Impaired efferocytosis by monocytes and monocyte-derived macrophages in patients with poorly controlled type 2 diabetes

Abstract

BACKGROUND

Deficient efferocytosis (i.e., phagocytic clearance of apoptotic cells) by macrophages has been frequently reported in experimental models of type 2 diabetes (T2D).

AIM

To translate these findings to humans by testing whether the efferocytosis capacity of blood monocytes and monocyte-derived macrophages is impaired in T2D patients.

METHODS

Overall, 30 patients with poorly controlled T2D [glycosylated hemoglobin (HbA1c) ≥ 8.0%] and 30 age- and sex-matched control subjects were enrolled in the study. The efferocytosis capacities of peripheral blood monocytes and monocyte-derived macrophages were assessed by flow cytometry and immunostaining. Macrophage membrane CD14 expression was examined by flow cytometry. Metabolic factors such as 25(OH)D and immune factors such as interleukin-1β were also measured.

RESULTS

The mean monocyte efferocytosis index in the diabetes group was significantly lower than that in the control group. Notably, efferocytosis remained impaired after monocytes differentiated into macrophages. Additionally, the percentages of classical monocytes (CD14⁺⁺CD16^- monocytes) and CD14⁺ macrophages were significantly lower in the diabetes group. Multivariate linear regression analysis in diabetes patients demonstrated that the monocyte efferocytosis index was independently associated with the HbA1c level, and that the macrophage efferocytosis index was significantly associated with the percentage of CD14⁺ macrophages.

CONCLUSION

Impaired efferocytosis was observed in T2D patients, with poor glycemic control affecting both blood monocytes and monocyte-derived macrophages. The efferocytosis index was negatively associated with metrics of glycemic control, and glucotoxicity may impact efferocytosis through reducing CD14 expression on both monocytes and macrophages.

Key Words: Type 2 diabetes; Efferocytosis; Monocyte; Macrophage

Core Tip: This study compared the efferocytosis function of blood monocytes and monocyte-derived macrophages in type 2 diabetes (T2D) patients with poor glycemic control. The results revealed that both monocytes and macrophages exhibited impaired efferocytosis. Additionally, the percentages of classical monocytes (CD14⁺⁺CD16^- monocytes) and CD14⁺ macrophages were significantly lower in the diabetes group. Multivariate linear regression analysis demonstrated that the monocyte efferocytosis index was independently associated with both the glycosylated hemoglobin level and the macrophage efferocytosis index showed a significant association with the percentage of CD14⁺ macrophages. These findings suggest that glucotoxicity may impact efferocytosis by reducing CD14 expression on both monocytes and macrophages in T2D patients.

INTRODUCTION

Type 2 diabetes (T2D) is a prevalent metabolic disease associated with significant morbidity and mortality[1]. In addition to insulin resistance and impaired insulin secretion, T2D is characterized by chronic low-grade inflammation, manifested by elevated levels of proinflammatory cytokines in the bloodstream and tissues[2,3]. Monocytes/macrophages, as key components of the immune system, have been implicated in contributing to the chronic inflammation observed in diabetes[4,5]. Mechanistically, elevated glucose levels promote the infiltration and accumulation of monocytes and macrophages in islet and insulin target tissues, resulting in the induction of a proinflammatory phenotype in macrophages[6]. Consequently, these macrophages produce increased levels of proinflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α, which activate the nuclear factor (NF)-κB pathway, ultimately leading to β-cell dysfunction and insulin resistance[6]. Moreover, macrophages contribute to the tissue damage associated with diabetes through the production of reactive oxygen species (ROS) and metalloproteinases[7]. Nevertheless, the precise mechanisms by which monocytes and macrophages are linked to chronic inflammation in T2D have not been fully elucidated.

Several factors can potentially impact monocyte/macrophage function, ranging from increased apoptosis and altered subset distributions to impaired phagocytosis. In recent years, researchers have reported that the phagocytic clearance of apoptotic cells, which is termed efferocytosis, is essential for maintaining homeostasis[8]. Efferocytosis is initiated primarily by ‘eat-me’ signals, particularly phosphatidylserine (PS), which is expressed on apoptotic cells. The recognition of PS by efferocytes activates a range of signaling pathways that subsequently modulate cytoskeleton remodeling, ensuring the engulfment of apoptotic cells by macrophages[3]. In addition to apoptotic cell removal, efferocytosis can stimulate the secretion of anti-inflammatory cytokines and inhibit the production of proinflammatory cytokines[9,10]. Conversely, deficiencies in efferocytosis lead to the accumulation of secondary necrotic cells, thus promoting inflammation[11]. Macrophages are the predominant efferocytes in this process[12]. Evidence from human studies and animal models has shown that reduced clearance of apoptotic cells is associated with chronic inflammatory diseases, such as chronic obstructive pulmonary disease (COPD), atherosclerosis (AS) and coronary heart disease[13-15]. Thus, impaired apoptotic cell engulfment by monocytes/macrophages may contribute to chronic inflammation in T2D.

