A novel method for autophagy detection in primary cells: impaired levels of macroautophagy in immunosenescent T cells

Autophagy is a conserved constitutive cellular process, responsible for the degradation of dysfunctional proteins and organelles. Autophagy plays a role in many diseases such as neurodegeneration and cancer; however, to date, conventional autophagy detection techniques are not suitable for clinical samples. We have developed a high throughput, statistically robust technique that quantitates autophagy in primary human leukocytes using the Image stream, an imaging flow cytometer. We validate this method on cell lines and primary cells knocked down for essential autophagy genes. Also, using this method we show that T cells have higher autophagic activity than B cells. Furthermore our results indicate that healthy primary senescent CD8(+) T cells have decreased autophagic levels correlating with increased DNA damage, which may explain features of the senescent immune system and its declining function with age. This technique will allow us, for the first time, to measure autophagy levels in diseases with a known link to autophagy, while also determining the contribution of autophagy to the efficacy of drugs.     

A novel image-based cytometry method for autophagy detection in living cells

Autophagy is an important cellular catabolic process that plays a variety of important roles, including maintenance of the amino acid pool during starvation, recycling of damaged proteins and organelles, and clearance of intracellular microbes. Currently employed autophagy detection methods include fluorescence microscopy, biochemical measurement, SDS-PAGE and western blotting, but they are time consuming, labor intensive, and require much experience for accurate interpretation. More recently, development of novel fluorescent probes have allowed the investigation of autophagy via standard flow cytometry. However, flow cytometers remain relatively expensive and require a considerable amount of maintenance. Previously, image-based cytometry has been shown to perform automated fluorescence-based cellular analysis comparable to flow cytometry. In this study, we developed a novel method using the Cellometer image-based cytometer in combination with Cyto-ID^® Green dye for autophagy detection in live cells. The method is compared with flow cytometry by measuring macroautophagy in nutrient-starved Jurkat cells. Results demonstrate similar trends of autophagic response, but different magnitude of fluorescence signal increases, which may arise from different analysis approaches characteristic of the two instrument platforms. The possibility of using this method for drug discovery applications is also demonstrated through the measurement of dose-response kinetics upon induction of autophagy with rapamycin and tamoxifen. The described image-based cytometry/fluorescent dye method should serve as a useful addition to the current arsenal of techniques available in support of autophagy-based drug discovery relating to various pathological disorders.     

A novel image-based cytometry method for autophagy detection in living cells

Autophagy is an important cellular catabolic process that plays a variety of important roles, including maintenance of the amino acid pool during starvation, recycling of damaged proteins and organelles, and clearance of intracellular microbes. Currently employed autophagy detection methods include fluorescence microscopy, biochemical measurement, SDS-PAGE and western blotting, but they are time consuming, labor intensive, and require much experience for accurate interpretation. More recently, development of novel fluorescent probes have allowed the investigation of autophagy via standard flow cytometry. However, flow cytometers remain relatively expensive and require a considerable amount of maintenance. Previously, image-based cytometry has been shown to perform automated fluorescence-based cellular analysis comparable to flow cytometry. In this study, we developed a novel method using the Cellometer image-based cytometer in combination with Cyto-ID^® Green dye for autophagy detection in live cells. The method is compared with flow cytometry by measuring macroautophagy in nutrient-starved Jurkat cells. Results demonstrate similar trends of autophagic response, but different magnitude of fluorescence signal increases, which may arise from different analysis approaches characteristic of the two instrument platforms. The possibility of using this method for drug discovery applications is also demonstrated through the measurement of dose-response kinetics upon induction of autophagy with rapamycin and tamoxifen. The described image-based cytometry/fluorescent dye method should serve as a useful addition to the current arsenal of techniques available in support of autophagy-based drug discovery relating to various pathological disorders.     

