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Aims

A recent large clinical study demonstrated the association between intermediate CD14++CD16+monocytes and cardiovascular events. However, whether that monocyte subset contributes to the pathogenesis of atrial fibrillation (AF) has not been clarified. We compared the circulating monocyte subsets in AF patients and healthy people, and investigated the possible role of intermediate CD14++CD16+monocytes in the pathophysiology of AF.

Methods and results

This case–control study included 44 consecutive AF patients without systemic diseases referred for catheter ablation at our hospital, and 40 healthy controls. Patients with systemic diseases, including structural heart disease, hepatic or renal dysfunction, collagen disease, malignancy, and inflammation were excluded. Monocyte subset analyses were performed (three distinct human monocyte subsets: classical CD14++CD16−, intermediate CD14++CD16+, and non-classical CD14+CD16++monocytes). We compared the monocyte subsets and evaluated the correlation with other clinical findings. A total of 60 participants (30 AF patients and 30 controls as an age-matched group) were included after excluding 14 AF patients due to inflammation. Atrial fibrillation patients had a higher proportion of circulating intermediate CD14++CD16+monocytes than the controls (17.0 ± 9.6 vs. 7.5 ± 4.1%, P < 0.001). A multivariable logistic regression analysis demonstrated that only the proportion of intermediate CD14++CD16+monocytes (odds ratio: 1.316; 95% confidence interval: 1.095–1.582, P = 0.003) was independently associated with the presence of AF. Intermediate CD14++CD16+monocytes were negatively correlated with the left atrial appendage flow during sinus rhythm (r= −0.679, P = 0.003) and positively with the brain natriuretic peptide (r = 0.439, P = 0.015).

Conclusion

Intermediate CD14++CD16+monocytes might be closely related to the pathogenesis of AF and reflect functional remodelling of the left atrium.

Atrial fibrillation, Inflammation, Pathophysiology of atrial fibrillation, Monocyte subsets, CD14++CD16+monocytes
What's new?

  • The proportion of intermediate CD14++CD16+monocytes was higher in the atrial fibrillation (AF) patients without obvious comorbidities than in healthy controls, and it was independently associated with the presence of AF.

  • Intermediate CD14++CD16+monocytes were negatively correlated with the left atrial appendage flow velocity during sinus rhythm and positively with the brain natriuretic peptide level.

  • Intermediate CD14++CD16+monocytes might be closely related to the pathogenesis of AF and reflect functional remodelling of the left atrium.

Introduction

Observational clinical and ex vivo studies have established a strong association between atrial fibrillation (AF) and inflammation. In endomyocardial biopsies of the right atrial septum from AF patients, inflammatory infiltrates such as monocytes and macrophages are commonly seen.1 Inflammatory infiltrates of leukocytes and macrophages in the atrium of patients with AF are significantly greater than in patients in sinus rhythm.2 Various inflammatory markers and mediators such as C-reactive protein (CRP), tumour necrosis factor (TNF)-α, interleukin (IL)-2, IL-6, IL-8, monocyte chemoattractant protein (MCP)-1, and myeloperoxidase (MPO) have been linked with the presence or outcome of AF.3–5 Immune cells such as monocytes and macrophages produce and secrete inflammatory cytokines, which then act upon the resident cells of the atria including both myocytes and fibroblasts.6 In patients who have received cardiac or cardiopulmonary bypass surgery, the perioperative monocyte activation is significantly associated with postoperative AF.7

A substantial heterogeneity of monocytes is reflected by the differential surface expression of the lipopolysaccharide (LPS) receptor (CD14) and the low-affinity Fcγ-III receptor (CD16).8 The existence of three distinct monocyte subsets is acknowledged by a recent consensus, namely, classical CD14++CD16−monocytes, intermediate CD14++CD16+monocytes, and non-classical CD14+CD16++monocytes.9,10 Classical CD14++CD16−monocytes phenotypically resemble mouse myeloid differentiation antigen Ly6Chigh monocytes because they express high levels of C–C chemokine receptor CCR2, CD62L, and CD64, and low levels of CX3CR1. The second, non-classical CD14+CD16++ population most closely resembles the Ly6Clow monocytes both phenotypically and functionally, expressing low levels of CCR2 and high levels of CX3CR1, and exhibiting patrolling behaviour.11 The third, intermediate CD14++CD16+population, is the main producer of reactive oxygen species.10 Cluster analyses indicate that this intermediate CD14++CD16+population tracks with CD16 monocytes and resembles Ly6Chigh more than Ly6Clow monocytes.11 However, most previous studies did not distinguish between intermediate CD14++CD16+ and non-classical CD14+CD16++monocytes but subsumed these two subsets as CD16 monocytes. Consequently, the intermediate monocyte subset was by far the most poorly characterized monocyte subset until recently. Although numerically the smallest monocyte subpopulation and seemingly displaying just an intermediate immunophenotype, the CD14++CD16+monocytes, are a clearly distinguishable subset.9,10 A recent large clinical study demonstrated the association of intermediate CD14++CD16+monocytes with cardiovascular events in subjects from the general population referred for elective coronary angiography.12

We hypothesized that intermediate CD14++CD16+monocytes contribute to the pathogenesis of AF. To this end, we compared the circulating monocyte subsets in AF patients and healthy people, and investigated the possible role of intermediate CD14++CD16+monocytes in the development of AF.

Methods

Study population

This case–control study included 44 consecutive AF patients, without any obvious co-morbidities, referred for catheter ablation at the Kobe University hospital, and 40 people were recruited at the Kenko Life Plaza, Hyogo Health Service Association (Institution of Medical Examination) to represent a control group with no cardiovascular health problems. This study was conducted to assess the possible role of intermediate CD14++CD16+monocytes in AF patients without any obvious risk factors of AF including inflammation, diabetes mellitus (DM), or any other systemic or structural heart diseases.13 We hypothesized that even unknown inflammation that is reflected only by an elevation of the CRP would seem to have an influence on the immune system, which would result in an increase in the monocytes and a change in the proportion of the monocyte subsets. Therefore, patients with systemic diseases, including structural heart disease, hepatic disease, renal disease (serum creatinine levels >1.5 mg/dL), collagen disease, malignancy, and unknown inflammation (CRP >0.3 mg/dL) were excluded. This study complied with the Declaration of Helsinki with regard to an investigation in humans, and the protocol for this study was approved by the Ethics Committee of Kobe University (No. 1318) and the Kenko Life Plaza, Hyogo Health Service Association. We also obtained written informed consent from all the participants.

