg/low and CD14⁺HLA-DR^high monocytes. CD10^neg/CD10^pos neutrophils and CD14⁺HLA-DR^neg/low/CD14⁺HLA-DR^high monocytes were isolated from patients with AMI (n=3–4). *p<0.05.

Figure 5

Download asset Open asset

Multiple intercorrelations among CD10^neg neutrophils, HLA-DR^neg/low monocytes and immune-inflammation 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-α, 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 6

Download asset Open asset

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^posLy6G^posCD101^neg (CD101^neg) and CD11b^posLy6G^posCD101^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.

Figure 7

Download asset Open asset

Immature CD101^neg neutrophils are present in mediastinal lymph nodes draining the heart after acute MI.

(A) Flow cytometric analysis of neutrophil subsets in mediastinal lymph nodes at steady state and 3 hr after reperfusion (AMI). (B) Cytospins of mediastinal lymph node cell suspensions stained with Wright's Giemsa. Mice were subjected to 1 hr of coronary occlusion and heart-draining lymph nodes were isolated 3 hr after reperfusion. Data are presented as mean ± SEM (n=3). *p<0.05.

Figure 8 with 2 supplements

Download asset Open asset

Elevated IFN-γ levels in patients with expanded CD10^neg neutrophils and increased frequency of CD4⁺CD28^null T-cells.

(A) Gating strategy identifying CD4⁺CD28^null T-cells. (B) Scatter plot showing IFN-γ levels according to frequency of CD10^neg neutrophils and CD4⁺CD28^null T-cells. (C) Principal component analysis (PCA) showing clustering according to circulating levels of IFN-γ, CD10^neg neutrophils and CD4⁺CD28^null T-cells. Patients were stratified based on frequency of CD10^neg neutrophils (≤10% or >10%) and frequency of CD4⁺CD28^null T-cells (≤0.5% or >0.5%). (D) Frequency of CD4⁺CD28^null T-cells in patients with acute coronary syndrome stratified according to cytomegalovirus (CMV) serostatus. (E) circulating IFN-γ levels stratified according to CMV serostatus. UA (n=11), NSTEMI (n=13), and STEMI (n=34). *p≤0.05.

Figure 8—figure supplement 1

Download asset Open asset

(A) CD4⁺CD28^null T-cell frequency distribution (log₁₀-transformed CD4⁺ T-cell fractions) of CMV^± (top, n=58), CMV^- (middle, n=23), and CMV⁺ (bottom, n=35) acute coronary syndrome (ACS) patients. CD4⁺CD28^null T-cells displayed a bimodal distribution related to CMV-seropositivity. (B) Boxplots show the log₁₀-transformed frequency of CD4⁺CD28^null T-cells in CMV^--UA, CMV^- (blue) and CMV⁺ (red) -ACS patients. Expansion index (dotted line) was calculated as UQ+1.5xIQR of CMV^--UA patients chosen as reference group. CD4⁺CD28^null T-cell frequency more than 0.5% was considered as an index of expansion; UQ (upper quantile), IQR (inter-quantile range). (C) CD10^neg neutrophils (CD10^neg) frequency distribution (log₁₀-transformed) of CMV^± (top, n=71), CMV^- (middle, n=31), and CMV⁺ (bottom, n=40) ACS patients. (D) Boxplots show the log₁₀-transformed frequency of CD10^neg in UA, NSTEMI and STEMI patients. Expansion index (dotted line) was calculated as UQ+1.5xIQR of UA patients. According, patients with CD10^neg frequency more than 10% had expansion. (E) Scaled frequency of CD4⁺CD28^null T-cells and CD10^neg neutrophils stratified by criteria of cell expansion. Hierarchical clustering performed on columns highlights the relationship among CD10^neg neutrophils, CD4⁺CD28^null T-cells, IFN-γ production and CMV seropositivity.

Figure 8—figure supplement 2

Download asset Open asset

Relationship among the peripheral expansion of immature CD10^neg neutrophils, CMV IgG titers, CD4⁺CD28^null T-cells, and circulating levels of IFN-γ and IL-12 in patients with AMI.

