†These authors contributed equally.
Academic Editor: Jerome L. Fleg
Background: Regulatory T (Treg) cells are a class of anti-inflammatory
lymphocyte subpopulations with a potential protective effect against
atherosclerosis, whereas T helper 17 (Th17) cells have been reported to possess
proatherogenic activity. It was believed that disturbed circulating Treg/Th17
balance was associated with the onset and progression of atherosclerosis. This
study is designed to probe the regulative action of serum Nod-like receptor
protein 3 (NLRP3) on the Treg/Th17 balance in patients with atherosclerosis.
Methods: Fifty-two patients with coronary atherosclerosis and stenosis
degrees of more than 50% were assigned to the coronary artery
disease (CAD) group, and an equal number of people without coronary
atherosclerosis were assigned to the control group (assessed by
coronary angiography). Peripheral blood mononuclear cells (PBMCs) from two group
patients were extracted and cultivated. The calculation of the Treg/Th17 ratio
and quantitative analysis of the Treg and Th17 cell frequencies were performed
through flow cytometry. Real-time fluorescence quantitative polymerase chain
reaction (RT-PCR) was executed for the quantitative mRNA detection of the fork
head-winged helix transcription factor (Foxp3) and the retinoic acid-related
orphan nuclear receptor C (RORC) in PBMCs. Enzyme-linked immunosorbent assays
were applied to measure the serum level of NLRP3, interleukin (IL)-10,
IL-1
Atherosclerosis is characterized as a chronic inflammatory disease with an
intricate pathological process in which both innate and adaptive
immunoinflammatory mechanisms are involved [1]. The inflammasome is known as an
important component of innate immunity and can be activated by a series of
pathogen- or injury-associated molecular patterns [2]. The Nod-like receptor
(NLR) family of proteins represents a fourteen-member subset that acts as an
essential component of the inflammasome, and its fourteen members are known as
NLRP1~14. As a pattern recognition receptor (PRR) belonging to
the NLR family, Nod-like receptor protein 3 (NLRP3) is directly induced by
multiple external stimuli which activates cysteinyl aspartate specific proteinase
1 (caspase-1) from pro-caspase-1. Once activated, the latter leads to increased
synthesis and secretion of interleukin (IL)-1
Since atherosclerosis is increasingly recognized as a chronic inflammatory disease, the crucial role of the interaction between multiple leukocyte subsets and inflammatory cytokines in atherogenesis has also been established. Similarly, the correlation between atherosclerosis and Treg/Th17 imbalance was recently revealed: under inflammatory conditions, the mutual transformation between Treg cells and Th17 cells is activated, which may lead to an alteration of the Treg/Th17 ratio and subsequently the promotion of atherosclerosis progression [5]. In addition, in our previous study, patients with acute myocardial infarction and coronary artery stenosis showed higher blood level of NLRP3, but the exact mechanism through which NLRP3 impacts atherosclerosis remains unclear. Interestingly, high expression of NLRP3 has been shown to promote the progression of inflammatory diseases by mediating the Treg/Th17 imbalance. Thus, we hypothesized that NLRP3 disturbs the Treg/Th17 balance by positively regulating the conversion of Tregs to Th17 cells, thereby promoting the overexpression of inflammatory factors and driving atherosclerotic plaque formation. The primary objective of this work was to investigate the potential impact of NLRP3 on the peripheral Treg/Th17 balance through the investigation and analysis of the correlation between serum Treg/Th17 ratio and NLRP3 level, intending to identify novel targets and a theoretical basis for atherosclerosis prevention and management.
The coronary artery disease (CAD) group is constituted with 52 patients who were diagnosed with coronary atherosclerosis admitting by the Department of Cardiology at Wuhan Central Hospital and the First College of Clinical Medical Sciences at China Three Gorges University between January 2018 and December 2020. Following were the inclusion criteria: (1) one or more coronary arteries with more than 50% atherosclerosis diagnosed by coronary angiography and (2) no anti-inflammatory drugs or immunosuppressants for half a year. Moreover, the patients’ exclusion were performed as following criteria: (1) patients with severe cardiac-cerebral vascular diseases, such as stroke and myocardial infarction; (2) patients with hepatorenal insufficiency or other metabolic diseases; (3) patients with cancer; (4) patients with all kinds of acute and chronic infectious diseases; and (5) patients with a history of surgery within half a year.