The clinical parameters (age, sex, atherosclerosis, and AF risk factors) were assessed. Atherosclerosis and the AF risk factors included hypertension (blood pressure ≥140/90 mmHg, and/or a history of anti-hypertensive medications), DM (fasting plasma glucose ≥126 mg/dL, casual plasma glucose ≥200 mg/dL, and/or a history of anti-diabetic medications), dyslipidemia [low-density lipoprotein cholesterol (LDL-C) ≥140 mg/dL, triglycerides (TG) ≥150 mg/dL, high-density lipoprotein cholesterol (HDL-C) <40 mg/dL, and/or a history of anti-dyslipidemia medications), high-sensitivity (hs)-CRP, current or past smoking, alcohol consumption, and the body mass index (BMI). In AF patients, additional examinations, including an echocardiogram (transthoracic and transoesophageal echocardiogram), contrast enhancement computed tomography for the left atrium (LA) volumetry, and measurements of the brain natriuretic peptide (BNP) level, were performed. Atrial fibrillation that lasted <7 days was defined as paroxysmal AF, and that ≥7 days as persistent AF.

Blood sampling and staining of the mononuclear leukocytes

Fresh anticoagulated peripheral blood samples were collected from all subjects during the preparation before the catheter ablation of AF. In order to confirm the difference in the monocyte distribution, blood samples directly drawn from the inferior vena cava (IVC), coronary sinus (CS), and LA were collected after the transseptal procedure and before the radio frequency applications in 11 patients.

Human peripheral blood mononuclear cells (PBMC) of AF patients and healthy volunteers were obtained in EDTA-coated tubes and prepared by a Ficoll gradient centrifugation. The antibodies used in this study were FITC-anti-CD14 and Alexa Flour 647-anti-CD16, both from BD (Becton, Dickinson and Company). We used an Attune flow cytometer (Beckman Coulter, Brea, CA). The analysis was performed with FlowJo7.6 software (Tree Star, Ashland, OR). The cytometric analysis was performed by a physician who was not informed of the patient's clinical background.

Statistical analysis

Continuous variables are expressed as the mean ± standard deviation and were compared by two-sample t-tests or the Mann–Whitney U tests. Categorical variables were compared using the χ2 test. An ANOVA was compared by blood samples directly drawn from the IVC, CS, and LA. A multivariable logistic regression model was used to determine the contributors to AF. We used standard methods to estimate the sample size for the multivariable logistic regression analysis, with at least 10 outcomes required for each included independent variable.14 With the presence of AF in 44 patients at the initial recruitment, we performed a multivariable logistic regression analysis with 3–4 variables. The variable selection was performed using a stepwise procedure. Those variables that had a P value of <0.1 in the univariate analysis and were clinically influential factors for AF (age, sex, hypertension, dyslipidemia, current smoking, renal function, and the hs-CRP) were included in the variable selection. In the case of variables that were expected to be collinear (i.e. the proportion of the monocyte subset), we included only the variables that had shown the strongest association with the presence of AF in the univariate analysis into the multivariable analysis. The associations between the continuous variables were assessed using a Pearson or Spearman rank correlation test. Statistical analyses were performed using the SPSS statistical software program (version 20.0, SPSS, Inc., Chicago, IL, USA). A two-sided P value of <0.05 was considered to indicate statistical significance.

Results

Baseline characteristics of the enrolled persons

As a result, 14 AF patients were excluded [6 patients due to unknown inflammation (CRP >0.3 mg/dL), 4 to a reduced LVEF (LVEF <50%), 3 to an infection, and 1 to DM), a total of 60 subjects (30 AF patients and 30 selected controls as an age-matched group) were included in the analysis. The 30 AF patients consisted of 17 with paroxysmal AF and 13 with persistent AF. Two AF patients and one control had an impaired glucose tolerance (fasting plasma glucose ≥110 and <126 mg/dL). The baseline characteristics and clinical data of the two groups (controls vs. AF patients) are summarized in Table 1. The AF patients had a higher BMI, TG level, and proportion of anti-arrhythmic and anti-coagulant drugs, and they had a lower diastolic blood pressure (dBP), total cholesterol (T-chol), and HDL-C level.

Table 1
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Baseline characteristics of the control and AF patients

VariableControl (healthy people)AF patientsP-value
N = 30N = 30
Age (years)61.1 ± 5.562.4 ± 9.70.153
Sex [male (%)]22 (73.3)25 (83.3)0.347
BMI22.0 ± 2.224.0 ± 3.30.013
sBP (mmHg)121.7 ± 15.6117.6 ± 11.00.496
dBP (mmHg)81.7 ± 10.175.9 ± 7.80.020
Current or past smoking (%)10 (33.3)11 (36.7)0.787
Alcohol consumption [ethanol (g/day)]19.9 ± 24.010.5 ± 20.10.136
CHADS 2 score0.13 ± 0.350.33 ± 0.660.187
Hypertension (%)4 (13.3)6 (20.0)0.488
Dyslipidemia (%)13 (43.3)7 (23.3)0.100
Laboratory parameters
 Leukocytes (cells/μL)5403.3 ± 1424.15713.3 ± 1429.40.404
 Hb (mg/dL)14.7 ± 1.414.1 ± 1.30.103
 eGFR creatinine (mL/min/1.73 m2)75.1 ± 10.969.0 ± 15.80.084
 T-chol (mg/dL)207.2 ± 29.8191.1 ± 27.20.033
 LDL-C (mg/dL)128.5 ± 34.9121.7 ± 29.40.418
 HDL-C (mg/dL)63.2 ± 16.950.5 ± 13.70.002
 TG (mg/dL)97.1 ± 58.6138.0 ± 64.80.001
 Fasting plasma glucose (mg/dL)95.8 ± 8.895.1 ± 9.20.912
 hs-CRP (mg/dL)0.046 ± 0.0840.060 ± 0.0760.090
Cytometric analysis
 monocytes (cells/μL)103.2 ± 66.9162.1 ± 127.50.152
 CD14++CD16−monocytes (%)79.3 ± 8.668.5 ± 15.00.004
 CD14++CD16+monocytes (%)7.5 ± 4.117.0 ± 9.6<0.001
 CD14+CD16++monocytes (%)11.8 ± 5.913.4 ± 7.70.559
Medication
 Anti-arrhythmic medication (%)0 (0.0)22 (73.3)<0.001
 Anti-coagulation therapy (%)0 (0.0)30 (100%)<0.001
VariableControl (healthy people)AF patientsP-value
N = 30N = 30
Age (years)61.1 ± 5.562.4 ± 9.70.153
Sex [male (%)]22 (73.3)25 (83.3)0.347
BMI22.0 ± 2.224.0 ± 3.30.013
sBP (mmHg)121.7 ± 15.6117.6 ± 11.00.496
dBP (mmHg)81.7 ± 10.175.9 ± 7.80.020
Current or past smoking (%)10 (33.3)11 (36.7)0.787
Alcohol consumption [ethanol (g/day)]19.9 ± 24.010.5 ± 20.10.136
CHADS 2 score0.13 ± 0.350.33 ± 0.660.187
Hypertension (%)4 (13.3)6 (20.0)0.488
Dyslipidemia (%)13 (43.3)7 (23.3)0.100
Laboratory parameters
 Leukocytes (cells/μL)5403.3 ± 1424.15713.3 ± 1429.40.404
 Hb (mg/dL)14.7 ± 1.414.1 ± 1.30.103
 eGFR creatinine (mL/min/1.73 m2)75.1 ± 10.969.0 ± 15.80.084
 T-chol (mg/dL)207.2 ± 29.8191.1 ± 27.20.033
 LDL-C (mg/dL)128.5 ± 34.9121.7 ± 29.40.418
 HDL-C (mg/dL)63.2 ± 16.950.5 ± 13.70.002
 TG (mg/dL)97.1 ± 58.6138.0 ± 64.80.001
 Fasting plasma glucose (mg/dL)95.8 ± 8.895.1 ± 9.20.912
 hs-CRP (mg/dL)0.046 ± 0.0840.060 ± 0.0760.090
Cytometric analysis
 monocytes (cells/μL)103.2 ± 66.9162.1 ± 127.50.152
 CD14++CD16−monocytes (%)79.3 ± 8.668.5 ± 15.00.004
 CD14++CD16+monocytes (%)7.5 ± 4.117.0 ± 9.6<0.001
 CD14+CD16++monocytes (%)11.8 ± 5.913.4 ± 7.70.559
Medication
 Anti-arrhythmic medication (%)0 (0.0)22 (73.3)<0.001
 Anti-coagulation therapy (%)0 (0.0)30 (100%)<0.001