(A) Principal component analysis (PCA) showing clustering according to circulating levels of IFN-γ, CD10^neg neutrophils and CMV IgG titers. Patients were stratified based on frequency of CD10^neg neutrophils (≤10% or >10%) and titer of CMV IgG (≤0.55 or >0.55 AU/mL). (B) Scaled frequency of CD10^neg neutrophils stratified by criteria of cell expansion and scaled levels of CMV seropositivity. Hierarchical clustering performed on columns highlights the relationship among CD10^neg neutrophils, CD4⁺CD28^null T-cells, IFN-γ, IL-12 levels and CMV seropositivity. *p<0.05, IL-12 belonging to IIIrd vs. first, IInd, and IVth cluster. (C) Spearman-correlation matrix of CMV IgG titers (AU/mL), CD16⁺CD66b⁺CD10^neg neutrophils (%), CD4⁺CD28^null T-cells (%) and circulating levels of IFN-γ, and IL-12. 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 9 with 1 supplement

Download asset Open asset

CD10^neg neutrophils enhance IFN-γ production by CD4⁺ T-cells via induction of interleukin-12.

(A) IFN-γ and (B) interleukin-12 production by CD4⁺ T-cells stimulated with anti-CD3/CD28 beads and co-cultured for 24 hr in absence (CD4⁺) or presence of CD10^pos neutrophils (CD10^pos+CD4⁺), CD10^neg neutrophils (CD10^neg+CD4⁺) using a transwell system or cultured with cell-free supernatants derived from CD10^pos neutrophils (csf-CD10^pos+CD4⁺), CD10^neg neutrophils (csf-CD10^neg+CD4⁺), CD10^neg neutrophils in the presence of neutralizing anti-IL-12 antibody (csf-CD10^neg+CD4⁺+IL12Ab). CD4⁺CD28^null T-cells were stimulated with anti-CD3/CD28 beads (CD4⁺CD28^neg) and cultured with cell-free supernatants derived from CD10^neg neutrophils (csf-CD10^neg+CD4⁺CD28^neg). CD10^neg/CD10^pos neutrophils, CD4⁺ T-cells and CD4⁺CD28^null T-cells were isolated from CMV-seronegative (CMV^-) or CMV-seropositive (CMV⁺) patients with AMI (n=3–5). Data are represented as fold-change to respective CD3/CD28-stimulated cells and presented as mean ± SEM. *p≤0.05.

Figure 9—figure supplement 1

Download asset Open asset

IFN-γ production by CD4⁺ T-cells from CMV-seropositive AMI patients stimulated with anti-CD3/CD28 beads and cultured for 24 hr in absence (CD4⁺) or presence of cell-free supernatants derived from CD10^pos neutrophils (csf-CD10^pos+CD4⁺) or CD10^neg neutrophils (csf-CD10^neg+CD4⁺).

D10^neg/CD10^pos neutrophils were isolated from CMV-seronegative patients with AMI. Data are represented as fold-change to respective CD3/CD28-stimulated cells. (n=5). *p<0.05 vs. CD4⁺, csf-CD10^pos+CD4⁺.

Figure 10

Download asset Open asset

Immature CD10^neg neutrophils and HLA-DR^neg/low monocytes inducing proinflammatory and adaptive immune responses emerge in patients with large acute myocardial infarction.

Author response image 1

Download asset Open asset

Tables

Table 1

General traits.

		UA (N=11)	NSTEMI (N=16)	STEMI (N=44)
Age (years)		63.3±2.5	64.9±3.4	60.6±1.7
Gender	Male/Female	9/2	14/2	36/8
BMI (kg/m²)		27.5±0.9	28.2±0.9	27.5±0.7
Blood analyses	LDL (mg/dL)	92.2±19.2	95.5±9.5	138.2±7.3
	CK (IU/L)	120.0 (87.0–444.0)	189.0 (126.8–377.0)	373.5 (110.5–931.2)
	CK_max (IU/L)	120.0 (86.5–440.0)	403.5 (150.5–578.2)	1343.5 (574.8–1917.0)
	CK-MB (IU/L)	19.0 (17.0–22.0)	32.0 (23.5–57.0)	47.0 (24.5–91.5)
	LVEF (%)	53.3±3.7	54.1±2.2	51.0±1.5
	Troponin (ng/L)	12.8 (5.3–22.7)	99.0 (36.7–273.5)	337.0 (84.0–962.0)
	Creatinine (µmol/L)	83.0±4.5	88.6±4.8	98.5±8.3
	CRP (mg/L)	1.6 (0.7–3.4)	1.8 (1.1–4.3)	2.5 (1.2–4.7)

