Type 2 diabetes (T2DM) and its complications are associated with a systemic low-grade inflammation manifested by higher systemic levels of proinflammatory cytokines, such as IL-6, IL-1
It was suggested previously that the primary trigger of immune cell activation and thus low grade inflammation could be both acute and prolonged periods of hyperglycemia in obese patients and other patients at risk of T2DM development [
To test this hypothesis, we aimed to investigate the changes in lymphocyte and monocyte populations in association with genetic predisposition to T2DM and the response of these cells to short-term hyperinsulinemia in healthy FDR of T2DM when compared to control subjects.
38 lean men participated in the study. Two groups of subjects matched for age and BMI were recruited: (1) nondiabetic first-degree relatives of T2DM patients (FDR;
All subjects were generally healthy and nonobese and did not use any prescription drugs, as determined by medical history and laboratory findings. The exclusion criteria for both groups were weight change more than 3 kg within 3 months preceding the study, smoking, hypertension, diabetes, hyperlipidemia, illicit drug, or alcohol abuse. Subjects were examined at a certified laboratory starting at 8 am after an overnight fast. Body weight and waist and hip circumferences were measured, and body composition was assessed by bioimpedance (QuadScan 4000, Bodystat, Douglas, British Isles). The study was approved by the Ethical Committee of the Third Faculty of Medicine, Charles University, in Prague, and all subjects gave their informed consent before the start of the study.
The euglycemic-hyperinsulinemic clamp was performed according to the de Fronzo method [
The subpopulation of blood cells representing lymphocytes and monocytes was analyzed according to their size and granularity. To detect specific surface antigens, the whole blood samples were stained with fluorescence-labelled monoclonal antibodies (FITC-conjugated antibody CD4, CD14; PE-conjugated antibody CD3, and CD163; PerCP-conjugated antibody CD45; APC-conjugated antibody CD8, CD36, TLR2, and CD196; APC-Cy7-conjugated antibody CD16; PE-Cy7-conjugated antibody CCR2 and CD25; BV421-conjugated antibody CD183; and CD194; all except CD163 from BD Bioscience, US; CD163 from Exbio, CZ) for 30 min at room temperature. After staining, erythrocytes were lysed by erythrocyte lysis buffer for 15 min at room temperature. The cells were then washed with PBS and analyzed on a FACSVerse flow cytometer and by BD FACSuite Software 1.0.6 (BD Biosciences). The number of immune cells in the analyzed populations was expressed as the percentage of gated events related to CD45, CD45+/14+ (monogate), or CD3+ (lymphogate) events. Background was set up to 5% of positive cells of isotype control.
T cells were distinguished by standard FSC/SSC position “lymphogate” and by positivity for pan T cell marker CD3. CD4-positive T cells were considered T helper (TH) cells and CD8-positive cells as T cytotoxic (Tc). TH1 cells were defined as CD4+/194-/196-/183+ (positive for CXCR3), TH2 were defined as CD4+/194+/196-/183- (positive for CCR4), and TH17 were defined as CD4+/194+/196+/183- (positive for CCR4/CCR6). Regulatory T cells (Tregs) were identified as CD4/CD25high/CD127low cells. Monocytes were distinguished by standard FSC/SSC position and by positivity for CD45, CD14, and CD16 markers.
Plasma concentrations of glucose, insulin, lipids, and nonesterified fatty acids (NEFA) were determined using standard biochemical methods. Cytokines in the plasma were analyzed using xMAP technology (High Sensitivity Human T-cell Kit: IL-4, IL-6, IL-10, IL-12, IL-17A, and TNF
Data are presented as the
The anthropometric and biochemical parameters of the subjects are listed in Table
The anthropometrical and biochemical parameters of the 2 groups of subjects.
Controls ( |
FDR ( |
||
---|---|---|---|
Age (years) | 0.469 | ||
Weight (kg) | 0.515 | ||
BMI (kg/m2) | 0.401 | ||
Waist (cm) | 0.341 | ||
Fat mass (%) | 0.117 | ||
Fat-free mass (kg) | 0.948 | ||
HDL (mmol/l) | 0.313 | ||
TG (mmol/l) | 0.808 | ||
Total cholesterol (mmol/l) | 0.209 | ||
Ureic acid ( |
0.725 | ||
Glucose (mmol/l) | 0.019 | ||
Insulin (mU/l) | 0.018 | ||
HOMA-IR | 0.015 | ||
MFFM (mg/kgFFM/min) | 0.461 |
Data are presented as the
At the fasting state (baseline), the T lymphocyte populations in the blood were not different between the groups, except for TH17 lymphocytes, which were higher in FDR (Figure
Effect of hyperinsulinemia on lymphocyte populations in the blood in FDR and in control subjects. Lymphocyte populations are expressed as the percentage of positive cells related to CD45/lymphogate.
Correlations between clinical parameters and circulating immune cells at baseline in all subjects.