The data are the mean value ± SD or n (%).

AF, atrial fibrillation; BMI, body mass index; eGFR, estimated glomerular filtration rate; Hb, haemoglobin; HbA1c, haemoglobin A1c; hs-CRP, high-sensitivity C-reactive protein; LDL-C, low-density lipoprotein cholesterol; T-chol, total cholesterol; TG, triglycerides.

Table 1
Open in new tab

Baseline characteristics of the control and AF patients

VariableControl (healthy people)AF patientsP-value
N = 30N = 30
Age (years)61.1 ± 5.562.4 ± 9.70.153
Sex [male (%)]22 (73.3)25 (83.3)0.347
BMI22.0 ± 2.224.0 ± 3.30.013
sBP (mmHg)121.7 ± 15.6117.6 ± 11.00.496
dBP (mmHg)81.7 ± 10.175.9 ± 7.80.020
Current or past smoking (%)10 (33.3)11 (36.7)0.787
Alcohol consumption [ethanol (g/day)]19.9 ± 24.010.5 ± 20.10.136
CHADS 2 score0.13 ± 0.350.33 ± 0.660.187
Hypertension (%)4 (13.3)6 (20.0)0.488
Dyslipidemia (%)13 (43.3)7 (23.3)0.100
Laboratory parameters
 Leukocytes (cells/μL)5403.3 ± 1424.15713.3 ± 1429.40.404
 Hb (mg/dL)14.7 ± 1.414.1 ± 1.30.103
 eGFR creatinine (mL/min/1.73 m2)75.1 ± 10.969.0 ± 15.80.084
 T-chol (mg/dL)207.2 ± 29.8191.1 ± 27.20.033
 LDL-C (mg/dL)128.5 ± 34.9121.7 ± 29.40.418
 HDL-C (mg/dL)63.2 ± 16.950.5 ± 13.70.002
 TG (mg/dL)97.1 ± 58.6138.0 ± 64.80.001
 Fasting plasma glucose (mg/dL)95.8 ± 8.895.1 ± 9.20.912
 hs-CRP (mg/dL)0.046 ± 0.0840.060 ± 0.0760.090
Cytometric analysis
 monocytes (cells/μL)103.2 ± 66.9162.1 ± 127.50.152
 CD14++CD16−monocytes (%)79.3 ± 8.668.5 ± 15.00.004
 CD14++CD16+monocytes (%)7.5 ± 4.117.0 ± 9.6<0.001
 CD14+CD16++monocytes (%)11.8 ± 5.913.4 ± 7.70.559
Medication
 Anti-arrhythmic medication (%)0 (0.0)22 (73.3)<0.001
 Anti-coagulation therapy (%)0 (0.0)30 (100%)<0.001
VariableControl (healthy people)AF patientsP-value
N = 30N = 30
Age (years)61.1 ± 5.562.4 ± 9.70.153
Sex [male (%)]22 (73.3)25 (83.3)0.347
BMI22.0 ± 2.224.0 ± 3.30.013
sBP (mmHg)121.7 ± 15.6117.6 ± 11.00.496
dBP (mmHg)81.7 ± 10.175.9 ± 7.80.020
Current or past smoking (%)10 (33.3)11 (36.7)0.787
Alcohol consumption [ethanol (g/day)]19.9 ± 24.010.5 ± 20.10.136
CHADS 2 score0.13 ± 0.350.33 ± 0.660.187
Hypertension (%)4 (13.3)6 (20.0)0.488
Dyslipidemia (%)13 (43.3)7 (23.3)0.100
Laboratory parameters
 Leukocytes (cells/μL)5403.3 ± 1424.15713.3 ± 1429.40.404
 Hb (mg/dL)14.7 ± 1.414.1 ± 1.30.103
 eGFR creatinine (mL/min/1.73 m2)75.1 ± 10.969.0 ± 15.80.084
 T-chol (mg/dL)207.2 ± 29.8191.1 ± 27.20.033
 LDL-C (mg/dL)128.5 ± 34.9121.7 ± 29.40.418
 HDL-C (mg/dL)63.2 ± 16.950.5 ± 13.70.002
 TG (mg/dL)97.1 ± 58.6138.0 ± 64.80.001
 Fasting plasma glucose (mg/dL)95.8 ± 8.895.1 ± 9.20.912
 hs-CRP (mg/dL)0.046 ± 0.0840.060 ± 0.0760.090
Cytometric analysis
 monocytes (cells/μL)103.2 ± 66.9162.1 ± 127.50.152
 CD14++CD16−monocytes (%)79.3 ± 8.668.5 ± 15.00.004
 CD14++CD16+monocytes (%)7.5 ± 4.117.0 ± 9.6<0.001
 CD14+CD16++monocytes (%)11.8 ± 5.913.4 ± 7.70.559
Medication
 Anti-arrhythmic medication (%)0 (0.0)22 (73.3)<0.001
 Anti-coagulation therapy (%)0 (0.0)30 (100%)<0.001

The data are the mean value ± SD or n (%).

AF, atrial fibrillation; BMI, body mass index; eGFR, estimated glomerular filtration rate; Hb, haemoglobin; HbA1c, haemoglobin A1c; hs-CRP, high-sensitivity C-reactive protein; LDL-C, low-density lipoprotein cholesterol; T-chol, total cholesterol; TG, triglycerides.

