The authors have declared that no competing interests exist.
Conceived and designed the experiments: LZ QW FZ. Performed the experiments: BW SD. Analyzed the data: ZD. Contributed reagents/materials/analysis tools: CG YS. Wrote the paper: WB XS LZ.
Interleukin (IL)-35 is a newly identified immune negative molecule which is secreted by CD4+Foxp3+ T regulatory cells (Tregs) and contributes to their suppressive capacity. Early data have shown that IL-35 inhibits development of several autoimmune diseases. However, the role of IL-35 in atherosclerosis, a lipid-driven chronic inflammatory disease in arterial wall, remains to be investigated. Here, we found that IL-35 was involved in atherosclerosis in apolipoprotein E-deficient (ApoE−/−) mice. ApoE−/− mice with established atherosclerotic lesion displayed a lower level of IL-35 compared to age-matched wild type C57BL/6 mice without plaque. However, IL-35 expression increased significantly in ApoE−/− mice with attenuated plaque. More importantly, we found that modulation of ER stress treated by chemical chaperone, 4-Phenyl butyric acid (PBA)
Interleukin (IL)-35 is a newly identified inhibitory cytokine produced by CD4+Foxp3+ T regulatory cells (Tregs) and is required for suppressive function of Tregs
Endoplasmic reticulum (ER) is an organelle in which newly synthesized proteins are correctly folded and assembled. Once it is perturbed by various pathological conditions, unfolded proteins will accumulate and result in endoplasmic reticulum stress (ER stress), also defined as the unfolded protein response (UPR). Accumulating evidence indicates that the UPR is chronically activated in atherosclerotic lesions by oxidative stress, high levels of intracellular cholesterol and saturated fatty acids
The subunits of IL-35, p35 and EBI3, are synthesized and assembled in ER
Male C57BL/6 wild type mice and male ApoE−/− mice were purchased from Beijing University and fed on a high-fat diet (0.25% cholesterol and 15% cocoa butter) from 8 weeks of age to 16 weeks to induce atherosclerotic plaques. All animal studies were approved by the Animal Care and Utilization Committee of Shandong University, China.
ApoE−/− mice (n = 10 for each group) were fed on a high-fat diet (0.25% cholesterol and 15% cocoa butter) from 8 weeks of age. Two weeks after high-fat diet, mice were divided into two groups randomly, one group, named as PBA, was injected intraperitoneally with PBA (P21005, Sigma-Aldrich, St. Louis, MO, USA) in phosphate buffered saline (PBS), 100 mg/kg, one time per three days for 5 weeks. The other group was injected intraperitoneally with PBS as control. Mice were sacrificed for analysis one week after the last treatment.
Mice were weighed at the end of experiment. Total plasma cholesterol (TCH), total triglycerides (TGs) and high-density lipoprotein (HDL) levels were determined with automated enzymatic technique (7080, HI TACH, Japan). Low-density lipoprotein (LDL) was detected with an automated chemically modified technique (Roche Modular DPP System, Roche, Switzerland).
Proteins obtained from separate thoracic and abdominal aorta, were separated by SDS-PAGE and blotted on PVDF membranes. Membranes were probed with primary antibodies, Phospho-eIF2a (p-eIF2a) (#3597 Cell Signaling Technology, USA), eIF2a (#9722 Cell Signaling Technology, USA), spliced XBP-1 (sXBP-1) (ab37152, Abcam, Hong Kong) and β-actin (Bioworld, Georgia, USA) overnight at 4°C, respectively, followed by secondary antibody conjugated with peroxidase (A0208, Beyotime, China) for one hour at room temperature. After washing, signals were visualized using electrochemiluminescence (Pierce Biotechnology, Rockford, IL, USA) and autoradiography.