Data are presented as mean ± SEM or as median (IQR). LDL, low density lipoprotein; CK, creatine kinase; CK_max, maximum CK; CK-MB, creatine kinase-myocardial band; LVEF, left ventricular ejection fraction; CRP, C-reactive protein.

Table 2

Leukocyte count and monocyte subsets.

	CTR (N = 17)	UA (N=11)	NSTEMI (N=16)	STEMI (N=44)	p (K-W)
Neutrophil (10³/µL)	3.25 (2.74–3.42)	4.05 (3.65–4.56)*	5.72 (4.80–7.79)*†	6.13 (5.18–7.04)*†	<0.0001
Monocyte (10³/µL)	0.65 (0.53–0.74)	0.74 (0.49–0.80)	0.88 (0.72–1.03)*†	0.99 (0.77–1.25)*†	<0.001
Lymphocyte (10³/µL)	2.20 (1.96–2.47)	1.82 (1.55–1.98)	1.97 (1.77–2.49)	2.07 (1.58–2.64)	0.30
Lymphocyte/neutrophil ratio	0.70 (0.57–0.79)	0.45 (0.38–0.56)*	0.32 (0.28–0.44)*	0.35 (0.27–0.45)*	<0.0001
Eosinophil (10³/µL)	0.16 (0.10–0.30)	0.15 (0.12–0.16)	0.21 (0.14–0.34)	0.12 (0.06–0.20)	0.08
Classical monocyte (n/µL)	476 (334–583)	332 (244–388)	510 (454–719)†	505 (388–666)†	<0.05
Intermediate monocyte (n/µL)	130 (73–145)	186 (131–367)*	204 (144–310)*	249 (167–442)*	<0.001
Non-classical monocyte (n/µL)	48 (30–64)	50 (37–67)	64 (47–108)	102 (60–138)*†	<0.001

Data are presented as median (IQR). Kruskal-Wallis (K-W) test; *p<0.05 vs. Control (CTR); †p<0.05 vs. UA.

Table 3

Immune inflammation markers.

	UA (N=11)	NSTEMI (N=10)	STEMI (N=26)	p (K-W)
MMP-9 (ng/mL)	429 (320-461)	447 (324-597)	544 (466-758)*	<0.01
S100A8/A9 (ng/mL)	7332 (4638–9461)	13802 (9152–21066)*	17352 (8592–27830)*	<0.05
NGAL (ng/mL)	264 (198-318)	328 (211-473)	417 (312-653)*	<0.05
MPO (ng/mL)	221 (153-337)	323 (158-443)	389 (230-487)*	0.05
IL-6 (pg/mL)	11.2 (9.2–21.1)	30.6 (24.5–57.4)*	47.7 (22.0–102.1)*	<0.01
TNF-α (pg/mL)	1.8 (1.3–15.7)	4.6 (2.9–7.2)	12.1 (5.0–21.8)	0.14
IL-1ß (pg/mL)	2.4 (2.3–2.9)	4.2 (2.4–7.9)	10.0 (2.5–16.4)*	0.05

Data are presented as median (IQR). Kruskal-Wallis (K-W) test; *p<0.05 vs. UA.

Table 4

CD4⁺ T-cells Subsets.

	UA (N=11)	NSTEMI (N=13)	STEMI (N=34)	p (K-W)
NAIVE (n/µL)	440 (338-511)	497 (328-567)	382 (312-627)	n.s.
CM (n/µL)	300 (233-364)	278 (242-328)	334 (210-478)	n.s
EM (n/µL)	122 (91-140)	114 (86-137)	102 (80-165)	n.s.
EMRA (n/µL)	55 (30-77)	42 (24-66)	47 (29-109)	n.s.
CD4⁺CD28^null (n/µL)	4 (3–39)	5 (4–28)	10 (1-38)	n.s.