In aged primary T cells,mitochondrial stress contributes to telomere attrition measured by a novel imaging flow cytometry assay

The decline of the immune system with age known as immune senescence contributes to inefficient pathogen clearance and is a key risk factor for many aged‐related diseases. However, reversing or halting immune aging requires more knowledge about the cell biology of senescence in immune cells. Telomere shortening, low autophagy and mitochondrial dysfunction have been shown to underpin cell senescence. While autophagy has been found to control mitochondrial damage, no link has been made to telomere attrition. In contrast, mitochondrial stress can contribute to telomere attrition and vice versa. Whereas this link has been investigated in fibroblasts or cell lines, it is unclear whether this link exists in primary cells such as human lymphocytes and whether autophagy contributes to it. As traditional methods for measuring telomere length are low throughput or unsuitable for the analysis of cell subtypes within a mixed population of primary cells, we have developed a novel sensitive flow‐FISH assay using the imaging flow cytometer. Using this assay, we show a correlation between age and increased mitochondrial reactive oxygen species in CD8⁺ T‐cell subsets, but not with autophagy. Telomere shortening within the CD8⁺ subset could be prevented in vitro by treatment with a ROS scavenger. Our novel assay is a sensitive assay to measure relative telomere length in primary cells and has revealed ROS as a contributing factor to the decline in telomere length.     

Differential use of autophagy by primary dendritic cells specialized in cross-presentation

Antigen-presenting cells survey their environment and present captured antigens bound to major histocompatibility complex (MHC) molecules. Formation of MHC-antigen complexes occurs in specialized compartments where multiple protein trafficking routes, still incompletely understood, converge. Autophagy is a route that enables the presentation of cytosolic antigen by MHC class II molecules. Some reports also implicate autophagy in the presentation of extracellular, endocytosed antigen by MHC class I molecules, a pathway termed “cross-presentation.” The role of autophagy in cross-presentation is controversial. This may be due to studies using different types of antigen presenting cells for which the use of autophagy is not well defined. Here we report that active use of autophagy is evident only in DC subtypes specialized in cross-presentation. However, the contribution of autophagy to cross-presentation varied depending on the form of antigen: it was negligible in the case of cell-associated antigen or antigen delivered via receptor-mediated endocytosis, but more prominent when the antigen was a soluble protein. These findings highlight the differential use of autophagy and its machinery by primary cells equipped with specific immune function, and prompt careful reassessment of the participation of this endocytic pathway in antigen cross-presentation.     

Regulation of autophagy as a therapy for immunosenescence-driven cancer and neurodegenerative diseases: The role of exercise

Aging is one of the risk factors for the development of low-grade inflammation morbidities, such as several types of cancer and neurodegenerative diseases, due to changes in the metabolism, hormonal secretion, and immunosenescence. The senescence of the immune system leads to improper control of infections and tissue damage increasing age-related diseases. One of the mechanisms that maintain cellular homeostasis is autophagy, a cell-survival mechanism, and it has been proposed as one of the most powerful antiaging therapies. Regular exercise can reestablish autophagy, probably through AMP-activated protein kinase activation, and help in reducing the age-related senescence diseases. Therefore, in this study, we discuss the effects of exercise training in immunosenescence and autophagy, preventing the two main age-related disease, cancer and neurodegeneration.     

A new genetic model for calcium induced autophagy and ER-stress in Drosophila photoreceptor cells

Cytoplasmic Ca²⁺ overload is known to trigger autophagy and ER-stress. Furthermore, ER-stress and autophagy are commonly associated with degenerative pathologies, but their role in disease progression is still a matter of debate, in part, owing to limitations of existing animal model systems. The Drosophila eye is a widely used model system for studying neurodegenerative pathologies. Recently, we characterized the Drosophila protein, Calphotin, as a cytosolic immobile Ca²⁺ buffer, which participates in Ca²⁺ homeostasis in Drosophila photoreceptor cells. Exposure of calphotin hypomorph flies to continuous illumination, which induces Ca²⁺ influx into photoreceptor cells, resulted in severe Ca²⁺-dependent degeneration. Here we show that this degeneration is autophagy and ER-stress related. Our studies thus provide a new model in which genetic manipulations trigger changes in cellular Ca²⁺ distribution. This model constitutes a framework for further investigations into the link between cytosolic Ca²⁺, ER-stress and autophagy in human disorders and diseases.     