In recent years, studies on mouse models of T2D have provided evidence of impaired efferocytosis in peritoneal macrophages, bone marrow-derived macrophages, and wound macrophages[16-18]. However, data regarding efferocytosis by monocytes or macrophages in human T2D are currently lacking, and such data are crucial for advancing clinical applications. Interestingly, studies have revealed that T2D patients exhibit a notable increase in the number of circulating apoptotic neutrophils and endothelial progenitor cells, suggesting impaired efferocytosis by phagocytic cells in the bloodstream[19,20]. Furthermore, increasing evidence indicates that the phagocytosis of bacteria by neutrophils[21] and monocytes[22] is impaired in T2D patients and is related to glycemic control. On the basis of these findings, we hypothesize that efferocytosis of blood monocytes and monocyte-derived macrophages is impaired in patients with poorly controlled T2D. Efferocytosis function was assessed using flow cytometry and immunofluorescence, with a focus on calculating the proportion of monocytes/macrophages engulfing apoptotic cells.

In addition, we analyzed monocyte subsets in both diabetes patients and control subjects by flow cytometry for CD14 and CD16 expression. Furthermore, we explored the potential relationships between the efferocytosis index and clinical and experimental characteristics, such as glycosylated hemoglobin (HbA1c) levels, monocyte subsets, and inflammatory factors.

MATERIALS AND METHODS

Study design

This study was conducted at Xinhua Hospital Affiliated with Shanghai Jiao Tong University School of Medicine and involved participants aged between 35 and 65 years. A total of 30 subjects with T2D and 30 healthy control subjects were recruited between June 2020 and July 2021. To reduce the confounding effects of age, sex, and inflammatory processes on efferocytosis, all the subjects were age and sex matched, and none had had acute inflammatory diseases within 2 weeks or a clinical history of COPD or rheumatologic diseases. Patients with poorly controlled T2D were recruited from the outpatient diabetes clinic at our hospital and had HbA1c levels > 8.0%. The diagnosis of diabetes was based on the guidelines provided by the American Diabetes Association (ADA) in 2020. No definition of “poorly controlled” diabetes is universally accepted, and various HbA1c thresholds have been proposed in the literature, including values greater than 7.5%, 8.0%, and 9.0%[23,24]. Because the ADA recommends an HbA1c target of up to 8.0% as a less stringent goal, our study defines poorly controlled T2D as having an HbA1c level exceeding 8.0%.

The patients included in the study had never received anti-inflammatory drugs, had no macroproteinuria, no severe diabetic eye disease, no history of ketosis and no insulin therapy requirement for 3 years after diagnosis. Control subjects were selected from the health examination population and had a fasting plasma glucose (FPG) level < 5.6 mmol/L and HbA1c < 5.7%, per the ADA criteria. Prior to participating in the study, all the subjects provided written informed consent in accordance with the guidelines and principles of the Ethics Committee of Xinhua Hospital Affiliated with Shanghai Jiao Tong University School of Medicine.

Analytical methods

Anthropometric measurements were taken with the subject wearing light clothing and standing barefoot. Body weight and height were measured to the nearest 0.1 kg and 0.1 cm, respectively. Waist circumference was measured at the midpoint between the costal margins and the iliac crests. Body mass index (BMI) was calculated as the weight in kilograms divided by the square of the height in meters (kg/m²). Blood pressure was obtained between 9 am and 6 pm in a quiet, private room and was assessed twice in the right arm after a 15-minute rest in a seated position using a standard mercury sphygmomanometer. The participants were asked not to speak to the research staff or use a smartphone during the measurements or for any of the preceding 5-minute rest periods.

Fasting venous blood samples were collected after a minimum of 10 hours of fasting. Plasma glucose levels were determined using the glucose oxidase method, whereas HbA1c was measured using high-performance liquid chromatography. Serum insulin levels were assessed using the radioimmunoassay. Lipid profiles, liver enzyme profiles, and creatinine levels were analyzed using a Hitachi 7080 analyzer. Circulating C-reactive protein levels were measured with an ELISA kit following the manufacturer's recommendations. The serum 25-hydroxy vitamin D (25(OH)D) concentration was detected via an automated electro chemiluminescent immunoassay from Roche.

