Autophagy In Peripheral Blood Mononuclear Cells Research Articles

Autophagy Activation in Peripheral Blood Mononuclear Cells of Peritoneal Dialysis Patients

The complete systemic deregulated biological network in patients on peritoneal dialysis (PD) is still only partially defined. High-throughput/omics techniques may offer the possibility to analyze the main biological fingerprints associated with this clinical condition. We applied an innovative bioinformatic analysis of gene expression microarray data (mainly based on support vector machine (SVM) learning) to compare the transcriptomic profile of peripheral blood mononuclear cells (PBMCs) of healthy subjects (HS), chronic kidney disease (CKD) patients, and patients on PD divided into a microarray group (5 HS, 9 CKD, and 10 PD) and a validation group (10 HS, 15 CKD, and 15 PD). Classical well-standardized biomolecular approaches (western blotting and flow cytometry) were used to validate the transcriptomic results. Bioinformatics revealed a distinctive PBMC transcriptomic profiling for PD versus CKD and HS (n= 419 genes). Transcripts encoding for key elements of the autophagic pathway were significantly upregulated in PD, and the autophagy related 5 (ATG5) reached the top level of discrimination [-Log10 P-value= 11.3, variable importance in projection (VIP) score= 4.8, SVM rank:1]. Protein levels of ATG5 and microtubule associated protein 1 light chain 3 beta (LC3B), an important constituent of the autophagosome, validated microarray results. In addition, the incubation of PBMCs of HS with serum of patients on PD upregulated both proteins. Autophagy in PBMCs from patients on PD was attenuated by N-acetyl-cysteine or Resatorvid treatment. Our data demonstrated, for the first time, that the autophagy pathway is activated in immune-cells of patients on PD, and this may represent a novel therapeutic target.

Autophagic response to exercise in peripheral blood mononuclear cells from young men is intensity-dependent and is altered by exposure to environmental heat.

Autophagy is essential to maintaining cellular homeostasis in all eukaryotic cells and to tolerance of acute stressors such as starvation, heat, and recovery after exercise. Limited information exists regarding the exercise intensity-dependent autophagic response in humans, and it is unknown how environmental heat stress may modulate this response. Therefore, we evaluated autophagy and accompanying pathways of cellular stress [the heat-shock response (HSR), apoptosis, and acute inflammation] in peripheral blood mononuclear cells (PBMCs) from 10 young men (mean [SD]; 22 [2] years) before, immediately after and up to 6-h postexercise recovery from 30 min of low-, moderate-, and high-intensity semirecumbent cycling [40%, 55%, and 70% of maximal oxygen consumption (V̇o2max), respectively] in a temperate environment (25°C) and at 70% of V̇o2max in a hot environment (40°C). Changes in protein content were analyzed via Western blot. Each increase in exercise intensity was associated with elevations in mean body temperature. LC3-II increased after moderate-intensity exercise, with further increases after high-intensity exercise (P < 0.05). However, an increase in beclin-2 and ULK1, with a decrease in p62 was only observed after high-intensity exercise, which was paralleled by elevated TNF-α and cleaved-caspase-3, with the HSR peaking at 6 h after exercise (P < 0.05). When exercise was performed in the heat, greater LC3-II and cleaved-caspase-3 accumulation were observed; however, beclin-2 declined in recovery (P < 0.05). Therefore, our findings indicate that autophagy in PBMCs during exercise may be associated with greater heat strain exhibited during increasing exercise intensities, which is modulated by exposure to heat.

Intravenous immunoglobulin mediates anti-inflammatory effects in peripheral blood mononuclear cells by inducing autophagy

Autophagy plays an important role in the regulation of autoimmune and autoinflammatory responses of the immune cells. Defective autophagy process is associated with various autoimmune and inflammatory diseases. Moreover, in many of these diseases, the therapeutic use of normal immunoglobulin G or intravenous immunoglobulin (IVIG), a pooled normal IgG preparation, is well documented. Therefore, we explored if IVIG immunotherapy exerts therapeutic benefits via induction of autophagy in the immune cells. Here we show that IVIG induces autophagy in peripheral blood mononuclear cells (PBMCs). Further dissection of this process revealed that IVIG-induced autophagy is restricted to inflammatory cells like monocytes, dendritic cells, and M1 macrophages but not in cells associated with Th2 immune response like M2 macrophages. IVIG induces autophagy by activating AMP-dependent protein kinase, beclin-1, class III phosphoinositide 3-kinase and p38 mitogen-activated protein kinase and by inhibiting mammalian target of rapamycin. Mechanistically, IVIG-induced autophagy is F(ab′)2-dependent but sialylation independent, and requires endocytosis of IgG by innate cells. Inhibition of autophagy compromised the ability of IVIG to suppress the inflammatory cytokines in innate immune cells. Moreover, IVIG therapy in inflammatory myopathies such as dermatomyositis, antisynthetase syndrome and immune-mediated necrotizing myopathy induced autophagy in PBMCs and reduced inflammatory cytokines in the circulation, thus validating the translational importance of these results. Our data provide insight on how circulating normal immunoglobulins maintain immune homeostasis and explain in part the mechanism by which IVIG therapy benefits patients with autoimmune and inflammatory diseases.

