- Academic Editor
Background: Type 1 diabetes mellitus (T1DM) is an autoimmune disease
characterized by immune response mediated islet beta cells destruction. However,
the mechanisms that cause immune response in TIDM are still under investigation.
Therefore, the goal of this study was to investigate the role of advanced
glycation end products (AGEs) in the regulation of the immune response in
peripheral blood mononuclear cells (PBMCs) from patients with T1DM.
Methods: PBMCs isolated from T1DM patients and control subjects were
used in the current study. Cytokines, AGEs related to glyoxalase 1 (GLO1),
methylglyoxal (MG)-derived AGEs were assessed longitudinally. Results:
The results of published T1DM PBMC microarray datasets using random-effects
meta-analysis models revealed immune responses in the PBMCs of patients with T1DM
compared with control subjects. Moreover, the activity of GLO1, which is the key
MG-metabolizing enzyme, was significantly reduced in PBMCs from T1DM patients. We
confirmed that, compared to the control subjects, GLO1 expression and activity
were markedly decreased and MG-derived AGEs were significantly accumulated in the
PBMCs from T1DM patients. In addition, phytohemagglutinin stimulated the
secretion of tumor necrosis factor alpha (TNF-
Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by the
destruction of pancreatic beta cells [1]. In the context of T1DM, peripheral
blood mononuclear cells (PBMCs) play a role in the autoimmune destruction of
pancreatic beta cells [2]. Insulitis, the infiltration of PBMCs in islets, is an
indicator of beta cell destruction in T1DM [3]. The exact mechanism by which
PBMCs contribute to the destruction of beta cells is not fully understood.
However, it may involve pro-inflammatory cytokines such as interleukin 1 beta
(IL-1
Methylglyoxal (MG), a highly reactive and toxic compound, is one of the precursors of advanced glycation end products (AGEs) [9]. Glyoxalase 1 (GLO1) is an enzyme that plays a critical role in the detoxification of MG [10] by preventing the formation of AGEs and reducing oxidative stress [11]. In patients with type 1 diabetes mellitus (T1DM), 67.5% showed preferentially high binding to MG–Lys–Cu2+ modified human DNA [12]. Peppa et al. [13] showed that MG-mediated protein structural alterations enhance immunogenicity in T1DM. In addition, inhibition of MG-mediated AGEs in non-obese diabetic mice was shown to significantly reduce insulitis and delay the onset of diabetes [13]. Those studies indicate that MG-mediated AGEs increase the progress of T1DM.
AGEs can activate the PBMCs of the immune system, promoting the immune response
and the release of pro-inflammatory cytokines and autoantibodies [14]. Dietary
AGEs increase circulating B cells and natural killer cells, as well as serum
levels of C-reactive protein in healthy subjects [15]. AGEs functionally
stimulate immune reactions in macrophages, mast cells, and B cells. The
inflammatory effects of AGEs are through activation of the nuclear factor kappa B
(NF-
In this study, the results of microarray meta-analysis of circulating PBMCs
showed that patients with T1DM exhibited an increase of immune response and
reduction of GLO1 expression, compared with that of control subjects. Our
experimental data further confirmed that diabetic patients had increased AGE
levels in PBMCs, which was indicated by a lower expression of GLO1 and higher
levels of intercellular MG-derived AGEs. Furthermore, pro-inflammatory cytokines,
such as IFN-
Ficoll was purchased from Shanghai Chemical Company (Shanghai, China). MG,
phytohemagglutinin (PHA), and S-p-bromobenzylglutathione cyclopentyl diester
(BrBzGCp2) were from Sigma-Aldrich (St. Louis, MO, USA). TRIzol was purchased
from Invitrogen (Carlsbad, CA, USA). MG-H1 antibody was obtained from Novus
Biologicals (Littleton, CO, USA). The BeyoRT™ III First Strand
cDNA Synthesis Kit; BeyoFast™ SYBR Green qPCR Mix; anti-mouse IgG
horseradish peroxidase (HRP)-linked secondary antibody; and enzyme-linked
immunosorbent assay (ELISA) kits for pro-inflammatory cytokines IL-1
The study was approved by the Affiliated Hospital of Gansu Medical College
(Lanzhou, Gansu province, China) and conducted in accordance with the principles
of the Declaration of Helsinki. Diabetic patients were recruited from the
Affiliated Hospital of Gansu Medical College. Informed consent was obtained from
the patients. A total of 12 males and 7 females between the ages of 9 and 45
years old with newly diagnosed T1DM (
Fasting human venous blood was collected in sterile heparinized tubes between
08:00 and 10:30 am. PBMCs were isolated using Ficoll gradients within 4 h after
the blood was drawn, and then washed with 1
Isolated PBMCs from control subjects were added to RPMI 1640 containing 10%
FBS. Cells (2
The measurement of GLO1 activity followed the protocol described by Qian et al. [10]. Cellular lysate from PBMCs was added to a hemithioacetal solution, which was prepared by pre-incubation with methylglyoxal and glutathione. The production of S-lactoylglutathione mediated by GLO1 was determined at an absorbance of 235 nm. GLO1 activity was calculated by the change in S-lactoylglutathione concentration.