The control group included 52 individuals who did not have coronary artery
stenosis as determined by coronary angiography. The baseline parameters,
including sex, age, or blood biochemical indices had no difference in both groups
(p
Characteristics | Control group | CAD group | t/ |
p value |
---|---|---|---|---|
(n = 52) | (n = 52) | |||
Male [n (%)] | 28 (53.8) | 25 (48.1) | 0.346 | 0.56 |
Current Smoking [n (%)] | 16 (30.8) | 22 (42.3) | –1.069 | 0.29 |
Hypertension [n (%)] | 14 (26.9) | 17 (32.7) | 0.414 | 0.52 |
Age [( |
57.8 |
59.3 |
1.493 | 0.22 |
Fasting BG [( |
4.6 |
4.5 |
1.126 | 0.26 |
Serum Cr [( |
77.2 |
80.6 |
–1.035 | 0.30 |
TC [( |
4.1 |
4.0 |
0.536 | 0.59 |
TG [( |
1.5 |
1.4 |
0.678 | 0.50 |
HDL-C [( |
1.2 |
1.3 |
–1.613 | 0.11 |
LDL-C [( |
2.2 |
2.3 |
–0.599 | 0.551 |
Note: BG, blood glucose; Cr, creatinine; LDL-C, low density lipoprotein cholesterol; TC, total cholesterol; TG, triacylglycerol; HDL-C, high density lipoprotein cholesterol. |
All participants had their fasting venous blood samples drawn the morning of the
day following admission. Density gradient centrifugation was performed to
separate peripheral blood mononuclear cells (PBMCs) from the obtained blood
sample. Resuspend the cell pellet with a moderate amount of phosphate buffer saline (PBS) and adjust the
concentration of PBMCs to 2
Gene | Sequence |
---|---|
Foxp3 | Forward primer: 5′- AACAGCACATTCCCAGAGTTCC -3′ |
Reverse primer: 5′- CATTGAGTGTCCGCTGCTTC -3′ | |
RORC | Forward primer: 5′- CCGAGGATGAGATTGCCCTCT -3′ |
Reverse primer: 5′- GGTGGCAGCTTTGCCAGGAT -3′ | |
GAPDH | Forward primer: 5′-CCACATCGCTCAGACACCAT-3′ |
Reverse primer: 5′-CCAGGCGCCCAATACG-3′ |
For another experiment, isolate serum from coagulated blood samples collected in
nonanticoagulant tubes by centrifugation (3000 r/min, 20 min) min after 30min of
standing. Collect upper serum and store it at –80 °C. Fasting blood glucose,
blood lipids, and serum creatinine were measured by the laboratory department of
Wuhan Central Hospital. The serum concentrations of NLRP3, IL-10, TGF-1, IL-1,
IL-17A, and IL-23 were tested via the enzyme-linked immunosorbent test (ELISA).