With a focus on paroxysmal and persistent AF patients, the clinical and biochemistry parameters for both groups are summarized in Table 2. The left ventricular ejection fraction (LVEF) was lower, and the BNP level and left atrial volume were higher in the persistent AF patients than that in the paroxysmal AF patients. The left atrial appendage (LAA) flow measured during sinus rhythm in the paroxysmal AF patients (N = 15) had a tendency to be higher than that in the persistent AF patients (N = 3) (61.9 ± 20.4 vs. 37.7 ± 14.0 cm/s, P = 0.067). However, there was no difference in the LAA flow between them during an AF rhythm (51.5 ± 3.5 vs. 34.8 ± 13.3 cm/s, 2 paroxysmal vs. 10 persistent AF patients, P = 0.121).

Table 2
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Baseline characteristics of the paroxysmal and persistent AF patients

VariableParoxysmal AFPersistent AFP-value
N = 17N = 13
Age (years)62.4 ± 10.262.5 ± 9.60.869
Sex [male (%)]15 (88.2)10 (76.9)0.410
Duration of AF (months)49.9 ± 44.538.2 ± 35.20.536
Laboratory parameters
 Leukocytes (μ/L)5458.8 ± 1531.86046.2 ± 1264.00.272
 Hb (mg/dL)13.8 ± 1.014.6 ± 1.60.114
 eGFR creatinine (mL/min/1.73 m2)69.3 ± 17.368.5 ± 14.20.245
 T-chol (mg/dL)187.8 ± 27.8195.5 ± 27.00.453
 LDL-C (mg/dL)121.8 ± 32.4121.5 ± 26.40.974
 HDL-C (mg/dL)49.9 ± 13.551.4 ± 14.60.650
 TG (mg/dL)134.8 ± 66.6142.2 ± 64.70.621
 Fasting plasma glucose93.4 ± 8.297.4 ± 10.20.239
 hs-CRP (mg/dL)0.040 ± 0.0420.069 ± 0.1060.869
 BNP (pg/mL)58.9 ± 95.475.5 ± 42.20.009
Echocardiogram parameters
 LVEF (%)64.8 ± 4.060.2 ± 4.60.009
 LA diameter (mm)39.7 ± 5.643.0 ± 6.80.158
 Rhythm during TEE [SR (%)]15 (88.2)3 (23.1)<0.001
 LAA flow during SR [cm/s (N = 18)]61.9 ± 20.4 (N = 15)37.7 ± 14.0 (N = 3)0.067
 LAA flow during AF [cm/s (N = 12)]51.5 ± 3.5 (N = 2)34.8 ± 13.3 (N = 10)0.121
CECT parameters
 LA volume (mL)96.5 ± 27.7146.9 ± 57.10.004
Cytometric analysis
 Monocytes (cells/μL)135.9 ± 72.2178.5 ± 164.10.414
 CD14++CD16−monocytes (%)71.7 ± 14.562.4 ± 15.00.065
 CD14++CD16+monocytes (%)14.0 ± 8.421.3 ± 10.30.017
 CD14+CD16++monocytes (%)13.1 ± 7.615.1 ± 7.80.525
VariableParoxysmal AFPersistent AFP-value
N = 17N = 13
Age (years)62.4 ± 10.262.5 ± 9.60.869
Sex [male (%)]15 (88.2)10 (76.9)0.410
Duration of AF (months)49.9 ± 44.538.2 ± 35.20.536
Laboratory parameters
 Leukocytes (μ/L)5458.8 ± 1531.86046.2 ± 1264.00.272
 Hb (mg/dL)13.8 ± 1.014.6 ± 1.60.114
 eGFR creatinine (mL/min/1.73 m2)69.3 ± 17.368.5 ± 14.20.245
 T-chol (mg/dL)187.8 ± 27.8195.5 ± 27.00.453
 LDL-C (mg/dL)121.8 ± 32.4121.5 ± 26.40.974
 HDL-C (mg/dL)49.9 ± 13.551.4 ± 14.60.650
 TG (mg/dL)134.8 ± 66.6142.2 ± 64.70.621
 Fasting plasma glucose93.4 ± 8.297.4 ± 10.20.239
 hs-CRP (mg/dL)0.040 ± 0.0420.069 ± 0.1060.869
 BNP (pg/mL)58.9 ± 95.475.5 ± 42.20.009
Echocardiogram parameters
 LVEF (%)64.8 ± 4.060.2 ± 4.60.009
 LA diameter (mm)39.7 ± 5.643.0 ± 6.80.158
 Rhythm during TEE [SR (%)]15 (88.2)3 (23.1)<0.001
 LAA flow during SR [cm/s (N = 18)]61.9 ± 20.4 (N = 15)37.7 ± 14.0 (N = 3)0.067
 LAA flow during AF [cm/s (N = 12)]51.5 ± 3.5 (N = 2)34.8 ± 13.3 (N = 10)0.121
CECT parameters
 LA volume (mL)96.5 ± 27.7146.9 ± 57.10.004
Cytometric analysis
 Monocytes (cells/μL)135.9 ± 72.2178.5 ± 164.10.414
 CD14++CD16−monocytes (%)71.7 ± 14.562.4 ± 15.00.065
 CD14++CD16+monocytes (%)14.0 ± 8.421.3 ± 10.30.017
 CD14+CD16++monocytes (%)13.1 ± 7.615.1 ± 7.80.525

The data are the mean value ± SD.

AF, atrial fibrillation; BNP, brain natriuretic peptide; eGFR, estimated glomerular filtration rate; Hb, haemoglobin; HbA1c, haemoglobin A1c; hs-CRP, high-sensitivity C-reactive protein; LDL-C, low-density lipoprotein cholesterol; SR, sinus rhythm; T-chol, total cholesterol; TEE, transoesophageal echocardiogram; TG, triglycerides; LA, left atrium; LAA, left atrial appendage; LVEF, left ventricular ejection fraction.