An engineered novel lentivector specifically transducing dendritic cells and eliciting robust HBV-specific CTL response by upregulating autophagy in T cells

Dendritic cells (DCs) play a predominant role in initiating cell immune responses. Here we generated a DC-targeting lentiviral vector (LVDC-UbHBcAg-LIGHT) and evaluated its capacity to elicit HBV-specific cytotoxic T lymphocyte (CTL) responses. DC-SIGN-mediated specific transduction using this construct was confirmed in DC-SIGN-expressing 293T cells and ex vivo-cultured bone marrow cells. LVDC-UbHBcAg-LIGHT-loaded DCs were highly effective in inducing HBV-specific CTLs. Mechanistic studies demonstrated autophagy blocking led to a significant increase in apoptosis and obvious inhibition of CD8 + T cells entry into S-phase, correspondingly attenuated LVDC-UbHBcAg-LIGHT-loaded DC-induced T cell responses. This observation was supported by accumulation of pro-apoptotic proteins and the main negative cell cycle regulator-CDKN1B that otherwise would be degraded in activated T cells where autophagy preferentially occured. Our findings revealed an important role of autophagy in the activation of T cells and suggested LVDC-UbHBcAg-LIGHT may potentially be used as a therapeutic strategy to combat persistent HBV infection with higher security.     

ROS-induced autophagy in cancer cells assists in evasion from determinants of immunogenic cell death

Calreticulin surface exposure (ecto-CALR), ATP secretion, maturation of dendritic cells (DCs) and stimulation of T cells are prerequisites for anticancer therapy-induced immunogenic cell death (ICD). Recent evidence suggests that chemotherapy-induced autophagy may positively regulate ICD by favoring ATP secretion. We have recently shown that reactive oxygen species (ROS)-based endoplasmic reticulum (ER) stress triggered by hypericin-mediated photodynamic therapy (Hyp-PDT) induces bona fide ICD. However, whether Hyp-PDT-induced autophagy regulates ICD was not explored. Here we showed that, in contrast to expectations, reducing autophagy (by ATG5 knockdown) in cancer cells did not alter ATP secretion after Hyp-PDT. Autophagy-attenuated cancer cells displayed enhanced ecto-CALR induction following Hyp-PDT, which strongly correlated with their inability to clear oxidatively damaged proteins. Furthermore, autophagy-attenuation in Hyp-PDT-treated cancer cells increased their ability to induce DC maturation, IL6 production and proliferation of CD4⁺ or CD8⁺ T cells, which was accompanied by IFNG production. Thus, our study unravels a role for ROS-induced autophagy in weakening functional interaction between dying cancer cells and the immune system thereby helping in evasion from ICD prerequisites or determinants.     

Ultrastructure of autophagy in plant cells

Just as with yeasts and animal cells, plant cells show several types of autophagy. Microautophagy is the uptake of cellular constituents by the vacuolar membrane. Although microautophagy seems frequent in plants it is not yet fully proven to occur. Macroautophagy occurs farther away from the vacuole. In plants it is performed by autolysosomes, which are considerably different from the autophagosomes found in yeasts and animal cells, as in plants these organelles contain hydrolases from the onset of their formation. Another type of autophagy in plant cells (called mega-autophagy or mega-autolysis) is the massive degradation of the cell at the end of one type of programmed cell death (PCD). Furthermore, evidence has been found for autophagy during degradation of specific proteins, and during the internal degeneration of chloroplasts. This paper gives a brief overview of the present knowledge on the ultrastructure of autophagic processes in plants.     

