Introduction
The autoimmune regulator (AIRE) is a transcriptional regulator, and mutations in this gene cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) characterized by chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency. APECED is classified as type IV primary immunodeficiencies (PID) by the International Union of Immunological Societies (IUIS) [
1]. Since AIRE is expressed in medullary thymic epithelial cells (mTEC) and can promote ectopic expression of a wide array of peripheral tissue antigens (PTA) in thymus, studies have focused on central tolerance and have addressed the role of AIRE in negative selection [
2]. AIRE is restrictively expressed in peripheral monocyte/dendritic cell lineage [
3] as well as in mTEC. Recently, signs of abnormal immune regulation have been revealed in AIRE deficient mice or patients with APECED by elevated numbers of blood monocytes [
4], increased antigen presenting cell mediated T cell activation [
4] and downregulated multiple immunologic receptors [
5], suggesting that AIRE plays a role in the regulation of immune responses. In the progress of immune responses, a highly conserved homeostatic cellular mechanism, autophagy, is confirmed to be equally important to innate and adaptive immunity by various studies. Autophagy can have a broad range of effects on innate and adaptive immunity, illustrated by antimicrobial defense, expression of pattern recognition receptors (PRR), secretion of cytokines, antigen presentation, development of lymphocytes and thymic selection [
6]. However, whether AIRE plays a role in autophagy in immune cells is unclear. Monocytes have been considered as precursor cells of macrophages and dendritic cells, and play an important role in both adaptive and innate immunity. Based on the expression of AIRE in monocytes, we explored the link between AIRE and autophagy in THP-1 human monocytes.
Materials and methods
Cell culture
For the isolation of viable monocytes from human peripheral blood, a special Ficoll based cell separation medium (Fico/Lite Monocytes, Atlanta Biologicals, USA) was used. The human acute monocytic leukemia cell line THP-1 was a gift from Dr. Guo-Hong LIN (Huazhong University of Science and Technology, China) and cultured in RPMI1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 0.05 mmol/L 2-mercaptoethanol, 100 IU/mL penicillin and 100 μg/mL streptomycin incubated at 37°C in a humidified 5% CO2 incubator. For differentiation of THP-1 cells, 100 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma, USA) was used. Cell autophagy was induced by Earle’s balanced salt solution (EBSS) for 2 h.
Transfection and RNA interference (RNAi)
pEGFP-AIRE plasmid was kindly provided by Prof. Mitsuru Matsumoto (University of Tokushima, Japan) [
7]. The empty pEGFP-C
3 vector was purchased from Clontech (BD Biosciences Clontech, USA). All plasmids were confirmed by restriction enzyme digestion and DNA sequencing. AIRE small interfering RNA (siRNA) (sc-37669) and control siRNA-A (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). One day before transfection, THP-1 cells were plated in culture medium containing differentiation inducing agent. For transient plasmid expression or RNAi analysis, cells were transfected in a 6-well tissue culture plate with 2 μg of the indicated plasmid DNA in 5 μL of Lipofectamine 2000 (Invitrogen, USA) and 50 pmol of the indicated siRNA according to the manufacturer’s instructions. Forty-eight hours after transfection, cells were harvested for further treatment.
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from THP-1 cells treated with TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instruction. Total RNA was reversely transcribed into complementary DNA using PrimeScript RT reagent kit (Takara, Japan). PCR reactions were performed on ThermoCycler (Biometra, Germany) using Premix Ex Taq kit (Takara, Japan). Primers used for amplifications were as follows: G3PDH, 5'-ACCACAGTCCATGCCATCAC-3' (forward) and 5'-TCCACCACCCTGTTGCTGTA-3' (reverse); β-actin, 5'-CATGTACGTTGCTATCCAGGC-3' (forward) and 5'-CTCCTTAATGTCACGCACGAT-3' (reverse); AIRE, 5'-CCAGGCTCTCAACTGAAGGC-3' (forward) and 5'-CGAACTTGCTGGGAGTGTAGAA-3' (reverse).