Data are presented as median (IQR). NAIVE, CCR7⁺CD45RA⁺; CM, CCR7⁺CD45RA^-; EM, CCR7^-CD45RA^-; EMRA, CCR7^-CD45RA⁺. Kruskal-Wallis (K-W) test; n.s., not significant.

Appendix 1—key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
biological sample (Homo sapiens)	Peripheral Blood Leukocytes	Medizinische Hochschule Hannover		Freshly isolated from Human
biological sample (Mus musculus)	Peripheral Blood, Heart, Lymph Node, Bone Marrow Leukocytes	Medizinische Hochschule Hannover		Freshly isolated from Mus musculus
antibody	anti-human CD14-APC-H7 (Mouse monoclonal)	BD Biosciences	Cat# 560180 RRID:AB_1645464	1:50
antibody	anti-human HLA-DR-FITC (Mouse monoclonal)	BioLegend	Cat# 307604 RRID:AB_314682	1:30
antibody	anti-human HLA-DR-PE (Mouse monoclonal)	BioLegend	Cat# 307606 RRID:AB_314684	1:30
antibody	anti-human HLA-DR-PerCP/Cy5.5 (Mouse monoclonal)	BioLegend	Cat# 307630 RRID:AB_893567	1:30
antibody	anti-human CD16- BV605 (Mouse monoclonal)	BioLegend	Cat# 302040 RRID:AB_2562990	1:50
antibody	anti-human CX3CR1-PE (Mouse monoclonal)	BioLegend	Cat# 341604 RRID:AB_1595456	1:50
antibody	anti-human CCR2-PerCP/Cy5.5 (Mouse monoclonal)	BioLegend	Cat# 357204 RRID:AB_2562004	1:50
antibody	anti-human CD66b-FITC (Mouse monoclonal)	BioLegend	Cat# 305104 RRID:AB_314496	1:30
antibody	anti-human CD10-BV421 (Mouse monoclonal)	BioLegend	Cat# 312218 RRID:AB_2561833	1:20
antibody	anti-human CD10-PE (Mouse monoclonal)	BioLegend	Cat# 312203 RRID:AB_314914	1:20
antibody	anti-human CD3-APC-H7 (Mouse monoclonal)	BD Biosciences	Cat# 56076 RRID:AB_1645475	1:30
antibody	anti-human CD4-APC (Mouse monoclonal)	BD Biosciences	Cat# 561840 RRID:AB_10895807	1:30
antibody	anti-human CD28-BV421 (Mouse monoclonal)	BioLegend	Cat# 302930 RRID:AB_2561910	1:30
antibody	anti-human CCR7-Alexa Fluor 488 (Mouse monoclonal)	BioLegend	Cat# 353206 RRID:AB_10916389	1:30
antibody	anti-human CD45RA-PE (Mouse monoclonal)	BioLegend	Cat# 304108 RRID:AB_314412	1:30
antibody	anti-human CD114-PE (Mouse monoclonal)	BioLegend	Cat# 346106 RRID:AB_2083867	1:25
antibody	anti-human CD177-FITC (Mouse monoclonal)	BioLegend	Cat# 315804 RRID:AB_2072603	1:25
antibody	anti-human CD11b-PerCP/Cy5.5 (Mouse monoclonal)	BioLegend	Cat# 301328 RRID:AB_10933428	1:25
antibody	anti-human CD101-PE (Mouse monoclonal)	BioLegend	Cat# 331011 RRID:AB_2716106	1:25
antibody	anti-human MERTK-APC (Mouse monoclonal)	BioLegend	Cat# 367612 RRID:AB_2687289	1:25
antibody	anti-human CD163-FITC (Mouse monoclonal)	BD Biosciences	Cat# 563697 RRID:AB_2738379	1:25
antibody	anti-IL-12	R and D Systems	Cat# MAB219 RRID:AB_2123616	4 µg/mL