A new genetic model for calcium induced autophagy and ER-stress in Drosophila photoreceptor cells

Cytoplasmic Ca²⁺ overload is known to trigger autophagy and ER-stress. Furthermore, ER-stress and autophagy are commonly associated with degenerative pathologies, but their role in disease progression is still a matter of debate, in part, owing to limitations of existing animal model systems. The Drosophila eye is a widely used model system for studying neurodegenerative pathologies. Recently, we characterized the Drosophila protein, Calphotin, as a cytosolic immobile Ca²⁺ buffer, which participates in Ca²⁺ homeostasis in Drosophila photoreceptor cells. Exposure of calphotin hypomorph flies to continuous illumination, which induces Ca²⁺ influx into photoreceptor cells, resulted in severe Ca²⁺-dependent degeneration. Here we show that this degeneration is autophagy and ER-stress related. Our studies thus provide a new model in which genetic manipulations trigger changes in cellular Ca²⁺ distribution. This model constitutes a framework for further investigations into the link between cytosolic Ca²⁺, ER-stress and autophagy in human disorders and diseases.     

Trehalose inhibits cell proliferation and amplifies long-term temozolomide- and radiation-induced cytotoxicity in melanoma cells: A role for autophagy and premature senescence

Cutaneous melanomas frequently metastasize to the brain, with temozolomide (TMZ) plus radiotherapy (RT) offering little control of these lesions. We tested whether trehalose, a natural glucose disaccharide proved to induce autophagy, could enhance the effect of TMZ and ionizing radiation (IR). In two melanoma cell lines (A375 and SK-Mel-28), which greatly differ in chemosensitivity and radiosensitivity, trehalose significantly inhibited short-term cell proliferation and also enhanced IR-induced cytostasis. Interestingly, in TMZ-resistant SK-Mel-28 cells, trehalose was more effective than TMZ, and combined trehalose + TMZ further reduced cell proliferation. In long-term experiments, colony-forming capacity was dramatically reduced by trehalose, and even more by combined trehalose + TMZ or trehalose + IR. In resistant SK-Mel-28 cells, although growth was inhibited most with trehalose + TMZ + IR-6 Gy combined treatment, it is notable that trehalose + TMZ treatment was also very effective. Along with a direct antiproliferative effect, two further mechanisms may explain how trehalose potentiates TMZ- and IR-induced effects: the remarkable trehalose-stimulated autophagy in A375 cells, which were sensitive to TMZ- and IR-induced apoptosis; and the notable trehalose-stimulated premature senescence in SK-Mel-28 cells, which were resistant to apoptosis and less prone to autophagy. In normal melanocytes, trehalose induced a minor autophagy and cell proliferation inhibition, without affecting cell viability; moreover, when trehalose was used in combination with TMZ, the slight TMZ-induced cytotoxicity was not significantly reinforced. Together, our results suggest that trehalose, a safe nutrient supplement able to cross the blood–brain barrier, is a promising candidate, worthy to be further explored in vivo, to augment the therapeutic efficacy of TMZ and RT in melanoma brain metastases.     

Role of autophagy in inhibiting the proliferation of A549 cells by type III interferon

Interferons (IFNs) have anti‐viral and anti‐tumour effects. Type III interferon, as a member of the recently discovered interferon family, has been proved to inhibit tumour proliferation and promote the apoptosis of various tumour cells. However, whether type III IFN could inhibit the proliferation of lung cancer was not clear. In this study, we found that interferon λ (IFN λ) could inhibit the proliferation of A549 cells and induce autophagy and apoptosis of A549 cells. IFN λ could promote the expression of autophagy gene Beclin1 and interfere the expression of autophagy gene Beclin1 with small interfering RNA, thus inhibiting the effect of type III interferon on anti‐proliferation and promoting apoptosis of lung cancer cell. These results suggested that IFN λ could inhibit the proliferation of A549 cells by activating autophagy pathway, and IFN λ might be one of the potential therapeutic drugs for lung cancer.     