Peripheral blood monocyte collection, isolation and differentiation into macrophages

Human monocytes were isolated from the peripheral blood of both diabetic patients and control subjects. Mononuclear cells were isolated from the blood samples using a Ficoll gradient (Cytiva/Global Life Sciences Solutions, Marlborough, MA, United States). The isolated peripheral blood mononuclear cells were subsequently incubated in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C. After a 2-hour incubation, nonadherent cells were removed by washing with PBS, and the adherent monocytes were allowed to mature into macrophages by cultivation for 7 days in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 10 ng/mL macrophage colony-stimulating factor (M-CSF; R&D Systems, Minneapolis, United States). To ensure the best differentiation conditions, monocytes were seeded at a density of 3-5 × 10⁵ cells per well. The culture medium was replenished with fresh medium on days 3 and 6, and the experiments were conducted on day 7 after differentiation.

Quantification of efferocytosis

Blood monocytes or monocyte-derived macrophages were cultured in 12-well plates. To generate apoptotic cells, Jurkat cells were labeled with CFSE (Thermo Fisher Scientific, MA, United States) at room temperature for 10 minutes (for flow cytometry) or with CMPTX (T4ermoFisher Scientific) at 37°C and 5% CO₂ for 30 minutes (for immunofluorescence). Apoptosis was induced in Jurkat cells by exposure to UV irradiation (254 nm, UVS-26, 0.02 J/s/cm²) for 30 minutes, followed by incubation at 37°C for 3 hours to allow for apoptotic cell formation. Approximately 40% of cells were early apoptotic[25].

The monocytes or monocyte-derived macrophages were transferred to RPMI medium without FBS and incubated with CFSE-labeled apoptotic Jurkat T cells at a ratio of 1:10 for 2 hours. The efferocytosis activity of monocytes/macrophages, indicated by the proportion of monocytes/macrophages engulfing apoptotic cells, was determined using flow cytometry. The gating strategy used for efferocytosis is shown in Supplementary Figure 1. The efferocytosis index was calculated as follows: (number of CFSE⁺CD14⁺ monocytes/number of total CD14⁺ monocytes) × 100 (%) or (number of CFSE⁺CD11b⁺ macrophages/number of total CD11b⁺ macrophages) × 100 (%).

To visualize efferocytosis, macrophages were stained with a FITC-conjugated anti-CD11b monoclonal antibody (mAb) (BioLegend, United States) and incubated with apoptotic Jurkat T cells labeled with CellTracker™ Red CMPTX (Thermo Fisher Scientific) at a 1: 10 ratio for 2 hours. After the removal of unbound target cells by washing with cold PBS, the uptake of apoptotic Jurkat T cells by macrophages was assessed using a fluorescence microscopy (Leica Microsystems, Wetzlar, Germany). The cells were counted in three replicate wells, with 200 macrophages per well for each condition. The efferocytosis index was determined as the percentage of macrophages containing at least one bound or ingested apoptotic cell relative to the total number of macrophages counted.

Identification and characterization of monocyte subsets

Monocyte subsets were identified using flow cytometry. To distinguish classical, intermediate and nonclassical monocytes, cells were stained with APC-conjugated anti-CD14 mAbs (BioLegend, United States) and PE-conjugated anti-CD16 mAbs (BioLegend, United States). A standardized gating strategy was employed for the analysis, following established guidelines[26]. The relative frequencies of these monocyte subsets are expressed as the percentage of the total monocyte gate.

Surface expression of CD14 on monocyte-derived macrophages

Human monocyte-derived macrophages were digested with trypsin-EDTA at 37 °C and 5% CO₂ for 10 minutes and resuspended in 100 μL of PBS. Then, the macrophages were stained with APC-conjugated anti-CD14 mAbs (BioLegend, United States) according to standard laboratory procedures. Surface marker analysis was carried out on monocyte-derived macrophages on day 7 of differentiation, which were used in the efferocytosis assay.

Analysis of plasma cytokines by cytometric bead array (CBA). Venous blood samples were collected from participants after an overnight fast of at least 10 hours. The samples were collected in EDTA-containing tubes and centrifuged at 4 °C. The resulting plasma was stored at -80 °C until analysis. The levels of plasma inflammatory cytokines, including TNF-α, IL-1β, IL-6, IL-8, IL-10 and IL-12, were measured using a CBA human inflammatory cytokine kit following the manufacturer's instructions.