Table 2
Open in new tab

Baseline characteristics of the paroxysmal and persistent AF patients

VariableParoxysmal AFPersistent AFP-value
N = 17N = 13
Age (years)62.4 ± 10.262.5 ± 9.60.869
Sex [male (%)]15 (88.2)10 (76.9)0.410
Duration of AF (months)49.9 ± 44.538.2 ± 35.20.536
Laboratory parameters
 Leukocytes (μ/L)5458.8 ± 1531.86046.2 ± 1264.00.272
 Hb (mg/dL)13.8 ± 1.014.6 ± 1.60.114
 eGFR creatinine (mL/min/1.73 m2)69.3 ± 17.368.5 ± 14.20.245
 T-chol (mg/dL)187.8 ± 27.8195.5 ± 27.00.453
 LDL-C (mg/dL)121.8 ± 32.4121.5 ± 26.40.974
 HDL-C (mg/dL)49.9 ± 13.551.4 ± 14.60.650
 TG (mg/dL)134.8 ± 66.6142.2 ± 64.70.621
 Fasting plasma glucose93.4 ± 8.297.4 ± 10.20.239
 hs-CRP (mg/dL)0.040 ± 0.0420.069 ± 0.1060.869
 BNP (pg/mL)58.9 ± 95.475.5 ± 42.20.009
Echocardiogram parameters
 LVEF (%)64.8 ± 4.060.2 ± 4.60.009
 LA diameter (mm)39.7 ± 5.643.0 ± 6.80.158
 Rhythm during TEE [SR (%)]15 (88.2)3 (23.1)<0.001
 LAA flow during SR [cm/s (N = 18)]61.9 ± 20.4 (N = 15)37.7 ± 14.0 (N = 3)0.067
 LAA flow during AF [cm/s (N = 12)]51.5 ± 3.5 (N = 2)34.8 ± 13.3 (N = 10)0.121
CECT parameters
 LA volume (mL)96.5 ± 27.7146.9 ± 57.10.004
Cytometric analysis
 Monocytes (cells/μL)135.9 ± 72.2178.5 ± 164.10.414
 CD14++CD16−monocytes (%)71.7 ± 14.562.4 ± 15.00.065
 CD14++CD16+monocytes (%)14.0 ± 8.421.3 ± 10.30.017
 CD14+CD16++monocytes (%)13.1 ± 7.615.1 ± 7.80.525
VariableParoxysmal AFPersistent AFP-value
N = 17N = 13
Age (years)62.4 ± 10.262.5 ± 9.60.869
Sex [male (%)]15 (88.2)10 (76.9)0.410
Duration of AF (months)49.9 ± 44.538.2 ± 35.20.536
Laboratory parameters
 Leukocytes (μ/L)5458.8 ± 1531.86046.2 ± 1264.00.272
 Hb (mg/dL)13.8 ± 1.014.6 ± 1.60.114
 eGFR creatinine (mL/min/1.73 m2)69.3 ± 17.368.5 ± 14.20.245
 T-chol (mg/dL)187.8 ± 27.8195.5 ± 27.00.453
 LDL-C (mg/dL)121.8 ± 32.4121.5 ± 26.40.974
 HDL-C (mg/dL)49.9 ± 13.551.4 ± 14.60.650
 TG (mg/dL)134.8 ± 66.6142.2 ± 64.70.621
 Fasting plasma glucose93.4 ± 8.297.4 ± 10.20.239
 hs-CRP (mg/dL)0.040 ± 0.0420.069 ± 0.1060.869
 BNP (pg/mL)58.9 ± 95.475.5 ± 42.20.009
Echocardiogram parameters
 LVEF (%)64.8 ± 4.060.2 ± 4.60.009
 LA diameter (mm)39.7 ± 5.643.0 ± 6.80.158
 Rhythm during TEE [SR (%)]15 (88.2)3 (23.1)<0.001
 LAA flow during SR [cm/s (N = 18)]61.9 ± 20.4 (N = 15)37.7 ± 14.0 (N = 3)0.067
 LAA flow during AF [cm/s (N = 12)]51.5 ± 3.5 (N = 2)34.8 ± 13.3 (N = 10)0.121
CECT parameters
 LA volume (mL)96.5 ± 27.7146.9 ± 57.10.004
Cytometric analysis
 Monocytes (cells/μL)135.9 ± 72.2178.5 ± 164.10.414
 CD14++CD16−monocytes (%)71.7 ± 14.562.4 ± 15.00.065
 CD14++CD16+monocytes (%)14.0 ± 8.421.3 ± 10.30.017
 CD14+CD16++monocytes (%)13.1 ± 7.615.1 ± 7.80.525

The data are the mean value ± SD.

AF, atrial fibrillation; BNP, brain natriuretic peptide; eGFR, estimated glomerular filtration rate; Hb, haemoglobin; HbA1c, haemoglobin A1c; hs-CRP, high-sensitivity C-reactive protein; LDL-C, low-density lipoprotein cholesterol; SR, sinus rhythm; T-chol, total cholesterol; TEE, transoesophageal echocardiogram; TG, triglycerides; LA, left atrium; LAA, left atrial appendage; LVEF, left ventricular ejection fraction.

Cytometric analysis of the monocyte subset

There was no significant difference in the distribution of monocyte subsets among the blood samples directly drawn from the IVC, CS, and LA in 11 AF patients. In detail, the classical CD14++CD16−monocytes were 60.8 ± 15.6% in the IVC, 61.9 ± 17.0% in the CS, and 64.3 ± 18.5% in the LA (P = 0.897), the intermediate CD14++CD16+monocytes were 18.9 ± 9.4% in the IVC, 17.6 ± 11.5% in the CS, and 18.6 ± 11.2% in the LA (P = 0.954), and the non-classical CD14+CD16++monocytes were 18.7 ± 8.6% in the IVC, 19.0 ± 8.5% in the CS, and 16.2 ± 9.4% in the LA (P = 0.737).

The mean monocyte count was 103.2 ± 66.9 cells/μL in the control group and 162.1 ± 127.5 cells/μL in the AF patients (P = 0.152), of which 79.3 ± 8.6% in the controls and 68.5 ± 15.0% in the AF patients were classical CD14++CD16−monocytes (P = 0.004), 7.5 ± 4.1% in the controls and 17.0 ± 9.6% in the AF patients were intermediate CD14++CD16+monocytes (P < 0.001), and 11.8 ± 5.9% in the controls and 13.4 ± 7.7% in the AF patients were non-classical CD14+CD16++monocytes (P = 0.559). Atrial fibrillation patients had a higher proportion of intermediate CD14++CD16+monocytes and lower proportion of classical CD14++CD16−monocytes than the controls (Figure 1 shows a representative example of the two groups).
Flow cytometric analysis of monocyte subpopulations. Depicted are classical CD14++CD16−monocytes (upper left), intermediate CD14++CD16+monocytes (upper right), and non-classical CD14+D16++monocytes (lower right) (representative examples, (A): control, (B): AF patient).
Figure 1

Flow cytometric analysis of monocyte subpopulations. Depicted are classical CD14++CD16−monocytes (upper left), intermediate CD14++CD16+monocytes (upper right), and non-classical CD14+D16++monocytes (lower right) (representative examples, (A): control, (B): AF patient).