antibody	Isotype control	R and D Systems	Cat# MAB002 RRID:AB_357344	4 µg/mL
antibody	CD16/CD32 Mouse BD Fc Block RUO (Rat monoclonal)	BD Biosciences	Cat# 553142 RRID:AB_394657	1:50
antibody	anti-mouse CD45-PerCP/Cy5.5 (Mouse monoclonal)	BioLegend	Cat# 109828 RRID:AB_893350	1:100
antibody	anti-mouse CD45-PerCP/Cy5.5 (Rat monoclonal)	BD Biosciences	Cat# 550994 RRID:AB_394003	1:100
antibody	anti-mouse F4/80-BV421 (Rat monoclonal)	BD Biosciences	Cat# 565411 RRID:AB_2734779	1:100
antibody	anti-mouse F4/80-APC (Rat monoclonal)	BioLegend	Cat# 123116 RRID:AB_893481	1:100
antibody	anti-mouse CD11b-Super Bright 600 (Rat monoclonal)	eBioscience	Cat# 63-0112-82 RRID:AB_2637408	1:100
antibody	anti-mouse CD11b- PerCP/Cy5.5 (Rat monoclonal)	BD Biosciences	Cat# 550993 RRID:AB_394002	1:100
antibody	anti-mouse CD4-Alexa Fluor 488 (Rat monoclonal)	BioLegend	Cat# 100423 RRID:AB_389302	1:100
antibody	anti-mouse CD115-Brilliant Violet 605 (Rat monoclonal)	BioLegend	Cat# 135517 RRID:AB_2562760	1:100
antibody	anti-mouse CD115-Alexa Fluor 488 (Rat monoclonal)	BioLegend	Cat# 135512 RRID:AB_11218983	1:100
antibody	anti-mouse Ly-6G-APC (Rat monoclonal)	BioLegend	Cat# 127613 RRID:AB_1877163	1:100
antibody	anti-mouse Ly-6G-PE (Rat monoclonal)	BD Biosciences	Cat# 551461 RRID:AB_394208	1:200
antibody	anti-mouse Ly-6G-APC-H7 (Rat monoclonal)	BD Biosciences	Cat# 565369 RRID:AB_2739207	1:100
antibody	anti-mouse CXCR2-Alexa Fluor 647 (Mouse monoclonal)	BioLegend	Cat# 149604 RRID:AB_2565563	1:100
antibody	anti-mouse CXCR2-PE (Rat monoclonal)	BioLegend	Cat# 149609 RRID:AB_2565689	1:100
antibody	anti-mouse CD101-Alexa Fluor 647 (Rat monoclonal)	BD Biosciences	Cat# 564473 RRID:AB_2738821	1:100
antibody	anti-mouse CD101-PE (Rat monoclonal)	eBioscience	Cat# 12-1011-80 RRID:AB_1210729	1:100
antibody	anti-MMP-9 (Goat polyclonal)	R and D Systems	Cat# AF909 RRID:AB_355706	1:100
antibody	anti IL-1ß (Rabbit polyclonal)	Abcam	Cat# ab9722 RRID:AB_308765	1:100
antibody	anti-goat IgG (H+L) Cross-Adsorbed Secondary Antibody Alexa Fluor 488 (Donkey polyclonal)	Invitrogen	Cat# A-11055 RRID:AB_2534102	1:500
antibody	anti-rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor 488 (Goat polyclonal)	Invitrogen	Cat# A-11034 RRID:AB_2576217	1:500
commercial assay or kit	MACSxpress Whole Blood Neutrophil Isolation	Miltenyi Biotec	Cat# 130-104-434
commercial assay or kit	PrepEase RNA Spin	Affymetrix	Cat# PN78766
commercial assay or kit	RNeasy Plus Mini	QIAGEN	Cat# 74134
commercial assay or kit	Dynabeads Untouched Human T-cells	Invitrogen	Cat# 11344D
commercial assay or kit	CellTrace Violet Cell Proliferation	Invitrogen	Cat# C34571