Briefly, peripheral blood samples from both diabetic patients and control subjects were centrifuged at 4 °C and 3000 rpm/min for 10 minutes. Human inflammatory cytokine capture beads were mixed and resuspended in serum enhancement buffer for 30 minutes at room temperature in the dark. Subsequently, 50 µL of the mixed capture beads and 50 µL of each sample were added to the assay tubes. The tubes were incubated for 1.5 hours at room temperature in the dark. The bead pellet was then washed by centrifugation at 200 × g for 5 minutes, and the supernatant was discarded, leaving approximately 100 µL of liquid in each assay tube. Fifty microliters of PE-conjugated detection antibodies were added to the assay tubes, followed by another 1.5-hour incubation at room temperature in the dark. Finally, the assay tubes were resuspended in 300 µL of wash buffer. The data were analyzed using FCAP Array software and FlowJo data analysis software. Protein concentrations were calculated based on the median fluorescence intensity (MFI).

Statistical analysis

Statistical analyses were conducted using SPSS version 27 software. The normality of the data was assessed for all variables. The results are presented as the mean ± SD when variables were normally distributed or medians with IQRs when they were not normally distributed. Differences between diabetes patients and control subjects were evaluated using two-tailed student's t test for parametric data or the Wilcoxon test for nonparametric data. A generalized linear model (GLM) was employed to identify differences in the efferocytosis index between the groups while adjusting for potential confounding factors. Univariate linear regressions were used to test for associations of the efferocytosis index with clinical, demographic and experimental characteristics. Variables that exhibited significant associations in the univariate analyses were included in the multivariate analysis models via a stepwise backward method. For multiple comparisons, groups were compared by one-way ANOVA or the Kruskal-Wallis rank test for normally and nonnormally distributed continuous variables, respectively. Statistical significance was defined as a P value < 0.05 (two-tailed). The significance levels for differences between control and patient macrophages were set at ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001 for different conditions.

Study approval

The study procedures for the human participants were approved by the ethics committee of Xinhua Hospital Affiliated with Shanghai Jiao Tong University School of Medicine. All the participants provided written informed consent prior to enrollment.

RESULTS

Main clinical, biological, and hemodynamic parameters in diabetic and control subjects

The clinical and demographic characteristics of the 30 diabetes patients and 30 control subjects are shown in Table 1. The diabetes subjects were almost identical in age and blood pressure to the control subjects but were significantly more obese (P < 0.05), with higher FPG and HbA1c levels (P < 0.001). Plasma hypersensitive C-reactive protein [hsCRP values (P = 0.011) and IL-1β levels (P = 0.031)] were significantly greater in T2D patients than in control subjects (Table 1).

Table 1 Clinical characteristics of type 2 diabetes or healthy control.

		Control subjects (n = 30)	Type 2 diabetes (n = 30)	P value
	Sex (F/M)	9/21	9/21	/
	Age (years)	50 ± 10	50 ±10	0.925
	Waist-to-hip ratio	0.90 ± 0.09	0.98 ± 0.04	0.010
	BMI (kg/m2)	22.83 ± 2.41	25.37 ± 4.14	0.016
	Systolic blood pressure (mmHg)	129 ± 12	132 ± 16	0.441
	Diastolic blood pressure (mmHg)	77 ± 10	80±10	0.221
	Mean arterial blood pressure (mmHg)	95.2 ± 9.7	97.6 ± 10.4	0.340
Glucose metabolism factors	Fasting plasma glucose (mmol/L)	5.07 ± 0.42	11.36 ± 0.43	< 0.001
	HbA1c (%)	5.1± 0.4	11.6 ± 2.3	< 0.001
	HbA1c (mmol/mol)	32 ± 4	104 ± 25	< 0.001
Lipid metabolism factors	Total cholesterol (mmol/L)	4.21 ± 0.77	4.78 ± 1.39	0.043
	Triglycerides (mmol/L)	1.44 ± 0.76	2.04 ± 1.94	0.077
	HDL cholesterol (mmol/L)	1.14 ± 0.26	1.38 ± 1.56	0.426
	LDL cholesterol (mmol/L)	2.77 ± 0.93	2.64 ± 0.89	0.483
	Apolipoprotein A (mmol/L)	1.23 ± 0.23	1.13 ± 0.31	0.419
	Apolipoprotein B (mmol/L)	0.89 (0.29)	0.97 (0.38)	0.294
	ApoA/ApoB ratio	1.43 (0.55)	1.18 (0.64)	0.110
Bone metabolism parameters	B-CTX (ng/mL)	0.49 (0.41)	0.45 (0.36)	0.053
	P1NP (ng/mL)	57.48 (28.55)	31.98 (21.56)	0.003
	Osteocalcin (ng/mL)	16.84 (6.36)	10.45 (6.03)	< 0.001
	25(OH)D (nmol/L)	50.59 ± 20.70	45.79 ± 20.73	0.605
	PTH (pmol/L)	3.86 (1.78)	3.15 (2.20)	0.079
Inflammatory factors	hsCRP (mg/L)	0.50 (1.50)	2.00 (4.00)	0.011
	TNF-α (pg/mL)	1.00 (1.08)	0.83 (0.91)	0.575
	IL-1β (pg/mL)	0.17 (0.95)	0.66 (1.61)	0.031
	IL-6 (pg/mL)	2.25 (3.27)	3.11 (2.18)	0.149
	IL-8 (pg/mL)	5.37 (5.05)	8.82 (11.90)	0.063
	IL-12 (pg/mL)	1.14 (1.38)	1.24 (1.03)	0.881
	IL-10 (pg/mlL)	0.22 (0.97)	0.24 (0.72)	0.360