Open in new tabDownload slide
As for the subtypes of AF, the mean monocyte count was 135.9 ± 72.2 cells/μL in the paroxysmal AF patients and 178.5 ± 164.1 cells/μL in the persistent AF patients (P = 0.414), of which 71.7 ± 14.5% in the paroxysmal AF patients and 62.4 ± 15.0% in the persistent AF patients were classical CD14++CD16−monocytes (P = 0.065), 14.0 ± 8.4% in the paroxysmal AF patients and 21.3 ± 10.3% in the persistent AF patients were intermediate CD14++CD16+monocytes (P = 0.017), and 13.1 ± 7.6% in the paroxysmal AF patients and 15.1 ± 7.8% in the persistent AF patients were non-classical CD14+CD16++monocytes (P = 0.525). There was no significant difference in the total monocyte cell count between the paroxysmal and persistent AF patients, though a proportion of the intermediate CD14++CD16+monocytes was higher in the persistent AF patients than in paroxysmal AF patients (Figure 2).
Comparison of the proportion of CD14++CD16+monocytes. (A) Comparison between the controls and AF patients. (B) Comparison among the controls, and paroxysmal and persistent AF patients. Data are presented as a box and whisker plot with the median and 25–75th percentiles (boxes) and 10–90th percentiles (whiskers).
Figure 2

Comparison of the proportion of CD14++CD16+monocytes. (A) Comparison between the controls and AF patients. (B) Comparison among the controls, and paroxysmal and persistent AF patients. Data are presented as a box and whisker plot with the median and 25–75th percentiles (boxes) and 10–90th percentiles (whiskers).

Open in new tabDownload slide

Intermediate CD14++CD16+monocytes as a contributor to atrial fibrillation

A univariate logistic regression analysis revealed that among the clinical, biochemistry, and other parameters, the BMI [odds ratio (OR): 1.318; 95% confidence interval (CI): 1.054–1.647, P = 0.016), dBP (OR: 0.923; 95% CI: 0.860–0.990, P = 0.025), T-chol (OR: 0.980; 95% CI: 0.961–0.999, P = 0.040), HDL-C (OR: 0.946; 95% CI: 0.910–0.984, P = 0.006), TG (OR: 1.012; 95% CI: 1.002–1.022, P = 0.023), eGFR (OR: 0.965; 95% CI: 0.927–1.006, P = 0.092), total monocyte count (OR: 1.006; 95% CI: 1.000–1.012, P = 0.068), and proportion of classical CD14++CD16−monocytes (OR: 0.924; 95% CI: 0.875–0.975, P = 0.004) and intermediate CD14++CD16+monocytes (OR: 1.341; 95% CI: 1.126–1.597, P = 0.001) influenced the presence of AF. As the result of a variable selection, three variables (BMI, HDL-C, and the proportion of CD14++CD16+ monocytes) were included in the multivariable analysis. The multivariable logistic regression analysis demonstrated that only the proportion of intermediate CD14++CD16+monocytes (OR: 1.316; 95% CI: 1.095–1.582, P = 0.003) was independently associated with the presence of AF (Table 3).

Table 3
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Univariate and multivariate logistic regression analyses as contributors to AF

VariableUnivariate analysis
Multivariate analysis
OR (95% CI)P-valueOR (95% CI)P-value
BMI (kg/m2)1.318 (1.054–1.647)0.0161.195 (0.895–1.595)0.226
dBP (mmHg)0.923 (0.860–0.990)0.025
T-chol (mg/dL)0.980 (0.961–0.999)0.040
HDL-C (mg/dL)0.946 (0.910–0.984)0.0060.962 (0.913–1.014)0.149
TG (mg/dL)1.012 (1.002–1.022)0.023
eGFR (mL/min/1.73 m2)0.965 (0.927–1.006)0.092
Monocytes (cells/μL)1.006 (1.000–1.012)0.068
CD14++CD16−monocytes (%)0.924 (0.875–0.975)0.004
CD14++CD16+monocytes (%)1.341 (1.126–1.597)0.0011.316 (1.095–1.582)0.003
VariableUnivariate analysis
Multivariate analysis
OR (95% CI)P-valueOR (95% CI)P-value
BMI (kg/m2)1.318 (1.054–1.647)0.0161.195 (0.895–1.595)0.226
dBP (mmHg)0.923 (0.860–0.990)0.025
T-chol (mg/dL)0.980 (0.961–0.999)0.040
HDL-C (mg/dL)0.946 (0.910–0.984)0.0060.962 (0.913–1.014)0.149
TG (mg/dL)1.012 (1.002–1.022)0.023
eGFR (mL/min/1.73 m2)0.965 (0.927–1.006)0.092
Monocytes (cells/μL)1.006 (1.000–1.012)0.068
CD14++CD16−monocytes (%)0.924 (0.875–0.975)0.004
CD14++CD16+monocytes (%)1.341 (1.126–1.597)0.0011.316 (1.095–1.582)0.003

The abbreviations are as in Tables 1 and 2.

Table 3
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Univariate and multivariate logistic regression analyses as contributors to AF

VariableUnivariate analysis
Multivariate analysis
OR (95% CI)P-valueOR (95% CI)P-value
BMI (kg/m2)1.318 (1.054–1.647)0.0161.195 (0.895–1.595)0.226
dBP (mmHg)0.923 (0.860–0.990)0.025
T-chol (mg/dL)0.980 (0.961–0.999)0.040
HDL-C (mg/dL)0.946 (0.910–0.984)0.0060.962 (0.913–1.014)0.149
TG (mg/dL)1.012 (1.002–1.022)0.023
eGFR (mL/min/1.73 m2)0.965 (0.927–1.006)0.092
Monocytes (cells/μL)1.006 (1.000–1.012)0.068
CD14++CD16−monocytes (%)0.924 (0.875–0.975)0.004
CD14++CD16+monocytes (%)1.341 (1.126–1.597)0.0011.316 (1.095–1.582)0.003
VariableUnivariate analysis
Multivariate analysis
OR (95% CI)P-valueOR (95% CI)P-value
BMI (kg/m2)1.318 (1.054–1.647)0.0161.195 (0.895–1.595)0.226
dBP (mmHg)0.923 (0.860–0.990)0.025
T-chol (mg/dL)0.980 (0.961–0.999)0.040
HDL-C (mg/dL)0.946 (0.910–0.984)0.0060.962 (0.913–1.014)0.149
TG (mg/dL)1.012 (1.002–1.022)0.023
eGFR (mL/min/1.73 m2)0.965 (0.927–1.006)0.092
Monocytes (cells/μL)1.006 (1.000–1.012)0.068
CD14++CD16−monocytes (%)0.924 (0.875–0.975)0.004
CD14++CD16+monocytes (%)1.341 (1.126–1.597)0.0011.316 (1.095–1.582)0.003

The abbreviations are as in Tables 1 and 2.

Correlation coefficients of the monocyte subtypes with other factors in the atrial fibrillation patients

The association of the monocyte subtypes with the other clinical and laboratory parameters (especially the duration of AF, echocardiogram parameters, BNP level, and LA volume in the AF patients) was assessed (Table 4). The intermediate CD14++CD16+monocytes exhibited a significant negative correlation with the LAA flow during sinus rhythm (r: −0.679; P = 0.003) and positive correlation with the BNP level (r: 0.439; P = 0.015); however, they were not significantly correlated with the LAA flow during AF (r: −0.252; P = 0.385).