Expression of autophagy-modulating genes in peripheral blood mononuclear cells from familial clustering patients with chronic hepatitis B virus infection.

We previously found that genetic factors are associated with a familial predisposition for developing liver cirrhosis and hepatocellular carcinoma during chronic hepatitis B virus (HBV) infection. Autophagy has been shown to play a role in HBV replication and the course of disease. More than 190 host genes have been identified that modify the process of autophagy, but which of these genes are involved in chronicity of HBV infection and how this occurs remains unclear. Chronic hepatitis B (CHB) patients were recruited to investigate the expression of autophagy-modulating genes in peripheral blood mononuclear cells (PBMCs). mRNA prepared from PBMCs from members of two families with clustering HBV infection, including 11 CHB patients and nine healthy spouses, was hybridized to high-density oligonucleotide arrays. Immunoblot analysis was used to determine the level of autophagy. Of the 192 autophagy-modulating genes, 18 were found to be differently expressed. Of these, 11 displayed decreased expression in CHB patients, while seven displayed increased expression compared to those in healthy controls. Functional analysis showed that these genes are closely involved in initiation, nucleation, elongation of phagophores, formation of autophagosomes, transportation to lysosomes, and the process of degradation. Western blot analysis revealed inhibited autophagy in PBMCs based on decreased lipidation of LC3II. A differential expression profile of autophagy-modulating genes was observed, and decreased autophagy in PBMCs could be closely associated with chronicity of HBV infection, suggesting a novel strategy for the treatment of patients with chronic HBV infection.

Resveratrol ameliorates gouty inflammation via upregulation of sirtuin 1 to promote autophagy in gout patients.

Resveratrol exerts an anti-inflammatory effect on collagen-induced arthritis and osteoarthritis in rats via activation of sirtuin 1 (SIRT1). Autophagy can be induced by resveratrol and leads to amelioration of interleukin-1 beta (IL-1β) release in vitro. We aimed to determine the anti-inflammatory mechanisms of resveratrol in patients with gout. Blood samples were obtained from patients with acute gout, intercritical gout (IG) and healthy controls (HC). The mRNA and protein levels of SIRT1 and nuclear factor-kappa B (NF-kB) p65 were determined in peripheral blood mononuclear cells (PBMCs) lysate from these patients. In the in vitro experiment, SIRT1, autophagy-related genes (beclin-1 and microtubule-associated protein 1 light-chain 3) and key genes involved in the gouty inflammatory pathway, including NF-κB p65, NLR family pyrin domain containing 3 (NLRP3), caspase-1 and IL-1β, were determined in PBMCs lysate or plasma from IG patients exposed to monosodium urate (MSU) crystals with or without resveratrol. The mRNA and protein levels of SIRT1 were downregulated in PBMCs from gout patients in comparison with HC. In the in vitro experiment, the protein levels of SIRT1 were downregulated in PBMCs from IG patients exposed to MSU crystals and were restored by resveratrol in a dose-dependent manner. Furthermore, high doses of resveratrol ameliorated the release of the inflammatory cytokine IL-1β. In addition, the mRNA levels of NLRP3 and NF-κB p65 were regulated by resveratrol, but caspase-1 and IL-1β were not. Furthermore, resveratrol promoted MSU-induced autophagy in PBMCs from patients with gout. These findings suggest that resveratrol ameliorates gouty inflammation via upregulation of SIRT1 to promote autophagy in patients with gout.

Pregnancy History Influences the Level of Autophagy in Peripheral Blood Mononuclear Cells From Pregnant Women.