Data are mean ± SD, n (%), or median (IQR). P values indicate differences between type 2 diabetes patients (n = 30) and healthy control (n = 30). HbA1c: Glycosylated hemoglobin; BMI: Body mass index; HDL: High-density lipoprotein; LDL: Low-density lipoprotein; hsCRP: Hypersensitive C-reactive protein.

Impaired efferocytosis by monocytes and monocyte-derived macrophages in T2D patients

The monocyte efferocytosis index (i.e., the percentage of monocytes phagocytizing apoptotic cells relative to all monocytes) was significantly lower in diabetes patients than in control subjects (Figure 1A), indicating a defective monocyte efferocytosis capacity in diabetes patients. Because obesity influences monocyte/macrophage polarization and impairs efferocytosis function[27-29], we adjusted for BMI and the waist-to-hip ratio by a GLM. The results revealed that the difference remained significant even after adjusting for BMI and the waist-to-hip ratio (P < 0.001). Similarly, the macrophage efferocytosis index was significantly lower in diabetes patients than in control subjects (Figure 1B and C), and the difference persisted after adjusting for BMI and the waist-to-hip ratio (P = 0.030).

Figure 1 Impaired efferocytosis function of monocytes and monocyte-derived macrophages in type 2 diabetes patients. A: Efferocytosis by monocytes in control subjects and type 2 diabetes patients determined by flow cytometry. The efferocytosis index was calculated as (number of CFSE⁺CD14⁺ monocytes/number of total CD14⁺ monocytes) × 100 (%). Data are presented as medians and quartiles or mean ± SD; B and C: Efferocytosis by monocyte-derived macrophages in control subjects and diabetic patients determined by flow cytometry or by immunofluorescence. The efferocytosis index was determined as (number of CFSE⁺CD11b⁺ monocytes/number of total CD11b⁺ monocytes) × 100 (%). The average number of efferosomes per efferocyte was calculated as the amount of efferocytosed apoptotic material per macrophage containing at least one bound or ingested apoptotic cell(s). Scale bar: 20 μm. The data are the mean ± SD. ^bP < 0.01 from paired t test (B) or unpaired t test (C), ^cP < 0.001 from the Wilcoxon matched pair test (for monocyte subsets) or paired t test (for the monocyte efferocytosis index). T2D: Type 2 diabetes.

Decreased CD14 expression in monocytes and macrophages in T2D patients

We next investigated the possible mechanism underlying the defective efferocytosis function of monocytes/macrophages in diabetes patients. Human blood monocytes consist of at least three subsets, namely, classical (CD14⁺⁺CD16⁻), nonclassical (CD14⁺CD16⁺⁺) and intermediate (CD14⁺⁺CD16⁺) monocytes. Among these subsets, classical monocytes are known to possess the highest phagocytic capacity[30]. We therefore investigated whether the percentages of monocyte subtypes differed between the two groups of patients. As shown in Supplementary Table 1 and Figure 2A, although the total monocyte numbers did not significantly differ between diabetes patients and control subjects, the percentage of classical (CD14⁺⁺CD16^-) monocytes was significantly lower in diabetes patients than in control subjects. Macrophages also express CD14, which is an efferocytosis receptor in macrophages[31] and is involved in tethering apoptotic cells to macrophages and interacting with other molecules within the phagocytic synapse[32]. In our study, the percentage of CD14⁺ macrophages and the MFI of CD14 were significantly lower in diabetes patients than in control subjects (Figure 2B and C).

Figure 2 Percentage of classical monocytes and macrophage CD14 expression were decreased in type 2 diabetes patients. A: Percentages of different monocyte subsets (classical, intermediate and nonclassical monocytes); B: The percentage of CD14⁺ macrophages in control subjects and diabetes patients was determined by flow cytometry; C: The median fluorescence intensity of CD14 in macrophages from control subjects and diabetes patients. The data are the mean ± SD. ^aP < 0.05, ^bP < 0.01 from paired t test. T2D: Type 2 diabetes; MFI: Median fluorescence intensity.