Table 4
Open in new tab

Correlation coefficients of the monocyte subtypes with other factors in the AF patients

VariableCD14++CD16−monocytes (%) (classical monocyte)
CD14++CD16+monocytes (%) (intermediate monocyte)
CD14+CD16++monocytes (%) (non-classical monocyte)
rP-valuerP-valuerP-value
Age−0.2460.1910.3500.0580.0650.732
BMI−0.0820.6680.0130.9460.1960.299
sBP0.0000.999−0.0120.9490.1050.580
dBP−0.2160.2510.1240.5130.1820.335
Leukocytes−0.1450.4430.3230.082−0.0430.823
Hb−0.0410.8310.1400.459−0.0100.958
eGFR creatinine0.2460.189−0.1680.376−0.1990.291
T-chol−0.2320.2180.2290.2230.1560.411
LDL-C−0.3230.0810.2500.1830.2190.244
HDL-C0.1410.459−0.0030.986−0.2390.203
TG0.0430.8220.0210.912−0.1560.409
FBS0.1240.515−0.1470.439−0.1370.471
hs-CRP−0.0890.6420.1270.5030.0200.918
BNP−0.4640.0100.4390.0150.5020.005
Duration of AF−0.0220.909−0.0500.7940.1130.554
LVEF0.2080.270−0.2340.212−0.0970.610
LA diameter−0.2070.2820.0810.6750.2460.199
LAA flow (sinus rhythm)0.3870.112−0.6790.003−0.1030.685
LAA flow (AF rhythm)−0.0010.997−0.2520.385−0.0330.919
LA volume−0.1630.3890.1070.5720.0400.833
VariableCD14++CD16−monocytes (%) (classical monocyte)
CD14++CD16+monocytes (%) (intermediate monocyte)
CD14+CD16++monocytes (%) (non-classical monocyte)
rP-valuerP-valuerP-value
Age−0.2460.1910.3500.0580.0650.732
BMI−0.0820.6680.0130.9460.1960.299
sBP0.0000.999−0.0120.9490.1050.580
dBP−0.2160.2510.1240.5130.1820.335
Leukocytes−0.1450.4430.3230.082−0.0430.823
Hb−0.0410.8310.1400.459−0.0100.958
eGFR creatinine0.2460.189−0.1680.376−0.1990.291
T-chol−0.2320.2180.2290.2230.1560.411
LDL-C−0.3230.0810.2500.1830.2190.244
HDL-C0.1410.459−0.0030.986−0.2390.203
TG0.0430.8220.0210.912−0.1560.409
FBS0.1240.515−0.1470.439−0.1370.471
hs-CRP−0.0890.6420.1270.5030.0200.918
BNP−0.4640.0100.4390.0150.5020.005
Duration of AF−0.0220.909−0.0500.7940.1130.554
LVEF0.2080.270−0.2340.212−0.0970.610
LA diameter−0.2070.2820.0810.6750.2460.199
LAA flow (sinus rhythm)0.3870.112−0.6790.003−0.1030.685
LAA flow (AF rhythm)−0.0010.997−0.2520.385−0.0330.919
LA volume−0.1630.3890.1070.5720.0400.833

The abbreviations are as in Tables 1 and 2.

Table 4
Open in new tab

Correlation coefficients of the monocyte subtypes with other factors in the AF patients

VariableCD14++CD16−monocytes (%) (classical monocyte)
CD14++CD16+monocytes (%) (intermediate monocyte)
CD14+CD16++monocytes (%) (non-classical monocyte)
rP-valuerP-valuerP-value
Age−0.2460.1910.3500.0580.0650.732
BMI−0.0820.6680.0130.9460.1960.299
sBP0.0000.999−0.0120.9490.1050.580
dBP−0.2160.2510.1240.5130.1820.335
Leukocytes−0.1450.4430.3230.082−0.0430.823
Hb−0.0410.8310.1400.459−0.0100.958
eGFR creatinine0.2460.189−0.1680.376−0.1990.291
T-chol−0.2320.2180.2290.2230.1560.411
LDL-C−0.3230.0810.2500.1830.2190.244
HDL-C0.1410.459−0.0030.986−0.2390.203
TG0.0430.8220.0210.912−0.1560.409
FBS0.1240.515−0.1470.439−0.1370.471
hs-CRP−0.0890.6420.1270.5030.0200.918
BNP−0.4640.0100.4390.0150.5020.005
Duration of AF−0.0220.909−0.0500.7940.1130.554
LVEF0.2080.270−0.2340.212−0.0970.610
LA diameter−0.2070.2820.0810.6750.2460.199
LAA flow (sinus rhythm)0.3870.112−0.6790.003−0.1030.685
LAA flow (AF rhythm)−0.0010.997−0.2520.385−0.0330.919
LA volume−0.1630.3890.1070.5720.0400.833
VariableCD14++CD16−monocytes (%) (classical monocyte)
CD14++CD16+monocytes (%) (intermediate monocyte)
CD14+CD16++monocytes (%) (non-classical monocyte)
rP-valuerP-valuerP-value
Age−0.2460.1910.3500.0580.0650.732
BMI−0.0820.6680.0130.9460.1960.299
sBP0.0000.999−0.0120.9490.1050.580
dBP−0.2160.2510.1240.5130.1820.335
Leukocytes−0.1450.4430.3230.082−0.0430.823
Hb−0.0410.8310.1400.459−0.0100.958
eGFR creatinine0.2460.189−0.1680.376−0.1990.291
T-chol−0.2320.2180.2290.2230.1560.411
LDL-C−0.3230.0810.2500.1830.2190.244
HDL-C0.1410.459−0.0030.986−0.2390.203
TG0.0430.8220.0210.912−0.1560.409
FBS0.1240.515−0.1470.439−0.1370.471
hs-CRP−0.0890.6420.1270.5030.0200.918
BNP−0.4640.0100.4390.0150.5020.005
Duration of AF−0.0220.909−0.0500.7940.1130.554
LVEF0.2080.270−0.2340.212−0.0970.610
LA diameter−0.2070.2820.0810.6750.2460.199
LAA flow (sinus rhythm)0.3870.112−0.6790.003−0.1030.685
LAA flow (AF rhythm)−0.0010.997−0.2520.385−0.0330.919
LA volume−0.1630.3890.1070.5720.0400.833

The abbreviations are as in Tables 1 and 2.