commercial assay or kit	OpTmizer CTS T-Cell Expansion	Gibco	Cat# A1048501
commercial assay or kit	Dynabeads Human T-Activator CD3/CD28	Gibco	Cat# 11131D
commercial assay or kit	VybrantDyeCycle Violet/SYTOX AADvanced Apoptosis	Invitrogen	Cat# A35135
commercial assay or kit	Apotracker Green	Biolegend	Cat# 427402
commercial assay or kit	MojoSort Human CD4 T Cell Isolation	BioLegend	Cat# 480009
commercial assay or kit	CD28 MicroBead	Miltenyi Biotec	Cat# 130-093-247
commercial assay or kit	LEGENDplex	BioLegend	Cat# 740180
commercial assay or kit	LEGENDplex	BioLegend	Cat# 740589
commercial assay or kit	LEGENDplex	BioLegend	Cat# 740929
commercial assay or kit	CMV-IgG-ELISA PKS	Medac Diagnostika	Cat# 115-Q-PKS
commercial assay or kit	Human IFN-gamma Quantikine ELISA	R and D Systems	Cat# DIF50
commercial assay or kit	iScript Reverse Transcription Supermix	Bio-Rad	Cat# 1708840
commercial assay or kit	SsoAdvanced Universal SYBR Green Supermix	Bio-Rad	Cat# 172–5271
chemical compound, drug	VersaLyse Lysing Solution	Beckman Coulter	Cat# A09777
chemical compound, drug	Ficoll-Paque Premium	SIGMA	Cat# GE17-5442-02
chemical compound, drug	ß-mercaptoethanol	Sigma Aldrich	Cat# 63689
chemical compound, drug	Heat Inctivated Fetal Bovin Serum	Gibco	Cat# A3840001
chemical compound, drug	May Grünwald Stain Solution	Polysciences	Cat# 24981–1
chemical compound, drug	May Grünwald Giemsa Phosphate Buffer	Polysciences	Cat# 25032–1
chemical compound, drug	Wright Giemsa Stain Solution	Polysciences	Cat# 24985–1
chemical compound, drug	Wright Giemsa Stain Phosphate Buffer	Polysciences	Cat# 24984–1
chemical compound, drug	Wright-Giemsa Stain Set	Astral Diagnostics	Cat# 5585
chemical compound, drug	Recombinant Human M-CSF Protein	R and D Systems	Cat# 216-MC-005	20 ng/mL
chemical compound, drug	Recombinant Human IFN-gamma Protein	R and D Systems	Cat# 285-IF	20 ng/mL
chemical compound, drug	Recombinant Human IL-4	Miltenyi Biotec	Cat# 130-093-920	20 ng/mL
chemical compound, drug	Dexamethasone	Sigma Aldrich	Cat# D1756	1 µM
chemical compound, drug	PenStrep	Gibco	Cat# 15140122
chemical compound, drug	L-Glutamine	Gibco	Cat#25030081
chemical compound, drug	Human S100A9	Invitrogen	Cat# A42590	2 ng/mL
chemical compound, drug	RBC Lysis Buffer	BioLegend	Cat# 420301
chemical compound, drug	Liberase	Roche Diagnostics	Cat# 5466202001
chemical compound, drug	Cytofix/Cytoperm Fixation/Permeabilization Kit	BD Biosciences	Cat# 554714
other	ChemoTxDisposable Chemotaxis System	Neuro Probe	Cat# NRP-106–8
other	Transwell Inserts	Thermo Scientific	Cat# 140620