Contributing factors related to monocyte and macrophage efferocytosis in the total sample population

Univariate linear regression analysis of the entire sample population revealed significant relationships between the monocyte efferocytosis index and variables such as the HbA1c level, FPG, percentage of classical monocytes, waist-to-hip ratio and 25(OH)D concentration (Figure 3A-E). No significant associations were found between the monocyte efferocytosis index and BMI, plasma lipids or plasma inflammatory factors. Following adjustment for these associated factors, multivariate linear regression analysis revealed significant and independent associations between monocyte efferocytosis and HbA1c levels (β = -3.792, P < 0.001) and the percentage of classical monocytes (β = 0.367, P = 0.014; Supplementary Table 2).

Figure 3 Parameters correlated with the efferocytosis index of monocytes and monocyte-derived macrophages in the total sample population. A-E: Univariate regression analysis of the relationships between each variable and the efferocytosis indices of monocytes; F-J: Monocyte-derived macrophages in the total sample population. The regression line (black line) and corresponding 95%CI (gray area) are shown for each univariate regression analysis. FPG: Fasting plasma glucose.

The macrophage efferocytosis index, on the other hand, was negatively correlated with the HbA1c, FPG, and serum IL-1β levels while positively correlated with the percentage of CD14⁺ macrophages (Figure 3F-J). Subsequent stepwise linear regression analysis indicated that the macrophage efferocytosis index was solely correlated with the percentage of CD14⁺ macrophages (β = 0.575, P < 0.001, Supplementary Table 3).

Factors related to monocyte and macrophage efferocytosis in T2D

In the diabetes group, the monocyte efferocytosis index was significantly related to the HbA1c level and the 25(OH)D concentration (Supplementary Figure 2A and B). Further multivariate linear regression analysis, adjusting for age, validated the correlation between the monocyte efferocytosis index and HbA1c levels (β = -2.729, P = 0.017) and 25(OH)D concentrations (β = 0.314, P = 0.013, Supplementary Table 4).

In contrast, the macrophage efferocytosis index in diabetes patients was positively correlated with the CD14⁺ macrophage ratio, while displaying a negative correlation with concentration of serum IL-1β (Supplementary Figure 2C and D). Notably, the macrophage efferocytosis index was not significantly correlated with glucose metabolism parameters such as the FPG, 2-hour glucose and HbA1c levels (P > 0.05). Subsequent multivariate linear regression analysis demonstrated that the macrophage efferocytosis index was correlated solely with the percentage of CD14⁺ macrophages (β = 0.534,P = 0.001; Supplementary Table 5).

To further investigate the impact of HbA1c, the diabetes group was stratified into two subgroups: One with an HbA1c lower than 11.0% (D1) and the other with an HbA1c of at least 11.0% (D2). The D2 and D1 groups did not significantly differ in age, BMI, inflammatory factors and monocyte subsets (Supplementary Table 6 and Figure 4A-C). Interestingly, the monocyte efferocytosis index was lower in the D1 group than in the control group and further decreased in the D2 group compared with the D1 group, while whereas macrophage efferocytosis index was not significantly different between the latter two groups (Figure 4D and E).

Figure 4 The monocyte efferocytosis index further decreased in the type 2 diabetes patients with glycosylated hemoglobin levels greater than 11.0%. A and B: The number and percentage of monocytes, percentage of monocyte subsets; C-E: efferocytosis index of monocytes and monocyte-derived macrophages in healthy controls and in diabetes patients with glycosylated hemoglobin levels lower than 11.0% (D1) or greater than 11.0% (D2). Scale bar: 20 μm. ^aP < 0.05,^cP < 0.001 from paired t test.

DISCUSSION

In this study, we provide evidence of impaired efferocytosis capacity in the peripheral blood monocytes and monocyte-derived macrophages of patients with T2D. Our findings indicate a significant reduction in the efferocytosis index in diabetes patients, and this impairment is associated with glycemic control. Previous studies have revealed a broad spectrum of alterations in circulating monocytes from diabetes patients, ranging from shortened telomeres[33], increased apoptosis[22], and impaired chemotaxis[34] to reduced phagocytosis of beads and pathogenic bacteria[22,35]. Our data further demonstrate that these abnormal monocytes also exhibit impaired phagocytosis of apoptotic cells, which is termed efferocytosis. These findings suggest that the monocyte population in the peripheral blood, which adheres to the vascular endothelium in T2D patients, enters the vascular wall and undergoes macrophage and foam cell transformation, is characterized by impaired efferocytosis. Because chronic vascular disease, particularly macroangiopathy, is a leading cause of morbidity and mortality in T2D, with AS being the underlying pathology, investigating the role of impaired macrophage efferocytosis in AS progression is crucial. Recent studies have shown that macrophage efferocytosis is impaired in AS, resulting in increased accumulation of apoptotic cells within plaques and secondary necrosis, thereby exacerbating the atherosclerotic process[36]. The findings from our study support the initial hypothesis that efferocytosis by peripheral blood monocyte-differentiated macrophages is significantly reduced in patients with T2D. These findings suggest that differentiated macrophages in T2D patients are at increased risk of hypofunction, leading to increased AS and plaque instability.