Isolated PBMCs (2
The concentrations of IL-1
Indirect ELISAs with anti-MG-H1 antibody were carried out following the protocol described by Moraru et al. [19]. In brief, cell lysate (20 µL) in ammonium bicarbonate 100 mM pH 9.6 into a 96-well plate. The plate was incubated at room temperature for 2 h and the wells were washed with PBS. Then the wells were blocked in 5% (w/v) bovine serum albumin and incubated at room temperature for 2 h. The wells were incubated overnight with an anti-MG-H1 antibody, followed by washing and then incubation with anti-rabbit IgG, HRP-linked secondary antibody at room temperature for 1 h. The absorbance at 450 nm was measured by a microplate reader.
RNA from PBMCs was isolated by TRIzol according to the manufacturer’s
instructions. RNA concentration was measured following cDNA synthesis using the
BeyoRT™ III First Strand cDNA Synthesis Kit. The PCR reactions
contained 10 pM sense and 10 pM antisense oligonucleotides with
BeyoFast™ SYBR Green qPCR Mix. The human GLO1 forward primer was
5
The National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) was used to explore the expression profiles in microarray datasets. The keywords ‘type 1 diabetes’, ‘autoimmune diabetes’, ‘mononuclear’, and ‘PBMC’ were applied for this search. The inclusion criteria were: (1) gene expression profiling of circulating PBMCs; (2) patients with newly diagnosed T1DM and suitable control subjects; and (3) clear microarray platforms. In summary, the current study identified three GEO datasets (GSE55098, GSE29142, and GSE9006). The study included a total of 64 newly diagnosed patients with T1DM and 44 control subjects. The microarray platforms were Affymetrix Human Genome U133 Plus 2.0 Array, Phalanx Human OneArray, and Affymetrix Human Genome U133A Array.
All microarray datasets were downloaded from the NCBI GEO datasets using the R package GEOquery. Individual datasets were re-analyzed using the R package limma version 3.42.2 (Parkville, Victoria, Australia). The results of log fold change (logFC), p values, and the left and right limits of the confidence interval for logFC from individual datasets were exported for further meta-analysis. Meta-analysis was performed with the MetaVolcanoR package [20] by combining the P values using the random method. Differentially expressed genes (DEGs) from random analysis between patients with T1DM and control subjects were used for the functional analysis. For the enrichment analysis, the R package, clusterProfiler (Guangzhou, China) was used for the analysis of Gene Ontology (GO) biological process [21].
Statistical analyses were performed with GraphPad Prism 8 (Dotmatics, Boston, MA, USA). Due to the limitation
of patients, the significant analysis was conducted with the Mann–Whitney U
Test. Correlation analysis was performed by simple linear regression analysis.
p
Three studies were recruited for this meta-analysis study. GSE55098 [22]
included 12 patients with newly diagnosed T1DM (defined as treatment with insulin
for
Activation of the immune response in peripheral blood mononuclear cells (PBMCs) from patients with type 1 diabetes mellitus (T1DM). (A) Differentially expressed genes (DEGs) of published microarray datasets. Red: upregulated DEGs; blue: downregulated DEGs; grey: no significant difference genes. (B) Forest plot of top 1% DEGs in PBMCs between patients with T1DM and control subjects using meta-analysis random effect analysis. (C) Gene Ontology (GO) term biological process analysis of upregulated DEGs of PBMCs from T1DM patients compared with control subjects. (D) GO term biological process analysis of downregulated DEGs of PBMCs from T1DM patients compared with control subjects.
Then we performed random analysis based on the results of limma analysis from individual studies. Fig. 1B shows the top 1% of both up- and downregulated DEGs of PBMCs from patients with T1DM. To identify DEGs related to the functional biological group, using GO biological process analysis, the results from upregulated DEGs in T1DM further confirmed that the development of T1DM was related to the immune response (Fig. 1C). The top pathways of downregulated DEGs in T1DM showed the inhibition of mitochondrial electron transport chain activity (Fig. 1D).