Western blotting
After treatment, the cells were washed with phosphate buffered saline (PBS) and harvested in mammalian protein extraction reagent (M-PER) (Thermo Scientific, USA) containing protease inhibitor cocktail (Roche, Switzerland). Protein concentration was measured using the Bradford Protein Assay Kit (TIANGEN, China). Thirty micrograms of total protein were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5.0% nonfat milk in Tris buffered saline, with Tween® 20 (TBST) and probed respectively with 1∶1000 dilution of anti-LC3B (Sigma, USA), 1∶200 dilution of anti-AIRE (Santa Cruz, USA), 1∶1000 dilution of anti-p62/SQSTM1 (Sigma, USA), 1∶200 dilution of anti-actin (Sigma, USA) and 1∶5000 dilution of horseradish peroxidase (HRP) conjugated anti-rabbit IgG (Thermo Scientific, USA) antibodies. Bands were visualized with BeyoECL Plus Kit (Beyotime, China).
Immunofluorescence
After transfection and/or starvation, cells were fixed with 4% (weight/volume) paraformaldehyde for 10 min and were made permeable with 0.1% Triton X-100 for 5 min. Then cells were blocked with 0.5% goat serum and 1% bovine serum albumin (Amersham Biosciences, USA) in PBS at room temperature for 1 h followed by incubation with primary antibody against LC3B at 4°C overnight. Cells were then probed with TRITC conjugated ImmunoPure goat anti-rabbit IgG (Thermo Scientific) for 1 h at room temperature and were mounted with aqueous mounting medium containing DAPI (Roche, USA). Fluorescent signals were analyzed using a fluorescence microscope (Olympus IX-71, Japan). Macroautophagy was quantified by counting the number of light chain 3 (LC3) positive dots or vacuoles on a per cell basis.
Statistical analysis
Statistical significance between groups was evaluated with analysis of variance (ANOVA) and paired t test. P values less than 0.05 were considered statistically significant. Results were presented as .
Results
AIRE was expressed in THP-1 human monocytes
Because AIRE had been shown to be expressed in human blood monocytes [
3], we examined expression of AIRE in THP-1 human monocytes by using RT-PCR. As shown in Fig. 1a, a specific transcription of AIRE was detected in blood monocytes and THP-1 cells. When we tested the expression of AIRE protein by Western blotting analysis, only trace amounts of protein could be detected in blood monocytes and THP-1 cells (Fig. 1b).
Overexpression of AIRE promoted the process of autophagy
To address the function of AIRE in monocytes, THP-1 cells were transiently transfected with an expression vector which contained full length human AIRE cDNA. Forty-eight hours after transfection, we observed the predominant location of AIRE in the nuclear dots by using fluorescence microscopy (Fig. 1c), as previously described [
7]. The expression of AIRE was confirmed by RT-PCR (Fig. 1d) and Western blotting (Fig. 1b).
As LC3 is the most widely used protein marker for detecting autophagic organelles, and the amount of LC3-II correlates with the extent of autophagosome formation, immunoblotting of LC3-II can be used to determine autophagy induction [
8]. In contrast to the empty vector transfected cells, the conversion of LC3-I to LC3-II was increased in AIRE transfected cells (Fig. 2a). To visualize autophagosome formation and indicate the extent of autophagy within cells, we measured formation and localization of LC3 positive autophagosomes by indirect immunofluorescence staining (Fig. 2b). Compared with cells from simulated transfection, the amount of LC3 positive vesicles per cell was significantly increased in AIRE transfected cells (
P<0.05) (Fig. 2c). However, there was no difference in total LC3 immunofluorescence staining between AIRE transfected cells and positive control (starved cells) (Fig. 2b and 2c).
To confirm that the observed LC3 positive vesicles indeed resulted from autophagy induced by AIRE, we treated AIRE transfected cells with wortmannin, which inhibits the activity of class III phosphatidylinositol 3-kinase required for autophagosome formation [
9]. In the presence of wortmannin, the decreased level of LC3-II from AIRE transfected cells and positive control was detected (Fig. 2a), demonstrating that AIRE induced autophagy was responsible for the formation of LC3-positive vesicles.