Additionally, macrophage efferocytosis plays a significant role in wound healing by promoting the accumulation of M2 reparative macrophages at the wound site[37,38] and wound macrophage efferocytosis has been shown to be impaired in diabetic mice[18,37,38]. Notably, promoting macrophage efferocytosis attenuates diabetic complications[39-42]. For example, statins and CD47-blocking antibodies have been found to ameliorate AS by promoting macrophage efferocytosis[40,41], and targeting the membrane transporter SLC7A11 has shown promise in improving efferocytosis and wound healing in diabetic contexts[43,44]. In conclusion, defective efferocytosis in monocytes and monocyte-derived macrophages may serve as a predictor of diabetic complications and a therapeutic target. Future studies are warranted to explore the relationship between the efferocytosis function of monocytes/macrophages and diabetic complications in patients with diabetes.

Multiple factors, including proinflammatory factors such as TNF-α[45] and HMGB1[46] and metabolites such as GAPDH[47] and high glucose[48], can affect monocyte and macrophage efferocytosis. The present study revealed a significant independent association between the monocyte efferocytosis index and HbA1c level in the overall study population and, specifically, in diabetes patients. Additionally, both the monocyte and macrophage efferocytosis indices were negatively correlated with FPG in the total sample. Previous in vitro studies have shown that elevated glucose concentrations impair macrophage efferocytosis and induce inflammation[48]. The underlying mechanism may be that high glucose leads to lysosomal permeabilization and the production of ROS, which results in efferocytosis dysfunction, inflammasome activation and cytokine secretion in macrophages. Furthermore, our previous research indicated that advanced glycation end products inhibit macrophage efferocytosis through the RAGE/RhoA/ROCK signaling pathway[25]. Therefore, uncontrolled glucose metabolism may impair the efferocytosis function of monocytes and macrophages in T2D patients.

Another potential factor associated with the monocyte efferocytosis index is vitamin D. Our study revealed an independent positive correlation between the monocyte efferocytosis index and serum 25(OH)D (or calcitriol) levels. As the main form of vitamin D, 25(OH)D serves as an indicator of vitamin D status and plays a crucial role in regulating calcium and phosphorus metabolism, as well as modulating the immune system[49]. Numerous studies have reported an association between low vitamin D levels and an increased risk of infections and autoimmune diseases[50]. Mechanistically, upon binding to the vitamin D receptor, vitamin D regulates the expression of several genes essential for innate immune defense, including those encoding cytokines, chemokines, antimicrobial peptides, and pattern recognition receptors. However, few studies have investigated the effect of vitamin D on the phagocytic function of immune cells. Some studies have demonstrated that vitamin D enhances immunoglobulin- and complement-dependent phagocytosis by human monocytes[51], whereas others have shown that vitamin D inhibits the phagocytosis of apoptotic T cells by human myeloid cells[52]. These contrasting findings may be attributed to different cell types and vitamin D concentrations. Our clinical evidence suggests that vitamin D may promote efferocytosis in human blood monocytes, thus revealing a novel role of vitamin D in immune modulation. In recent years, researchers have discovered that CD14 is significantly induced by vitamin D in both peripheral blood mononuclear cells and human monocytic cell lines[53,54]. Because the macrophage efferocytosis index is positively associated with CD14 expression in both monocytes and macrophages, vitamin D may enhance efferocytosis through the upregulation of CD14 expression. To validate this hypothesis, studies involving vitamin D supplementation in diabetes patients or experimental data regarding the effects of vitamin D treatment on monocytes and macrophages are warranted.

Human monocytes can be classified into three distinct subsets: Classical (CD14⁺⁺CD16⁻), nonclassical (CD14⁺CD16⁺⁺) and intermediate (CD14⁺⁺CD16⁺) monocytes[26]. Among these subsets, classical monocytes are known to possess the highest phagocytic capacity[30]. However, studies investigating monocyte subsets in diabetes patients have produced conflicting results. Some studies have reported a reduction in the frequency of classical monocytes in T2D patients, which negatively correlates with glycemic control[55]. Conversely, other studies have reported no alterations in the distributions of these three monocyte subsets in T2D patients[56]. In our study, we observed a significant decrease in the percentage of the classical monocyte subset in T2D patients, and this decrease was positively correlated with the monocyte efferocytosis index in the entire sample population. Hence, the reduced frequency of classic monocytes may be partly responsible for impaired monocyte efferocytosis in T2D patients.