Maternal immune responses are altered during pregnancy and differ between nulliparous and multiparous women. The influence of a prior gestation on autophagy in peripheral blood mononuclear cells (PBMCs) from pregnant women has not been determined and is the subject of this investigation. Peripheral blood mononuclear cells were isolated from 212 pregnant women and immediately lysed in the presence of protease inhibitors, and the extent of autophagy was determined by quantitation of the concentration of p62 (sequestosome-1) in the lysates by enzyme-linked immunosorbent assay (ELISA). In PBMCs, the p62 level is inversely related to the extent of autophagy. The level of the stress-inducible 70-kDa heat shock protein (hsp70), an inhibitor of autophagy, was also measured in the lysates by ELISA. Data were analyzed by the Spearman rank correlation, Mann-Whitney U test, or Kruskal-Wallis test, as appropriate. The p62 concentration in PBMCs increased (autophagy decreased) with the number of previous live ( P = .0322), preterm ( P = .0143), or term ( P = .0418) deliveries. The p62 level was lower (autophagy higher) in women with a prior spontaneous pregnancy loss but no deliveries as compared to women with their first conception ( P = .0087). The intracellular hsp70 concentration correlated with the p62 level ( P < .0001). Multiparity is associated with a reduced level of autophagy in PBMCs. Dysregulated autophagy might be one mechanism leading to spontaneous abortion in nulliparous women.

Evidence for the link between defective autophagy and inflammation in peripheral blood mononuclear cells of type 2 diabetic patients.

Autophagy was shown to modulate inflammation in immune cells. This study was designed to evaluate the association between autophagy and inflammation in peripheral blood mononuclear cells (PBMCs) of type 2 diabetic (T2D) and non-diabetic (ND) subjects. The autophagy markers were measured by real-time PCR and western blot. The gene expression of pro- and anti-inflammatory cytokines was assessed by real-time PCR. Reduced transcription of BECN1 and LAMP2 and unchanged expression of MAP1LC3B and ATG5 were observed in PBMCs of T2D patients. Decreased LC3B-II and increased p62/SQSTM1 levels were found in PBMCs of diabetic patients. The p-mTOR level was higher in PBMCs of diabetic patients. An increase in both IL-1β and TNF-α gene expression, along with a decrease in the expression of IL-10, was observed in PBMCs of T2D patients. TNF-α mRNA expression was inversely correlated with the mRNA expression of BECN1 and LAMP2. TNF-α and IL-1β expression were negatively correlated with the protein levels of LC3B-II. TNF-α and IL-1β expression had also a positive correlation with protein level of p62. IL-10 mRNA expression was positively correlated with the mRNA expression of BECN1 and LAMP2 and protein levels of LC3B-II and negatively correlated with protein level of p62. In addition, p-mTOR level was positively correlated with IL-1β and TNF-α mRNA expression. The results revealed a reduced autophagy in PBMCs of T2D patients that is liked with an enhanced inflammation. The suppression of autophagy in PBMCs of diabetic patients may be associated with the activation of the mTOR signaling.

Correlation between autophagy levels in peripheral blood mononuclear cells and clinical parameters in patients with chronic obstructive pulmonary disease.

Autophagy serves a role in the pathogenesis of chronic inflammatory diseases. The aim of the present study was to compare the autophagy levels in the peripheral blood mononuclear cells (PBMCs) of patients with chronic obstructive pulmonary disease (COPD) and healthy individuals and to assess the association between autophagy and the clinical parameters of COPD. Samples of peripheral blood from 20patients with stable COPD and 20 healthy controls were collected. PBMCs were harvested using Ficoll density gradient centrifugation. Levels of the autophagy‑associated proteins ubiquitin‑binding protein p62 (p62), microtubule‑associated proteins 1A/1B light chain 3A (LC3I/II) and beclin‑1 in PBMCs were detected by western blotting. Enzyme‑linked immunosorbent assay kits were used to detect the serum concentrations of interleukin (IL)‑6, IL‑8 and tumor necrosis factor (TNF)‑α. Associations between the levels of autophagy and forced expiratory volume in 1sec % predicted (FEV1%) and pro‑inflammatory factors were assessed. Western blotting demonstrated that the protein expression of p62 was decreased, but LC3II/I and beclin‑1 levels increased in patients with COPD compared with healthy controls. Serum levels of IL‑6, IL‑8 and TNF‑α were increased in patients with COPD. The extent of PBMC autophagy was negatively correlated with FEV1% predicted, but positively correlated with levels of pro‑inflammatory cytokines. The levels of autophagy in PBMCs in patients with COPD were increased and were negatively correlated with FEV1% predicted and positively correlated with circulating levels of pro‑inflammatory cytokines. Autophagy may serve a role as a biomarker of the severity of COPD or as a therapeutic target for treatment of COPD.