Silencing of AIRE downregulated autophagy
To further determine the effect of AIRE on cell autophagy, siRNA was used to silence AIRE expression in THP-1 cells by cotransfecting plasmid DNA and siRNA. The effectiveness of silencing was confirmed by RT-PCR, fluorescence microscopy and Western blotting. First, although AIRE expression was detectable with RT-PCR and Western blotting assays, signals were very low in AIRE siRNA transfected cells compared with those in control siRNA transfected cells (Fig. 1d and 1b). In addition, AIRE siRNA also strongly inhibited AIRE-GFP fusion protein expression (Fig. 1c).
In contrast to overexpression of AIRE, knockdown of AIRE by specific siRNA impaired starvation induced autophagy. Immunoblot analysis demonstrated that AIRE siRNA reduced the upregulation of LC3-II level induced by starvation (Fig. 3a). Moreover, the production of extensive autophagic vacuoles induced by starvation was significantly inhibited (P<0.05) (Fig. 3b and 3c).
Degradation of protein p62/SQSTM1 was not regulated by AIRE
The protein p62/SQSTM1 is known to bind to both ubiquitin and LC3 and is degraded along with LC3-II in autolysosomes [
10]. The level of p62 increases when autophagy is inhibited and decreases as autophagy is induced [
11]. Therefore, the level of p62 can be used as an alternative or complementary method for the detection of autophagy by immunoblot analysis. In this report, we did not find significant difference in p62 protein expression between control and AIRE transfected cells (Fig. 4).
Discussion
Recently, a report [
12] showed that AIRE
+ mTEC subpopulation has a high turnover. AIRE does not directly affect the division of mTEC but, rather, has an effect on their apoptosis, suggesting AIRE is a proapoptotic factor. Since autophagic cell death and apoptosis, which are the two different forms of programmed cell death, share many common regulators [
13], it is worth hypothesizing that AIRE is included in these factors and may regulate autophagy. Furthermore, it was reported [
14] that AIRE function as E3 ubiquitin ligase, a feature similar to the Atg5-Atg12/Atg16L1 complex, which is crucial for Atg8/LC3 lipidation in autophagy [
15], and thus we presume AIRE might equally have a role in modulating autophagy. Actually, in our experiment, by using Western blotting we confirmed that the level of LC3-II was elevated by overexpressing AIRE in THP-1 cells, while wortmannin (an autophagy inhibitor) decreased the conversion of LC3-I to LC3-II. After silencing AIRE, the opposite changes in LC3-II amounts appeared during starvation. In addition, it was further supported by the indirect immunofluorescence staining analysis in which the expression level of LC3 positive vesicles was AIRE dependent. It suggested a strong likelihood that AIRE should change the function of monocytes by regulating autophagy under the conditions of stress. However, whether AIRE promotes autophagy as E3 ubiquitin ligase deserves a more detailed examination. Moreover, the direct binding of p62/SQSTM1 to Atg8/LC3 has been documented in the degradation of ubiquitinated protein aggregates by autophagy [
10]. In addition, the overexpression of p62/SQSTM1 is observed in the differentiation of myelogenous cells followed by autophagy [
16]. It is identical to our findings in PMA treated THP-1 cells, which indicated p62/SQSTM1 might be a regulated target in the process of autophagy. However, the expression of endogenous p62/SQSTM1 protein remained unchanged when AIRE was introduced into or knocked out from THP-1 cells. Thus, it should be further explored whether p62/SQSTM1 protein is involved in THP-1 monocyte signaling pathways engaged in autophagy mediated by AIRE.
Recently, increasing attention has been paid to the chronic mucocutaneous candidiasis in APECED. Although there is some disagreement, it is definite that no difference between APECED patients and healthy subjects is observed in the expression of PRR [
17] (recognition of microorganism) and the phagocyte function [
18]. Considering the importance of autophagy in antimicrobial defense, the defect of AIRE resulting in impaired autophagy may contribute to perseverative fungal infection. Certainly, this alternative explanation needs further clinical and experimental studies to confirm.
To conclude, it is first reported here that AIRE is involved in regulating the autophagy in THP-1 human monocytes, which will give more clues for a more advanced exploration of the role of AIRE in immune regulation. However, the exact molecular mechanism underlying how AIRE modulates autophagy remains unknown, and hence further studies should be conducted.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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