The determinants of macrophage efferocytosis are not identical to those of monocyte efferocytosis. In our study, membrane CD14 expression was significantly lower in macrophages from diabetes patients than in those from control subjects, and the macrophage efferocytosis index was significantly associated with the percentage of CD14⁺ macrophages. Macrophages also express CD14, which not only functions as a pattern recognition receptor during the phagocytosis of bacteria but also plays a significant role in the clearance of apoptotic cells by both human and murine macrophages[31,57]. CD14 is an efferocytosis receptor in macrophages[31]^, and researchers have reported that the ‘eat-me signal’ PS may provide an apoptotic-cell-associated ligand for CD14. CD14 is involved in tethering apoptotic cells to macrophages and interacting with other molecules within the phagocytic synapse[32]. Combined with the finding that the monocyte efferocytosis index was positively correlated with the percentage of classical monocytes, which are strongly positive for CD14, we hypothesized that glucotoxicity may impair efferocytosis by reducing CD14 expression on both monocytes and macrophages. CD14 exists in two forms: A membrane-bound form on the cell surface (mCD14) and a soluble variant in circulation (sCD14). Because previous studies have reported no significant difference in the sCD14 concentration between patients with T2D and healthy individuals, our study focused on the expression of mCD14. Few studies have examined the effects of glucose or its metabolic products on CD14. Although high glucose levels may increase CD14 expression induced by lipopolysaccharides (LPS) or oxidized low-density lipoprotein[58,59], high glucose alone does not significantly influence CD14 mRNA or protein expression[59]. Therefore, exploring the mechanisms by which high glucose affects mCD14 expression in monocytes and macrophages during efferocytosis is both interesting and meaningful. Additional experimental data will be necessary in the future to further investigate this phenomenon.

Efferocytosis is an anti-inflammatory process, and phagocytic monocytes/macrophages are capable of secreting the anti-inflammatory factors IL-10 and TGF-β while inhibiting the production of LPS-induced inflammatory factors, such as TNF-α, IL-1β, and IL-6[25,32]. T2D is characterized by chronic inflammation, and monocytes with impaired efferocytosis in diabetes patients may contribute to an enhanced inflammatory environment in the vasculature. In our study, diabetes patients presented significantly increased serum IL-1β concentrations and hsCRP levels. Macrophage efferocytosis in diabetes patients was significantly and negatively correlated with the serum IL-1β concentration. These findings suggest that, in the diabetic environment, efferocytosis may lose its anti-inflammatory effects, resulting in elevated serum IL-1β concentrations. Importantly, inflammatory factors present in the bloodstream may also inhibit macrophage efferocytosis[60]. Further investigation is essential to elucidate whether inflammatory factors such as IL-1β play a significant role in CD14-dependent efferocytosis.

This study has certain limitations, particularly in understanding the underlying mechanism of impaired efferocytosis in T2D patients. Although we established an independent correlation between HbA1c, FPG, and efferocytosis, the cause-effect relationship still warrants further clarification. To address this issue, a comparison of the efferocytosis index in T2D patients before and after systematic glycemic control is necessary. Furthermore, our study revealed a significant positive correlation between macrophages and monocyte efferocytosis (Figure 3F). However, the disparity in terms of macrophage efferocytosis between diabetic patients and healthy subjects was not as pronounced or significant as that in monocyte efferocytosis. This discrepancy may be attributed to the use of uniform fetal bovine serum and low-sugar media during the induction of differentiation. A study by Katz et al[35] employed serum from diabetic patients to mimic the in vivo environment, which could provide valuable insights for our future investigations. Finally, because different monocyte subsets possess distinct phagocytic functions and our findings indicate that impaired monocyte efferocytosis might be associated with a reduced frequency of classical monocytes, exploring the variations in efferocytosis among different monocyte subsets between diabetes patients and control subjects would be intriguing.

CONCLUSION

In conclusion, our study provides evidence of significant impairment in efferocytosis among blood monocytes and differentiated macrophages in T2D patients. Hyperglycemia, the toxic effects of HbA1c, and a reduced frequency of classical monocytes are likely contributing factors to this impaired efferocytosis mechanism.

ACKNOWLEDGEMENTS

The authors thank all study participants and acknowledge the laboratory assistance from the Research Center of Xinhua Hospital.