Expression analysis of protein homeostasis pathways in the peripheral blood mononuclear cells of sporadic amyotrophic lateral sclerosis patients

Misfolded protein aggregates are the hallmark of Amyotrophic Lateral Sclerosis (ALS) which suggests involvement of protein homeostasis pathways in etiology of ALS. However, status of protein homeostasis in peripheral blood of ALS is not well established. We analyzed expression levels of key genes of proteostasis pathways in peripheral blood mononuclear cells (PBMCs) of sporadic ALS (sALS) patients and healthy controls. Increased protein carbonylation was observed in patients reflecting oxidative damage in PBMCs. We observed increased transcript and protein levels of GRP78 suggesting Endoplasmic reticulum (ER) insult to cells. Further, significant upregulation of spliced XBP1 and two stress sensors: IRE1α/ERN1 and ATF6 indicated induction of unfolded protein response (UPR). Genes involved in autophagosome initiation (ULK1, ULK2, ATG13); nucleation and elongation (BECLIN1, ATG7, ATG16L1, ATG5, ATG10) and vesicular trafficking genes were significantly increased in patients. Increased lipidation of LC3 validated induction of autophagy. Accumulation of low molecular weight ubiquitinated proteins in patients suggested deregulation of proteasome (UPS) pathway. In addition, cytosolic chaperones (HSP70 and HSP27) and HSF1 were elevated in patients. Increased TDP43 indicated role of TDP43 in disease pathology. Our findings suggest that there is oxidative insult and upregulation of UPR, vesicular trafficking and autophagy in PBMCs of sALS patients.

Palmitate-induced impairment of autophagy turnover leads to increased apoptosis and inflammation in peripheral blood mononuclear cells

Previous works have linked high concentrations of palmitate to cellular toxicity by autophagy modulation. However, the ways in which palmitate regulates inflammation and apoptosis in peripheral blood mononuclear cells (PBMCs), has not been well characterized. In the present study, we therefore aimed to investigate the role autophagy in inflammatory responses and apoptotic death of PBMCs treated with palmitate. 0.5mM palmitate increased the level of LC3-II at 6h, peaked at 12h and then decreased at 24h. The protein level of p62 was significantly increased at 6h and 12h, suggesting an impairment of autophagic flux in palmitate-treated PBMCs. Inhibiting autophagy with chloroquine (CQ) and 3-Methyladenine (3-MA) significantly augmented palmitate-induced PBMCs apoptotic death as demonstrated by increased cleaved PARP level and increased the percentage of apoptotic (YO-PRO-1 positive and PI negative) cells. Furthermore, CQ pretreatment exacerbated palmitate-induced TNF-α and IL-6 mRNA expression in PBMCs. Moreover, induction of autophagy by pretreatment of PBMCs with rapamycin resulted in a distinct increase of palmitate-induced apoptosis. The induction of autophagy also led to a further increase in palmitate-induced expression of TNF-α and IL-6. These results indicate that the excess palmitate could impair autophagy, hence contributing to palmitate-induced-inflammation and apoptosis in PBMCs. Therefore, dysregulated autophagy in PBMCs may provide a novel mechanism that connects diet-induced obesity to low grade inflammation in patients with type 2 diabetes.

Inflammatory Stress on Autophagy in Peripheral Blood Mononuclear Cells from Patients with Alzheimer's Disease during 24 Months of Follow-Up.

Recent findings indicate that microglia in Alzheimer’s disease (AD) is senescent whereas peripheral blood mononuclear cells (PBMCs) could infiltrate the brain to phagocyte amyloid deposits. However, the molecular mechanisms involved in the amyloid peptide clearance remain unknown. Autophagy is a physiological degradation of proteins and organelles and can be controlled by pro-inflammatory cytokines. The purpose of this study was to evaluate the impact of inflammation on autophagy in PBMCs from AD patients at baseline, 12 and 24 months of follow-up. Furthermore, PBMCs from healthy patients were also included and treated with 20 μM amyloid peptide 1–42 to mimic AD environment. For each patient, PBMCs were stimulated with the mitogenic factor, phytohaemagglutin (PHA), and treated with either 1 μM C16 as an anti-inflammatory drug or its vehicle. Autophagic markers (Beclin-1, p62/sequestosome 1 and microtubule-associated protein-light chain 3: LC3) were quantified by western blot and cytokines (Interleukin (IL)-1β, Tumor necrosis Factor (TNF)-α and IL-6) by Luminex X-MAP® technology. Beclin-1 and TNF-α levels were inversely correlated in AD PBMCs at 12 months post-inclusion. In addition, Beclin-1 and p62 increased in the low inflammatory environment induced by C16. Only LC3-I levels were inversely correlated with cognitive decline at baseline. For the first time, this study describes longitudinal changes in autophagic markers in PBMCs of AD patients under an inflammatory environment. Inflammation would induce autophagy in the PBMCs of AD patients while an anti-inflammatory environment could inhibit their autophagic response. However, this positive response could be altered in a highly aggressive environment.