After sacrificing, mice were perfused with PBS through the left ventricle and hearts were fixed in 4% paraformaldehyde solution overnight and embedded in optimal cutting temperature (OCT) (Sakura Finetek, Torrance, CA, USA) medium or paraffin. Aortic root sections were cut from the embedded hearts. Serial cryosection of 6∼10 µm or paraffin-section of 4∼6 µm were dissected along the longitudinal direction of aortic root vessels using cryotome (HM550, Thermo scientific, USA). Five sections spaced 80 µm apart of each aorta root were stained with oil red “O” staining (O0625, Sigma-Aldrich, USA). Corresponding frozen sections were subjected to immunohistochemical staining according to respective antibody protocols, including rat anti-mouse macrophage Moma-2 (MCA519A647, AbD serotec, Oxford, U.K.), rabbit polyclonal to α- smooth muscle actin (ab5694, Abcam, Hong Kong), rat anti-mouse CD3 (MCA500G, AbD Serotec, Oxford, U.K.), rabbit anti-mouse Foxp3 (BA2032-1, BOSTER, China) and rabbit anti Phospho-PERK (p-PERK, 3179, Cell Signaling Technology, Danvers, MA, USA) to detect the levels of macrophage, smooth muscle cell, CD3+ T cell, Foxp3+ cells and p-PERK in lesion respectively. Alexa Fluor 555-labeled goat anti-rabbit IgG (A0462. Beyotime, China) was used to perform indirect immunofluorescence assays of Foxp3. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) was performed on paraffin sections with TUNEL kit (Roche, USA) according to manufacturer’s instructions. Images were captured using an Olympus microscope (IX71; Olympus Corporation, Tokyo, Japan). The area of plaque and positive staining were measured using Image-Pro Plus software (Media Cybernetics, Bethesda, MD, USA).
Total RNA was isolated from thoracic and abdominal aorta using TRIzol reagent (15596-026, Invitrogen, Carlsbad, California, USA). Reverse transcription was performed with RT-PCR quick master mix (PCR-311, TOYOBO, Japan) to get cDNA, and real-time quantitative polymerase chain reactions were performed with Ultra SYBR mixture (CW0956, CW Bio, China) using CFX 96 Real-Time Detection System (Bio-RAD, USA). Sequences of related gene specific primers were included in
Genes | Primers | Sequences |
IFN-γ | Forward | |
Reverse | ||
TNF-α | Forward | |
Reverse | ||
IL-17A | Forward | |
Reverse | ||
IL-23 | Forward | |
Reverse | ||
TGF-β | Forward | |
Reverse | ||
IL-10 | Forward | |
Reverse | ||
EBI3 | Forward | |
Reverse | ||
IL-12α | Forward | |
Reverse | ||
IL-12β | Forward | |
Reverse | ||
p28 | Forward | |
Reverse | ||
Foxp3 | Forward | |
Reverse |
Splenocytes isolated from PBA-treated and control mice were washed with PBS and stained with PE-Cy5-conjugated anti-CD4 (Anti-Mouse CD4 PE-Cyanine5, eBioscience, San Diego, CA, USA) and then were fixed and perforated by the fixation and permeabilization kit (eBioscience, San Diego, CA, USA) and then stained intracellularly with PE-conjugated anti-Foxp3 monoclonal antibodies (Anti-Foxp3 PE, eBioscience, San Diego, CA, USA). Flow cytometric analysis was performed using a Cytomics FC500 (Beckman Coulter, Brea, CA, USA) and the data were analyzed by CXP2.0 software.
Serum was collected for detection of TNF-α, IFN-γ, IL-17 and IL-10 using a mouse cytometric bead array (Cytometric Bead Array, BD Biosciences, San Jose, CA, USA) and for assays of IL-23 (EMC114.48, NeoBioscience, China), and TGF-β (BMS608/4, eBioscience, San Diego, CA, USA) using ELISA according to manufacturer’s instructions. IL-35 in serum was quantified using IL-35 ELISA kits (RapidBio Lab, USA) according to the manufacturer's instruction and the sensitivity of this assay is ≥1.0 pg/ml.
All analyses were done by SPSS 11.0 (SPSS, Chicago, IL,USA). Data are expressed as mean ± SEM. Unpaired
Previous researches have demonstrated that ER stress is involved in atherosclerosis and recovery of ER function is believed to be a critical factor for improvement of atherosclerosis
Representative photomicrographs and the quantitative analysis of smooth muscle cell (SMC), CD3, macrophage (Mφ), and TUNEL in lesion of aortic root of ApoE−/− mice. The aortic root sections were stained with rabbit polyclonal to α- smooth muscle actin, rat anti-mouse macrophage Moma-2 and rat anti mouse CD3. The content of macrophage, smooth muscle cell, CD3 T cell in lesion was analyzed respectively by immunohistochemistry. For detection of cell apoptosis, sections were incubated with anti-TUNEL antibody and the content of apoptotic cells was analyzed by immunofluorescence. (n = 10 per group), *p<0.05, **p<0.01, ***p<0.001.