As shown in Table 1, patients with T1DM had significantly higher levels of
fasting blood glucose and hemoglobin A1C compared with control subjects. The
correlation between fasting blood glucose and glycated hemoglobin was positive (r
= 0.7081, p
Control | T1DM | |
Sex (male/female) | 35 (23/12) | 19 (12/7) |
Age (years) | 24.45 |
20.84 |
Fasting glucose (mM/L) | 4.87 |
9.90 |
HbA1c (%) | 5.07 |
6.77 |
GAD+ (N) | 14 | |
IAA+ (N) | 7 |
* p
Accumulation of cellular Advanced glycation end products (AGEs)
in peripheral blood mononuclear cells (PBMCs) from patients with type 1 diabetes
mellitus (T1DM). (A) Cellular levels of methylglyoxal (MG)-derived AGEs were
measured by indirect enzyme-linked immunosorbent assay (ELISA), as described in
Materials and Methods. * indicates the comparison with control subjects,
as determined by Mann–Whitney U Test. p
T1DM is considered an autoimmune disease. Therefore, the pro-inflammatory
cytokines were determined in the serum of patients with T1DM. As shown in Fig. 3A, there was no significant difference in circulating levels of IL-1
The activation of immune response in PBMCs from patients with T1DM. (A) Serum cytokine levels from patients with T1DM and control subjects were determined by commercial ELISA kits. (B) Supernatant cytokine levels were measured in phytohemagglutinin (PHA)-treated PBMCs from patients with T1DM and control subjects by commercial ELISA kits. * indicates the comparison with control subjects, as determined by Mann–Whitney U Test. The correlation of secreted cytokines induced by PHA in PBMCs from T1DM and cellular MG-derived AGEs (C), GLO1 activity (D) were analyzed. * indicates the comparison with control subjects, as determined by simple linear regression analysis.
To explore whether cellular AGEs aggravate the immune response in T1DM,
correlation analysis was performed between pro-inflammatory cytokines such as
IFN-
To further evaluate the role of MG-derived AGEs on pro-inflammatory secretion, MG and a GLO1 inhibitor, BrBzGCp2, was applied in the current study. Fig. 4A shows that treatment of PBMCs with 20 µM MG significantly reduced cell viability compared with vehicle. To further explore the effect of MG on the accumulation of cellular AGEs, the results showed that 10 µM MG markedly enhanced the accumulation of MG-derived AGEs (Fig. 4B). The effect of BrBzGCp2 on GLO1 activity was also determined in PBMCs from control subjects. The results of Fig. 4C showed that 1.5 µM BrBzGCp2 led to a significant reduction of PBMC viability compared with that of vehicle. However, we did not observe a significant difference in cell viability between 0.5 and 1 µM BrBzGCp2. To determine whether BrBzGCp2 induces cellular AGE accumulation, PBMCs were treated with the vehicle, 0.5 and 1 µM BrBzGCp2 for 24 h. The 0.5 µM BrBzGCp2 effectively increased cellular MG-derived AGE accumulation (Fig. 4D). Therefore, in the current study, 10 µM MG and 0.5 µM BrBzGCp2 were applied to PBMCs.
Accumulation of cellular AGEs by MG and GLO1 inhibitor enhanced
immune response in mononuclear cells. (A) Cell viability of MG-treated
mononuclear cells from control subjects (n = 10) was determined. * indicates the
comparison with control subjects, as determined by the Mann–Whitney U Test. (B)
Cellular MG-derived AGEs in MG-treated PBMCs were determined by indirect ELISA. *
indicates the comparison with control subjects, as determined by Mann–Whitney U
Test. (C) Cell viability of GLO1 inhibitor – S-4-Bromobenzylglutathione
cyclopentyl diester (BrBzGCp2) treated mononuclear cells from control subjects (n
= 10) was determined. * indicates the comparison with control subjects, as
determined by the Mann–Whitney U Test. (D) Cellular MG-derived AGEs in
BrBzGCp2-treated PBMCs were determined by indirect ELISA. * indicates the
comparison with control subjects, as determined by Mann–Whitney U Test. (E)
TNF-
Finally, we examined the effects of MG and BrBzGCp2 on the regulation of the
immune response in PBMCs from control subjects. Fig. 4E shows that treatment of
PBMCs with 10 µM MG or 0.5 µM BrBzGCp2 led to an
increase in TNF-
It is well-known that T1DM is an autoimmune disease [25, 26]; however, the immune response in T1DM is still under investigation. Therefore, in this study, we explored the role of AGEs in the regulation of the immune response in the PBMCs from patients with T1DM. Our results showed the following. (1) Using GO biological process analysis based on the meta-analysis of published GEO datasets, upregulated DEGs in PBMCs from patients with T1DM showed an increased inflammatory response. The downregulated DEGs indicated the inhibition of mitochondrial electron chain transport in patients with T1DM. (2) In agreement with the results of meta-analysis, our results further confirmed that MG-derived AGE level significantly increased and the expression of GLO1, which is the key enzyme that detoxicates MG into S-2-hydroxyacylglutathione [10], was markedly reduced in PBMCs from T1DM. Moreover, the enhancement of AGEs was positively correlated to inflammatory cytokines secretion. (3) Exposure to MG and a GLO1 inhibitor in PBMCs from control subjects increased inflammatory cytokine secretion. In summary, our study indicated that in the PBMCs of patients with T1DM, the accumulation of cellular AGEs enhances the immune response. This may decrease the destruction of islet beta cells.