Additional files

Transparent reporting form: https://cdn.elifesciences.org/articles/66808/elife-66808-transrepform-v2.pdf
Download elife-66808-transrepform-v2.pdf

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Mendeley

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

Daniela Fraccarollo
Jonas Neuser
Julian Möller
Christian Riehle
Paolo Galuppo
Johann Bauersachs

(2021)

Expansion of CD10^neg neutrophils and CD14⁺HLA-DR^neg/low monocytes driving proinflammatory responses in patients with acute myocardial infarction

eLife 10:e66808.

https://doi.org/10.7554/eLife.66808

Share this article

Cite this article

Increased circulating levels of CD14+HLA-DRneg/low monocytes in patients with AMI.

Time course analysis of subset-frequencies and circulating levels of monocytes in patients with unstable angina (UA; n=11), non-ST-elevation MI (NSTEMI, n=16), and ST-elevation MI (STEMI, n=44).

Monocyte subset-frequencies.

CD14+HLA-DRneg/low monocytes from patients with AMI are not immunosuppressive but exhibit an inflammatory phenotype.

Expression of genes dysregulated in circulating monocytes and infarct macrophages in a mouse model of reperfused AMI.

Circulating normal-density CD10neg neutrophils increase in patients with AMI.

Increased frequency of CD10negneutrophils in patients with AMI.

Immature CD10neg neutrophils from patients with AMI express high amounts of MMP-9 and S100A9 and display resistance to apoptosis.

Expression of genes regulated in circulating neutrophils and infarct neutrophils in a mouse model of reperfused AMI.

Immature CD101neg neutrophils are rapidly released into the bloodstream after reperfusion in a mouse model of AMI.

Interactions between CD10negneutrophils and CD14+HLA-DRneg/lowmonocytes.

Multiple intercorrelations among CD10neg neutrophils, HLA-DRneg/low monocytes and immune-inflammation markers.

Immature CD101neg 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.

Immature CD101neg neutrophils are present in mediastinal lymph nodes draining the heart after acute MI.

Elevated IFN-γ levels in patients with expanded CD10neg neutrophils and increased frequency of CD4+CD28null T-cells.

Relationship among the peripheral expansion of immature CD10neg neutrophils, CMV IgG titers, CD4+CD28null T-cells, and circulating levels of IFN-γ and IL-12 in patients with AMI.

CD10neg neutrophils enhance IFN-γ production by CD4+ T-cells via induction of interleukin-12.

IFN-γ production by CD4+ T-cells from CMV-seropositive AMI patients stimulated with anti-CD3/CD28 beads and cultured for 24 hr in absence (CD4+) or presence of cell-free supernatants derived from CD10pos neutrophils (csf-CD10pos+CD4+) or CD10neg neutrophils (csf-CD10neg+CD4+).

Immature CD10neg neutrophils and HLA-DRneg/low monocytes inducing proinflammatory and adaptive immune responses emerge in patients with large acute myocardial infarction.

General traits.

Leukocyte count and monocyte subsets.

Immune inflammation markers.

CD4+ T-cells Subsets.

Transparent reporting form

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

Increased circulating levels of CD14⁺HLA-DR^neg/low monocytes in patients with AMI.

CD14⁺HLA-DR^neg/low monocytes from patients with AMI are not immunosuppressive but exhibit an inflammatory phenotype.

Circulating normal-density CD10^neg neutrophils increase in patients with AMI.

Increased frequency of CD10^negneutrophils in patients with AMI.

Immature CD10^neg neutrophils from patients with AMI express high amounts of MMP-9 and S100A9 and display resistance to apoptosis.

Immature CD101^neg neutrophils are rapidly released into the bloodstream after reperfusion in a mouse model of AMI.

Interactions between CD10^negneutrophils and CD14⁺HLA-DR^neg/lowmonocytes.

Multiple intercorrelations among CD10^neg neutrophils, HLA-DR^neg/low monocytes and immune-inflammation markers.

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.

Immature CD101^neg neutrophils are present in mediastinal lymph nodes draining the heart after acute MI.

Elevated IFN-γ levels in patients with expanded CD10^neg neutrophils and increased frequency of CD4⁺CD28^null T-cells.

Relationship among the peripheral expansion of immature CD10^neg neutrophils, CMV IgG titers, CD4⁺CD28^null T-cells, and circulating levels of IFN-γ and IL-12 in patients with AMI.

CD10^neg neutrophils enhance IFN-γ production by CD4⁺ T-cells via induction of interleukin-12.

IFN-γ production by CD4⁺ T-cells from CMV-seropositive AMI patients stimulated with anti-CD3/CD28 beads and cultured for 24 hr in absence (CD4⁺) or presence of cell-free supernatants derived from CD10^pos neutrophils (csf-CD10^pos+CD4⁺) or CD10^neg neutrophils (csf-CD10^neg+CD4⁺).

Immature CD10^neg neutrophils and HLA-DR^neg/low monocytes inducing proinflammatory and adaptive immune responses emerge in patients with large acute myocardial infarction.

CD4⁺ T-cells Subsets.