Weight (g) | TG (mmol/L) | TCH (mmol/L) | LDL (mmol/L) | HDL (mmol/L) | |
Control | 30.26±0.85 | 47.74±1.16 | 4.33±1.24 | 7.72±0.17 | 11.25±1.26 |
PBA | 33.10±1.60 | 46.51±1.39 | 3.93±0.96 | 7.65±0.29 | 10.49±2.44 |
P value | 0.17 | 0.52 | 0.82 | 0.82 | 0.79 |
The data are given as the mean
We further investigated the change of cytokines in lesion after ER stress modulation by real-time PCR. As shown in
The expression of cytokine mRNAs in the thoracic and abdominal aorta was quantified by real -time PCR analysis and normalized to β-actin. Fold-changes in expression in PBA treated mice relative to controls are shown.
The change in circulating cytokines was detected by cytometric bead array for TNF-α, IFN-γ, IL-17 and IL-10, and by ELISA for IL-23, TGF-β and IL-35. As shown in
Serum was obtained from ApoE−/− mice with or without PBA treatment and the concentrations of
It has been reported that IL-35 is not only an effector molecule of Tregs but also an inducer of Tregs. Therefore, we detected the effect of PBA treatment on Treg cells. The spleen cells were collected from PBA treatment or control group and stained with CD4 and Foxp3 specific antibodies, and then the proportion of Tregs (CD4+ Foxp3+) in CD4+ T cells was analyzed by Flow Cytometry. As shown in
The above results indicate that modulation of ER stress by PBA attenuates atherosclerotic lesion and upregulates level of IL-35 both in arterial wall and in circulation. To further confirm the association of IL-35 with atherosclerosis, we detected the change of serum IL-35 level in development process of plaque in ApoE−/− mice compared with wild type C57BL/6 mice. As shown in
Here, we find that IL-35 is involved in atherosclerosis in ApoE−/− mice. ApoE−/− mice with atherosclerotic lesion have lower levels of IL-35 but the level increases in mice with attenuated plaque treated with PBA. More importantly, we find that modulation of ER stress by PBA treatment mainly upregulates immune negative regulating molecules, IL-35 as well as IL-10 and the transcription factor Foxp3, but has no obvious impact on pro-inflammatory molecules, such as TNF-α, IFN-γ, IL-17 and IL-23, which provides a new insight into the benefits of ER stress recovery to attenuated plaque.
To date, little is known about the role of IL-35 in atherosclerosis. Although previous research shows that expression of EBI3 and p35 is detected in human atheroma plaques, but other subunits, IL-27 α/p28 can also be found in plaque
Atherosclerotic plaques, particularly advanced lesions, contain a large amount of toxic lipids (such as saturated fatty acids or free cholesterol) and pro-inflammatory cytokines (TNF-α, IFN-γ, IL-6, IL-12 etal). The pathophysiological environment can activate UPR which in turn upregulates expression of inflammatory genes (such as IFN-γ and IL-17) accelerating the progress of atherosclerotic plaque
Although our research did not confirm the causal relationship of IL-35 with atherosclerosis absolutely, it provided a new direction for the research about atherosclerosis. Because IL-35 comprises two subunits, p35 and EBI3, which take part in the composition of IL-12 and IL-27 respectively, it is difficult to produce recombined IL-35 and neutralizing antibody or knockdown/knockout related genes without influencing other cytokines. We believe that further research should overcome those difficulties and the detailed mechanism of IL-35 influencing atherosclerosis would be elucidated in future.
Collectively, our results indicate that IL-35 is involved in the atherosclerosis. The modulation of ER stress by PBA is able to upregulate cytokine IL-35 which may contribute to attenuation of plaque in ApoE−/− mice.
Thanks for the language editing of professor Wanjun Chen of US National Institutes of Health.