NLRP3 (Wuhan Feien Biotechnology, No. eh4202), IL-10 (Xinbosheng Biotechnology,
No. ehc009.96), TGF-
IBM SPSS Statistics 22 (IBM Corp., Armonk, NY, USA) was used for all data
analyses. Measurement data are presented with the formation of (
Compared to the control group, individuals with coronary atherosclerosis had
significantly lower Treg cell frequencies and Treg/Th17 ratios in their PBMCs
(p
Group | n | Treg (%) | Th17 (%) | Treg/Th17 |
---|---|---|---|---|
Control | 52 | 6.1 |
1.2 |
5.1 |
CAD | 52 | 3.8 |
2.3 |
1.4 |
t value | 16.586 | –13.604 | 39.774 | |
p value |
Foxp3 is known as a specific transcription factor which acts as a central
effector in regulatory T cells growth and differentiation. RORC is regarded as a
Th17 transcription factor. In support of those previously determined definitions
of Foxp3 and RORC, in this study, we observed a marked improvement of RORC mRNA
expression and an obvious reduction of Foxp3 mRNA in the CAD group (p
The mRNA expression of RORC and Foxp3 in
PBMCs. (A) In the CAD group, a considerably higher amount of RORC mRNA was found
as compared to the control (n = 5, p
As shown in Table 4, the serum concentrations of levels of NLRP3 and Th17
cell-related inflammatory cytokines (IL-1
Group | n | NLRP3 (ng/mL) | IL-10 (pg/mL) | TGF- |
IL-1 |
IL-17A (pg/mL) | IL-23 (pg/mL) |
---|---|---|---|---|---|---|---|
Control | 52 | 2.2 |
21.5 |
472.5 |
14.4 |
19.1 |
44.2 |
CAD | 52 | 5.6 |
11.9 |
235.5 |
26.8 |
54.6 |
83.6 |
t value | –30.411 | 13.347 | 37.989 | –13.376 | –28.131 | –31.990 | |
p value | |||||||
Note: NLRP3, Nod-like receptor protein 3; IL-10, Interleukin 10; TGF- |
The negative correlation between the serum concentration of NLRP3 and the
Treg/Th17 ratio is shown in Fig. 2 (n = 52, r = –0.699, p
Correlation analysis on serum NLRP3 level and Treg/Th17 in
patients with coronary atherosclerosis (n = 52, r = –0.699, p
NLRP3 has been identified as a sensor component of the NLPR3 inflammasome that
responds to various external stimuli and promotes inflammasome assembly [6]. The
assembled NLRP3 inflammasome facilitates the cleavage and activation of
caspase-1, which directly binds to the IL-1
Interestingly, as two of the CD4+ T lymphocyte cell subsets, it has been
thoroughly described in experimental animal research and clinical studies that
Treg and Th17 cells exhibit crucial but diverse roles in atherosclerosis
progression, in which Treg cells and related anti-inflammatory cytokines
(TGF-
In addition, the regulatory effect of NLRP3 on the Treg/Th17 cell
differentiation and its strong correlation with the progression of multiple
immune diseases has been revealed in recent studies [19, 20, 21]. For instance, high
expression of NLRP3 has also been observed in psoriatic lesions. Moreover,
skin-specific NLRP3 suppression can inhibit the proliferation and chemotaxis of
keratinocytes through the downregulation of IL-1
This study finds that serum NLRP3 level may reflect the Treg/Th17 ratio in individuals with atherosclerosis, however, some limitations remain existing. Significantly, the current findings of this study reveal a negative correlation between serum NLRP3 level and the ratio of Treg/Th17, which can be applied to the risk assessment of atherosclerosis. However, the negative correlation between NLRP3 and the Treg/Th17 ratio does not prove its causation, more evidence is required to examine NLRP3 as a key regulator for Treg/Th17 balance in further study. Moreover, the number of included patients with coronary atherosclerosis was only 52. Design of multicenter and a larger number of included patients might have provided more trustworthy results to support the correlation between serum NLRP3 and Treg/Th17 ratio.
In conclusion, the serum level of NLRP3 is inversely correlated with Treg/Th17 ratio in atherosclerosis. This finding provides a new idea for the research on immune mechanisms of atherosclerosis, clinical risk assessment of atherosclerosis and the prevention and treatment of atherosclerotic disease. Further investigations are required for the precise role and underlying mechanism of NLRP3 acting on the Treg/Th17 balance.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
JY, ZXF and XG designed the research study. ZXF, YFH, JCW and LHD performed the research. LHD, CJY and JCW provided help and advice on the data analysis. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.
The study was approved by the local ethics committee (Wuhan Central Hospital and the First College of Clinical Medical Sciences at China Three Gorges University, Number: 2018YYEC-004). All patients gave their informed consent to participate in this study.
Not applicable.
The present study was supported by the Natural Science Foundation of Yichang (grant no. A20-2-004) and supported by the application and basic research project from the science and technology department of Qinghai province (Grant No.2022-ZJ-758).
The authors declare no conflict of interest.