The expression and significance of microtubule-associated protein 1 light chain 3-related protein-1 (LC3) mRNA in peripheral blood mononuclear cells in patients with systemic lupus erythematosus

Increasing evidence supports the involvement of autophagy in the etiopathology of autoimmune diseases. Systemic lupus erythematosus (SLE) is a potentially fatal autoimmune disease characterized by production of multiple autoantibodies through poorly understood mechanism. In order to explore the role of autophagy in the development of SLE, the expression of autophagy related gene microtubule-associated protein 1 light chain 3 (MAPLC3) in peripheral blood mononuclear cells (PBMCs) was measured in patients with SLE. The mRNA levels of LC3 in PBMCs from 56 SLE patients and 45 healthy individuals were detected by real-time quantitative polymerase chain reaction (qPCR) technique. Autophagy in PBMCs was also determined by flow cytometry (FACs) in 20 SLE patients and 15 healthy controls. The correlation between LC3 mRNA expression and disease activity of SLE (SLEDAI) was then analyzed. The mRNA level of LC3 (RQ) in SLE patients was obviously downregulated compared with that in healthy population (1.30 ± 0.10 vs 1.35 ± 0.09; P = 0.029), paralleled with the decreased autophagy rate detected by flow cytometry in PBMCs of SLE patients [(2.21 ± 1.07) % vs (9.91 ± 4.01) %;P = 0.047]. Moreover, LC3 mRNA expression level was negatively correlated with SLEDAI (r = -0.337, P = 0.023). However, when the clinical features of 27 SLE patients with decreased LC3 mRNA expression (RQ<1.351) were compared with those of other 29 SLE patients with normal or high LC3 mRNA expression (RQ>1.351), increasing rates of arthritis, serositis, hematological abnormalities were noted in patients with decreased LC3 mRNA expression yet without statistically significance. However, there was a significant difference between two groups in the incidence of renal involvement (P = 0.028). The impaired autophagy due to downregulated LC3 mRNA level in SLE patients indicates that autophagy plays a role in mediating the occurrence and development of SLE.

Induction of the 70 kDa heat shock protein stress response inhibits autophagy: possible consequences for pregnancy outcome

Aim: The induction of heat shock protein synthesis and activation of autophagy are intracellular processes stimulated under adverse conditions. We evaluated the relationship between intracellular concentrations of the inducible 70 kDa heat shock protein (hsp70) and autophagy induction in human peripheral blood mononuclear cells (PBMCs) following exposure to sera from pregnant and non-pregnant women.Methods: Autophagy was induced in PBMCs by incubation for 48 h with sera from 42 pregnant women at mid-gestation and 45 non-pregnant women. Intracellular concentrations of hsp70 and p62 were measured by ELISA. p62 is a cytoplasmic protein that is consumed during autophagy induction. Its concentration in the cytoplasm is inversely proportional to the extent of autophagy induction (high p62 = low autophagy).Results: The p62 concentration was highly correlated with the hsp70 level utilizing sera from both pregnant (Spearman r = 0.4731, p = 0.0015) and non-pregnant (Spearman r = 0.6214, p < 0.0001) women. Median p62 (7.4 ng/ml versus 2.7 ng/ml, p < 0.0001) and hsp70 (7.0 ng/ml versus 3.5 ng/ml, p = 0.0022) levels were higher when PBMCS were incubated with sera from pregnant women.Conclusion: The extent of autophagy in PBMCs is inversely proportional to the intracellular hsp70 concentration and sera from pregnant women induces hsp70 and inhibits autophagy to a greater extent than does sera from non-pregnant women. A stress response that induces hsp70 has the potential to interfere with autophagy-related events.