Clinical parameter | Immune population | Correl. coef. | |
---|---|---|---|
FM | CD45/3/4+ | 0.016 | 0.399 |
CD4/CD8 ratio | 0.020 | 0.387 | |
Insulin | Classical monocytes | 0.026 | 0.376 |
CD4/CD8 ratio | 0.048 | 0.387 | |
HOMA-IR | Classical monocytes | 0.029 | 0.369 |
CD4/CD8 ratio | 0.035 | 0.348 | |
MFFM | CD45/14/163+ | 0.045 | 0.341 |
IL-4 | CD45/3/4+ | 0.014 | -0.422 |
CD4/CD8 ratio | 0.010 | -0.451 | |
Intermediate monocytes | 0.020 | 0.418 |
Data are presented as Pearson’s correlation coefficient and
The monocyte populations in the blood did not differ between the groups at fasting conditions (Figures
Effect of hyperinsulinemia on monocyte populations in the blood in FDR and in control subjects: (a) classical, intermediate, and nonclassical monocytes expressed as the percentage of positive cells related to CD45/14+; (b) monocyte populations expressing markers TLR2, CD163, CCR2, and CD36 expressed as the percentage of positive cells related to CD45/14+; (c) expression of markers TLR2, CD163, CCR2, and CD36 on monocyte subsets expressed as MFI
The concentrations of cytokines in the plasma before and after the hyperinsulinemic clamp in the 2 groups of subjects.
Controls ( |
FDR ( | |||
---|---|---|---|---|
Basal | Clamp | Basal | Clamp | |
IL-4 (pg/ml) | ||||
IL-6 (pg/ml) | ||||
IL-10 (pg/ml) | ||||
IL-12 (pg/ml) | ||||
IL-17 (pg/ml) | ||||
TNF |
||||
MCP-1 (pg/ml) |
Data are presented as the
In response to hyperinsulinemia, a decrease in the relative content of CD4+ T helper cells and an increase of relative content of CD8+ cytotoxic T cells were detected in both groups of subjects. Thus, the CD4/CD8 ratio decreased from
No change in the relative content of classical (CD45/14+/16-) and nonclassical monocytes (CD45/14low/16+) was observed in response to hyperinsulinemia in either group. Intermediate monocyte (CD45/14+/16+) count decreased after 2 h of sustained hyperinsulinemia in the control group only (Figure
An increased of expression (expressed as MFI) of the TLR2 scavenger receptor was detected on monocytes in response to hyperinsulinemia in both groups of subjects (Figure
No difference in the plasma levels of IL-4, IL-6, IL-10, IL-12, IL-17, TNF
In this study, we investigated the relative content and polarization of circulating monocyte and T lymphocyte populations and the effect of hyperinsulinemia on these immune cells, in respect to genetic predisposition to T2DM.
Compared to control subjects without genetic predisposition to T2DM, FDR entering our study exhibited mildly higher glucose and insulin fasting levels (and concomitantly higher HOMA-IR index) representing early risk factors for the development of T2DM. These findings were in agreement with the previously described difference between nonobese FDR and control subjects [
Moreover, the higher percentage of baseline Th17 lymphocytes found in the FDR group fits to the concept of the proinflammatory state preceding/contributing to diabetes onset, since TH17 T-cells producing IL-17 were associated with proinflammatory reactions and diabetic complications [
On the other hand, there was no difference between the two groups in relative distribution of classical and nonclassical monocyte populations, while the “intermediate” CD14+/16+ monocytes were lower in the FDR group. Intermediate monocytes express more than 80% of genes and surface markers at levels between classical and nonclassical monocytes [
In order to investigate further regulation of circulating lymphocytes and monocytes, we investigated responses of these immune cell populations to insulin action; namely, we studied these populations during short-term hyperinsulinemia induced by hyperinsulinemic-euglycemic clamp. The decrease of the CD4/CD8 ratio of T-lymphocytes evoked by short-term hyperinsulinemia in both groups of subjects suggests a marked role of insulin in the balance of these two cell populations. In fact, the insulin-stimulated decrease of CD4+ T-cells was accompanied by a decline of the relative content of TH2 and TH17 subpopulations in the FDR group to the levels seen in the control group. This could suggest that in the absence of elevated glucose, hyperinsulinemia is able to normalize the balance between TH subsets, while under more physiological or chronic conditions, this protective role of insulin may be masked or overridden by the proinflammatory effects of glucose and other metabolites/factors. On the other hand, hyperinsulinemia drove the decline of intermediate monocytes in control subjects to the level seen in FDR. Although the function of intermediate monocytes in metabolism is questionable as mentioned above, this result could suggest that both acute and chronic hyperinsulinemia is an important regulator of this monocyte subtype.
Further, an increase of expression (MFI) of TLR2, a well-characterized immune scavenger receptor, on monocytes was observed in response to hyperinsulinemia. TLR2 was shown to trigger low-grade chronic inflammation and activation of macrophages, present in obesity, T2DM, or atherosclerosis [
Finally, our study did not show any effect of the genetic predisposition on fasting plasmatic levels of various cytokines similarly as described previously [
In conclusion, we demonstrated a shift in the peripheral CD4/CD8 lymphocytes ratio, TH17 cells, and intermediate monocytes in subjects genetically predisposed to T2DM in comparison to controls. The imbalance in these immune cell populations might be features of genetic predisposition to T2DM and may play a role in pathogenesis of T2DM. Interestingly, short-term hyperinsulinemia affected mostly the immune cell populations deregulated in FDR subjects, which suggests an important interplay between immune system homeostasis and insulin levels.
The (lymphocyte and monocyte populations measured by flow cytometry and plasma cytokine levels) data used to support the findings of this study are included within the article. The original (raw) data of this study are available from the corresponding author upon request.
The authors declare no conflict of interest.
We thank Jana Potočková and Iveta Humlová (3rd Faculty of Medicine, Charles University, Prague) for the excellent technical assistance. The work is supported by grant GACR 16-14048S of the Grant Agency of the Czech Republic and programme PROGRES Q36 of Charles University.