In our study, we first performed meta-analysis based NCBI GEO datasets using random effect analysis. The results from upregulated DEGs showed that the activation of neutrophils (Fig. 1C), one of the main cell types involved in T1DM [27]. The downregulated DEGs also demonstrated the inhibition of the mitochondrial electron chain transport (Fig. 1D). The mitochondrial electron chain transport, including complex I, III and IV, is the major focus of immunometabolism [28]. Exposure of rats to rotenone, a mitochondrial complex I inhibitor, causes systemic oxidative stress and the release of pro-inflammatory cytokines [29]. The mitochondrial complex III inhibitor, antimycin A, also contributes to the pro-inflammatory response [30]. In summary, the results from our meta-analyses further supported the hypothesis that T1DM is an immune response disease.
Cellular AGEs and the immune response are strongly interconnected [31, 32]. AGEs can function as inflammatory stimulators that trigger inflammatory signaling [33], including the secretion of pro-inflammatory cytokines [34]. A study revealed that the AGE receptor binds to high mobility group box 1 (HMGB1) and macrophage antigen-1 (Mac-1). The activation of HMGB1 and Mac-1 propagates inflammation [35, 36]. Overexpression of GLO1 improves the pro-inflammatory response in experimental diabetic nephropathy [37]. Results of Fig. 2A confirmed the accumulation of MG-derived AGEs in PBMCs from patients with T1DM. Fig. 2C,D shows that GLO1 expression and activity in PBMCs from patients with T1DM were significantly reduced, which were in agreement with single gene expression from random analysis (Fig. 2B). Furthermore, PHA-stimulated pro-inflammatory cytokine release was positively correlated with the levels of MG-H1, and negatively correlated with GLO1 activity in PBMCs from T1DM (Fig. 3B–D).
The effect of the accumulation of cellular AGEs on the immune response is also
detected in different diseases. Exposure of neutrophils to MG or inhibition of
GLO1 activity using RNA interference in healthy keratinocytes or using GLO1
inhibitor, BrBzGCp2 in rat bone marrow mesenchymal stem cells also demonstrated
that impaired GLO1 activity enhanced the expression of TNF-
In summary, our results showed that the accumulation of AGEs by hyperglycemia in diabetic patients was positively correlated with the immune response in PBMCs. The exposure of MG or inhibition of GLO1 also stimulated the secretion of pro-inflammatory cytokines. After the onset of T1DM, the accumulation of AGEs caused by hyperglycemia and inhibition of GLO1 increased the secretion of pro-inflammatory cytokines. The joint effects of glycation and the immune response in T1DM may accelerate the progression of T1DM and related complications. Therefore, the treatment of T1DM may include alleviating hypoglycemia and improving the immune response. However, there are several limitations in the current study (1) the limited numbers of T1DM patients: we only recruited 19 T1DM patients. (2) The age difference between T1DM. In current study, the age of patients was from 9 to 45 years old. Therefore, the increase of recruited T1DM patients will contribute to understand the correlation between AGEs and immune response in T1DM.
Data of meta-analysis from T1DM patients (GSE55098, GSE29142, and GSE9006) are from GEO datasets, National Center for Biotechnology Information. Clinical data were not available due to patients’ information.
LY performed the research. YQ and LY performed meta-analysis and analyzed the data. SL provided scientific advice and patients collection. DS designed the study, collected patients for this study, and wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.
The ethical committee of Affiliated Hospital of Gansu Medical College has approved this study [2023]IEC (027). And all patients have been formed consent.
Not applicable.
This research received no external funding.
The authors declare no conflict of interest.
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