Enhanced autophagy blocks angiogenesis via degradation of gastrin-releasing peptide in neuroblastoma cells

Neuroblastoma is characterized by florid vascularization leading to rapid tumor dissemination to distant organs; angiogenesis contributes to tumor progression and poor clinical outcomes. We have previously demonstrated an increased expression of gastrin-releasing peptide (GRP) and its receptor, GRPR, in neuroblastoma and that GRP activates the PI3K-AKT pathway as a proangiogenic factor during tumor progression. Interestingly, AKT activation phosphorylates MTOR, a critical negative regulator of autophagy, a cellular process involved in the degradation of key proteins. We hypothesize that inhibition of GRPR enhances autophagy-mediated degradation of GRP and subsequent inhibition of angiogenesis in neuroblastoma. Here, we demonstrated a novel phenomenon where targeting GRPR using shRNA or a specific antagonist, RC-3095, decreased GRP secretion by neuroblastoma cells and tubule formation by endothelial cells in vitro. Furthermore, shGRPR or RC-3095 treatment enhanced expression of proautophagic proteins in human neuroblastoma cell lines, BE(2)-C, and BE(2)-M17. Interestingly, rapamycin, an inhibitor of MTOR, enhanced the expression of the autophagosomal marker LC3-II and GRP was localized within LC3-II-marked autophagosomes in vitro as well as in vivo, indicating autophagy-mediated degradation of GRP. Moreover, overexpression of ATG5 or BECN1 attenuated GRP secretion and tubule formation, whereas opposite effects were observed with siRNA silencing of ATG5 and BECN1. Our data supported the role of autophagy in the degradation of GRP and subsequent inhibition of angiogenesis. Therefore, activation of autophagy may lead to novel antivascular therapeutic strategies in the treatment of highly vascular neuroblastomas.     

Effect of sodium butyrate on autophagy and apoptosis in Chinese hamster ovary cells

Sodium butyrate (NaBu), which is widely used in recombinant Chinese hamster ovary cell (rCHO) cultures for high-level expression of therapeutic proteins, is known to induce apoptosis in a dose-dependent manner. Lately, the significance of autophagy has increased in the field of CHO cell culture due to the fact that autophagy is related to the programmed cell death mechanism. To determine the effect of NaBu on autophagy as well as apoptosis of rCHO cells, rCHO cells producing erythropoietin were subjected to NaBu treatment. NaBu treatment up to 5 mM increased cleaved forms of PARP, caspase-3, and Annexin V positive population, confirming the previous results that NaBu induces apoptosis. Concurrently, NaBu treatment increased the level of accumulation of the autophagic marker, LC3-II, independently of nutrient depletion, suggesting that NaBu induces autophagy. To elucidate the potential role of autophagy induced by NaBu, a representative autophagy inducer (rapamycin) or an inhibitor (bafilomycin A1) was added to cultures together with NaBu. It was found that autophagy had the potential role of a positive cell survival mechanism under NaBu treatment. Furthermore, gradual reduction in mitochondrial membrane potential/mass and recruitment of a mitophagy protein, Parkin, to the mitochondria were observed under NaBu treatment, suggesting that this positive function of autophagy might be mediated by the autophagic removal of damaged mitochondria. Taken together, autophagy was observed in rCHO cell culture under NaBu treatments and the results obtained here support the positive effects of autophagy induced by NaBu treatments.     

A novel real-time PCR-based method for the detection of Listeria monocytogenes in food

AIMS: A new real-time PCR-based method was developed for the detection of Listeria monocytogenes in food. METHODS AND RESULTS: A two-step enrichment involving a 24-h incubation in half-Fraser broth followed by a 6-h subculture in Fraser broth was used, followed by cell lysis and real-time PCR with primers and a TaqMan probe previously developed in our laboratory. When the method was evaluated with 144 naturally contaminated food samples, 44 were detected as positive by the PCR-based method and 42 by the standard method EN ISO 11290-1. With 61 food samples artificially contaminated at a level of 10(0) CFU per 25 g, 61 and 58 positive samples were detected by the respective methods. CONCLUSIONS: The developed real-time PCR-based method facilitated the detection of L. monocytogenes in food on the next day after the sample reception, with a reduction of false-positive results because of dead bacterial cells and false-negative results because of PCR inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: The method can be used for L. monocytogenes detection in food as a faster alternative to current methods.     