Discussion

The major findings of this study are as below:

  1. Comparing the AF patients without obvious comorbidities to healthy controls, the AF patients had a higher proportion of circulating intermediate CD14++CD16+monocytes than the controls. A multivariable logistic regression analysis demonstrated that only the proportion of intermediate CD14++CD16+monocytes (OR: 1.316; 95% CI: 1.095–1.582, P = 0.003) was independently associated with the presence of AF.

  2. Intermediate CD14++CD16+monocytes were negatively correlated with the LAA flow during sinus rhythm and positively with the BNP level; however, there was no correlation with the other parameters including the AF duration and LA volume.

Inflammation in atrial fibrillation

Various inflammatory markers and mediators such as the CRP, TNF-α, IL-2, IL-6, IL-8, and MCP-1 have a relationship with the presence or outcome of AF.3–5 Recent studies indicated that activated inflammatory cells and inflammatory mediators might confer a proarrhythmic state by promoting endothelial damage in patients after cardiac surgery.7

The existence of three distinct monocyte subsets is acknowledged by a recent consensus, namely, classical CD14++CD16−monocytes, intermediate CD14++CD16+monocytes, and non-classical CD14+CD16++monocytes. The intermediate CD14++CD16+population, which is the main producer of reactive oxygen species, predicts the future cardiovascular events in patients with coronary artery disease.12 It has been reported that Ly6Chigh monocytes, which cluster together with human intermediate CD14++CD16+monocytes, have a proatherosclerotic role in murine studies.15,16 Indeed, the intermediate CD14++CD16+monocytes correlated with the prevalent cardiovascular risk factors and CRP in their study. In the present study, selected AF patients with a low-grade hs-CRP and no obvious comorbidities had a higher proportion of intermediate CD14++CD16+monocytes than the healthy controls.

Although patients who were diagnosed with DM were excluded from the present study, two AF patients and one of the controls had an impaired glucose tolerance. Recent studies reported that DM is associated with an increased risk of subsequent AF; however, the mechanisms underlying the relationship between DM and AF remain speculative.13 Nevertheless, the fasting plasma glucose level and other co-morbidity parameters, including dyslipidemia and hypertension, did not influence the presence of AF in this study, and only the increase in the intermediate CD14++CD16+monocytes proportion was independently associated with the presence of AF.

Potential mechanisms of intermediate CD14++CD16+monocytes in the pathogenesis of atrial fibrillation

Rapid atrial activation in AF results in a calcium overload in atrial myocytes, often leading to cell apoptosis and inducing a low-grade inflammatory response.17 Inflammation of the atrial myocardium, associated with monocyte activation, might contribute to the formation of AF.7 The inflammation process might modify the cell death, increase the atrial fibrosis, and change the atrial conduction properties, which are related to mechanoelectrical remodelling.1 An increased TNF-α level was noted in AF patients, which contributed to inflammation, atrial fibrosis, and the development of atrial arrhythmias in an animal model.18 The persistence of AF results in mechanical atrial remodelling, including an atrial enlargement and LAA flow depression.19 The biologic significance of these monocyte subsets is not completely understood. However, in a recent study, classical CD14++CD16−monocytes appeared to be the principal source of antigen-presenting dendritic cells, non-classical monocytes suppressed the secretion of inflammatory cytokines, and intermediate CD14++CD16+monocytes were the main producer of reactive oxygen species and were presumed to mainly exist in bone marrow and transform into classical and non-classical populations in the blood.20 In the present study, the persistent AF patients had a significantly larger LA than the paroxysmal AF patients, and the mean proportion of intermediate CD14++CD16+monocytes was higher in the persistent AF patients than in the paroxysmal AF patients. The proportion of intermediate CD14++CD16+monocytes had a negative correlation with the LAA flow during sinus rhythm; however, it was not correlated with the LAA flow during an AF rhythm. The LAA flow during an AF rhythm ordinarily decreases more than during sinus rhythm, and the correlation between intermediate CD14++CD16+monocytes and the LAA flow during sinus rhythm, not an AF rhythm, might reflect its affection for the atrial systolic function. Considered all together, although the mechanisms that contribute to an increased expression of intermediate CD14++CD16+monocytes is still unknown, it is speculated that intermediate CD14++CD16+monocytes contribute to the progression of remodelling of the atrium and AF. Due to the nature of a cross-sectional study such as the present study, a prospective cohort is needed to clarify the issue of which intermediate CD14++CD16+monocytes contribute to the initiation or progression of AF.

Study limitations

There seems to be some limitations to this study. First, this study was limited by the small number of participants. Second, a loss of some cells occurs during extracting mononuclear leukocytes from blood samples due to the nature of the flow cytometry procedure, so the total monocyte cell count seemed to be less than its true value. This fact may potentially influence the proportion of each monocyte subset. Third, although the fasting plasma glucose level, dyslipidemia, and hypertension did not influence the presence of AF in this study, we did not assess the severity of atherosclerosis including with carotid ultrasonography, the ankle-brachial pressure index, and the pulse wave velocity. Fourth, we did not perform an analysis of the other inflammation/oxidative stress markers including the MPO, which has been identified as a significant risk factor for AF and a crucial prerequisite for structural remodelling of the myocardium.4 The information of the association between these markers and different monocyte subsets might strengthen the findings of the present study. Fifth, the information regarding the association among the detailed degrees of atrial remodelling (i.e. the voltage mapping of the right and left atria), and the proportion of intermediate CD14++CD16+monocytes and its change over the time course was not assessed in the present study. Sixth, we could not determine whether the elevation in the intermediate CD14++CD16+monocytes reflected the extent of the monocyte infiltration into the atrial endothelium. Finally, the results of this study were observational in nature, so we could not provide a mechanistic explanation for the effect of the specific monocyte subsets on the pathogenesis of AF. Due to the nature of a cross-sectional study such as the present study, a prospective cohort with a larger sample size is needed to clarify the issue of which intermediate CD14++CD16+monocytes contribute to the initiation or progression of AF and to strengthen the findings.

Conclusions

To the best of our knowledge, our present study, comparing AF patients with no obvious comorbidities and healthy controls, is the first to show a significant association between an increased proportion of intermediate CD14++CD16+monocytes and AF. In addition, a preferential increase in the circulating intermediate CD14++CD16+monocytes was also found to be independently associated with the presence of AF, and was negatively correlated with the LAA flow during sinus rhythm. These findings suggest that this subset of monocytes might be closely related to the pathogenesis of the initiation and progression of AF.

Acknowledgements

The authors are grateful to Dr Katsuji Ikekubo and Tsutomu Kamino of the Kenko Life Plaza, Hyogo Health Service Association for their assistance in collecting the control volunteer data.

Conflict of interest: The Section of Arrhythmias, Division of Cardiovascular Medicine, Department of Internal Medicine, at Kobe University Graduate School of Medicine is financially supported by Medtronic and St Jude Medical.

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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: [email protected].
Topic:
Issue Section:
Clinical Research > Atrial fibrillation
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