Targeting AMPK-ULK1-mediated autophagy for combating BET inhibitor resistance in acute myeloid leukemia stem cells

Bromodomain and extraterminal domain (BET) inhibitors are promising epigenetic agents for the treatment of various subsets of acute myeloid leukemia (AML). However, the resistance of leukemia stem cells (LSCs) to BET inhibitors remains a major challenge. In this study, we evaluated the mechanisms underlying LSC resistance to the BET inhibitor JQ1. We evaluated the levels of apoptosis and macroautophagy/autophagy induced by JQ1 in LSC-like leukemia cell lines and primary CD34⁺ CD38^? leukemic blasts obtained from AML cases with normal karyotype without recurrent mutations. JQ1 effectively induced apoptosis in a concentration-dependent manner in JQ1-sensitive AML cells. However, in JQ1-resistant AML LSCs, JQ1 induced little apoptosis and led to upregulation of BECN1/Beclin 1, increased LC3 lipidation, formation of autophagosomes, and downregulation of SQSTM1/p62. Inhibition of autophagy by pharmacological inhibitors or knockdown of BECN1 using specific siRNA enhanced JQ1-induced apoptosis in resistant cells, indicating that prosurvival autophagy occurred in these cells. Independent of MTOR signaling, activation of the AMPK (p-Thr172)-ULK1 (p-Ser555) pathway was found to be associated with JQ1-induced autophagy in resistant cells. AMPK inhibition using the pharmacological inhibitor compound C or by knockdown of PRKAA/AMPKα suppressed autophagy and promoted JQ1-induced apoptosis in AML LSCs. These findings revealed that prosurvival autophagy was one of the mechanisms involved in the resistance of AML LSCs to JQ1. Targeting the AMPK-ULK1 pathway or inhibition of autophagy could be an effective therapeutic strategy for combating resistance to BET inhibitors in AML and other types of cancer.     

A one-step dual-labeling method for antigen detection in mast cells

This paper describes a one-step light microscopy method for demonstrating the antigen contents of unequivocally identified mast cells. It is based on the differential metachromatic properties of proteoglycans, mostly heparin and chondroitin sulfate, and 1-naphthol in the presence of toluidine blue in an acidic medium. Proteoglycans occur in all mast cells and 1-naphthol is used to demonstrate the peroxidase activity of the sections treated by the horseradish peroxidase-labeled avidin–biotin complex method for antigen detection. Granules containing proteoglycans present the classical metachromatic reaction by appearing purplish-red, while granules containing antigen appear a brilliant green. When both types of granules are distinct inside the cell, single- and double-stained cells can be accurately separated and counted. We hope that this new procedure will contribute to a further identification of mast cell mediator contents and to a better understanding of the physiology of this cellular population.     

Oxidative stress induces autophagy in response to multiple noxious stimuli in retinal ganglion cells

Retinal ganglion cells (RGCs) are the only afferent neurons that can transmit visual information to the brain. The death of RGCs occurs in the early stages of glaucoma, diabetic retinopathy, and many other retinal diseases. Autophagy is a highly conserved lysosomal pathway, which is crucial for maintaining cellular homeostasis and cell survival under stressful conditions. Research has established that autophagy exists in RGCs after increasing intraocular pressure (IOP), retinal ischemia, optic nerve transection (ONT), axotomy, or optic nerve crush. However, the mechanism responsible for defining how autophagy is induced in RGCs has not been elucidated. Accumulating data has pointed to an essential role of reactive oxygen species (ROS) in the activation of autophagy. RGCs have long axons with comparatively high densities of mitochondria. This makes them more sensitive to energy deficiency and vulnerable to oxidative stress. In this review, we explore the role of oxidative stress in the activation of autophagy in RGCs, and discuss the possible mechanisms that are involved in this process. We aim to provide a more theoretical basis of oxidative stress-induced autophagy, and provide innovative targets for therapeutic intervention in retinopathy.